Biosensor with external reader

Abstract

The present disclosure provides devices for sensing an analyte in a biological sample of a subject. Related methods are also disclosed.

Description

BIOSENSOR WITH EXTERNAL READERCROSS-REFERENCE

[0001] This application claims benefit of U.S. Provisional Patent Application No. 63 / 618,272 filed January 5, 2024, U.S. Provisional Patent Application No. 63 / 633,545 filed April 12, 2024, and U.S. Provisional Patent Application No. 63 / 661,558 filed June 18, 2024, each of which applications is entirely incorporated herein by reference.BACKGROUND

[0002] Analytes, such as ribonucleic acid (RNA), deoxyribonucleic acid (DNA), proteins, small molecules, or peptides, may be detected in a body of a subject and may be correlated with a health or physiological condition of the subject. However, most assays to detect such analytes are generally performed with low temporal frequency and require testing in a laboratory facility following sample collection.SUMMARY

[0003] The ability to measure and quantify analytes in real-time has many transformative applications in medicine such as in precision drug dosing, measuring the pharmacokinetic and pharmacodynamics of drugs during clinical trials, and for diagnosing and monitoring various diseases. As an exemplar, continuous glucose monitors (CGMs) have revolutionized the treatment and management of diabetes. Unfortunately, CGM technology is not broadly generalizable to measure other molecules since CGMs leverage naturally occurring enzymes which only exist for a few privileged molecules (e.g., glucose, lactate, ketones, and alcohol). Furthermore, the development of percutaneous and implantable biosensors is often stifled by the high costs and regulatory burden associated with developing a sterilizable biosensor system and protecting the biosensor from degradation and fibrotic encapsulation by the immune system. Therefore, there is a clear unmet need for strategies to develop real-time biosensors using analyte binding probes that can be developed against a large variety of molecules and that ease the manufacturing and regulatory burdens that plague other continuous biosensing technologies. The invention described herein is a miniaturized biosensing platform which can be configured to sense a large variety of analyte with molecular precision and which significantly eases many constraints typically associated with percutaneous biosensors by conducting the molecular recognition outside of the body.

[0004] In an aspect, the present disclosure provides a device for sensing an analyte in a biological sample of a subject, comprising a sensing domain that is a region, wherein molecular sensing occurs by having molecular sensors come in contact with an analyte carrying fluid, and that comprises of molecules that receives an excitation optical beam from a reader and emits the emission beam back to the reader, whereby, the emitted beam to the reader encodes the information on the concentration of the analyte that is being detected in the analyte carrying fluid; an apparatus that comprises the sensing domain and allows the interaction of the interstitial fluid with the sensing domain; an arrangement which allows the interaction of the interstitial fluid from the biological sample with the apparatus holding the sensing domain; an optical reader that is miniaturized, comprises an excitation optical beam that comes out of the optical reader and couples to the apparatus comprising the sensing domain and collects an emitted optical beam from the apparatus, processes the emitted beam into electrical signal and provides a user readable data; and a structural arrangement which allows optical coupling between the optical reader and the apparatus holding the sensing domain. In some embodiments, the apparatus comprises a microfluidic chip, wherein the microfluidic chip comprises one or more microfluidic channels. In some embodiments, the optical reader and the microfluidic chip are in close proximity to each other and held together for the duration of the measurement. In some embodiments, the microfluidic chip is comprised of areas that contain sensing domain and that are aligned with excitation and emission optical beam from and to the optical reader. In some embodiments, the microfluidic chip along with the optical reader reside outside the human body. In some embodiments, the microfluidic channels flow dialysate in it that is in contact with the areas of the sensing domain and interacts with it freely. In some embodiments, the sensing domains have molecular sensors that are designed to sense the analytes carried by the dialysate. In some embodiments, there are multiple sensing domains consisting of different molecular sensors designed to sense different analytes and placed along different parts of the microfluidic channels within the same microfluidic chip. In some embodiments, the coupling of the optical beams from the optical reader to the areas of different sensing domains along the microfluidic channels allow simultaneous sensing of multiple analyte concentrations continuously. In some embodiments, the arrangement that allows the interaction between the interstitial fluid and the apparatus holding the sensing domain is a pumped fluidic system such as a microdialysis unit. In some embodiments, the microdialysis unit interacts with a microfluidic unit using tubing. In some embodiments, the microdialysis unit comprises of a pump which is pumping a perfusate through a tubing that inserts into the interstitial fluid in the biologicalsample, the perfusate interacts and collects the analytes smaller than a certain size from the interstitial fluid and passes it out of the biological sample into the microfluidic chip using the tubing that connects the microdialysis unit to the microfluidic chip. In some embodiments, the analytes collected from the Interstitial fluid of the biological sample and pushed into the microfluidic channels of the microfluidic chip interact with the sensing domains in the microfluidic chip and transduce the optical signal that is coupled with the optical reader. In some embodiments, the area containing the sensing domain is larger than the spot size of the probing optical beam from the optical reader. In some embodiments, the area containing the sensing domain is uniform in its spatial density of the sensing molecule. In some embodiments, the area containing the sensing domain is non-uniform in its spatial density of the sensing molecules. In some embodiments, the smaller spot size than the area containing the sensing domain provides alignment tolerance between the reader and the microfluidic chip. In some embodiments, the apparatus holding the sensing domain is a microneedle array, wherein there is a base of the array which holds at least one microneedle in it. In some embodiments, the microneedle array is inserted into the biological sample and is in contact with the interstitial fluid of the biological sample. In some embodiments, the microneedle is hollow and the sensing domain resides inside the microneedle. In some embodiments, the arrangement that allows the interaction between the interstitial fluid of the biological sample and the sensing domain are holes at the tip of the microneedles. In some embodiments, the holes are arranged near the tip along the circumference of the microneedles. In some embodiments, the hole is the central lumen of the microneedle. In some embodiments, wherein there are holes both along the circumference and the central lumen. In some embodiments, the sensing domain resides at the tip of the microneedles. In some embodiments, wherein there is a multitude of microneedles that have different sensing domains designed to sense different analytes. In some embodiments, the multitude of microneedles containing different sensing domains are held together by a base to form a microneedle array that interacts with a probing optical beam from a reader. In some embodiments, an optical reader is optically coupled to the microneedle array. In some embodiments, the optical reader that is optically coupled to the microneedle array is abutting or in close proximity of the microneedle array. In some embodiments, the microneedles of the microneedle array are at least partially inserted in the biological sample to be in contact with the interstitial fluid while the optical reader abutting the microneedle array and the base of the microneedle array reside above the skin of the biological sample. In some embodiments, the microneedle and the optical reader system are designed to collect data for different targetanalytes in a multiplexed way. In some embodiments, the optical reader consists of at least one light source, a light detector, an excitation beam path to carry light from the source to the output of the reader, an emission beam path to collect light from a device that is coupled with the optical reader and bring it to the light detector, beam shaping elements in both the excitation and the emission optical paths such as lenses to collimate or focus light, optical filters in both the excitation and emission paths, optical apertures for ensuring off-angle light rejection, electronics circuits to drive light sources and to process collected light, and a mechanical housing to hold the reader together. In some embodiments, the optical reader consists of at least 1 laser source that is designed to emit light that in turn is designed to pass efficiently to the sensing domain and excite a light sensitive molecule in the sensing domain. In some embodiments, the light sensitive molecule is a fluorophore that is attached to an aptamer switch and the aptamer switch is designed to bind to a target analyte and upon binding goes through a conformational change in its structure that modulates the light coming out of the fluorophore and coupling into the optical reader. In some embodiments, the optical reader consists of at least 1 light detector that is designed to collect emitted light that in turn is emitted from the sensing domain and carries information on the concentration of the target analyte that the sensing domain is designed to sense. In some embodiments, the light detector has integrated electronics that processes the light signal and converts optical into electrical signals. In some embodiments, the light detector is an ambient light sensor that is made of silicon and is responsive to wavelengths from 400 nm to 1100 nm. In some embodiments, the light detector is an image sensor integrated chip. In some embodiments, the emitted light is at 630 nm and is emitted by a laser. In some embodiments, the emitted light is at 680 nm and is emitted by a laser that may either be an edge emitter or a VCSEL. In some embodiments, the light emitter at 680 nm is a VCSEL. In some embodiments, there is at least one excitation beam path that consists of a light source, a collimating lens, a dichroic filter, apertures, and a focusing lens that focuses light out of the reader and into the sensing domain, henceforth sensing domain focusing lens, wherein the focusing lens serves the dual purpose of focusing the light and collecting the emission light. In some embodiments, the device comprises an emission path that entails a longer wavelength than the excitation wavelength and that partially overlaps with the excitation path in going through the same focusing lens and the dichroic filter, has its own focusing lens, henceforth detector focusing lens, that is located after the emission path passes through the dichroic filter that focuses light on a detector. In some embodiments, the dichroic filter is designed to substantially reflect excitation light but to substantially transmit the emission light at a longer wavelength. In some embodiments, thedetector focusing lens distance from the detector is adjusted to obtain an appropriate spot size on the detector. In some embodiments, the spot size is configured to target specific pixels or a multitude of pixels on the detector. In some embodiments, some pixels have filters that make them more sensitive to either the excitation or the emission wavelengths. In some embodiments, the pixels which allow the excitation light to pass through are used to monitor the background spurious light that contributes to reducing the dynamic range of the sensor. In some embodiments, the device is configured to have multiple optical beams coming out of it by having a multitude of light emitters, multiple detectors, multiple beam paths for both excitation and emission, lenses, filters, and apertures, and wherein each beam interacts with a separate sensing domain allowing a functional ability to measure multiple analytes.

[0005] In another aspect, the present disclosure provides a device for sensing a biological sample of a subject, comprising: a probe comprising an inner lumen and an outer lumen, wherein the inner lumen is concentric with the outer lumen, wherein the inner lumen is fluidically coupled to the outer lumen, and wherein the probe outer lumen is fluidically coupled to a sensing domain; a piercing element configured to pierce a body surface of a subject to provide the probe in contact with a biological sample within the body of the subject; and a pump fluidically coupled to the inner lumen of the probe, wherein the sensing domain and the pump are provided adjacent to the body surface of the subject. In some embodiments, an end of the inner lumen is fluidically coupled to an end of the outer lumen. In some embodiments, the biological sample within the body of the subject diffuses into the fluid through the inner lumen of the probe, through the outer lumen of the probe, or a combination thereof. In some embodiments, the inner lumen of the probe, the outer lumen of the probe, or a combination thereof, comprise a semipermeable membrane, and wherein the biological sample diffuses through the semipermeable membrane into the fluid forced through the inner lumen of the probe, into the fluid forced through the outer lumen of the probe, or a combination thereof. In some embodiments, the biological sample is sensed by the sensing domain when: (i) the biological sample diffuses into the fluid forced through the inner lumen or the fluid forced through the outer lumen of the probe, and (ii) the biological sample couples to a molecular sensor of the sensing domain, the subject comprises a human subject. In some embodiments, the sensing domain comprises a microfluidic chip, wherein the microfluidic chip comprises one or more microfluidic channels. In some embodiments, the molecular sensor is disposed within a channel of the one or more microfluidic channels. In some embodiments, the piercing element is a retractable needle. In some embodiments, the body surface of the subject comprises a skin surface of the subject. In some embodiments, thedevice further comprises a wearable housing, wherein the wearable housing comprises the sensing domain and the pump. In some embodiments, the wearable housing comprises the probe, the piercing element, the sensing domain, and the pump, and wherein the wearable housing covers at least a portion of the body surface of the subject. In some embodiments, the wearable housing is provided in contact with the body surface of the subject. In some embodiments, the probe is provided within the piercing element. In some embodiments, the probe is concentric with the piercing element. In some embodiments, the wearable housing comprises an adhesive, wherein the adhesive couples the wearable housing to the body surface of the subject. In some embodiments, the fluid comprises a dialysate, the dialysate comprises a dialysate buffer. In some embodiments, the pump is fluidically coupled to the outer lumen of the probe. In some embodiments, the pump is configured to force a fluid through the inner lumen or the outer lumen of the probe through to the sensing domain. In some embodiments, the probe comprises an open flow microperfusion probe.

[0006] In another aspect, the present disclosure provides a system comprising a biosensor with an external reader.

[0007] In another aspect, the present disclosure provides a method for biosensing with an external reader.

[0008] In another aspect, the present disclosure provides a device for sensing an analyte in a biological sample of a subject, comprising a semipermeable membrane with an inlet and outlet fluidic path, wherein the semipermeable membrane is contacted with interstitial fluid of a subject allowing analytes to diffuse across the semipermeable membrane; a dialysate buffer which is connected to the inlet of the semi-permeable membrane; a mechanism of providing flow of the dialysate buffer through the device either continuously or at scheduled intervals; a flow-cell with an inlet and outlet, the inlet of the flow-cell is attached to the outlet of the semi-permeable membrane; analyte binding probes which are contained within the flow-cell and configured to change signal in response to analyte binding; and a detector operatively coupled to the flow-cell, wherein the detector is configured to detect a change in signal from the analyte binding probes. In some embodiments, two or more analytes are measured using different analyte binding probes. In some embodiments, the multiple analyte binding probes are spatially separated. In some embodiments, the multiple analyte binding probes are labeled with spectrally different optical reporters. In some embodiments, one of the analyte binding probes measures a reference molecule. In some embodiments, the reference molecule is used to correct for recovery rates <100%. In some embodiments, the two or more analytes are measured and used to correct for recovery rate. In someembodiments, the alteration in recovery rate is caused by the foreign body response (e.g., fibrotic encapsulation). In some embodiments, the signal from two different analyte probes are used ratiometrically. In some embodiments, an exogenous biocompatible molecule is included within the dialysate buffer to serve as a reference molecule that decreases in concentration with increased recovery rate, and this decrease is measured to correct for changes in recovery rate. In some embodiments, the analyte binding probe is an aptamer switch. In some embodiments, the analyte binding probe is a dual-antibody switch. In some embodiments, the analyte binding probe is antibody-aptamer chimeric switch. In some embodiments, the analyte binding probe is a protein-based switch composed of two or more analyte binding regions. In some embodiments, the detector detects a change in optical signal. In some embodiments, the optical signal is fluorescence intensity. In some embodiments, the optical signal is FRET ratio. In some embodiments, the optical signal is fluorescence lifetime. In some embodiments, the detector is an electrical detector measuring an electrochemical signal generated by a change in distance between a redox reporter and a surface of an electrode caused by a target-binding induced conformational change of an analyte binding probe. In some embodiments, the detector is detecting an enzymatic electrochemical signal measuring a change in enzymatic reaction rate. In some embodiments, the analyte binding probes immobilized on a surface within the flow cell. In some embodiments, the analyte binding probes are contained within a polymeric matrix within the flow cell. In some embodiments, the analyte binding probe is attached to a bead or particle within the flow cell. In some embodiments, the analyte is vancomycin. In some embodiments, the analyte is methoxy. In some embodiments, the device is used to measure pharmacokinetic and / or pharmacodynamic properties of the drug. In some embodiments, a reference molecule is DHEA-S. In some embodiments, the analyte measured is cortisol. In some embodiments, the reference molecule included within the dialysate buffer is maltose. In some embodiments, the dialysate fluid is recirculated through the device. In some embodiments, the device contains a battery configured to supply power to the device for at least 4 hours. In some embodiments, the device consists of a re-usable part and disposable part. In some embodiments, the reusable part comprises a pump, optical detector, and light source. In some embodiments, the disposable part comprises a semi-permeable probe, dialysate, and flow cell containing analyte binding probes. In some embodiments, a portion or the entirety of the dialysate is spatially separated and stored periodically as fractions. In some embodiments, the dialysate is stored within tubes or vials. In some embodiments, the dialysate is stored within a length of tubing. In some embodiments, the dialysate is stored within a microfluidic chip. In someembodiments, the fractions are separated into different containers. In some embodiments, the fractions are separated by segments of air or gas. In some embodiments, the fractions are separated by a valve. In some embodiments, the fractions are separated by a size exclusion membrane. In some embodiments, the fractions are separated by use of a fraction collector.

[0009] In another aspect, the present disclosure provides a device for sensing an analyte in a biological sample of a subject, comprising a piercing element configured to pierce a body surface of a subject, to thereby bring the piercing element in contact with a biological sample, wherein the piercing element comprises an inlet and an outlet, and wherein the outlet is fluidically coupled to a sensing domain; a pump fluidically coupled to the inlet; wherein the sensing domain is configured to receive a fluid from the piercing element, and wherein the sensing domain and the pump are provided adjacent to the body surface of the subject. In some embodiments, the device comprises a second pump fluidically coupled to the outlet. In some embodiments, the second pump provides a negative pressure through the outlet. In some embodiments, the pump provides a positive pressure through the inlet. In some embodiments, the pump provides a flow rate of fluid at from about 0.001 pL / min to about 10 pL / min. In some embodiments, the pump provides a flow rate of fluid at from about 0.05 pL / min to about 2 pL / min. In some embodiments, the sensing domain and the pump are provided on the body surface of the subject. In some embodiments, the device comprises a wearable housing, wherein the wearable housing comprises the sensing domain and the pump, and wherein the wearable housing covers at least a portion of the body surface of the subject. In some embodiments, a portion of the wearable housing may be coupled to the body surface of the subject with an adhesive. In some cases, the body surface may comprise a skin surface of the subject.

[0010] In another aspect, the present disclosure provides a device for sensing an analyte in a biological sample of a subject, comprising a piercing element configured to pierce a body surface of a subject, to thereby bring the piercing element in contact with a biological sample, wherein the piercing element comprises a semipermeable membrane comprising an inlet and an outlet, and wherein the outlet is fluidically coupled to a sensing domain; a dialysate buffer fluidically coupled to the inlet; and an optical reader optically coupled to the sensing domain, wherein the optical reader and the sensing domain are provided adjacent to the body surface of the subject. In some embodiments, the dialysate buffer is sterile. In some embodiments, the dialysate buffer comprises electrolytes in interstitial fluid of the biological sample. In some embodiments, the dialysate buffer comprises a reference molecule to determine the degree of equilibration between the dialysate buffer and the interstitial fluid. In some embodiments, thedialysate buffer comprises one or more molecules that alter an osmotic pressure between the interstitial fluid and the dialysate buffer. In some embodiments, the one or more molecules that alter the osmotic pressure are albumin or large molecular-weight dextran. In some embodiments, the dialysate buffer comprises one or more molecules that promote collection of the analyte. In some embodiments, the dialysate buffer comprises an anti-inflammatory compound. In some embodiments, the analyte is a lipophilic molecule. In some embodiments, the semipermeable membrane comprises pores reduce a concentration of one or more molecules with a molecular weight of at least about 6 kDa, at least about 10 kDa, at least about 50 kDa, at least about 100 kDa, at least about 150 kDa, or at least about 200 kDa, from diffusing into the dialysate buffer compared to a semipermeable membrane without the pores. In some embodiments, the sensing domain comprises a microfluidic chip, wherein the microfluidic chip comprises one or more microfluidic channels. In some embodiments, the microfluidic chip further comprises one or more flow cells fluidically coupled to the piercing element. In some embodiments, the one or more flow cells contain an analyte binding probe. In some embodiments, the one or more flow cells are calibrated using a solution containing the analyte. In some embodiments, a flow rate of the one or more flow cells are adjusted at a scheduled frequency. In some embodiments, the flow rate of the one or more flow cells are adjusted according to a change of signal generated by the sensing domain. In some embodiments, the change of signal generated by the sensing domain at various flow rates of the one or more flow cells are used for correcting or calibrating the device. In some embodiments, the analyte binding probe is configured to provide a change in an emitted optical signal by the analyte binding probe when the analyte binding probe comes in contact with the analyte in the biological sample of the subject. In some embodiments, the analyte binding probe receives an excitation optical beam from the optical reader and emits the emission beam back to the optical reader. In some embodiments, the excitation optical beam is defocused at a plane of the sensing domain up to an area of 1 square mm. In some embodiments, the excitation optical beam is defocused through the inclusion of material between the flow cell and the optoelectronic reader. In some embodiments, a perfusate exiting from the microfluidic chip is collected in a container. In some embodiments, the container is coupled to the sensing domain. In some embodiments, a portion of the perfusate is collected in one or more containers. In some embodiments, a size of the portion of the perfusate is from about 5 pL to about 50 pL. In some embodiments, a size of the portion of the perfusate is from about 0.05 pL to about 500 pL. In some embodiments, the portion of the perfusate is collected in from about 1 minute to 24 hours. In some embodiments, the portionof the perfusate is collected in from about 5 minute to 6 hours. In some embodiments, the perfusate in the container is examined under a microscope. In some embodiments, the perfusate in the container mixes with an analyte binding probe that receives an excitation optical beam. In some embodiments, the piercing element is concentric with the semipermeable membrane. In some embodiments, the inlet and the outlet are located at one end of the piecing element, and the semipermeable membrane is located at the other of the piercing element. In some embodiments, the piercing element is disposed colinear with the semipermeable membrane. In some embodiments, the sensing domain comprises non- molecular sensors. In some embodiments, the non-molecular sensors comprise humidity sensors, motion sensors, temperature sensors, or a combination thereof. In some embodiments, wherein the sensing domain comprises molecular sensors. In some embodiments, the sensing domain comprises an electrical detector measuring an electrochemical signal generated by a change in charge configuration near an electrode surface due to the addition or subtraction of charge due to analyte binding or target-binding induced conformational change of an analyte binding probe or a combination of these effects. In some embodiments, the sensing domain comprises an electrical detector measuring an electrochemical signal generated by a change in electron transfer rate between a redox reporter and a surface of an electrode caused by a target-binding induced conformational change of an analyte binding probe. In some embodiments, the sensing domain comprises an electrical detector measuring an electrochemical signal generated by a change in electron transfer rate between a redox reporter immobilized within a molecular pendulum construct and an electrode surface caused by analyte binding to the molecular pendulum which modulates mass and drag of the pendulum. In some embodiments, the sensing domain comprises an optical detector measuring a change in mobility of a particle generated by binding of an analyte to a receptor on the particle or microfluidic chip surface which modulates a binding interaction between analyte or receptors functionalized on both the particle and the surface and thus the degree of motion of the particle relative to the surface. In some embodiments, the sensing domain is calibrated using a solution, wherein the solution produces a measurable fluorescent signal. In some embodiments, the optical reader comprises a light emitter. In some embodiments, the light emitter is on a printed circuit board (PCB) and emits laser beam that is actively aligned with the optical reader. In some embodiments, the active alignment is performed by registering an aperture array to reader mechanics that support an optical path of the laser beam. In some embodiments, the active alignment is performed by optimizing excitation power through an aperture array that is registered tooptical path of the laser beam. In some embodiments, the sensing domain and the optical reader are provided on the body surface of the subject. In some embodiments, the device comprises a wearable housing, wherein the wearable housing comprises the sensing domain and the optical reader, and wherein the wearable housing covers at least a portion of the body surface of the subject.

[0011] In another aspect, the present disclosure provides a device for sensing an analyte in a biological sample of a subject, comprising a piercing element configured to pierce a body surface of a subject, to thereby bring the piercing element in contact with a biological sample, wherein the piercing element comprises a semipermeable membrane, wherein the piercing element comprises an inlet and an outlet, wherein the outlet is fluidically coupled to a sensing domain, wherein the sensing domain comprises a microfluidic chip comprising one or more flow cells fluidically coupled to the piercing element, and wherein the one or more flow cells contain an analyte binding probe; a pump fluidically coupled to the inlet, wherein the pump provides a positive pressure to push a dialysate through the inlet and to the semipermeable membrane; and an optical reader optically coupled to the sensing domain, wherein the analyte binding probe receives an excitation optical beam from the optical reader and emits the emission beam back to the optical reader.

[0012] In another aspect, the present disclosure provides a method for sensing an analyte in a biological sample of a subject, the method comprising piercing a body surface of a subject, to thereby bring a piercing element in contact with a biological sample; pumping a dialysate buffer through an inlet of the piercing element; providing interstitial fluid of the biological sample in contact with the dialysate buffer, wherein a perfusate passes through the outlet of the piecing element; contacting the perfusate with a sensing domain and collecting the perfusate in an output reservoir, wherein the sensing domain is fluidically coupled to an outlet of the piercing element; collecting an emitted optical beam from the sensing domain via an optical reader, wherein the optical reader is optically coupled to the sensing domain; processing the emitted beam into electrical signal; and providing a user readable data. In some embodiments, a recovery rate of the analytes is at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the method further comprises a second pump fluidically coupled to the outlet. In some embodiments, the second pump provides a negative pressure through the outlet. In some embodiments, the pump provides a positive pressure through the inlet. In some embodiments, the pump provides a flow rate of fluid at from about 0.001 pL / min to about 10 pL / min. In some embodiments, the pump provides a flow rate of fluid at from about 0.05pL / min to about 2 pL / min. In some embodiments, the dialysate buffer is sterile. In some embodiments, the dialysate buffer comprises electrolytes in interstitial fluid of the biological sample. In some embodiments, the dialysate buffer comprises a reference molecule. In some embodiments, a degree of equilibration between the dialysate buffer and the interstitial fluid is determined by comparing the reference molecule in the dialysate buffer to the reference molecule in the interstitial fluid. In some embodiments, the dialysate buffer comprises one or more molecules that alter an osmotic pressure between the interstitial fluid and the dialysate buffer. In some embodiments, the one or more molecules that alter the osmotic pressure are albumin or large molecular-weight dextran. In some embodiments, the dialysate buffer comprises one or more molecules that promote collection of the analyte. In some embodiments, the analyte is a lipophilic molecule. In some embodiments, the semipermeable membrane comprises pores that reduce a concentration of one or more molecules with a molecular weight of at least about 6 kDa, at least about 10 kDa, at least about 50 kDa, at least about 100 kDa, at least about 150 kDa, or at least about 200 kDa, from diffusing into the dialysate buffer compared to a semipermeable membrane without the pores. In some embodiments, the sensing domain comprises a microfluidic chip, wherein the microfluidic chip comprises one or more microfluidic channels. In some embodiments, the microfluidic chip further comprises one or more flow cells fluidically coupled to the piercing element. In some embodiments, the one or more flow cells contain an analyte binding probe. In some embodiments, the analyte binding probe is configured to provide a change in an emitted optical signal by the analyte binding probe when the analyte binding probe comes in contact with the analyte in the biological sample of the subject. In some embodiments, the analyte binding probe receives an excitation optical beam from the optical reader and emits the emission beam back to the optical reader. In some embodiments, a perfusate exiting from the microfluidic chip is collected in a container. In some embodiments, the container is coupled to the sensing domain. In some embodiments, a portion of the perfusate is collected in one or more containers. In some embodiments, a size of the portion of the perfusate is from about 5 pL to about 50 pL. In some embodiments, a size of the portion of the perfusate is from about 0.05 pL to about 500 pL. In some embodiments, the portion of the perfusate is collected in from about 1 minute to 24 hours. In some embodiments, the portion of the perfusate is collected in from about 5 minute to 6 hours. In some embodiments, the perfusate in the one or more containers is examined under a microscope. In some embodiments, the perfusate in the one or more containers mixes with an analyte binding probe that receives an excitation optical beam. In some embodiments, the piercing element is concentric with thesemipermeable membrane. In some embodiments, the inlet and the outlet are located at one end of the piecing element, and the semipermeable membrane is located at the other of the piercing element. In some embodiments, the piercing element is disposed colinear with the semipermeable membrane. In some embodiments, the inlet is located at one end of the piercing element, and the outlet is located at the other end of the piercing element.

[0013] In another aspect, the present disclosure provides for a device for sensing an analyte in a biological sample of a subject, the device comprising: a piercing element configured to pierce a body surface of a subject, to thereby bring the piercing element in contact with a biological sample, wherein the piercing element comprises an inlet and an outlet, and wherein the outlet is fluidically coupled to a sensing domain; and a pump fluidically coupled to the inlet; wherein the sensing domain is configured to receive a fluid from the piecing element, and wherein the sensing domain and the pump are provided adjacent to the body surface of the subject. In some embodiments, the microdialysis probe is inserted into the skin of the subject through the use of an applicator. In some embodiments, the microdialysis probe is of a concentric design.

[0014] A variety of minimally invasive, transdermal biosensors are available for analyte detection, but these biosensors face multiple limitations, as recognized herein, including prolonged response time, lack of precise specificity, high background noise, and lack of multiplexed detection capability. Such biosensors may not have the appropriate response functionality or ligand specificity for detection of an analyte in interstitial fluid, such as skin interstitial fluid. Recognized herein is an unmet need for improved biosensors which can perform an assay for analyte detection at the point of care which can deliver a result to a subject shortly following sample collection, and improved biosensors which can perform multiplexed or continuous assays for ongoing monitoring of an analyte, including the concentration of an analyte. Disclosed herein are biosensors for minimally invasive, continuous, real-time measurement of one or more target analytes in the body of a subject from a biological sample. In some cases, the biological sample is interstitial fluid (e.g., skin interstitial fluid).

[0015] Provided herein is a device for sensing an analyte in a biological sample of a subject, comprising: a support; a piercing element coupled to the support, wherein the piercing element is configured to pierce a body surface of the subject when the device is coupled to the body surface, to thereby bring the piercing element in contact with the biological sample; an analyte binding probe on or within the piercing element, wherein the analyte binding probe is configured to provide a change in an optical signal when the analyte binding probecomes in contact with the analyte in the biological sample of the subject; a detector operatively coupled to the support, wherein the detector is configured to detect the optical signal; and at least one of: an optical excitation path comprising a waveguide and a light coupler and: i) comprising one or more focusing elements configured to focus light from a light source towards the light coupler, ii) a reflective cladding layer surrounding the waveguide configured to reflect the light back inside the waveguide and guide the light toward the piercing element; or iii) the light coupler being configured to couple light from a light source into the waveguide; or an optical emission path comprising: i) apertures in a reflective cladding layer surrounding a waveguide configured to transmit an emission light from the analyte probe towards the detector, or ii) one or more focusing elements configured to focus emission light from the analyte binding probe towards the detector; or comprising a waveguide, an optical excitation path comprising the waveguide, and an optical emission path comprising the waveguide, wherein the waveguide transmits light from a light source to the analyte binding probe, and wherein the waveguide is configured to permit light to be transmitted through the waveguide from the analyte binding probe to the detector; or wherein the analyte binding probe is comprised in a hydrogel matrix in the sensing domain, wherein the analyte binding probe is comprised on one or more beads comprised by the hydrogel matrix, or wherein the analyte binding probe has a dissociation constant of at least 1 pM with respect to the analyte. In some embodiments, including a light source, wherein the light source emits light at wavelengths of 600 nm to 750 nm. In some embodiments, including a light source, wherein the light source emits light at wavelengths of 350nm to 1500nm. In some embodiments, the light source emits light within a 50 nm band within the wavelengths of 600 nm to 750 nm. In some embodiments, the light source emits light within a 50 nm band within the wavelengths of 350nm to 1500nm. In some embodiments, the optical waveguide is configured to transmit light at wavelengths of 350 nm to 1500 nm. In some embodiments, the optical emission path is configured to reject light from the optical excitation path. In some embodiments, the optical emission path is configured to reject at least 75% of light from the optical excitation path. In some embodiments, the optical emission path is configured to reject light that is a which is a same wavelength as light emitted by the light source. In some embodiments, the optical emission path is configured to reject light which is a same wavelength through the optical waveguide from the light source In some embodiments, the optical excitation path and the optical emission path are configured to minimize interference with each other. In some embodiments, the optical excitation path and the optical emission path are partially co-located within the optical waveguide. In some embodiments, theexcitation path is configured to transmit light in a vertical direction and a horizontal direction. In some embodiments, the emission path is configured to transmit light in a vertical direction. In some embodiments, the emission path is configured to transmit light only in a vertical direction. In some embodiments, the reflective cladding layer comprises an air gap. In some embodiments, at least a portion of the air gap is positioned between a base of the piercing element and the optical waveguide. In some embodiments, the air gap is configured to reflect light towards the piercing element. In some embodiments, the air gap has a lower refractive index than the waveguide. In some embodiments, the waveguide comprises a refractive index of greater than 1. In some embodiments, the waveguide comprises a geometrical core that is made of a dielectric material, wherein the reflective cladding surrounding the dielectric core is a reflective metal or a dielectric material of lower refractive index that the dielectric core, optionally, wherein the reflective metal comprises Al or Ag. In some embodiments, the waveguide comprises a geometrical cross section that is a rectangle. In some embodiments, the two dimensions of the rectangle range from 100 microns to 5 mm. In some embodiments, the optical waveguide is configured to only transmit the excitation light to the piercing element, and wherein the optical waveguide is a dielectric waveguide comprising the reflective cladding, wherein light is configured to enter the optical waveguide within a narrow range of angles around the surface normal of the top, light-source facing side of the waveguide. In some embodiments, the range of the angles is 40-50 degrees as measured from the surface normal of the bottom coupling surface of the waveguide. In some embodiments, including a light coupler. In some embodiments, the light coupler is configured to transmit light from the light source to a waveguide. In some embodiments, the waveguide comprises a dielectric core, wherein the reflective cladding surrounds the dielectric core. In some embodiments, the reflective cladding comprises a metal, Al, or Ag. In some embodiments, the reflective cladding comprises an aperture between the light source and the light coupler and the aperture is defined by the absence of the reflective cladding. In some embodiments, the dielectric core surrounded by the reflective cladding is configured to confine light within the waveguide. In some embodiments, the waveguide comprises a protruding portion which extends into a base of the piercing element. In some embodiments, the waveguide comprises a connecting portion which extends into a base of the piercing element. In some embodiments, the excitation path comprises connecting materials between the waveguide and the location of the analyte probe that optically extracts light from the waveguide towards the piercing element and the analyte probe. In some embodiments, the connecting material is an extension of the dielectric core material of the waveguide. In some embodiments, theconnecting material is different than the core of the waveguide and comprises a substantially similar refractive index as the core of the wave guide, and, optionally, comprises multiple layers. In some embodiments, the piercing element comprises a reflective material about a base of the piercing element. In some embodiments, the optical excitation path guides light from a light source through the protruding portion of waveguide and to the reflective material about the base of the piercing element, wherein the reflective material about the base of the piercing element reflects the light to the analyte binding probe on or within the piercing element. In some embodiments, the optical excitation path is configured to reject light which does not enter the waveguide at a critical angle. In some embodiments, the critical angle is between ±20 degrees as measured from a vector normal to a base of the piercing element. In some embodiments, the critical angle is an angle which is approximately 0 degrees from the vector normal to the base of the piercing element. In some embodiments, including an opaque layer comprising apertures, wherein light must enter the optical excitation path at the critical angle to pass through the apertures. In some embodiments, the optical emission path comprises a first focusing element configured to focus at least part of the emission light from the analyte binding probe into a first beam. In some embodiments, the first beam comprises light rays that are substantially parallel to each other and substantially perpendicular to the surface of the detector. In some embodiments, the first beam is configured to be incident on one or more pixels of the detector. In some embodiments, including a second focusing element configured to focus the first beam towards the detector. In some embodiments, a diameter of the second beam coming from the second focusing element is configured to be a smaller size diameter than the first beam when the second beam crosses the plane of the detector and is incident on one or more pixels of the detector, thereby improving the signal to noise ratio of the detector. In some embodiments, an optical path of the first beam is isolated from an optical path of the excitation light. In some embodiments, including an optical filter between the first beam and the detector which does not transmit a portion of light that falls on the filter. In some embodiments, the portion of the light blocked from transmission by filter has a wavelength which is a same wavelength as the light source. In some embodiments, the optical filter is a dichroic filter, an absorptive filter, or a combination thereof. In some embodiments, the first focusing element transmits light to the filter at an angle within approximately + / - 25 degrees with respect to a surface normal vector of the filter. In some embodiments, the first focusing element transmits light to the filter at an angle of approximately 0 degrees with respect to the surface normal vector of the filter. In some embodiments, the portion of light not transmitted through the filter comprises wavelengths oflight less than 700 nm in wavelength. In some embodiments, the optical filter substantially transmits emission light wavelengths coming from the analyte probe, while substantially blocking light which is a similar wavelength as excitation light. In some embodiments, the optical filter is configured to block at least 99, 99.9, 99.99, 99.999, 99.9999, 99.99999, 99.99999% of light having a same wavelength as light from the light source transmitted through the excitation path. In some embodiments, having a same wavelength as light from the light source transmitted through the emission path. In some embodiments, including a second optical filter, wherein the second optical filter is a dichroic filter, an absorptive filter, or a combination thereof. In some embodiments, the first optical filter and the second optical filter in combination transmit emission light while substantially blocking light which is a similar wavelength as excitation light. In some embodiments, including a light source. In some embodiments, the light source is a downward firing light source positioned above the pierceable member. In some embodiments, including an opaque layer comprising apertures, wherein light must enter the optical excitation path at the critical angle to pass through the apertures. In some embodiments, the light source is positioned on a first side of the opaque layer, and wherein the detector is positioned on a opposite side of the opaque layer. In some embodiments, including an opaque material with an aperture in the emission path that is configured to block transmission of excitation light that does not strike the optical filter within approximately + / -25 degrees with respect to a vector normal to the surface of the optical filter. In some embodiments, the optical excitation path further comprises an optical filter between the light source and the light coupler. In some embodiments, the optical filter is a dichroic filter, an absorptive filter, or a combination thereof. In some embodiments, the analyte binding probe is an aptamer switch, or an antibody switch. In some embodiments, the light coupler comprises a faceted mirror. In some embodiments, the faceted mirror is configured to direct light towards the waveguide. In some embodiments, the faceted mirror is configured to direct light from a section of free space optics towards the waveguide. In some embodiments, the faceted mirror is partially metalized. In some embodiments, the faceted mirror is fully metalized. In some embodiments, the faceted mirror is non-metalized. In some embodiments, including an optoelectronics system, the optoelectronics system comprising the detector, wherein the optoelectronics system is operatively coupled to the support, and wherein the optoelectronic system is configured to detect the change in the optical signal using the detector. In some embodiments, including an excitation light source. In some embodiments, the excitation light source comprises a laser, or an LED. In some embodiments, including an electrical system coupled to the detector that processes the changeand generates an electrical signal related to the concentration of the analyte. In some embodiments, the optical excitation path comprises a section of free space optics between a light source and a waveguide. In some embodiments, the optical emission path comprises a section of free space optics between where the emission light starts from the analyte probe area and a detector. In some embodiments, the device further comprises a battery. In some embodiments, the light source is configured to increase a single battery cycle life of the device. In some embodiments, the detector is configured to increase a single battery cycle life of the device. In some embodiments, the optical excitation path is configured to increase light coupling efficiency from the excitation light source to the analyte probe, and is configured to increase a single battery cycle life of the device. In some embodiments, the optical emission path is configured to increase light coupling efficiency from the analyte probe to a detector, and is configured to increase a single battery cycle life of the device. In some embodiments, the piercing element comprises a structural domain, a barrier domain, and a sensing domain. In some embodiments, the hydrogel matrix is based on PEG or PVA. In some embodiments, the PEG-based hydrogel is comprised of PEGDMA monomer subunits with a weight of about 750 Daltons to 20 kilodaltons at weight percentages of about 5 % to about 30 %. In some embodiments, the PEGDMA monomer subunits are photocrosslinked or chemically crosslinked. In some embodiments, the analyte binding probe is comprised in a sensing domain comprising a hydrogel matrix comprising one or more particles comprising the analyte binding probe. In some embodiments, the particles comprise a polymer, optionally, wherein the polymer is polystyrene. In some embodiments, the particles are magnetic. In some embodiments, the particles comprise a diameter of about 100 nm to about 1000 microns. In some embodiments, the particles comprise a diameter of at least 100 nm. In some embodiments, the particles comprise a diameter of up to about 100 microns. In some embodiments, the particles comprise a diameter of about 1 micron to about 50 microns. In some embodiments, the number of particles in a single sensor is about 1 particle to about 10A9particles. In some embodiments, the number of particles in a single sensor is at least 10A3 particles. In some embodiments, the number of particles in a single sensor is at least 10A5 particles. In some embodiments, the number of particles in a single sensor is about 10A5 particles to about 10A8 particles. In some embodiments, the single sensor is an individual member of the piercing element. In some embodiments, the single sensor is located in a well separate from the piercing element, and is fluidically connected to the piercing element. In some embodiments, a concentration of particles in the sensing domain ranges from 10A3 particles to 10A9 particles / mL. In some embodiments, aconcentration of particles in the sensing domain ranges from 10A5 particles to 10A8 particles / mL. In some embodiments, the concentration of analyte binding probes on a surface of the one or more particles is about 10A6 to about 5*10A13 analyte binding probes per cmA2. In some embodiments, the concentration of analyte binding probes on a surface of the one or more particles is about 10Al 0 to about 10Al 3 analyte binding probes per cmA2. In some embodiments, the analyte binding probe is an aptamer, wherein the concentration of analyte binding probes on a surface of the one or more particles is about 10A6 to about 5*10A13 DNA strands per cmA2. In some embodiments, the analyte binding probe is an aptamer, wherein the concentration of analyte binding probes on a surface of the one or more particles is about 10A1° DNA strands / cmA2to about 10A13DNA strands / cmA2. In some embodiments, the analyte binding probe is an aptamer, wherein an average spacing of the analyte binding probes ranges from about 1.4 nm to about 10 micrometers between DNA strands. In some embodiments, the analyte binding probe is an aptamer, wherein an average spacing of the analyte binding probes ranges from about 3 nm to about 100 nm between DNA strands. In some embodiments, including a light source. In some embodiments, the device is configured to be placed in optical communication with an external light source. In some embodiments, including two or more analyte binding probes on or within the piercing element, each of the two or more analyte binding probes configured to provide a change in an optical signal when the two or more analyte binding probes contact a second analyte in the biological sample of the subject. In some embodiments, including two or more analyte binding probes on or within the piercing element, each of the two or more analyte binding probes configured to provide a change in an optical signal when the two or more analyte binding probes contact a second analyte in the biological sample of the subject. In some embodiments, the analyte binding probe comprises a fluorophore conjugated to an aptamer configured to bind to the analyte, wherein the analyte binding probe is configured to contact the biological sample of the subject and provide the change in an optical signal when the piercing element is inserted into a skin of a subject. In some embodiments, including a multiplexed array of analyte binding probes on or within the piercing element, each of the multiplexed array of analyte binding probes configured to provide a change in an optical signal when the multiplexed array of analyte binding probe contacts a subsequent analyte in the biological sample of the subject. In some embodiments, the subsequent analyte is a different analyte, the multiplexed array of analyte binding probes is configured to detect a plurality of analytes in the biological sample of the subject. In some embodiments, the piercing element comprises a plurality of piercing elements. In some embodiments, the plurality of piercing elements defines subsets of piercingelements, wherein each subset of piercing elements comprises a different analyte binding probe configured to detect a different analyte in the biological sample of the subject. In some embodiments, the subsets of piercing elements comprising the different analyte binding probes comprises the multiplexed array of analyte binding probes. In some embodiments, the waveguide comprises a polymer glassy matrix comprising dispersed photoluminescent particles. In some embodiments, the polymer comprises silicones, polysiloxanes, silsequioxanes, or combinations thereof. In some embodiments, the polymer comprises: polymethylmethacrylate (PMMA), polystyrene (PS), polycarbonate (PC), polyurethane (PU), epoxy resin, deuterated and halogenated poly acrylates, fluorinated polyimides, perfluorocyclobutyl (PFCB) aryl ether polymers, nonlinear optical polymers, benzocyl cobutene (BCB), perfluorovinyl ether cyclopolymer (CYTOP), tetrafluoroethylene and perfluorovinyl ether copolymer (Teflon AF), silicone, fluorinated poly (arylene ether sulfide), poly(pentafluorostyrene), fluorinated dendrimers, fluorinated hyperbranched polymers, or combinations thereof. In some embodiments, the piercing element is coupled to the support using a lock and key attachment. In some embodiments, the piercing element is coupled to the support using a mortise and tenon attachment. In some embodiments, the piercing element is coupled to the support using a dovetail attachment. In some embodiments, the piercing element is coupled to the support using a magnetic attachment. In some embodiments, the piercing element is coupled to the support using an adhesive. In some embodiments, the piercing element is coupled to the support using one or more elastically deformable attachment elements. In some embodiments, the piercing element is coupled to the support using one or more elastically deformable attachment elements configured to rebound once inserted into the support. In some embodiments, the piercing element is removably coupled to the support. In some embodiments, the piercing element comprises a microneedle array which is removably coupled to the support. In some embodiments, the optical light guide is removably coupled to the support. In some embodiments, the analyte binding probe coupled to the optical reporter is an oligonucleotide probe. In some embodiments, the oligonucleotide probe is an aptamer. In some embodiments, the aptamer is coupled to a displacement strand, wherein the displacement strand is partially complementary to the aptamer. In some embodiments, the displacement strand is coupled to a second optical reporter. In some embodiments, the second optical reporter is a fluorophore or a quencher. In some embodiments, the aptamer is coupled to a linker moiety placed between the aptamer and the displacement strand. In some embodiments, the linker moiety is a nucleotide acid moiety, a peptide nucleic acid (PNA) moiety, a peptide moiety, a disulfide bond, aphosphodiester linkage, or a polymer. In some embodiments, one or more sidewalls of the piercing element are reflective. In some embodiments, one or more sidewalls of each microneedle of the microneedle array are reflective. In some embodiments, the piercing element comprises a structural domain, a barrier domain, and a sensing domain. In some embodiments, the structural domain is positioned on a surface of the support and extends outward from the surface to define a needle, and defines an interior space, wherein the sensing domain is contained within the interior space. In some embodiments, the structural domain encapsulates the barrier domain. In some embodiments, the structural domain encloses the sensing domain. In some embodiments, the sensing domain encapsulates the analyte binding probe within a matrix of the sensing domain, wherein the barrier domain comprises a hydrogel, a polymer, or combinations thereof. In some embodiments, the sensing domain comprises the analyte binding probe. In some embodiments, the sensing domain comprises the analyte binding probe in a hydrogel matrix. In some embodiments, the analyte binding probe is bound to one or more beads in the hydrogel matrix. In some embodiments, the barrier domain comprises a hydrogel, a polymer, or combinations thereof. In some embodiments, the structural domain encapsulates the sensing domain, wherein the sensing domain is configured to control transfer of the analyte to the analyte binding probe. In some embodiments, the barrier domain is configured to control transfer of the analyte to sensing domain via diffusion. In some embodiments, the sensing domain and / or the barrier domain comprise a plurality of pores. In some embodiments, the analyte binding probe is contained within the sensing domain at a concentration of about 10 pM to about 1 mM. In some embodiments, the analyte binding probe is contained within the sensing domain at a concentration of about 1 nM to about 1 uM. In some embodiments, the detector comprises a semiconductor material. In some embodiments, the detector comprises a semiconductor photodetector. In some embodiments, the detector comprises a semiconductor photodetector comprising a silicon photomultiplier chip, an avalanche photodiode, a metal-semiconductor- metal photodiode, a CMOS sensor array, a CCD (charge coupled device), a lateral semiconductor photodiode, an array of photodetectors, an ambient light sensor, an array of ambient light sensors, or combinations thereof, or combinations thereof. In some embodiments, the semiconductor material or the semiconductor photodetector comprises a p- n junction. In some embodiments, the semiconductor material or the semiconductor photodetector comprises Si, Ge, InGaAs, GaAs, InP, Ge, SiGe, InGaN, GaN, or combinations thereof. In some embodiments, the detector comprises a silicon photomultiplier (SiPM) detector. In some embodiments, the piercing element is configured to transmit light withinthe piercing element towards the analyte binding probe. In some embodiments, the piercing element is configured to guide light emitting from the analyte binding probe out of the piercing element. In some embodiments, the piercing element is configured to transmit light using a reflective metal coating on a surface within the piercing element, optionally, wherein the reflective metal coating comprises Ag or Au. In some embodiments, the piercing element is made of a reflective metal configured to reflect light, optionally, wherein the reflective metal comprises stainless steel, Ti, Au Ag, or combinations thereof. In some embodiments, the piercing element comprise a polymer, a plastic polymer, or polymer coated with reflective metal, or combinations thereof, optionally wherein the reflective metal is optionally Ag, Au, or combinations thereof. In some embodiments, the reflective metal coating is configured to increases an efficiency of light coupling from a top of the piercing element towards the analyte binding probe within the piercing element. In some embodiments, the reflective metal coating is configured to increases an efficiency of light coupling from the analyte binding probe towards the detector. In some embodiments, the piercing element is configured to guide light using a dielectric material comprised within the piercing element. In some embodiments, the dielectric material is a dielectric waveguide with a core and cladding. In some embodiments, the dielectric waveguide is an optical fiber. In some embodiments, the second waveguide guides an excitation light towards the analyte binding probe, and an emission light from the analyte binding probe towards the detector. In some embodiments, the analyte binding probe is located above the piercing elements. In some embodiments, the piercing element comprises a plurality of piercing members held together by a base. In some embodiments, the plurality of piercing members is at least partially hollow. In some embodiments, at least one of the plurality of piercing members provides a fluorescence signal of known magnitude to establish a reference signal as to the change in optical signal from the analyte binding probe under a given biological condition. In some embodiments, the given biological conditions comprise a pH value, a temperature, a salt concentration, or combinations thereof. In some embodiments, at least two of the piercing members comprise a same analyte binding probe. In some embodiments, at least two of the piercing members comprise a same analyte binding probe in a same concentration. In some embodiments, at least two of the piercing members comprise two different analyte binding probes that detect two different analytes. In some embodiments, the change in optical signal when the analyte binding probe comes in contact with the analyte is detected using fluorescence resonance energy transfer (FRET) or a time resolved fluorescence (TRF) or change in optical emission intensity. In some embodiments, the change in analyte concentration when the analytebinding probe comes in contact with the analyte is detected using a change in intensity of the fluorescence. In some embodiments, the analyte binding probe comprises a quantum dot. In some embodiments, the analyte binding probe comprises an aptamer conjugated to a quantum dot. In some embodiments, the optical coupler is an optical grating. In some embodiments, the optical grating is a diffraction element configured to guide light from a free space into the waveguide, or from the waveguide into a free space. In some embodiments, the optical grating is configured to direct light from the waveguide toward the piercing element. In some embodiments, the optical grating is configured to direct light from the piercing element towards the waveguide. In some embodiments, the piercing element is separable from the device. In some embodiments, the piercing element is configured to store the biological sample for subsequent analysis.

[0016] Aspects disclosed herein provide a method for sensing an analyte in a biological sample of a subject, comprising: piercing a skin of the subject to contact a biological sample, and bringing the biological sample in contact with an analyte binding probe; inducing a conformational change in the analyte binding probe by binding a target analyte with the analyte binding probe; applying a light source to the analyte binding probe to produce an optical signal; measuring a presence, a lack of presence, an increase, or a decrease of the optical signal to determine the presence or concentration of the target analyte in the sample. In some embodiments, the method includes passing light through an optical waveguide from a light source to the analyte binding probe. In some embodiments, the method includes passing light through an optical waveguide from the analyte binding probe to a detector. In some embodiments, applying the light source to the analyte binding probe comprises transmitting light through an optical excitation path comprising a waveguide and a light coupler and: i) transmitting light through one or more focusing elements and focusing light from a light source towards the light coupler, ii) transmitting light through a waveguide and reflecting it within the waveguide using a reflective cladding layer surrounding the waveguide, thereby guiding the light toward the piercing element or iii) transmitting light from a light source, through the light coupler, and into the waveguide. In some embodiments, measuring a presence, a lack of presence, an increase, or a decrease of the optical signal comprises transmitting light through an optical emission path comprising: i) apertures in a reflective cladding layer surrounding a waveguide, transmitting light through the apertures towards the detector, or ii) one or more focusing elements configured to focus emission light from the analyte binding probe towards the detector, transmitting lights through the one or more focusing elements towards the detector. In some embodiments, applying a light sourceto the analyte binding probe to produce an optical signal comprises transmitting light about an optical excitation path comprising a waveguide, and an optical emission path comprising the waveguide, the waveguide transmitting light from the light source to the analyte binding probe, and the transmitting light through the waveguide from the analyte binding probe to a detector. In some embodiments, applying the light source to the analyte binding probe and / or measuring a presence, a lack of presence, an increase, or a decrease of the optical signal comprises the waveguide transmitting light from a light source to the analyte binding probe, and the waveguide transmitting light from the analyte binding probe to the detector. In some embodiments, the analyte binding probe is comprised in a sensing domain comprising a hydrogel matrix comprising one or more particles comprising the analyte binding probe. In some embodiments, the analyte binding probe has a dissociation constant of at least 1 pM with respect to the analyte. In some embodiments, piercing the skin of the subject comprises piercing the skin of the subject with a piercing element that is removable from a device. In some embodiments, piercing the skin of the subject comprises storing the biological sample in the piercing element for subsequent analysis. In some embodiments, measuring the presence, the lack of presence, the increase, or the decrease of the optical signal to determine the presence or concentration of the target analyte in the sample occurs using a device coupled to a piercing element. In some embodiments, measuring the presence, lack of presence, increase of optical signal, or decrease of optical signal to determine the concentration of the target analyte in the biological sample occurs while the piercing element is attached to the body of a subject. In some embodiments, measuring the presence, lack of presence, increase of optical signal, or decrease of optical signal to determine the concentration of the target analyte in the biological sample occurs while the piercing element is removed from the body of a subject.

[0017] Provided herein is a device for sensing an analyte in a biological sample of a subject, comprising: a support; a piercing element coupled to the support, wherein the piercing element is configured to pierce a body surface of the subject when the device is coupled to the body surface, to thereby bring the piercing element in contact with the biological sample; an analyte binding probe on or within the piercing element, wherein the analyte binding probe is configured to provide a change in an optical signal when the analyte binding probe comes in contact with the analyte in the biological sample of the subject; and a detector operatively coupled to the support, wherein the detector is configured to detect the optical signal. In some embodiments, the device further comprises a light source. In some embodiments, the device is configured to be placed in optical communication with anexternal light source. In some embodiments, the device further comprises two or more analyte binding probes on or within the piercing element, each of the two or more analyte binding probes configured to provide a change in an optical signal when the two or more analyte binding probes contact a second analyte in the biological sample of the subject. In some embodiments, the device further comprises a multiplexed array of analyte binding probes on or within the piercing element, each of the multiplexed array of analyte binding probes configured to provide a change in an optical signal when the multiplexed array of analyte binding probe contacts a subsequent analyte in the biological sample of the subject. In some embodiments, the subsequent analyte is a different analyte, the multiplexed array of analyte binding probes is configured to detect a plurality of analytes in the biological sample of the subject. In some embodiments, the piercing element comprises a plurality of piercing elements. In some embodiments, the plurality of piercing elements defines subsets of piercing elements, wherein each subset of piercing elements comprises a different analyte binding probe configured to detect a different analyte in the biological sample of the subject. In some embodiments, the subsets of piercing elements comprising the different analyte binding probe comprises the multiplexed array of analyte binding probes. In some embodiments, the support comprises an optical light guide, or an optical waveguide. In some embodiments, the optical light guide comprises a 2-dimensional array of light guides. In some embodiments, the optical light guide comprises a light guide core. In some embodiments, the optical light guide comprises a light guide core. In some embodiments, the optical light guide comprises a coupling region. In some embodiments, the optical light guide comprises a coupling region where the optical light guide contacts the microneedle. In some embodiments, the optical light guide comprises a coupling region positioned at a base of the microneedle. In some embodiments, the coupling region is in contact with the base of the microneedle, or is adjacent to the base of the microneedle. In some embodiments, light passes through the coupling region to the analyte binding probe. In some embodiments, the coupling region comprises a higher refractive index than the light guide core. In some embodiments, the coupling region comprises a higher refractive index than the light guide core, and refracts light towards the aptamer binding probe. In some embodiments, the coupling region comprises a lower refractive index than the light guide core, and refracts light towards the detector. In some embodiments, the coupling region comprises a same refractive index as the light guide core. In some embodiments, the optical light guide comprises one or more reflectors. In some embodiments, the reflectors are positioned in contact with a base of the piercing element. In some embodiments, the reflectors surround a base of the piercingelement. In some embodiments, the reflectors form an array about a base of the piercing element. In some embodiments, the reflectors are adjacent to a base of the piercing element. In some embodiments, the reflectors are configured to reflect light towards the piercing element. In some embodiments, the reflectors are configured focus light towards the analyte binding probe. In some embodiments, the reflectors comprise a reflective material on a sidewall of a structure. In some embodiments, the 2-dimensional array comprises dichroic mirrors. In some embodiments, the 2-dimensional array comprises gratings. In some embodiments, the 2-dimensional array is etched. In some embodiments, the light guide comprises a glass or a polymer. In some embodiments, the optical light guide comprises a polymer glassy matrix comprising dispersed photoluminescent particles. In some embodiments, the polymer comprises silicones, polysiloxanes, silsequioxanes, or combinations thereof. In some embodiments, the polymer comprises: polymethylmethacrylate (PMMA), polystyrene (PS), polycarbonate (PC), polyurethane (PU), epoxy resin, deuterated and halogenated poly acrylates, fluorinated polyimides, perfluorocyclobutyl (PFCB) aryl ether polymers, nonlinear optical polymers, benzocyl cobutene (BCB), perfluorovinyl ether cyclopolymer (CYTOP), tetrafluoroethylene and perfluorovinyl ether copolymer (Teflon AF), silicone, fluorinated poly (arylene ether sulfide), poly(pentafluorostyrene), fluorinated dendrimers, fluorinated hyperbranched polymers, or combinations thereof. In some embodiments, the optical light guide is configured to: couple to a light source, to guide at least a portion of light from the light source along a length of the optical light guide, and to divert at least a portion of the light from the light guide to the piercing element. In some embodiments, the support comprises an optical light guide is configured to provide a light transmission efficiency of at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 50, 60, 65, 70, 75, 80, 85, 90, 95, or 99% transmission efficiency. In some embodiments, the support comprises an optical light guide is configured to provide a light transmission efficiency of at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 50, 60, 65, 70, 75, 80, 85, 90, 95, or 99 % transmission efficiency from the light source to the piercing element. In some embodiments, the optical light guide is configured to collect an emission light of the optical reporter from the analyte binding probe, to guide at least a portion of the emission light to the detector. In some embodiments, the support comprises an optical light guide is configured to provide an essentially losses light transmission from the piercing element to the detector. In some embodiments, the support comprises an optical light guide is configured to provide a light transmission efficiency of at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 50, 60, 65, 70, 80, 85, 90, 95, or 99 % transmission efficiency from thepiercing element to the detector. In some embodiments, the optical light guide defines a plurality of paths from the light source to the piercing element. In some embodiments, the light source is configured to provide light in different wavelengths. In some embodiments, the light source comprises a plurality of light sources each configured to provide a different wavelength of light. In some embodiments, the device is configured to be coupled to an external light source each configured to provide a different wavelength of light. In some embodiments, the plurality of paths from the light source to the piercing element comprises one or more distinct paths from the light source to the multiplexed array of analyte binding probes. In some embodiments, multiplexed array of analyte binding probes is spatially multiplexed across the piercing element. In some embodiments, at least one of the plurality of paths is configured to guide light from one of the plurality of light sources with a specific wavelength of light to one of the analyte binding probes in the multiplexed array of analyte binding probes which is configured to be responsive to the specific wavelength of light. In some embodiments, the plurality of paths from the light source to the piercing element comprises one or more distinct paths from the light source to the subsets of piercing elements. In some embodiments, at least one of the plurality of paths is configured to guide light from one of the plurality of light sources with a specific wavelength of light to one of the analyte binding probes in the subsets of piercing elements. In some embodiments, the optical light guide comprises one or more switches configured to block one or more of the plurality of paths. In some embodiments, the piercing element is coupled to the support using one or more holes. In some embodiments, the piercing element is coupled to the support using one or more insertable elements configured to attach to a corresponding one or more receiving elements. In some embodiments, the piercing element is coupled to the support using a lock and key attachment. In some embodiments, the piercing element is coupled to the support using a mortise and tenon attachment. In some embodiments, the piercing element is coupled to the support using a dovetail attachment. In some embodiments, the piercing element is coupled to the support using a magnetic attachment. In some embodiments, the piercing element is coupled to the support using an adhesive. In some embodiments, the piercing element is coupled to the support using one or more elastically deformable attachment elements. In some embodiments, the piercing element is coupled to the support using one or more elastically deformable attachment elements configured rebound once inserted into the support. In some embodiments, the piercing element is removably coupled to the support. In some embodiments, the piercing element comprises a microneedle array which is removably coupled to the support. In some embodiments, the optical light guide is removably coupled tothe support. In some embodiments, the optical light guide is removably coupled to the support, and is removable from the support while remaining coupled to the light guide. In some embodiments, the optical light guide comprises a first portion and a second portion, wherein the first portion is removably coupled to the support, and the second portion is coupled to the piercing element. In some embodiments, the optical light guide comprises a first portion and a second portion removably coupled to each other. In some embodiments, the second portion is removably coupled to the support, wherein the second portion is coupled to the piercing element, In some embodiments, the second portion is removable from the first portion. In some embodiments, the support does not comprise an optical light guide. In some embodiments, the device comprises a free space optical system. In some embodiments, the device comprises a free space optical system configured to transfer light from the light source to the analyte binding probe. In some embodiments, the device comprises a free space optical system configured to transfer light from the analyte binding probe to the detector. In some embodiments, the free space optical system comprises a light source coaxially aligned with the analyte binding probe. In some embodiments, the free space optical system comprises a light source vertically integrated with the analyte binding probe. In some embodiments, the free space optical system comprises a light source directly coupled to the piercing element. In some embodiments, the detector is directly coupled to the piercing element. In some embodiments, there is a light path between the analyte binding probe and the detector. In some embodiments, a light source is directly coupled to the analyte binding probe. In some embodiments, there is a light path between the analyte binding probe and the light source. In some embodiments, there is an unobstructed light path between the analyte binding probe and the light source. In some embodiments, the light source is a plurality of light-emitting diodes (LEDs) or lasers. In some embodiments, the light source is a plurality of light-emitting diodes (LEDs) or lasers, each of the plurality of light-emitting diodes (LEDs) or lasers configured to deliver a different wavelength of light. In some embodiments, the light source has wavelength of at least about 200 nanometers. In some embodiments, the light source has wavelength of at most about 2 micrometers. In some embodiments, the analyte binding probe is coupled to an optical reporter. In some embodiments, the optical reporter is a fluorophore or a quencher. In some embodiments, the analyte binding probe coupled to the optical reporter is an oligonucleotide probe. In some embodiments, the oligonucleotide probe is an aptamer. In some embodiments, the aptamer is coupled to a displacement strand, wherein the displacement strand is partially complementary to the aptamer. In some embodiments, the displacement strand is coupled to a second optical reporter. In someembodiments, the second optical reporter is a fluorophore or a quencher. In some embodiments, the aptamer is coupled to a linker moiety placed between the aptamer and the displacement strand. In some embodiments, the linker moiety is a nucleotide acid moiety, a peptide nucleic acid (PNA) moiety, a peptide moiety, a disulfide bond, a phosphodiester linkage, or a polymer. In some embodiments, the analyte binding probe coupled to the optical reporter is a polynucleotide probe. In some embodiments, the analyte binding probe comprises an antibody. In some embodiments, the analyte binding probe is configured to undergo a conformational change when the analyte interacts with the analyte binding probe. In some embodiments, the conformation is configured to provide the change in the optical signal. In some embodiments, the change in the optical signal is in a function of a concentration of the analyte. In some embodiments, the optical signal is the emission light. In some embodiments, the analyte binding probe is configured to detect one or plurality of analytes. In some embodiments, the analyte is an antibody. In some embodiments, the analyte is a oligonucleotide, mRNA, RNA, DNA, cDNA, a lipid, lipid particle, an exosome, a viral particle, or combinations thereof. In some embodiments, the analyte is a protein. In some embodiments, the analyte is a small molecule. In some embodiments, the small molecule is a drug. In some embodiments, the piercing element is a needle. In some embodiments, the piercing element is a microneedle. In some embodiments, the needle is configured to penetrate a stratum corneum of the subject. In some embodiments, the piercing element is configured to penetrate into a dermis of the subject. In some embodiments, one or more sidewalls of the piercing element are reflective. In some embodiments, one or more sidewalls of the needle are reflective. In some embodiments, the needle comprises a structural domain, a barrier domain, and a sensing domain. In some embodiments, the structural domain is positioned on a surface of the support and extends outward from the surface to define a needle, and defines an interior space, wherein the sensing domain is contained withing the interior space. In some embodiments, the needle comprises a well. In some embodiments, the sensing domain is contained within the well. In some embodiments, the barrier domain coats an exterior surface of the structural domain. In some embodiments, the structural domain defines one or more opening on a lateral face of the needle. In some embodiments, the structural domain defines pyramidal needles. In some embodiments, the structural domain defines pyramidal needles comprising an opening on each face of the pyramidal needles. In some embodiments, the barrier domain coats the openings. In some embodiments, the barrier domain contacts a portion of the sensing domain. In some embodiments, the barrier domain contacts a portion of the sensing domain at the openings. In some embodiments, the needlecomprises a structural domain, and the sensing domain. In some embodiments, the structural domain encapsulates the barrier domain. In some embodiments, the structural domain encloses the sensing domain. In some embodiments, the sensing domain encapsulates the analyte binding probe within a matrix of the sensing domain, wherein the barrier domain comprises a hydrogel, a polymer, or combinations thereof. In some embodiments, the sensing domain comprises the analyte binding probe. In some embodiments, the sensing domain comprise the analyte binding probe in a hydrogel matrix. In some embodiments, the sensing domain encapsulates the analyte binding probe in a hydrogel matrix. In some embodiments, the analyte binding probe is attached to hydrogel by conjugation methods comprising: DBCO-Azide, BCN-Tetrazine, biotin-streptavidin, EDC, NHS / EDC, thiol maleimide, DBCO-N3, DBCO-DHPA, BCN-N3, or any combination thereof. In some embodiments, the hydrogel is produced from Cu-click reactions. In some embodiments, the hydrogel is produced from Cu-free click reaction. In some embodiments, the sensing domain encapsulates the analyte binding probe within a matrix of the sensing domain. In some embodiments, the barrier domain comprises a hydrogel, a polymer, or combinations thereof. In some embodiments, the structural domain encapsulates the sensing domain, wherein the sensing domain is configured to control transfer of the analyte to analyte binding probe. In some embodiments, the barrier domain coats a portion of the sensing domain, wherein the barrier domain is configured to control transfer of the analyte to sensing domain. In some embodiments, the barrier domain is configured to control transfer of the analyte to sensing domain via diffusion. In some embodiments, the sensing domain is configured to control transfer of the analyte to analyte binding probe via diffusion. In some embodiments, the sensing domain comprises a plurality of pores. In some embodiments, the barrier domain comprises a plurality of pores. In some embodiments, the structural domain comprises one or more openings on an exterior surface of the structural domain. In some embodiments, the one or more openings is configured to allow the analyte to contact the sensing domain. In some embodiments, the sensing domain defines a passage connecting the one or more openings to the analyte binding probe. In some embodiments, the needle comprises a plurality of openings. In some embodiments, the needle comprises a structural domain comprising a hollow region of the needle. In some embodiments, the hollow region is orientated in a longitudinal direction. In some embodiments, the plurality of openings are positioned on a lateral face of the needle. In some embodiments, the plurality of openings are positioned on opposing lateral faces of the needle. In some embodiments, the sensing domain extends longitudinally into the needle. In some embodiments, the sensing domain extendslongitudinally into the needle and defines a passage connecting the one or more openings to the analyte binding probe. In some embodiments, there is one opening on a lateral surface of the needle. In some embodiments, the structural domain is directly in contact with the sensing domain. In some embodiments, the structural domain and the sensing domain extends longitudinally from the device. In some embodiments, the sensing domain is positioned orthogonally to a longitudinal axis of the needle. In some embodiments, the sensing domain is positioned orthogonally to a longitudinal axis of the needle, wherein the sensing domain is positioned parallel to a longitudinal axis of the needle. In some embodiments, the sensing domain is positioned on the exterior surface of the structural domain. In some embodiments, the barrier domain is positioned on the exterior surface of the structural domain, and wherein the sensing positioned within an interior space of the structural domain. In some embodiments, the barrier domain is positioned on the exterior surface of the structural domain, positioned within an interior space of the structural domain, and wherein the sensing domain is positioned within the structural domain an in contact with the barrier domain positioned within an interior space of the structural domain. In some embodiments, the barrier domain is positioned on the exterior surface of the structural domain and wherein the barrier domain is positioned within an interior space of the structural domain, and wherein the sensing domain is positioned within the structural domain orthogonally to a longitudinal axis of the needle, and is in contact with the barrier domain positioned within an interior space of the structural domain. In some embodiments, the barrier domain is positioned on the exterior surface of the structural domain and wherein the barrier domain is positioned within an interior space of the structural domain, and wherein the sensing domain is positioned throughout a matrix of the barrier domain. In some embodiments, the barrier domain is partially within an interior space of the structural domain. In some embodiments, the sensing domain is within an interior space of the structural domain and is exposed to an exterior surface of the needle through the one or more openings, and wherein the analyte binding probe is positioned throughout a matrix of the sensing domain. In some embodiments, the barrier domain is positioned on the exterior surface of the structural domain and wherein the sensing domain is positioned within an interior space of the structural domain, and wherein the analyte binding probe is positioned throughout a matrix of the sensing domain, wherein there are at least two opening on opposing lateral faces of the needle. In some embodiments, the analyte binding probe is contained within the sensing domain at a concentration of about 1 nM to about 1 mM. In some embodiments, the analyte binding probe is contained within the sensing domain at a concentration of about 1 nM to about 1 uM. In some embodiments,the analyte binding probe is distributed substantially uniformly dispersed throughout the barrier domain. In some embodiments, the needle has a length of at least about 3 millimeters. In some embodiments, the needle has a length of at least about 1 millimeter. In some embodiments, the needle has a length of at least about 500 micrometers. In some embodiments, the needle has a length of at least about 100 micrometers. In some embodiments, the needle is a 3-dimensional printed needle. In some embodiments, the needle is a hydrogel needle. In some embodiments, the needle is a polymer. In some embodiments, the needle comprises a cavity filled with a hydrogel matrix. In some embodiments, the barrier domain comprises a hydrogel matrix. In some embodiments, wherein the microneedle is solid. In some embodiments, the microneedle has a hollow core. In some embodiments, the microneedle is porous. In some embodiments, the microneedle is a swellable microneedle. In some embodiments, the device comprises a plurality of piercing elements, wherein the plurality of piercing elements comprises the piercing element. In some embodiments, at least one piercing element of the plurality of piercing element is coupled to a control reference probe. In some embodiments, the device is configured to perform a single time-point measurement of the analyte. In some embodiments, the device is configured to perform continuous, real-time measurement of the analyte. In some embodiments, the device is configured to present some information related to the analyte. In some embodiments, the information includes a presence of the analyte. In some embodiments, the information includes a concentration of the analyte. In some embodiments, the detector comprises a semiconductor material. In some embodiments, the detector comprises a semiconductor photodetector. In some embodiments, the detector comprises a semiconductor photodetector comprising a silicon photomultiplier chip, an avalanche photodiode, a metal-semiconductor- metal photodiode, a CMOS sensor array, a CCDs (charge coupled device), a lateral semiconductor photodiode, an array of photodetectors, or combinations thereof. In some embodiments, the semiconductor material or the semiconductor photodetector comprises an p-n junction. In some embodiments, the semiconductor material or the semiconductor photodetector comprises Si, Ge, InGaAs, GaAs, InP, Ge, SiGe, InGaN, GaN, or combinations thereof. In some embodiments, the detector comprises a silicon photomultiplier (SiPM) detector. In some embodiments, the SiPM detector has a dimension up to about 1 cm. In some embodiments, the SiPM detector has a dimension up to about 3 millimeters. In some embodiments, the device further comprises a battery. In some embodiments, the device further comprises a wireless communication configured to communicate with an external device to exchange the information. In some embodiments, the device is a skin patch. Insome embodiments, the skin patch is removable. In some embodiments, the device is configured to emit the optical signal when the analyte contacts the analyte binding probe. In some embodiments, the device is configured to result in a decrease in optical signal when the analyte binding probe is bound to the analyte. In some embodiments, the device is configured to emit the optical signal in a default configuration when not in contact with the analyte. In some embodiments, the analyte binding probe emits the optical signal, and does not emit the optical signal when bound to the analyte. In some embodiments, the analyte binding probe emits the optical signal, and undergoes the conformational change when bound to the analyte as to not emit the optical signal. In some embodiments, the analyte binding probe emits the optical signal, and undergoes the conformational change when bound to the analyte as to emit a reduced optical signal. In some embodiments, the device measures a concentration of the analyte in the biological sample of the subject by measuring a decrease in the optical signal. In some embodiments, the device further comprises an LED array. In some embodiments, the device further comprises an LED driver. In some embodiments, the device further comprises a temperature sensor. In some embodiments, the device further comprises an MCU, a Bluetooth low energy model, or a CMOS image sensor, or combinations thereof.

[0018] Provided herein is a method for sensing an analyte in a biological sample of a subject, comprising: (a) piercing a skin of the subject to contact a biological sample; (b) bringing the biological sample in contact with an analyte binding probe; (c) inducing a conformational change in the analyte binding probe by binding a target analyte with the analyte binding probe; (d) applying a light source to the analyte binding probe to produce an optical signal, and € measuring a presence, a lack of presence, an increase, or a decrease of the optical signal to determine the presence or concentration of the target analyte in the sample. In some embodiments, the method further comprises passing light through an optical waveguide from a light source to the analyte binding probe. In some embodiments, the method further comprises passing light through the optical waveguide from the analyte binding probe to a detector.

[0019] Another aspect of the present disclosure provides a non-transitory, computer-readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

[0020] Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0021] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0022] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0024] Figure 1: Various configurations of microdialysis probes 1) Concentric where the inlet and outlet are located at the distal end of the probe, the semipermeable membrane is located at the proximal end of the probe and it joins the inlet and outlet which are concentric tubes for some specified length 2) Loop where the inlet and outlet are located at the distal end of the probe, the semipermeable membrane is located at the proximal end of the probe, and it joins the inlet and outlet which are two separate tubes 3) Linear where the inlet is located at the distal end of the probe and the outlet is located at the proximal end of the probe (or vice versa) and the semipermeable membrane is along the length of the probe between the inlet and outlet.

[0025] Figure 2: The analyte binding probe may be immobilized in a number of ways 1) Directly chemically attached to the surface 2) Via ligand binding 3) Embedded in asemipermeable holding material (such as hydrogel) 4) Attached to small particles that are embedded in a semipermeable holding material.

[0026] Figure 3: A representation of a portable tethered system intended for use in a hospital or clinical setting. The control unit, including battery power and user interface, is mounted on the infusion stand. The control unit is connected to a wearable unit, containing the multiplexed optical measurement system and disposable patch, via electrical wires and fluidic tubing.

[0027] Figure 4: A block diagram of the components in a portable tethered system.

[0028] Figure 5: The disposable and reusable components of Example 1. The reusable component contains the optical measurement system, while the disposable component contains the microfluidic chip.

[0029] Figure 6: The disposable component of the wearable system. The microfluidic chip is fixtured in a disposable part that also includes features for fluidic connection to the microdialysis, alignment to the wearable optical measurement system, and an Output Reservoir. For simplicity, the optical black coating and apertures have been omitted in this figure.

[0030] Figure 7: Exploded views of the disposable part of the wearable system in Example 1.

[0031] Figure 8: Cross-sectional views of the disposable part from the wearable system of Example 1.

[0032] Figure 9: The PCB arrangement including light sources.

[0033] Figure 10: Active alignment of Light source. On the back of the rigid sections, there are “laser flap handle” components which are used for active alignment of the laser to the optical components of the excitation path in the optical measurement system.

[0034] Figure 11: Cross section of the optical path of the reusable part and optical measurement system of the embodiment described in Example 1. The shading indicates the excitation light path, and the emission light path.

[0035] Figure 12: Illumination system and detection system timing diagram reflecting the operation of the embodiment described in Example 1. The illumination system illuminates the sensing spots before the measurement begins in the detection system, and the detection system completes the measurement prior to deactivating the illumination system. The intermeasurement period may be adjusted to the needs of the application.

[0036] Figure 13: A representation of a wearable system. The wearable system may be worn on different parts of the body including but not limited to the arm, the leg, the abdomen, or the back.

[0037] Figure 14: The disposable and reusable components of Example 2. The reusable component contains the optical measurement system, while the disposable component contains the microfluidic chip.

[0038] Figure 15: A block diagram of the components in a wearable system where the pump is single-use and contained in the disposable portion of the device.

[0039] Figure 16: The disposable components of Example 2 including the flow cell, reservoirs, pump, and probe.

[0040] Figure 17: The disposable components of Example 2 showing attachment features and extended probe.

[0041] Figure 18: Flow Cell portion of the disposable component in Example 2.

[0042] Figure 19: A block diagram of the components in a wearable system where the pump, (or at minimum the driving mechanism of the pump) is contained in the reusable portion of the device.

[0043] Figure 20: An exemplary output of a temperature calibration with the optical measurement system mated to an inert flow cell. (Top) Temperature data collected during calibration by the optical measurement system via an in-built temperature sensor. (Bottom) Uncorrected data (shown in dark gray, 202) displays an inverse dependence with temperature. This dependence can be characterized and corrected out to generated data that is unresponsive to temperature changes (shown in light gray, 200)

[0044] Figure 21: An additional embodiment of a sensor system containing a reusable optoelectronics reader and a disposable assembly containing the microfluidic chip, collection vial, and various tubing and fittings. The assembled unit comprising the reusable reader and disposable assembly is compatible with being attached to a microdialysis probe and external pump in order to take biosensor measurements.

[0045] Figure 22: Exploded view of the components from the example embodiment in Figure 21.

[0046] Figure 23: A depiction of a completed flow cell assembly described in Example 4. Spots of conjugated DNA are depicted as shaded circles for illustrative purposes and are not visible in actual flow cells.

[0047] Figure 24: Sample data output from the calibration routine described in Example 5. The flow cell used in this experiment contained 3 spots of caffeine aptamer switch (channels1-3, 208, 204, and 214, respectively) and 4 spots of fluorophore-labeled DNA used as a control strand (channels 4-7, 216, 210, 212, 206, respectively).

[0048] Figure 25: Continuous and real-time molecular measurement of caffeine in human subjects. Shows the normalized signals for multiplexed caffeine 218 and positive control 220 sensors on a single device. The experiment is described in Example 6.

[0049] Figure 26: Predicted biosensor concentrations of the data in Figure 25 by using information from the in vitro calibration. Accuracy of the biosensor can be evaluated by comparing the measured biosensor concentration to the concentration of the perfusate collected in the waste vial at 30 minute intervals (denoted as stars).

[0050] Figure 27: Schematic of typical microdialysis setup. In conventional microdialysis experiments, a syringe pump is connected to a syringe filled with a sterile buffer. The syringe is connected to a microdialysis probe which is inserted into the dermis of a subject. A portion of the probe inserted in the patient consists of a semi-permeable membrane which allows equilibrium of small molecule analytes in the dermal ISF into the microdialysis probe. Fluid from the outlet of the microdialysis probe is then collected in aliquots over time for subsequent analysis and measurement using conventional laboratory instrumentation.

[0051] Figure 28: Block diagram of device components. The device consists of several key components and are separated into two parts- a re-usable part and a disposable part. The two components can be locked together when in use, and then separated so that the disposable part can be disposed of, and the re-usable part can be configured with a new disposable component. Optionally, a new battery can used for each use of the device, or a rechargeable battery contained in re-useable complement can be charged between device uses. The reusable part contains the pump, which is used to drive the sterile buffer through the microdialysis membrane and flow cell, and the light source, detector, electronics, and communication / data transmission hardware. The re-usable part is configured to read the optical signal from the analyte binding probes contained in the flow cell in order to measure the concentration of one or more of the patient’s analytes. The disposable component flows a sterile buffer through the microdialysis probe, which is in contact with the dermis of a subject. A semi-permeable membrane is contacted with the subjects ISF and allows diffusion of some analytes from the ISF into the microdialysis probe. The fluid then flows into a flow cell where it is contacted with one or more analyte binding probes regions. The analyte binding probe regions contain optically responsive molecular switches which change in fluorescence signal bound to an analyte molecule and interrogated optically. The re-usable component measures the fluorescence signal of the molecular switches by deliveringexcitation light, and then using a detector to measure and quantify emission light from the aptamer switches. Light sources such as LEDs and lasers can be used. Detectors such as CMOS image sensors, ambient light sensors, SiPM, and avalanche photodetectors can be used. To separate excitation light from the emission light, filters, such as dichroic or absorptive filters can be used. Once passed through the analyte binding probe regions, the fluid can be collected into a waste compartment.

[0052] Figures 29A-29C: Example of body -worn device. Figure 29A shows an overview of components. In the device, a reusable battery-powered pump is used to drive the dialysate through the fluidic system. The outlet of the pump is connected to the inlet of a disposable linear catheter with a semipermeable membrane. The semipermeable membrane portion of the fluidic path is embedded in the dermis of the posterior upper arm. The outlet of the fluidic path with semipermeable membrane is attached to the inlet of the detection flow cell. This disposable detection flow cell is clipped into a reusable detector element, which is adhered to the skin of the posterior upper arm. Figure 29B shows a cross-section of linear catheter. The semipermeable membrane is embedded in the dermis and allows analytes below the molecular weight cutoff of the membrane to exchange freely between the dialysate and interstitial fluid. As the dialysate is driven by the pump, analytes that diffuse into the tubing flow out of the outlet and into the next section of the system, the detection flow cell. Figure 29C shows a cross section of detector flow cell and detector element. The detector flow chamber is disposable and snaps into a housing, which contains reusable optical elements. Within the detection flow cell chamber are spatially multiplexed detection regions. The detection regions are comprised of analyte binding probes covalently attached to particles, which are sterically trapped within a semipermeable polymer matrix. The polymer matrix is immobilized within the detection chamber via chemical interactions with the chamber. The detector outputs excitation light that interacts with luminescent molecules on the analyte binding probes, and collects and quantifies emission light from the luminescent molecules on the analyte binding probes. As analytes from the semipermeable membrane flow into the detection chamber, they can diffuse into the polymer matrix and interact with the analyte binding probes, inducing binding events that lead to measurable changes in signal recorded by the optical detection elements. Fluid flows out of the detection chamber into an on-board waste reservoir. The measurements taken by the detector are stored by the detector and can be wirelessly transmitted to a separate device for analysis and interpretation of measurements.

[0053] Figure 30: Example of flow cell with analyte binding probes. The fluid from the semi-permeable membrane is connected to the inlet of the flow cell and the outlet of the flowcell goes to a waste reservoir. In the fluidic path of the flow cell consists of 3 analyte binding regions (Al, A2, and A3), in which aptamer switches are immobilized. Specifically, the optically responsive optical switches are conjugated to polystyrene beads and entrapped in a hydrogel matrix. Each analyte binding region contains an analyte binding probe for a different molecular target. The flow cell is configured such that the analyte binding probe regions Al -A3 can be optically excited and the subsequent emission light from the fluorescently-labeled aptamer switches can be measured with a detector. The light source and detector for this is contained within the re-usable portion of the device and can be coupled temporarily to the flow cell, which is contained in the disposable portion of the device.

[0054] Figure 31: Proof-of-concept microdialysis benchtop device: Real-time glucose sensing with a benchtop device using a 30 mm linear 20 kDa cutoff microdialysis membrane. In this case a syringe pump drives the buffer through the microdialysis probe and into a flow cell (or microfluidic sample chamber). Binding probes such as aptamer switches are conjugated to magnetic particles, which are immobilized in the chamber using a permanent magnet adhered to the top of the microfluidic chip. The outlet of the microfluidic chip is attached to a waste reservoir. The microfluidic chip is affixed to the stage of an inverted fluorescence microscope. The microscope delivers light from an LED light engine to excite luminescent molecules within the binding probes. The emission light of the luminescent molecules is imaged by a camera attached to the microscope. The images can be used to quantify the signal of the binding probes over time, which in some embodiments are mapped to concentrations using a calibration curve.

[0055] Figures 32A-32B: In vitro sensing of glucose with microdialysis benchtop device. Figure 32A Detection of glucose with microdialysis probe in buffer. The proof-of-concept microdialysis benchtop device was used with a glucose-specific optically-responsive aptamer switch. The setup was run with a fluid flow rate of 10 pL / min, and the portion of the linear microdialysis probe containing the semi-permeable membrane was submerged in buffer and read for 30 minutes. After 30 minutes, a highly concentrated solution of glucose (~1 M) was spiked into the reservoir which contained the buffer and microdialysis tube to raise the overall concentration of glucose in solution to approximately 200 mM. The glucose diffuses through the semipermeable membrane and then into the flow cell of the device. The aptamer switches respond to the increase and glucose and result in an increase in fluorescence signal. After 60 minutes, the glucose solution in the reservoir containing the microdialysis membrane was exchanged several times with buffer and a reduction in fluorescent signal is seen as expected. Figure 32B The above experiment was repeated, except in this case themicrodialysis probe was inserted into a portion of pig skin. The pig skin was submerged in buffer and then concentrated glucose (~1 mM) was added to the buffer at the first red line to raise the concentration of the surrounding solution to approximately 200 mM. As seen previously, the signal increases due to diffusion of glucose into the pig skin, and then across the microdialysis membrane to the flow cell sensor. At the second red line the buffer is exchanged several times to remove glucose from the solution surrounding the pig skin.

[0056] Figure 33 represents an exemplary device to detect analyte(s) of interest, wherein the support does not include a waveguide, in accordance with an example embodiment.

[0057] Figures 34A - 34B represents a top view of an exemplary device of an array (Figure 34A) comprising a light source, a detection circuit, and a photodetector (Figure 34B).

[0058] Figure 35 represent an exemplary device to detect analytes of interest with a waveguide.

[0059] Figures 36A - 36H illustrate different microneedle configurations comprising a structural domain, a barrier domain, a sensing domain, and one or more analyte binding sensors.

[0060] Figures 37A-37C show an exemplary device to detect analytes of interest with exemplary waveguides. In Figure 37A shown is a coupling embodiment between the light guide and the microneedle where the coupling region of the microneedle is substantially index-matched with the light guide core; as light bounces around it continues to travel without deflection into the microneedles and reaches the sensing domain in the microneedles. In Figure 37B shown is a coupling embodiment between the light guide and the microneedle where the coupling region of the microneedle has a higher index material than the core of the light guide, turning the light inward toward the microneedle. In Figure 37C, reflective sidewalls about a base of a light guide base aid in directing more light toward the detector situated at the top of the waveguide through reflection from the sidewalls.

[0061] Figures 38A-38C show an exemplary device to detect analytes of interest with exemplary removable sensing portions. In Figure 38A, the light guide is part of the reusable sensor hardware; in Figure 38B the light guide is part of disposable microneedle array; and in Figure 38C the light guide is partially in both sensor hardware and in the microneedle array.

[0062] Figures 39A-39B show exemplary piercing elements.

[0063] Figure 40 shows a flowchart illustrating exemplary detection methods using the devices of the present disclosure.

[0064] Figure 41 shows a graph illustrating the phase shift of output light which may be produced from an exemplary device when used to detect an analyte as compared to the emitted light waveform without an analyte response, and input light waveform.

[0065] Figure 42 depicts an exemplary circuit diagram showing electrical couplings between various components of the device.

[0066] Figure 43 depicts computer systems that are programmed to implement methods of the disclosure.

[0067] Figure 44 depicts an exploded cross-sectional view of an exemplary device where the light source is positioned directly above a molecular sensing domain, without a light guide.

[0068] Figure 45 depicts the construction of an exemplary disposable microneedle array patch, which includes an array, a window, and an adhesive patch.

[0069] Figure 46 depicts a cross section of the patch in Figure 45, comprising an array base with microneedles, an analyte binding probe comprised in the hydrogel matrix in each needle, and a transparent window with conical features that seal the top of the needles and optically couple to the gel.

[0070] Figure 47 depicts an exploded view of an exemplary durable sensor device that comprises an enclosure, an image sensor board, an optical filter, an LED board with apertures, and a sensor window.

[0071] Figure 48 depicts an isomeric cross section of an assembled sensor device as shown in Figure 47.

[0072] Figure 49 depicts a portion of the LED array on the LED board, and the row and column lines used for addressing the LEDs.

[0073] Figure 50 depicts a cross section of a sensor device as shown in Figure 47. This figure particularly shows the arrangement of the optical elements and the paths of the illumination and emission light.

[0074] Figure 51 shows the schematic of the optical sensor with generic building blocks.

[0075] Figure 52 shows a schematic circuit diagram of one possible embodiment of a detection system, with generic building blocks.

[0076] Figure 53 shows the microneedle array for one embodiment.

[0077] Figure 54 is a schematic block diagram showing different components of the optical sensor.

[0078] Figure 55 shows a method of coupling the optical modes in the lightguide with the needle sensing domains.

[0079] Figure 56 shows a specific embodiment of Figure 55, where the sidewall of the base is substantially absorptive and only light rays at a substantially non-grazing angle at the core / cladding interface directly couple into the microneedle sensing domain.

[0080] Figure 57 shows total internal reflectance at the protrusion area and the base sidewall.

[0081] Figure 58 shows an embodiment where the protrusion area refractive index is higher than the refractive index of the core of the lightguide.

[0082] Figure 59 shows light being emitted from the microneedle sensing molecules toward the photodetector and Sensing PC board.

[0083] Figure 60 shows light being emitted from the microneedle sensing molecules toward the photodetector and Sensing PC board, with a possible geometrical stack for capturing the emitted light.

[0084] Figure 61 shows different paths the emitted light can take depending on the incidence angle at the upper core / cladding interface and their relative refractive indices.

[0085] Figure 62 shows an embodiment where the sidewall of the base of the microneedles is substantially absorptive.

[0086] Figure 63 shows an embodiment where the sidewall of the base of the microneedles is substantially reflective and specular.

[0087] Figure 64 shows an embodiment where the sidewall of the base of the microneedle is a dielectric stack capable of both refraction and TIR.

[0088] Figure 65 is a diagram of the sensing light collection stack above the lightguide core.

[0089] Figure 66 shows the addition of a spatial filter above the lightguide and before the optical long-pass filter.

[0090] Figure 67 shows an embodiment where the spatial filter used to cutoff substantially non-normal angles is placed after the optical long-pass filter.

[0091] Figure 68 shows the optical filter characteristics at various angles.

[0092] Figure 69 shows a microneedle array with needles going into human skin. The microneedles are hollow and hold target molecule sensing analyte probes.

[0093] Figures 70A-B show the packaged form of an exemplary sensor that uses guided light. Figure 70A shows the front view of the sensor. Figure 70B shows the back view of the sensor.

[0094] Figures 71A-B show the durable, reusable part of the sensor depicted in Figure 70. Figure 71A shows the packaged reusable part from the backside. Figure 71B shows an exploded view of the main layers and components inside the reusable part of the sensor.

[0095] Figures 72-74 show cross sectional views of all of the layers of both the reusable and disposable parts of the sensor depicted in Figures 70A-70B.

[0096] Figure 72 shows a cross sectional view of the reusable and disposable parts of the sensor depicted in Figures 70A-70B.

[0097] Figure 73 shows a magnified view of Figure 72 with more detailed depictions of the layers and showing all of the layers described in detail in the disclosure.

[0098] Figure 74 shows a slightly tilted view of the sensor depicted in Figure 72 with the same layers depicted in Figure 73.

[0099] Figure 75 depicts an exploded view of the disposable portion of the sensor depicted in Figure 72. It comprises the microneedle array and a plastic waveguide structure with special turning mirrors at both ends of the waveguide.

[0100] Figures 76A and 76B depicts individual pieces of the disposable patch in greater detail. They shows two different views from the top down of the disposable part of the image sensor.

[0101] Figure 77 is a schematic showing the acceptance angle range for an embodiment where the cladding of the lightguide is air everywhere.

[0102] Figure 78 is a diagram of the reusable fully assembled sensor.

[0103] Figure 79 is an opened-up view of the fully assembled sensor.

[0104] Figure 80 is an exploded view of the components inside the reusable sensor.

[0105] Figure 81 shows an enlarged cross-sectional view of the emission path.

[0106] Figure 82 shows an oblique view of the twin opposing lens design.

[0107] Figure 83 shows another oblique view of the twin opposing lens design.

[0108] Figure 84 shows the pin and needle arrays of the twin opposing lens design.

[0109] Figure 85 shows the sensor foci array of the twin opposing lens design.

[0110] Figure 86 shows a simulation of the excitation light launched into the waveguide and then coupled into the microneedles.

[0111] Figure 87 shows a simulation of emission light launched through the double lens design.

[0112] Figure 88 shows another simulation of emission light launched through the double lens design.

[0113] Figure 89 shows another simulation of emission light launched through the double lens design.

[0114] Figure 90 shows a twin lens focusing light on a small pixel of the image sensor.DETAILED DESCRIPTION

[0115] Continuous and real-time measurement of analytes in-vivo enables many applications towards predictive medicine, precision drug dosing, analysis of pharmacokinetics and pharmacodynamics during therapeutic drug development, and diagnosis and monitoring of various diseases. Continuous glucose monitors (CGMs) are the most prominent example of a real-time, in-vivo molecular monitoring technology, and have revolutionized the management of diabetes treatment. However, the core technology utilized by CGMs is not generalizable, as they use naturally occurring enzymes which exist only for very few molecules (e.g., glucose, lactate, ketones, and alcohol). A further challenge lies in developing percutaneous and implantable biointerfaces which may pass regulatory muster, be adequately sterilized, and be robust to degradation and fibrotic encapsulation when used in vivo. Developing new devices to satisfy these requirements may be costly, and there is a clear unmet need for a biosensor system and corresponding instrumentation that may leverage established and approved biointerfaces while expanding the dictionary of molecules which may be monitored in real time. The disclosure described herein is a biosensor system that integrates microdialysis biointerfaces with a fluidic pump and optical biosensors that is a sensitive and wearable measurement system. Such a device enables the continuous, in-vivo measurement of a large variety of analytes, and expands the data available to study physiological processes at the molecular level.

[0116] In many situations it is desirable to monitor the concentration of analytes periodically over a time interval. For example, many hormones, such as cortisol and melatonin, vary in concentration throughout the day and are known to have diurnal and / or ultradian rhythm. Dysregulation of these hormones may form the basis of a variety of disorders such as Cushing’s Disease, Addison’s disease, mild autonomous cortisol secretion (MACS), and insomnia. However, since current diagnostic tests are single time point assays conducted with low temporal resolution, the diagnosis and treatment of these conditions may be challenging. Another case where measuring analytes with high temporal resolution is important is in drug dosing and characterizing the pharmacokinetics and pharmacodynamics of drugs during clinical trials. Unfortunately, there are no existing solutions to meet these important needs.

[0117] Microdialysis is a technique for measuring analytes in dermal interstitial fluid (ISF) in a research setting. However, current microdialysis techniques are not suitable for real-time biosensing applications since the dialysate eluent from the device may need to be collectedinto various aliquots, and then sent to a laboratory for testing. Furthermore, microdialysis of dermal ISF may need the patient to remain largely immobile during the duration of the sample collection. This present disclosure provides a device which combines several concepts in order to enable the measurement of several analytes simultaneously (henceforth, multiplexed) with high temporal resolution (minutes to hours). These frequent measurements of analyte concentration may be used to capture the dynamic nature and trend profiles of specific analytes and this information may be used for a variety of impactful and transformative applications. Furthermore, unlike the enzymatic detection methods that are used in CGMs, the device provided herein is compatible with several types of analyte binding probes which may be rapidly generated against nearly any target molecule de novo. Importantly, to collect data at scale, the associated measurement device may be a wearable and may include its own pump, such that the user is not bound to a benchtop instrument and unable to exhibit normal behaviors that may influence underlying physiology. In some cases, the device may be provided adjacent to a body surface of the subject. In some cases, the device may be provided on the body surface of the subject. Finally, the ability to measure multiple molecules in a single device enables methods to correct for dilution effects which constrain the parameters under which microdialysis-based sensors may perform (e.g., flowrate, membrane area, measurement time-lags). The device is composed of several subsystems which may include, but are not limited to, a microdialysis probe, an input reservoir, a multiplexed optical measurement system, a microfluidic chip, an Output Reservoir, and a fluidic pump. In some embodiments, the multiplexed optical measurement system (or the optical reader) and fluidic pump are contained in a reusable, wearable device. In some embodiments, the microdialysis probe, the Input Reservoir, the Output Reservoir and the microfluidic chip are contained in a disposable component that mates with the reusable device. This arrangement allows a cost effective implementation of the wearable device where the expensive components are reused and the inexpensive components are disposed of after use. The device can also be used to measure other biofluids. For example, the device can be used to measure blood, saliva, urine, and cerebrospinal fluid (CSF). The probe can be inserted into various organs such as the brain, kidney, liver, eye, and muscle. The sensor system can also be used to measure samples in non-biological samples, such as waste water or fluids from industrial processes. For example, the sensor could be used to measure fermentation processes or chemical process reactions.

[0118] In some cases, the device may comprise a width of about 30 mm to about 58 mm. In some cases, the device may comprise a width of about 30 mm to about 32 mm, about 30 mmto about 34 mm, about 30 mm to about 36 mm, about 30 mm to about 38 mm, about 30 mm to about 40 mm, about 30 mm to about 42 mm, about 30 mm to about 46 mm, about 30 mm to about 48 mm, about 30 mm to about 50 mm, about 30 mm to about 54 mm, about 30 mm to about 58 mm, about 32 mm to about 34 mm, about 32 mm to about 36 mm, about 32 mm to about 38 mm, about 32 mm to about 40 mm, about 32 mm to about 42 mm, about 32 mm to about 46 mm, about 32 mm to about 48 mm, about 32 mm to about 50 mm, about 32 mm to about 54 mm, about 32 mm to about 58 mm, about 34 mm to about 36 mm, about 34 mm to about 38 mm, about 34 mm to about 40 mm, about 34 mm to about 42 mm, about 34 mm to about 46 mm, about 34 mm to about 48 mm, about 34 mm to about 50 mm, about 34 mm to about 54 mm, about 34 mm to about 58 mm, about 36 mm to about 38 mm, about 36 mm to about 40 mm, about 36 mm to about 42 mm, about 36 mm to about 46 mm, about 36 mm to about 48 mm, about 36 mm to about 50 mm, about 36 mm to about 54 mm, about 36 mm to about 58 mm, about 38 mm to about 40 mm, about 38 mm to about 42 mm, about 38 mm to about 46 mm, about 38 mm to about 48 mm, about 38 mm to about 50 mm, about 38 mm to about 54 mm, about 38 mm to about 58 mm, about 40 mm to about 42 mm, about 40 mm to about 46 mm, about 40 mm to about 48 mm, about 40 mm to about 50 mm, about 40 mm to about 54 mm, about 40 mm to about 58 mm, about 42 mm to about 46 mm, about 42 mm to about 48 mm, about 42 mm to about 50 mm, about 42 mm to about 54 mm, about 42 mm to about 58 mm, about 46 mm to about 48 mm, about 46 mm to about 50 mm, about 46 mm to about 54 mm, about 46 mm to about 58 mm, about 48 mm to about 50 mm, about 48 mm to about 54 mm, about 48 mm to about 58 mm, about 50 mm to about 54 mm, about 50 mm to about 58 mm, or about 54 mm to about 58 mm. In some cases, the device may comprise a width of about 30 mm, about 32 mm, about 34 mm, about 36 mm, about 38 mm, about 40 mm, about 42 mm, about 46 mm, about 48 mm, about 50 mm, about 54 mm, or about 58 mm. In some cases, the device may comprise a width of at least about 30 mm, about 32 mm, about 34 mm, about 36 mm, about 38 mm, about 40 mm, about 42 mm, about 46 mm, about 48 mm, about 50 mm, or about 54 mm. In some cases, the device may comprise a width of at most about 32 mm, about 34 mm, about 36 mm, about 38 mm, about 40 mm, about 42 mm, about 46 mm, about 48 mm, about 50 mm, about 54 mm, or about 58 mm.

[0119] In some cases, the device may comprise a length of about 50 mm to about 60 mm. In some cases, the device may comprise a length of about 50 mm to about 51 mm, about 50 mm to about 52 mm, about 50 mm to about 53 mm, about 50 mm to about 54 mm, about 50 mm to about 55 mm, about 50 mm to about 56 mm, about 50 mm to about 57 mm, about 50 mm to about 58 mm, about 50 mm to about 59 mm, about 50 mm to about 60 mm, about 51 mmto about 52 mm, about 51 mm to about 53 mm, about 51 mm to about 54 mm, about 51 mm to about 55 mm, about 51 mm to about 56 mm, about 51 mm to about 57 mm, about 51 mm to about 58 mm, about 51 mm to about 59 mm, about 51 mm to about 60 mm, about 52 mm to about 53 mm, about 52 mm to about 54 mm, about 52 mm to about 55 mm, about 52 mm to about 56 mm, about 52 mm to about 57 mm, about 52 mm to about 58 mm, about 52 mm to about 59 mm, about 52 mm to about 60 mm, about 53 mm to about 54 mm, about 53 mm to about 55 mm, about 53 mm to about 56 mm, about 53 mm to about 57 mm, about 53 mm to about 58 mm, about 53 mm to about 59 mm, about 53 mm to about 60 mm, about 54 mm to about 55 mm, about 54 mm to about 56 mm, about 54 mm to about 57 mm, about 54 mm to about 58 mm, about 54 mm to about 59 mm, about 54 mm to about 60 mm, about 55 mm to about 56 mm, about 55 mm to about 57 mm, about 55 mm to about 58 mm, about 55 mm to about 59 mm, about 55 mm to about 60 mm, about 56 mm to about 57 mm, about 56 mm to about 58 mm, about 56 mm to about 59 mm, about 56 mm to about 60 mm, about 57 mm to about 58 mm, about 57 mm to about 59 mm, about 57 mm to about 60 mm, about 58 mm to about 59 mm, about 58 mm to about 60 mm, or about 59 mm to about 60 mm. In some cases, the device may comprise a length of about 50 mm, about 51 mm, about 52 mm, about 53 mm, about 54 mm, about 55 mm, about 56 mm, about 57 mm, about 58 mm, about 59 mm, or about 60 mm. In some cases, the device may comprise a length of at least about 50 mm, about 51 mm, about 52 mm, about 53 mm, about 54 mm, about 55 mm, about 56 mm, about 57 mm, about 58 mm, or about 59 mm. In some cases, the device may comprise a length of at most about 51 mm, about 52 mm, about 53 mm, about 54 mm, about 55 mm, about 56 mm, about 57 mm, about 58 mm, about 59 mm, or about 60 mm.

[0120] In some cases, the device may comprise a thickness of about 12 mm to about 30 mm. In some cases, the device may comprise a thickness of about 12 mm to about 14 mm, about 12 mm to about 16 mm, about 12 mm to about 18 mm, about 12 mm to about 20 mm, about12 mm to about 22 mm, about 12 mm to about 24 mm, about 12 mm to about 26 mm, about12 mm to about 28 mm, about 12 mm to about 30 mm, about 14 mm to about 16 mm, about14 mm to about 18 mm, about 14 mm to about 20 mm, about 14 mm to about 22 mm, about14 mm to about 24 mm, about 14 mm to about 26 mm, about 14 mm to about 28 mm, about14 mm to about 30 mm, about 16 mm to about 18 mm, about 16 mm to about 20 mm, about16 mm to about 22 mm, about 16 mm to about 24 mm, about 16 mm to about 26 mm, about16 mm to about 28 mm, about 16 mm to about 30 mm, about 18 mm to about 20 mm, about18 mm to about 22 mm, about 18 mm to about 24 mm, about 18 mm to about 26 mm, about18 mm to about 28 mm, about 18 mm to about 30 mm, about 20 mm to about 22 mm, about20 mm to about 24 mm, about 20 mm to about 26 mm, about 20 mm to about 28 mm, about20 mm to about 30 mm, about 22 mm to about 24 mm, about 22 mm to about 26 mm, about22 mm to about 28 mm, about 22 mm to about 30 mm, about 24 mm to about 26 mm, about24 mm to about 28 mm, about 24 mm to about 30 mm, about 26 mm to about 28 mm, about26 mm to about 30 mm, or about 28 mm to about 30 mm. In some cases, the device may comprise a thickness of about 12 mm, about 14 mm, about 16 mm, about 18 mm, about 20 mm, about 22 mm, about 24 mm, about 26 mm, about 28 mm, or about 30 mm. In some cases, the device may comprise a thickness of at least about 12 mm, about 14 mm, about 16 mm, about 18 mm, about 20 mm, about 22 mm, about 24 mm, about 26 mm, or about 28 mm. In some cases, the device may comprise a thickness of at most about 14 mm, about 16 mm, about 18 mm, about 20 mm, about 22 mm, about 24 mm, about 26 mm, about 28 mm, or about 30 mm.Microdialysis Probe

[0121] In some embodiments, the microdialysis probe has an inlet and outlet (the inlet is located at the distal end of the probe and the outlet is located at the proximal end of the probe, or vice versa) and contains a segment of semipermeable membrane in between, henceforth a linear probe. In some embodiments a loop microdialysis probe is used (the inlet and outlet are located at the distal end of the probe and the fluid paths are approximately parallel). In some embodiments a concentric microdialysis probe is used (the inlet and outlet are colocated at the distal end of the probe and the fluid paths are concentric), henceforth a concentric probe. Figure 1 shows the different microdialysis probe configurations.

[0122] In some embodiments, the probe is open flow microperfusion probe (also known as OFM probe and sampling technique), which allows for membrane-free microsampling. Membrane-free OFM probes feature an exchange area with no diffusion barrier. This means that sampling is not affected by the size or lipophilicity of the analytes.

[0123] In some embodiments, an applicator is used to insert the probe into the dermis of a subject. The applicator may be similar to those leveraged by existing CGMs where a needle punctures the skin and may leave behind the microdialysis probe in the dermis or may be colocated with the microdialysis probe. In some cases, the device may be applied by a trained health care worker, and in other cases the patient may apply the device themselves in point- of-use settings such as in the subject’s home. In some embodiments, the microdialysis probe may be inserted into the patient using a minimally invasive surgical procedure. For example, a 21 Gauge needle may be inserted through a portion of the dermis, with an entrance and exithole in the dermis, and the microdialysis probe may be inserted into the dermis such that the semipermeable membrane is contained entirely in the patient’s dermis. In some cases, a topical anesthetic may be used.

[0124] In some embodiments the semipermeable membrane comprises a microarray patch (also known as microneedle array patch or MAP) with at least one microneedle that is held together by a base. In some embodiments at least one microneedle in the array contains a portion of semipermeable membrane inside the needle. In some embodiments, the microneedle itself is a semipermeable membrane in which the dialysate flows across the top of the MAP or throughout the MAP. In some embodiments, the semipermeable membrane comprises a pin or “concentric” microdialysis probe. In some embodiments, the semipermeable membrane comprises a linear microdialysis probe. In some embodiments, an applicator, such as those used in CGM devices, uses a retractable needle to insert the probe into the dermis of the subject. In other embodiments, the probe is surgically inserted into the dermis of the subject.

[0125] In some embodiments the semipermeable membrane is configured as a size exclusion membrane such that molecules above a molecular weight cutoff cannot diffuse freely and are impeded across the semipermeable membrane. For example, a 20 kDa size exclusion membrane will allow small molecules (e.g., molecules <5 kDa) to diffuse unimpeded, but will prevent larger molecules, such as antibodies, from diffusing across the membrane. On the other hand, membranes with larger molecular weight cutoffs (e.g., 80 kDa, 100 kDa, 150 kDa, 200 kDa) may be used which will allow larger proteins to pass through the membrane, but still exclude larger molecules.

[0126] The semipermeable membrane may be comprised of a material that allows the diffusion of some molecules from the physiological fluid of the subject wearing the device and the dialysate buffer that is flowed through the microdialysis probe of the device. The membrane may be made from a variety of different hydrogel and / or polymeric materials. Such hydrogels may be created from a variety of materials, including but not limited to, PEG (polyethylene glycol), PVA (poly vinyl alcohol), or agarose. Such hydrogels may be crosslinked using a variety of mechanisms such as photo-crosslinking, chemically cross linking, thermal crosslinking, (e.g., APS / TEMED, glutaraldehyde) or physical crosslinking. The membrane may also be made from sheets of films of natural and / or synthetic polymers that contain a network of sub-microscopic holes of pores in which molecules of certain sizes may pass through. These semipermeable membranes may be made of many materials,including but not limited to, cellulose acetate, potassium ferrocyanide, polysulfone, and / or polyethersulfone.Applicators

[0127] An applicator is a critical component in the wearable biosensing system, enabling users to self-apply the device efficiently, reliably, and with minimal discomfort. By facilitating consistent and precise placement of the probe, such as a microdialysis probe, applicators enhance the usability and accessibility of the device, making it suitable for a broad range of users, including those without medical training. The design of the applicator directly contributes to the adoption of the biosensor system in real-world environments, allowing seamless integration into daily life. Furthermore, applicators with user-friendly designs, automated features, or integrated functions ensure that probe placement is accurate, reducing risks of improper insertion, discomfort, or device failure.

[0128] This invention can leverage different types of applicators to insert the microdialysis probe into the dermis of the patient. In most embodiments, the microdialysis probe is concentric in design since concentric probes are more amenable to insertion with an applicator than linear probes since only a single puncture site into the skin must be created. In some embodiments a needle-based applicator is used. These applicators use a needle to puncture the skin, enabling insertion of the probe, which remains in place after the needle is withdrawn. This approach mirrors the mechanisms used in continuous glucose monitors (CGMs) and ensures reliable probe deployment. Needle-based applicators can incorporate depth control features to guarantee proper placement without excessive penetration, improving safety and comfort for the user.

[0129] In some embodiments, the applicator is built directly into the wearable device, offering an all-in-one solution for application and monitoring. The integration of the applicator and microdialysis probe into a single housing can increase the usability of the device. In some embodiments the applicator can be actuated mechanically by having the user manually activate the applicator by pressing a button, twisting a knob, pressing the device against their skin, or other means. These actions deliver a controlled mechanism to insert the probe effectively.

[0130] In some embodiments the applicator can be triggered by automated or software- controlled means. Smart devices may allow the applicator to be triggered via software commands, such as through a mobile application or remote control. This capability isparticularly useful for users who require precise actuation or remote assistance in device setup.

[0131] In some embodiments the applicator is deployed by using a spring-based mechanism. A spring-based mechanism may be employed to drive the needle or probe into the skin with controlled force. Spring-loaded designs can be used as standalone units or integrated into the wearable device. In some embodiments a press-and-deploy applicator can be used, where the applicator deploys the probe upon pressing the device against the skin. The applicator may use a combination of mechanical pressure and needle release to insert the probe smoothly. Such designs are particularly advantageous for single-handed application and can be paired with disposable units for hygiene. In some embodiments motorized applicators can be used. Motorized systems can drive the insertion of the probe at a controlled speed and angle, which could allow for enhanced placement precision.

[0132] In some embodiments, the entire applicator and probe system is disposable. In other embodiments, a portion of the applicator can be re-used, alongside a disposable portion. For example, for a needle-based applicator, the probe and insertion needle could be disposable, while a re-settable spring-loaded mechanical assembly could be re-used. In some embodiments, the probe and applicator are housed in a single, integrated unit. The combined design simplifies handling and minimizes the number of steps required during application, reducing the potential for user error.

[0133] The applicators of the present invention can be configured in a variety of ways. In some embodiments, the applicator and biosensor device are separate. In such designs, the applicator is used solely to insert the probe, which is then connected externally to the biosensor device. Other embodiments combine the applicator and biosensor in a single housing, allowing for streamlined operation and reduced handling complexity.Input Reservoir

[0134] The Input Reservoir supplies the system with the dialysate buffer that is pumped through the fluid path and is used to analyze analyte concentrations.

[0135] In some embodiments, the reservoir (henceforth, dialysate reservoir or cartridge) is used to hold the dialysate buffer where the pump draws dialysate buffer from the dialysate reservoir. In other embodiments, the Input Reservoir is utilized as part of the pumping mechanism Input Reservoir may be a reservoir that is of fixed volume, or of variable volume (for example deformable or transformable such as a syringe).

[0136] In some embodiments, the Input Reservoir may come ready to use (pre-filled and sterilized with a connector / adaptor). In some embodiments, users may fill the Input Reservoir with dialysate buffer.

[0137] In some embodiments, the dialysate buffer contains molecules, such as salts and sugars (which may include sodium chloride, potassium chloride, magnesium chloride, dextran, mannitol) to maintain osmotic balance.

[0138] In some embodiments, the dialysate buffer is driven through the flow path of the device. In some embodiments, the fluid is flowed through the system by the use of a pump. Other mechanisms, such as an applied vacuum may be used to provide the driving force. The flow rate of the dialysate buffer through the system may be adjusted to meet the needs of equilibrating target analyte concentrations in the ISF with the concentrations in the dialysate, and are likely to be from 10 pL per minute to 10 uL per minute. In some embodiments, flow rates are from 1 nL per minute to 5 uL per minute.Microfluidic Chip

[0139] The eluent from the outlet of the microdialysis probe flows into the inlet of the microfluidic chip or flow cell. The microfluidic chip is a mating device that:1. allows interaction of the fluid with analytes from the subject with the sensing molecules2. allows the light from an optical measurement system or an optical reader to probe the sensing molecules by providing some level of transparency to all the wavelengths involved in sensing. In some embodiments, the optical reader may be in close proximity to the microfluidic chip.

[0140] The chip may be optically transmissive such that excitation light may be transmitted to the analyte binding probes, and any emitted light may be transmitted back to a photosensitive device. Materials may include glass, silicon, or polymers such as PDMS, PMMA, PC, etc.

[0141] In the flow cell, the eluent is contacted with analyte binding probes provided herein such that the analyte binding probes may bind to the analytes that are flowing in the eluent. The region in which the analyte binding probes interact with the analyte is referred to as the “sensing domain.” In some embodiments, the analyte binding probes are immobilized directly on a surface within the flow cell in a region referred to as a “sensing spot.” In some embodiments, the analyte binding probes are contained within a polymeric matrix (such as a hydrogel). In some embodiments, the analyte binding probes are covalently immobilizedwithin the polymeric matrix, and in other cases they are entrapped (e.g., by steric hindrance). In some embodiments, the analyte binding probes are contained on beads or particles that are located within the flow cell. Figure 2 shows methods of immobilizing analyte binding probes.

[0142] In some embodiments, the fluid in the device may be flowed over different regions of the flow cell which contain different types of analyte binding probes configured to have affinity to different target molecules. In some embodiments, this enables measurement of multiple analytes simultaneously in a multiplexed measurement. The fluid may be flowed serially through the various analyte binding regions, or the fluid may be split over different analyte binding regions in parallel. In part, the decision to split the fluid path in parallel channels may be done to reduce the pressure and / or resistance of flow through the flow cell.Output Reservoir

[0143] The eluent that exits the microfluidic chip is deposited into an Output Reservoir.

[0144] In some embodiments, the eluent is collected and the Output Reservoir is designed such that its internal volume is at least equal or greater than that of the Input Reservoir. In some such embodiments the collected eluent is to be disposed of and the reservoir is designed to be removable and disposable. In some embodiments, the collected eluent is to be saved for further analysis and the Output Reservoir maintains a series of discrete samples.

[0145] In some embodiments, the Output Reservoir is designed to allow the evaporable contents of the eluent to evaporate through a semipermeable membrane at a suitable rate, thereby reducing the size of the Output Reservoir.Analyte Binding Probes

[0146] The present disclosure provides sensors for analyte detection. An analyte binding probe may comprise polypeptide or nucleic acid sequences, antibodies, peptides, proteins, physiochemical detectors, enzymes, artificial binding proteins, or combinations thereof. In some embodiments, the analyte sensor comprises a polypeptide or peptide sequence such as an antibody. For example, an analyte sensor may comprise single stranded deoxyribonucleic acid (ssDNA), double stranded deoxyribonucleic acid (dsDNA), ribonucleic acid (RNA), nucleic acids in some cases with modified bases, and the like. The analyte sensor may be an oligonucleotide probe and the analyte may be a complementary target nucleic acid. In another embodiment, the binding domain may be a dsDNA strand specific to a target enhancer protein target. In some embodiments, the analyte sensor may comprise a nucleicacid sequence comprising an aptamer. These binding probes may be engineered to contain luminescent reporters and quenchers, such that they may modulate luminescence upon binding to an analyte molecule. In some embodiments, the mode of luminescence is continuous wave fluorescence, whereby the sensing domain may be illuminated for a short duration (and repeated illuminated periodically at closely spaced time intervals suitable for the desired time resolution for the application), while the light emitted by the luminescent reporters is measured. The duration of illumination may be from 10s of microseconds to several 10s of seconds. The magnitude of light emitted from the fluorescence encodes the information on the analyte concentration. In other embodiments, the reporter and quencher may be configured to generate signals by time-resolved fluorescence, decay curve monitoring or phosphorescence. The analyte binding probes are localized to sensing spots such that they may be addressed directly by the measurement system. In some embodiments, the binding probes are covalently linked to the substrate of a microfluidic chip that is mated to the optical reader. In other embodiments, the binding probes may be suspended in a hydrogel that may be bound or physically constrained to the correct location. In other embodiments, the binding probes may be freely diffusing but confined by a size-exclusive membrane.Multiplexed Optical Measurement System- Measuring multiple analyte concentrations simultaneously

[0147] The multiplexed optical measurement system contains the components to carry out a quantitative reading of the emitted luminescence from the analyte binding probes in multiple spatially segregated sensing spots. These spatially segregated sensing spots may encode information on multiple analytes. This includes an illumination system and a detection system which are comprised of optical, electronic, and mechanical components.

[0148] The illumination system is comprised of an optical path that begins with a light source powered by an electrical circuit. In some embodiments the light source may be comprised of LEDs, EELs, SLEDs, or VCSELs. The light source emits light at a wavelength that corresponds to the excitation spectrum of the optical reporter used in the analyte binding probe. This light may be referred to as “excitation light.” Optical filters may be used in the excitation light path to separate the excitation light from any undesired light that may be generated or captured in the system. The excitation light is then guided to the sensing spot through optical components that may include lenses, apertures, optical filters, mirrors, light guides, waveguides, optical fibers, or other components. The excitation light may be focused on the sensing spot or may be defocused by intentionally having the sensing area not exactlyin the focal plane of the focusing lens of the excitation path. In some embodiments, the excitation spot may be defocused such that it excites a portion of the sensing spot with an area up to 1 square mm. In some embodiments, the de-focusing may be achieved by inserting materials of fixed thickness between the microfluidic chip and its mount.

[0149] In some embodiments, the sensing molecules may be inside the microfluidic chip which is in close proximity to the optical reader and the excitation light from the optical reader passes through the transparent part of the microfluidic channel to get to the sensing domain. The transparent part of the microfluidic channel may be made of glass. Subsequently, the emitted light coding the information on the concentration of the analyte that the sensing molecule is configured to sense is passed back through the glass of the microfluidic channel and coupled back into the optical reader and guided to a detector inside the optical reader.

[0150] In some embodiments, the illumination system may provide excitation light at multiple wavelengths. This may be for the purpose of increasing the number of analytes that may be able to be sensed in a smaller spatial area or for providing references at various wavelengths. The multiple wavelengths may arise from different light sources or may be produced from a single source using devices such as Tunable lasers.

[0151] In the sensing spot, the excitation light is absorbed and partially converted to luminescence, which will be referred to as “emission light.” The emission light is measured in the detection system, which collects the emission light and delivers it to a photosensitive device via optical components which may include lenses, apertures, optical filters, mirrors, light guides, waveguides, optical fibers, or other components. The photosensitive device fully or partially comprises an optical detector and may be one or several photodiodes, avalanche photodiodes, image sensors, silicon photomultipliers, light sensors, or custom integrated circuits which include photosensitive devices. The detection system uses the optical detector to convert the optical signal to an electrical system, which is then digitized and reported to the user, who may be a clinician, researcher, patient or another professional.

[0152] As the excitation light is much higher in magnitude than the luminescence, the device may include strategies to eliminate any excitation light that may find its way into the detection system. These strategies may include the use of lens, filters, apertures, mirrors, optical coatings, absorbing materials and light-trapping geometries.

[0153] A key feature of the device is the ability to measure multiple analytes in the flow cell by leveraging different analyte binding probes in the same device and having an optical measurement system that is able to perform this measurement. In some embodiments, theanalyte binding probes are spatially separated, such as in different compartments within the flow cell of the device, so that the signal from each analyte binding probe may be spatially falling on different detectors or pixels of a detector that are found within an array of pixels. In some embodiments, the optical measurement system enables this by having multiple combinations of illumination and detection systems, referred to as “channels” to correspond to individual sensing spots. In other embodiments, the optical measurement system may share channels between multiple spots. In other embodiments, the analyte binding probes are labeled with spectrally orthogonal fluorophores such that the signals from the various analyte binding probes may be spectrally demultiplexed. In some embodiments, both spatial and spectral multiplexing may be leveraged.Additional Sensors and Corrections

[0154] In some embodiments, the reader includes further sensing elements. In some embodiments those additional sensors may measure temperature, orientation, motion, humidity, altitude, pressure, or some combination thereof. In some embodiments, optical signals may be processed in conjunction with other data streams to improve the accuracy of the molecular measurement. In some embodiments, information from these sensors may be used alone, or in conjugation with combination with other sensor and / or biosensor elements in order to provide information regarding the health status, diagnosis, and / or treatment of a subject.

[0155] In some embodiments, where components of the optical path are sensitive to temperature, the temperature data may be used to apply a correction to the optical measurements. In some embodiments, the temperature-sensitive components may include the light emitter or the detector. In some embodiments, data from single or multiple temperature sensors may be used. In some embodiments, where multiple temperature sensors are used, the temperature sensors may be distributed around the PCB to obtain localized corrections for individual optical channels. In an exemplary embodiment, the correction can be generated by placing the unit in a temperature-controlled chamber with an inert chip assembled to the reader. In such an embodiment, the temperature may be swept while taking optical measurements. In some embodiments, these temperature measurements may then be used to ratiometrically adjust optical measurements when interrogating optical analyte binding probes.Fraction collection and storage of dialysate

[0156] In some embodiments, the dialysate is stored for subsequent analysis. In some embodiments, separating the fractions is to prevent diffusion of analytes from one fraction of dialysate to another, so that the composition of the dialysate may be analyzed over a period of time. In some embodiments, the dialysate is periodically stored into spatially separated volumes over time. The periodic storage of dialysate is advantageous since it may allow validation of the biosensor performance. For example, the fractions of dialysate may be related to the time interval of collection, and then the concentration of a given analyte in the collected fraction may be measured using standard analytical methods and then compared to the signal from the biosensor. Using such a comparison, information regarding the accuracy and precision of the device may be measured over time. The collection of dialysate fractions also allows additional analytical methods (e.g., ELISAs, LC-MS, GC-MS, etc.) to be performed. These additional analytical techniques may complement the real-time data obtained with the biosensor by providing additional data on analytes and biomarkers, such as proteomic and metabolic information.

[0157] There are many methods to spatially separate and store fractions of dialysate. In some embodiments, the dialysate may be collected into a series of tubes using a fraction collector. One example of a fraction collector is where the outlet tubing is held above a series of vials or containers and the dialysate flows into a given vial that is placed below the outlet tubing. Over time, the vial underneath the tubing may be changed in an automatic fashion periodically in order to collect a time-series of dialysate into the series of vials or containers. In some embodiments, other methods are employed to spatially separate and store fractions of dialysate which may be more amenable to miniaturization.

[0158] An alternative method is to store the fractions of dialysate into a length of tubing. The fractions in the tubing may be spatially separated from each other by incorporating a segment of air (or another gas), which prevents the diffusion of analytes between the fractions of dialysate. These types of separation methods have been described in the literature, for example in Thomas J. Upton, et al. (2023) High resolution daily profiles of tissue adrenal steroids by portable automated collection. Science Translational Medicine, related patent: https: / / patents.soosle.com / patent / US20150238132Al / en which are hereby incorporated by reference in their entirety.

[0159] In some embodiments, the fraction collection occurs within a microfluidic chip. In some embodiments, this is the same microfluidic chip that the real-time biosensing occurs in,and in some embodiments it is separate from where the real-time biosensing occurs. The fractions may be spatially separated using a variety of methods within the microfluidic chip. In some embodiments, segments of air or another gas are used to spatially separate the fraction of dialysate. In some embodiments, valves and / or actuators within the microfluidic device may spatially separate the fractions of dialysate into different compartments within the flow cell. For example, valves may be controlled mechanically to open or close at given time intervals, or passive valves may be configured to stop fluid flow after a compartment fills up. In some embodiments, the valves are check valves. In some embodiments, where analytes are insoluble in a second phase that is immiscible with water, spatial separation may be achieved through two phase droplet generation where water droplets are generated within a flow of the second phase using droplet generation microfluidics.Dialysate Buffer

[0160] In some embodiments the dialysate buffer contains molecules, such as dextran, to alter the osmotic pressure. The dialysate buffer is driven through the flow path of the device. In some embodiments the fluid is flown through the system by the use of a pump. Other mechanisms, such as an applied vacuum can be used to provide the driving force. The flow rate of the dialysate buffer through the system can be any value, although it will likely be between 10 pL per minute and 10 uL per minute. Flow rates between 1 nL per minute and 5 uL per minute are likely preferred. In some embodiments, the dialysate contains reference molecules that can be measured by the biosensor in order to measure recovery across the microdialysis membrane. In some embodiments, the reference molecule measurements can be used to correct or adjust the concentrations and / or signals of other molecules being measured by the sensor. In some embodiments, the reference molecule is endogenous. In some embodiments, the reference molecule is exogenous.

[0161] In some embodiments, the dialysate contains anti-inflammatory agents in order to reduce inflammation or other aspects of the foreign body or immune response. In some embodiments, the anti-inflammatory agents can be incorporated onto the surface or within the portion of the microdialysis probe and / or tubing which is contacted with the biological sample of the subject. In some embodiments, the anti-inflammatory is dexamethasone acetate, or another similar dexamethasone compound. In some embodiments, the antiinflammatory agent can consist of one or a combination of the following compounds: hydrocortisone, prednisolone, triamcinolone, ibuprofen, ketoprofen, indomethacin, aspirin, sirolimus (rapamycin), tacrolimus, infliximab, adalimumab, anakinra, azithromycin, vitaminE (tocopherol), N-acetylcysteine, and curcumin. These agents may be selected based on their ability to modulate inflammatory pathways, reduce oxidative stress, or inhibit immune cell activation and recruitment, thereby minimizing inflammation and improving the biocompatibility of the device. The specific choice and formulation of these agents may vary depending on the intended application, the duration of use, and the particular biological environment of the subject.Analyte recovery and methods to correct for dilution effects

[0162] One critical parameter in microdialysis systems is the recovery percent, or the amount analyte that is contained in the eluent from the microdialysis tubing in contrast to the concentration of the analyte in the dermal ISF. If the recovery is 100%, the concentration measured in the eluent of the microdialysis setup is the same as the concentration in the subject's dermal ISF. However, if insufficient time is given to allow the analytes in the ISF to diffuse through the semi-permeable membrane in the microdialysis probe, then the recovery will be less than 100% and quantification of the analyte in the microdialysis eluent will not be representative of the analyte concentration in the subject. Many factors can affect the recovery rate, such as the properties of the semi-permeable membrane (e.g., molecular weight size cutoff), the properties of the analyte, the length and diameter of the semi-permeable membrane, and the flow rate of the dialysate buffer through the microdialysis probe. Furthermore, when a material such as a microdialysis probe is implanted, the foreign body response can change the ability of analytes to diffuse through the membrane through mechanisms such as fibrotic encapsulation.

[0163] In some embodiments, one or more reference molecules are measured by the device in addition to the target analyte(s). In some cases the reference molecule can be an analyte with known concentration, or that is known to be stable in concentration. For example, DHEA-S can be measured as a reference molecule as it is known to be relatively stable in concentration over time. By comparing the signal from a target molecule, such as cortisol, against a reference such as DHEA-S, differences in recovery rate over time can be accounted for. In some embodiments the reference molecule is endogenous, and in some embodiments it is exogenous.

[0164] In some embodiments, the reference molecules are known analytes that are introduced at a controlled concentration within the dialysate buffer. In some of these embodiments, the molecule is chosen to have a molecular weight similar to the analyte of interest so that it will diffuse across the microdialysis semi-permeable membrane at the same rate as the target. Inother embodiments, the reference molecule is a larger or smaller molecular weight than that of the target analyte. In some of these cases, the molecule may be too large to diffuse across the membrane and will remain within the microdialysis tubing. In some embodiments, multiple reference molecules of varying molecular weights can be used. In some cases retrodialysis (also known as delivery method) can be used to calibrate the recovery using the equation recovery(%) = 100 - (Cdial / Cperf-100), where Cdial is the concentration of the reference molecule in the dialysate and Cperf is the concentration in the perfusate.

[0165] When a reference molecule is used that can permeate the membrane, the concentration of the reference molecule in the dialysate buffer will decrease due to its diffusion across the membrane and into ISF, and this decrease will be dependent on the rate of analyte exchange across the membrane, thus allowing for differences in the recovery rate with time to be accounted for. For example, a biocompatible molecule at a similar molecular weight to the target (such as maltose as a proxy for cortisol) could be added within the dialysate, and by comparing the signal from a target molecule to the proportional decrease in signal from this reference molecule retrieved from microdialysis, differences in recovery rate over time can be accounted for.

[0166] In some embodiments, two analytes can be measured, and a ratio can be taken of the signal from each. This ratiometric approach can also be used to account for situations where equilibration is not achieved between analytes in the dermal ISF and dialysate in the microdialysis probe. Furthermore, in many clinical applications ratios between biomarkers are known to be especially informative, such as in the case of creatinine and urea.

[0167] The ability to correct for recovery rate by measuring reference molecules, or by comparing two molecules ratiometrically not only allows more precise quantification of analytes over prolonged periods of time, but also allows faster flow rates or shorter membranes to be used by the device since the recovery percentage of the analyte does not need to be near 100%. This can be beneficial since shorter membranes can allow for the development of less invasive probes and since faster flow rates can increase the temporal resolution of the measuring system.Calibration

[0168] In some embodiments, the microfluidic chip may be calibrated before and / or after use. For example, one or more aqueous solutions containing one or more of the analytes may be flowed through the microfluidic chip and the response from the analyte-binding probes characterized. The responses from these signals can be stored or associated with the specificmicrofluidic chip and used to convert the signal from the biosensor device into concentration values that are being measured. In some embodiments, the solutions may contain different pH or salt concentrations. In some embodiments, the solutions may cause dehybridization or unfolding of the molecular switches (for example by increasing pH, increasing temperature, or adding an organic solvent such as DMSO or DMF). In some embodiments, the response of the analyte binding probe may be evaluated as a function of temperature.

[0169] In some embodiments, calibration measurements may be used to create a linear fit correlating signal to concentration. In some embodiments, other higher order polynomial equations can be fitted. In some embodiments, Langmuir isotherms or other molecular binding models can be used. In some embodiments, thermodynamic models can be used, while in other cases information from on and off-rates can be used to fit kinetic models. In some embodiments, the calibration signals can be used to establish maximum and minimum signals of the unbound and bound states of the analyte binding probes.

[0170] In some embodiments, the flow cells are factory calibrated prior to packaging. After calibration, the flow cells may be stored in a dried state. To dry the flow cells a process like evaporation, washing with non-aqueous solvents (such as acetone or ethanol), or freeze- drying may be used. In some embodiments, the flow cells are stored in a solution. The solution may contain properties that prevent bacterial growth. In some embodiments, the device may be calibrated immediately prior to use by the end user. In some embodiments, this may be the only calibration that occurs. In some embodiments, calibration prior to use may occur in addition to the factory calibration. In some embodiments, calibrating or quality control solutions can be used to ensure the device itself is functioning. In some embodiments, these calibration solutions may contain fluorescent dyes.Variable Flowrate

[0171] In some embodiments, the flowrate of the pump may be changed between two or more flowrates, and measurements are taken with the biosensor device at each flow rate. The analysis of biosensor response as a function of flowrate in the device has many uses for establishing accurate and precise quantification over long durations of sensor use. The ability to obtain long duration measurements is a key feature of percutaneous biosensors and an important enabling innovation.

[0172] In some embodiments, the flowrate of the pump can be altered in order to reduce the recovery rate of an analyte across the microdialysis probe, and thus to decrease the concentration of the analyte reaching the sensor. In some embodiments, the reduction inconcentration is at least 50%, 25%, 10%, 5%, or 1%. In some cases, measurements of the biosensor at these reduced analyte concentrations can be used to re-baseline the sensor or to re-calibrate the signal. In some embodiments, the sensor reading at the reduced recovery can be used to create a new baseline for the unbound analyte binding probes, and can be used to re-establish a baseline to correct for baseline drift and other artifacts.

[0173] In some embodiments, the sensor response as a function of flowrate can be used to estimate recovery across the microdialysis membrane, or similarly, can be used to evaluate the change in recovery rate of the sensor as a function of time due to effects like foreign body response and fibrotic encapsulation. In some embodiments, the no-net-flux method or dynamic no-net-flux method can be used to correct for differences in recovery rate. In some cases, loss=gain methods (retrodialysis) can be used to measure recovery rate by measuring a reference molecule or calibrator added to the perfusate. In some embodiments, the flow rate can be changed and measured to ensure that the system is fully equilibrated and that the concentration of the analyte does not change by a significant amount. In some embodiments, the method of flow variation can be used and the sensor readings are used to fit or derive parameters from the following exponential function: Cdial = C0(l-exp(-rA / F)) where Cdial is the concentration of the compound in the microfluidic chip of the sensor (or collected perfusate), CO is its concentration in the periprobe fluid, r is the mass transport coefficient, A is the surface area of the microdialysis membrane, and F specifies the chosen flow rate.

[0174] In some embodiments, the flowrate can be periodically increased in order to re-prime and dislodge any potential bubbles and / or particulates from the system (e.g., microdialysis probe, tubing, flow cell). The periodic changes in flowrate can occur over any timescale. In some embodiments, the increase in flowrate can range from short amounts of time (seconds) to longer durations (tens of minutes). In some embodiments, the increase in flowrate is not conducted on a set period of time, but can be triggered by a sensor. For example, it could be triggered by a flow rate, pressure, or optical sensor that can detect a change in the system, such as the emergence of an air bubble. In some embodiments, both periodic and triggered repriming can be used in combination.

[0175] In some embodiments, the flow rate can be decreased or stopped when biosensor readings are not being taken in order to conserve power and extend the duration the device can be used without recharging or changing the battery.Antigen Binding Probes: Aptamers

[0176] Sometimes referred to as “synthetic antibodies,” aptamers may be pre-selected singlestranded oligonucleotide (e.g., DNA or RNA) or peptide molecules that bind to specific target molecules including proteins and peptides with affinities and specificities that are comparable to antibodies. These molecules can assume a variety of shapes due to their propensity to form helices and single-stranded loops with specific binding pockets, explaining their versatility in binding to diverse targets. Their specificity and characteristics are not directly determined by their primary sequence but by their tertiary structure which can be analogous to the globular shape of tRNA. Aptamers have a wide range of applications including diagnostics and therapeutics and can be chemically synthesized using known techniques. Furthermore, aptamers can offer a number of advantages over traditional antibodies including avoiding the need to specifically know the precise epitopes or biomarkers themselves. Finally, aptamers may be typically non-immunogenic, easy to synthesize, characterize, modify, and exhibit high specificity and affinity for their target antigen.

[0177] The aptamer may be nucleic acid or peptide molecules that bind to a specific target molecule. In some embodiments, binding of the target analyte to the aptamer induces conformational changes in the aptamer. In some embodiments, the aptamer may bind to various molecular targets, for example, small molecules, macromolecules, metabolites, proteins, carbohydrates, metals, nucleic acids, cells, tissues, and organisms.

[0178] By using a variety of selection techniques, aptamers can be selected to find targets, e.g., on a surface or inside a cell of interest, without the need to identify the precise biomarker or epitopes themselves. In many cases, the aptamer identification process can begin with a large random pool of oligonucleotides or peptides that are systematically subjected to negative and positive rounds of selection against a target, e.g., a protein molecule, to filter out low affinity or unspecific binders. The remaining aptamers can be collected and propagated, e.g., PCR amplified, and used in subsequent rounds of selection. This selection process, referred to as Systemic Evolution of Ligands by Exponential Enrichment or SELEX, is commonly used for selecting and identifying highly targeted aptamers. A variant of this methodology, known as cell-SELEX, has been developed for aptamers that are capable of recognizing whole living cells.Aptamer Switch

[0179] In some embodiments, the antigen binding probe may comprise at least one of the four elements: a single-stranded oligonucleotide; a short, complementary DNA sequence tothe oligonucleotide; a linking moiety that conjugates the oligonucleotide with the DNA sequence; and luminescent molecules.

[0180] In some embodiments, the single oligonucleotide has a first and second terminus, wherein the first terminus can be attached to a luminescent molecule and a second terminus attached to a piercing element. In some embodiments, the single oligonucleotide further comprises a short, partially complementary DNA sequence, wherein the short, partially complementary DNA sequence further comprises a second luminescent molecule.

[0181] In some embodiments, the single-stranded oligonucleotide has a first and second terminus, where the first terminus can be attached to the linking moiety. The linking moiety can be also attached to the first terminus of a short DNA strand having a partially complementary sequence to the oligonucleotide, where the short DNA strand has a first and second terminus. Luminescent molecules are attached to the second termini of the oligonucleotide and the short DNA strand.

[0182] In some embodiments, the oligonucleotide can be an aptamer. Aptamers are nucleic acid molecules that bind to a specific target molecule such as small molecules, proteins, nucleic acids, cells, tissues, and organisms. In some embodiments, aptamers are singlestranded oligonucleotides exhibiting high affinity and specificity toward any given target molecule. The aptamer disclosed herein may be any suitable size.

[0183] In some embodiments, the size of the aptamer as disclosed herein can be about 5 nucleotides to about 250 nucleotides. In some embodiments, the probe may further comprise a short, complementary DNA sequence that, in the absence of a target analyte, can hybridize with complete or partial complementary to a portion of the aptamer. In some embodiments, the aptamer and complementary DNA sequence described herein may have one or more mismatched nucleotides.Linking Moieties

[0184] In some embodiments the analyte sensor may further comprise a flexible linker region that attaches the oligonucleotides (e.g., aptamer) to the short DNA sequence. In some cases, the linker moiety can be a nucleotide acid moiety that does not bind to either the oligonucleotide, the short DNA sequence, a peptide nucleic acid (PNA) moiety, a peptide moiety, a disulfide bond, a phosphodiester linkage, or a polymer such as a polyethylene glycol (PEG) moiety. The linker region disclosed herein can be about 2 residues in length to about 45 residues in length. In some embodiments, a linker can be a homopolymeric polynucleotide. An intramolecular linker can be about 5 nucleotides to about 60 nucleotides.Antigen Binding Probes: Peptides

[0185] In some embodiments, an analyte sensor may be a polypeptide-based probe employing intramolecular signal transduction. In some embodiments, the polypeptide-based probe contains at least one of the following elements: a polypeptide with an antigen binding region, a linking moiety, and a luminescent molecule. In some embodiments, the polypeptide- based probe is a protein-based affinity reagent. In some embodiments, the polypeptide-based probe comprises nanobodies, antibody fragments, peptides, cysteine-knot proteins (knottins), or combinations thereof.

[0186] In some embodiments, a polypeptide with an antigen binding domain is further conjugated with a luminescent molecule. In some embodiments, a polypeptide with an antigen binding domain is attached to a linking moiety. The linking moiety can be also attached to a second polypeptide with another antigen binding domain, and the luminescent molecules are attached to the first and second polypeptides. In some cases, when the two antigen binding domains bind to the molecular target, they become closer in proximity and can alter the optical signal via a fluorophore / quencher or FRET-type interaction to produce an optical signal.

[0187] Antibody fragments which recognize specific epitopes can be generated by known techniques. Antibody fragments are antigen binding portions of an antibody, such as F(ab')2, Fab', F(ab)2, Fab, Fv, scFv and the like. F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule and Fab' fragments can be generated by reducing disulfide bridges of the F(ab')2 fragments. Alternatively, Fab' expression libraries can be constructed (Huse et al., 1989, Science, 246:1274-1281) to allow rapid and easy identification of monoclonal Fab' fragments with the desired specificity. F(ab)2 fragments may be generated by papain digestion of an antibody.

[0188] In some embodiments, the polypeptide is a single-chain polypeptide. In some embodiments of any of the single-chain polypeptides described herein, the single-chain polypeptide can be or include a BiTe, a (scFv)2, a nanobody, a nanobody -HS A, a DART, a TandAb, a scDiabody, a scDiabody-CH3, scFv-CH-CL-scFv, a HSAbody, scDiabody-HSA, or a tandem-scFv.

[0189] In some embodiments, the polypeptide is a multi-chain polypeptide. In some embodiments, the multi-chain polypeptide can be or can include an antibody, a Dual scFab, a F(ab’)2, a diabody, a crossMab, a DAF (two-in-one), a DAF (four-in-one), a DutaMab, a DT- IgG, a knobs-in-holes common light chain, a knobs-in-holes assembly, a charge pair, a Fab-arm exchange, a SEEDbody, a LUZ-Y, a Fcab, a rA-body, an orthogonal Fab, a DVD-IgG, a IgG(H)-scFv, a scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, a triple body, a miniantibody, a minibody, a TriBi minibody, scFv-CH3 KIH, Fab-scFv, a F(ab’)2-scFv2, a scFv-KIH, a Fab-scFv-Fc, a tetravalent HC Ab, a scDiabody-Fc, a Diabody-Fc, a tandem scFv-Fc, an Intrabody, a dock and lock, a ImmTAC, an IgG-IgG conjugate, a Cov-X-Body, or a scFvl-PEG-scFv2.

[0190] In some embodiments, the antigen-binding domain is humanized or human.

[0191] The antibodies of use can be of various isotypes, such as human IgGl, IgG2, IgG3, or IgG4. The antibodies or fragments thereof can be chimeric human-mouse, humanized (human framework and murine hypervariable (CDR) regions), or fully human, as well as variations thereof, such as half-IgG4 antibodies (referred to as “unibodies”). The antibodies or fragments thereof may be designed or selected to comprise human constant region sequences that belong to specific allotypes, which may result in reduced immunogenicity when administered to a human subject. Preferred allotypes for administration include a non-Glml allotype (nGlml), such as Glm3, Glm3,l, Glm3,2, or Glm3,l,2. More preferably, the allotype is selected from the group consisting of the nGlml, Glm3, nGlml, 2, and Km3 allotypes.

[0192] In some embodiments, non-limiting examples of analyte binding probe includes a DARPin, an affibody, a monobody, a nanobody, a diabody, an antibody (including a monospecific or bispecific antibody); a cell-targeting oligopeptide including but not limited to RGD integrin-binding peptides, de novo designed binders, a bicycle peptide, conotoxins, small molecules such as folic acid, and a virus that binds to the cell surface.Antibody-bait switches

[0193] In some embodiments, the analyte sensor is a single antibody construct comprising: an antibody or a binding fragment thereof comprising a first label, wherein the antibody or the binding fragment thereof is linked to a blocking analyte and a second label via a linker, wherein: in the absence of a target analyte, the blocking analyte binds to the antibody or the binding fragment thereof, and in the presence of the target analyte, the target analyte competes with the blocking analyte for binding to the antibody or the binding fragment thereof, and the first and second labels interact to generate a detectable readout that changes depending on whether the blocking analyte is bound by the antibody or the binding fragment thereof, or is not bound by the antibody or the binding fragment thereof. In someembodiments, the second label is conjugated to the linker. In some embodiments, the second label is conjugated to the blocking analyte. In some embodiments, the first label is a fluorophore and the second label is a quencher. In some embodiments, the first label is a quencher and the second label is a fluorophore. In some embodiments, the first label is a donor fluorophore and the second label is an acceptor fluorophore. In some embodiments, the first label is an acceptor fluorophore and the second label is a donor fluorophore. In some embodiments, the detectable readout is an optical signal, an electrical signal, an electrochemical signal, a nuclear magnetic resonance signal, or a biological signal. In some embodiments, the optical signal is a fluorescent signal. In some embodiments, binding of the target analyte to the antibody or the binding fragment thereof increases or decreases the detectable readout.

[0194] In some embodiments, the analyte sensor is a single antibody construct comprising: an antibody or a binding fragment thereof comprising a redox reporter, wherein the antibody or the binding fragment thereof is linked to a blocking analyte and a sensing electrode; wherein: in the absence of a target analyte, the blocking analyte binds to the antibody or the binding fragment thereof, and in the presence of the target analyte, the target analyte competes with the blocking analyte for binding to the antibody or the binding fragment thereof, and the redox reporter and the sensing electrode interact to generate an electrical signal that changes depending on whether the blocking analyte is bound by the antibody or the binding fragment thereof, or is not bound by the antibody or the binding fragment thereof. In some embodiments, the antibody or the binding fragment thereof is linked to the blocking analyte and the sensing electrode via a linker. In some embodiments, the antibody or the binding fragment thereof is linked to the blocking analyte by a first linker and the antibody or the binding fragment thereof is linked to the sensing electrode via a second linker. In some embodiments, the antibody or the binding fragment thereof is linked to the sensing electrode via a gold-thiol bond. In some embodiments, the sensing electrode is a gold electrode.Dual-antibody switches

[0195] In some embodiments the analyte sensor is a dual antibody construct comprising: (a) two detecting strands, wherein a first detecting strand comprises a first antibody or a binding fragment thereof, and a second detecting strand comprises a second antibody or a binding fragment thereof; and (b) a first label and a second label, wherein the detecting strands or portions thereof are complementary and hybridize to each other or one or more scaffold strands, and wherein in the presence of the target analyte, the first antibody and the secondantibody bind to two different epitopes on the target analyte, and the first and second labels interact with each other to generate a detectable readout compared to when there is an absence of the target analyte.

[0196] In some embodiments, the two detecting strands hybridize to a scaffold strand, thereby linking the two detecting strands. In some embodiments, the two detecting strands hybridize to a single scaffold strand. In some embodiments, two detecting strands hybridize to separate scaffold strands which scaffold strands hybridize to each other. In some embodiments, the two detecting strands hybridize to each other, thereby linking the two detecting strands. In some embodiments, the first label is linked to the first antibody and the second label is linked to the second antibody. In some embodiments, the first label and / or second label is linked to a scaffold strand. In some embodiments, the first label is linked to a first label oligonucleotide that is hybridized to a scaffold strand and second label is linked to a second label oligonucleotide that is hybridized to a scaffold strand. In some embodiments, the first label is linked to a first scaffold strand and the second label is linked to a second scaffold strand. In some embodiments, in the presence of the target analyte, a portion of the first scaffold strand and a portion of the second scaffold strand hybridize to each other.

[0197] In some embodiments, the antibody construct generates fluorescence as a detectable readout when it binds to a target analyte. In some embodiments, one of the first and second labels on the antibody construct can be a fluorophore and the other of the first and second labels can be a quencher. In this case, the blocking analyte binds to the antibody in the absence of the target analyte and the fluorescence signal is quenched. In some embodiments, one of the first and second labels on the antibody construct can be a donor fluorophore and the other of the first and second labels can be an acceptor fluorophore and the first and second labels can form a FRET pair. In some embodiments, when the antibody is bound by the blocking analyte in the absence of a target analyte, the first and second labels are in proximity of each other to produce a FRET signal. The disappearance or reduction of the FRET fluorescence can serve as a signal of target analyte binding. In other words, in the absence of the target analyte, the fluorescent signal from the acceptor fluorophore can serve as the detectable signal. In the presence of the target analyte and the formation of the antibody -target analyte complex, the donor fluorophore and the acceptor fluorophore are not in proximity of each other to produce a FRET signal and the fluorescent signal of the donor fluorophore can serve as the detectable readout.Antibody-aptamer switches

[0198] In certain embodiments, the analyte sensors comprise a capture agent that binds to a first epitope of an analyte, and an aptamer that binds to a second epitope of the analyte, where the aptamer is stably associated with the capture agent and produces a detectable signal upon cooperative binding of the capture agent and aptamer to the analyte. The capture agent mediates initial analyte binding due to its higher affinity, thereby increasing the local analyte concentration and leading to cooperative binding and signaling by the aptamer, e.g., a stranddisplacement (SD) aptamer switch. In certain embodiments this design leads to an enhancement in sensitivity as compared to the aptamer alone, and the molecular sensor may exhibit reversible binding, enabling repeated measurements and retaining remarkable sensitivity even in interferent-rich samples.

[0199] A variety of suitable capture agents may be employed, non-limiting examples of which include a receptor (e.g., when the analyte is a ligand of the receptor), a ligand (e.g., when the analyte is a receptor for the ligand), a small molecule, an antibody, a T-cell receptor (TCR), etc. In some instances, the capture agent is an antibody. By “antibody” is meant an antibody or immunoglobulin of any isotype (e.g., IgG (e.g., IgGl, IgG2, IgG3, or IgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of a tetramer which in turn is composed of two dimers of a heavy and light chain polypeptide); single chain antibodies (e.g., scFv); fragments of antibodies (e.g., fragments of whole or single chain antibodies) which retain specific binding to the analyte. In certain embodiments, the antibody is an IgG, Fab, scFv, scFv- Fc, scFv-CH3, scFv-zipper, scFab, VHH / VH, or diabody.

[0200] In some embodiments, the detectable signal is an optically-detectable signal. For example, the detectable signal may be a fluorescent signal, e.g., where the aptamer or a displacement strand (if present) is labeled with a fluorophore. Non-limiting examples of approaches for the production of fluorescent signals upon cooperative binding of the capture agent and aptamer to the analyte include fluorophore-quencher interactions, Forster resonance energy transfer (FRET), and the like. In certain embodiments, the aptamer comprises a quencher moiety that quenches the fluorophore in a first conformation when the molecular sensor is not bound to the analyte, and upon cooperative binding of the capture agent and aptamer to the analyte, the aptamer assumes a second confirmation in which the fluorophore assumes an unquenched state.

[0201] In some embodiments, the aptamer is stably associated with the capture agent. As used herein, “stably associated” means a physical association between two entities in which the mean half-life of 25 association is one day or more in PBS at 4°C. In some embodiments, the physical association between the two entities has a mean half-life of one day or more, oneweek or more, one month or more, including six months or more, e.g., 1 year or more, in PBS at 4°C. According to some embodiments, the stable association arises from a covalent bond between the two entities, a non- covalent bond between the two entities (e.g., an ionic or metallic bond), or other forms of chemical attraction, such as hydrogen bonding, Van der Waals forces, and the like. The linkage between the aptamer and capture agent (e.g., antibody) can take the form of either direct conjugation of the aptamer to the capture agent, or hybridization of part of the aptamer sequence to an anchor DNA sequence conjugated to the capture agent. As such, the linker region may be composed of a combination of regions of the anchor strand and the aptamer strand.Analyte Detection

[0202] In some embodiments, the analyte binding domain can change its conformation (e.g., Aptamer Switch) upon binding to a target analyte. In some embodiments, an analyte sensor may be an antibody switch sensor or any other sensor that may undergo conformational rearrangement upon interaction with one or more analytes that modulates the optical signal. Suitable optical signals which can be used as an assay readout include any optical signal which can be generated by a proximity assay, such as those generated by fluorescence resonance energy transfer (FRET), fluorescence polarization, fluorescence quenching, phosphorescence technique, luminescence enhancement, luminescence quenching, diffraction or plasmon resonance, all of which are known techniques.

[0203] For example, in the absence of a target molecule, the analyte detection is in a position such that a first label (e.g., fluorophore) is in close proximity to a second label (e.g., quencher), the detectable optical read-out is quenched (e.g., reduced) by the second label. Conversely, when the analyte binding domain binds to its target molecule, the binding between binding domain and target molecule induces a conformational change, such that the first label (e.g., fluorophore) is away from the second label (e.g., quencher) resulting in the increase of detectable optical read-out.

[0204] Efficient and complete quenching of the fluorescence emitted from the fluorophore by the quencher depends in part on the overlap between the fluorophore emission and quencher absorption spectra. For example, the fluorophore coumarin emits at emission wavelength around 472 nm and can be paired with quencher (QSY35) which absorbs at a wavelength of around 475 nm. In another example, fluorophore Alexa 532 emits at emission wavelength around 554 nm and can be paired with quencher QSY7 which absorbs at wavelength around 560 nm. In yet another example, fluorophore Alex 647 emits at an emission wavelengtharound 665 nm and can be paired with quencher QSY 21 which absorbs at wavelength around 661 nm.

[0205] In some embodiments, the substrate incorporates assay components that generate an optical readout using FRET. In this assay format, a pair of fluorophores are used wherein one serves as a donor chromophore and the other acts as an acceptor chromophore. With respect to the fluorescence emission spectrum, the emission spectrum of the donor chromophore overlaps with the absorption spectrum of the acceptor chromophore, such that when the donor and acceptor chromophores are brought into close proximity, a proportion of the energy which normally would produce fluorescence emitted by the donor chromophore (following irradiation with incident radiation of a wavelength absorbed by the donor chromophore) will be non-radiatively transferred to the adjacent acceptor chromophore, a process known in the art as fluorescence resonance energy transfer, with the result that a proportion of the fluorescent signal emitted by the donor chromophore is quenched, that the lifetime of the fluorescence is changed, and, in some instances, that the acceptor chromophore emits fluorescence. The acceptor chromophore may, however, be a non-fluorescent dye. Fluorescence resonance energy transfer generally only occurs when the donor and acceptor chromophores are brought into close proximity by the binding of, for instance, an analyte to an aptamer, which causes a conformational change which brings the donor and acceptor chromophores together. Thus, in the presence of analyte, the amount of quenching can be increased (resulting in a measurable decrease in the intensity of the fluorescent signal emitted by the donor chromophore or an increase in the intensity of the signal emitted by the acceptor chromophore). The intensity or lifetime of the fluorescent signal emitted from the donor chromophore thus correlates with the concentration of target analyte in the interstitial fluid bathing the sensor.

[0206] The sensor can be adapted for the detection or quantitative measurement of any target analyte present in interstitial fluid such as glucose (in connection with the long-term monitoring of diabetics), urea (in connection with kidney disease or dysfunction), lactate (in connection with assessment of muscle performance in sports medicine), ions such as sodium, calcium or potassium and therapeutic drugs whose concentration in the blood must be closely monitored, such as, for example, digoxin, theophylline or immunosuppressant drugs. The above analytes are listed by way of example only and it is to be understood that the precise nature of the analyte to be measured is not material.

[0207] The sensor can be interrogated transcutaneously using optical means, for example, no physical connection may be required between the sensor and the optical means. When thesensor incorporates the technique of fluorescence resonance energy transfer, the optical means can supply a first beam of incident radiation at a wavelength within the absorption spectrum of the donor chromophore and a second beam of incident radiation at a wavelength within the absorption spectrum of the acceptor chromophore. In addition, the optical means can be capable of measuring optical signals generated in the sensor at two different wavelengths; wavelength 1 within the emission spectrum of the donor chromophore (the signal generated in connection with the measurement of analyte and wavelength 2 in the emission spectrum of the acceptor chromophore (which could be the analyte signal or the internal reference or calibration signal).

[0208] Optical means suitable for use in remote interrogation of the sensor can include a simple high-throughput fluorimeter comprising an excitation light source such as, for example, a light-emitting diode (for example blue, green or red), an excitation light filter (for example a dichroic, dye filter) and a fluorescent light detector for example (PIN diode, Silicon photo-multiplier, Avalanche photodiode, image sensor configuration).Electrochemical detection

[0209] In some embodiments the analyte binding probes can be configured as electrochemical sensors. For example, an aptamer switch can be immobilized on an electrode (e.g., gold electrode) and labeled with a redox reporter, such as methylene blue. Upon analyte binding, the analyte binding probes undergoes a change in conformational state which causes the distance between the redox reporter and the surface of the electrode to change. This change in distance causes a change in electrical signal in response to the target analyte which can be used to measure the analyte concentration.FET detection

[0210] In some embodiments, the analyte binding probes can be configured as field effect sensors. For example, an aptamer switch can be immobilized on an electrode (e.g., silicon oxide, silicon nitride, hafnium oxide, gold, etc.). Upon analyte binding, the analyte binding probes can generate a change in electric potential at the electrode by changing the charge distribution near the surface of the electrode. In some embodiments, this change in charge distribution may be achieved by the analyte binding probe undergoing a change in conformational state. In some embodiments, this change in charge distribution may be achieved by the addition or subtraction of charge from the proximity of the analyte to the surface of the electrode. In some embodiments, both effects may occur. This change incharge distribution causes a change in electrical signal in response to the target analyte which can be used to measure the analyte concentration.Enzymes sensors

[0211] Although the preferred embodiment of the device leverages affinity -based analyte binding probes, more conventional enzymatic approaches can also be used in the device. The enzymatic approaches can be used either alone, or in combination, with the other analyte binding probes and signaling mechanisms described in this application.Other Electrochemical Sensors

[0212] In some embodiments, the microfluidic chip contains an electrode that is coated with a material. The electrode coating may include a quaternary ammonium, sulfonated tetrafluoroethylene, polyethyleneimine-functionalized carbon nanotube, or combinations thereof. In some embodiments a sweeping voltage is applied to the electrode and a current is measured to detect the presence or concentration of one substance within the bodily fluid of a subject.Molecular Pendulum Sensors

[0213] In some embodiments, the analyte binding probes can be configured as molecular pendulum sensors. In the function of a molecular pendulum sensor, an analyte-binding receptor is attached to one end of a short double-stranded DNA sequence that is attached to the sensor surface at its other end. This double-stranded DNA sequence may contain a redoxactive reporter that allows generation of electrochemical signals by an electrode sensor surface. Voltages applied to the electrode exert forces on the double-stranded DNA due to its charge, and thus induce motion of the molecular pendulum construct consisting of the double-stranded DNA and analyte-binding receptor, and in turn this motion can be detected through electrochemical signals measured at the electrode. Binding of the analyte to the pendulum modulates its motion under these conditions due to increased drag from the bound analyte, thus changing electron transfer and electrochemical signaling. In some embodiments, electrochemical signaling can be measured through chronoamperometry. In some embodiments, the redox reporter is ferrocene. In some embodiments, the analyte-binding receptor is an antibody. In some embodiments, the analyte-binding receptor is a nucleic acid aptamer.

[0214] In some embodiments, analyte binding by the pendulum sensor can be disrupted to induce analyte dissociation and thus reset the sensor into the unbound state. This may offer increased sensing kinetics for pendulum sensors designed with analyte-binding receptors that have slow off rates. In some embodiments, the binding can be disrupted by applying a voltage square wave of specified amplitude and frequency to the sensing electrode, which induces rapid oscillation of the pendulum and thus disrupts the receptor binding structure or disrupts its binding to the analyte. In some embodiments this disruption is performed intermittently to reset the sensor, followed by subsequent measurements of re-binding of the analyte.Particle Mobility Sensors

[0215] In some embodiments, the analyte binding probes can be configured as particle mobility sensors. In a particle mobility sensor, the motion of a nano- or micro-particle that is functionalized with analyte-binding receptors is monitored to determine the binding state of the receptors by changes in the degree of motion upon analyte binding. The particle motion is measured optically, for example using microscopy, and the motion over time is analyzed to determine the concentration of analyte. In some embodiments, the particle may be functionalized with a first analyte-binding receptor and a microfluidic chip surface is functionalized with a second analyte-binding receptor that binds a separate region of the analyte from the first receptor. In some embodiments, analyte binding to both receptors may cause the particle to be confined in its motion, allowing detection of binding through decreased mobility. In some embodiments, the particle may be functionalized with an analyte-binding receptor and the microfluidic chip surface is functionalized with a molecule that is similar to or identical to the analyte of interest. In some embodiments, in the absence of analyte the receptor on the particle may bind to the surface analyte, limiting particle motion, whereas presence of analyte can competitively bind with the receptor, disrupting surface binding and thus increasing the particle motion. In some embodiments, the particle may be free to diffuse within a sensing chip. In some embodiments, the particle may be tethered to the surface through a long tether that allows substantial motion but forms a linkage between the particle and the surface.

[0216] Responsive to the unmet need for improved biosensors which can perform an assay for analyte detection at the point of care which can deliver a result to a subject shortly following sample collection, and improved biosensors which can perform multiplexed or continuous assays for ongoing monitoring of an analyte; disclosed herein are biosensors forminimally invasive, continuous, real-time measurement of one or more target analytes in the body of a subject from a biological sample, and monitoring of concentration of one or more target analytes in the body of a subject from a biological sample. The present disclosure provides devices for detection of one or more analytes at the point of care, and for ongoing monitoring of analytes at the point of care. The device may utilize optical detection modalities to detect the presence and concentration of an analyte in a sample, and provide for structures to distribute light to a detection element of the device for detection of one or more target analytes. Various aspects of the present disclosure describe a system and method to monitor analytes in a subject from a bodily fluid of a subject, for example, from skin interstitial fluid (ISF). The detection of these analytes (e.g., biomarkers) may include a plurality of piercing elements that can penetrate into the skin, a support attached to the piercing element, and an analyte binding probe.

[0217] In the present disclosure, solutions to the problem of detecting small concentrations of analytes in the interstitial fluid utilizing a wearable patch with an optical sensor are presented. Previous implementations of wearable patches with optical sensors have suffered from a low quantity of light, and a loss of light during transmission about optical excitation and optical emission paths, resulting in lower sensitivity of analyte detection. The present disclosure proposes solutions to this problem by improving the optical coupling efficiency in the optical sensor. For example, some embodiments optimize the optical excitation path by utilizing a waveguide and an optical coupler and comprising one or more focusing elements configured to focus light from a light source towards the light coupler. Some embodiments optimize the optical excitation path with a reflective cladding layer surrounding the waveguide, which is configured to reflect the light back inside the waveguide and guide the light toward the piercing element. Some embodiments optimize the optical excitation path by configuring the light coupler to couple light from a light source into the waveguide.

[0218] Some embodiments of the present disclosure optimize the optical emission path, and allow the detection of very small levels of emission light, which is in some cases orders of magnitude small than an excitation light. In some embodiments, apertures in a reflective cladding layer surrounding a waveguide are configured to transmit an emission light from the analyte probe towards the detector. In some embodiments, one or more focusing elements are configured to focus emission light from the analyte binding probe towards the detector. In some embodiments, the optical excitation path and the optical emission path are configured to minimize interference with each other. In some embodiments, optical filters in combination transmit emission light while substantially blocking light which is a same wavelength asexcitation light. The present disclosure also provides solutions to the problem of how to develop a sensor that is sufficiently small enough to comprise a wearable patch. In some embodiments, a solution to this problem is to design a waveguide that can transmit both excitation and emission light in one device. This allows for the sensor to be more effectively miniaturized and compacted. However, utilizing a waveguide which is both a component of the optical emission path and optical excitation path presents problems associated with the detection of the emission light, which is in some cases orders of magnitude small than an excitation light. In some cases, the waveguide transmits light from a light source to the analyte binding probe, and waveguide is configured to permit light to be transmitted through the waveguide from the analyte binding probe to the detector. In some cases, the waveguide transmits light from a light source to the analyte binding probe through a directed path in the wave guide, in which light is reflected using one or more reflective walls. In some cases, the waveguide transmits light from the analyte binding probe to a detector using an optical emission path that is directly aligns the analyte binding probe with the detector about the optical emission path.

[0219] The present disclosure also provides solutions to problems related to detecting analytes in more flexible or sensitive ways. In some embodiments, the analyte binding probes that bind to the analyte can be attached to one or more particles within a hydrogel matrix inside the piercing element. Having the particles physically entrapped by the hydrogel solves the problem of decoupling any conjugation chemistry compatibility requirements between the hydrogel and the aptamers. In addition, the user can QC / QA the particles prior to inserting into the device, which has advantages for reproducibility and consistency during the manufacturing process.

[0220] Previous implementations of wearable patches with optical sensors have suffered from a low quantity of light and a loss of light, resulting in lower sensitivity of analyte detection. Optimizing the optical excitation path is one feature in the present disclosure that provides the beneficial technical effect of improving the optical coupling efficiency in the optical sensor. Improving the optical coupling efficiency has the beneficial technical effect of decreasing the power level and battery size needed in the device. In some embodiments, the optical excitation path is optimized by utilizing a waveguide and an optical coupler and comprising one or more focusing elements configured to focus light from a light source towards the light coupler. Some embodiments optimize the optical excitation path with a reflective cladding layer surrounding the waveguide, which is configured to reflect the light back inside the waveguide and guide the light toward the piercing element. Someembodiments optimize the optical excitation path by configuring the light coupler to couple light from a light source into the waveguide.

[0221] Optimizing the optical emission path can provide the beneficial technical effect of allowing the device to detect smaller amounts of emission light. This allows for a smaller detector, which would allow for the beneficial technical effect of improving the miniaturizability and compactness of the device. In some embodiments, apertures in a reflective cladding layer surrounding a waveguide are configured to transmit emission light from the analyte probe towards the detector. In some embodiments, one or more focusing elements are configured to focus emission light from the analyte binding probe towards the detector. In some embodiments, the optical excitation path and the optical emission path are configured to minimize interference with each other. In some embodiments, optical filters in combination transmit emission light while substantially blocking light which is a same wavelength as excitation light.

[0222] In some embodiments, the present disclosure comprises a waveguide that can transmit both excitation and emission light in a single device. This has the beneficial technical effect of allowing for the sensor to be more efficiently miniaturized and compacted, and possibly creating a sensor that is sufficiently small enough to comprise a wearable patch. However, utilizing a waveguide which is both a component of the optical emission path and optical excitation path presents problems associated with the detection of the emission light, which is in some cases orders of magnitude small than an excitation light. In some cases, the waveguide transmits light from a light source to the analyte binding probe, and waveguide is configured to permit light to be transmitted through the waveguide from the analyte binding probe to the detector. In some cases, the waveguide transmits light from a light source to the analyte binding probe through a directed path in the wave guide, in which light is reflected using one or more reflective walls. In some cases, the waveguide transmits light from the analyte binding probe to a detector using an optical emission path that is directly aligns the analyte binding probe with the detector about the optical emission path.

[0223] The present disclosure also provides beneficial technical effects related to detecting analytes in more flexible or sensitive ways. In the present disclosure, the analyte binding probes that bind to the analyte can be attached to one or more particles within a hydrogel matrix inside the piercing element. Having the particles physically entrapped by the hydrogel provides the beneficial technical effect of decoupling any conjugation chemistry compatibility requirements between the hydrogel and the aptamers. In addition, the user can-n -QC / QA the particles prior to inserting into the device, which has beneficial technical effects of reproducibility and consistency during the manufacturing process.

[0224] In some embodiments, the piercing element may be removable, interchangeable, or disposable. In some embodiments, the piercing element may comprise needles. In some embodiments, the needle may comprise microneedles. In some embodiments, the piercing element is configured to pierce a body surface of the subject when the device is coupled to the body surface, to thereby bring the piercing element in contact with the biological sample. In some embodiments, the piercing element comprises an analyte binding probe on or within the piercing element, wherein the analyte binding probe is configured to provide a change in an optical signal when the analyte binding probe comes in contact with the analyte in the biological sample of the subject. In some embodiments, the device comprises a detector operatively coupled to the support, wherein the detector is configured to detect the optical signal. In some embodiments, the device may comprise two or more analyte binding probes within the piercing element, where each of the two or more analyte binding probes provides a change in an optical signal when the two or more analyte binding probes contacts a second analyte in the biological sample of the subject. In some embodiments, the device uses a multiplexed array of analyte binding probes on or within the piercing element, each of the multiplexed array of analyte binding probes configured to provide a change in an optical signal when the multiplexed array of analyte binding probe contacts a subsequent analyte in the biological sample of the subject. In some embodiments, the subsequent analyte is a different analyte, and the multiplexed array of analyte binding probes is configured to detect a plurality of analytes in the biological sample of the subject. In some embodiments, the piercing element comprises a plurality of piercing elements which defines subsets of piercing elements, wherein each subset of piercing elements comprises a different analyte binding probe configured to detect a different analyte in the biological sample of the subject. In some embodiments, the subsets of piercing elements comprising the different analyte binding probes comprises the multiplexed array of analyte binding probes. The multiplexed array of analyte binding probes can be spatially multiplexed within the piercing element.

[0225] The device may comprise a light source, configured to shine one or more beams of light of one or more wavelengths to the analyte binding probe. The support may comprise an optical light guide or an optical waveguide, which can guide or collect a portion of optical signal (e.g., light) to and from the piercing element. The optical signal detected may comprise a) fluorescence intensity b) FRET ratio, c) time-resolved fluorescence (via phosphorescence), d) decay curve monitoring (detecting the lifetime of the phosphorescence reporter), or e)combinations thereof. The optical light guide or optical waveguide may define one or more optical paths connecting the light source to the analyte binding probe, and the analyte binding probe to the detector. In another embodiment, the support may attach directly to the piercing element, and the light source may attach directly to the piercing element, and there may not be an optical light guide or optical waveguide. The optical waveguide may comprise a 2- dimensional array of light guides. The 2-dimensional array may comprise dichroic mirrors, and the 2-dimensional array may comprise gratings, or be etched. The optical waveguide may comprise a polymer glassy matrix comprising dispersed photoluminescent particles. The polymers may include: polysiloxanes, silsequioxanes, polymethylmethacrylate (PMMA), polystyrene (PS), polycarbonate (PC), polyurethane (PU), epoxy resin, deuterated and halogenated polyacrylates, fluorinated polyimides, perfluorocyclobutyl (PFCB) aryl ether polymers, nonlinear optical polymers, benzocyl cobutene (BCB), perfluorovinyl ether cyclopolymer (CYTOP), tetrafluoroethylene and perfluorovinyl ether copolymer (Teflon AF), silicone, fluorinated poly(arylene ether sulfide), poly(pentafluorostyrene), fluorinated dendrimers, fluorinated hyperbranched polymers, or combinations thereof. The optical waveguide may couple to a light source, to guide at least a portion of light from the light source along a length of the optical light guide, and to divert at least a portion of the light from the light guide to the piercing element. The optical light guide may provide a light transmission efficiency of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99 % transmission efficiency, and may provide the same from the light source to the piercing element. The optical waveguide may collect an emission light of the optical reporter from the analyte binding probe, to guide at least a portion of the emission light to the detector. The optical waveguide may provide an essentially lossless light transmission from the piercing element to the detector, or provide a light transmission efficiency of at least 10 % transmission efficiency from the piercing element to the detector. The optical waveguide may provide an essentially lossless light transmission from the piercing element to the detector, or provide a light transmission efficiency of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99 from the piercing element to the detector.

[0226] The optical waveguide may define a plurality of paths from the light source to the piercing element. The light source may provide light in different wavelengths, and the light source may have a plurality of light sources each configured to provide a different wavelength of light. The optical light guide may include a plurality of paths from the light source to the piercing element and comprises one or more distinct paths from the light source to the multiplexed array of analyte binding probes. The at least one of the plurality of pathsmay guide light from one of the plurality of light sources with a specific wavelength of light to one of the analyte binding probes in the multiplexed array of analyte binding probes which is responsive to the specific wavelength of light. The plurality of paths from the light source to the piercing element may include one or more distinct paths from the light source to the subsets of piercing elements. The plurality of paths may guide light from one of the plurality of light sources with a specific wavelength of light to one of the analyte binding probes in the subsets of piercing elements. The optical waveguide may include one or more switches configured to block one or more of the plurality of paths.

[0227] The device may have the piercing element coupled to the support using one or more holes, one or more insertable elements configured to attach to a corresponding one or more receiving elements, a lock and key attachment, a mortise and tenon attachment, a dovetail attachment, a magnetic attachment, an adhesive, one or more elastically deformable attachment elements, the elastically deformable attachment elements rebounding once inserted into the support. The piercing element may be removably coupled to the support.

[0228] The light source may include light-emitting diodes (LEDs) or lasers; or a plurality of light-emitting diodes (LEDs) or lasers, each of the plurality of light-emitting diodes (LEDs) or lasers configured to deliver a different wavelength of light.

[0229] The analyte binding probe may be coupled to an optical reporter, where the optical reporter is a fluorophore or a quencher. The analyte binding probe may be an oligonucleotide probe, or an aptamer. The aptamer may be coupled to a displacement strand, wherein the displacement strand is partially complementary to the aptamer. The displacement strand may be coupled to a second optical reporter, where the second optical reporter is a fluorophore or a quencher, and wherein the aptamer is coupled to a linker moiety placed between the aptamer and the displacement strand. The linker moiety can be a nucleotide acid moiety, a peptide nucleic acid (PNA) moiety, a peptide moiety, a disulfide bond, a phosphodiester linkage, or a polymer. The analyte binding probe may undergo a conformational change when the analyte interacts with the analyte binding probe, thereby providing a change in optical signal, for example, an emission light, or an emission light which is a function of a concentration of the analyte.

[0230] The piercing element may include a structural domain, a barrier domain, a sensing domain, and the analyte binding probe. The structural domain can encapsulate the barrier domain, and the barrier domain can encapsulate the analyte binding probe, for example, within a matrix of the barrier domain, where the barrier domain includes a hydrogel, a polymer, or combinations thereof. The barrier domain may control transfer of the analyte toanalyte binding probe, for example, via diffusion. A variety of configurations may be utilized for the piercing element and the structural domain, the barrier domain, and the analyte binding probe, for example: where the structural domain includes one or more openings on an exterior surface of the structural domain; where the one or more openings are configured to allow the analyte to contact the barrier domain; where the barrier domain defines a passage connecting the one or more openings to the analyte binding probe; where the needle includes a structural domain with a plurality of openings; where the needle includes a structural domain comprising a hollow region of the needle; where the hollow region is orientated in a longitudinal direction; where the plurality of openings are positioned on a lateral face of the needle; where the plurality of openings are positioned on opposing lateral faces of the needle; where the barrier domain extends longitudinally into the needle; where the barrier domain extends longitudinally into the needle and defines a passage connecting the one or more openings to the analyte binding probe; where there is one opening on a lateral surface of each needle; where the structural domain is directly in contact with the analyte binding probe; where the structural domain and the analyte binding probe extend longitudinally from the device; where the analyte binding probe is positioned orthogonally to a longitudinal axis of the needle; the analyte binding probe is positioned orthogonally to a longitudinal axis of the needle, where the barrier domain is positioned parallel to a longitudinal axis of the needle; the barrier domain is positioned on the exterior surface of the structural domain; where the barrier domain is positioned on the exterior surface of the structural domain, and positioned within an interior space of the structural domain; where the barrier domain is positioned on the exterior surface of the structural domain, positioned within an interior space of the structural domain, and where the analyte binding probe is positioned within the structural domain and in contact with the barrier domain positioned within an interior space of the structural domain; where the barrier domain is positioned on the exterior surface of the structural domain and where the barrier domain is positioned within an interior space of the structural domain, and where the analyte binding probe is positioned within the structural domain orthogonally to a longitudinal axis of the needle, and is in contact with the barrier domain positioned within an interior space of the structural domain; where the barrier domain is positioned on the exterior surface of the structural domain and where the barrier domain is positioned within an interior space of the structural domain, and where the analyte binding probe is positioned throughout a matrix of the barrier domain; where the barrier domain is within an interior space of the structural domain, and where the analyte binding probe is positioned throughout a matrix of the barrier domain; where the barrier domain is within aninterior space of the structural domain and is exposed to an exterior surface of the needle through the one or more openings, and where the analyte binding probe is positioned throughout a matrix of the barrier domain; where the barrier domain is positioned on the exterior surface of the structural domain and where the barrier domain is positioned within an interior space of the structural domain, and where the analyte binding probe is positioned throughout a matrix of the barrier domain, where there are at least two openings on opposing lateral faces of the needle, or combinations thereof. The analyte binding probe is comprised within the barrier domain at a concentration of about 1 nM to about 1 mM, and at a about 1 nM to about 1 uM. In some cases, the analyte binding probe is substantially uniformly dispersed throughout the barrier domain.

[0231] In some cases, the device may have a signal off configuration where the device is configured to not emit an optical signal when the analyte does not contact the analyte binding probe, or will emit a reduced signal when the analyte contacts the analyte binding probe. For example, the analyte binding probes may emit an optical signal in a default configuration when not in contact with an analyte; undergo a conformational change when bound to an analyte as to not emit an optical signal or will undergo a conformational change when bound to an analyte as to emit a reduced optical signal; and the device can measure a concentration of the analyte in the biological sample of the subject by measuring a decrease in the optical signal.

[0232] The reader device can detect a change in an optical signal when an analyte binds to the analyte binding probe. In some embodiments, the device may include its own optical sensor, control electronics, and a communication system to exchange data. The analyte information (e.g., analyte concentration, presence) can be sent from the reader to a display device. The display device could be, for example, a wearable, laptop, desktop, handheld, or tablet computer, a mobile phone, or a subsystem of such a device, such as CPU or a display. In some embodiments, the device may comprise the display. The display device can store the data received from the reader, perhaps process the data, and generate display(s) based on the received and / or processed data. In some embodiments, the device may comprise a computer- readable medium configured to induce operation of the device and record the results of one or more tests. The device may comprise a battery, capacitor, a hybrid electrochemical cell or any other suitable power source to provide power to the various components of the device.

[0233] The device may be utilized to detect a variety of target analytes for a number of varying consumer and commercial purposes. For instance, the device may be configured as to detect glucose as a measure of metabolic health, fitness, or diabetes; cortisol as a measure ofstress, sleep, fitness, or meditation; potassium or sodium as a measure of kidney function or dehydration; bilirubin or bile acids as a measure of liver health; lactate as a measure of fitness, infection, metabolic health, or sepsis; 3 -hydroxybutyrate as a measure of ketosis; creatinine as a measure of dehydration, kidney function / health; serum amyloid A (SAA) as a general marker of inflammation, infection, or sepsis; uric acid as a measure of gout, dehydration, kidney health; urea as a measure of liver health, kidney health, dehydration, or fitness; concentration of a therapeutic agent in a patient; hydration levels, and other analytes for various purposes.Example Systems

[0234] Figure 33 is a block diagram of a device 1-100 for sensing an analyte in a biological sample of a subject, comprising: a support 1-106; a power source 1-101; an array of piercing elements 104 coupled to said support, wherein said piercing element can be configured to pierce a body surface of said subject when said device is coupled to said body surface, thereby bringing said piercing element in contact with said biological sample. Each piercing element can be further coupled to a sensor interface module 1-103 comprising a light source 1-108, a detection circuitry 1-110 and a photodetector 1-105. When an analyte binds to a binding probe, the analyte binding probe may be configured to provide a change in an optical signal. The generated optical signal may be detected by the detector 1-102, and notifies the wearer of the presence of the analyte via a controller 1-102.

[0235] Figure 35 is a block diagram of a device 3-111 for sensing an analyte in a biological sample of a subject, comprising; a support 3-106; a power source 3-101; an array of piercing elements 3-104 coupled to said support, wherein said piercing element can be configured to pierce a body surface of said subject when said device can be coupled to said body surface, to thereby bring said piercing element in contact with said biological sample. Each piercing element can be coupled to an analyte binding probe on or within said piercing element, wherein said analyte binding probe can be configured to provide a change in an optical signal when said analyte binding probe comes in contact with said analyte in said biological sample of said subject; and a detector 3-102 operatively coupled to said support, wherein said detector can be configured to detect said optical signal. For example, the analyte binding probe may undergo a conformational change from a first configuration to a second configuration when it binds to a particular analyte, and the analyte binding probe may be fluorescent when exposed to light in the second configuration. The device further comprises an optical waveguide 3-109 coupled to said support. The optical waveguide defines a lightpath between the light source and the analyte binding probe, and from the analyte binding probe to the detector. A light source 3-108 from within the device can be illuminated and applied to optical waveguide, which applies the light source to the analyte binding sensor operatively coupled to a fluorophore and generate optical signal. The generated optical signal can be detected by the detector 3-102, and notifies the wearer of the presence of the analyte via a controller 3-102.

[0236] The piercing element 3-104 may comprise a structural domain 3-112, a barrier domain 3-113, and one or more analyte binding sensors 3-114 with various configurations as shown in Figures 36A-36H.Piercing Element

[0237] The present disclosure provides a device which includes a plurality of piercing elements having a base end and a tip wherein the piercing elements are configured to penetrate into the skin to contact interstitial fluid, or other bodily fluids. In some embodiments, the piercing element may include one or more skin insertion objects, such as needles, microneedles, lancets, blades, knives, protrusions, or other suitable objects.

[0238] In some embodiments, the piercing element may comprise an analyte binding sensor having a detectable optical label and configured to interact with a target analyte present in interstitial fluid, and a structural domain. In some embodiments, the piercing element may comprise an analyte binding sensor, a structural domain, and a barrier domain. The barrier domains are configured such that the interstitial fluid can readily diffuse into the microneedle and interact with the analyte binding probe that may be dispersed throughout the microneedles or contained in a polymer matrix within an interior hollow space of the microneedles. In some embodiments, it is advantageous for the assay components to have a restricted diffusion in order to minimize their loss from the substrates into the bloodstream. This can be achieved by ensuring that the biodegradable material has a pore size that permits the diffusion of low molecular weight analytes such as glucose, but not diffusion of the assay components themselves. The assay components may be of high molecular weight, such as proteins or polymers, in order to restrict their loss from the sensor.

[0239] The piercing element may comprise a structural domain, a sensing domain, a barrier domain, and / or analyte binding domain and may be configured in various geometries. The structural domain can be positioned on a surface of the support and extend outward from the surface to define a needle, and define an interior space, wherein the sensing domain is contained withing the interior space. The needle can define a well within, where the sensingdomain is contained within the well. The barrier domain may comprise a thin layer of a higher molecular weight hydrogel, and the barrier domain can coat an exterior surface of the structural domain. The structural domain can define one or more openings on a lateral face of the needle. In some embodiments, the sensing domain encapsulates the analyte binding probe. In some embodiments, the sensing domain entraps the analyte binding probe. In some embodiments, the structural domain encapsulates the analyte binding probe within a matrix of the sensing domain within a polymer or hydrogel matrix. Any suitable polymer may be used including hydrogels. As used herein, the term “entrap” and variations thereof is used interchangeably with “encapsulate” and is used to mean that the aptamer is immobilized within or on the constituents of the matrix. As used herein, “matrix” refers to essentially a three-dimensional environment which has at least one antigen binding probe immobilized therein for the purpose of measuring a detectable signal from analyte-analyte binding probe interaction. The relationship between the constituents of the matrix and the analyte binding probes includes, but are not limited to, covalent, ionic, and Van der Waals interactions and combinations thereof. The spatial relationship between the matrix and the aptamers includes heterogeneous and homogeneous distribution within and / or upon any or all of the matrix volume.

[0240] In some embodiments, the structural domain encapsulates said barrier domain, wherein said barrier domain can be configured to control transfer of said analyte to analyte binding probe. In some embodiments, the structural domain comprises one or more openings on an exterior surface of the structural domain. The one or more openings can be configured to allow said analyte to contact said barrier domain. In some embodiments, the barrier domain defines a passage connecting the one or more openings to the analyte binding probe. In some embodiments, the needle comprises a structural domain with a plurality of openings. In some embodiments, the needle comprises a structural domain comprising a hollow region of the needle. In some embodiments, the hollow region can be orientated in a longitudinal direction. In some embodiments, the plurality of openings can be positioned on a lateral face of the needle. In some embodiments, the plurality of openings can be positioned on opposing lateral faces of the needle. In some embodiments, the sensing domain extends longitudinally into the needle. In some embodiments, the sensing domain extends longitudinally into the needle and defines a passage connecting the one or more openings to the sensing domain with one opening on a lateral surface of the needle. In some embodiments, the structural domain can be directly in contact with the sensing domain. In some embodiments, the structural domain and the sensing domain extend longitudinally from the device. In some embodiments,the sensing domain can be positioned orthogonally to a longitudinal axis of the needle. In some embodiments, the sensing domain can be positioned orthogonally to a longitudinal axis of the needle, wherein the barrier domain can be positioned parallel to a longitudinal axis of the needle. In some embodiments, the barrier domain can be positioned on the exterior surface of the structural domain. In some embodiments, the barrier domain can be positioned on the exterior surface of the structural domain, and positioned within an interior space of the structural domain. In some embodiments, the barrier domain can be positioned on the exterior surface of the structural domain, positioned within an interior space of the structural domain, and wherein the sensing domain can be positioned within the structural domain in contact with the barrier domain positioned within an interior space of the structural domain. In some embodiments, the barrier domain can be positioned on the exterior surface of the structural domain, positioned within an interior space of the structural domain, and wherein the sensing domain can be positioned within the structural domain orthogonally to a longitudinal axis of the needle, and can be in contact with the barrier domain positioned within an interior space of the structural domain. In some embodiments, the barrier domain can be positioned on the exterior surface of the structural domain, positioned within an interior space of the structural domain, and wherein the sensing domain can be positioned throughout a matrix of the barrier domain. In some embodiments, the barrier domain can be within an interior space of the structural domain, and wherein the sensing domain can be positioned throughout a matrix of the barrier domain. In some embodiments, the barrier domain can be within an interior space of the structural domain and can be exposed to an exterior surface of the needle through the one or more openings, and wherein the sensing domain can be positioned throughout a matrix of the barrier domain. In some embodiments, the barrier domain can be positioned on the exterior surface of the structural domain, positioned within an interior space of the structural domain, and wherein the sensing domain can be positioned throughout a matrix of the barrier domain, wherein there are at least two opening on opposing lateral faces of the needle.

[0241] In some embodiments, the piercing element may be a microneedle. The microneedle can have straight or tapered shafts. In some embodiments, the diameter of the microneedle can be greatest at the base end of the microneedle and taper to a point or tip at the end distal to the base. The micro-needle can also be fabricated to have a shaft that includes both a straight (un-tapered) portion and a tapered portion. In some cases, the microneedle comprises a beveled edge to improve insertion into the skin.

[0242] In some embodiments, the length of the microneedle can be about 50 pm to about 5 mm. In some embodiments, the length of the microneedle can be about 50 pm to about 100m, about 50 pm to about 200 pm, about 50 pm to about 400 pm, about 50 pm to about 600 pm, about 50 pm to about 800 pm, about 50 pm to about 1 mm, about 50 pm to about 1.5 mm, about 50 pm to about 2 mm, about 50 pm to about 3 mm, about 50 pm to about 4 mm, about 50 pm to about 5 mm, about 100 pm to about 200 pm, about 100 pm to about 400 pm, about 100 pm to about 600 pm, about 100 pm to about 800 pm, about 100 pm to about 1 mm, about 100 pm to about 1.5 mm, about 100 pm to about 2 mm, about 100 pm to about 3 mm, about 100 pm to about 4 mm, about 100 pm to about 5 mm, about 200 pm to about 400 pm, about 200 pm to about 600 pm, about 200 pm to about 800 pm, about 200 pm to about 1 mm, about 200 pm to about 1.5 mm, about 200 pm to about 2 mm, about 200 pm to about 3 mm, about 200 pm to about 4 mm, about 200 pm to about 5 mm, about 400 pm to about 600 pm, about 400 pm to about 800 pm, about 400 pm to about 1 mm, about 400 pm to about 1.5 mm, about 400 pm to about 2 mm, about 400 pm to about 3 mm, about 400 pm to about 4 mm, about 400 pm to about 5 mm, about 600 pm to about 800 pm, about 600 pm to about 1 mm, about 600 pm to about 1.5 mm, about 600 pm to about 2 mm, about 600 pm to about 3 mm, about 600 pm to about 4 mm, about 600 pm to about 5 mm, about 800 pm to about 1 mm, about 800 pm to about 1.5 mm, about 800 pm to about 2 mm, about 800 pm to about 3 mm, about 800 pm to about 4 mm, about 800 pm to about 5 mm, about 1 mm to about 1.5 mm, about 1 mm to about 2 mm, about 1 mm to about 3 mm, about 1 mm to about 4 mm, about 1 mm to about 5 mm, about 1.5 mm to about 2 mm, about 1.5 mm to about 3 mm, about 1.5 mm to about 4 mm, about 1.5 mm to about 5 mm, about 2 mm to about 3 mm, about 2 mm to about 4 mm, about 2 mm to about 5 mm, about 3 mm to about 4 mm, about 3 mm to about 5 mm, or about 4 mm to about 5 mm. In some embodiments, the length of the microneedle can be about 50 pm, about 100 pm, about 200 pm, about 400 pm, about 600 pm, about 800 pm, about 1 mm, about 1.5 mm, about 2 mm, about 3 mm, about 4 mm, or about 5 mm. In some embodiments, the length of the microneedle can be at least about 50 pm, about 100 pm, about 200 pm, about 400 pm, about 600 pm, about 800 pm, about 1 mm, about 1.5 mm, about 2 mm, about 3 mm, or about 4 mm. In some embodiments, the length of the microneedle can be at most about 100 pm, about 200 pm, about 400 pm, about 600 pm, about 800 pm, about 1 mm, about 1.5 mm, about 2 mm, about 3 mm, about 4 mm, or about 5 mm.

[0243] The cross-sectional dimensions of the micro-needles can be about 1 pm to about 500 pm. The cross-sectional dimensions of the micro-needles can be about 1 pm to about 50 pm, about 1 pm to about 100 pm, about 1 pm to about 150 pm, about 1 pm to about 200 pm, about 1 pm to about 250 pm, about 1 pm to about 300 pm, about 1 pm to about 350 pm, about 1 pm to about 400 pm, about 1 pm to about 450 pm, about 1 pm to about 500 pm,about 50 pm to about 100 pm, about 50 pm to about 150 pm, about 50 pm to about 200 pm, about 50 pm to about 250 pm, about 50 pm to about 300 pm, about 50 pm to about 350 pm, about 50 pm to about 400 pm, about 50 pm to about 450 pm, about 50 pm to about 500 pm, about 100 pm to about 150 pm, about 100 pm to about 200 pm, about 100 pm to about 250 pm, about 100 pm to about 300 pm, about 100 pm to about 350 pm, about 100 pm to about 400 pm, about 100 pm to about 450 pm, about 100 pm to about 500 pm, about 150 pm to about 200 pm, about 150 pm to about 250 pm, about 150 pm to about 300 pm, about 150 pm to about 350 pm, about 150 pm to about 400 pm, about 150 pm to about 450 pm, about 150 pm to about 500 pm, about 200 pm to about 250 pm, about 200 pm to about 300 pm, about 200 pm to about 350 pm, about 200 pm to about 400 pm, about 200 pm to about 450 pm, about 200 pm to about 500 pm, about 250 pm to about 300 pm, about 250 pm to about 350 pm, about 250 pm to about 400 pm, about 250 pm to about 450 pm, about 250 pm to about 500 pm, about 300 pm to about 350 pm, about 300 pm to about 400 pm, about 300 pm to about 450 pm, about 300 pm to about 500 pm, about 350 pm to about 400 pm, about 350 pm to about 450 pm, about 350 pm to about 500 pm, about 400 pm to about 450 pm, about 400 pm to about 500 pm, or about 450 pm to about 500 pm. The cross-sectional dimensions of the micro-needles can be about 1 pm, about 50 pm, about 100 pm, about 150 pm, about 200 pm, about 250 pm, about 300 pm, about 350 pm, about 400 pm, about 450 pm, or about 500 pm. The cross-sectional dimensions of the micro-needles can be at least about 1 pm, about 50 pm, about 100 pm, about 150 pm, about 200 pm, about 250 pm, about 300 pm, about 350 pm, about 400 pm, or about 450 pm. The cross-sectional dimensions of the micro-needles can be at most about 50 pm, about 100 pm, about 150 pm, about 200 pm, about 250 pm, about 300 pm, about 350 pm, about 400 pm, about 450 pm, or about 500 pm. The length can be selected for the particular application, accounting for both an inserted and uninserted portion, and the target bodily fluid to be assayed.

[0244] An array of microneedles can include a mixture of microneedles having, for example, various lengths, outer diameters, inner diameters, cross-sectional shapes, and spacings between the microneedles, for example, varying depending on the target patient, target fluid, or target assay. Generally, the microneedles are sized to avoid or minimize contact with nerve endings in the biological tissue, such as the dermis, thereby eliminating or reducing pain when the microneedles are inserted, for example into the skin. The array of microneedles may penetrate into a dermis of the subject.

[0245] The microneedles can be oriented perpendicular or at an angle to the substrate. In some embodiments, the microneedles are oriented perpendicular to the substrate to providestructural strength and to permit ease of insertion into the tissue. An array of microneedles can include a mixture of microneedle orientations, heights, spacings, or other parameters. This variation in an array can be useful, for example, if different microneedles are to provide different sensing or insertion functions.

[0246] In another embodiment, the microfabricated microneedles can be formed from a polymer that includes the analyte binding probe. The microneedles of the device can be constructed from a variety of polymeric materials, including biocompatible and / or biodegradable polymers. Representative polymers include, without limitation, biodegradable polymers of hydroxy acids such as lactic acid and glycolic acid polylactide, polyglycolide, polylactide-co-glycolide, and copolymers with PEG, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valeric acid), and poly(lactide-co-caprolactone). Representative non-biodegradable polymers include polycarbonate, polymethacrylic acid, ethylenevinyl acetate, polytetrafluoroethylene (TEFLON™), and polyesters. In one embodiment, the micro-needles can be constructed from materials that are optically transparent and do not interfere with the optical detection of target analytes.

[0247] The piercing element may be a removable and replaceable element of the device. Different piercing elements may comprise different analyte binding probes for detection of different analytes using the same device. In some cases, it may be desirable for the piercing element to be a replaceable part of the device as to permit optimal analyte detection over an extended period. In some cases, there may be a foreign body response to the piercing element, which may negatively impact the ability of the analyte binding probe to detect the target analyte. In such cases, it may be desirable to utilize a replaceable piercing element as to permit for highly specific detection of the target analyte which is not negatively impacted by a foreign body response to the piercing element.

[0248] The piercing element may comprise an adhesive to aid in attaching the device to the skin of the user. The adhesive may surround the microneedle array such that when adhered to the skin a light-tight seal is formed around the array to prevent inadvertent excitation of the fluorophore in the microneedles. In some embodiments, the device may further comprise an adhesive on one or more surfaces of the device in contact with a skin of the user.Hydrogel Polymers

[0249] In some embodiments, the structural domain, sensing domain and / or the barrier domain may comprise a polymer matrix. The structural domain may comprise a higher molecular and higher density weight hydrogel, as compared to the barrier domain. The barrierdomain may comprise a higher molecular and higher density weight hydrogel, as compared to the sensing domain.

[0250] The polymer matrix can be in any desirable form or shape including one or more of disk, fiber, cylinder, patch, nanoparticle, microsphere, porous polymer, open cell foam, and combinations thereof providing it permits permeability to analyze. The polymer matrix may additionally prevent leaching of the analyte binding domain from the sensing mechanism. In some cases, the polymer matrix is substantially transparent and permits light from optical sources or any other interrogating signals to or from the reporter group bound to the aptamer to pass through the biosensor. When used in an in vivo application, the biosensor will be exposed to a substantially physiological range of analyte and determination or detection of a change in analyte concentration would be desired whereas the determination or detection includes continuous, programmed, and episodic detection means.

[0251] In some embodiments, the polymer matrix comprises an optical transmittance of about 10 % to about 99 %. In some embodiments, the polymer matrix comprises an optical transmittance of about 10 % to about 15 %, about 10 % to about 25 %, about 10 % to about 50 %, about 10 % to about 75 %, about 10 % to about 80 %, about 10 % to about 85 %, about 10 % to about 90 %, about 10 % to about 95 %, about 10 % to about 97 %, about 10 % to about 98 %, about 10 % to about 99 %, about 15 % to about 25 %, about 15 % to about 50 %, about 15 % to about 75 %, about 15 % to about 80 %, about 15 % to about 85 %, about 15 % to about 90 %, about 15 % to about 95 %, about 15 % to about 97 %, about 15 % to about 98 %, about 15 % to about 99 %, about 25 % to about 50 %, about 25 % to about 75 %, about 25 % to about 80 %, about 25 % to about 85 %, about 25 % to about 90 %, about 25 % to about 95 %, about 25 % to about 97 %, about 25 % to about 98 %, about 25 % to about 99 %, about 50 % to about 75 %, about 50 % to about 80 %, about 50 % to about 85 %, about 50 % to about 90 %, about 50 % to about 95 %, about 50 % to about 97 %, about 50 % to about 98 %, about 50 % to about 99 %, about 75 % to about 80 %, about 75 % to about 85 %, about 75 % to about 90 %, about 75 % to about 95 %, about 75 % to about 97 %, about 75 % to about 98 %, about 75 % to about 99 %, about 80 % to about 85 %, about 80 % to about 90 %, about 80 % to about 95 %, about 80 % to about 97 %, about 80 % to about 98 %, about 80 % to about 99 %, about 85 % to about 90 %, about 85 % to about 95 %, about 85 % to about 97 %, about 85 % to about 98 %, about 85 % to about 99 %, about 90 % to about 95 %, about 90 % to about 97 %, about 90 % to about 98 %, about 90 % to about 99 %, about 95 % to about 97 %, about 95 % to about 98 %, about 95 % to about 99 %, about 97 % to about 98 %, about 97 % to about 99 %, or about 98 % to about 99 %, including increments therein. In someembodiments, the polymer matrix comprises an optical transmittance of about 10 %, about 15 %, about 25 %, about 50 %, about 75 %, about 80 %, about 85 %, about 90 %, about 95 %, about 97 %, about 98 %, or about 99 %. In some embodiments, the polymer matrix comprises an optical transmittance of at least about 10 %, about 15 %, about 25 %, about 50 %, about 75 %, about 80 %, about 85 %, about 90 %, about 95 %, about 97 %, or about 98 %. In some embodiments, the polymer matrix comprises an optical transmittance of at most about 15 %, about 25 %, about 50 %, about 75 %, about 80 %, about 85 %, about 90 %, about 95 %, about 97 %, about 98 %, or about 99 %.

[0252] The polymer matrix may be prepared from biocompatible materials or incorporate materials capable of minimizing adverse reactions with the body. Adverse reactions for implants include inflammation, protein fouling, tissue necrosis, immune response and leaching of toxic materials.

[0253] Hydrogels as polymers are particularly useful. As used herein, the term “hydrogel” is used to indicate a water-insoluble, water-containing polymer network. Numerous hydrogels may be used in the present invention. The hydrogels may be, for example, polysaccharides such as agarose, dextran, carrageenan, alginic acid, starch, cellulose, or derivatives of these such as, e.g., carboxymethyl derivatives, or a water-swellable organic polymer such as, e.g., polyvinyl alcohol, polyacrylic acid, polyacrylamide, polyethylene glycol, copolymers of styrene and maleic anhydride, copolymers of vinyl ether and maleic anhydride and derivates thereof. Derivatives providing for covalently crosslinked networks are preferred. Synthesis and biomedical and pharmaceutical applications of hydrogels have been described by a number of researchers. An exemplary hydrogel matrix derived from a water-soluble, UV cross linkable polymer comprises poly(vinyl alcohol), N-methyl-4(4'-formylstyryl)pyridinium methosulphate acetal (CAS Reg. No. [107845-59-0]) available from PolyScience Warrington, Pa.

[0254] The polymers that are to be used in the hydrogel matrices may be functionalized. That is, the polymers or monomers comprising the polymers can possess reactive groups such that the hydrogel matrices are amenable to chemical reactions, e.g., covalent attachment. As used herein, a “reactive group” is a chemical group that can chemically react with a second group. The reactive group of the polymer or monomers comprising the polymer may itself be an entire chemical entity or it may be a portion of an entire chemical entity, including, but not limited to, single atoms or ions. Further, the second group with which the reactive group can be capable of reacting can be the same or different from the reactive group of the polymer or monomers comprising the polymers. Examples of reactive groups include, but are not limitedto, halogens, amines, amides, aldehydes, acrylates, vinyls, hydroxyls and carboxyls. In one embodiment, the polymers or monomers comprising the polymers of the hydrogel should be functionalized with carboxylic acid, sulfate, hydroxy or amine groups. In some embodiments, the polymers or monomers comprising the polymers of the hydrogel are functionalized with one or more acrylate groups. In some embodiments, the acrylate functional groups are terminal groups. The reactive groups of the polymers or monomers comprising the polymers of the matrix may be reactive with any component of the matrix portion of the biosensor, such as, but not limited to, another polymer or monomer within the matrix, a binding protein, and an additive.

[0255] Suitable polymers which may be used in the present disclosure include, but are not limited to, one or more of the polymers selected from the group consisting of poly (vinyl alcohol), polyacrylamide, poly (N-vinyl pyrolidone), polyethylene oxide) (PEO), hydrolyzed polyacrylonitrile, polyacrylic acid, polymethacrylic acid, poly(hydroxyethyl methacrylate), polyurethane polyethylene amine, poly(ethylene glycol) (PEG), cellulose, cellulose acetate, carboxy methyl cellulose, alginic acid, pectinic acid, hyaluronic acid, heparin, heparin sulfate, chitosan, carboxymethyl chitosan, chitin, collagen, pullulan, gellan, xanthan, carboxymethyl dextran, chondroitin sulfate, cationic guar, cationic starch as well as salts and esters thereof. The polymers of the hydrogel matrix may also comprise polymers of two or more distinct monomers. Monomers used to create copolymers for use in the matrices include, but are not limited to, acrylate, methacrylate, methyl methacrylate, methacrylic acid, alkylacrylates, phenylacrylate, hydroxyalkylacrylates, hydroxyalkylmethacrylates, aminoalkylacrylates, aminoalkylmethacrylates, alkyl quaternary salts of aminoalkylacrylamides, alkyl quaternary salts of aminoalkylmethacrylamides, and combinations thereof. Polymer components of the matrix may, of course, include blends of other polymers.

[0256] In some embodiments, the hydrogel can be comprised of polyethylene glycol) dimethacrylate (PEGDMA). PEGDMA is commercially available in a variety of molecular weights. For example, PEGDMA is available from at least Aldrich Chemical Co. (Milwaukee, Wis. USA) and from Polysciences, Inc. (Warrington, Pa., USA) and can be synthesized in an assortment of molecular weights. In some embodiments, the hydrogel can be comprised of polymers formed from Cu-click reactions, copper free click reactions (e.g. DBCO-Azide, BCN-Tetrazine), EDC, NHS / EDC, thiol maleimide, etc ), DBCO-N3, DBCO- DHPA, BCN-N3 f, or combinations thereof.

[0257] In some embodiments, the hydrogels comprise PEGDMA and at least one acrylate. As used herein, the term acrylate is well understood in the art. Specifically, acrylates are compounds, including but not limited to, polymers, comprising the acrylic group(HC2=CH — C(=O). Examples of acrylates include, but are not limited to, acrylic acid, ethyl acrylate, methacrylic acid, methyl methacrylic acid and acrylamides. In another specific embodiment, the hydrogels comprise more than one acrylate. In a more specific embodiment, the hydrogels comprise a mixture of methacrylate and methyl methacrylate.

[0258] The polymers used in the hydrogel matrices can be modified to contain nucleophilic or electrophilic groups. In some embodiments, the polymers may further comprise polyfunctional small molecules that do not contain repeating monomer units but are polyfunctional, z.e., containing two or more nucleophilic or electrophilic functional groups. These polyfunctional groups may readily be incorporated into conventional polymers by multiple covalent bond-forming reactions. For example, PEG can be modified to contain one or more amino groups to provide a nucleophilic group. Examples of other polymers that contain one or more nucleophilic groups include, but are not limited to, polyamines such as ethylenediamine, tetramethylenediamine, pentamethylenediamine, hexamethylenediamine, bis-(2-hydroxyethyl)amine, bis-(2-aminoethyl)amine, and tris-(2-aminoethyl)amine. Examples of electrophilic groups include, but are not limited to, succinimide esters, epoxides, hydroxybenzotriazole esters, oxycarbonylimidazoles, nitrophenyl carbonates, tresylates, mesylates, tosylates, carboxylates, and isocyanates. In one embodiment, the composition comprises a bis-amine-terminated poly(ethylene) glycol.

[0259] In some embodiments, the polymers may be crosslinked, either physically or chemically, to form a hydrogel. Physical crosslinking includes, but is not limited to, such non-chemical processes as radiation treatment such as electron beams, gamma rays, x-rays, ultraviolet light, anionic and cationic treatments. The crosslinking of the polymers may also comprise chemical crosslinking, such as covalent crosslinking. For example, a chemical crosslinking system may include, but is not limited to, the use of enzymes, which is well- known in the art. Another example of the chemical covalent crosslinking comprises the use of peroxide. Chemical crosslinking may occur when a crosslinking reagent reacts with at least two portions of a polymer to create a three-dimensional network. Covalent crosslinking may also occur when multifunctional monomers are used during the crosslinking process. For example, an acrylate monomer may be polymerized with a bifunctional acrylate monomer to form a crosslinked polymer. Any crosslinking reagent will be suitable for the present invention, provided the crosslinking reagent will at least partially dissolve in water or anorganic solvent and can form the crosslinked polymer. For example, if the polymer is an amine-terminated PEG, the crosslinking reagent should be capable of reacting with the PEG- amine groups and be substantially soluble in water. In another example, (hydroxyethyl methacrylate) and methacrylic acid monomers can be polymerized with poly(ethylene glycol)-bis-alkylacrylate crosslinking agent in water or in dimethylformide to form polymeric hydrogels.

[0260] If the crosslinked polymers are functionalized with nucleophilic groups, such as amines (primary, secondary and tertiary), thiols, thioethers, esters, nitrites, and the like, the crosslinking reagent can be a molecule containing an electrophilic group. Examples of electrophilic groups have been described herein. Likewise, if polymers to be crosslinked are functionalized with electrophilic groups, the crosslinking reagent can be a molecule containing a nucleophilic group. It is understood that one skilled in the art can exchange the nucleophilic and electrophilic functional groups as described above without deviating from the scope of the present embodiment. It is also understood that the binding molecule can provide the requisite nucleophilic and electrophilic functional groups. For example, where the binding molecule is a protein, the nucleophilic and electrophilic functional groups may be present as naturally occurring amino acids in the protein, or may be introduced to the protein using chemical techniques described herein. Other general methods for preparing or crosslinking polymers to form hydrogel matrices are well known in the art.

[0261] In some embodiments, the analyte binding probe can be encapsulated within a hydrogel (e.g., entrapped within a hydrogel). In some cases, analyte binding probe can be held within hydrogel through steric interactions and / or intermolecular forces. In some cases, the analyte binding probe is covalently bound to the hydrogel. The attachment of the analyte binding probes to the hydrogel should not interfere with the binding of the analyte binding probes to the target ligand. Furthermore, the attachment of the analyte binding probes to the hydrogel should be resistant to degradation. The functional group in one embodiment, a polymer or other component of the hydrogel, serves to couple the analyte binding probe to the hydrogel. The coupling of the analyte binding probe to the hydrogel can be accomplished in any number of ways. For example, coupling reactions between the hydrogel and binding molecule include, but are not limited to, diazonium coupling, isothiocyano coupling, hydrazide coupling, amide formation, disulfide coupling, maleic anhydride coupling, thiolactone coupling, and dichlotriazine coupling. These coupling reactions between two functional groups are well documented, and are considered well known to those skilled in the art. For example, an amino functional group in an analyte binding probe can be covalentlycoupled to a carboxyl functional group of one or more components of a hydrogel using coupling agents such as l-ethyl-3 -(3 -dimethylaminopropyl) carbodiimide hydrochloride (EDC) or dicyclohexylcarbodiimide (DCC). It is understood that the amino and carboxyl functional groups of the binding molecule and one or more components of the hydrogel as described above can be transposed without deviating from the scope of the embodiment.

[0262] In some embodiments, the analyte binding probes may be bound encapsulated non- covalently within polymer matrix or scaffold by any suitable means that allows analyte- induced conformational change of the analyte binding probes, retention of the analyte binding probes within the polymer to prevent loss or leaching of the analyte binding probe, and to provide a stable, continuous and reversible sensor response to changing concentrations of the target analyte of interest. For instance, well-established processes for enzyme immobilization in hydrogels may be used.

[0263] In some embodiments, the substrate can be made of a biodegradable material or polymer. In another embodiment, the substrate can be coated or embedded within a matrix of biodegradable material or polymer. In some embodiments, the substrate can be retained by an envelope of biodegradable material or polymer, or may be separately covered with biodegradable material or polymer.

[0264] In some embodiments, the substrate is suspended within the matrix which comprises the analyte binding probe for detecting or measuring analytes in interstitial fluid. In some embodiments, low molecular weight analytes, such as glucose, can freely diffuse into the matrix from the surrounding interstitial fluid.

[0265] Alternatively, the substrate can be made from a solid or gel-like polymer biodegradable material within which the sensor components are mounted or distributed. When injected or implanted cutaneously this solid polymer sensor hydrates and swells, and target analyte penetrates through the structure to encounter the substrate.

[0266] Biodegradable materials suitable for use in the coating of the substrate or the construction of the substrate include cross-linked proteins such as human albumin, fibrin gels, polysaccharides such as starch or agarose, polylactides (PLA) such as poly (DL-lactide), polyglycolides (PGA) such as poly (DL-glycolide), poly(lactide-co-glycolides) (PLGA), polyanhydrides, polyorthoesters, fatty acid / cholesterol mixtures that form semi-solid derivates, hyaluronates and liquid crystals of monoolein and water. These materials have the advantage that they are broken down into biologically acceptable molecules which are metabolized and removed from the body via normal pathways.Microneedle Configuration

[0267] In another embodiment, the device may comprise a piercing element which may include an array of microfabricated microneedles. The microneedles may comprise a well or have an interior hollow space, wherein the analyte binding probes are encapsulated in a polymer matrix contained in the hollow interior space. The polymer matrix of the hollow interior space can be the same or different from the polymer forming the microneedles. The analyte binding probe can be bound covalently or non-covalently to the wall of the hollow interior space or to the polymer matrix within the hollow interior space. In some cases, the analyte binding probe can be sterically trapped within the matrix. In some cases, the analyte binding probe can be held within the matrix with a combination of different interactions (e.g., Van der Waals forces, other intermolecular interactions). In some cases, the microneedles may comprise a pyramidal structure which define a plurality of faces about the exterior surface of the microneedles. In some cases, the microneedles may comprise opening about the plurality of faces about the exterior surface of the microneedles, for example, as illustrated in Figures 39A-39B.

[0268] In some embodiments, each microneedle can be associated with a plurality of types of analyte binding probes, each type directed to a specific target analyte, to provide a spatially defined microneedle array to allow for the detection of a multitude of different analytes, such as Na+, K+, glucose, etc., in a controlled spatial array. In some embodiments, the interaction with the target analyte can occur in or on the microneedle. In some embodiments, the analyte binding sensor is located in the microneedle and contacts the interstitial fluid containing the analyte. The detectable change may be indicated optically, e.g., based on fluorescence emitted by the nanosensors when optically interrogated. The analyte binding probe can be located inside the surface of a porous microneedle, and / or embedded within the microneedle but exposed to interstitial fluid. In another embodiment, the microneedle array having an analyte binding probe can be readily removed by simply removing it from the transdermal patch (e.g., consumable). The piercing element can be coupled to the support using one or more holes, using one or more insertable elements configured to attach to a corresponding one or more receiving elements, using a lock and key attachment, using a mortise and tenon attachment, using a dovetail attachment, using a magnetic attachment, using an adhesive, using one or more elastically deformable attachment elements, using one or more elastically deformable attachment elements configured and rebound once inserted into the support, or combinations thereof. In some embodiments, the piercing element alone is removable from the device, as is illustrated in Figure 38A. In some embodiments, both the light guide and thepiercing element are removably coupled to the support, for example, as illustrated in Figure 38B where the light guide is coupled to the piercing element, and the light guide is removable from the support. In some embodiments, the light guide comprises first portion and a second portion, where the second portion is coupled to the piercing element, and wherein the first portion and the second portion are separable from one another as to remove the piercing element and the second portion of the light guide from the support, as is illustrated in Figure 38CAnalyte Binding Probe

[0269] The present disclosure provides sensors for analyte detection. An analyte binding probe can comprise polypeptide or nucleic acid sequences, antibodies, peptides, proteins, physiochemical detectors, enzymes, artificial binding proteins, or combinations thereof. In some embodiments, the analyte sensor comprises a polypeptide or peptide sequence such as an antibody. For example, an analyte sensor may comprise single stranded deoxyribonucleic acid, double stranded DNA (dsDNA), ribonucleic acid (RNA), nucleic acids in some cases with modified bases, and the like. The analyte sensor may be an oligonucleotide probe and the analyte may be a complementary target nucleic acid. In another embodiment, the binding domain can be a dsDNA strand specific to a target enhancer protein target. In some embodiments, the analyte sensor may comprise a nucleic acid sequence comprising an aptamer.Antigen Binding Probe: Aptamers

[0270] Sometimes referred to as “synthetic antibodies,” aptamers may be pre-selected singlestranded oligonucleotide (e.g., DNA or RNA) or peptide molecules that bind to specific target molecules including proteins and peptides with affinities and specificities that are comparable to antibodies. These molecules can assume a variety of shapes due to their propensity to form helices and single-stranded loops with specific binding pockets, explaining their versatility in binding to diverse targets. Their specificity and characteristics are not directly determined by their primary sequence but by their tertiary structure which can be analogous to the globular shape of tRNA. Aptamers have a wide range of applications including diagnostics and therapeutics and can be chemically synthesized using known techniques. Furthermore, aptamers can offer a number of advantages over traditional antibodies including avoiding the need to specifically know the precise epitopes or biomarkers themselves. Finally, aptamers may be typically non-immunogenic, easy to synthesize, characterize, modify, and exhibithigh specificity and affinity for their target antigen.

[0271] The aptamer may be nucleic acid or peptide molecules that bind to a specific target molecule. In some embodiments, binding of the target analyte to the aptamer induces conformational changes in the aptamer. In some embodiments, the aptamer may bind to various molecular targets, for example, small molecules, macromolecules, metabolites, proteins, carbohydrates, metals, nucleic acids, cells, tissues, and organisms.

[0272] By using a variety of selection techniques, aptamers can be selected to find targets, e.g., on a surface or inside a cell of interest, without the need to identify the precise biomarker or epitopes themselves. In many cases, the aptamer identification process can begin with a large random pool of oligonucleotides or peptides that are systematically subjected to negative and positive rounds of selection against a target, e.g., a protein molecule, to filter out low affinity or unspecific binders. The remaining aptamers can be collected and propagated, e.g., PCR amplified, and used in subsequent rounds of selection. This selection process, referred to as Systemic Evolution of Ligands by Exponential Enrichment or SELEX, is commonly used for selecting and identifying highly targeted aptamers. A variant of this methodology, known as cell-SELEX, has been developed for aptamers that are capable of recognizing whole living cells.Aptamer Switch

[0273] In some embodiments, the antigen binding probe may comprise at least one of the four elements: a single-stranded oligonucleotide; a short, complementary DNA sequence to the oligonucleotide; a linking moiety that conjugates the oligonucleotide with the DNA sequence; and luminescent molecules.

[0274] In some embodiments, the single oligonucleotide has a first and second terminus, wherein the first terminus can be attached to a luminescent molecule and a second terminus attached to a piercing element. In some embodiments, the single oligonucleotide further comprises a short, partially complementary DNA sequence, wherein the short, partially complementary DNA sequence further comprises a second luminescent molecule.

[0275] In some embodiments, the single-stranded oligonucleotide has a first and second terminus, where the first terminus can be attached to the linking moiety. The linking moiety can be also attached to the first terminus of a short DNA strand having a partially complementary sequence to the oligonucleotide, where the short DNA strand has a first and second terminus. Luminescent molecules are attached to the second termini of the oligonucleotide and the short DNA strand.

[0276] In some embodiments, the oligonucleotide can be an aptamer. Aptamers are nucleic acid molecules that bind to a specific target molecule such as small molecules, proteins, nucleic acids, cells, tissues, and organisms. In some embodiments, aptamers are singlestranded oligonucleotides exhibiting high affinity and specificity toward any given target molecule. The aptamer disclosed herein may be any suitable size.

[0277] In some embodiments, the size of the aptamer as disclosed herein can be about 5 nucleotides to about 250 nucleotides. In some embodiments, the size of the aptamer as disclosed herein can be about 5 nucleotides to about 25 nucleotides, about 5 nucleotides to about 50 nucleotides, about 5 nucleotides to about 75 nucleotides, about 5 nucleotides to about 100 nucleotides, about 5 nucleotides to about 125 nucleotides, about 5 nucleotides to about 150 nucleotides, about 5 nucleotides to about 175 nucleotides, about 5 nucleotides to about 200 nucleotides, about 5 nucleotides to about 225 nucleotides, about 5 nucleotides to about 250 nucleotides, about 25 nucleotides to about 50 nucleotides, about 25 nucleotides to about 75 nucleotides, about 25 nucleotides to about 100 nucleotides, about 25 nucleotides to about 125 nucleotides, about 25 nucleotides to about 150 nucleotides, about 25 nucleotides to about 175 nucleotides, about 25 nucleotides to about 200 nucleotides, about 25 nucleotides to about 225 nucleotides, about 25 nucleotides to about 250 nucleotides, about 50 nucleotides to about 75 nucleotides, about 50 nucleotides to about 100 nucleotides, about 50 nucleotides to about 125 nucleotides, about 50 nucleotides to about 150 nucleotides, about 50 nucleotides to about 175 nucleotides, about 50 nucleotides to about 200 nucleotides, about 50 nucleotides to about 225 nucleotides, about 50 nucleotides to about 250 nucleotides, about 75 nucleotides to about 100 nucleotides, about 75 nucleotides to about 125 nucleotides, about 75 nucleotides to about 150 nucleotides, about 75 nucleotides to about 175 nucleotides, about 75 nucleotides to about 200 nucleotides, about 75 nucleotides to about 225 nucleotides, about 75 nucleotides to about 250 nucleotides, about 100 nucleotides to about 125 nucleotides, about 100 nucleotides to about 150 nucleotides, about 100 nucleotides to about 175 nucleotides, about 100 nucleotides to about 200 nucleotides, about 100 nucleotides to about 225 nucleotides, about 100 nucleotides to about 250 nucleotides, about 125 nucleotides to about 150 nucleotides, about 125 nucleotides to about 175 nucleotides, about 125 nucleotides to about 200 nucleotides, about 125 nucleotides to about 225 nucleotides, about 125 nucleotides to about 250 nucleotides, about 150 nucleotides to about 175 nucleotides, about 150 nucleotides to about 200 nucleotides, about 150 nucleotides to about 225 nucleotides, about 150 nucleotides to about 250 nucleotides, about 175 nucleotides to about 200 nucleotides, about 175 nucleotides to about 225 nucleotides, about 175 nucleotides to about 250 nucleotides, about200 nucleotides to about 225 nucleotides, about 200 nucleotides to about 250 nucleotides, or about 225 nucleotides to about 250 nucleotides. In some embodiments, the size of the aptamer as disclosed herein can be about 5 nucleotides, about 25 nucleotides, about 50 nucleotides, about 75 nucleotides, about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, or about 250 nucleotides. In some embodiments, the size of the aptamer as disclosed herein can be at least about 5 nucleotides, about 25 nucleotides, about 50 nucleotides, about 75 nucleotides, about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, or about 225 nucleotides. In some embodiments, the size of the aptamer as disclosed herein can be at most about 25 nucleotides, about 50 nucleotides, about 75 nucleotides, about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, or about 250 nucleotides. In some embodiments, the probe may further comprise a short, complementary DNA sequence that, in the absence of a target analyte, can hybridize with complete or partial complementary to a portion of the aptamer. In some embodiments, the aptamer and complementary DNA sequence described herein may have one or more mismatched nucleotides. One or more mismatched nucleotide may be 1 or more mismatched nucleotides; 2 or more mismatched nucleotides; 3 or more mismatched nucleotides; 4 or more mismatched nucleotides; 5 or more mismatched nucleotides; 6 or more mismatched nucleotides; 7 or more mismatched nucleotides; 8 or more mismatched nucleotides; 9 or more mismatched nucleotides; 10 or more mismatched nucleotides; 12 or more mismatched nucleotides; 14 or more mismatched nucleotides; 16 or more mismatched nucleotides; 18 or more mismatched nucleotides; or 20 or more mismatched nucleotides. The length of a short, complementary DNA sequence can be about 2 nucleotides to about 45 nucleotides. The length of a short, complementary DNA sequence can be about 2 nucleotides to about 4 nucleotides, about 2 nucleotides to about 6 nucleotides, about 2 nucleotides to about 8 nucleotides, about 2 nucleotides to about 10 nucleotides, about 2 nucleotides to about 15 nucleotides, about 2 nucleotides to about 20 nucleotides, about 2 nucleotides to about 25 nucleotides, about 2 nucleotides to about 30 nucleotides, about 2 nucleotides to about 35 nucleotides, about 2 nucleotides to about 40 nucleotides, about 2 nucleotides to about 45 nucleotides, about 4 nucleotides to about 6 nucleotides, about 4 nucleotides to about 8 nucleotides, about 4 nucleotides to about 10 nucleotides, about 4 nucleotides to about 15 nucleotides, about 4 nucleotides to about 20 nucleotides, about 4 nucleotides to about 25 nucleotides, about 4 nucleotides to about 30 nucleotides, about 4 nucleotides to about 35 nucleotides, about 4 nucleotides to about 40nucleotides, about 4 nucleotides to about 45 nucleotides, about 6 nucleotides to about 8 nucleotides, about 6 nucleotides to about 10 nucleotides, about 6 nucleotides to about 15 nucleotides, about 6 nucleotides to about 20 nucleotides, about 6 nucleotides to about 25 nucleotides, about 6 nucleotides to about 30 nucleotides, about 6 nucleotides to about 35 nucleotides, about 6 nucleotides to about 40 nucleotides, about 6 nucleotides to about 45 nucleotides, about 8 nucleotides to about 10 nucleotides, about 8 nucleotides to about 15 nucleotides, about 8 nucleotides to about 20 nucleotides, about 8 nucleotides to about 25 nucleotides, about 8 nucleotides to about 30 nucleotides, about 8 nucleotides to about 35 nucleotides, about 8 nucleotides to about 40 nucleotides, about 8 nucleotides to about 45 nucleotides, about 10 nucleotides to about 15 nucleotides, about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 25 nucleotides, about 10 nucleotides to about 30 nucleotides, about 10 nucleotides to about 35 nucleotides, about 10 nucleotides to about 40 nucleotides, about 10 nucleotides to about 45 nucleotides, about 15 nucleotides to about 20 nucleotides, about 15 nucleotides to about 25 nucleotides, about 15 nucleotides to about 30 nucleotides, about 15 nucleotides to about 35 nucleotides, about 15 nucleotides to about 40 nucleotides, about 15 nucleotides to about 45 nucleotides, about 20 nucleotides to about 25 nucleotides, about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 35 nucleotides, about 20 nucleotides to about 40 nucleotides, about 20 nucleotides to about 45 nucleotides, about 25 nucleotides to about 30 nucleotides, about 25 nucleotides to about 35 nucleotides, about 25 nucleotides to about 40 nucleotides, about 25 nucleotides to about 45 nucleotides, about 30 nucleotides to about 35 nucleotides, about 30 nucleotides to about 40 nucleotides, about 30 nucleotides to about 45 nucleotides, about 35 nucleotides to about 40 nucleotides, about 35 nucleotides to about 45 nucleotides, or about 40 nucleotides to about 45 nucleotides. The length of a short, complementary DNA sequence can be about 2 nucleotides, about 4 nucleotides, about 6 nucleotides, about 8 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, or about 45 nucleotides. The length of a short, complementary DNA sequence can be at least about 2 nucleotides, about 4 nucleotides, about 6 nucleotides, about 8 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, or about 40 nucleotides. The length of a short, complementary DNA sequence can be at most about 4 nucleotides, about 6 nucleotides, about 8 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, or about 45 nucleotides.Linking Moieties

[0278] In some embodiments the analyte sensor may further comprise a flexible linker region that attaches the oligonucleotides (e.g., aptamer) to the short DNA sequence. In some cases, the linker moiety can be a nucleotide acid moiety that does not bind to either the oligonucleotide, the short DNA sequence, a peptide nucleic acid (PNA) moiety, a peptide moiety, a disulfide bond, a phosphodiester linkage, or a polymer such as a polyethylene glycol (PEG) moiety. The linker region disclosed herein can be about 2 residues in length to about 45 residues in length. The linker region disclosed herein can be about 2 residues in length to about 4 residues in length, about 2 residues in length to about 6 residues in length, about 2 residues in length to about 8 residues in length, about 2 residues in length to about 10 residues in length, about 2 residues in length to about 15 residues in length, about 2 residues in length to about 20 residues in length, about 2 residues in length to about 25 residues in length, about 2 residues in length to about 30 residues in length, about 2 residues in length to about 35 residues in length, about 2 residues in length to about 40 residues in length, about 2 residues in length to about 45 residues in length, about 4 residues in length to about 6 residues in length, about 4 residues in length to about 8 residues in length, about 4 residues in length to about 10 residues in length, about 4 residues in length to about 15 residues in length, about 4 residues in length to about 20 residues in length, about 4 residues in length to about 25 residues in length, about 4 residues in length to about 30 residues in length, about 4 residues in length to about 35 residues in length, about 4 residues in length to about 40 residues in length, about 4 residues in length to about 45 residues in length, about 6 residues in length to about 8 residues in length, about 6 residues in length to about 10 residues in length, about 6 residues in length to about 15 residues in length, about 6 residues in length to about 20 residues in length, about 6 residues in length to about 25 residues in length, about 6 residues in length to about 30 residues in length, about 6 residues in length to about 35 residues in length, about 6 residues in length to about 40 residues in length, about 6 residues in length to about 45 residues in length, about 8 residues in length to about 10 residues in length, about 8 residues in length to about 15 residues in length, about 8 residues in length to about 20 residues in length, about 8 residues in length to about 25 residues in length, about 8 residues in length to about 30 residues in length, about 8 residues in length to about 35 residues in length, about 8 residues in length to about 40 residues in length, about 8 residues in length to about 45 residues in length, about 10 residues in length to about 15 residues in length, about 10 residues in length to about 20 residues in length, about 10 residues in lengthto about 25 residues in length, about 10 residues in length to about 30 residues in length, about 10 residues in length to about 35 residues in length, about 10 residues in length to about 40 residues in length, about 10 residues in length to about 45 residues in length, about 15 residues in length to about 20 residues in length, about 15 residues in length to about 25 residues in length, about 15 residues in length to about 30 residues in length, about 15 residues in length to about 35 residues in length, about 15 residues in length to about 40 residues in length, about 15 residues in length to about 45 residues in length, about 20 residues in length to about 25 residues in length, about 20 residues in length to about 30 residues in length, about 20 residues in length to about 35 residues in length, about 20 residues in length to about 40 residues in length, about 20 residues in length to about 45 residues in length, about 25 residues in length to about 30 residues in length, about 25 residues in length to about 35 residues in length, about 25 residues in length to about 40 residues in length, about 25 residues in length to about 45 residues in length, about 30 residues in length to about 35 residues in length, about 30 residues in length to about 40 residues in length, about 30 residues in length to about 45 residues in length, about 35 residues in length to about 40 residues in length, about 35 residues in length to about 45 residues in length, or about 40 residues in length to about 45 residues in length. The linker region disclosed herein can be about 2 residues in length, about 4 residues in length, about 6 residues in length, about 8 residues in length, about 10 residues in length, about 15 residues in length, about 20 residues in length, about 25 residues in length, about 30 residues in length, about 35 residues in length, about 40 residues in length, or about 45 residues in length. The linker region disclosed herein can be at least about 2 residues in length, about 4 residues in length, about 6 residues in length, about 8 residues in length, about 10 residues in length, about 15 residues in length, about 20 residues in length, about 25 residues in length, about 30 residues in length, about 35 residues in length, or about 40 residues in length. The linker region disclosed herein can be at most about 4 residues in length, about 6 residues in length, about 8 residues in length, about 10 residues in length, about 15 residues in length, about 20 residues in length, about 25 residues in length, about 30 residues in length, about 35 residues in length, about 40 residues in length, or about 45 residues in length.

[0279] In some embodiments, a linker can be a homopolymeric polynucleotide. An intramolecular linker can be about 5 nucleotides to about 60 nucleotides. An intramolecular linker can be about 5 nucleotides to about 10 nucleotides, about 5 nucleotides to about 15 nucleotides, about 5 nucleotides to about 20 nucleotides, about 5 nucleotides to about 25 nucleotides, about 5 nucleotides to about 30 nucleotides, about 5 nucleotides to about 35nucleotides, about 5 nucleotides to about 40 nucleotides, about 5 nucleotides to about 45 nucleotides, about 5 nucleotides to about 50 nucleotides, about 5 nucleotides to about 55 nucleotides, about 5 nucleotides to about 60 nucleotides, about 10 nucleotides to about 15 nucleotides, about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 25 nucleotides, about 10 nucleotides to about 30 nucleotides, about 10 nucleotides to about 35 nucleotides, about 10 nucleotides to about 40 nucleotides, about 10 nucleotides to about 45 nucleotides, about 10 nucleotides to about 50 nucleotides, about 10 nucleotides to about 55 nucleotides, about 10 nucleotides to about 60 nucleotides, about 15 nucleotides to about 20 nucleotides, about 15 nucleotides to about 25 nucleotides, about 15 nucleotides to about 30 nucleotides, about 15 nucleotides to about 35 nucleotides, about 15 nucleotides to about 40 nucleotides, about 15 nucleotides to about 45 nucleotides, about 15 nucleotides to about 50 nucleotides, about 15 nucleotides to about 55 nucleotides, about 15 nucleotides to about 60 nucleotides, about 20 nucleotides to about 25 nucleotides, about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 35 nucleotides, about 20 nucleotides to about 40 nucleotides, about 20 nucleotides to about 45 nucleotides, about 20 nucleotides to about 50 nucleotides, about 20 nucleotides to about 55 nucleotides, about 20 nucleotides to about 60 nucleotides, about 25 nucleotides to about 30 nucleotides, about 25 nucleotides to about 35 nucleotides, about 25 nucleotides to about 40 nucleotides, about 25 nucleotides to about 45 nucleotides, about 25 nucleotides to about 50 nucleotides, about 25 nucleotides to about 55 nucleotides, about 25 nucleotides to about 60 nucleotides, about 30 nucleotides to about 35 nucleotides, about 30 nucleotides to about 40 nucleotides, about 30 nucleotides to about 45 nucleotides, about 30 nucleotides to about 50 nucleotides, about 30 nucleotides to about 55 nucleotides, about 30 nucleotides to about 60 nucleotides, about 35 nucleotides to about 40 nucleotides, about 35 nucleotides to about 45 nucleotides, about 35 nucleotides to about 50 nucleotides, about 35 nucleotides to about 55 nucleotides, about 35 nucleotides to about 60 nucleotides, about 40 nucleotides to about 45 nucleotides, about 40 nucleotides to about 50 nucleotides, about 40 nucleotides to about 55 nucleotides, about 40 nucleotides to about 60 nucleotides, about 45 nucleotides to about 50 nucleotides, about 45 nucleotides to about 55 nucleotides, about 45 nucleotides to about 60 nucleotides, about 50 nucleotides to about 55 nucleotides, about 50 nucleotides to about 60 nucleotides, or about 55 nucleotides to about 60 nucleotides. An intramolecular linker can be about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 45 nucleotides, about 50 nucleotides, about 55 nucleotides, or about 60 nucleotides. An intramolecular linker can be at least about 5nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 45 nucleotides, about 50 nucleotides, or about 55 nucleotides. An intramolecular linker can be at most about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 45 nucleotides, about 50 nucleotides, about 55 nucleotides, or about 60 nucleotides.Photoluminescent Label

[0280] A photoluminescent label (e.g., fluorophore label) can be any chemical moiety that exhibits an absorption maximum at or beyond 280 nm, and when covalently attached to the analyte binding probe or other reagent retains its spectral properties. In some embodiments, the photoluminescent label can be any molecule that provides luminescent indication of analyte sensor and target molecule binding. In some cases, the luminescent molecule is a fluorophore, quencher, fluorescence resonance energy transfer (FRET) or FRET pair. A fluorophore molecule may be a molecule that has the ability to absorb energy from light, transfer this energy internally, and emit the energy as light within a characteristic wavelength range. In some embodiments, an analyte binding probe may comprise a fluorophore and a quencher, which absorbs excitation energy from a fluorophore. Suppression of emission from a fluorophore may occur as a result of the formation of a complex between the fluorophore and the quencher, where the absorption spectra of the two molecules change upon formation of the complex. When the two molecules are not in close proximity with each other, fluorescence emission may be restored. In some embodiments, the quencher may be another fluorophore or a nonfluorescent molecule. Fluorophores of the present disclosure include, without limitation; a pyrene, an anthracene, a naphthalene, an acridine, a stilbene, an indole or benzindole, an oxazole or benzoxazole, a thiazole or benzothiazole, a 4-amino-7- nitrobenz-2-oxa-l,3-diazole (NBD), a cyanine, a carbocyanine a carbostyryl, a porphyrin, a salicylate, an anthranilate, an azulene, a perylene, a pyridine, a quinoline, a borapolyazaindacene (including any corresponding compounds disclosed, a xanthene, an oxazine or a benzoxazine, a carbazine, a phenalenone, a coumarin (including an corresponding compounds;...

Claims

CLAIMS1. A device for sensing a biological sample of a subject, comprising: a probe comprising an inner lumen and an outer lumen, wherein the inner lumen is concentric with the outer lumen, wherein the inner lumen is fluidically coupled to the outer lumen, and wherein the probe outer lumen is fluidically coupled to a sensing domain; a piercing element configured to pierce a body surface of a subject to provide the probe in contact with a biological sample within the body of the subject; and a pump fluidically coupled to the inner lumen of the probe, wherein the sensing domain and the pump are provided adjacent to the body surface of the subject.

2. The device of claim 1, wherein an end of the inner lumen is fluidically coupled to an end of the outer lumen.

3. The device of any one of the preceding claims, wherein the biological sample within the body of the subject diffuses into the fluid through the inner lumen of the probe, through the outer lumen of the probe, or a combination thereof.

4. The device of any one of the preceding claims, wherein the inner lumen of the probe, the outer lumen of the probe, or a combination thereof, comprise a semipermeable membrane, and wherein the biological sample diffuses through the semipermeable membrane into the fluid forced through the inner lumen of the probe, into the fluid forced through the outer lumen of the probe, or a combination thereof.

5. The device of any one of the preceding claims, wherein the biological sample is sensed by the sensing domain when: (i) the biological sample diffuses into the fluid forced through the inner lumen or the fluid forced through the outer lumen of the probe, and (ii) the biological sample couples to a molecular sensor of the sensing domain.

6. The device of any one of the preceding claims, wherein the subject comprises a human subject.

7. The device of any one of the preceding claims, wherein the sensing domain comprises a microfluidic chip, wherein the microfluidic chip comprises one or more microfluidic channels.

8. The device of any one of the preceding claims, wherein the molecular sensor is disposed within a channel of the one or more microfluidic channels.

9. The device of any one of the preceding claims, wherein the piercing element is a retractable needle.

10. The device of any one of the preceding claims, wherein the body surface of the subject comprises a skin surface of the subject.

11. The device of any one of the preceding claims, further comprising a wearable housing, wherein the wearable housing comprises the sensing domain and the pump.

12. The device of any one of the preceding claims, wherein the wearable housing comprises the probe, the piercing element, the sensing domain, and the pump, and wherein the wearable housing covers at least a portion of the body surface of the subject.

13. The device of any one of the preceding claims, wherein the wearable housing is provided in contact with the body surface of the subject.

14. The device of any one of the preceding claims, wherein the probe is provided within the piercing element.

15. The device of any one of the preceding claims, wherein the probe is concentric with the piercing element.

16. The device of any one of the preceding claims, wherein the wearable housing comprises an adhesive, wherein the adhesive couples the wearable housing to the body surface of the subject.

17. The device of any one of the preceding claims, wherein the fluid comprises a dialysate.

18. The device of any one of the preceding claims, wherein the dialysate comprises a dialysate buffer.

19. A device for sensing an analyte in a biological sample of a subject, comprising: a semipermeable membrane with an inlet and outlet fluidic path, wherein the semipermeable membrane is contacted with interstitial fluid of a subject allowing analytes to diffuse across the semipermeable membrane; a dialysate buffer which is connected to the inlet of the semipermeable membrane; a mechanism of providing flow of the dialysate buffer through the device either continuously or at scheduled intervals; a flow cell with an inlet and outlet, the inlet of the flow cell is attached to the outlet of the semipermeable membrane; an analyte binding probes which are contained within the flow cell and configured to change signal in response to analyte binding; and a detector operatively coupled to the flow cell, wherein the detector is configured to detect a change in signal from the analyte binding probes.

20. The device of claim 19, wherein two or more analytes are measured using different analyte binding probes.

21. The device of claim 20, wherein the multiple analyte binding probes are spatially separated.

22. The device of claim 20, wherein the multiple analyte binding probes are labeled with spectrally different optical reporters.

23. The device of claim 19, wherein one of the analyte binding probes measures a reference molecule.

24. The device of claim 23, wherein the reference molecule is used to correct for recovery rates <100%.

25. The device of claim 20, wherein the two or more analytes are measured and used to correct for recovery rate.

26. The device of claim 25, wherein the alteration in recovery rate is caused by the foreign body response (e.g., fibrotic encapsulation).

27. The device of claim 20, wherein the signal from two different analyte probes are used ratiometrically.

28. The device of claim 19, wherein an exogenous biocompatible molecule is included within the dialysate buffer to serve as a reference molecule that decreases in concentration with increased recovery rate, and this decrease is measured to correct for changes in recovery rate.

29. The device of claim 19, wherein the analyte binding probe is an aptamer switch.

30. The device of claim 19, wherein the analyte binding probe is a dual-antibody switch.

31. The device of claim 19, wherein the analyte binding probe is antibody-aptamer chimeric switch.

32. The device of claim 19, wherein the analyte binding probe is a protein-based switch composed of two or more analyte binding regions.

33. The device of claim 19, wherein the detector detects a change in optical signal.

34. The device of claim 19, wherein the optical signal is fluorescence intensity.

35. The device of claim 19, wherein the optical signal is FRET ratio.

36. The device of claim 19, wherein the optical signal is fluorescence lifetime.

37. The device of claim 19, wherein the detector is an electrical detector measuring an electrochemical signal generated by a change in distance between a redox reporter and a surface of an electrode caused by a target-binding induced conformational change of an analyte binding probe.

38. The device of claim 19, wherein the detector is detecting an enzymatic electrochemical signal measuring a change in enzymatic reaction rate.

39. The device of claim 19, wherein the analyte binding probes immobilized on a surface within the flow cell.

40. The device of claim 19, wherein the analyte binding probes are contained within a polymeric matrix within the flow cell.

41. The device of claim 19, wherein the analyte binding probe is attached to a bead or particle within the flow cell.

42. The device of claim 19, wherein the analyte is vancomycin.

43. The device of claim 19, wherein the analyte is methoxy.

44. The device of claim 19, wherein the device is used to measure pharmacokinetic and / or pharmacodynamic properties of the drug.

45. The device of claim 23, wherein the reference molecule is DHEA-S.

46. The device of claim 19, wherein the analyte measured is cortisol.

47. The device of claim 23, wherein the reference molecule included within the dialysate buffer is maltose.

48. The device of claim 19, wherein the dialysate fluid is recirculated through the device.

49. The device of claim 19, wherein the device contains a battery configured to supply power to the device for at least about 4 hours.

50. The device of claim 19, wherein the device comprises a re-usable part and disposable part.

51. The device of claim 50, wherein the reusable part comprises a pump, optical detector, and light source.

52. The device of claim 50, wherein the disposable part comprises a semi-permeable probe, dialysate, and flow cell containing analyte binding probes.

53. The device of claim 19, wherein a portion or the entirety of the dialysate buffer is spatially separated and stored periodically as fractions.

54. The device of claim 19, wherein the dialysate buffer is stored within tubes or vials.

55. The device of claim 19, wherein the dialysate buffer is stored within a length of tubing.

56. The device of claim 19, wherein the dialysate buffer is stored within a microfluidic chip.

57. The device of claim 53, wherein the fractions are separated into different containers.

58. The device of claim 53, wherein the fractions are separated by segments of air or gas.

59. The device of claim 53, wherein the fractions are separated by a valve.

60. The device of claim 53, wherein the fractions are separated by a size exclusion membrane.

61. The device of claim 53, wherein the fractions are separated by use of a fraction collector.

62. A device for sensing an analyte in a biological sample of a subject, comprising: a piercing element configured to pierce a body surface of a subject, to thereby bring the piercing element in contact with a biological sample, wherein the piercing element comprises an inlet and an outlet, and wherein the outlet is fluidically coupled to a sensing domain; and a pump fluidically coupled to the inlet; wherein the sensing domain is configured to receive a fluid from the piecing element, and wherein the sensing domain and the pump are provided adjacent to the body surface of the subject.

63. The device of claim 62, further comprising a second pump fluidically coupled to the outlet.

64. The device of claim 63, wherein the second pump provides a negative pressure through the outlet.

65. The device of any one of claims 62-64, wherein the pump provides a positive pressure through the inlet.

66. The device of any one of claims 62-65, wherein the pump provides a flow rate of fluid at from about 0.001 pL / min to about 10 pL / min.

67. The device of any one of claims 62-66, wherein the pump provides a flow rate of fluid at from about 0.05 pL / min to about 2 pL / min.

68. The device of any one of claims 62-67, wherein the sensing domain and the pump are provided on the body surface of the subject.

69. The device of any one of claims 62-68, further comprising a wearable housing, wherein the wearable housing comprises the sensing domain and the pump, and wherein the wearable housing covers at least a portion of the body surface of the subject.

70. A device for sensing an analyte in a biological sample of a subject, comprising: a piercing element configured to pierce a body surface of a subject, to thereby bring the piercing element in contact with a biological sample, wherein the piercing element comprises a semipermeable membrane comprising an inlet and an outlet, and wherein the outlet is fluidically coupled to a sensing domain; a dialysate buffer fluidically coupled to the inlet; and an optical reader optically coupled to the sensing domain, wherein the optical reader and the sensing domain are provided adjacent to the body surface of the subject.

71. The device of claim 70, wherein the dialysate buffer is sterile.

72. The device of claim 70 or 71, wherein the dialysate buffer comprises electrolytes in interstitial fluid of the biological sample.

73. The device of any one of claims 70-72, wherein the dialysate buffer comprises a reference molecule to determine the degree of equilibration between the dialysate buffer and the interstitial fluid.

74. The device of any one of claims 70-73, wherein the dialysate buffer comprises one or more molecules that alter an osmotic pressure between the interstitial fluid and the dialysate buffer.

75. The device of claim 74, wherein the one or more molecules that alter the osmotic pressure are albumin or large molecular-weight dextran.

76. The device of any one of claims 70-75, wherein the dialysate buffer comprises one or more molecules that promote collection of the analyte.

77. The device of any one of claims 70-76, wherein the dialysate buffer comprises an antiinflammatory compound.

78. The device of any one of claims 70-77, wherein the analyte is a lipophilic molecule.

79. The device of any one of claims 70-78, wherein the semipermeable membrane comprises pores that reduce a concentration of one or more molecules with a molecular weight of at least about 6 kDa, at least about 10 kDa, at least about 50 kDa, at least about 100 kDa, at least about 150 kDa, or at least about 200 kDa, from diffusing into the dialysate buffer compared to a semipermeable membrane without the pores.

80. The device of any one of claims 70-79, wherein the sensing domain comprises a microfluidic chip, wherein the microfluidic chip comprises one or more microfluidic channels.

81. The device of any one of claims 70-80, wherein the microfluidic chip further comprises one or more flow cells fluidically coupled to the piercing element.

82. The device of claim 81, wherein the one or more flow cells contain an analyte binding probe.

83. The device of claims 81 or 82, wherein the one or more flow cells are calibrated using a solution containing the analyte.

84. The device of any one of claims 81-83, wherein a flow rate of the one or more flow cells are adjusted at a scheduled frequency.

85. The device of any one of claims 81-84, wherein the flow rate of the one or more flow cells are adjusted according to a change of signal generated by the sensing domain.

86. The device of claim 85, wherein the change of signal generated by the sensing domain at various flow rates of the one or more flow cells are used for correcting or calibrating the device.

87. The device of claim 82, wherein the analyte binding probe is configured to provide a change in an emitted optical signal by the analyte binding probe when the analyte binding probe comes in contact with the analyte in the biological sample of the subject.

88. The device of any one of claims 70-87, wherein the analyte binding probe receives an excitation optical beam from the optical reader and emits the emission beam back to the optical reader.

89. The device of any one of claims 70-88, wherein the excitation optical beam is defocused at a plane of the sensing domain up to an area of 1 square mm.

90. The device of claim 70-89, wherein the excitation optical beam is defocused through the inclusion of material between the flow cell and the optoelectronic reader.

91. The device of claims 80 or 81, wherein a perfusate exiting from the microfluidic chip is collected in a container.

92. The device of claim 91, wherein the container is coupled to the sensing domain.

93. The device of claims 91 or 92, wherein a portion of the perfusate is collected in one or more containers.

94. The device of claim 93, wherein a size of the portion of the perfusate is from about 5 pL to about 50 pL.

95. The device of claim 93, wherein a size of the portion of the perfusate is from about 0.05 pL to about 500 pL.

96. The device of any one of claims 93-95, wherein the portion of the perfusate is collected in from about 1 minute to 24 hours.

97. The device of any one of claims 93-95, wherein the portion of the perfusate is collected in from about 5 minute to 6 hours.

98. The device of any one of claims 91-97, wherein the perfusate in the container is examined under a microscope.

99. The device of any one of claims 91-98, wherein the perfusate in the container mixes with an analyte binding probe that receives an excitation optical beam.

100. The device of any one of claims 96-99, wherein the piercing element is concentric with the semipermeable membrane.

101. The device of any one of claims 70-100, wherein the inlet and the outlet are located at one end of the piecing element, and the semipermeable membrane is located at the other of the piercing element.

102. The device of any one of claims 70-101, wherein the piercing element is disposed colinear with the semipermeable membrane.

103. The device of any of the preceding claims, wherein the sensing domain comprises non- molecular sensors.

104. The device of any of the preceding claims, wherein the non-molecular sensors comprise humidity sensors, motion sensors, temperature sensors, or a combination thereof.

105. The device of any one of claims 19-104, wherein the sensing domain comprises molecular sensors.

106. The device of any one of claims 62-105, wherein the sensing domain comprises an electrical detector measuring an electrochemical signal generated by a change in charge configuration near an electrode surface due to the addition or subtraction of charge due to analyte binding or target-binding induced conformational change of an analyte binding probe or a combination of these effects.

107. The device of any one of claims 62-105, wherein the sensing domain comprises an electrical detector measuring an electrochemical signal generated by a change in electron transfer rate between a redox reporter and a surface of an electrode caused by a target-binding induced conformational change of an analyte binding probe.

108. The device of any one of claims 62-105, wherein the sensing domain comprises an electrical detector measuring an electrochemical signal generated by a change in electron transfer rate between a redox reporter immobilized within a molecular pendulum construct and an electrode surface caused by analyte binding to the molecular pendulum which modulates mass and drag of the pendulum.

109. The device of any one of claims 62-105, wherein the sensing domain comprises an optical detector measuring a change in mobility of a particle generated by binding of an analyte to a receptor on the particle or microfluidic chip surface which modulates a binding interaction between analyte or receptors functionalized on both the particle and the surface and thus the degree of motion of the particle relative to the surface.

110. The device of any of the preceding claims, wherein the sensing domain is calibrated using a solution, wherein the solution produces a measurable fluorescent signal.

111. The device of any one of claims 70-110, wherein the optical reader comprises a light emitter.

112. The device of claim 111, wherein the light emitter is on a printed circuit board (PCB) and emits laser beam that is actively aligned with the optical reader.

113. The device of claim 112, wherein the active alignment is performed by registering an aperture array to reader mechanics that support an optical path of the laser beam.

114. The device of claims 112 or 113, wherein the active alignment is performed by optimizing excitation power through an aperture array that is registered to optical path of the laser beam.

115. The device of any one of claims 70-114, wherein the sensing domain and the optical reader are provided on the body surface of the subject.

116. The device of any one of claims 70-115, further comprising a wearable housing, wherein the wearable housing comprises the sensing domain and the optical reader, and wherein the wearable housing covers at least a portion of the body surface of the subject.

117. The device of claim 116, wherein a portion of the wearable housing is coupled to the body surface of the subject with an adhesive.

118. A device for sensing an analyte in a biological sample of a subject, comprising: a piercing element configured to pierce a body surface of a subject, to thereby bring the piercing element in contact with a biological sample, wherein the piercing element comprises a semipermeable membrane, wherein the piercing element comprises an inlet and an outlet, wherein the outlet is fluidically coupled to a sensing domain, wherein the sensing domain comprises a microfluidic chip comprising one or more flow cells fluidically coupled to the piercing element, and wherein the one or more flow cells contain an analyte binding probe; a pump fluidically coupled to the inlet, wherein the pump provides a positive pressure to push a dialysate through the inlet and to the semipermeable membrane; and an optical reader optically coupled to the sensing domain, wherein the analyte binding probe receives an excitation optical beam from the optical reader and emits the emission beam back to the optical reader.

119. A method for sensing an analyte in a biological sample of a subject, the method comprising: piercing a body surface of a subject, to thereby bring a piercing element in contact with a biological sample; pumping a dialysate buffer through an inlet of the piercing element; providing interstitial fluid of the biological sample in contact with the dialysate buffer, wherein a perfusate passes through the outlet of the piecing element;contacting the perfusate with a sensing domain and collecting the perfusate in an output reservoir, wherein the sensing domain is fluidically coupled to an outlet of the piercing element; collecting an emitted optical beam from the sensing domain via an optical reader, wherein the optical reader is optically coupled to the sensing domain; processing the emitted beam into electrical signal; and providing a user readable data.

120. The method of claim 119, wherein a recovery rate of the analytes is at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

121. The method of claims 119 or 120, further comprising a second pump fluidically coupled to the outlet.

122. The method of claim 121, wherein the second pump provides a negative pressure through the outlet.

123. The method of any one of claims 119-122, wherein the pump provides a positive pressure through the inlet.

124. The method of any one of claims 119-123, wherein the pump provides a flow rate of fluid at from about 0.001 pL / min to about 10 pL / min.

125. The method of any one of claims 119-124, wherein the pump provides a flow rate of fluid at from about 0.01 pL / min to about 2 pL / min.

126. The method of any one of claims 119-125, wherein the dialysate buffer is sterile.

127. The method of any one of claims 119-126, wherein the dialysate buffer comprises electrolytes in interstitial fluid of the biological sample.

128. The method of any one of claims 119-127, wherein the dialysate buffer comprises a reference molecule.

129. The method of claim 128, wherein a degree of equilibration between the dialysate buffer and the interstitial fluid is determined by comparing the reference molecule in the dialysate buffer to a reference molecule in the interstitial fluid.

130. The method of any one of claims 119-129, wherein the dialysate buffer comprises one or more molecules that alter an osmotic pressure between the interstitial fluid and the dialysate buffer.

131. The method of claim 130, wherein the one or more molecules that alter the osmotic pressure are albumin or large molecular-weight dextran.

132. The method of any one of claims 119-131, wherein the dialysate buffer comprises one or more molecules that promote collection of the analyte.

133. The method of any one of claims 119-132, wherein the analyte is a lipophilic molecule.

134. The method of any one of claims 119-133, wherein the semipermeable membrane comprises pores that reduce a concentration of one or more molecules with a molecular weight of at least about 6 kDa, at least about 10 kDa, at least about 50 kDa, at least about 100 kDa, at least about 150 kDa, or at least about 200 kDa, from diffusing into the dialysate buffer compared to a semipermeable membrane without the pores.

135. The method of any one of claims 119-134, wherein the sensing domain comprises a microfluidic chip, wherein the microfluidic chip comprises one or more microfluidic channels.

136. The method of any one of claims 119-135, wherein the microfluidic chip further comprises one or more flow cells fluidically coupled to the piercing element.

137. The method of claim 136, wherein the one or more flow cells contain an analyte binding probe.

138. The method of claim 137, wherein the analyte binding probe is configured to provide a change in an emitted optical signal by the analyte binding probe when the analyte binding probe comes in contact with the analyte in the biological sample of the subject.

139. The method of claims 137 or 138, wherein the analyte binding probe receives an excitation optical beam from the optical reader and emits the emission beam back to the optical reader.

140. The method of claim any one of claims 135-139, wherein a perfusate exiting from the microfluidic chip is collected in a container.

141. The method of claim 140, wherein the container is coupled to the sensing domain.

142. The method of any one of claims 119-141, wherein a portion of the perfusate is collected in one or more containers.

143. The method of claim 142, wherein a size of the portion of the perfusate is from about 5 pL to about 50 pL.

144. The method of claim 142, wherein a size of the portion of the perfusate is from about 0.05 pL to about 500 pL.

145. The method of any one of claims 142-144, wherein the portion of the perfusate is collected in from about 1 minute to 24 hours.

146. The method of any one of claims 142-144, wherein the portion of the perfusate is collected in from about 5 minute to 6 hours.

147. The method of any one of claims 142-146, wherein the perfusate in the one or more containers is examined under a microscope.

148. The method of any one of claims 142-147, wherein the perfusate in the one or more containers mixes with an analyte binding probe that receives an excitation optical beam.

149. The method of any one of claims 119-148, wherein the piercing element is concentric with the semipermeable membrane.

150. The method of any one of claims 119-149, wherein the inlet and the outlet are located at one end of the piecing element, and the semipermeable membrane is located at the other of the piercing element.

151. The method of any one of claims 119-148, wherein the piercing element is disposed colinear with the semipermeable membrane.

152. The method of claim 151, wherein the inlet is located at one end of the piercing element, and the outlet is located at the other end of the piercing element.

153. A device for sensing an analyte in a biological sample of a subject, comprising: a sensing domain that is a region, wherein molecular sensing occurs by having molecular sensors come in contact with an analyte carrying fluid, and that comprises of molecules that receives an excitation optical beam from a reader and emits the emission beam back to the reader, whereby, the emitted beam to the reader encodes the information on the concentration of the analyte that is being detected in the analyte carrying fluid; an apparatus that comprises the sensing domain and allows the interaction of the interstitial fluid with the sensing domain; an arrangement which allows the interaction of the interstitial fluid from the biological sample with the apparatus holding the sensing domain; an optical reader that is miniaturized, comprises an excitation optical beam that comes out of the optical reader and couples to the apparatus comprising the sensing domain and collects an emitted optical beam from the apparatus, processes the emitted beam into electrical signal and provides a user readable data; and, a structural arrangement which allows optical coupling between the optical reader and the apparatus holding the sensing domain.

154. The device of claim 153, wherein the apparatus comprises a microfluidic chip, wherein the microfluidic chip comprises one or more microfluidic channels.

155. The device of claim 154, wherein the optical reader and the microfluidic chip are in close proximity to each other and held together for the duration of the measurement.

156. The device of claim 154, wherein the microfluidic chip is comprised of areas that contain sensing domain and that are aligned with excitation and emission optical beam from and to the optical reader.

157. The device of claim 154, wherein the microfluidic chip along with the optical reader reside outside the human body.

158. The device of claim 154, wherein the microfluidic channels flow dialysate in it that is in contact with the areas of the sensing domain and interacts with it freely.

159. The device of claim 153, wherein the sensing domains have molecular sensors that are designed to sense the analytes carried by the dialysate.

160. The device of claim 153, wherein there are multiple sensing domains consisting of different molecular sensors designed to sense different analytes and placed along different parts of the microfluidic channels within the same microfluidic chip.

161. The device of claim 160, wherein the coupling of the optical beams from the optical reader to the areas of different sensing domains along the microfluidic channels allow simultaneous sensing of multiple analyte concentrations continuously.

162. The device of claim 153, wherein the arrangement that allows the interaction between the interstitial fluid and the apparatus holding the sensing domain is a pumped fluidic system such as a microdialysis unit.

163. The device of claim 162, wherein the microdialysis unit interacts with a microfluidic unit using tubing.

164. The device of claim 162, wherein the microdialysis unit comprises of a pump which is pumping a perfusate through a tubing that inserts into the interstitial fluid in the biological sample, the perfusate interacts and collects the analytes smaller than a certain size from the interstitial fluid and passes it out of the biological sample into the microfluidic chip using the tubing that connects the microdialysis unit to the microfluidic chip.

165. The device of claim 162, wherein the analytes collected from the interstitial fluid of the biological sample and pushed into the microfluidic channels of the microfluidic chip interact with the sensing domains in the microfluidic chip and transduce the optical signal that is coupled with the optical reader.

166. The device of claim 160, wherein the area containing the sensing domain is larger than the spot size of the probing optical beam from the optical reader.

167. The device of claim 160, wherein the area containing the sensing domain is uniform in its spatial density of the sensing molecule.

168. The device of claim 160, wherein the area containing the sensing domain is non-uniform in its spatial density of the sensing molecules.

169. The device of claim 166, wherein the smaller spot size than the area containing the sensing domain provides alignment tolerance between the reader and the microfluidic chip.

170. The device of claim 153, wherein the apparatus holding the sensing domain is a microneedle array, wherein there is a base of the array which holds at least one microneedle in it.

171. The device of claim 170, wherein the microneedle array is inserted into the biological sample and is in contact with the interstitial fluid of the biological sample.

172. The device of claim 170, wherein the microneedle is hollow and the sensing domain resides inside the microneedle.

173. The device of claim 153, wherein the arrangement that allows the interaction between the interstitial fluid of the biological sample and the sensing domain are holes at the tip of the microneedles.

174. The device of claim 173, wherein the holes are arranged near the tip along the circumference of the microneedles.

175. The device of claim 173, wherein the hole is the central lumen of the microneedle.

176. The device of claim 173, wherein there are holes both along the circumference and the central lumen.

177. The device of claim 172, wherein the sensing domain resides at the tip of the microneedles.

178. The device of claim 172, wherein there is a multitude of microneedles that have different sensing domains designed to sense different analytes.

179. The device of claim 178, wherein the multitude of microneedles containing different sensing domains are held together by a base to form a microneedle array that interacts with a probing optical beam from a reader.

180. The device of claim 179, wherein an optical reader is optically coupled to the microneedle array.

181. The device of claim 180, wherein the optical reader that is optically coupled to the microneedle array is abutting or in close proximity of the microneedle array.

182. The device of claim 181, wherein the microneedles of the microneedle array are at least partially inserted in the biological sample to be in contact with the interstitial fluid while the optical reader abutting the microneedle array and the base of the microneedle array reside above the skin of the biological sample.

183. The device of claim 179, wherein the microneedle and the optical reader system are designed to collect data for different target analytes in a multiplexed way.

184. The device of claim 153, wherein the optical reader consists of at least one light source, a light detector, an excitation beam path to carry light from the source to the output of the reader, an emission beam path to collect light from a device that is coupled with the optical reader and bring it to the light detector, beam shaping elements in both the excitation and the emission optical paths such as lenses to collimate or focus light, optical filters in both the excitation and emission paths, optical apertures for ensuring off-angle light rejection, electronics circuits to drive light sources and to process collected light, and a mechanical housing to hold the reader together.

185. The device of claim 184, wherein the optical reader consists of at least one laser source that is designed to emit light that in turn is designed to pass efficiently to the sensing domain and excite a light sensitive molecule in the sensing domain.

186. The device of claim 185, wherein the light sensitive molecule is a fluorophore that is attached to an aptamer switch and the aptamer switch is designed to bind to a target analyte and upon binding goes through a conformational change in its structure that modulates the light coming out of the fluorophore and coupling into the optical reader.

187. The device of claim 184, wherein the optical reader consists of at least one light detector that is designed to collect emitted light that in turn is emitted from the sensing domain and carries information on the concentration of the target analyte that the sensing domain is designed to sense.

188. The device of claim 187, wherein the light detector has integrated electronics that processes the light signal and converts optical into electrical signals.

189. The device of claim 188, wherein the light detector is an ambient light sensor that is made of silicon and is responsive to wavelengths from 400 nm to 1100 nm.

190. The device of claim 187, wherein the light detector is an image sensor integrated chip.

191. The device of claim 185, wherein the emitted light is at 630 nm and is emitted by a laser.

192. The device of claim 185, wherein the emitted light is at 680 nm and is emitted by a laser that may either be an edge emitter or a VCSEL.

193. The device of claim 192, wherein the light emitter at 680 nm is a VCSEL.

194. The device of claim 184, wherein there is at least one excitation beam path that consists of a light source, a collimating lens, a dichroic filter, apertures, and a focusing lens that focuses light out of the reader and into the sensing domain, henceforth sensing domainfocusing lens, wherein the focusing lens serves the dual purpose of focusing the light and collecting the emission light.

195. The device of claim 184 that comprises an emission path that entails a longer wavelength than the excitation wavelength and that partially overlaps with the excitation path in going through the same focusing lens and the dichroic filter, has its own focusing lens, henceforth detector focusing lens, that is located after the emission path passes through the dichroic filter that focuses light on a detector.

196. The device of claim 194, wherein the dichroic filter is designed to substantially reflect excitation light but to substantially transmit the emission light at a longer wavelength.

197. The device of claim 184, wherein the detector focusing lens distance from the detector is adjusted to obtain an appropriate spot size on the detector.

198. The device of claim 197, wherein the spot size is configured to target specific pixels or a multitude of pixels on the detector.

199. The device of claim 198, wherein some pixels have filters that make them more sensitive to either the excitation or the emission wavelengths.

200. The device of claim 199, wherein the pixels which allow the excitation light to pass through are used to monitor the background spurious light that contributes to reducing the dynamic range of the sensor.

201. The device of claim 184, that is configured to have multiple optical beams coming out of it by having a multitude of light emitters, multiple detectors, multiple beam paths for both excitation and emission, lenses, filters, and apertures, and wherein each beam interacts with a separate sensing domain allowing a functional ability to measure multiple analytes.

202. A system comprising a biosensor with an external reader.

203. A method for biosensing with an external reader.

204. The device of any one of claims 1-18, wherein the pump is fluidically coupled to the outer lumen of the probe.

205. The device of any one of claims 1-18, wherein the pump is configured to force a fluid through the inner lumen or the outer lumen of the probe through to the sensing domain.

206. The device of any one claims 1-18, wherein the probe comprises an open flow microperfusion probe.

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