Factory and in-vivo calibration of electrochemical aptamer sensors

Abstract

A method of making sensors with a plurality of aptamers and performing continuous measurements with the sensors is provided. The method involves fabricating a plurality of sensors where each sensor has at least one electrode with a plurality of aptamers attached to the electrode surface. A portion of the plurality of sensors is calibrated and a portion of the plurality of aptamer sensors is identified as being commercial quality ("the product portion"). At least one set of calibration data gathered from the calibration of the plurality of sensors is associated mathematically to the product portion.

Description

FACTORY AND IN-VIVO CALIBRATION OF ELECTROCHEMICAL APTAMER SENSORSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 531,047, filed August 7, 2023, and U.S. Provisional Application Serial No. 63 / 561,278, filed March 4, 2024, which applications are hereby incorporated by reference in their entirety.FIELD OF THE INVENTION

[0002] This invention relates generally to aptamer sensors, and more specifically to aptamer sensors which provide a calibrated measurement of at least one analyte.BACKGROUND OF THE INVENTION

[0003] This section is intended to introduce the reader to various aspects of art that may be related to various aspects of the present invention, which are described and / or claimed below. This discussion is believed to be helpful in providing the reader with background information to facilitate a better understanding of various aspects of the present invention. Accordingly, it should be understood that these statements are to be read in this light, and not as admissions of prior art.

[0004] Aptamer sensors can identify the presence and / or concentration of an analyte of interest via the use of an aptamer sequence that specifically binds to the analyte of interest. These sensors include aptamers attached to an electrode or to an optical surface, wherein each of the aptamers has a redox active molecule (redox tag) or fluorescent molecule and a quencher (optical tags) attached thereto. A redox couple can transfer electrical charge to or from the electrode, and a fluorescent molecule can provide photons to a photodetector. When an analyte binds to the aptamer, the aptamer changes shape, changing the electron transfer for the redox tag or the fluorescence of the optical tag. This results in a measurable change that can be translated to a measure of presence or concentration of the analyte. When used in this manner, aptamers are an example of an affinity -based biosensor. These aptamer sensors can be incorporated into a wearable or even an implantable biosensor with form factors and use case scenarios similar to continuous glucose monitors.

[0005] A major unresolved challenge for aptamer sensors and other affinity -based biosensors is calibration of the sensors. Techniques that have been proposed for ‘calibration free’ aptamer sensor operation recognize that methods are needed to overcome the very high sensor-to-sensor variability when fabricating sensors and significant sensor drift (accuracy degradation) with aptamer sensors while they operate continuously. Historically for aptamer sensors, a lack of highlyreproducible manufacturability, coupled with rapid degradation of aptamer sensors, has directed research and development teams to focus on methods to avoid prior calibration of sensors because such approaches would not provide adequate accuracy during use of the sensors in real-world applications such as wearable or implantable continuous molecular monitoring.

[0006] Aptamer sensors and other affinity -based biosensors have challenges related to calibration of the sensors for additional reasons. Aptamer sensors are distinct from prior art sensors and calibration schemes such as those used for continuous glucose monitors. Continuous glucose monitors have sensitivities (change in measurement vs. change in glucose concentration) that are highly dependent on factors such as electrode area or enzyme area and to diffusion rate through a protective membrane. While aptamer sensors also have a sensitivity, it is not necessarily highly dependent on factors such as electrode area or diffusion rate through a protective membrane. Therefore, aptamer sensors, for example, have different in-vitro and in-vivo calibration needs. For example, for glucose sensors, foreign body response can limit glucose flux to an electrochemical sensor and reduce the sensor current even if the glucose level in interstitial fluid is unchanged. However, for aptamer sensors, diffusion limiting factors affect lag times for analytes and, if the analytes change slowly in their concentration in the body, such changes in lag times may not have any meaningful effect on sensor accuracy. Furthermore, in-vivo calibration of aptamer sensors brings additional challenges not seen with glucose sensors. Aptamer sensors can measure analytes that are large enough, that have enough cellular receptor density in tissue, or which have lipophilicity or other factors that further challenge their in-vivo correlation between blood and interstitial fluid and therefore between blood and the aptamer sensor measurement. Therefore, aptamer sensors present a unique set of challenges that must be resolved for continuous monitoring applications, especially if economical batch calibration is to be achieved, and if the user of the device is to have minimum or ideally no required finger-prick calibration steps during use of the device. Furthermore, the degradation methods for aptamer sensors are unique compared to the degradation methods for glucose sensors and their degradation is often multi-modal in nature, presenting additional challenges when attempting to predict degradation such that sensitivity of the sensor can be adjusted in real-time to maintain a reasonably calibrated measurement.

[0007] Novel approaches for aptamer sensors which reduce or eliminate these drawbacks could provide significant benefits for enabling accurate monitoring devices for analytes beyond glucose.SUMMARY OF THE INVENTION

[0008] Certain exemplary aspects of the invention are set forth below. It should be understood that these aspects are presented merely to provide the reader with a brief summary of certain formsthe invention might take and that these aspects are not intended to limit the scope of the invention. Indeed, the invention may encompass a variety of aspects that may not be explicitly set forth below.

[0009] Many of the drawbacks and limitations stated above can be resolved by creating novel and advanced interplays of chemicals, materials, sensors, electronics, microfluidics, algorithms, computing, software, systems, and other features or designs, in a manner that affordably, effectively, conveniently, intelligently, or reliably brings sensing technology into proximity with biofluid and analytes.

[0010] Certain embodiments of the disclosed invention include sensors as simple individual elements. It is understood that many sensors require two or more electrodes, counter electrodes, reference electrodes, or additional supporting technology or features which are not captured in the description herein. Sensors measure a characteristic of an analyte. Sensors are preferably electrical in nature, but may also include optical, chemical, mechanical, or other known biosensing mechanisms. Sensors can be in duplicate, triplicate, or more, to provide improved data and readings. Sensors may provide continuous or discrete data and / or readings. Certain embodiments of the disclosed invention show sub-components of what would be sensing devices with more subcomponents needed for use of the device in various applications, which are known (e.g., a battery, antenna, adhesive), and for purposes of brevity and focus on inventive aspects, such components may not be explicitly shown in the diagrams or described in the embodiments of the disclosed invention. For example, electrochemical aptamer sensors typically require separate working, counter, and reference electrodes, but the present invention simply focuses its discussion on the working electrode which is coupled to the sensing transducing bioreceptor element in the form of a mixed monolayer of aptamers and blocking molecules.

[0011] In one aspect of the present invention, a method of making sensors with a plurality of aptamers and performing continuous measurements with the sensors is provided. The method involves fabricating a plurality of sensors. Each sensor has at least one electrode and the electrode has an electrode surface. Next, attaching a plurality of aptamers to the electrode surface. The aptamers have one or more attached redox tags. Also, the attached redox tags provide electron transfer with the electrode. Then, applying a blocking layer to the electrode surface. The blocking layer has a blocking layer surface and a plurality of pathways supporting the electron transfer between the electrode and the redox tags. Next, calibrating a portion of the plurality of sensors (“the calibration portion”), producing at least one set of calibration data. Then, identifying a portion of the plurality of aptamer sensors as being commercial quality (“the product portion”). The plurality of sensors is fabricated in a batch process. Also, at least one set of calibration data gathered from the calibration of the plurality of sensors is associated mathematically to the product portion. The product portion is capable of providing accurate or precise continuous measurement.

[0012] In one embodiment, the calibration portion and the product portion are the same portion. In another embodiment, the calibration portion and the product portion are the same portion and are one sensor. In one embodiment, the calibration portion comprises at least three sensors. In another embodiment, the calibration data gathered from the calibration portion is at least in part in-vitro data. In one embodiment, the calibration data gathered from the calibration portion is at least in part in-vivo data.

[0013] In another aspect of the present invention, a device for continuous measurement of at least one analyte in a test fluid is provided. The device includes one or more sensors made using the method described above. In one embodiment, the electrode area has a standard deviation less than a value selected from the group consisting of 0.02, 0.01, 0.05, and 0.02 pm2. In another embodiment, the electrode area has a standard deviation less than a value selected from the group consisting of 20, 10, 5, and 2% of electrode area. In one embodiment, the device also includes at least one time point for calibration during manufacturing of the device, wherein the time point is after sensor fabrication. In another embodiment, the device also includes at least one time point for calibration during manufacturing of the device, wherein the time point is after sensor shelfstabilization. In one embodiment, the device also includes at least one time point for calibration during manufacturing of the device, wherein the time point is after sensor sterilization.

[0014] In another embodiment, the calibration data is data collected over a period of time selected from the group consisting of 1, 3, 7, 10, and 14 days. In one embodiment, the calibration data is comprised of redox tag current for either a single time point or multiple time points versus time. In another embodiment, the calibration data is comprised of redox peak potential for either a single time point or multiple time points versus time. In one embodiment, the calibration data is comprised of oxygen reduction current either a single time point or multiple time points versus time. In another embodiment, the calibration data is comprised of redox tag density or total number of redox tags for either a single time point or multiple time points versus time. In one embodiment, the calibration data is comprised of electrical capacitance or electrical impedance for either a single time point or multiple time points versus time. In another embodiment, the calibration data is comprised of temporal or frequency response of redox tag current for either a single time point or multiple time points versus time.

[0015] In one embodiment, the calibration data is comprised of a chronoamperometric response for either a single time point or multiple time points versus time. In another embodiment, the calibration data is comprised of electrode surface area for either a single time point or multiple time points versus time. In one embodiment, the calibration data is comprised of titration response for either a single time point or multiple time points versus time. In another embodiment, the calibration data includes statistical data. In one embodiment, the continuous measurementcomprises a continuous measurement method selected from the group consisting of square wave voltammetry, continuous square wave voltammetry, kinetic differential measurement, calibration free measurement, impedance spectroscopy, differential pulse voltammetry, chronoamperometry, and combinations thereof.

[0016] In one embodiment, the calibration data includes at least one variable in-vivo parameter. In another embodiment, the calibration data includes at least one in-vivo correlation between interstitial fluid and blood. In one embodiment, the calibration data further comprises calibration data gathered during use of the product portion of the sensors. In another embodiment, the method also includes auto-calibration data gathered during use of the product portion of the sensors. In one embodiment, the method also includes auto-calibration data gathered during use of the product portion of the aptamer sensors, wherein the auto-calibration data is frequency or temporal response of the sensors gathered during use of the sensor.

[0017] In another embodiment, the method also includes auto-calibration data gathered during use of the product portion of the aptamer sensors, wherein the auto-calibration data is electrode surface area. In one embodiment, the calibration data further includes in-vivo calibration data gathered after or near the point of steady state concentration of an analyte in interstitial fluid. In another embodiment, the calibration data further includes a plurality of sets of calibration data gathered at a plurality of analyte concentrations. In one embodiment, the method also includes at least one measurement waveform that alters the electron transfer characteristics through the blocking layer and therefore further comprises at least a first stabilization period. The calibration data is at least in part comprised of at least one measurement of the first stabilization period. In another embodiment, the method also includes a plurality of waveforms wherein at least one of the plurality of waveforms alters the electron transfer characteristics through the monolayer and further comprising a second stabilization period that occurs after each application of at least one of the plurality of waveforms.

[0018] In one embodiment, at least one of the waveforms is an analyte measurement waveform, a sensor diagnostic waveform, or a sensor preserving waveform. In another embodiment, the plurality of waveforms is periodic. In one embodiment, the method also includes a calibration portion calibration portion with at <+ / - 2% standard deviation over a period of time selected from the group consisting of 7, 14, 21, 30, and 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version. In another embodiment, the method also includes a calibration portion calibration portion with at <+ / - 3% standard deviation over a period of time selected from the group consisting of 7, 14, 21, 30, and 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version. In oneembodiment, the method also includes a calibration portion calibration portion with at <+ / - 5% standard deviation over a period of time selected from the group consisting of 7, 14, 21, 30, and 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

[0019] In another embodiment, the method also includes a calibration portion calibration portion with at <+ / - 10% standard deviation over a period of time selected from the group consisting of 7, 14, 21, 30, and 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version. In one embodiment, the method also includes a calibration portion calibration portion with at <+ / - 20% standard deviation over a period of time selected from the group consisting of 7, 14, 21, 30, and 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version. In another embodiment, the method also includes a product portion with at <+ / - 5% accuracy over a period of time selected from the group consisting of 7, 14, 21, 30, and 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

[0020] In one embodiment, the method also includes a product portion with at <+ / - 10% accuracy over a period of time selected from the group consisting of 7, 14, 21, 30, and 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version. In another embodiment, the method also includes a product portion with at <+ / - 20% accuracy over a period of time selected from the group consisting of 7, 14, 21, 30, and 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version. In one embodiment, the method also includes a product portion of aptamer sensors with a change in a zero gain frequency that is less than at least one of <+ / -5%, <+ / - 10%, <+ / -20%, <+ / - 40%, <+ / -80% of the zero grain frequency over a period of time selected from the group consisting of 7, 14, 21, 30, and 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version. In another embodiment, the method also includes an oxygen reduction current measurable at a potential of -0.5V which increases by at least one of <10%, <20%, <50%, <100% of the initial oxygen reduction current over a period of time selected from the group consisting of 7, 14, 21, 30, and 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

[0021] In another aspect of the present invention, a method of creating a factory calibrated batch of aptamer sensors is provided. The method involves fabricating the aptamer sensors in a batch,then calibrating a portion of the aptamer sensors (“the calibration portion”), producing at least one set of calibration data. Next, identifying a portion of the aptamer sensors as being commercial quality (“the product portion”). Then assigning the calibration data to the product portion of the aptamer sensors. In one embodiment, the calibration data includes sensor response measured over multiple days. In another embodiment, the calibration data includes a single point calibration captured after a period of time selected from the group consisting of 1, 2, and 3 hours of sensor operation.

[0022] In one embodiment, the calibration data includes a single point calibration and a predicted calibration curve based on previous or historical measurement data. In another embodiment, the calibration data further comprises a measurement selected from the group consisting of aptamer density, frequency map, square wave voltammetry, cyclic voltammetry, intermittent pulseamperometry, chronoamperometry, continuous square wave voltammetry, electrical impedance, oxygen reduction current and combinations thereof. In one embodiment, the calibration data is modified using auto-calibration during use, and wherein the factory calibrated aptamer sensors further comprise a known or expected or predicted standard deviation for the sensor response; wherein auto-calibration occurs when the sensor response is changing greater than at least one of the standard deviation or twice the standard deviation of the sensor response In another embodiment, the auto-calibration shifts the frequency response utilized to identify at least one of the signal OFF frequency with maximum sensor response, the zero or non-responsive frequency, or the signal ON frequency with maximum response. In one embodiment, the calibration data includes a frequency response, and during use the device measures at least one of the following to recalibrate the frequency response: change in aptamer density, electrical impedance, oxygen reduction current. In another embodiment, the calibration data set is obtained in a human, only after or near the point of steady state of concentration of the analyte in interstitial fluid.

[0023] In one embodiment, factory calibration of the sensors is conducted immediately after fabrication of the sensors. In another embodiment, factory calibration of the sensors is conducted after shelf-stabilization of the sensors. In one embodiment, factory calibration of the sensors is conducted after attachment to electronics. In another embodiment, factory calibration of the sensors is conducted after sterilization. In one embodiment, factory calibration of the sensors is conducted at one or more time points and one or more measures to provide a sensor longevity calibration dataset. In another embodiment, factory calibration of the sensors is conducted at a single point and the calibration is captured at a time after at least one of 1 , 2, or 3 hours of operation.

[0024] In one embodiment, calibration data includes at least one measurement parameter. In another embodiment, the measurement parameter is sensitivity. In one embodiment, the sensitivity includes a plurality of different sensitivities for a plurality of different measurements.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The objects and advantages of the disclosed invention will be further appreciated in light of the following detailed descriptions and drawings in which:

[0026] FIG. 1 is a schematic of one embodiment of a device in accordance with principles of the present invention.

[0027] FIG. 2 is a schematic of one embodiment of a device in accordance with principles of the present invention.

[0028] FIG. 3 is a schematic of one embodiment of a device in accordance with principles of the present invention.

[0029] FIG. 4 is a schematic of one embodiment of a plurality of sensors in accordance with principles of the present invention.

[0030] FIG. 5A is an illustration of square wave voltammograms for redox tag currents for several different electrode areas.

[0031] FIG. 5B is an illustration of square wave voltammograms for redox tag currents for several different electrode areas, which have been normalized.

[0032] FIG. 6 is an illustration of a Langmuir-Isotherm curve of redox peak current vs. concentration with standard deviation.

[0033] FIG. 7 is a flow chart of a factory calibration process.

[0034] FIG. 8A is an illustration of a sensor reading over time.

[0035] FIG. 8B is an illustration of a sensor reading over time.

[0036] FIG. 9 is an illustration of a sensor reading over time that includes multiple manual recalibration events.

[0037] FIG. 10 is an illustration of a graph showing analyte measure over time.

[0038] FIG. 11 A is an illustration of a graph showing sensor response vs. square wave frequencies for 1 day.

[0039] FIG. 1 IB is an illustration of a graph showing sensor response vs. square wave frequencies for 3 days.

[0040] FIGs 12A-12D are a series of data sets showing the parameters used in Equation 1.

[0041] FIG. 13 A is an illustration of a graph showing analyte measured over time.

[0042] FIG. 13B is an illustration of a graph showing analyte measured over time.

[0043] FIG. 14 is an illustration of a graph showing analyte measured over time.

[0044] FIG. 15A is an illustration of a graph showing analyte measured over time.

[0045] FIG. 15B is an illustration of a graph showing analyte measured over time.

[0046] FIG. 16 is example data for Figure 15.

[0047] FIG. 17 shows example data for factory calibration.

[0048] FIG. 18 is a system level diagram of an example system.

[0049] FIG. 19 is a device level diagram of an example device.

[0050] FIG. 20 is a graph showing in-vivo data collected for a cortisol sensor inserted subcutaneously in a rat.

[0051] FIG. 21 is Equation 1.DEFINITIONS

[0052] As used herein, “continuous sensing” with a “continuous sensor” means a sensor that changes in response to changing concentration of at least one solute in a solution such as an analyte. Similarly, as used herein, “continuous monitoring” means the capability of a device to provide multiple measurements of an analyte over time. A “continuous measurement’ may also be used in this context.

[0053] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, pH, size, concentration or percentage is meant to encompass variations of ±20% in some embodiments, ±10% in some embodiments, ±5% in some embodiments, ±1% in some embodiments, ±0.5% in some embodiments, and ±0.1% in some embodiments from the specified amount, as such variations are appropriate to perform the disclosed method.

[0054] As used herein, the term “aptamer” means a molecule that undergoes a conformation or binding change as an analyte binds to the molecule, and which satisfies the general operating principles of the sensing method as described herein. Such molecules are, e.g., natural or modified DNA, RNA, or XNA oligonucleotide sequences, spiegelmers, peptide aptamers, affimers and other forms of affinity -based biosensors. Modifications may include substituting unnatural nucleic acid bases for natural bases within the aptamer sequence, replacing natural sequences with unnatural sequences, or other suitable modifications that improve sensor function, but which behave analogous to traditional aptamers. Two or more aptamers bound together can also be referred to as an aptamer (i.e., not separated in solution). Aptamers can have molecular weights of at least 1 kDa, 10 kDa, or 100 kDa.

[0055] As used herein, the term “sensing monolayer” means at least a plurality of aptamers on a working electrode, which may also include a plurality of molecules or mixtures of molecules that form a non-monolayer protective layer or monolayer protective layer of the surface, where the protection is from interferents, degradation, or other factors that can degrade accuracy and / or longevity of the sensor.

[0056] As used herein, a “protective membrane” refers to one or more layers or materials which protect a sensor blocking layer from fouling and is permeable to at least electrical charge transfer. A protective membrane may optionally also be selectively permeable to additional components in a test fluid such as, for example, at least one analyte, wherein the presence of the at least one analyte allows the sensor to operate properly while the protective membrane protects against performance reduction due to fouling, or some combination thereof.

[0057] As used herein, the term “continuous measurement” means data collected by a device recording a plurality of readings over a period of time during which the sensing occurs. A continuous measurement includes measurements ranging from constant to intermittent to periodic (e.g. every day from 5 AM to 10 AM).

[0058] As used herein, the term “analyte” means any solute in biofluid in or from the body which can be measured using a sensor. Analytes can be small molecules, proteins, peptides, electrolytes, acids, bases, antibodies, molecules with small molecules bound to them, DNA, RNA, drugs, chemicals, pollutants, or other solutes in a solution or fluid.

[0059] As used herein, a “sensor,” “sensing device” or “device” comprises at least one sensor based on at least one aptamer and at least one sample solution during use of the sensor. Devices can sense multiple samples and be in multiple configurations such as a device to measure blood, or a microneedle or in-dwelling sensor needle to measure interstitial fluid, or a device to measure saliva, tears, sweat, or urine sensor, or a device to measure water pollutants or food processing solutes, or other devices which measure at least one analyte found in a sample solution.

[0060] As used herein, a “redox current” or “redox signal” comprises the total redox current between the plurality of redox tags attached to aptamers on the sensor and the electrode for a given sensor, the electrode typically referred to as the working electrode, as measured using techniques such as square wave voltammetry, chronoamperometry, or other suitable methods. The redox current is measured as the amplitude of the faradic redox-tag peak current minus the background current amplitude outside the redox peak. Said in another way, if the measurement is a voltammogram from square wave voltammetry then than the redox current is the peak height of redox current as measured above the background current as if there were no redox tags.

[0061] As used herein, “signal gain” or “sensor response” comprises a change in the redox current or other type of signal due to a change in concentration of the analyte as measured using techniques such as square wave voltammetry, chronoamperometry, or other suitable methods.

[0062] As used herein, “background current” comprises the current measured that is not the redox current. Background current can be due to capacitive charging, faradic currents, oxygen reduction currents, other redox active species, etc.

[0063] As used herein, “normalized current” is measured current normalized to a value of current. For example, normalized to redox current at the beginning of testing of the sensor (such as t=Os the sensor is placed into test solution such as serum, or t=Os the collection of data begins even if the sensor was already in serum). Or, for example, normalizing the background current for a batch of sensors to a common value of background current for all the sensors.

[0064] As used herein, a “electron transfer rate” comprises the measured rate or time at which electrons are transferred between the redox tag and the working electrode.

[0065] As used herein, a “frequency response” refers to a change in redox current or sensor response measured from the device as a function of measurement frequency, such as the frequency used for a square wave voltammetry measurement. A change in frequency response can also be related to a change in the electron transfer rate. Frequency response can also be interpreted from a chronoamperometric curve such as that used in the measurement method of continuous square wave voltammetry.

[0066] As used herein, a “non-responsive frequency” is a measurement frequency where the redox current does not respond to an increase or decrease in concentration of the analyte. Non- responsive frequency response can also be interpreted from a chronoamperometric curve such as that used in the measurement method of continuous square wave voltammetry.

[0067] As used herein, “sensor accuracy” is the maximum difference that will exist between the actual value (which must be measured by a primary or good secondary standard) and the indicated value at the output of the sensor. The accuracy can be expressed either as a percentage of full scale or in absolute terms. Additional statistical methods, as needed, may be included to interpret accuracy.

[0068] As used herein, “test fluid” is interstitial fluid or a suitable proxy for the test fluid such as serum.

[0069] As used herein, “a calibration portion” of aptamer sensors is for a batch of sensors fabricated together or fabricated using the same or similar processes (also may be referred to as a batch), that after fabrication are tested for at least one measurement at the factory in-vitro or in- vivo to provide calibration data to improve use of the sensors during use as a product outside of the factory. After fabrication the testing may also be a continuous measurement, which in many cases may be preferred if the sensor degrades in a manner that is not highly predictable which may reduce accuracy during use of the sensor. Calibration may mean supporting accuracy and / or precision for the duration of use of the sensor, or other measures which help maintain the accuracy or precision of the sensors during use. For example, a sensor for a hormone may need only maintain precision in percent changes upward or downward in hormone levels instead of representing accurate absolute concentrations.

[0070] As used herein, “a product portion” of aptamer sensors is for a batch of sensors fabricated together or fabricated using the same or similar processes, that after fabrication are used as product by end-users for continuous measurement. The term product does not necessarily require commercial sale and generally is only limited to use of the sensors outside of the factory in applications. Precision is at least in part determined by standard deviation and hence standard deviation is important to both accuracy and precision.

[0071] As used herein “calibration data” could be a single data point, a curve, multiple data points, a code, an algorithm, a formula, or any other mathematical correction factor or other type of corretion that enables factory calibration of a sensor. Calibration data can be represented, stored, or used in numerous ways, including for example an adjustable resistor or capacitor, so long as it satisfies the purpose of enabling factory calibration of the sensor.

[0072] As used herein, “electrode area” is the area of a working electrode that is electrochemically active and which can therefore support redox electron transfer. A rough or porous gold electrode can have an electrode area that is multiple times greater than the observable linearly calculated area from electrode length times width, or 3.14 times radius squared, or other measurement.

[0073] As used herein, “batch process” means a process used to fabricate or manufacture a plurality of sensors using the same or a similar process such that the sensors operate the same or similarly during use. The same or similar operation may include accuracy, precision, degradation, or other measures during use.

[0074] As used herein, “sensor shelf-stabilization” means any methods, materials, designs, or other elements or features used to preserve a sensor to reduce degradation during the period between manufacturing of the sensor and end-use of the sensor. For example, this period may include time stored in a warehouse, or on a delivery truck, or in a commercial store such as a pharmacy.

[0075] As used herein, “sensor sterilization” means any methods, materials, designs, or other elements or features used to sterilize a sensor before its end use. For example, sensor sterilization may include electron-beam, gamma irradiation, steam, UV-radiation, heat, ethylene oxide, or other suitable methods.

[0076] As used herein, “auto-calibration data” means data collected during use of the sensor which is used to improve, maintain, correct, alert on, or otherwise have impact on the accuracy, precision or other performance feature of the sensor. For example, auto-calibration data may include frequency or temporal response to find a zero or non-responsive frequency that may change during use (the collected auto-calibration data is used for auto-calibrating the measurement and / or reported data). For example, auto-calibration data may include a direct or indirect measurement of electrode surface area such as electrical capacitance or a cyclic voltammogram to collect allredox charge and calculate the number of aptamers with redox tags on the sensors. For example, auto-calibration data could also provide an alert to the user or into stored data that the sensor has lost accuracy or precision or other performance features. For example, auto-calibration data could be used to continually report a direct measure or estimate of the accuracy, precision, or other performance feature of the sensor during its use (the collected auto-calibration data is autocalibrating the reporting of the performance of the sensor).DETAILED DESCRIPTION OF THE INVENTION

[0077] One or more specific embodiments of the present invention will be described below. In an effort to provide a concise description of these embodiments, all features of an actual implementation may not be described in the specification. It should be appreciated that in the development of any such actual implementation, as in any engineering or design project, numerous implementation-specific decisions must be made to achieve the developers’ specific goals, such as compliance with system-related and business-related constraints, which may vary from one implementation to another. Moreover, it should be appreciated that such a development effort might be complex and time consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure.

[0078] Certain embodiments of the disclosed invention show sensors as simple individual elements. It is understood that many sensors require two or more electrodes, reference electrodes, or additional supporting technology or features which are not captured in the description herein. Sensors can be arranged in duplicate, triplicate, or more, to provide improved data and readings. Sensors may provide continuous or discrete data and / or readings. Certain embodiments of the disclosed invention show sub-components of what would be sensing devices with more subcomponents needed for use of the device in various applications, which are known (e.g., a reference or counter electrode, a battery, antenna, adhesive), and for purposes of brevity and focus on inventive aspects, such components may not be explicitly shown in the diagrams or described in the embodiments of the disclosed invention. All ranges of parameters disclosed herein include the endpoints of the ranges.

[0079] Unless otherwise stated, like numerals may refer to like features across the figures presented herein.

[0080] With reference to FIG. 1, in embodiments of the present invention a device 111, 113 or 115 are configured to measure at least one analyte in the body in interstitial fluid or blood, and are configured on skin 12 (111, 113) or implanted in the body (115). Each device has a housing 110, 112, 114, which may contain electronics and other required components for a sensing device. Each device has a sensor 100, 102, 104. In the case of sensor 100, the sensor is inserted into the skin.In the case of sensor 102, the sensor 102 is coupled to biofluid 14 in the skin via porous or hollow microneedles 190. Sensors may be electrochemical or optical based sensors such as aptamers or other affinity -based sensors. Biofluid 14 may be interstitial fluid, blood, or another type of biofluid such as cerebrospinal fluid or other biofluids.With reference to FIG. 2, where like numerals refer to like features, in embodiments of the present invention a sensor 200 like that in FIG. 1 (e.g. 100, 102, 104) carries a plurality of aptamers 224 and uses an electrochemical measurement of sensor signal. An example aptamer 224 sequence is the aptamer for vancomycin (identified as “SEQ ID NO. 1” below) with a carbon thiol linker on the 5' end for attachment to the electrode 220 and methylene blue redox tag 270 on the 3’ end. Embodiments of the invention may include other aptamers known to persons having ordinary skill in the art than those aptamers disclosed here, either as a substitution for or in addition to the vancomycin aptamer disclosed above. Embodiments of the invention may include other aptamer tagging schemes such as dual reporter tagging to correct for sensor drift, tags with different redox potentials, two or more tags that can provide redox quenching, or other suitable tags, methods of tagging, and methods of measuring the tags in response to changing analyte concentrations. An example working electrode 220 is gold and carries the aptamers 224 and a protective monolayer or blocking layer 222. In different embodiments, the protective monolayer or blocking layer 222 is mercaptohexanol or mercaptooctanol. The sensor 200 may further comprise a protective membrane or other protective material (not shown) such as polybetaine. The aptamers 224 and blocking layer 222 together form a sensing monolayer. When analyte 280 binds to the aptamer 224 the aptamer changes shape and the availability of the redox tag 270 for electron transfer with electrode 220 is altered, resulting in a sensor response. As illustrated in FIG. 2, increasing analyte 280 will result in an increase in redox current for the sensor response. However, the present invention is not so limited to this specific example and may include decreasing redox current or other sensor responses to increasing analyte 280. Such sensors can be fabricated and measure the target analyte for a week or more reliably.

[0081] With reference to FIG. 3, where like numerals refer to like features, in embodiments of the present invention a sensor 300 like that in FIG. 1 (e.g. 100, 102, 104) carries a plurality of aptamers 324 and uses optical measurement of sensor signal. The aptamers 324 carry a fluorescent tag 370 and a fluorescent quencher 372. Alternate fluorescent tags are possible including those that have resonance transfer, changes in ratios of fluorescent tags, changes in fluorescence lifetime, and other fluorescent tags that can, with the aptamer 324, result in an optical signal that changes in response to changing concentrations of analyte 380. Fluorescent tag 370 is excited by photons of light 390 in waveguide 350 which may be a glass or polymer fiber or any other suitable lightguiding material and geometry. When analyte 380 binds to aptamers 324, the aptamer changesshape and changes the fluorescence of fluorescent tag 370 when excited by source light 390, and the fluorescent emitted light is coupled into waveguide 350 and propagates to an optical detector (resulting in a signal gain or sensor response). Such sensors are similar to molecular beacons used in assays. They can be fabricated and used to measure the target analyte continuously.

[0082] With reference to embodiments of the present invention, FIGs 2 and 3 are non-limiting examples of aptamer sensors. They are provided to illustrate examples of the types of aptamer sensors that will be further presented in embodiments of the present invention.Factory Calibration - Fabrication and Characterization Methods

[0083] With reference to FIG. 4, where like numerals refer to like features, in embodiments of the present invention, a plurality of sensors 400 can be fabricated in a batch on a substrate 416. In one embodiment, the substrate can be a 125 pm plastic sheet such as PET or Kapton. For example, electrochemical sensors 200 can be batch fabricated by depositing and patterning titanium as an adhesion layer and then gold as a working electrode surface 220. If desired, an electrical insulator can be deposited and patterned, such as SU-8 or dielectric inks used in electrochemical sensor test strips. This is followed by incubation of aptamers 224 and blocking molecules 222 and coated with a protective hydrogel such as UV-cured polybetaine and optionally preserved with a coating of trehalose for dry storage. These example methods and designs for fabrication of sensor 400 are examples only and non-limiting to the present invention. The sensors 400 can then be cut from the substrate 416 using a laser, razor blade or other tooling and then attached to devices such as those illustrated in FIG. 1. Alternatively, sensors can be fabricated individually if desired, although likely resulting in increased manufacturing cost. Sensors may also be optical in nature as taught for FIG. 3. For example, waveguide 350 could be a planar waveguide with a titanium adhesion layer on the PET followed by an optical cladding coating of silicon dioxide (refractive index n=1.46) or cytop fluoropolymer (n=1.34) with adhesion promoter followed by coating with polymethylmethacrylate (n=1.49) or SiON (n=1.8) as the waveguide core and then coated with aptamers 324 and additional chemistry (such as antifouling chemistry, hydrogels, etc.) as needed for individual applications. For optical sensors, the term ‘current’ as used herein can be exchanged with the term ‘signal’ or ‘intensity’ which measure photons of light instead of electrons.

[0084] With reference to FIG. 5A in embodiments of the present invention, where like numerals refer to like features, a plurality of sensors are fabricated in a batch and at least 3 sensors from the batch are set aside for factory calibration measurements. For electrochemical sensors as illustrated in FIG. 2, FIG. 5A further illustrates square wave voltammograms 560, 562, 564 for the redox tag 270 currents vs. background current 560a, 562a, 564a. Methylene blue typically has a redox peak current near -0.3V vs. an Ag / AgCl reference electrode. As shown in FIG. 5A, typically sensorsfrom batch fabrication have differing background currents 560a, 562a, 564a due to differing total electrode 220 surface areas or other factors. This variable background current can be addressed in one or more ways. For example, photolithography of a high-precision electrical insulator, which may be a photo-definable polymer such as SU-8, on vacuum deposited gold can be used to achieve electrode areas with a target of 317 pm x316 pm or 0.1 pm2with a standard deviation less than at least one of 0.02, 0.01, 0.05, 0.02 pm2or expressed as a percentage 20, 10, 5, or 2% of electrode area. Alternate methods include laser ablation or other suitable electrode patterning methods. Alternately, as shown in FIG. 5B, the background currents that existed in FIG. 5A are optionally first mathematically normalized to a common value 561a or referred to as ib. The factory calibration process may then proceed as illustrated in FIG. 7. As non-limiting examples, the factory calibration process may commence immediately after fabrication, or after shelf-stabilization with a material such as trehalose, or after attachment to electronics, or after sterilization, all of which can impact the sensor response after fabrication. For example, shelf-stabilization with trehalose, and e-beam sterilization can each cause a loss of 10-50% sensor response.

[0085] In various embodiments of the present invention, the factory calibration process may next include one or more of the following steps:(1) minimizing differences in background currents via high-precision electrode areas during fabrication, or by normalizing the sensor background currents (561a) as illustrated in FIG. 5B. The background currents can then be statistically merged and interpreted as a representative voltammogram 561 (such as a statistical mean). If electrode areas are highly reproducible and sensing monolayers highly reproducible across the batch of sensors, then such normalization may not be required;(2) titrating a calibration portion of the sensors over a plurality of concentrations to capture a portion of the sensor response vs. analyte concentration in a test fluid such as human serum at 33 degrees Celsius (or other temperatures such as 35 or 37 degrees Celsius), which increases or decreases the redox peak current as illustrated in 561b, 561c and can then be plotted as a statistically analyzed (such as mean) Langmuir-Isotherm curve 600 of redox peak current vs. concentration 663 with standard deviation 663a as shown in FIG. 6. Unlike exogenous analytes (e.g. drugs) for endogenous analytes (e.g. insulin) the serum may need to be depleted of the analyte using enzymes, antibody capture, denaturization, or other suitable methods;(3) storing the curve FIG. 6 as a calibration curve or data set or algorithm or other suitable calibration feature or element, using methods such as digital archival storage;(4) assigning a common code from the batch, and associating that code with a saved calibration curve from the calibration portion sensors;(5) providing the product portion of the sensors to users. When the sensor is activated for use, the electronics worn by the user, the reader or sensor electronics, or software or firmware or memory already has the code or calibration data set. Alternately the code is entered or provided and the calibration data set is accessed as needed from the cloud, a reader, or other suitable storage location for the calibration data set. Alternately, the calibration data could be loaded onto the devices at the factory and later utilized during use of the device for measurements. Alternatively, the software uploads the calibration curve without a code if the fabrication of sensors in manufacturing has a very tight standard deviation. Alternately the calibration data exists and is used in the cloud. Alternately the calibration data exists and is used in another computing device other than the sensor or the sensor reader. Other calibration methods that can access and utilize the calibration data set, algorithms, or other calibration feature or elements even if not explicitly mentioned here are suitable. The calibration data is then ready to be used to maintain sensor accuracy as will be illustrated later in examples such as FIG. 13;(6) measuring analyte concentrations in the body using the product portion of the sensors (FIG. 1). The raw sensor signal is calibrated via the calibration data or curve into a precise or accurate measurement of the analyte. For example, if the raw sensor signal redox peak is 3.3 pA, and the calibration curve provides that 3.3 pA corresponded to 53 pM vancomycin, then the measurement will then be set to report 53 pM vancomycin. Based on the standard deviation 663a or other suitable data, the sensor may also report an accuracy, confidence, or other statistical measure relevant to the measurement.(7) lastly, reporting the calibrated measurements from the sensor to the cloud, user, doctor or other end location for the calibrated measurements.

[0086] In one embodiment, the present invention involves a method of creating a factory calibrated batch of aptamer sensors. The method involves fabricating multiple sensors in a batch or with similar methods. Then, creating calibration data from at least 3 sensors comprising a calibration portion. Next, assigning the calibration data to the product portion of the sensors. Finally, using the calibration data during at least 0.5, 1, 3, 7, 10, or 14 days of measurement with the remaining sensors to report a quantified measurement of at least one analyte.

[0087] The above example is a single point factory calibration. For most forms of aptamer sensors, the sensor characteristics also change over time during operation. Two non-limiting examples are shown in FIGs 8 A and 8B. These examples can be representative of redox current, normalized redox current, kinetic differential measurements, calibration free measurements (as will be discussed later in FIG. 13), other measures, or could be other measurable responses such as chronoamperometry which measures changes in time for redox electron transfer instead of magnitude of redox current. In FIG. 8A, which represents an example with a 6-carbon linkedmonolayer and a polybetaine protective membrane, fouling by solutes such as albumin is suppressed and during initial operation the sensor redox peak current 865a increases as the gold surface and monolayer remodels during early (hours) of electrochemical measurement. Longer term (days), typically the redox peak current gradually decreases as the sensing monolayer is lost from the sensor due to desorption. In FIG. 8B, which represents an example with an 8-carbon linked mercapto-octanol blocking layer with weak or minimal hydrogel protection, fouling by solutes such as albumin occurs rapidly. During initial operation, the sensor redox peak current 865b decreases as foulants impede or alter redox electron transfer. Longer term (days), typically the redox peak current also gradually decreases as aptamer is lost from the sensor due to desorption or as fouling continues to occur over time. Therefore, in embodiments of the present invention, the calibration process described for FIG. 7 can be repeated at one or more time points and one or more measures to provide a sensor longevity calibration dataset comprising part or all of curves 865a, 865b, which can further be used to enable accurate measurements over longer term operation of the sensors. An illustrative example is later taught for FIG. 13 which similarly applies to FIG. 8 and other embodiments of the present invention. Therefore, the present invention includes a method where the calibration includes the sensor response over at least one of 1, 3, 7, 10, or 14 days. Alternately, a single point calibration could be captured, after 1, 2, or at least 3 hours, which is suitable for sensors that are highly stable long-term but which have a significant initial change in response. In additional embodiments of the present invention, sensors may have highly variable initial responses but predictable longer-term responses (rates of degradation or change in sensor response). In this case, a single point calibration can be used to initially calibrate the sensors, and the longer-term response of the sensors need not be calibrated for every batch of sensors because it is highly predictable, and as a result, sensor calibration includes both a factory calibrated batch calibrated curve and a predicted calibration curve based on previous or historical measurement data.

[0088] With respect to embodiments of the present invention, there are other factors that affect sensor calibration and accuracy and which can be measured at the time of factory calibration. These factors include: electrochemically active electrode surface area, aptamer density (measured by total redox charge in a cyclic voltammogram), frequency map, measures such as square wave voltammetry, cyclic voltammetry, intermittent pulse-amperometry, chronoamperometry, continuous square wave voltammetry, electrical impedance where lower or resistance or higher capacitance indicate a more porous blocking layer 222, effects of variable pH and salinity, effects of temperature, oxygen reduction current beyond -0.3 V vs. a Ag / AgCl reference electrode, and other measures.Calibration Free Operation vs Factory Calibration

[0089] In the context of the present invention, there is a difference between ‘calibration -free’ operation and ‘factory calibration’. Calibration-free methods, as will be detailed in later sections, are dependent on electron transfer characteristics between a redox tag and a working electrode. In reality, calibration free methods benefit from factory calibration, because electron transfer characteristics for the sensor, and other factors, will change over time during in-vivo operation for days or weeks in duration. In combination with factory calibration, the purpose of calibration-free techniques arguably changes. It is no longer to be the method to maintain sensor accuracy, but rather to simply provide superior data that feeds into the factory calibration model, formula, algorithm, or method that then, in turn, maintains sensor accuracy. Any calibration method is only as good as the data fed into it. For example, a calibration method that relies only on change in absolute redox peak current measured would not be optimally accurate, because absolute current has significant variability that reflects much more than just the response of aptamers to changing analyte concentration. Conversely, square-wave voltammetry when used with multiple frequency or continuous square wave operation can extract superior data that has less variability, and which is more dependent on the response of aptamers to changing analyte concentrations. Ultimately, some of the leading factors that must be ‘calibrated for’ are temperature, concentration, in some cases pH or salinity, and sensor response to waveforms, and how these all change with time. Calibration-free methods do not account for all the ways that the sensor changes with time, nor were they originally developed or intended to account for all the ways that the sensor changes with time. This is especially true, because at least in the field of aptamer sensors, it was only very recently that sensors have been fabricated with enough longevity that longer term changes in sensor function and performance could be observed for the first time.

[0090] With reference to embodiments of the present invention, the following measures are nonlimiting examples of measures that may be included for factory calibration as either a one-time measurement for batch calibration or multiple time points, including time periods up to 2 weeks of operation or more:

[0091] A) Redox tag current at a single time-point or vs. time, and at multiple analyte concentrations or measurement parameters (such as square wave frequency), measured as redox peak vs. baseline as described in FIG. 5B.

[0092] B) Redox tag peak current potential at a single time-point or vs. time. Knowing the voltage location of the peak redox potential informs not only the scanning window, but also can be a measure of sensor degradation, For example, the redox peak can shift to larger potentials with sensor fouling. In another example, as a protective monolayer such as mercaptooctanol is lostredox gage peak potential can shift to smaller potentials, or for example, as a reference or counter electrode changes or degrades over time.

[0093] C) Oxygen reduction current at a single time-point or vs. time, measured for example at - 0.5 V as an increase in background current. As oxygen reduction current increases it can be evidence that the protective monolayer is degrading or becoming more porous which can shift the frequency response by allowing more rapid electron transfer rates. It can also alter interpretation of baseline current vs. redox peak current as the baseline is no longer flat.

[0094] D) Redox tag density or total number of redox tags at a single time point or vs. time, measured, for example, using cyclic voltammetry which integrates the measured redox charge and which then, with a known electrode surface area, gives redox tag density and therefore aptamer density. If aptamers in redox currents desorb over time they can give a false signal decrease that is not due to less analyte but simply due to less redox tags being available for electron transfer.

[0095] E) Electrical capacitance or impedance at a single time point or vs. time, which can give a measure of fouling and / or protective monolayer degradation, both of which for example can shift frequency response for a sensor or the sensor response. Electrical capacitance can be measured using simple electrical impedance methods or can be determined from the capacitance-dominated current measured less than a ms or less than several ms after a square wave pulse is applied.

[0096] F) Frequency response, electron transfer rates, sensor response at a time point sampled during continuous square wave, or other types of temporal or frequency response of redox tag current at a single time point or vs. time which directly affects sensor accuracy as described herein.

[0097] G) Chronoamperometric response of redox tag current at a single time point or vs. time, which not only affects chronoamperometric measurement but also the accuracy of the sampling points chosen for continuous square wave voltammetry.

[0098] H) Electrode surface area, also known as electrochemically active surface area, may be measured at the time of manufacturing. For gold electrodes, a cyclic voltammogram can be measured in sulfuric acid to calculate the gold electrode surface area from the charge passed in the gold oxide reduction peak. This measurement of electrode surface area is typically performed before addition of the sensing monolayer. Other methods for measuring electrode surface area after addition of the sensing monolayer may include, for example, electrical capacitance, or cyclic voltammetry to obtain the total aptamers and redox tags on the sensor surface, which if deposited with tight standard deviation across a batch of sensors will be representative of the electrode surface area (more electrode surface area = more total number of aptamers and redox tags = more total charge transfer measured during a cyclic voltammetry cycle).

[0099] I) Sensor response vs. analyte concentration, also known as a titration response at a single time point or vs. time, may be measured using cyclic voltammetry, differential pulse voltammetry,square wave voltammetry, continuous square wave voltammetry, kinetic differential measurement between ‘signal on’ and ‘signal off sensor response as illustrated in FIG. 11, two or more frequencies as described in FIGs 11A-11B and FIGs 12A-12D, chronoamperometry, or other suitable methods. It is generally important to calibrate sensor response against the change in redox current using a single frequency (or timing sample point for continuous square wave voltammetry) or two or more frequencies or sample points (also called kinetic differential measurement) and use the batch calibration data to correct for the change in redox current over time. Additionally, the frequency response may shift over time as illustrated in FIGs 11 A and 1 IB and be corrected for as well via calibration measurement and then during use of the sensor.

[0100] J) Standard deviation, precision, accuracy, or other statistical methods for any of the abovedescribed measures can be performed to allow the sensing system to report to end users, to databases, or otherwise report the ongoing precision, accuracy, or other statistical information important to determine confidence in the data being measured by the sensors.

[0101] Additional factory calibration measures may include a variable in-vivo parameter, such as salinity, temperature, or pH. However, if those measures are used for calibration, they must include a suitable measurement method or a dedicated sensor for salinity, temperature, or pH on the sensor itself during use. If temperature, pH, or salinity are not known then their effects as they may change during operation on sensor response cannot be corrected for during sensor operation.

[0102] All of the above measure data can be translated into formulas, algorithms, look up tables, correction factors, data sets, or other suitable methods that then allow the sensor measurement, accuracy, precision, or other measures related to sensor measurement and operation to correct for sensor drift, degradation, changes, or other factors that reduce confidence in the measured data.

[0103] Factory calibration may further include blood correlation to interstitial fluid for a particular analyte, especially for large analytes that can be more dilute in interstitial fluid than in blood. For example, if the analyte is a protein, for example cytokine that has an interstitial fluid concentration that is only 50% of blood levels, then factory calibration could involve limited testing on humans to determine this 50% dilution. Then users could be provided a corrected or calibrated reading of 2X the factory calibrated measurement value tested in-vitro in human serum where all of the added cytokine used to determine sensor response would be available to the sensor (unlike in the body with interstitial fluid). Practically, such a blood to interstitial fluid correlation could, but would unlikely, be performed with every batch of sensors created and would be determined with a limited set of human subjects as little as one time. For example, end-users of the devices could be used who also have blood draws analyzed and such data fed back into the calibration algorithms over time.

[0104] Factory calibration may further include potentiostat (electronics) specific parameters that differ from sensor type to sensor type (e.g. protein vs small molecule sensors, long vs. short aptamers), differ during the measurement of sensors during in-vivo use, differ over time due to changes in the sensor, or for other reasons and uses. For example, a typical set of available parameter ranges for a potentiostat with square wave voltammetry or continuous square wave voltammetry may be:

[0105] Initial Potential (V) from -10 - +10 V, for example -0.05V,

[0106] Final Potential (V) from -10 - +10 V, for example, -0.50V,

[0107] Potential Step (V) from 0.001 - 0.05 V which is the staircasing voltage increment, for example 1 mV, or 5 mV,

[0108] Amplitude (V) from 0.001 - 0.5 which is the square wave amplitude, half peak-to-peak, for example 35 mV,

[0109] Frequency (Hz) from 1 - 100000 which is the square wave frequency, for example 10Hz, or 40Hz, or 300 Hz,

[0110] Quiet Time (sec) from 0 - 100000 which is the quiescent time before potential scan, which can be no time to any amount of time required,

[0111] Sensitivity (A / V) from 10'12- 0.1 which is the sensitivity scale used during redox current measurement (sampling), for example ~10'4to 10'7A / V for a working electrode with ~mm2area,

[0112] Measurement width (sec), which is the duration of time over which the redox current measurement (sample) is collected and averaged (if applicable), for example a single data point, 5 data points, or other number of data points or durations of measurement.

[0113] There are similar or other parameters commonly known for chronoamperometry, cyclic voltammetry, or other measurement techniques which do not need to be explicitly listed to be included within embodiments of the present invention.

[0114] Many commercial products such as glucose monitors do not require factory calibration of measurement parameters like those listed above because there is no need to adjust them. However, aptamer sensors present several unique cases where factory calibration data includes at least one measurement parameter. For example, without fouling protection aptamer sensors directly exposed to blood or interstitial fluid can change their redox current change by about 10X during the first several hours of use, suggesting that a sensitivity change of 10X would be beneficial to maintaining the highest precision of redox current measurement and ultimately the highest accuracy. Therefore, the optimal sensitivity could be measured at the factory during calibration and utilized as taught herein to adjust the measurement sensitivity during use. For example, starting with a sensitivity of 10'6and non-linearly adjusting it in between scans down to a sensitivity of 10'7over the first 6 hours of operation. As another example, an aptamer sensor for lactate can use a verysmall aptamer with an allowable aptamer density on the electrode that is 10X greater than an aptamer sensor for a protein, which has a much larger aptamer. Also, due to the size of the protein it is more easily overcrowded on the sensor surface. Therefore, the sensitivity for lactate sensing could be 10X less sensitive than the sensitivity for the protein, because the lactate sensor could have 10X more redox current. For example, during continuous square wave voltammetry (see next section for more details) multiple redox current measurements are sampled at time points such as 3 ms and 100 ms during which the redox current magnitude differs by approximately 10X. Therefore, the ideal measurement sensitivity would be 10X less sensitive for the 3 ms measurement vs. the 100 ms measurement. For example, factory calibration data includes at least one measurement parameter that is measurement sensitivity and the measurement sensitivity includes a plurality of different sensitivities for a plurality of different measurements. In practice, implementing a plurality of sensitivities can be achieved by programming the electronics to adjust the sensitivity during a measurement, such as using a first sensitivity for redox current sampled at 3 ms, then increasing sensitivity by 10X for the 100 ms sampled measurement. Alternately, in practice one could perform multiple measurements, such as measuring redox currents at 3 ms for a first square wave voltammetry measurement scan, then measuring redox currents at 100 ms for a second square wave voltammetry measurement scan where the sensitivity is 10X more sensitive for the second measurement scan than for the first measurement scan.Factory Calibration - Recalibration During Use

[0115] With reference to FIG. 9 in embodiments of the present invention, a sensor may be manually calibrated (or ‘recalibrated’) at the point of care or point of use (the calibration data is modified using manual calibration at the point of use). For example, as illustrated in FIG. 9, consider the non-limiting example of an NT-proBNP sensor for a heart-failure patient in the hospital. NT-proBNP blood tests are regularly ordered in hospital for heart failure and can be used to improve the accuracy of the sensor. In addition, patients could have at-home tests or followup appointments after discharge from the hospital which could be used to calibrate the sensor. In addition, at home care and prolonged use of the sensor could require additional calibration, even using at-home testing capabilities (such as finger-prick tests). FIG. 9 illustrates a sensor reading over time that includes multiple manual recalibration events at three example time points, 967a, 967b, 967c. At 967a for example, the concentration reported for NT-proBNP is increased because the sensor was underrepresenting the NT-proBNP concentration. While FIG. 9 does not show retractive recalibration, it could be applied using averaging or other mathematical methods such than there are no discontinuities in the analyte measurement plot over time (this could appear as a smooth continuous line that is appropriately shifted as needed due to recalibration events).Factory Calibration - Auto-calibration During Use

[0116] With reference to embodiments of the present invention, while manual calibration is highly feasible, it is less desirable than auto-calibration. Aptamer sensors are known to have multiple approaches that claim to provide ‘calibration free’ operation, such as taught by Plaxco et al in U.S. Patent Application Publication 2020 / 0182820 Al. An additional such ‘calibration free’ method, amongst other possible methods, was recently taught by White et al. in “Abeykoon SW, White RJ. Continuous Square Wave Voltammetry for High Information Content Interrogation of Conformation Switching Sensors. ACS Meas Sci Au. 2022.” The calibration provides a concentration value, the concentration value calculated according to Equation 1 shown in FIG. 21. In Equation 1, [T] is the concentration of the analyte (or target), KD is the aptamers dissociation constant for the target, i is a constant comprising the redox peak current at the measurement frequency, a is a constant comprising the ratio of output signal at the minimally (or non- responsive) frequency and target-free redox peak signal at the measurement frequency, i\R is the redox peak at the at the minimally (or non -responsive) frequency, and y is a constant comprising the ratio of analyte-saturated output signal to analyte-free output signal. These measures and parameters are illustrated in FIGs 12A-12D, as taught by Plaxco et al.

[0117] However, such calibration free methods inherently assume stable electron transfer kinetics and stable peak and non-responsive frequencies of measurement for square wave voltammetry (assumes that the frequencies chosen for i or for INR do not change), or assume stable sampling time points for continuous square wave voltammetry. Ultimately, for most aptamer sensors these frequencies are not stable and shift during operation. Embodiments of the present invention teach methods that allow the measurement system to automatically recalibrate the accuracy of a ‘calibration free’ sensing method as illustrated in FIGS. 10 and 11. Again, as stated previously, the purpose of using ‘calibration free’ methods in the present invention might not necessarily be to avoid further calibration, but rather to get superior data collection that ultimately improves sensor accuracy.

[0118] A first example of autocalibration is as follows. In a first embodiment of auto-calibration, the present invention requires the sensor to have a measured or known or expected or predicted standard deviation 1063a in addition to the actual sensor measurement of the analyte 1063. Secondly, in a first embodiment of auto-calibration the present invention requires the sensor to change in sensor response or analyte measurement 1063 at least greater than the standard deviation 1063a or at least twice the standard deviation 1063a, such as time points 1068 and 1069. Therefore, the calibration data can be modified using auto-calibration during use. In addition, the factory calibrated aptamer sensors further include a known or expected or predicted standard deviation forthe sensor response; and autocalibration occurs when the sensor response is changing by an amount greater than the standard deviation of the sensor response. As an example of this requirement, cortisol or melatonin or NT-proBNP have large daily fluctuations which would satisfy this requirement for cortisol or melatonin or NT-proBNP sensors. An additional example of this requirement are doses of drugs that are orally taken and measured with a sensor, which would satisfy this requirement as well. As illustrated in FIGS. 11A and 11B, at t=l days the peak responsive frequency for signal OFF 1190a, the non-responsive frequency 1190b, and the peak responsive frequency for signal ON 1190c can all change to new frequencies at 3 days or other durations as illustrated as 1191a, 1191b, 1191c. When the sensor is changing in response (changing in redox peak current) to a degree greater than the standard deviation of the sensors (FIG. 10, 1068, 1069), a full or partial scan of sensor response vs. frequencies can be collected and optionally plotted or analyzed as shown like that in FIGs 11 A-l IB and the frequencies determined based on what frequencies have maximum change in redox peak current as analyte concentration changes (1190a, 1191a, 1190c, 1191c) and based on what frequencies have minimum or no change in redox peak current as analyte concentration changes (1190b, 1191b). These new frequencies can then be stored in the device, software, or other location and used to reprogram and maintain the accuracy of ‘calibration free’ methods. Said differently, for example, the non-responsive frequency such as frequency 1190b, 1191b can simply be refound or redetermined during use of the device by measuring at what frequency the redox current is not changing when the responsive frequencies are confirming that the analyte is changing in concentration. In yet another embodiment, measurement of only the shift in the non-responsive frequency 1191b can be obtained, again because during periods of significant change in sensor response 1068, 1069, at the non-responsive frequency 1090b, 1191b the sensor response should not be changing and is therefore identifiable. New knowledge of the shift in the non-responsive frequency 1191b can then be used to shift the peak frequency for the signal OFF 1191a or signal ON 1191c frequencies (assuming the whole frequency response shifts similarly or predictably with respect to shift in the non-responsive frequency). For example, if the zero frequency increases by 10%, the signal ON frequency could be assumed to shift by 10% (or some other predetermined value based on sensor aging tests). For this same autocalibration method but applied to continuous square wave voltammetry, the measurements are just time points (inverse frequency) and method and end result are adequately analogous for purposes of embodiments of the present invention. These same frequencies or time points can also be used to inform kinetic differential measurements which increase sensor signal to noise by subtracting the signal OFF response from the signal ON response or by ratios of signal ON to signal OFF responses.

[0119] With reference to embodiments of the present invention, additional forms of autocalibration may be utilized. Sensing devices illustrated in FIG. 1 can be fitted or utilized to additionally measure at least one of: change in aptamer density (via a cyclic voltammogram) which can impact frequency response, electrical impedance (via electrical impedance measurement over a plurality of measurement frequencies) which can impact charge transfer rates and frequency response and capacitive background current, temperature or pH or salinity (with dedicated sensors known to those skilled in the art) which can affect sensor response, oxygen reduction current (via square wave voltammetry or amperometry) which can impact background current and which indicates a change in the blocking layer density which further impacts charge transfer rates and frequency response and capacitive background current, or other possible measures. For example, with electrical impedance measurement, if charge transfer resistance (Rct) decreases by 20% then the peak measurement frequency can shift by an increase of 55Hz and the sensor calibration of redox peak current decrease by 5%. All of this can be automatically used to correct the calibration of the device in real time as the sensor changes during operation over time.

[0120] With reference to embodiments of the present invention, embodiments of the present invention may also apply to optical sensors as illustrated in FIG. 3. For example, batch calibration applies to optical sensors using similar methods where fluorescence signal or peak intensity replaces redox peak current, such as normalizing the background fluorescence for all sensors in a batch for minimum or maximum sensor response to target analyte (zero or saturated target), or such as pulling 3 sensors from a batch of 12 sensors for factory calibration.

[0121] With reference to embodiments of the present invention unlike glucose sensors which are diffusive flux dependent, aptamer sensors are concentration equilibrium dependent and may require calibration for analyte dilution of blood vs. interstitial fluid values. For example, the blood values for an analyte can be measured from a single individual or a plurality of individuals to obtain a mean value, and these blood levels can be used to calibrate the interstitial fluid sensed values into blood values. For example, an albumin sensor for cardiovascular diseases will, in some patients, have an albumin level in interstitial fluid that is approximately half that of blood. Therefore, first a batch of sensors can be calibrated as taught in previous embodiments, and then one or more blood measurements of albumin can be used to calibrate the sensor response in interstitial fluid (such as a 2X multiple) such that the sensor is able to predict blood levels of albumin. Similar calibration can be used for Tmax and Cmax for partitioning, for example of orally dosed drugs. Because aptamers are affinity based and dependent on concentration, sensor calibration may require adequate time such as minutes or hours to reach steady-state or near-steady state concentrations (steady concentrations or fluxes of analyte throughout the body, or steady concentrations after healing of the sensor insertion site has occurred) before a calibration readingis recorded between blood and interstitial fluid vs. attempting to calibrate while concentrations are changing. Therefore, an embodiment of the present invention includes factory calibration, that is obtained in a human, only after or near the point of steady state of concentration of the analyte in interstitial fluid.Kinetic Differential Measurement for Reduced Sensor Drift and Greater Sensor Accuracy

[0122] With reference to embodiments of the present invention, kinetic differential measurement may be used as illustrated in FIG. 13A. In the plot of FIG. 13A, normalized redox peak current is based on the signal ON redox current 1360a, but the present invention need not be so limited. Kinetic differential measurement provides a continuous measurement 1360a, that is the difference between the signal ON measurement 1362a and the signal OFF measurement 1364a. While the signal ON 1362a and signal OFF 1364a measurements typically decrease over time in redox current, the measurement 1360a is more stable and has less drift as taught in “Ferguson BS, Hoggarth DA, Maliniak D, Ploense K, White RJ, Woodward N, Hsieh K, Bonham AJ, Eisenstein M, Kippin TE, Plaxco KW, Soh HT. Real-time, aptamer-based tracking of circulating therapeutic agents in living animals.”

[0123] The present invention further provides a calibration over time as well for kinetic differential measurement as illustrated in FIG. 13B. Assume for example the sensors of FIG. 13A are the calibration portion of the sensors, then the sensors of FIG. 13B may be the product portion of the sensors shown for the non-limiting example of an analyte that might not change over time (such as a drug monitor on drug users who are not using the drug and the sensing device captures that drug compliance). Simply, the calibration provides a more accurate measurement over time 1360B. Calibration curves may be also be captured and used for multiple sensor response levels corresponding to multiple analyte concentrations. Example curves are illustrated in FIG. 14 where 1460-90 is the calibration curve for 90% of the maximum sensor response, 1460-50 is the calibration curve for 50% of the sensor response (sometimes also known as the Kd or binding affinity for the sensor), and 1460-0 could be calibration curve for no analyte or immeasurable levels of the analyte or for 10% of the maximum sensor response.

[0124] Example experimental data for two batches of sensors is shown in FIG. 17 for a noncalibrated set of data which is the batch portion of a batch of sensors (n=3) and a product portion (n=l) of the batch of sensors all plotted for a single stable concentration of analyte for an aptamer sensor fabricated as described herein and measured with a constant waveform, which as will be discussed in the next section cannot always be assumed. In FIG. 17 the outer dotted line for each plot represents the standard deviation.Calibration and / or Accounting for Sensor Stabilization

[0125] One of the challenges for affinity biosensors like those described herein, is that measurement technique can require time to stabilize to an accurate measurement value. For example, periods of several hours are often needed with electrochemical aptamer sensors like those described herein. Stabilization is a poorly addressed problem in the field of affinity biosensors for at least two reasons: (1) academic or other research studies can permit a device to stabilize either in-vitro or in-vivo before accurate data can be collected and are not subject to commercial requirements of having accurate data as quickly as possible after start of sensor use; (2) affinity biosensors have notoriously suffered from short operational longevity making it difficult to see longer term needs to restabilize the sensor after initial stabilization. Now that more stable sensor chemistries such as 8-carbon linked monolayers (such as mercaptooctanol) can be implemented on optimized gold and with proper fouling protection, sensor stabilization needs now face a new set of challenges. Two example challenges are as follows.

[0126] A first challenge is initial sensor stabilization, as illustrated in FIG. 15A. A sensor response curve for a constant concentration of analyte is shown for initial placement into serum or interstitial fluid for a sensor that will experience fouling 1562 (for example, using no hydrogel protection). Fouling stabilization can, in part, be resolved by prefouling the sensor as described in application WO2021067779A1 “Shelf-Stable, Ready-to-use, Electrochemical Aptamer Sensors.” However, a long-lasting sensor (days to weeks or more) cannot tolerate fouling if it is to be maximally accurate in measurement, and additional slow fouling occurs over periods of days or more without fouling protection. Even a sensor that does not have fouling and which is long lasting for days or weeks may have an initial stabilization as illustrated in FIG. 15 A for the no fouling sensor response curve 1560. An example of such a sensor is an aptamer sensor on sodium-hydroxide roughened gold with a mercaptooctanol blocking layer and eight-carbon linkage for the aptamer and with polybetaine hydrogel protection,.

[0127] A second challenge is sensor stabilization during operation after initial stabilization, as illustrated in FIG. 15B and shown with real data in FIG. 16. FIG. 15B shows a sensor response curve that represents an aptamer biosensor that is subjected to a first waveform 1591 that is an analyte measurement waveform or a second waveform 1592 that, for example, is a sensor diagnostic waveform to determine the on-going health of the sensor and to therefore correct for sensor degradation and improve calibration over time such as auto-calibration as taught herein. For example, waveform 1591 could be square wave voltammetry or continuous square wave voltammetry, whereas waveform 1592 could be a frequency response like that taught in FIG. 11 or impedance spectroscopy to determine monolayer degradation through electrical capacitance or DC voltage at -0.5 V to determine monolayer degradation through the amount of oxygen reductioncurrent at the sensor. Each time waveform 1592 is applied it disturbs the sensor and in particular the sensing monolayer and any foulants attached thereto and could otherwise require the sensor to restabilize before accurate data can be collected. The downside of having to perform sensor stabilization is simple, there is less time over which accurate data can be collected. Without traditional sensor fouling of large solutes such as proteins, sensor stabilization can largely be due to reorganization of the sensing monolayer including the molecules bound to the surface, and solutes in the sample fluid such as interstitial fluid that incorporate in or near the monolayer itself but which are too small to be irreversible in binding to the monolayer or to impede freedom of movement for the aptamer. As the sensing monolayer or adjacent solutes reorganize, the reorganization alters the electron transfer characteristics through the monolayer.

[0128] Initial sensor stabilization, also referred to as a first stabilization period, in an embodiment of the present invention, can be resolved by calibrating against the sensor stabilization response to at least one waveform. For example, for an embodiment of the present invention, where a sensor does not experience significant fouling and where pre-fouling the sensor would not be beneficial (FIG. 15A no fouling sensor response curve 1560) the initial sensor response can be simply characterized from a calibration portion of sensors and applied in use to the product portion of sensors.

[0095] For sensor stabilization after initial sensor stabilization, which can be referred to as a second sensor stabilization period, in an embodiment of the present invention, several additional examples and details are now provided. Waveforms may be at least one of an analyte measurement waveform, a sensor diagnostic waveform, a sensor preserving waveform, or another type of waveform. For example, an aptamer sensor could have a mixed monolayer of aptamers for two or more analytes, where two or more redox tag species at different redox potentials are used to differentiate measurement of each of the aptamer populations, requiring different waveforms for each type of aptamer 1591, 1592, and the waveforms being different therefore disturbing the sensor monolayer. Or for example, waveform 1592 could be a sensor diagnostic waveform such as impedance spectroscopy or a DC potential measure of oxygen reduction current. Or for example, waveform 1592 could be a DC potential of -0.2V used to extend the lifetime of the sensor by suppressing oxidation of the monolayer chemistry and therefore requiring less electrical computational or analog power vs. continuously running a more complex waveform such as square wave voltammetry. The exact durations of the waveforms 1591 and 1592 shown in FIG. 15B and the exact impact on sensor response and the need for sensor stabilization is not limited to the example shown in FIG. 15B which is simply a single illustrative example. Furthermore, a plurality of waveforms may be more than just two waveforms (e.g. 3 different waveforms). Now, in the present embodiment, sensor stabilization after initial sensor stabilization can be resolved such thatmore data can be accurately collected in at least two methods. In a first method, the sensor response 1591 is characterized from a calibration portion of sensors and used to correct the product portion of sensors while the sensor is re-stabilizing each time during use (e.g. restabilizing after application of waveform 1592). In a second method, the waveforms are periodic. If for example two or more waveforms are applied at periodic intervals as illustrated in FIG. 15B, then the restabilization can be assumed to be identical or similar each time during at least a period of operation of multiple hours and, more ideally, for multiple days or weeks. If the sensor restabilization is similar each time because the waveforms are applied periodically, then accurate data can be collected even during periods where the sensor restabilizes each time because the trend for sensor restabilization is known or is the same or similar each time.

[0096] Embodiments of the present invention may be applied to sensors that do not foul or which have little change in sensor response to fouling, or to devices which foul and which have a significant change in sensor response due to fouling (albeit such devices typically have much shorter operational lifetimes or less accuracy in measurement). Foulants that are bound to the monolayer of the sensor can also re-arrange over time in response to a waveform and therefore cause a need for stabilization.Other Aptamer Sensors

[0097] The sensing or working electrode is comprised of an electrode material such as gold. The gold is then incubated with aptamers via thiol attachment to the gold electrode, and the aptamer includes a redox tag such as methylene blue. In between the aptamers, the gold is further incubated with a protective monolayer such as mercaptohexanol, mercaptoocotanol, or other suitable chemistry. A protective membrane such as polybetaine or other suitable material may be added to prevent fouling of the monolayer surface. The working electrode may be preserved in a preservative such as trehalose to enable dry storage. In a non-limiting but specific example, binding of aptamer to target causes a shape confirmation change which brings the redox tag closer to the electrode resulting in increased electron transfer (increased electrical current). As concentration of target increases, more binding of target to aptamer occurs, and more electron transfer occurs (more measurable electrical current). As concentration of target decreases, conversely, electrical current decreases. Devices as taught herein can insert the supports carrying the working electrodes into skin using one or methods such as those commonly deployed for the insertion of glucose sensors needles for continuous glucose monitors (such as a slotted insertion guide or other methods). An example of device fabrication and testing is as follows:Electrochemical measurements

[0098] Electrochemical measurements can be performed with a miniaturized potentiostat or performed by a benchtop CHI 620E potentiostat (Austin, Texas) connected to a 64-channel multiplexer in a standard three-electrode system with aptamer / alkylthiolate functionalized electrodes serving as working electrodes. The counter and reference electrodes can be inserted into the skin using a platinum counter electrode, and an Ag / AgCl reference electrode, or alternately the counter and reference electrodes can be a large gel-electrode-pad electrode on the surface of the skin as taught in application W02022067051A1 “Aptamer sensors with reference and counter voltage control.” Cyclic voltammograms can be recorded in a window from -0.1 V to -0.5 V at a scan rate of 100 mV / s. Square-wave voltammetry can be performed in a potential window from -0.1 V to -0.5 V at 25 mV amplitude at the optimal frequency of measurement for each aptamer.

[0099] Kinetic differential measurements, two frequency measurement, or continuous square wave voltammetry may be utilized to improve calibration-free operation of the sensors. Example in-vivo data collected for a cortisol sensor inserted subcutaneously in a rat is shown in FIG. 20, where bolus injections of cortisol are performed at 5 or 10 mg / kg.Additional Targets

[0100] The present invention applies generally to aptamer sensors and other types of affinity biosensors, and is therefore not limited to specific examples as taught herein. Available electrochemical sensors for analytes such as cortisol, vancomycin, phenylalanine, insulin, BNP, NT -proBNP, IL-6, C-peptide, C-Reactive protein, albumin, and sensors for other analyte targets may be incorporated in the present invention without limitation. Such aptamers can be obtained from the literature, by SELEX, or purchased from companies such as SOMAlogic or Base Pair Technologies and adapted into an aptamer sensor similar to that shown in FIG. 3 or other aptamer sensing configurations based on alternative switching mechanisms such as redox quenching, molecular pendulums, or other suitable methods.Ultra-Stable Sensor Performance Features

[0101] While the performance of a sensor can change significantly over time, factory calibration can correct for much of these changes. Aptamer sensors have unique calibration requirements compared to other types of continuous sensors. Some of the most important performance features for an aptamer sensor that is able to leverage factory calibration to maintain its performance are:

[0102] 1) maintaining adequate redox tag current vs. background signal, which for redox tag current, the aptamers and redox tags should stay attached to the monolayer, and for backgroundsignal the blocking layer portion of the monolayer should stay intact such that capacitive current and oxygen reduction current do not increase substantially;

[0103] 2) maintaining predictable electron transfer rates between the redox tags and the electrode, which can shift significantly if the sensing monolayer becomes more porous due to monolayer desorption or if electron transfer becomes impeded via fouling;

[0104] 3) potentially most importantly of all, the above factors may change some, but if they change in a predictable way then batch calibration must maintain a tight standard deviation for the calibration portion of the sensors. This is especially important because if sensor to sensor variation in performance is large, then it is much more difficult to economically or practically use calibration of every sensor for the duration of the intended use period (the calibration portion and product portion being one and the same) because that requires a significant amount of testing resources and may prematurely degrade or age the sensor product.The present invention provides for the first time, ultra stable standard deviation, capacitance, electron transfer rates, and oxygen reduction currents, in support of factory calibration, and ultimately therefore real-world accuracy of the product portion of aptamer sensors. The following tabular data demonstrates sensor standard deviation that is dramatically more stable than demonstrated before for aptamer sensors and which is more stable than what is often observed across continuous biosensors in general. The sensors are fabricated using methods as detailed herein. Example 2 shows one specific example of a sensor that enables ultra-stable performance.

[0105] In one embodiment, the present invention includes a calibration portion of aptamer sensors with at <+ / - 2% standard deviation for at least 10X range of measurement over at least one of 7 or 14 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

[0106] In another embodiment, the present invention includes a calibration portion of aptamer sensors with at <+ / - 3% standard deviation for at least 10X range of measurement over at least one of 7, 14, 21, 30, 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

[0107] In one embodiment, the present invention includes a calibration portion of aptamer sensors with at <+ / -5% standard deviation for at least 10X range of measurement over at least one of 7, 14, 21, 30, 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

[0108] In another embodiment, the present invention includes a calibration portion of aptamer sensors with at <+ / -10% standard deviation for at least 10X range of measurement over at least one of 7, 14, 21, 30, 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

[0109] In one embodiment, the present invention includes a calibration portion of aptamer sensors with at <+ / -20% standard deviation for at least 10X range of measurement over at least one of 7, 14, 21, 30, 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

[0110] In another embodiment, the present invention includes a product portion of aptamer sensors with at <+ / - 5% accuracy for at least 10X range of measurement over at least one of 7, 14, 21, 30, 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

[0111] In one embodiment, the present invention includes a product portion of aptamer sensors with at <+ / - 10% accuracy for at least 10X range of measurement over at least one of 7, 14, 21, 30, 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

[0112] In another embodiment, the present invention includes a product portion of aptamer sensors with at <+ / - 20% accuracy for at least 10X range of measurement over at least one of 7, 14, 21, 30, 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

[0113] In one embodiment, the present invention includes a product portion of aptamer sensors with a change in a zero gain frequency that is less than at least one of <+ / -5%, <+ / - 10%, <+ / -20%, <+ / -40%, <+ / -80% of the day 1 zero grain frequency over 7, 14, 21, 30, 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

[0114] In another embodiment, the present invention includes an oxygen reduction current measurable at a potential of -0.5V which increases by at least one of <10%, <20%, <50%, <100% of the initial oxygen reduction current over 7, 14, 21, 30, 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

[0115] The above example assumes that batches have a very tight distribution of the magnitude of redox current at a given concentration of analyte. For example, assume that the sensor has both signal ON and signal OFF responses and a non-responsive frequency where the current does not change. Changes in electrode area, aptamer density, or other factors can affect this magnitude of current from sensor to sensor. During factory calibration, if needed, several additional measures can be taken to provide performance like that specified above even if magnitude of current varies between sensors in the batch. For example, to inform the magnitude of current for each individual sensor, non-limiting options may include the following:

[0116] (1) during manufacturing the magnitude of current for each individual sensor in the product portion of sensors can be measured directly at the non-responsive frequency or at responsive frequencies where the analyte concentration in the test solution is known, or the magnitude of current may be indirectly determined through measures such as electrode surface area or electrical capacitance.

[0117] (2) during in-vivo use, the magnitude of current can be measured by measuring the product portion of sensors at the non-responsive frequency. As previously described herein, if needed the non-responsive frequency can be automatically determined during in-vivo use for most analytes by determining when the analyte is changing in concentration and measuring at what frequency no resulting change in current occurs.

[0118] (3) during in-vivo use, electrode active surface area can be measured using electrical capacitive or other methods during in-vivo use for each sensor, and is used to indirectly determine the magnitude of current. Other methods may include, for example, cyclic voltammetry to obtain the total aptamers and redox tags on the sensor surface, which if deposited with tight standard deviation across a batch of sensors will be representative of the electrode surface area (more electrode surface area = more total number of aptamers and redox tags = more total charge transfer measured during a cyclic voltammetry cycle). For example, within the first hours of in-vivo operation the electrode active surface area can be measured electrochemically or via electrical capacitance, and this measurement is used to normalize the measured current as previously described for FIG. 5.

[0119] (4) during in-vivo use, total redox tag density or number of redox tags can be measured at manufacturing or during operation of the sensor using cyclic voltammetry or other measures and used to indirectly determine the magnitude of current.

[0120] (5) other methods as needed or available for devices as described herein.Once the magnitude of current is known for each sensor, and because the percent change in current for each sensor is very predictable with a small standard deviation (see tables above), then the current for any given sensor can be normalized to its own known magnitude of current to then maintain standard deviations as described and specified in the tables below.Application to Redox Quenching

[0121] The present invention may also apply to redox quenching systems such as carminic acid or hemin tagged aptamers that are tagged for example at the end of a hybridized aptamer strand and with analyte binding the strand is broken and the redox activity of the redox tags is activated. In this switching construct for an aptamer sensor, a non-responsive frequency may also still apply to maintain calibration as do other techniques as described herein. One major differencewith redox quenching systems from the perspective of the present invention is their background redox tag current can be very low or close to zero such that the total change in redox current due to analyte binding can be much larger. Therefore, in one embodiment, the redox quenching systems of the present invention are a plurality of aptamers comprising one or more attached redox tags, wherein the attached redox tags provide electron transfer with the electrode.System Overview

[0122] With a basic understanding of the above-described sensors in place, reference is now drawn to FIG. 18, where like numerals do not refer to like features (the numbering for FIG. 18 is an 800 series of numbers and has no relation to FIG. 8). FIG. 18 illustrates a general diagram of a computer system 800 according to various aspects of the present disclosure. Like numerals in FIG. 8 do not necessarily refer to like features like that in the other figures. The computer system 800 comprises a plurality of hardware processing devices (designated generally by the reference 802) that are linked together by one or more network(s) (designated generally by the reference 804).

[0123] The network(s) 804 provides communications links between the various processing devices 802 and may be supported by networking components 806 that interconnect the processing devices 802, including for example, routers, hubs, firewalls, network interfaces, wired or wireless communications links and corresponding interconnections, cellular stations and corresponding cellular conversion technologies (e.g., to convert between cellular and TCP / IP, etc.). Moreover, the network(s) 804 may comprise connections using one or more intranets, extranets, local area networks (LAN), wide area networks (WAN), wireless networks (Wi-Fi), the Internet, including the world wide web, cellular and / or other arrangements for enabling communication between the processing devices 802, in either real time or otherwise (e.g., via time shifting, batch processing, etc.).

[0124] A processing device 802 can be any device capable of communicating with another processing device 802, e.g., via Bluetooth, Ultrawide band, near field communication (NFC), via one or more radio frequencies (RF) or via any other form of wired or wireless communication, over the network 804, or combinations thereof.

[0125] Some examples of processing devices 802 are cellular devices (including cellular mobile telephones (i.e., smartphones)), tablet computers, netbook computers, notebook computers, personal computers, servers, cloud devices, edge devices, etc.

[0126] Also, in certain contexts and roles, a processing device 802 is intended to be a wearable monitoring device. Examples of a wearable monitoring device include a purpose-driven appliance, Internet of Things (loT) device, special purpose device, etc. A processing device 802 implementedas a wearable monitoring device is schematically illustrated in FIG. 8 as a wearable device mounted to a patient’s arm solely for convenience of illustration. In practical applications, the wearable monitoring device can attach to other parts of a patient’s body.

[0127] In some embodiments, the wearable monitoring device can communicate locally (e.g., to a smart phone) via Bluetooth, ultrawide band, via one or more radio frequencies (RF) or via any other form of wired or wireless communication. In other embodiments, the wearable monitoring device can communicate across a network, e.g., via Wi-Fi and / or communicate locally to another processing device 802.

[0128] The illustrative computer system 800 also includes a processing device implemented as a server 812 (e.g., a web server, file server, and / or other processing device) that supports an analysis engine 814 and corresponding data sources (collectively identified as data sources 816). The analysis engine 114 and data sources 116 provide the resources to implement and store data related to collecting and aggregating data from wearable monitoring devices, captured events, combinations thereof, etc., as described in greater detail herein.

[0129] In an exemplary implementation, the data sources 116 are implemented by a collection of databases that store various types of information. Solely by way of example, the data sources 816 can include device data 818, e.g., data related to wearable monitoring devices, including configuration data, version data, software versioning and control, data generated from wearing a wearable monitoring device, etc. The data sources 816 can also include medical data 820, e.g., medical research, etc., used to calibrate, tune, design, modify, etc., wearable monitoring devices. The data sources 816 can also optionally include user data 822, e.g., data regarding the patients that are wearing the wearable monitoring devices, where such data is collected. As yet further examples, the data sources 816 can include platform data 824, e.g., data used by the analysis engine 814, e.g., computer drivers, GUI information, algorithms for processing physiological conditions, etc. As yet a further example, the data sources 816 can optionally include miscellaneous data 826, e.g., any data needed by the analysis engine 814 that is not otherwise accounted for above.

[0130] Considering FIG. 18 as an environment used by wearable monitoring devices, in some embodiments, the processing of physiological data of a corresponding patient wearing the wearable monitoring device (e.g., biochemical sensing with additional sensing modalities that enhance patient care or health and wellness) can be carried out entirely on a processing device 802 (such as a wearable monitoring device itself); on a processing device 802 such as a smartphone, by the analysis engine 814, or via combinations thereof (e.g., by distributing processing tasks among two or more processing devices).

[0131] With specific regard to a processing device 802 implemented as a wearable monitoring device (see processing device 802 schematically attached to a patient’s arm), it may be desirableto carry out all of the processing on the wearable monitoring device itself. In this regard, a corresponding device such as a smartphone can optionally provide a graphical user interface for displaying dashboard measurement results, but all processing is carried out on the wearable monitoring device itself.

[0132] In other embodiments, the smart phone can carry out some processing, e.g., to compare computed data to dashboard thresholds, to carry out algorithms, rules, or other processing, as described more fully herein.

[0133] In still other embodiments, the analysis engine 814 can collect data from each wearable monitoring device, e.g., for trend analysis of patient data, for device state of health monitoring (e.g., to detect faults in the wearable devices themselves), for battery charge level monitoring, for versioning (such as to carry out software updates), etc.

[0134] In some embodiments, the analysis engine 814 is controlled by a third party, e.g., the manufacturer of the wearable monitoring devices that are implemented in the environment.

[0135] In some embodiments, the analysis engine 814 schematically represents integration into an electronic health record system, e.g., to connect a patient to the patient’s doctor so that the doctor can access the electronic data generated by a corresponding wearable monitoring device.

[0136] Calibration codes or data and methods as taught herein can be stored in any device in the system 800 which can store memory and which can also communicate that calibration in a manner such that the end data shared with the user is corrected for by calibration before the data is shared with the user.Monitoring Device

[0137] Referring now to FIG. 19, an example wearable monitoring device 900 is schematically illustrated, according to aspects of the present disclosure where like numerals do not refer to like features (the numbering for FIG. 19 is a 900 series of numbers and has no relation to FIG. 9). The wearable monitoring device 900 can represent an example embodiment of a processing device 802 (FIG. 18), e.g., a wearable monitoring device as previously described.

[0138] The wearable monitoring device 900 includes a housing 910 that attaches to a patient. The housing can attach to the patient via an adhesive 904, a strap, or other securement.

[0139] The wearable monitoring device 900 also includes at least a first working electrode 920 and may include a second working electrode 922 and further may include a third working electrode 924 or even more working electrodes. Alternately electrodes 922 and 924 may be counter and reference electrodes. In some embodiments, one or more electrodes include an analyte detecting material, e.g., aptamers, such that continuous sensing can be carried out. Electrode 950, aspreviously described may be a gel electrode pad and serve the roles of a reference and counter electrode.

[0140] In practical applications, the housing 910 is couplable to the electrodes 920, 922, 924. As used herein, "couplable" is to be construed broadly to mean any one of permanently coupled, detachably coupled, temporarily coupled, user attachable, user detachable, user attachable and detachable, factory attachable, factory detachable, user attachable, factory attachable and detachable, or any combination thereof, unless specifically noted otherwise.

[0141] As illustrated, the housing 910 includes a potentiostat 991 that is communicably coupled to the electrodes 920, 922, 924 (or a combination thereof) using an optional multiplexer 990, or alternatively each of electrodes 920, 922, 924 can receive a direct dedicated connection to a potentiostat 991. In practical applications, the term “potentiostat” is to be interpreted broadly and is not limited to any particular number of sensors. For instance, the potentiostat can be implemented as a bipotentiostat, polypotentiostat, etc., depending upon the sensor configuration provided by the wearable monitoring device 900.

[0142] Additionally, wearable monitoring device 900 includes a controller 993 that is communicably coupled to memory 992. The controller 993 is also communicably coupled to a communication interface 994.

[0143] The controller 993 includes necessary electronics that enable the controller 993 to carry out the intended functionality of the wearable monitoring device. For instance, the controller 993 can include a processor, bus interface, ports, registers, memory, etc., that enables the wearable monitoring device 900 to carry out the functionality described more fully herein.

[0144] Also, as illustrated, the controller 992 is communicably coupled to one or more of the optional multiplexer 990, potentiostat 991, the memory 992, the transceiver s) 994, optional miscellaneous sensors 995, optional display / output 996, combinations thereof, etc.

[0145] The communication interface 994 may comprise, for example, at least one transceiver that communicates via Bluetooth, Wi-Fi, Ultrawideband, near field communication, combinations thereof, etc.

[0146] The optional display / output 996 can comprise a display screen, a dimensionally limited display screen, a touch screen, a haptic output, a light output, a speaker / alarm, or combinations thereof.

[0147] The controller 993 uses the potentiostat 991 to collect measurements from electrodes 920a, 920b, 920c, and stores the collected measurements in the memory 992. The controller 993 may further provide filtering, analysis, control, authorization, authentication, and other controller specific functions. The communication interface 994 facilitates coupling the wearable monitoring device 900 with an external computing device, e.g., a smartphone, a cloud computer, etc. In thisregard, the communication interface 994 can include one or more modalities, each with different data and / or authorizations. For instance, a patient may access data from the wearable monitoring device 900 on a smartphone 1006 as illustrated in FIG. 10, whereas a doctor may be able to access more detailed information from a cloud server and / or through electronic health records (see FIG. 9). In this regard, multiple modalities of communication may be utilized with wearable monitoring device 900.

[0148] In some embodiments, the adhesive 904 of the wearable monitoring device 900 is, or includes, a gel electrode 950 that is connected to at least one of the potentiostat 991, the controller 993, or the sensor 995. For example, a gel electrode 950 could be the counter or reference electrode for the electrodes 920, 922, 924.EXAMPLESMaterials

[0149] Sulfuric acid (96%, p.a.), Sodium hydroxide (98%, pellets), Pulverized phosphate buffered saline (PBS, pH 7.4), Tris-EDTA solution (TE buffer; pH: 8), Bovine Serum, tris(2- carboxyethyl) phosphine hydrochloride (TCEP; 98%), sodium azide (99.5%), l,6-d6-mercapto-l- hexanol (MCH; 98%) and 8-mercapto-l -octanol (MCO; 97%) were obtained from Sigma Aldrich (USA). 2-hydroxy-2methylpropiophenone (photo-initiator, purity: >96%) was purchased from TCI Chemicals. [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (monomer, purity: >95%) was obtained from Chem-Impex INC, USA. Absolute ethanol (100%; anhydrous) was purchased from Fisher Scientific (USA). Vancomycin hydrochloride (94.6%) and ethylene glycol-dimethacrylate (cross-linker, purity: >98%) were obtained from Alfa Aesar. The 3 ’-methylene blue and 5 ’-thiol modified oligonucleotide sequences were synthetized by Integrated DNA Technologies (IDT, USA). Example aptamer sequences are as follows:Sensor preparation

[0150] Gold electrodes with a titanium adhesion layer are deposited on PET or Kapton strips that provide a support for the electrodes such as support 460 in FIG. 4. The gold can be patterned via photolithography and chemical etching, and electrical insulators applied that are photo- definable or screen-printable. The gold can be further electroplated with additional gold and electrochemically roughened or cleaned or used as-is prior to aptamer and protective layer incubation. Electrochemical cleaning can be performed in a standard three-electrode electrochemical cell consisting of a gold working electrode, platinum counter electrode, and Ag / AgCl reference electrode by running 700 cyclic voltammetry scans in 0.5 M NaOH from - I V to -1.6 V at a scan rate of 1 V / s and subsequently 150 scans in 0.5 M H2SO4 solution from 0V to 1.6 V at 1 V / s. Once the electrochemical cleaning is complete, electrodes can be thoroughly rinsed with DI water, dried in a nitrogen stream (99.999% purity), and used for subsequent incubation. A lyophilized pellet of modified aptamer can be diluted down to a 100 pM stock solution using TE buffer and kept at -20 °C until use. Preparation of aptamer working solution can be performed by first mixing an aliquot of the 100 pM aptamer stock solution with equal volume of 0.5 M TCEP dissolved in Milli-Q water. The mixture can then be set aside for Ihr to ensure complete reduction of any disulfide aptamer molecules. The obtained solution can then be diluted to an intermediary concentration of ~4 uM with lx PBS / 2 mM MgCh buffer and the concentration confirmed via the absorbance measured at 260 nm using a Nanodrop UV / Vis Spectrophotometer. This solution can then be subsequently diluted to 500 nM with lx PBS / 2 mM MgCh buffer for incubation of aptamerfor one hour. The aptamer functionalized electrodes can then be rinsed with DI water and incubated overnight at room temperature in 5 mM MCH or MCO prepared in lx PBS. The functionalized sensors can then be rinsed with DI water prior to coating with trehalose for storage and ultimately then used for measurement. If electrochemical roughening and hydrogel protection is desired, prior to aptamer and MCH or MCO incubation, electrodes can be immersed in 5 M NaOH solution and subjected to 20 ms long alternating potential steps of -5 V and +0.8 V (vs. Hg / Hg2SO4, sat. Na2SO4) respectively, for a total duration of 6000s in an electrochemical cell consisting of a Kapton®-carbon counter and saturated Hg / Hg2SO4 reference electrode. Once the roughening is completed, the electrodes can be rinsed with copious amounts of DI water and aptamer and MCH or MCO, then incubated as described above. Modification of the sensors with an antibiofouling zwitterionic polybetaine-based hydrogel can be performed by drop-casting 1 pL of the aqueous mixture consisting of monomer / cross-linker / photo-initiator (2.8g / l .8 pl / 36pl respectively dissolved in 1 ml of DI water) over the sensor and exposing it to UV light ( : 280-450 nm, Bluewave LEDPrime UVA, Dynamax, USA) for 45 min.Example 1

[0151] The following is a non-limiting embodiment of the present invention which captures several of the techniques described above for maintaining calibration of a sensor during in-vivo use.

[0152] 1) 500 sensors are fabricated in a batch, shelf stabilized and sterilized, and 12 sensors are reserved as calibration portion of the sensors and 488 sensors as product portion of the sensors.

[0153] 2a) The calibration portion of the sensors is then tested in human serum at 34 °C at the factory for 15 days duration with an intended product portion of the sensors to have a maximum use of 14 days. The sensors are tested simultaneously at 50% analyte concentration, and the data captured and merged as the average and standard deviation using kinetic differential measurement using 10Hz continuous square wave voltammetry, every 5 minutes, and the maximum signal ON, signal OFF and non-responsive currents are measured and their time points (frequencies) of measurement (e.g equivalent to 10, 40, and 300 Hz).

[0154] 2b) On days 0, 1, 3, 5, 7, 10, 14 a titration curve is captured for the sensors with 10 titration points at 10% increments from 0% to 90% of the range of the sensor response which is typically around 80X change in concentration.

[0155] 3) The magnitude of current associated with 50% analyte concentration is stored as a calibration curve and a sensor response calibration curve is created based on a fit to the titration curves generated on days 0, 1, 3, 5, 7, 10, 14 (titration does not need to be continuously measured to create a calibration curve that includes sensor response of the titrated range).

[0156] 4) The product portion of sensors are then assigned a code that relates back to the data captured for the calibration portion of the sensors. The code retrieves full calibration data from the cloud (server). Alternately the sensors can be fully programmed with the calibration curves at the factory. The product portion of the sensors may also be assigned calibration data for in-vivo blood correlation that is tested for each batch of sensors with a limited number of human subjects, or less often, or even not-at-all in some cases.

[0157] 5) The product portion of the sensors are then applied to the user and after a half hour of stabilization the sensor briefly captures three times sequentially the total aptamer tag current with cyclic voltammetry and obtains an average to inform a measure of current magnitude for the individual sensor. Optionally, at 12 hours of sensor stabilization the sensor begins an autocalibration process where it starts to look for changes in analyte concentrations that are sufficient to determine the non-responsive frequency and the current magnitude at the non- responsive frequency and use that value to further calibrate the accuracy of the sensors over time. With continuous square wave voltammetry, the frequency of 10Hz does not need to be changed in product use to capture the non-responsive frequency because continuous square wave voltammetry can inherently capture all the frequency data in a single 10 Hz scan.Example 2

[0158] A sensor according to the present invention was fabricated as follows. Electrochemically NaOH roughened gold was coated with an MCO blocking layer after cortisol aptamer was linked to the gold via a thiol with an 8 carbon (8 methyl group) linker. UV-cured polybetaine hydrogel was applied. The sensor was tested continuously in 33 degrees Celsius serum, measured using square wave Kinetic differential measurement at 10 Hz for signal OFF and 300 Hz for signal and titrated at day 7, 14, 21, 30, 40, and 50. Concentration of cortisol in micromolar is ‘cone.’, standard deviation is ‘sd’, and the kinetic differential measurement is ‘KDM’. The concentrations of cortisol are high for this particular sensor such that for product portion of sensors a known zero concentration of cortisol would exist in between delivery of boluses of cortisol. The measurement range was from 0.5 micromolar to 500 micromolar. The resulting data is shown below:

[0159] Table 1 - Day 7 data

[0160] Table 2 - Day 14 data

[0161] Table 3 - Day 21 data

[0162] Table 4 - Day 30 data

[0163] Table 5 - Day 40 data

[0164] Table 6 - Day 50 data

[0165] Several aspects of this ultra-stable data set are highly illustrative. The 5 micromolar data is near the middle of the sensor range (half the maximum redox tag current measured) and is near the binding affinity value for the sensor for cortisol.

[0166] Table 7 - Table of Standard Deviation as a Percentage of Redox Tag Current

[0129] Although not described in detail herein, other steps which are readily interpreted from or incorporated along with the disclosed embodiments shall be included as part of the invention. The embodiments that have been described herein provide specific examples to portray inventive elements, but will not necessarily cover all possible embodiments commonly known to those skilled in the art.SEQUENCESSEQ ID NO: 1CGA GGG TAC CGC AAT AGT ACT TAT TGT TCG CCT ATT GTG GGT CGGSEQ ID N0:2GGA CGA CGC CAG AAG TTT ACG AGG ATA TGG TAA CAT AGT CGTSEQ ID N0:3CG ACC GCG TTT CCC AAG AAA GCA AGT ATT GGT TGG TCG

Claims

WHAT IS CLAIMED IS:

1. A method of making sensors comprising a plurality of aptamers and performing continuous measurements with the sensors, the method comprising: fabricating a plurality of sensors, each sensor comprising at least one electrode, the electrode having an electrode surface, attaching a plurality of aptamers to the electrode surface, the aptamers comprising one or more attached redox tags; wherein the attached redox tags provide electron transfer with the electrode; applying a blocking layer to the electrode surface; wherein the blocking layer comprises a blocking layer surface and a plurality of pathways supporting the electron transfer between the electrode and the redox tags; calibrating a portion of the plurality of sensors (“the calibration portion”), producing at least one set of calibration data; identifying a portion of the plurality of aptamer sensors as being commercial quality (“the product portion”); wherein the plurality of sensors is fabricated in a batch process; and further, wherein at least one set of calibration data gathered from the calibration of the plurality of sensors is associated mathematically to the product portion and that the product portion is capable of providing accurate or precise continuous measurement.

2. The method of claim 1 wherein the calibration portion and the product portion are the same portion.

3. The method of claim 1 wherein the calibration portion and the product portion are the same portion and are one sensor.

4. The method of claim 1 wherein the calibration portion comprises at least three sensors.

5. The method of claim 1 wherein the calibration data gathered from the calibration portion is at least in part in-vitro data.

6. The method of claim 1 wherein the calibration data gathered from the calibrationportion is at least in part in-vivo data.

7. A device for continuous measurement of at least one analyte in a test fluid comprising one or more sensors made using a method comprising: fabricating a plurality of sensors, each sensor comprising at least one electrode, the electrode having an electrode surface, attaching a plurality of aptamers to the electrode surface, the aptamers comprising one or more attached redox tags; wherein the attached redox tags provide electron transfer with the electrode; applying a blocking layer to the electrode surface; wherein the blocking layer comprises a blocking layer surface and a plurality of pathways supporting the electron transfer between the electrode and the redox tags; calibrating a portion of the plurality of sensors (“the calibration portion”), producing at least one set of calibration data; identifying a portion of the plurality of aptamer sensors as being commercial quality (“the product portion”); wherein the plurality of sensors is fabricated in a batch process; and further, wherein at least one set of calibration data gathered from the calibration of the plurality of sensors is associated mathematically to the product portion and that the product portion is capable of providing accurate or precise continuous measurement.

8. The device of claim 7, wherein the electrode area has a standard deviation less than a value selected from the group consisting of 0.02, 0.01, 0.05, and 0.02 pm2.

9. The device of claim 7, wherein the electrode area has a standard deviation less than a value selected from the group consisting of 20, 10, 5, and 2% of electrode area.

10. The device of claim 7, further comprising at least one time point for calibration during manufacturing of the device, wherein the time point is after sensor fabrication.

11. The device of claim 7, further comprising at least one time point for calibration during manufacturing of the device, wherein the time point is after sensor shelf-stabilization.

12. The device of claim 7, further comprising at least one time point for calibration duringmanufacturing of the device, wherein the time point is after sensor sterilization.

13. The method of claim 1, wherein the calibration data is data collected over a period of time selected from the group consisting of 1, 3, 7, 10, and 14 days.

14. The method of claim 1, wherein the calibration data is comprised of redox tag current for either a single time point or multiple time points versus time.

15. The method of claim 1, wherein the calibration data is comprised of redox peak potential for either a single time point or multiple time points versus time.

16. The method of claim 1, wherein the calibration data is comprised of oxygen reduction current either a single time point or multiple time points versus time.

17. The method of claim 1, wherein the calibration data is comprised of redox tag density or total number of redox tags for either a single time point or multiple time points versus time.

18. The method of claim 1, wherein the calibration data is comprised of electrical capacitance or electrical impedance for either a single time point or multiple time points versus time.

19. The method of claim 1, wherein the calibration data is comprised of temporal or frequency response of redox tag current for either a single time point or multiple time points versus time.

20. The method of claim 1, wherein the calibration data is comprised of a chronoamperometric response for either a single time point or multiple time points versus time.

21. The method of claim 1, wherein the calibration data is comprised of electrode surface area for either a single time point or multiple time points versus time.

22. The method of claim 1, wherein the calibration data is comprised of titration response for either a single time point or multiple time points versus time.

23. The method of claim 1, wherein the calibration data includes statistical data.

24. The method of claim 1, wherein the continuous measurement comprises a continuous measurement method selected from the group consisting of square wave voltammetry, continuous square wave voltammetry, kinetic differential measurement, calibration free measurement, impedance spectroscopy, differential pulse voltammetry, chronoamperometry, and combinations thereof.

25. The method of claim 1, wherein the calibration data includes at least one variable in- vivo parameter.

26. The method of claim 1, wherein the calibration data includes at least one in-vivo correlation between interstitial fluid and blood.

27. The method of claim 1, wherein the calibration data further comprises calibration data gathered during use of the product portion of the sensors.

28. The method of claim 1, further comprising auto-calibration data gathered during use of the product portion of the sensors.

29. The method of claim 1, further comprising auto-calibration data gathered during use of the product portion of the aptamer sensors, wherein the auto-calibration data is frequency or temporal response of the sensors gathered during use of the sensor.

30. The method of claim 1, further comprising auto-calibration data gathered during use of the product portion of the aptamer sensors, wherein the auto-calibration data is electrode surface area.

31. The method of claim 1, wherein calibration data further comprises in-vivo calibration data gathered after or near the point of steady state concentration of an analyte in interstitial fluid.

32. The method of claim 1, wherein calibration data further comprises a plurality of sets of calibration data gathered at a plurality of analyte concentrations.

33. The method of claim 1, further comprising at least one measurement waveform that alters the electron transfer characteristics through the blocking layer and therefore further comprises at least a first stabilization period; wherein the calibration data is at least in part comprised of at least one measurement of the first stabilization period.

34. The method of claim 1, further comprising a plurality of waveforms wherein at least one of the plurality of waveforms alters the electron transfer characteristics through the monolayer and further comprising at least a second stabilization period that occurs after each application of at least one of the plurality of waveforms.

35. The method of claim 34, wherein at least one of the waveforms is an analyte measurement waveform, a sensor diagnostic waveform, or a sensor preserving waveform.

36. The method of claim 34, wherein the plurality of waveforms is periodic.

37. The method of claim 1 further comprising a calibration portion calibration portion with at <+ / - 2% standard deviation over a period of time selected from the group consisting of 7, 14, 21, 30, and 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

38. The method of claim 1 further comprising a calibration portion calibration portion with at <+ / - 3% standard deviation over a period of time selected from the group consisting of 7, 14, 21, 30, and 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

39. The method of claim 1 further comprising a calibration portion calibration portion with at <+ / - 5% standard deviation over a period of time selected from the group consisting of 7, 14, 21, 30, and 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

40. The method of claim 1 further comprising a calibration portion calibration portion with at <+ / - 10% standard deviation over a period of time selected from the group consisting of 7, 14, 21, 30, and 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

41. The method of claim 1 further comprising a calibration portion calibration portion with at <+ / - 20% standard deviation over a period of time selected from the group consisting of 7, 14, 21, 30, and 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

42. The method of claim 1 further comprising a product portion with at <+ / - 5% accuracy over a period of time selected from the group consisting of 7, 14, 21, 30, and 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

43. The method of claim 1 further comprising a product portion with at <+ / - 10% accuracy over a period of time selected from the group consisting of 7, 14, 21, 30, and 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

44. The method of claim 1 further comprising a product portion with at <+ / - 20% accuracy over a period of time selected from the group consisting of 7, 14, 21, 30, and 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

45. The method of claim 1 further comprising a product portion of aptamer sensors with a change in a zero gain frequency that is less than at least one of <+ / -5%, <+ / -l 0%, <+ / -20%, <+ / -40%, <+ / -80% of the zero grain frequency over a period of time selected from the group consisting of 7, 14, 21, 30, and 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

46. The method of claim 1 further comprising an oxygen reduction current measurable at a potential of -0.5V which increases by at least one of <10%, <20%, <50%, <100% of the initial oxygen reduction current over a period of time selected from the group consisting of 7, 14, 21, 30, and 40 days of continuous biosensing in interstitial fluid or serum or an equivalent surrogate test fluid at a temperature experienced during operation for the product version.

47. A method of creating a factory calibrated batch of aptamer sensors comprising: fabricating the aptamer sensors in a batch; calibrating a portion of the aptamer sensors (“the calibration portion”), producing at least one set of calibration data; identifying a portion of the aptamer sensors as being commercial quality (“the product portion”); and assigning the calibration data to the product portion of the aptamer sensors.

48. The method of claim 47 wherein the calibration data includes sensor response measured over multiple days.

49. The method of claim 47 wherein the calibration data includes a single point calibration captured after a period of time selected from the group consisting of 1, 2, and 3 hours of sensor operation.

50. The method of claim 47 wherein the calibration data includes a single point calibration and a predicted calibration curve based on previous or historical measurement data.

51. The method of claim 47 wherein the calibration data further comprises a measurement selected from the group consisting of aptamer density, frequency map, square wave voltammetry, cyclic voltammetry, intermittent pulse-amperometry, chronoamperometry, continuous square wave voltammetry, electrical impedance, oxygen reduction current and combinations thereof.

52. The method of claim 47 wherein the calibration data is modified using auto-calibration during use, and wherein the factory calibrated aptamer sensors further comprise a known or expected or predicted standard deviation for the sensor response; wherein auto-calibrationoccurs when the sensor response is changing greater than at least one of the standard deviation or twice the standard deviation of the sensor response.

53. The method of claim 52 wherein the auto-calibration shifts the frequency response utilized to identify at least one of the signal OFF frequency with maximum sensor response, the zero or non-responsive frequency, or the signal ON frequency with maximum response.

54. The method of claim 47 wherein the calibration data includes a frequency response, and during use the device measures at least one of the following to recalibrate the frequency response: change in aptamer density, electrical impedance, oxygen reduction current.

55. The method of claim 47 wherein the calibration data set is obtained in a human, only after or near the point of steady state of concentration of the analyte in interstitial fluid.

56. The method of claim 47, wherein factory calibration of the sensors is conducted immediately after fabrication of the sensors.

57. The method of claim 47, wherein factory calibration of the sensors is conducted after shelf-stabilization of the sensors.

58. The method of claim 47, wherein factory calibration of the sensors is conducted after attachment to electronics.

59. The method of claim 47, wherein factory calibration of the sensors is conducted after sterilization.

60. The method of claim 47, wherein factory calibration of the sensors is conducted at one or more time points and one or more measures to provide a sensor longevity calibration dataset.

61. The method of claim 47, wherein factory calibration of the sensors is conducted at a single point and the calibration is captured at a time after at least one of 1, 2, or 3 hours of operation.

62. The method of claim 1 wherein calibration data includes at least one measurementparameter.

63. The method of claim 62 wherein the measurement parameter is sensitivity.

64. The method of claim 63 where the sensitivity includes a plurality of different sensitivities for a plurality of different measurements.

Images