Apparatus, method, and system for measuring enzyme activity and enzyme concentration

Abstract

A method (300) is described for determining the concentration (360a) and enzymatic activity (360b) of one or more target molecules in a sample solution. The method includes providing (310) an apparatus (100) including one or more detection elements (212). One or more detection elements (212) include a capture detection element functionalized with one or more molecules that binds a target molecule. One or more detection elements (212) include an interaction detection element functionalized with one or more molecules that are modified by a target molecule. The apparatus (100) also includes a sample addition zone (106) and a flowpath (112) connecting the sample addition zone (106) and the detection elements (212). The method also includes delivering the sample solution (320) to the sample addition zone (106).

Description

[0001] Attorney Docket No. 204606-0200-00 WO

[0002] TITLE OF THE INVENTION

[0003] Apparatus, Method, and System for Measuring Enzyme Activity and Enzyme Concentration

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims priority to U.S. Provisional Application No. 63 / 697,805 filed on September 23, 2024, incorporated herein by reference in its entirety.

[0006] FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0007] This invention was made with government support under 70NANB22H015 awarded by the National Institute of Standards and Technology. The government has certain rights in the invention.

[0008] BACKGROUND OF THE INVENTION

[0009] Enzyme assays are a cornerstone of basic biology, clinical diagnosis, and biomanufacturing. Typically, enzyme activity is measured, but concentration of the enzyme is also of interest, as are comparisons between concentration and activity.

[0010] Demand for quality, accurate, quick, and efficient biosensors is present in the healthcare setting. Assessing different demands in the healthcare field is critical when evaluating different diagnostic needs. Diagnostic assays can utilize a variety of approaches for diagnosis. Detection of an antigen through a corresponding antibody is an extremely common technique found throughout healthcare and is widespread, with the ELISA assay being a commonly used test, available commercially for a variety of concentration detections. However, in addition to quantification of concentration through antibody, enzymes can be studied through their enzymatic properties, i.e., the cleavage of a substrate. This requires some type of fluorescently tagged substrate through a UV-vis absorbance spectrometry measurement. Both are valuable measurements that currently require separate assays for study. The use of photonic biosensors as a means to rapidly and inexpensively detect clinically relevant analytes is of great potential, as it provides real time kinetic information.

[0011] Detection of enzymes via photonic devices is common in the field of photonic Attorney Docket No. 204606-0200-00 WO biosensors. The use of a variety of photonic crystals and waveguide based silicon and silicon nitride devices utilizing integrated photonic circuits can detect multiple enzymatic analytes with high accuracy at low limits of detection (Inan, H.; et al. Chem. Soc. Rev. 2017, 46 (2), 366-388). The detection of such enzymes has relied upon the use of antibody binding.

[0012] The ability to detect a single antibody using a photonic ring resonator has been demonstrated (Cognetti, J. S.; et al. Lab. Chip 2021, 21 (15), 2913-2921). This detection was then expanded by multiplexing the design (Bryan, M.R.; et al. ACS Sens. 2024, 9, 1799-1808). Detection via photonic multiplexing was therefore established as viable and useful tool to quantify antibody concentration.

[0013] Detection of proteins can be detected by immobilizing capture antibodies on ring resonators and observing the shift in ring resonator peaks.

[0014] However, there remains a need in the art for simultaneous measurement of both target molecule concentration and target molecule enzymatic activity. The present invention satisfies this unmet need.

[0015] SUMMARY OF THE INVENTION

[0016] Aspects of the invention relate to a method for determining the concentration and enzymatic activity of one or more target molecules in a sample solution. The method can include providing an apparatus including one or more detection elements. One or more detection elements may be a capture detection element functionalized with one or more molecules that binds a target molecule. One or more detection elements may be an interaction detection element functionalized with one or more molecules that are modified by a target molecule. The apparatus may also include a sample addition zone and a flowpath connecting the sample addition zone and the detection elements. The method may also include delivering the sample solution to the sample addition zone.

[0017] In some embodiments, the apparatus also includes one or more control detection elements functionalized with one or more control molecules having lower binding affinity to the one or more target molecules than the capture molecule or the interaction molecule. Attorney Docket No. 204606-0200-00 WO

[0018] In some embodiments, the apparatus also includes one or more capture control detection elements functionalized with one or more capture molecules that bind a control target molecule, and one or more interaction control detection elements functionalized with one or more control molecules having lower affinity for the one or more target molecules than the interaction molecule.

[0019] In some embodiments, the method also includes passing light through each of the detection elements, detecting the light after passing through each of the detection elements before and after each of the elements contacts a sample solution, determining the resonance wavelength of each of the detection elements before each of the detection elements contacts a sample solution based on the detected light, determining the resonance wavelength of each of the detection elements after each of the detection elements contacts the sample solution based on the detected light, calculating the shift in resonance wavelength of each of the detection elements before and after each of the detection elements contacts the sample solution, determining the concentration of the one or more target molecules based on the resonance wavelength shift of the capture detection elements, and determining the enzymatic activity of the one or more target molecules based on the resonance wavelength shift of the interaction detection elements.

[0020] In some embodiments, the determination of the concentration of the one or more target molecules is also based on the difference between the resonance wavelength shift of the capture detection elements and the resonance wavelength shift of the capture control detection elements.

[0021] In some embodiments, the determination of the enzymatic activity of the one or more target molecules is also based on the difference between the resonance wavelength shift of the interaction detection elements and the resonance wavelength shift of the control interaction detection elements.

[0022] In some embodiments, the contact between the one or more target molecules and the capture detection elements forms a complex comprising the one or more target molecules and the one or more capture molecules bound to the capture detection elements. Attorney Docket No. 204606-0200-00 WO

[0023] In some embodiments, contact between the one or more target molecules and the interaction detection elements causes at least one or more interaction molecules or interaction molecule portions to be released from the interaction detection elements.

[0024] In some embodiments, contact between the one or more target molecules and the one or more capture detection elements causes an increase in the resonance wavelength of the one or more capture detection elements.

[0025] In some embodiments, contact between the one or more target molecules and the one or more interaction detection elements causes a decrease in the resonance wavelength of the one or more interaction detection elements.

[0026] In some embodiments, the method also includes adding a modulator of the one or more target molecules to the sample solution.

[0027] In some embodiments, the method also includes diagnosing a subject based on the determination of the concentration and enzymatic activity of the one or more target molecules in the sample solution.

[0028] In some embodiments, the method also includes obtaining the sample solution from a subject.

[0029] In some embodiments, the method also includes administering one or more modulators of the one or more target molecules to the subject.

[0030] In some embodiments, the method also includes administering one or more modulators of the one or more target molecules to a subject based on the determination of the enzymatic activity of the one or more target molecules in the sample solution.

[0031] In some embodiments, the detection elements include at least one of a ring resonator, a double ring resonator, a cylindrical resonator, a spherical resonator, a spiral waveguide, a Vernier filter, a photonic crystal, and a Mach-Zehnder Interferometer (“MZI”).

[0032] In some embodiments, the one or more capture molecules is selected from one or more of the group consisting of: a protein, a peptide, an antibody, an antigen, an enzyme, a substrate, and a nucleic acid. Attorney Docket No. 204606-0200-00 WO

[0033] In some embodiments, the one or more interaction molecules is selected from one or more of the group consisting of: a protein, a peptide, an enzymatic substrate, a nucleic acid, a small molecule enzymatic substrate, a phosphopeptide, and a polymer.

[0034] In some embodiments, the one or more control molecules is selected from one or more of the group consisting of: a protein, a peptide, an antibody, an antigen, an enzyme, a substrate, and a nucleic acid.

[0035] In some aspects, the invention relates to a system for determining the concentration and enzymatic activity of one or more target molecules in a sample solution. The system can include an apparatus. The apparatus can include one or more detection elements. One or more detection elements may be a capture detection element functionalized with one or more molecules that binds a target molecule. One or more detection elements may be an interaction detection element functionalized with one or more molecules that are modified by a target molecule. The apparatus may also include a sample addition zone and a flowpath connecting the sample addition zone and the detection elements.

[0036] In some embodiments, the system also includes a light source configured to pass light through each of the detection elements, a photodetector configured to receive the light after passing through each of the set of one or more detection elements, a display, a processor communicatively connected to the apparatus, the light source, the photodetector, and the display, and a non-transitory computer readable medium with instructions stored thereon, which when executed by a processor perform steps including passing a light spectra through each of the one or more detection elements with the light source, detecting the light spectra that has passed through each of the one or more detection elements with the photodetector, analyzing the detected light spectra, and determining a resonance wavelength of each of the detection elements based on the analysis of the detected light spectra.

[0037] In some embodiments, the apparatus also includes one or more control detection elements functionalized with one or more control molecules having lower binding affinity to the one or more target molecules than the capture molecule or the interaction molecule. Attorney Docket No. 204606-0200-00 WO

[0038] In some embodiments, the apparatus also includes one or more capture control detection elements functionalized with one or more capture molecules that bind a control target molecule, and one or more interaction control detection elements functionalized with one or more control molecules having lower affinity for the one or more target molecules than the interaction molecule.

[0039] In some embodiments, the non-transitory computer readable medium with instructions stored thereon, which when executed by a processor also perform steps including determining the resonance wavelength of each of the detection elements before each of the detection elements contacts a sample solution based on the detected light spectra, determining the resonance wavelength of each of the detection elements after each of the detection elements contacts the sample solution based on the detected light spectra, calculating the shift in resonance wavelength of each of the detection elements before and after each of the detection elements contacts the sample solution, and determining the concentration of the one or more target molecules based on the resonance wavelength shift of the capture detection elements, and determining the enzymatic activity of the one or more target molecules based on the resonance wavelength shift of the interaction detection elements.

[0040] In some embodiments, the determination of the concentration of the one or more target molecules is also based on the difference between the resonance wavelength shift of the capture detection elements and the resonance wavelength shift of the capture control detection elements.

[0041] In some embodiments, the determination of the enzymatic activity of the one or more target molecules is also based on the difference between the resonance wavelength shift of the interaction detection elements and the resonance wavelength shift of the control interaction detection elements.

[0042] In some embodiments, the contact between the one or more target molecules and the capture detection elements forms a complex comprising the one or more target molecules and the one or more capture molecules bound to the capture detection elements. Attorney Docket No. 204606-0200-00 WO

[0043] In some embodiments, contact between the one or more target molecules and the interaction detection elements causes at least one or more interaction molecules or interaction molecule portions to be released from the interaction detection elements.

[0044] In some embodiments, contact between the one or more target molecules and the one or more capture detection elements causes an increase in the resonance wavelength of the one or more capture detection elements.

[0045] In some embodiments, contact between the one or more target molecules and the one or more interaction detection elements causes a decrease in the resonance wavelength of the one or more interaction detection elements.

[0046] In some embodiments, the non-transitory computer readable medium with instructions stored thereon, which when executed by a processor also perform steps including diagnosing a subject based on the determination of the concentration and enzymatic activity of the one or more target molecules.

[0047] In some embodiments, the non-transitory computer readable medium with instructions stored thereon, which when executed by a processor also perform steps including determining a treatment for a subject based on the determination of the enzymatic activity of the one or more target molecules in the sample solution.

[0048] In some embodiments, the detection elements include at least one of a ring resonator, a double ring resonator, a cylindrical resonator, a spherical resonator, a spiral waveguide, a Vernier filter, a photonic crystal, and a Mach-Zehnder Interferometer (“MZI”).

[0049] In some embodiments, the one or more capture molecules is selected from one or more of the group consisting of: a protein, a peptide, an antibody, an antigen, an enzyme, a substrate, and a nucleic acid.

[0050] In some embodiments, the one or more interaction molecules is selected from one or more of the group consisting of: a protein, a peptide, an enzymatic substrate, a nucleic acid, a small molecule enzymatic substrate, a phosphopeptide, and a polymer. Attorney Docket No. 204606-0200-00 WO

[0051] In some embodiments, the one or more control molecules is selected from one or more of the group consisting of: a protein, a peptide, an antibody, an antigen, an enzyme, a substrate, and a nucleic acid.

[0052] In some aspects, the invention relates to a photonic integrated circuit (PIC) having at least one first grating coupler, at least two second grating couplers, at least one waveguide between the first grating coupler and the second grating couplers, at least one detection element functionalized with one or more capture molecules that bind one or more target molecule disposed within the at least one waveguide, and at least one detection element functionalized with one or more interaction molecules that are modified by the one or more target molecules disposed within the at least one waveguide.

[0053] In some embodiments, the PIC also has at least one detection element functionalized with one or more control molecules disposed within the at least one waveguide, wherein the control molecule has lower binding affinity to the one or more target molecules than the capture molecule or the interaction molecule.

[0054] In some embodiments, the PIC also has at least one detection element functionalized with one or more capture molecules that bind a control target molecule, and at least one detection element functionalized with one or more control molecules having lower affinity for the one or more target molecules than the interaction molecule.

[0055] In some embodiments, the detection elements include at least one of a ring resonator, a double ring resonator, a cylindrical resonator, a spherical resonator, a spiral waveguide, a Vernier filter, a photonic crystal, and a Mach-Zehnder Interferometer (“MZI”).

[0056] In some embodiments, the one or more capture molecules is selected from one or more of the group consisting of: a protein, a peptide, an antibody, an antigen, an enzyme, a substrate, and a nucleic acid.

[0057] In some embodiments, the one or more interaction molecules is selected from one or more of the group consisting of: a protein, a peptide, an enzymatic substrate, a nucleic acid, a small molecule enzymatic substrate, a phosphopeptide, and a polymer. Attorney Docket No. 204606-0200-00 WO

[0058] In some embodiments, the one or more control molecules is selected from one or more of the group consisting of: a protein, a peptide, an antibody, an antigen, an enzyme, a substrate, and a nucleic acid.

[0059] In some aspects, the invention relates to a substrate having a sample addition zone in fluid communication with a wicking zone and a sample detection zone, wherein the sample detection zone is between the sample addition zone and the wicking zone, and wherein the sample addition zone is upstream of the wicking zone; and at least one photonic integrated circuit (PIC) positioned in the sample detection zone, disposed directly on a top or bottom surface of the substrate, optically coupled to a light source and a photodetector via a fiber bundle.

[0060] BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0062] Figure 1 A through Figure ID depict a schematic of a sensing concept used in an exemplary embodiment of an enzyme concentration and enzyme activity sensor and photonics integrated circuit (PIC) and a Class-Laminated Adhesive Microfluidics (GLAM) card. Figure 1A depicts a schematic of immobilized antibody capture of a target enzyme producing a resonance red-shift. Figure IB depicts a schematic of enzymatic cleavage of an immobilized substrate producing a resonance blue-shift. Assays of Figure 1A and Figure IB can operate simultaneously on neighboring rings of a photonic chip (PIC). Figure 1C depicts an 8-ring photonic integrated circuit (PIC) used in experiments of Example #1. Figure ID depicts a GLAM card used for running assays.

[0063] Figure 2 depicts a schematic of probe and substrate print geometry, along with orientation of the PIC relative to the direction of microfluidic flow after assembly into a Glass Laminated Adhesive Microfluidics (GLAM). Attorney Docket No. 204606-0200-00 WO

[0064] Figure 3 depicts results of an antibody capture assay of a serial dilution of Cathepsin-L (CTSL). Each point represents the average of 3 replicate assays, with error bars ± one standard deviation shown. Red line shows nonlinear least squares fit of the data to a logistic model. Calculated LLOD = 2.0 ng / mL.

[0065] Figure 4 depicts serial dilution Cathepsin-L response (enzymatic degradation of immobilized azocasein). To enable curve fitting, the absolute value of the net shift was used, instead of the negative shift. Each point represents the average of 3 replicate assays, with error bars ± one standard deviation. The red line shows the calculated logistic fit of the data. Calculated LLOD = 1.8 ng / mL.

[0066] Figure 5 depicts results showing dual photonic assay successfully reports changes due to changes in CTSL concentration and inhibition of CTSL activity in human serum. Baseline concentration and activity of pooled normal human serum (PNHS) alone, doped with 10 ng / mL CTSL, and doped with 10 ng / mL CTSL plus inhibitor were recorded. Serum doped with 10 ng / mL Cathepsin-B (CTSB) produces a response statistically indistinguishable from that produced by serum alone, demonstrating the specificity of the assay. Each value represents the average of 6 replicate measurements, with error bars plus / minus one standard deviation.

[0067] Figure 6A through Figure 6B depict the response from both capture antibody and azocasein on the multiplex chip using individual human serum samples. Error Bars = + / - 2 standard deviations. Figure 6A depicts the overall magnitude (absolute value) of each shift, as the red shift from capture antibody binding (left bar for each sample) and the blue shift from azocasein degradation (right bar for each sample) result in shifts that occur in opposite directions. Figure 6B depicts the magnitude and direction of each shift (net shift), as the red shift from capture antibody binding and the blue shift from azocasein degradation result in shifts that occur in opposite directions.

[0068] Figure 7 depicts the response on GLAM cards using doped 25 single-donor human serum samples assayed before and after being doped with Cathepsin-L for a Cathepsin-L concentration of 10 ng / mL. The x-axis displays the red (antibody capture) and blue (azocasein degradation) shifts measured simultaneously for each sample, while the y-axis shows the sample number (SN). Attorney Docket No. 204606-0200-00 WO

[0069] Figure 8 depicts the results of an ELISA for Cathepsin-L (CTSL). Black squares are known concentrations of cathepsin-L in buffer; red square is from the commercial pooled normal human serum, with the measured absorbance used to extract the concentration with the known ELISA dilution response. The ELISA assay had a linear range specified from 24 pg / mL to 6 ng / mL, within the expected range of CTSL in serum.

[0070] Figure 9 depicts the results of UV-Vis spectroscopy to confirm the enzymatic activity of Cathepsin-L.

[0071] Figure 10 depicts a schematic of a method for measuring a target molecule concentration and a target molecule enzymatic activity in a sample solution.

[0072] Figure 11 depicts a diagram of a photonic biosensor including a substrate (cassette, slide, glass slide, membrane, fibrous substrate, or test card) and a PIC in accordance with some embodiments.

[0073] Figure 12 depicts a diagram of an example micropillar layout for a fluid flow path of a biosensor in accordance with some embodiments.

[0074] Figure 13 depicts a cut-away of the exemplary substrate of Figure 11 at a section of a sample detection zone that includes an optical input port and an optical output port in accordance with some embodiments.

[0075] Figure 14 depicts a cut-away of the exemplary substrate of Figure 11 at a section of a sample detection zone that includes an optical input port and an optical output port in accordance with some embodiments.

[0076] Figure 15 depicts an exemplary PIC in accordance with some embodiments.

[0077] Figure 16 depicts an exemplary multiplex hub optical system in accordance with some embodiments.

[0078] Figure 17 depicts an exemplary computing environment in accordance with some of the embodiments.

[0079] Figure 18 depicts a schematic of the layers an example layered fluidic layout for a fluid flow path of a biosensor in accordance with some embodiments.

[0080] Figure 19 depicts a schematic of an assembled example layered fluidic layout for a fluid flow path of a biosensor in accordance with some embodiments.

[0081] Figure 20 depicts a comparison of resonance shifts observed between CTSL substrates azocasein and casein. This assay was performed using a previous-generation Attorney Docket No. 204606-0200-00 WO micropillar microfluidics card described in Bryan, Butt, et al (Bryan, M. R. et al. ACS Sens. 2024.). Azocasein shows better dynamic range at the lower concentration, while casein shows better dynamic range at higher concentration.

[0082] Figure 21 depicts a representative example of the reference-subtracted signal seen for an anti-Cathepsin-L antibody capture. Representative red-shift response as a function of time seen from antibody-mediated capture of Cathepsin-L (CTSL) on a ring resonator. Concentration of Cathepsin-L was 100 ng / mL in pH 5.8 modified (i.e., potassium-free) phosphate-buffered saline (mPBS) in assay wash buffer (AWB) mixed with fetal bovine serum (FBS) at a 4: 1 ratio (FB20).

[0083] Figure 22 depicts a representative example of the reference-subtracted signal seen for CTSL-mediated substrate cleavage resulting in a blue shift. Representative timedependent blue shift seen from substrate cleavage by CTSL of azocasein on a ring resonator. Concentration of CTSL was 100 ng / mL in pH 5.8 mPBS with FB20.

[0084] Figure 23 depicts a comparison of a PIC with individual trenches around each ring resonator (left) and a PIC with a unified trench which was used in Example #1 (right).

[0085] Figure 24 depicts a design file (GDS) image and optical microscope image of a PIC.

[0086] Figure 25 depicts a schematic representation of a multiplex optical hub photonic biosensing apparatus. Visible-wavelength light (VIS), infrared-wavelength light (IR), single-mode fiber (SMF), multi-mode fiber (MMF), and universal serial bus (USB) are depicts. A GLAM disposable card is shown resting on the optical hub stage.

[0087] Figure 26 depicts the results of a reference standard UV-Vis enzymatic assay to establish activity of CTSL used in Example #1 and to assess levels of CTSL in pooled normal human serum (PNHS) and FB20.

[0088] DETAILED DESCRIPTION

[0089] The following discussion omits or only briefly describes conventional features of devices, methods, and systems for measuring enzyme activity and enzyme concentration that are apparent to those skilled in the art. Those of ordinary skill may thus recognize that other elements may be desirable and / or necessary to implement the devices, systems, Attorney Docket No. 204606-0200-00 WO and methods described herein. It is noted that various examples are described in detail with reference to the drawings. Reference to these various examples does not limit the scope of the claims attached hereto. Additionally, any examples set forth in this specification are intended to be non-limiting and merely set forth some of the many possible implementations for the appended claims. Further, particular features described herein can be used in combination with other described features in each of the various possible combinations and permutations. As such, it is understood that the detailed description is exemplary and explanatory only and is not restrictive of the broad inventive concepts upon which the examples disclosed herein are based.

[0090] Unless otherwise specifically defined herein, all terms are to be given their broadest reasonable interpretation. This includes meanings implied from the specification as well as meanings understood by those skilled in the art and / or as defined in dictionaries, treatises, etc.

[0091] It is noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless otherwise specified. The terms “includes” and / or “including,” when used in this specification, specify the presence of stated features, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0092] Relative terms such as “horizontal,” “vertical,” “up,” “down,” “top,” and “bottom” as well as derivatives thereof (e.g., “horizontally,” “downwardly,” “upwardly,” etc.) should be construed to refer to the orientation as then-described or as shown in the drawing figure under discussion. These relative terms are for convenience of description and normally are not intended to require a particular orientation in actuality. Terms including “inwardly” versus “outwardly,” “longitudinal” versus “lateral” and the like are to be interpreted relative to one another or relative to an axis of elongation, or an axis or center of rotation, as appropriate. Terms concerning attachments, coupling and the like, such as “connected” and “interconnected,” refer to a relationship wherein structures are secured or attached to one another either directly or indirectly through intervening structures, as well as both movable or rigid attachments or relationships, unless expressly described otherwise. The phrases “operatively” or “operably connected” indicates such Attorney Docket No. 204606-0200-00 WO an attachment, coupling or connection that allows the pertinent structures to operate as intended by virtue of that relationship.

[0093] Reference throughout the specification to “exemplary”, “one example”, “an example” or “some examples” means that a particular feature, structure, or characteristic described in connection with at least one example of the subject matter disclosed. Thus, the appearance of the phrases “in one example”, “in an example” or “in some examples” in various places throughout the specification is not necessarily referring to the same example. Further, the particular features, structures or characteristics of “one example”, “an example” or “some examples” may be combined in any suitable manner with each other to form additional examples of such combinations. It is intended that examples of the disclosed subject matter cover modifications and variations thereof. Terms such as “first,” “second,” “third,” etc., merely identify one of a number of portions, components, steps, operations, functions, and / or points of reference as disclosed herein, and likewise do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation.

[0094] Moreover, throughout this disclosure, various aspects can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, 6, and any whole and partial increments there between. This applies regardless of the breadth of the range. As used herein, the term “about” in reference to a measurable value, such as an amount, a temporal duration, and the like, is meant to encompass variations of plus or minus 20%, plus or minus 10%, plus or minus 5%, plus or minus 1%, and plus or minus 0.1% of the specified value, as such variations are appropriate.

[0095] The terms “proximal,” “distal,” “anterior,” “posterior,” “medial,” “lateral,” “superior,” and “inferior” are defined by their standard usage indicating a directional Attorney Docket No. 204606-0200-00 WO term of reference. For example, “proximal” refers to a position that is situated nearer to the center of a body or point of attachment or interest, while “distal” refers to a position that is situated away from the center of the body or point of attachment or interest. In another example, “anterior” refers to the front of a body or structure, while “posterior” refers to the rear of a body or structure, in relation to a relative viewpoint. In another example, “medial” refers to the direction towards the midline of a body or structure, and “lateral” refers to the direction away from the midline of a body or structure. In some examples, “lateral” or “laterally” may refer to any sideways direction. In another example, “superior” refers to the top of a body or structure, while “inferior” refers to the bottom of a body or structure. It should be understood, however, that the directional term of reference may be interpreted within the context of a specific body or structure, such that a directional term referring to a location in the context of the reference body or structure may remain consistent as the orientation of the body or structure changes.

[0096] The present invention discloses novel systems, methods, and devices for simultaneously measuring enzymatic activity and enzyme concentration. It should be appreciated that aspects of the devices, systems, and methods disclosed herein may be operated in conjunction with other systems or devices, including any assays to measure analyte concentrations, any assays to measure enzymatic activity, any clinical diagnostic assays, drug administration systems or devices, and / or any medical treatments.

[0097] In some aspects, the invention relates to a device for determining the concentration of one or more target molecules and the enzymatic activity of one or more target molecules in a sample solution. In some embodiments, the concentration and enzymatic activity of the same target molecule in a sample solution is measured. In some embodiments, the device comprises one or more detection elements including ring resonators functionalized with any capture molecule or any plurality of capture molecules. In some embodiments, the capture molecule binds to a target molecule in the sample solution thereby generating a complex of the capture molecule and target molecule on the ring resonator. In some embodiments, the device comprises one or more ring resonators functionalized with an interaction molecule or a plurality of interaction molecules. In some embodiments, the interaction molecule is modified by a target molecule in a sample solution. For example, the target molecule may cleave some or all Attorney Docket No. 204606-0200-00 WO of the interaction molecule to release some or all of the interaction molecule from the ring resonator thereby decreasing the size or amount of the interaction molecule on the ring resonator. Changes in the size or amount of molecules, for example by formation of a capture molecule-target molecule complex or cleavage of interaction molecule on the ring resonator may be detected by the device and may refer to the concentration or enzymatic activity of a target molecule in a sample solution. For example, the resonant wavelength of a ring resonator may change based on the size or amount of molecules on the ring resonator. The resonant wavelength of a ring resonator may be measured at any point. For example, the resonant wavelength may be measured before and after the ring resonator is exposed to a sample solution or a control solution.

[0098] Referring to Figure 10, in some aspects, the invention relates to a method 300 of measuring a target molecule concentration and a target molecule enzymatic activity in a sample solution. In some embodiments, the method 300 comprises the steps of 310 providing a device with one or more detection elements, 320 delivering a sample solution to the one or more detection elements, 330 passing light through the one or more detection elements, 340 detecting spectra of the light after passing through the detection element, 350 determining a refractive index shift of the one or more detection elements, 360a determining the concentration of a target molecule in the sample solution based on the refractive index shift, and 360b determining the enzymatic activity of a target molecule in the sample solution based on the refractive index shift.

[0099] In some embodiments, any device or methods for using any device with one or more detection elements are provided. Exemplary devices and device components including detection elements, microfluidic slides, cassettes, membranes, fibrous substrates, test cards, photonic integrated circuits, laser sources, and other device components are described in Bryan at al. (Bryan, M.R.; et al. ACS Sens. 2024, 9, 1799- 1808), U.S. Patent Application 17 / 585,914, and International Patent Application No. PCT / US2023 / 066909 each of which is incorporated by reference in its entirety.

[0100] In some embodiments, the device is a photonic biosensor. The photonic biosensor may include a microfluidic slide, a glass slide, a cassette, a membrane, a fibrous substrate, or a test card. In some embodiments, the device may include at least one photonic integrated circuit (“PIC”) having at least one detection element. Attorney Docket No. 204606-0200-00 WO

[0101] The detection element may be connected to a fluid pathway provided on a slide, cassette, membrane, fibrous substrate, or test card. The example slide, cassette, membrane, fibrous substrate, or test card may also include an area for receiving a sample solution, where the fluid pathway uses wicking or capillary action (or other passive microfluidic transport structure) to passively pull a sample solution into contact with the one or more detection elements. Additionally, the slide, cassette, membrane, fibrous substrate, or test card may include optical ports for optically coupling with a light source and light detector of a laboratory analyzer, PoC device, or other analyte analysis device.

[0102] During use, in an example embodiment, a sample solution is applied to a receiving area surface, also referred to as a sample addition zone, of the example slide, cassette, membrane, fibrous substrate, or test card. A fluid pathway can include a passive microfluidic transport that causes the applied sample solution to flow to a detection zone and wicking zone. The detection zone includes a silicon-based PIC having one or more functionalized detection elements. In some embodiments, the detection element of the PIC is functionalized with one or more types of capture molecules, interaction molecules, or control molecules. In some embodiments, light is applied by a light source of an instrument to an input light port of the example slide, cassette, membrane, fibrous substrate, or test card, which directs the light through the PIC and the detection element. In some embodiments, light is applied through the example slide, cassette, membrane, fibrous substrate, or test card, which directs the light through the PIC and the detection element. In some embodiments, an output light port of the example slide, cassette, membrane, fibrous substrate, or test card receives the light after passing through the detection element. In some embodiments, output light is read directly through the example slide, cassette, membrane, fibrous substrate, or test card. Contact between the functionalized detection element and the fluid sample solution causes refractive light index changes, which are detected by a light detector or photosensor of the instrument. The degree of the refractive light index change is indicative of the presence of one or more target molecules, a concentration of one or more target molecules, and / or the enzymatic activity of one or more target molecules.

[0103] In some embodiments, a sample solution is delivered to the one or more detection elements of the device in any manner. In some embodiments, the sample solution is Attorney Docket No. 204606-0200-00 WO delivered to the sample zone such that it subsequently flows to the detection zone and then subsequently the wicking zone. In some embodiments, the sample solution is pipetted, dropped, injected, or poured into the sample addition zone. In some embodiments, the sample solution is directly pipetted or dropped onto the detection element.

[0104] Any solution may be used as a sample solution. In some embodiments, the sample solution is any biological fluid, serum, blood, whole blood, plasma, cerebral spinal fluid, tumor aspirate, infection site aspirate, Medium Enriched for Secreted Antibodies, saliva, urine, cell culture media, spent cell culture media, any cell lysate including human cell lysate, or any combination of biological fluids. In some embodiments, the sample solution is derived from a patient or human subject. The sample solution may be diluted before being added to the device. The sample solution may be diluted with any buffer solution, any saline solution, or water. The sample solution may be diluted to achieve a desired viscosity to promote flow. The sample solution may be diluted such that a target molecule concentration in the sample solution is reduced to a detectable range. In other words, dilution may be employed to reduce the concentration of the target molecule in the sample solution such that the capture molecule or interaction molecule of a detection sensor does not become saturated after contacting the sample solution. In some embodiments, any molecule, drug, inhibitor, catalyst or co-enzyme may be added to the sample solution to aid or inhibit the interaction of a target molecule with an interaction molecule.

[0105] In some embodiments, the sample solution or the device may be used at any temperature such that the enzymatic activity of a target molecule in the sample solution may be assayed at any temperature, for example the body temperature of a human or animal subject. In some embodiments, the sample solution is pH balanced such that the enzymatic activity of a target molecule may be assayed at any pH.

[0106] In light of the disclosure herein and without limiting the disclosure in any way, in one aspect of the present disclosure, which may be combined with any other aspect listed herein unless specified otherwise, a photonic biosensor apparatus includes a substrate having a sample addition zone, a wicking zone, and a detection zone located between the sample addition zone and the wicking zone. The substrate also includes a fluid pathway Attorney Docket No. 204606-0200-00 WO that fluidly couples (e g., in fluid communication with) the sample addition zone, the detection zone, and the wicking zone, an optical input port located at a section of the sample detection zone and configured to optically couple to a light source, and an optical output port located at the section of the sample detection zone and configured to optically couple to a light detector. The photonic biosensor apparatus also includes a photonic integrated circuit connected to the substrate at the section of the sample detection zone. The photonic integrated circuit includes a first grating coupler aligned with the optical input port, at least two second grating couplers aligned with the optical output port, at least one waveguide between the first grating coupler and the second grating coupler, and at least one detection element provided along the at least one waveguide and positioned to contact fluid sample solution within the fluid pathway at the section of the sample detection zone.

[0107] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the fluid pathway includes a sample addition zone, a detection zone, and a wicking zone. The fluid pathway may be configured such that a sample solution can be added to the sample addition zone and flow from the sample zone to the detection zone and then to the wicking zone. In other words, the detection zone may be positioned in the flow path in between the sample zone and the wicking zone. The fluid pathway may also include an enhancer zone positioned at any point in the fluid pathway.

[0108] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the sample zone is configured such that a sample solution may be applied and then flow to the detection zone and the wicking zone. The wicking zone may include any component that promotes the wicking or collection of the applied sample solution, for example micropillars, a micropillar reservoir, a wicking pad, a Whatman wicking pad, nitrocellulose paper, any absorbent material, or any reservoir.

[0109] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the at least one detection element includes at least one of a ring resonator, an optical ring resonator, a silicon nitride ring resonator, a double ring resonator, a cylindrical resonator, a spherical Attorney Docket No. 204606-0200-00 WO resonator, a spiral waveguide, a photonic crystal, or a Mach-Zehnder Interferometer (“MZI”). The at least one detection element may be functionalized with any molecule. At least one or more detection elements may be functionalized with different molecules. The detection element may have any dimensions. For example, the detection element may be a ring resonator having any diameter, for example about 198 pm or about 164 pm. Ring resonator dimensions can vary widely and may be dependent on the material that the ring resonator is constructed from.

[0110] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the at least one waveguide includes a silicon nitride waveguide. The at least one waveguide may have any dimensions. For example, a waveguide that comprises a ring resonator may be about 1.5 pm wide and about 220 nm tall.

[0111] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the photonic integrated circuit has a rectangular prism or cuboid shape.

[0112] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the photonic integrated circuit has a rectangular prism or cuboid shape.

[0113] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the first grating coupler is provided at a first side of a face of the photonic integrated circuit, the at least two second grating couplers are provided at a second, opposing side of the same face of the photonic integrated circuit, and the at least one detection element is positioned between the first side and the second side.

[0114] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the photonic integrated circuit has a length between 2-20 mm, a width between 0.25-10 mm, and a height between 0.1-5 mm.

[0115] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the substrate includes an enhancer zone or a conjugate zone between the detection zone and the sample addition Attorney Docket No. 204606-0200-00 WO zone along the fluid pathway, the enhancer zone or the conjugate zone includes at least one reagent for binding with the fluid sample solution.

[0116] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the optical input port includes a first tunnel through the substrate and the optical output port includes a second tunnel through the substrate.

[0117] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the first tunnel is located on a first side of the fluid pathway in the section and the second tunnel is located on an opposite, second side of the fluid pathway in the section.

[0118] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the substrate includes at least one of a slide, glass slide, cassette, membrane, fibrous substrate, or test card.

[0119] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the light source and the photodetector are included within a read head of at least one of a laboratory analyzer or a point-of-care (“PoC”) analyzer.

[0120] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, at least some of the fluid pathway includes micropillars or projections that are substantially vertical to the surface of the substrate and having a height, diameter, and reciprocal spacing such that lateral capillary flow of the fluid sample solution is achieved.

[0121] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the height is between 1-1000 pm, the diameter is between 10-100 pm, and the reciprocal spacing is between 5-100 pm.

[0122] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the detection zone is configured to provide at least one of fluorescence or colorimetric detection of one or more target molecules within the fluid sample solution. In some embodiments, a second antibody or other molecule may be used to increase mass of a detection element. For example, a solution containing a capture molecule may be delivered to a ring resonator to Attorney Docket No. 204606-0200-00 WO increase the mass of a bound capture molecule / target molecule complex such that smaller increases in refractive index shift could be increased. In some embodiments, an interaction molecule may be conjugated with a fluorescent molecule such that the fluorescent molecule is also cleaved upon interaction with the target molecule. Therefore, enzymatic activity of the target molecule may be secondarily confirmed or measured by a fluorescence readout.

[0123] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the photonic integrated circuit is connected to the substrate using at least one of a UV curable adhesive, physical stacking, or a tape / glue application.

[0124] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the optical input port is a first optical input port, the optical output port is a first optical output port, the section is a first section, and the photonic integrated circuit is a first photonic integrated circuit, and wherein the substrate further includes a second optical input port located at a second section of the sample detection zone and configured to optically couple to the light source, and a second optical output port located at the second section of the sample detection zone and configured to optically couple to the light detector.

[0125] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the apparatus further comprises a second photonic integrated circuit connected to the substrate at the second section, the second photonic integrated circuit including a first grating coupler aligned with the second optical input port, at least two second grating couplers aligned with the second optical output port, at least one waveguide between the first grating coupler and the second grating coupler, and at least one detection element provided along the at least one waveguide and positioned to contact fluid sample solution within the fluid pathway at the second section.

[0126] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the light source is a laser or a tunable laser. In some embodiments the light is passed through a polarization controller such that the light is linearly polarized with TE orientation relative to a waveguide that Attorney Docket No. 204606-0200-00 WO comprises a detection element on the device. Any coherent light source that is tunable within some range, for example 1-10 nm, of a central frequency may be used. In some embodiments, TM polarized light may be used.

[0127] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the at least one detection element of the first photonic integrated circuit is configured for the detection of a first target molecule and the at least one detection element of the second photonic integrated circuit is configured for the detection of a second target molecule.

[0128] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the at least one detection element of the first photonic integrated circuit is configured for the detection of a first target molecule, including a concentration of a first target molecule, and the at least one detection element of the second photonic integrated circuit is configured for the detection of the enzymatic activity of the first target molecule.

[0129] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the at least one detection element of the first photonic integrated circuit is configured for the detection of the enzymatic activity of a first target molecule, and the at least one detection element of the second photonic integrated circuit is configured for the detection of the enzymatic activity of a second target molecule.

[0130] In another aspect of the present disclosure, which may be used in combination with any other aspect listed herein unless stated otherwise, the at least one detection element of the first photonic integrated circuit is configured for the detection of nonspecific binding of components in a sample solution to a detection element, and the at least one detection element of the second photonic integrated circuit is configured for the detection of the concentration or enzymatic activity of a target molecule.

[0131] In some embodiments, the detection zone may include any number of PICs, for example silicon-based PICs, having any number of functionalized detection elements for example functionalized ring resonators. In some embodiments, one or more detection elements of a PIC are functionalized with one or more types of capture molecules. A capture molecule may be chosen, designed, or engineered to bind to a target molecule in Attorney Docket No. 204606-0200-00 WO the sample solution. The binding of a capture molecule to a target molecule in the sample solution may generate a complex of the capture molecule bound to the target molecule on the detection element. The capture molecule may have any affinity for the target molecule. The capture molecule may have a high affinity for the target molecule such that the capture molecule remains bound to the target molecule and the complex remains on the detection element after any number of washing or rinsing steps. The capture molecule may have a high affinity for the target molecule such that the capture molecule binds the target molecule after the ring resonator is incubated with a sample solution for a short period of time. For example, the time it takes for a specified volume of the sample solution to flow past the detection zone.

[0132] Suitable examples of target molecules include but are not limited to, nucleic acids (e.g., deoxyribonucleic acids and ribonucleic acids), aptamers, polypeptides (e.g., proteins and enzymes), antibodies, antigens, enzymes, proteases, nucleases, metabolites, small molecules, and lectins.

[0133] Suitable examples of capture molecules include but are not limited to, nucleic acids (e.g., deoxyribonucleic acids and ribonucleic acids), aptamers, polypeptides (e.g., proteins and enzymes), antibodies, antigens, and lectins. As will be appreciated by one of ordinary skill in the art, any molecule that can specifically associate with a target molecule can be used as a capture molecule. In certain embodiments, the target molecule and capture molecule represent a binding pair. In certain embodiments, the target molecule is an enzyme and the capture molecule is a substrate that covalently binds the enzyme. It will also be understood that binding pairs of target molecules and capture molecules described above can be reversed in several embodiments (e.g., in one embodiment an antibody that specifically binds to an antigen can be the target molecule and the antigen can be the capture molecule, whereas in another embodiment the antibody can be the capture molecule and the antigen can be the target molecule). The capture molecule may have a binding affinity for the target molecule such that there is a low probability of dissociation of a capture molecule / target molecule complex on the timescales at which the device and method are used. The capture molecule may irreversibly bind a target molecule. Attorney Docket No. 204606-0200-00 WO

[0134] In some embodiments, one or more of the PICs in the detection zone include one or more detection elements functionalized with one or more types of interaction molecules. An interaction molecule may be chosen, designed, or engineered such that it may be modified by a target molecule that is in the sample solution. Modifications of an interaction molecule by a target molecule may include any modifications that decrease the size or amount of molecules that remain on the detection element. For example, modifications of the interaction molecule may include cleavage or degradation of the interaction molecule. An interaction molecule that is degraded or cleaved may be released from the detection element. In some embodiments, some or all of the interaction molecule may be cleaved by the target molecule thereby releasing some or all of the interaction molecule from the detection element. For example, the target molecule may be an enzyme and the corresponding interaction molecule may be a cleavable substrate of the enzyme. For example, the target molecule may be a protease including a collagenase and the capture molecule may be collagen. In some embodiments, the target molecule may be a nuclease and the corresponding interaction molecule may be a nucleic acid comprising a sequence that is recognized by the nuclease. In some embodiments, the interaction molecule may include any number of moieties or peptide sequences that are recognized for cleavage or degradation by a target molecule. In some embodiments, the interaction molecule is a polymer that can be sequentially degraded by a target molecule.

[0135] The size or mass of an interaction molecule may be chosen to tune the detection range of the device, method, or system. For example, the interaction molecule may be designed or chosen such that a different mass is cleaved from the interaction molecule upon interaction with a target molecule. For example, a greater size of the cleavable or releasable portion of the interaction molecule would result in a greater change in mass bound to the detection element per interaction with a target molecule. Interaction molecules with a larger cleavable or releasable portion may result in greater shifts of refractive index per every target molecule interaction. For example, interaction molecules with a larger or more massive cleavable substrate may be chosen to accurately detect low concentrations of a target molecule or less enzymatically active target molecules. In another example, interaction molecules with a smaller or less massive cleavable substrate may be chosen to accurately detect high concentrations of a target molecule or more Attorney Docket No. 204606-0200-00 WO enzymatically active target molecules. In some embodiments, a device may contain one or more interaction molecule detection elements optimized for low concentrations of a target molecule or less enzymatically active target molecules and one or more interaction molecule detection elements optimized for high concentrations of a target molecule or more enzymatically active target molecules. In some examples, the target molecules is a nuclease and the interaction molecule is a nucleic acid. The nuclease may recognize a recognition site of a nucleic acid target molecule. The number of nucleotide bases on the detection element side or the cleavable side of the recognition site may be chosen for a desired mass of interaction molecule and desired mass of the cleavable portion of the interaction molecule. In some examples, the target molecule is Cathepsin-L and the interaction molecule may be casein or azocasein as in Figure 20. Casein and Azocasein are different sizes and may be more useful for accurately measuring different concentrations of cathepsin-L or cathepsin-L with different levels of enzymatic activity.

[0136] In some embodiments, the device includes one or more PICs that include one or more detection elements functionalized with one or more types of capture molecules specific for a target molecule and one or more PICs that include one or more detection elements functionalized with one or more types of interaction molecules specific for the same target molecule. In some embodiments, the device includes a PIC that includes one or more detection elements functionalized with one or more types of capture molecules specific for a target molecule and one or more detection elements functionalized with one or more types of interaction molecules specific for the same target molecule. In these embodiments, the concentration of a target molecule in the sample solution may be measured using a detection element functionalized with the capture molecule while the enzymatic activity of the same target molecule may be measured using the detection element functionalized with the interaction molecule. In this embodiment, the concentration and enzymatic activity of a target molecule is measured in the same assay in the same sample solution. In some examples, the concentration and enzymatic activity of a target molecule is measured simultaneously. In some examples, the concentration and enzymatic activity of a target molecule is measured using the same device or apparatus. In some embodiments, the concentration and enzymatic activity of a target molecule is measured in the same flow path of a device or chip. Attorney Docket No. 204606-0200-00 WO

[0137] In some embodiments, the concentration and enzymatic activity of a target molecule is measured using the same chip or substrate. For example, in one embodiment, the device and method are used to detect the concentration and enzymatic activity of cathepsin-L. In one embodiment, the device comprises a detection element comprising a ring resonator functionalized with an antibody that binds to cathepsin-L and further comprises a second detection element comprising a ring resonator functionalized with azocasein, which is cleaved upon interaction with cathepsin-L. The concentration and enzymatic activity of cathepsin-L in a biological fluid may relate to the presence or severity of Covid- 19 or any other disease, disorder, or injury.

[0138] In some embodiments, the concentration and enzymatic activity of a target molecule is measured using the same chip or substrate. For example, in one embodiment, the device and method are used to detect the concentration and enzymatic activity of a collagenase or matrix metalloproteinase. In some embodiments, the device comprises a detection element comprising a ring resonator functionalized with an antibody that binds to a collagenase, a matrix metalloproteinase (MMP), or any other protease that can recognize collagen, and further comprises a second detection element comprising a ring resonator functionalized with collagen, which is cleaved upon interaction with the collagenase or the matrix metalloproteinase. Any collagenase or MMP subtype concentration and enzymatic activity may be detected.

[0139] In some embodiments, the concentration and enzymatic activity of a target molecule is measured using the same chip or substrate. In some embodiments, the target molecule is alkaline phosphatase, cathepsin-D, MMP-9, lysozyme, tartrate-resistant acid phosphatase, amylase, and / or lipase.

[0140] In some embodiments, the device includes one or more PICs that include one or more detection elements functionalized with any molecule such that the detection element may act as a control. The control may be designed such that the change in refractive index due to nonspecific binding or nonspecific adherence of components of the sample solution to the detection element may be determined. The control detection element may be functionalized with a random molecule, any protein solution including serum or serum albumin, any solution known in the art for protein blocking, for example serum, albumin, or milk, any antibody specific for an antigen that is known to not be Attorney Docket No. 204606-0200-00 WO present or be in a low concentration in the sample solution of interest, any antibody specific for an antigen that has a known concentration in the sample solution of interest, or any immunoglobulin species. The control molecule may be chosen such that is has a lower binding affinity for the target molecule than a capture molecule or interaction molecule for the target species.

[0141] In some embodiments, the device includes one or more PICs that include one or more detection elements functionalized with any molecule such that the detection element may act as a capture molecule control detection element for a corresponding capture molecule detection element. For example, a capture molecule control detection element may be functionalized with any molecule with low binding affinity for any target molecule of interest. In some examples, a capture molecule control detection element is functionalized with any molecule that has a lower binding affinity to the target molecule than a corresponding capture molecule for the target molecule. In some embodiments, a capture molecule control detection element of a PIC is designed to correspond with a capture molecule detection element of a PIC. In some embodiments, a capture molecule control molecule may be a capture molecule for one or more control target molecules. A control target molecule may be any molecule that is not one of the one or more target molecules. A control target molecule may be known not to be present in the sample solution, be present in a negligible amount in the sample solution, or have a known concentration in the sample solution. For example, the capture molecule detection element may be functionalized with an antibody specific for a target molecule, and the corresponding capture molecule control detection element may be functionalized with an antibody of the same species and / or type that is instead specific to an antigen that is known not to be present, present in low concentrations, or present in a known concentration in the sample solution. For example, the capture molecule detection element may be functionalized with a single-stranded nucleic acid that binds with a nucleic acid target molecule, and the capture molecule control detection element may be functionalized with a single-stranded nucleic acid that binds with a nucleic acid molecule that is known not to be present, present in low concentrations, or present in a known concentration in the sample solution. In some embodiments, the capture molecule control detection element may be functionalized with a random molecule, any protein solution Attorney Docket No. 204606-0200-00 WO including serum or albumin, any solution known in the art for protein blocking, for example serum, albumin, or milk, any antibody specific for an antigen that is known to not be present or be in a low concentration in the sample solution of interest, or any immunoglobulin species.

[0142] In some embodiments, the device includes one or more PICs that include one or more detection elements functionalized with any molecule such that the detection element may act as an interaction molecule control detection element. The interaction molecule control detection element may be functionalized with a molecule with low affinity to a target molecule. The interaction molecule control detection element may correspond with an interaction molecule detection element of the PIC. For example, the interaction molecule control detection element may be functionalized with a molecule that is a similar size, structure, or composition to the interaction molecule of a corresponding interaction molecule detection element. In some examples, the interaction molecule control detection element may be functionalized with a molecule that has a lower binding affinity to the target molecule than the interaction molecule of a corresponding interaction molecule detection element. In some examples, the interaction molecule control detection element is functionalized with molecule that is not suitable for cleavage or degradation by a target molecule. In some examples, the interaction molecule control detection element may be functionalized with a random molecule, any serum, any serum albumin, any milk, or any protein blocking solution. In some examples, the interaction molecule control detection element may be functionalized with a nucleic acid. In some examples, the interaction molecule control detection element may be functionalized with a nucleic acid with a sequence of nucleotides such that it is known not to specifically bind any molecules in a sample solution to an appreciable level.

[0143] In some embodiments, the device includes one or more PICs that include at least one capture molecule detection element for a target molecule and at least one interaction molecule detection element for the same target molecule such that the enzymatic activity and concentration of the target molecule can be measured. In some embodiments, the device further includes a control molecule detection element such that the contribution of nonspecific binding to the detection element can be measured. Attorney Docket No. 204606-0200-00 WO

[0144] In some embodiments, the detection elements are in any position relative to the flow path of the sample solution. The detection elements may be of the same PIC or a different PIC. For example, the detection elements may be positioned as in Figure 2 and the sample solution may flow across the detection elements from left to right as depicted. Alternatively, the detection elements may be positioned as in Figure 2 and the sample solution may flow across the detection elements from right to left (i.e., flow may be opposite of the direction depicted in Figure 2). Capture molecule detection elements may be positioned upstream or downstream of interaction molecule detection elements for the same target molecule in relation to the sample solution flow path. In some embodiments, control detection elements are in any position relative to other detection elements and the sample solution flow path.

[0145] In some embodiments, any amount of a sample solution is provided to the sample addition zone. In some embodiments about 50 pL of a sample solution is provided to the sample addition zone. In some embodiments, the maximum sample solution amount allowed by a device sample addition zone is provided. In some embodiments, the flow path promotes passive flow at any rate. In some embodiments, the flow path promotes laminar flow at any rate.

[0146] Contact between the sample solution and the functionalized detection element may cause refractive light index changes, which are detected by a light detector or photosensor of the instrument. In other words, contact between the sample solution and the functionalized detection element may cause a change in the resonance peak wavelength of the functionalized detection element. The degree of the refractive light index change can be indicative of the presence of one or more target molecules, a concentration of one or more target molecules, and / or the enzymatic activity of a target molecule. The degree of the change in wavelength of the resonance peak of the detection element can be indicative of the presence of one or more target molecules and / or a concentration of one or more target molecules, or the enzymatic activity of a target molecule.

[0147] As in Figure 1, in the case when the detection element is functionalized with a capture molecule, contact with the target molecule and generation of a target molecule - capture complex on the detection element can cause a refractive index increase. A Attorney Docket No. 204606-0200-00 WO refractive index increase may also be referred to as a red shift in the resonance peak wavelength. A greater magnitude of the increase in refractive index can refer to a greater target molecule concentration in a sample solution. In the case when the detection element is functionalized with an interaction molecule, contact with the target molecule and subsequent degradation or cleavage of the interaction molecule from the detection element can cause a refractive index decrease. A refractive index decrease may also be referred to as a blue shift in the resonance peak. A greater magnitude of the decrease in refractive index can refer to greater enzymatic activity of a target molecule in a sample solution.

[0148] In some embodiments, output light spectra are detected and / or recorded after the light has passed through a PIC including at least one detection element. Light spectra may be detected by a detector. Spectral measurements may be recorded continuously or at any frequency. Spectral measurements may be recorded before, during, and after sample solution contacts a detection element of the device. Spectral measurements may be recorded over time so that target molecule - capture molecule binding kinetics and target molecule - interaction molecule reaction kinetics can be determined. Spectral measurements may be represented as the optical loss corresponding to a range of light wavelengths compared to input light.

[0149] Any method known in the art may be used to analyze recorded spectra and determine the resonance peak of a detection element. For example, a Lorentzian function or any other function may be fitted to the plot of optical loss of the input light in decibels vs light wavelength. Features of the plot including peak location, peak height, quality factor, chi-squared values, and peak fitting parameters may be extracted from the fitted function.

[0150] The resonance peak may be determined before and after sample solution contacts the detection element to determine the change in resonance peak wavelength due to contact with the sample solution. The measured change in resonance peak wavelength of a detection element before and after contacting a sample solution is referred to as a resonance shift. In some embodiments, the resonance wavelength shift of a control detection element is subtracted from the resonance wavelength shift of a capture molecule detection element or interaction molecule detection element. In some Attorney Docket No. 204606-0200-00 WO embodiments, resonance wavelength shift of a capture molecule control detection element is subtracted from the resonance wavelength shift of a corresponding capture molecule detection element. In some embodiments, resonance shift of an interaction molecule control detection element is subtracted from the resonance shift of a corresponding interaction molecule detection element. This subtraction may correct for the contributions to resonance wavelength shift due to nonspecific binding of sample solution components to a detection element or nonspecific release of detection element functionalized molecules.

[0151] In some embodiments, as in Figure 3 and Figure 4, a calibration is performed such that the relationship between resonance shift of a detection element and the concentration of a target molecule can be determined. A calibration can be performed by plotting resonance shift against sample solutions with a known concentration of a target molecule. Therefore any measured resonance shift of a detection element may correspond to a concentration of a target molecule in a sample solution.

[0152] In some embodiments, a subject may be diagnosed with a disease, disorder, or condition based on the determination of any number of target molecule concentrations, any number of target molecule enzymatic activities, or a combination of any measurements of target molecule concentration and enzymatic activity. In some embodiments, the severity or stage of a subject’s disease, disorder, or condition may be determined based on the determination of any number of target molecule concentrations, any number of target molecule enzymatic activities, or a combination of any measurements of target molecule concentration and enzymatic activity. In some embodiments, the disease, disorder, or condition is Covid- 19, influenza, fasciolosis, liver disease, cardiac disease, cancer, kidney failure, parasitic infection, carcinoma, cardiomyopathy, or pancreatic cancer.

[0153] For example, the target molecule may be cathepsin-L in the case of diagnosing the presence or severity of Covid- 19, kidney failure, parasitic infection, carcinoma, cardiomyopathy, or pancreatic cancer. For example, greater cathepsin-L concentrations and / or greater cathepsin-L enzymatic activity in a biological fluid, for example serum, of a subject may correspond to the presence or severity of Covid- 19, kidney failure, parasitic infection, carcinoma, cardiomyopathy, or pancreatic cancer in a subject. In some Attorney Docket No. 204606-0200-00 WO examples, the concentration and enzymatic activity of cathepsin-L can be used to assess if a subject has at one point in the past been infected with Covid- 19.

[0154] In some aspects, device includes or is used in combination with a cell culture system. In some examples, the device may monitor the concentration or enzymatic activity of a target molecule in a cell culture system in real time. For example, the device may monitor any target molecule that is secreted by cells in culture. In some examples, a microphysiological cell culture system, microfluidic cell culture system, or other cell culture system is in fluid communication with the device. For example, cell culture media or other fluid may be configured to flow past a cell culture and then subsequently flow to the device including the one or more detection elements of the device. The cell culture media or a target molecule in the cell culture media may be affected by interaction with the cells, for example the cell may secrete a target molecule, may modify a target molecule, may catabolize a target molecule, may metabolize a target molecule, or sequester a target molecule.

[0155] In some aspects, the device includes or is used to aid in biomanufacturing techniques. For example, cells may be engineered to produce, secrete, or metabolize a desired molecule. The device may be used to measure, optionally in real-time, the production or activity of a target molecule produced by cells.

[0156] In some aspects, the invention relates to a method of testing or confirming the activity or efficacy of an inhibitor of a target molecule. In some embodiments, an inhibitor is directly added to a sample solution before delivering the sample solution to one or more detection elements of a device. In this embodiment, the one or more detection elements of the device include one or more capture molecule detection elements in which capture molecule is specific for the target of the inhibitor, one or more interaction molecule detection elements in which the interaction molecule is a substrate of the target of the inhibitor, and one or more control detection elements. The concentration and enzymatic activity of the target molecule can be determined and used to assess the activity of the inhibitor. In some embodiments, the inhibitor is administered to a subject by any method including orally, by injection, by inhalation, or by application to the skin. In this embodiment, the sample solution is a biological fluid derived from the patient. The method may then be used to confirm or test the efficacy or activity of the Attorney Docket No. 204606-0200-00 WO inhibitor after administration to a subject. In some embodiments, the inhibitor may be readministered based on the determined target molecule concentration and target molecule enzymatic activity in the patient derived sample solution. For example, a greater measured enzymatic activity of a target molecule may inform a decision to readminister or increase the dosage of an administered inhibitor to a subject.

[0157] In some embodiments, the device and method may be used to assay the effect of known modulator or modulator candidates on a target molecule. Modulators or modulator candidates may include inhibitor or inhibitor candidates that may inhibit the activity of the target molecule; or activator or activator candidates that activate the activity of the target molecule. For example, any number of modulator candidates may be individually added to solutions containing the target molecule. In some examples, a biological fluid derived from a subject is aliquoted and a different modulator candidate is added to each aliquot. The aliquots may then be delivered to the device separately or to separate devices such that the effectiveness of each modulator or modulator candidate on a target molecule can be assayed. In some examples, the aforementioned process or a similar process may be used for personalized medicine. In some examples, a modulator is chosen to be administered to a subject based on its effect on a target molecule in a patient-derived biological fluid.

[0158] In some embodiments, the device and method may be used to aid in drug discovery. For example, any number of modulator candidates may be added individually to sample solutions containing a target molecule. A modulator candidate may be chosen for further study based on its effectiveness in modulating a target molecule. In some embodiments, the sample solution may be a buffer solution containing a target molecule. In some embodiments, the sample solution may be a biological fluid derived from an animal model in which a target molecule of interest may or may not be added to the sample solution. The animal may be a model animal for a disease or injury of interest. In some embodiments, the sample solution may be a biological fluid gathered from one or more human subjects. The human subjects may have a disease of interest, have an injury of interest, or have any characteristic of interest. For example, the human subjects may be chosen for any genetic profile, chosen for any known mutation in the genetic sequence Attorney Docket No. 204606-0200-00 WO coding for a target molecule, or chosen for having any allele that codes for a target molecule.

[0159] Additional features and advantages are described in, and will be apparent from, the following Detailed Description and the Figures. The features and advantages described herein are not all-inclusive and, in particular, many additional features and advantages will be apparent to one of ordinary skill in the art in view of the figures and description. Also, any particular embodiment does not have to have all of the advantages listed herein and it is expressly contemplated to claim individual advantageous embodiments separately. Moreover, it should be noted that the language used in the specification has been selected principally for readability and instructional purposes, and not to limit the scope of the inventive subject matter.

[0160] Figure 11 is a diagram of a photonic biosensor 100, according to an example embodiment of the present disclosure. The example biosensor 100 includes a substrate 102, which may include a slide, cassette, membrane, fibrous substrate, or test card. The substrate 102 may be constructed from glass, plastic, composites, cyclic olefin copolymers, polystyrene, polymethymethacrylate (“PMMA”), nylon, polycarbonate, or combinations thereof. Further, the substrate 102 may be manufactured via hot embossing, micro molding, or any other molding or printing method.

[0161] The substrate 102 may be a glass slide. The substrate 102 may be hydrophilic such that it is configured to drive capillary flow of a sample solution. The substrate may be coated with a hydrophilic coating, for example silicon dioxide. The substrate 102 may be an ultraviolet ozone treated glass slide. The substrate 102 may be designed or chosen such that it is not prone to molding errors, including debris, artifacts, or height differences, that may drive heterogeneous flow. The substrate 102 may be optically transparent at any light wavelength. For example, infrared wavelengths including 1550 nm. The substrate may be produced in any manner. For example, the substrate 102 may be die cut, molded, cut, laminated, grown, formed from a roll, and / or extruded. A cover for the substrate 102 may be included.

[0162] The example substrate includes a sample addition zone 106, a wicking zone 108, and a detection zone 110. The zones 106 to 110 are fluidly coupled together (e.g., in fluid communication) via a fluid pathway 112. The sample addition zone 106 includes a Attorney Docket No. 204606-0200-00 WO metering port for receiving a fluid sample solution. The example wicking zone 108 provides an area for flow control and / or waste collection. The wicking zone 108 provides a termination point of the fluid pathway, while the sample addition zone 106 provides a starting point. In some embodiments, the wicking zone 108 may be covered by tape support, which provides physical protection of accumulated fluid sample solution in the wicking zone 108. An entry section leading to the wicking zone 108 may be configured to pull the fluid sample solution into the wicking zone 108 to prevent the fluid sample solution from backing up through the detection zone 110.

[0163] The example substrate 102 may, in some embodiments, include an optical enhancer zone (e.g., a conjugate zone). The optical enhancer zone is located downstream from the sample addition zone 106. In some embodiments, the optical enhancer zone is located adjacent to the sample addition zone 106. Further, the optical enhancer zone is located upstream from the detection zone 110 and the wicking zone 108. The optical enhancer zone includes one or more reagents for binding with a fluid sample solution. In some embodiments, the fluid sample solution dissolves fluorescent labeled conjugates as the fluid sample solution flows through the optical enhancer zone.

[0164] The example detection zone 110 includes one or more test zones that are configured to capture a bound capture molecule / target molecule complex. The test zone may allow for the detection of a target molecule using fluorometric or colorimetric detection. For example, the test zone may be used in addition to a PIC detection element. The test zone may act as a confirmation of the readout of PIC detection element, or the test zone may allow for multiplexed measurement of different target molecules. The different test zones may provide for the detection of a target molecule or different target molecules. A concentration or presence of bound capture molecule / target molecule complex at each test zone may be measured using fluorescence or colorimetric detection. For example, a supplied capture molecule may be fluorescently tagged. In some instances, after a conjugate in the optical enhancer zone is completely dissolved, the fluid sample solution acts as a wash and removes unbound material into the wicking zone 108. After a complete wash by the fluid sample solution, the test zones of the detection zone 110 may be read with a fluorimeter or other optical analyzer. Attorney Docket No. 204606-0200-00 WO

[0165] In some embodiments, at least some of the fluid pathway 112, the sample addition zone 106, the optical enhancer zone, the detection zone 110, and / or the wi eking zone 108 may include a plurality of projections or micropillars. The example projections or micropillars are substantially vertical to a surface of the substrate 102 and have a height, diameter, and reciprocal spacing such that lateral capillary flow of the fluid sample solution is achieved. In some embodiments, the projections or micropillars have a height that is between 1-1000 pm, a diameter that is between 10-100 pm, and reciprocal spacing that is between 5-100 pm, preferably between 10-25 pm. In some instances, a base of the projections or micropillars may have a greater diameter compared to a top. In these instances, a diameter of the projections or micropillars may taper from the base to the top.

[0166] Example substrates are described further in U.S. Pat. Nos. 10,073,091, 9,689,870, 9,389,228, 9,285,361, 8,895,293, 8,821,812, 8,409,523, and 8,025,854, where each are hereby incorporated by reference in their entirety.

[0167] In some embodiments, the PIC 104 may be used in conjunction with one or more detector sections of the detection zone 110. The detector sections may be configured for colorimetric / digital detection and / or fluorescence detection. The sample addition zone 106 is configured to receive serum / plasma, whole blood, or other fluids for analysis by the PIC 104 and the detector sections. The optical enhancer zone may provide one or more capture options, including conjugate capture and / or mass enhancer capture. Further, the wi eking zone 108 may include one or more features such as fluid control and / or end of test detection. In some embodiments, the wi eking zone 108 may include a porous material to enhance fluid flow.

[0168] As described above in connection with Figure 11, the fluid pathway 112 may provide for fluid flow using micropillars. The micropillars may be placed in the sample addition zone 106, the wi eking zone 108, the detection zone 110, optical enhancer zone, the wash zone, and / or space between these zones along the fluid pathway 112. In other embodiments, capillary flow may be achieved without the use of micropillars. For example, the fluid pathway 112 and / or the zones 106, 108, and / or 110 may be achieved using texturing / surface patterning. Alternatively, capillary flow may be achieved using porous media (e.g., “paper in poly”, fiber materials, or thread / fabric bundles). In other embodiments, capillary flow may be provided using a thin film coating and / or various Attorney Docket No. 204606-0200-00 WO coated spreading layers and channel beads. Coatings to provide a wettable / hydrophilic surface for the fluid pathway 112 and / or the zones 106, 108, and / or 110 include oxygen plasma treatment, neutral atom beam bombardment, gas cluster ion beam bombardment, surface silanization, etc.

[0169] Figure 12 is a diagram of an example micropillar layout for a fluid flow path 112 of the substrate 102 for the biosensor 100 of Figure 11, according to an example embodiment of the present disclosure. Micropillars 1202 are placed within the fluid flow path 112, including at locations along the fluid flow path 112 that align with a functionalized detection element 212 of the PIC 104. The micropillars 1202 may include cylinders having 50 pm diameters. As shown, the micropillars 1202 are placed in a hexagonal array such that the micropillars 1202 are spaced apart by 100 pm. The spacing and size of the micropillars 1202 provides for capillary flow along the fluid flow path 112.

[0170] In other embodiments, the micropillars have a rectangular shape. In these other embodiments, the micropillars may be placed into rows, with 50 pm to 150 pm of space between adjacent micropillars. Further each adjacent row may be offset from each other. The offset may correspond to gaps of adjacent rows such that a micropillar in one row is aligned with a gap between micropillars in an adjacent row.

[0171] In some embodiments alignment channels 1102 and 1104 are formed in the substrate 102. The alignment channels 1102 and 1104 may include through holes or apertures, and are located adjacent to respective ports 202 and 204.

[0172] In an example, the photonic reader includes alignment pins. After the biosensor 100 is moved into a specified location within the laboratory instrumentation, the photonic reader and / or the substrate 102 is moved such that the alignment pins pass through the alignment channels 1102 and 1104. This provides fast alignment. In some embodiments after the rough alignment, the photonic reader and / or the substrate 102 may have alignment fine-tuned to ensure a light source and a light detector are optically aligned with the optical input port 202 and the optical output port 204.

[0173] It should be noted that while Figure 11 through Figure 14 show one input port 202 and one output port 204, in other embodiments, the substrate 102 may have more than one input port, no input ports, more than one output port, and / or no output ports. In some Attorney Docket No. 204606-0200-00 WO embodiments, a larger output port enables the use of multiple channels in the PIC 104 for multiplexing. In some embodiments, the use of multiple ports enables the use of multiple channels in the PIC 104 for multiplexing. For example, the substrate may have a single input port 202 and multiple output ports 204. In this example, the PIC 104 has multiple channels corresponding to the number of output ports 204. Light received via the input port 202 is split along the separate channels to provide different types of optical analysis. Additionally or alternatively, the substrate 102 may include multiple input ports 202 and multiple output ports 204 (and / or multiple pairs of alignment channels) to accommodate multiple PICs 104 placed at different locations along the flow path 112.

[0174] Figure 11 also shows approximate dimensions of an exemplary biosensor 100, which has a length of about 22 mm and a width of about 15 mm. In this example, the sample addition zone 106 has a width of 5.7 mm and a length of 21.2 mm. The flow path connecting the sample addition zone 106 and the downstream detection zone 110 has a width of 1.16 mm, and the wicking zone 108 has a diameter of 7.6 mm. It should be appreciated that the biosensor 100 may have alternative dimensions based on design and end-use application.

[0175] In some embodiments, the flow path connecting the sample addition zone 106 and the downstream detection zone 110 or any other zone or section of the device is a channel of any dimensions including height and width. In some embodiments, the flow path is cylindrical or rectangular. In some embodiments, the flow path is designed to reduce local target molecule depletion in the sample detection zone or near any detection elements.

[0176] In some embodiments, the alignment channels 1102 and 1104 and the ports 202 and 204 may be omitted. In these alternative embodiments, light coupling is provided directly to the waveguide 210 of the PIC 104. In an example, an input fiber connected to a light source is configured to align with a side of the PIC 104 to optically couple directly with the waveguide 210. An output fiber is placed on an opposing side of the PIC 104 to receive the light.

[0177] The example substrate 102 of Figure 11 also includes optical ports for noncontact optical coupling with a read head of a laboratory analyzer or PoC analyzer. Figure 13 and Figure 14 show a cut-away of the substrate 102 at a section 200 of the sample detection Attorney Docket No. 204606-0200-00 WO zone 110 that includes an optical input port 202 and an optical output port 204, according to an example embodiment of the present disclosure. The input port 202 is configured to optically couple to a light source and the output port 204 is configured to optically couple to a light detector. To provide non-contact optical alignment, the substrate 102 is positioned in an analyzer instrument such that a light source is directly aligned with the optical input port 202. Similarly, the positioning of the substrate 102 in the analyzer instrument causes the output port 204 to align with an optical detector. This non-contact coupling eliminates the need for complex optical coupling with the PIC 104.

[0178] The optical input port 202 includes a first tunnel through the substrate 102 and the optical output port 202 includes a separate, second tunnel through the substrate 102. While the tunnels are shown as being cylindrical, the tunnels may have other profiles, such as rectangular triangular, etc. In the illustrated embodiment, the input port 202 is shown as being on one side of the fluid pathway 112, while the output port 204 is shown as being on an opposite side of the fluid pathway 112. In other embodiments, the ports 202 and 204 maybe on a same side of the fluid pathway 112.

[0179] Figure 13 and Figurel4 also show an enlarged view of the PIC 104. Figure 13 shows a diagram of the PIC 104 prior to connection to the substrate 102. Figure 14 shows a diagram of the PIC 104 after placement on the substrate 102. The example PIC 104 includes a first grating coupler 206 that is aligned with the optical input port 202. The PIC 104 also includes a second grating coupler 208 that is aligned with the optical output port 204. The grating couplers 206 and 208 have shapes that conform to the circular profiles of the respective optical ports 202 and 204.

[0180] The grating couplers 206 and 208 include periodic etch structures that diffract light in a certain direction. In the illustrated example, the grating coupler 206 diffracts light from a vertical direction through the optical input port 202 to a horizontal direction through the PIC 104. Additionally, the grating coupler 208 diffracts light from a horizontal direction from the PIC 104 to a vertical direction through the optical output port 204. In other embodiments, the grating couplers may be replaced with mirrors or a reflective coating that directs light between the ports 202 and 204 and the PIC 104.

[0181] The example PIC 104 also includes at least one waveguide 210 between the first grating coupler 206 and the second grating coupler 208. Further, the PIC 104 includes at Attorney Docket No. 204606-0200-00 WO least one detection element 212 that are provided along the at least one waveguide 210. The detection element 212 is positioned to contact a fluid sample solution within the fluid pathway 112. It should be appreciated that the detection element 212 and / or the PIC 104 generally does not block fluid passage along the fluid pathway 112. Instead, a small space is provided between a floor of the fluid pathway 112 and the detection element 212 to enable a fluid sample solution to pass through. In some embodiments, the small space is between 10 pm and 5000 pm.

[0182] Also, as shown in Figure 13, the substrate 102 may include recess sections 220 and 222 around the ports 202 and 204 for receiving corresponding sides of the PIC 104. The recessed sections 220 and 222 enable the PIC 104 to be securely connected to the substrate 102. In some embodiments, at least one of a UV curable adhesive, physical stacking, or a tape / glue application is used to secure the PIC 104 to the substrate 102 at the recess sections 220 and 222.

[0183] In one embodiment, the PIC 104 is configured for front-side coupling wherein the PIC 104 is coupled to a top surface of the substrate 102 opposite the fiber bundle 120.

[0184] In one embodiment, the PIC 104 is configured for back-side coupling wherein the PIC 104 is coupled to a bottom surface of the substrate 102 between the substrate 102 and the fiber bundle 120.

[0185] In some embodiments, the photonic biosensor apparatus 100 includes a sample addition zone 106 in fluid communication with a wi eking zone 108 and a sample detection zone 110, wherein the sample detection zone 110 is between the sample addition zone 106 and the wi eking zone 108. In some embodiments, the biosensor apparatus 100 further includes at least one optical input port 202 disposed within the sample detection zone 110, wherein the optical input port 202 is configured to optically couple to a light source. In some embodiments, the biosensor apparatus 100 further includes at least one optical output port 204 disposed within the sample detection zone 110, wherein the optical output port 202 is configured to optically couple to a photodetector via a fiber bundle 120. In some embodiments, the biosensor apparatus 100 further includes at least one photonic integrated circuit (PIC) 104 disposed directly atop a substrate 102. Further details of the PIC 104 are described below. Attorney Docket No. 204606-0200-00 WO

[0186] In some embodiments, the fiber bundle 104 comprises a plurality of individual fibers. In some embodiments, the individual fibers of the fiber bundle 104 comprise multimode fibers and / or singlemode fibers. In some embodiments, the individual fibers of the fiber bundle 104 comprise one singlemode fiber and a at least one multimode fibers. In some embodiments, the individual fibers of the fiber bundle 104 are positioned in a hexagonal close-packing configuration. In some embodiments, the individual fibers of the fiber bundle 104 are positioned in a square close-packing configuration.

[0187] In some embodiments, the apparatus 100 is configured to detect three or more measurands simultaneously. In some embodiments, the apparatus 100 the apparatus is configured to detect three or more target molecules simultaneously. In some embodiments, the apparatus 100 the apparatus is configured to detect the enzymatic activity of three or more target molecules simultaneously. In some embodiments, the apparatus 100 the apparatus is configured to detect the concentration and the enzymatic activity of three or more target molecules simultaneously. In some embodiments, the apparatus is configured to detect the concentration and the enzymatic activity of any number of target molecules simultaneously.

[0188] In some embodiments, the substrate 102 comprises at least one of a slide, cassette, membrane, fibrous substrate, or test card. In some embodiments, the light source and the photodetector are included within a read head of at least one of a laboratory analyzer or a point-of-care (“PoC”) analyzer.

[0189] In some embodiments, the apparatus 100 further comprises at least one of a fluid pathway, a paper pathway, or a membrane pathway that fluidly couples the sample addition zone 106, the detection zone 110, and the wicking zone 108. In some embodiments, the fluid pathway includes micropillars or projections that are substantially vertical to the surface of the substrate and having a height between about 1 pm to 1000 pm, a diameter between about 10 pm to 100 pm, and a reciprocal spacing between the micropillars between about 5 pm to 100 pm such that lateral capillary flow of the fluid sample solution is achieved.

[0190] In some embodiments, the detection zone 110 is configured to provide at least one of fluorescence, refractive index shift, Raman signal, absorbance signal, plasmonic shift or colorimetric detection of one or more target molecules within the fluid sample Attorney Docket No. 204606-0200-00 WO solution. In some embodiments, the PIC 104 is connected to the substrate 102 using at least one of a UV curable adhesive, physical stacking, lamination or a tape / glue application.

[0191] Figure 15 shows an exemplary PIC 104. In the example shown, the PIC 104 includes at least one first (input) grating coupler 206, at least two second (output) grating couplers 208, at least one waveguide 210 between the first grating coupler 206 and the second grating couplers 208, and at least one detection element 212 disposed within the at least one waveguide 210. In some embodiments, the at least one first (input) grating coupler 206 is aligned with an optical input port 202. In some embodiments, the at least two second (output) grating couplers 208 aligned with an optical output port 204.

[0192] In some embodiments, the at least one detection element 212 can include at least one capture molecule, at least one interaction molecule, and / or at least one control molecule. In some embodiments, the capture molecule is configured to capture target molecules, such as proteins, antibodies, peptides, nucleic acids, and any other suitable substance. In some embodiments, the interaction molecule is configured to capture target molecules, such as proteins, antibodies, peptides, nucleic acids, and any other suitable substance. In some embodiments, the control molecule is configured to allow for nonspecific binding of sample solution components, such as proteins, antibodies, peptides, nucleic acids, and any other suitable substance. In some embodiments the at least one detection element 212 includes at least one of a ring resonator, a double ring resonator, a cylindrical resonator, a spherical resonator, a spiral waveguide, photonic crystal, a Mach-Zehnder Interferometer (“MZI”), and any other suitable design or combinations thereof.

[0193] In some embodiments, the at least one waveguide 210 comprises a silicon nitride waveguide. In some embodiments, the at least one waveguide 210 splits into a plurality of branches from the first grating coupler 206 to the at least two second grating couplers 208. In some embodiments, the at least one detection element 212 is positioned on one of the plurality of branches. In some embodiments, the at least one detection element 212 is positioned to contact a fluid sample solution within the sample detection zone 110.

[0194] In some embodiments, the PIC 104 comprises a rectangular prism or cuboid shape. In some embodiments, the PIC 104 has a length between 2-20 mm (e.g., 4 mm), a Attorney Docket No. 204606-0200-00 WO width between 0.25-10 mm (e g., 1 mm), and a height between 0.1-5 mm. In some embodiments, an array of PICs 104 may be connected to the substrate 102 for multiplex applications. In some embodiments, the PIC 104 can include 0 to 100, 1 to 20, 2 to 10, 2 or more, 5 or more, 8 or more, or any suitable number of detection elements 212. In some embodiments, a PIC 104 with zero detection elements 212 may be included in an array. In some embodiments, the PIC 104 may be used as a reference for light calibration and / or adjustment.

[0195] In some embodiments, the at least one detection element 212 has an extinction ratio greater than 5 dB under aqueous cladding. In some embodiments, each detection element 212 has a unique extinction ratio. In some embodiment, the spectral footprint of individual detection elements 212 sharing the same bus waveguide may be made distinguishable by altering the quality factor and / or extinction ratio, thus enabling the identification of control / experimental rings. In some embodiments, intentional reduction of the quality factor and / or extinction ratio broadens (or lessens the depth of) resonance dips in the spectrum, effectively labeling the ring in question based on the peak shape.

[0196] Figure 16 shows a ray diagram of an exemplary multiplex hub optical system of the biosensor 100. Light emitted from the output gratings 208 of the PIC 104 passes through the first surface of the hub output lens LISI, reflects 90-degrees off the angled interior surface of the hub and exits the second surface L1S2 into free space as a collimated beam. The beam then reflects 90 degrees toward lens L2, which focuses the beam on to facet of the fiber bundle 120. Contrary to the usual implementation of fiber bundles, in this case, each fiber is intended to capture light from a single light source within an array of discrete sources. The fibers of the fiber bundle 120 can be any suitable arrangement including hexagonal close packing and / or square close packing.

[0197] In some aspects of the present invention, software executing the instructions provided herein may be stored on a non-transitoiy computer-readable medium, wherein the software performs some or all of the steps of the present invention when executed on a processor.

[0198] Aspects of the invention relate to algorithms executed in computer software. Though certain embodiments may be described as written in particular programming languages, or executed on particular operating systems or computing platforms, it is Attorney Docket No. 204606-0200-00 WO understood that the system and method of the present invention is not limited to any particular computing language, platform, or combination thereof. Software executing the algorithms described herein may be written in any programming language known in the art, compiled or interpreted, including but not limited to C, C++, C#, Objective-C, Java, JavaScript, MATLAB, Python, PHP, Perl, Ruby, or Visual Basic. It is further understood that elements of the present invention may be executed on any acceptable computing platform, including but not limited to a server, a cloud instance, a workstation, a thin client, a mobile device, an embedded microcontroller, a television, or any other suitable computing device known in the art.

[0199] Parts of this invention are described as software running on a computing device. Though software described herein may be disclosed as operating on one particular computing device (e.g. a dedicated server or a workstation), it is understood in the art that software is intrinsically portable and that most software running on a dedicated server may also be run, for the purposes of the present invention, on any of a wide range of devices including desktop or mobile devices, laptops, tablets, smartphones, watches, wearable electronics or other wireless digital / cellular phones, televisions, cloud instances, embedded microcontrollers, thin client devices, or any other suitable computing device known in the art.

[0200] Similarly, parts of this invention are described as communicating over a variety of wireless or wired computer networks. For the purposes of this invention, the words “network”, “networked”, and “networking” are understood to encompass wired Ethernet, fiber optic connections, wireless connections including any of the various 802.11 standards, cellular WAN infrastructures such as 3G, 4G / LTE, or 5G networks, Bluetooth®, Bluetooth® Low Energy (BLE) or Zigbee® communication links, or any other method by which one electronic device is capable of communicating with another. In some embodiments, elements of the networked portion of the invention may be implemented over a Virtual Private Network (VPN).

[0201] Figure 17 and the following discussion are intended to provide a brief, general description of a suitable computing environment in which the invention may be implemented. While the invention is described above in the general context of program modules that execute in conjunction with an application program that runs on an Attorney Docket No. 204606-0200-00 WO operating system on a computer, those skilled in the art will recognize that the invention may also be implemented in combination with other program modules.

[0202] Generally, program modules include routines, programs, components, data structures, and other types of structures that perform particular tasks or implement particular abstract data types. Moreover, those skilled in the art will appreciate that the invention may be practiced with other computer system configurations, including handheld devices, multiprocessor systems, microprocessor-based or programmable consumer electronics, minicomputers, mainframe computers, and the like. The invention may also be practiced in distributed computing environments where tasks are performed by remote processing devices that are linked through a communications network. In a distributed computing environment, program modules may be located in both local and remote memory storage devices.

[0203] Figure 17 depicts an illustrative computer architecture for a computer 500 for practicing the various embodiments of the invention. The computer architecture shown in Figure 17 illustrates a conventional personal computer, including a central processing unit 550 (“CPU”), a system memory 505, including a random-access memory 510 (“RAM”) and a read-only memory (“ROM”) 515, and a system bus 535 that couples the system memory 505 to the CPU 550. A basic input / output system containing the basic routines that help to transfer information between elements within the computer, such as during startup, is stored in the ROM 515. The computer 500 further includes a storage device 520 for storing an operating system 525, application / program 530, and data.

[0204] The storage device 520 is connected to the CPU 550 through a storage controller (not shown) connected to the bus 535. The storage device 520 and its associated computer-readable media, provide non-volatile storage for the computer 500. Although the description of computer-readable media contained herein refers to a storage device, such as a hard disk or CD-ROM drive, it should be appreciated by those skilled in the art that computer-readable media can be any available media that can be accessed by the computer 500.

[0205] By way of example, and not to be limiting, computer-readable media may comprise computer storage media. Computer storage media includes volatile and nonvolatile, removable and non-removable media implemented in any method or technology Attorney Docket No. 204606-0200-00 WO for storage of information such as computer-readable instructions, data structures, program modules or other data. Computer storage media includes, but is not limited to, RAM, ROM, EPROM, EEPROM, flash memory or other solid state memory technology, CD-ROM, DVD, or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store the desired information and which can be accessed by the computer.

[0206] According to various embodiments of the invention, the computer 500 may operate in a networked environment using logical connections to remote computers through a network 540, such as TCP / IP network such as the Internet or an intranet. The computer 500 may connect to the network 540 through a network interface unit 545 connected to the bus 535. It should be appreciated that the network interface unit 545 may also be utilized to connect to other types of networks and remote computer systems.

[0207] The computer 500 may also include an input / output controller 555 for receiving and processing input from a number of input / output devices 560, including a keyboard, a mouse, a touchscreen, a camera, a microphone, a controller, a joystick, or other type of input device. Similarly, the input / output controller 555 may provide output to a display screen, a printer, a speaker, or other type of output device. The computer 500 can connect to the input / output device 560 via a wired connection including, but not limited to, fiber optic, ethernet, or copper wire or wireless means including, but not limited to, Bluetooth, Near-Field Communication (NFC), infrared, or other suitable wired or wireless connections.

[0208] As mentioned briefly above, a number of program modules and data files may be stored in the storage device 520 and RAM 510 of the computer 500, including an operating system 525 suitable for controlling the operation of a networked computer. The storage device 520 and RAM 510 may also store one or more applications / programs 530. In particular, the storage device 520 and RAM 510 may store an application / program 530 for providing a variety of functionalities to a user. For instance, the application / program 530 may comprise many types of programs such as a word processing application, a spreadsheet application, a desktop publishing application, a database application, a gaming application, internet browsing application, electronic mail application, messaging application, and the like. According to an embodiment of the present invention, the Attorney Docket No. 204606-0200-00 WO application / program 530 comprises a multiple functionality software application for providing word processing functionality, slide presentation functionality, spreadsheet functionality, database functionality and the like.

[0209] The computer 500 in some embodiments can include a variety of sensors 565 for monitoring the environment surrounding and the environment internal to the computer 500. These sensors 565 can include a Global Positioning System (GPS) sensor, a photosensitive sensor, a gyroscope, a magnetometer, thermometer, a proximity sensor, an accelerometer, a microphone, biometric sensor, barometer, humidity sensor, radiation sensor, or any other suitable sensor.

[0210] In some embodiments, the device includes a sample solution delivery card that is configured to introduce a sample solution to one or more detection elements of a PIC. In some embodiments, the sample delivery card is constructed as in Figure 18. Now referring to Figure 18, depicted is an exemplary embodiment of the layers of a sample solution delivery card. In some examples, the sample solution delivery card is constructed of layers. In some examples, all layers of the sample delivery card include alignment holes to aid in the alignment of the layers of the sample delivery card. In some examples, the sample solution delivery card includes a base layer. The base layer may be hydrophilic and / or transparent. In some examples, the sample delivery card includes an adhesive layer above the base layer. The adhesive layer may be adhesive on the bottom and top side. The adhesive layer may include an enhancer region and a fluid channel. In some examples, the sample solution delivery card includes a top cover layer. The top cover layer may be hydrophilic. The top cover layer may include a sample inlet, or sample addition zone, a PIC holder for PIC alignment, and a reservoir region. The reservoir region may also be referred to as a wicking zone. The PIC holder may be configured to hold a photonic chip and the reservoir holder may be configured to hold a reservoir pad. The sample solution delivery card may be configured such that a sample solution is added to the sample addition zone, flows through the fluid channel therefore contacting the photonic chip, and subsequently flows to the reservoir region containing a reservoir pad. Now referring to Figure 19, depicted is an exemplary embodiment of the sample solution delivery card constructed of the layers as depicted in Figure 18. Attorney Docket No. 204606-0200-00 WO

[0211] The aforementioned systems, processes and methods described herein may be utilized for desired applications as would be appreciated by those skilled in the art. For example, the aforementioned systems, processes and methods described herein can be utilized as a disposable multiplex assay in measuring the presence, level or enzymatic activity of target molecules, such as proteins, enzymes, antibodies, peptides, nucleic acids, antigens, viruses, metabolites, and any other suitable substance.

[0212] In one example, the aforementioned systems, processes, and methods described herein may be used as clinical diagnostics of various diseases such as SARS-CoV-2 and influenza. Furthermore, the aforementioned systems, processes and methods described herein can be utilized with any suitable system, such as those described in U.S. Patent Application No. 17 / 585,914, and hereby incorporated herein by reference in its entirety.

[0213] The disclosures of each and every patent, patent application, and publication cited herein are hereby each incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

[0214] EXPERIMENTAL EXAMPLES

[0215] The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0216] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore specifically point out exemplary embodiments of the present Attorney Docket No. 204606-0200-00 WO invention and are not to be construed as limiting in any way the remainder of the disclosure.

[0217] Example 1 : A Dual-Readout Photonic Sensor for Simultaneous Measurement of Enzyme Activity and Concentration

[0218] Enzyme assays are a cornerstone of basic biology and clinical diagnosis. Typically, enzyme activity is measured, but concentration of the enzyme is also of interest, as are comparisons between concentration and activity. In these situations, separate concentration (i.e. ELISA) and activity (i.e. absorbance) assays are required to fully quantify. In this Example, a multiplex disposable photonic biosensor for simultaneous measurement of enzyme activity and concentration is reported. Capture of the enzyme by a ring resonator-bound antibody produces a red shift in resonance, which can be referenced to a nonspecific binding control. At the same time, enzyme-mediated degradation of a ring-bound substrate produces a resonance blue shift, which can be referenced to a peptide inert to enzymatic cleavage. The dual assay was tested with human Cathepsin-L, dysfunction of which is a hallmark of several diseases, including COVID-19, kidney failure, and cancer. Both assays were found to be well-behaved analytically, with lower limits of detection of 2.0 ng / mL (concentration) and 1.8 ng / mL (activity), well within the range clinically relevant concentrations. Further assessment with a panel of 25 single-donor human serum samples confirmed utility of the assay in a complex, biologically relevant matrix. This approach therefore serves as a useful method for Cathepsin-L detection, and a prototype for other dual-mode photonic enzyme assays.

[0219] Quantification of enzymatic activity and enzyme concentration are crucial in research, medicine, biotechnology, and many other areas (Bisswanger, H. Enzyme Assays. Report. Enzymol. Data - STREND A Recomm. Beyond 2014, 1 (1), 41-55., Goddard, I.-P. et al. Trends Biotechnol. 2004, 22 (7), 363-370., Katsimpouras, C. et al. Chem. Biotechnol. Pharm. Biotechnol. 2021, 69, 91-102., Fiksdal, L. et al. Quality. Curr. Opin. Bio-technol. 2008, 19 (3), 289-294., Seitkalieva, A. V. et al. Environ. Monit. Assess. 2015, 188 (1), 70., Wang, I. ELECTROPHORESIS 2002, 23 (5), 713-718.). While often only one of these measurements is required (activity or concentration), there are other instances where knowledge of both activity and concentration is important. For example, in a variety of cancers, enzyme activity is known to change as concentration Attorney Docket No. 204606-0200-00 WO remains the same (Stefanini, M. Cancer 1985, 55 (9), 1931-1936.). Likewise, enzyme inhibition is a hallmark of many therapeutics; understanding the efficacy of inhibition is only possible when the concentration of the enzyme being inhibited is found to be constant (Del Prete, S. et al. Mol. Basel Switz. 2024, 29 (18).). Unfortunately, two separate assay formats are currently required for full quantification of an enzyme: a capture-based assay such as ELISA for enzyme concentration, and a separate assay to quantify the enzyme’s activity. This creates obvious disadvantages in workflow complexity, and increases the potential for error. The clinical need for a multimode biosensor able to address both detection mechanisms has been discussed (Chu, S. S. et al. Sensors 2022, 22 (14), 5200.).

[0220] The detection of enzyme presence or concentration has relied upon the use of antibody binding, which, while specific, fails to provide additional details about the activity of said enzyme. Some work towards detection of enzymatic activity has been achieved within the field of photonic crystals. The use of porous silicon was shown to be an effective metric of enzymatic activity, denoting such occurrences via a color change (Orosco, M. M.; et al. Adv. Mater. 2006, 18 (11), 1393-1396). While this lacks real time enzymatic sensing, it provides the proof that photonic crystals can detect enzymatic activity. This work was then expanded with real time monitoring of a mesoporous silicon double layer (Orosco, M. M.; et al. Nat. Nanotechnol. 2009, 4 (4), 255-258). This method traps protease and substrate with pores only small enough to capture the degraded substrate. This work is limited by size requirements, where the pore is small enough to restrict the entry of substrate but allow entry of degraded fragments. Additional work has used a similar mechanism with pores to measure enzymatic degradation of gelatin through an alumina photonic film (Nemati, M.; et al. Anal. Chem. 2015, 87 (17), 9016- 9024). The use of a photonic sensor to measure drug inhibition in real time has previously been demonstrated as well (Nemati, M.; et al. Anal. Chem. 2016, 88 (11), 5971-5980). Both antibody detection and enzymatic assays have been established in a variety of photonics platforms, and are common assays in medical diagnostics (Orosco, M. M.; et al. Adv. Mater. 2006, 18 (11), 1393-1396, Cognetti, J. S.; et al. Lab. Chip 2021, 21 (15), 2913-2921). Attorney Docket No. 204606-0200-00 WO

[0221] While combined assays for enzymatic activity and concentration have not been reported, significant effort has been devoted to the development of novel approaches for each separately. In particular, optical approaches including those based on photonic devices have been widely studied as such devices are readily manufactured, relatively inexpensive, and useful in a wide range of applications (Butt, J. N. et al. APL Photonics 2024, 9 (1), 016104., Bryan, M. R. et al. ACS Sens. 2024, 9 (4), 1799-1808., Pezzotti, G. et al. In Silicon Nitride Bioceramics; Bal, B. S., McEntire, B. J., Pezzotti, G., Eds.; Springer International Publishing: Cham, 2024; pp 415-431 ). Photonic sensing of enzymes via antibody capture is well known, but photonic structures have also been used for detection of enzymatic activity (Inan, H. et al., Chem. Soc. Rev. 2017, 46 (2), 366- 388.). For example, color changes in porous silicon have been used to report enzymatic activity (Orosco, M. M. et al., Adv. Mater. 2006, 18 (11), 1393-1396 ). Real-time monitoring of a mesoporous silicon double layer has been used to sense protease activity, as has enzymatic degradation of gelatin through an optically responsive alumina film (Orosco, M. M. et al Nat. Nanotechnol. 2009, 4 (4), 255-258., Nemati, M. et al. Anal. Chem. 2015, 87 (17), 9016-9024.). Liquid crystal biosensors have been used to measure enzyme activity in a number of medical and environmental applications (Sil, S. et al., Langmuir 2025, 41 (8), 4959-4975.). The use of a photonic sensor for real-time measurement of drug-dependent enzyme activity inhibition has been demonstrated (Nemati, M. et al., Anal. Chem. 2016, 88 (11), 5971-5980.). An assay employing singlewalled carbon nanotubes has been described for monitoring cholinesterase activity and inhibition in plasma, but the approach is limited to fluorescent substrates (Basu, S. et al., Small 2024, 20 (24), 2309481.).

[0222] In order to develop an assay capable of simultaneously quantifying enzyme concentration and activity in a sample, a recently developed “disposable photonics” ring resonator platform recently developed was used built upon (Bryan, M. R. et al., ACS Sens. 2024, 9 (4), 1799-1808., Cognetti, J. S. et al., Lab. Chip 2021, 21 (15), 2913- 2921.). Widely studied over many years by many groups as photonic biosensors21 27, a ring resonator consists of a circular waveguide placed near a linear (“bus”) waveguide such that light couples evanescently from the bus waveguide to the ring (Cardenosa- Rubio, M. C. et al., Environ. Monit. Assess. Biosens. Environ. Monit. Hum. Health 2019, Attorney Docket No. 204606-0200-00WO

[0223] 10, 38-46., Luchansky, M S. et al., Anal. Chem. 2010, 82 (5), 1975-1981., Guider, R. et al., Sens. Bio-Sens. Res. 2015, 6, 99-102., Qavi, A. J. et al., Cell Rep. Methods 2022, 2 (6)., Kindt, J. T. et al., Curr. Opin. Chem. Biol. 2013, 17 (5), 818-826., Luan, E. et al., Sensors 2018, 18 (10)., Bryan, M. R. et al., ACS Sens. 2023, 8 (2), 739-747.). Wavelengths of light satisfying the resonant condition (eq. 1 (below); netris the effective refractive index of the medium) circulate in the ring and are 180° out of phase with light in the bus waveguide, producing a drop in output power at the resonant wavelength (Arcs). Since the resonant wavelength is a function of the effective refractive index, molecules of interest binding to the ring via an immobilized capture probe produce a red-shift (increased effective refractive index) in proportion to the amount of material bound. Subtraction of the red-shift observed in a paired ring resonator functionalized with a control probe allows for correction for nonspecific binding and determination of the true binding signal (Bucukovski, J. et al., Anal. Chem. 2025, 97 (6), 3525-3535.). It was hypothesized that enzymatic cleavage of a sensor-bound peptide substrate would result in a blue-shift (decreased refractive index) due to the loss of mass. Combining this with antibody capture of the enzyme on a neighboring ring would enable simultaneous quantification of enzyme concentration and activity in a single test (Figure 1). Example shifts seen during assays are shown in Figure 21 (antibody capture of enzyme) and Figure 22 (enzymatic cleavage by the enzyme).

[0224] 27ineff

[0225] ^res m (1)

[0226] Sample delivery is a critical component of any diagnostic assay. In previous work, injection molded polystyrene micropillar cards were used to form the microfluidic channel and drive capillary flow of sample to the sensor surface. While effective, this approach relies on precisely molded parts, exact placement of multiple components, and limits sample volumes. In this Example, the microfluidic design was simplified to a format termed Glass Laminated Adhesive Microfluidics (GLAM). This approach layers doubled-sided adhesive, in which a channel layer has been defined, onto a glass microscope slide. This provides a simple, repeatable, and effective mechanism for fluid Attorney Docket No. 204606-0200-00 WO delivery to the sensor chip surface. The sensor chip (Figure 1C) is placed on the channel layer and is flanked by silicon chips to promote uniform flow. Sample volumes for the assay can be tuned by changing the composition and geometry of a reservoir pad that is placed downstream from the sensor chip (Figure ID).

[0227] The example target molecule Cathepsin L is now described.

[0228] Abnormal levels of Cathepsin-L have been noted as biomarkers for numerous diseases, including COVID-19, kidney failure, and several types of cancer. In each of these cases, Cathepsin-L requires quantification for its enzymatic activity and its concentration.

[0229] Cathepsin-L is a lysosomal cysteine protease, involved in protein processing and in breaking down cells during apoptosis prior to their recycling (Kirschke, H.; et al. TURK, V., VITALE, L., Eds.; Pergamon, 1981; pp 93-101, Felbor, U.; et al. EMBO J. 2000, 19 (6), 1187-1194). Elevation of cathepsin-L in disease relative to baseline levels has been observed in numerous pathologies, including kidney failure, parasitic infection, carcinoma, cardiomyopathy, pancreatic cancer, and infection with the Sars-CoV-2 virus, or COVID-19 (Piwkowska, A.; et al. Diagn. Basel 2022, 12 (5), Garsen, M.; et al. Kidney Int 2016, 90 (5), 1012-1022, Gonzales Santana, B.; et al. PLoS Negl. Trop. Dis. 2013, 7 (9), e2414, Ruan, J.; et al. PLOS ONE 2014, 9 (11), el 12136, Mehra, S.; et al. Mol Cell Biochem 2017, 428 (1-2), 139-147, Singh, N.; et al. World J Gastroenterol 2014, 20 (46), 17532-17540, Zhao, M.-M.; et al. Signal Transduct. Target. Ther. 2021, 6 (1), 134). In the context of COVID-19, the difference in Cathepsin-L concentration between individuals who develop severe cases vs. mild or asymptomatic cases, is sufficiently large to distinguish those at most risk for severe disease early in infection. Additionally, Cathepsin-L has quantifiable enzymatic activity (Katunuma, N.; et al. Turk, V., Vitale, L. J., Eds.; Pergamon, 1981; pp 83-92). Increased enzymatic activity has been found to be an additional factor in severe cases of COVID19, as well as being an indicator in numerous stages of cancer (Sudhan, D. R.; et al. Pharmacol Ther 2015, 155, 105-116). However, measuring concentration and enzymatic activity requires multiple assays. Since Cathepsin-L primarily targets disulfide bonds as its mechanism of action, numerous inhibitors have been found to exist, and they work at nearly 100% effectiveness even at extremely low concentrations (Steven, F. S. Proteinases and their Inhibitors; Turk, V., Attorney Docket No. 204606-0200-00 WO

[0230] Vitale, L. J., Eds.; Pergamon, 1981 ; pp 305-315). Cathepsin-L has also been shown to play a role in the activation of SARS-CoV-2, and its inhibition is a target of the commercial drug Paxlovid (Ashhurst, A. S.; et al. J. Med. Chem. 2022, 65 (4), 2956- 2970, Mondal, S.; et al. J. Am. Chem. Soc. 2022, 144 (46), 21035-21045).

[0231] Cathepsin-L has also been noted for its enzymatic affinity towards casein proteins and the dyed azocasein protein (Kirschke, H.; et al. Proteinases and their Inhibitors; TURK, V., VITALE, L., Eds.; Pergamon, 1981; pp 93-101). Cathepsin-L has also been noted for being the only protease to demonstrate high activity towards casein at pH levels between 5.5 and 6.5, allowing for the observation of enzymatic activity without potential interference with other assays (Kirschke, H.; et al. Proteinases and their Inhibitors; TURK, V., VITALE, L., Eds.; Pergamon, 1981; pp 93-101).

[0232] As an initial test of the dual-format enzyme assay, Cathepsin L (CTSL) was focused on. CTSL is a lysosomal cysteine protease, involved in protein processing and in breaking down cells during apoptosis prior to their recycling (Kirschke, H. et al., In Proteinases and their Inhibitors; TURK, V., VITALE, L., Eds.; Per-gamon, 1981; pp 93- 101., Felbor, U. et al., EMBO J. 2000, 19 (6), 1187-1194.). Elevation of CTSL in disease relative to baseline levels has been observed in numerous pathologies, including kidney failure, parasitic infection, carcinoma, cardiomyopathy, pancreatic cancer, and infection with the SARS-CoV-2 virus, the causative agent of COVID-19 (Piwkowska, A. et al., Diagn. Basel 2022, 12 (5)., Garsen, M. et al., Kidney Int 2016, 90 (5), 1012-1022., Gonzales Santana, B. et al., PLoS Negl. Trop. Dis. 2013, 7 (9), e2414., Ruan, J. et al., PLOS ONE 2014, 9 (11), el 12136., Mehra, S. et al., Mol Cell Biochem 2017, 428 (1-2), 139-147., Singh, N. et al., World J Gastroenterol 2014, 20 (46), 17532-17540., Zhao, M.-M. et al., Signal Transduct. Target. Ther. 2021, 6 (1), 134.). CTSL has also been shown to play a role in the activation of SARS-CoV-2 (Ashhurst, A. S. et al., J. Med. Chem. 2022, 65 (4), 2956-2970.). Differing levels of CTSL between severe vs. mild / asymptomatic cases of COVID-19 enable a metric for early risk assessment of the infection. The quantifiable CTSL enzymatic activity has also been shown to be an additional factor in severe cases of COVID-19, as well as being an indicator in numerous stages of cancer (Zhao, M.-M. et al., Signal Transduct. Target. Ther. 2021, 6 (1), 134., Katunuma, N. et al., In Proteinases and their Inhibitors; Turk, V., Vitale, L. J., Eds.; Attorney Docket No. 204606-0200-00 WO

[0233] Pergamon, 1981 ; pp 83-92., Sudhan, D. R. et al., Pharmacol Ther 2015, 1 5, 105-1 16 ). Numerous inhibitors of CTSL enzymatic activity are known; its inhibition is one of the targets of the commercial drug Paxlovid (Steven, F. S. In Proteinases and their Inhibitors; Turk, V., Vitale, L. J., Eds.; Pergamon, 1981; pp 305-315., Mondal, S. et al., J. Am. Chem. Soc. 2022, 144 (46), 21035-21045.).

[0234] Azocasein, a dye-conjugated derivative of casein, is the standard substrate for photometric CTSL assays, and was used as the immobilized enzyme substrate for the photonic assay of this Example (Kirschke, H. et al., In Proteinases and their Inhibitors; TURK, V., VITALE, L., Eds.; Pergamon, 1981; pp 93-101.). CTSL activity is typically measured at a pH between 5.5 and 6.5, and specificity for the substrate casein / azocasein is known to be high in this pH range (Kirschke, H. et al., In Proteinases and their Inhibitors; TURK, V., VITALE, L., Eds.; Pergamon, 1981; pp 93-101., Kirschke, H. In Handbook of Proteolytic Enzymes; 2013; Vol. 2, pp 1808-1817., Barrett, A. J. et al., In Methods in Enzymology; Proteolytic Enzymes, Part C; Academic Press, 1981; Vol. 80, pp 535-561.). It was anticipated that ring resonators bearing immobilized casein would show a blue-shift in proportion to the amount of substrate cleaved off the surface of the ring.

[0235] Materials and methods of the Example are now described.

[0236] Human CTSL and Cathepsin-B (CTSB) was expressed from human HEK 293 cells. Goat anti-fluorescein (anti-FITC) antibody and bovine serum albumin (BSA) were used as nonspecific binding and substrate controls. Gibco Premium Fetal Bovine Serum (FBS) was purchased from ThermoFisher (Waltham, MA). Assay wash buffer (AWB), which was mixed with FBS at a 4: 1 ratio to make FB20 and used to dilute serum samples, was composed of mPBS with 3 mM EDTA and 0.01% Tween-20. The diluent for antibody / antigen printing was modified (i.e., potassium-free) phosphate-buffered saline (mPBS) at a concentration of 0.20 M and a pH of 7.2. (3- glycidyloxypropyljtrimethoxysilane (GPTMS). The diluent for running assays was mPBS, mixed with FB20, at a pH of 5.8, adjusted to ensure CTSL enzymatic activity. A CTSL ELISA kit was used to confirm enzyme activity and was purchased from RayBiotech (Peachtree Comers, GA). A Perkin Elmer Enspire multimode 96 well plate reader was used for absorbance measurements for both the ELISA and enzyme assays. Attorney Docket No. 204606-0200-00 WO

[0237] Whole human blood samples were allowed to clot for 30 minutes after draw. Samples were then spun at 1200 x g for 5 min, and serum was pipetted off into a 15 mL conical tube and spun again for 10 minutes to remove any remaining cellular material. The serum was then aliquoted and stored at -80 °C until use.

[0238] Microring Resonators.

[0239] The silicon nitride microring resonators used in this work are consistent with designs of Bryan et al. but have been adapted to accommodate an increased number of sensing elements within the same chip footprint (Bryan et al., ACS Sens. 2024, 9 (4), 1799-1808). The resonators were fabricated with waveguides 1.5 pm wide and 220 nm tall, supporting a single transverse electric (TE) polarization mode. Optical modeling was performed using the finite difference (FD) method in OptoDesigner (Synopsys Photonic Design Suite). A 5.3 pm bottom oxide thickness was used to improve grating coupler performance relative to earlier versions. Each photonic integrated circuit (PIC) includes eight microring resonators, arranged as four channels with two rings per bus waveguide. To accommodate this within the 800 pm-wide usable region of the 1x4 mm PIC, the ring diameter was reduced from 198 pm to 164 pm, and the coupling gap was narrowed to 375 nm to compensate for increased bending losses and preserve near-critical coupling. These eight rings are integrated within a 1.3 pm-wide fully etched trench, replacing the previously reported individually etched sensing trenches (Figure 23). This new layout allows for higher integration density and improved fluidic compatibility. Photonic sensors were fabricated using the 300 mm AIM Photonics fabrication line (Fahrenkopf, N. M. et al., IEEE J. Sei. Top. Quantum Electron. 2019, 25 (5), 1-6.). Wafers were then diced by the Aim Photonics Testing and Packaging facility (Rochester, NY). An image of the PIC layout (GDS) and an optical microscope image of a representative PIC are provided in Figure 24. The average quality (Q) factor for ring resonators fabricated in this manner was found to be 22,206 ± 2,740.

[0240] Photonic Chip Functionalization. Prior to functionalization, sensor PICs were removed from the wafer and washed for 20 minutes in a 3 : 1 mixture of sulfuric acid and 25% hydrogen peroxide (“piranha” solution) Piranha solution is highly caustic and reacts violently with organics. Afterwards, the PICs were washed in nanopure water for 20 minutes before being dried under a stream of nitrogen gas. PICs were next placed in a Attorney Docket No. 204606-0200-00 WO chemical vapor deposition (CVD) system (Yield Engineering Systems, Fremont, CA) where approximately a monolayer of GPTMS was deposited on the surface with a thickness between 6-10 A as measured on satellite silicon / SiCh substrates by spectroscopic ellipsometry (J. A. Woolam VASE). Covalent attachment of proteins and peptides to the GPTMS functionalized surface was achieved by spotting directly on the rings using a sciFLEXARRAYER SX piezoelectric microarrayer (Scienion AG, Berlin, Germany), as described previously (Bryan, M. R. et al., ACS Sens. 2024, 9 (4), 1799- 1808.). The rings used for substrate cleavage were spotted with bovine serine albumin (BSA) as a control and azocasein or casein as a substrate at 1000 pg / mL. The rings used for antibody capture were spotted with CTSL monoclonal antibodies at 500 pg / mL and referenced to anti-fluorescein isothiocyanate (anti-FITC) as a non-specific binding control, spotted at 500 pg / mL. All rings received approximately 1 nL of the respective solution, which covers the entire ring. The print layout is shown in Figure 2. Sensor chips were maintained at 75% humidity overnight to allow reaction with GPTMS to proceed to completion, then used within a day. The average post-functionalization Q factor for representative rings was found to be 20,481 ± 410.

[0241] Assay Consumable Assembly. PICs were integrated with a simple, inexpensive glass microscope slide-based microfluidic card (GLAM) designed to provide passive flow of sample liquids into the photonic chip for analysis. 75 x 25 mm glass slides were used. Glass slides were treated before assembly with UV / ozone for five minutes to increase hydrophilicity. A section of 26 pM thick double-sided adhesive patterned using a laser cutter (Full Spectrum Laser, Hobby Series 20 x 12, Las Vegas, NV) was placed on the treated glass slide. This adhesive layer defines the microfluidic channel geometry and structures a channel height that facilitates efficient analyte delivery. The sensor chip was placed onto the double-sided adhesive such that there was conformal contact of the input and output gratings and the adhesive. The sensor region of the chip is located directly above the open channel. After placing the sensor chip, two silicon chips, also treated with UV / ozone, were placed on the adhesive flanking the sensor chip to stabilize flow. Finally, a strip of filter paper (QI, Whatman, Little Chalfont, UK) was placed downstream of the PIC, to facilitate continuous flow and provide a waste reservoir for the sample volumes used. Attorney Docket No. 204606-0200-00 WO

[0242] Apparatus Overview. The optical apparatus used for all photonic measurements follows the methods previously described and is shown in Figure 25 (Bryan, M. R. et al., ACS Sens. 2024, 9 (4), 1799-1808.). A tunable laser (Keysight 81606A0) is directed through polarization controllers (Thorlabs FPC561 with SMF-28 FC / PC connectors) to maximize transverse electrical (TE) polarization relative to the orientation of the silicon nitride waveguide. Light is directed through the input of the optical hub (15 X 15 X 16 mm; diamond-turned by Syntec Optics, Rochester, NY) and aligned to the grating input. Light from the four output gratings is collected by the output section of the optical hub and directed through a custom fiber bundle (IDIL Optics) of multimode fibers (Thorlabs FP200ERT) to four channels of the optical power meter (Keysight N7745A). Alignment of the PIC is achieved through an IR microscope. A 5x IR objective lens (Mitutoyo Plan Apo NIR 46-402) with on-axis illumination directs light though a long-pass dichroic mirror (Thorlabs DMLP950R) to the IR camera (WiDy InGaAs 650. Proper alignment is confirmed through visual inspection of the IR microscope image, maximization of output power relative to other alignments, and resonance spectra.

[0243] The tunable laser and optical power meter are connected to a computer via a general -purpose interface bus (GPIB) and are controlled by the Insertion Loss software of the Keysight Photonic Application Suite (N7700A). Spectral measurements were recorded by repeated wavelength sweeps. Ten-nm spectra were taken continuously at 1 pm resolution, centered on 1550 nm, with each spectral sweep taking approximately 6 s, as previously described (Klose, A. M. et al., Anal. Chem. 2021, 93 (40), 13580-13588.). Output spectra were processed automatically through custom software written in Python and deployed on Anaconda, as previously described (Bryan, M. R. et al., ACS Sens. 2024, 9 (4), 1799-1808.).

[0244] Sample Preparation and Assay Procedure. To make serial dilution samples, commercially obtained Pooled Normal Human Serum (PNHS) from 2018 was used for determination of detection limits. Pre-COVID-19 pandemic PNHS serum was used in order to avoid potential complications from the inadvertent inclusion of samples from subjects with undiagnosed COVID-19 disease in the pooled serum, which would lead to an unnaturally high baseline. 100 pL of FB20 was mixed with 900 pL mPBS at pH 5.8, or at the same ratio with different volumes, to make the sample diluent, FB2. A 100 Attorney Docket No. 204606-0200-00 WO pg / mL solution of Cathepsin-L was prepared in FB2, then diluted with FB2 in 10-fold increments to produce a dilution series down to a concentration of 100 pg / mL'1in a constant background matrix. These samples were used to generate the serial dilution responses. In all assays, 75 pL of fluid was added to the sample inlet of a GLAM card. Data collection started immediately. Assays were conducted at ambient laboratory temperature (22° C), and endpoints were measured at 5 minutes. Each sample was run 3 times. The data was then fit to a four-parameter logistic (4-PL) equation in Origin 2023 (OriginLab Corporation, Northampton, MA).

[0245] To test the effect of an enzyme inhibitor on the assay, unspiked serum samples used as controls were formed by mixing 90 pL of FB2 with 10 pL of serum. Spiked serum samples were made by mixing 80 pL of FB2 with 10 pL of serum and 10 pL of a 100 ng / mL cathepsin-L solution made in the same manner as the serial dilution measurements. Each sample was then vortexed to ensure it was fully mixed. The spiked sample was then split into an additional two samples, and 1 mg of the E-64 inhibitor was added to one of the two samples. Each sample was then vortexed to ensure it was fully mixed. Samples were allowed to incubate at 4° C for 3 hours prior to assay, and then run immediately. To test potential interference, 80 pL of FB2 with 10 pL of serum and 10 pL of a 100 ng / mL cathepsin-B solution, made in the same manner as the cathepsin-L solutions.

[0246] To assess the effect of background matrix variability, 25 single-donor serum samples were selected from individuals between age 18 and 35 without any known health conditions. Each sample was split into spiked and unspiked samples. Unspiked serum samples were formed by mixing 90 pL of FB2 with 10 pL of serum. Spiked serum samples were made by mixing 80 pL of FB2 with 10 pL of serum and 10 pL of a 100 ng / mL cathepsin-L solution made from the serial dilution measurements. Each sample was then vortexed to ensure it was fully mixed. As above, each sample was allowed to incubate at 4° C for 3 hours prior to assay.

[0247] Results of the Example are now described.

[0248] Serial Dilution Curve Generation for Cathepsin-L. The concentration and enzymatic activity of commercial CTSL used throughout this study were confirmed using reference-standard ELISA and UV-Vis enzymatic assays. Attorney Docket No. 204606-0200-00 WO

[0249] To confirm enzymatic activity of Cathepsin-L, 200 pL of 0.2 ng / mL azocasein in mPBS pH = 5.8 was mixed with 100 pL of cathepsin-L at varying concentrations. A UV- vis absorption peak of the azocasein was taken and a peak was identified at 440 nm. This is consistent with the information provided by the supplier, although the absorbance peak of azocasein can change due to the conjugated dye (Azocasein protease substrate 102110- 74-7. Sigmaaldrich(dot)com / US / en / product / sigma / a2765 (accessed 2025-04-21), Chu, S. S. et al., Sensors 2022, 22 (14), 5200). The absorbance was then measured at 440 nm every five seconds for one minute. The resulting shifts in absorbance were then analyzed, with the endpoint shift in absorbance to concentration. The Vmax can be extracted from these data by the molar absorbance of azocasein, which was not calculated because the supplier provided a range of values for the extinction coefficient (Azocasein protease substrate 102110-74-7. Sigmaaldrich(dot)com / US / en / product / sigma / a2765 (accessed 2025-04-21)). This UV-vis absorbance assay confirms that the concentrations of cathepsin-L used in these experiments exhibit enzymatic activity within a linear range.

[0250] The reference UV-Vis enzymatic assay was likewise used to establish the baseline concentration and activity of PNHS and FBS prior to doping in recombinant CTSL, and to confirm that cathepsin-B was inactive in this matrix (Figure 8 and Figure 26). The presence of Cathepsin-L in PNHS was confirmed with an ELISA assay, and found to be 1.97 ng / mL, and the spiked serum had a net concentration of 11.97 ng / mL. This encompasses the range of reported healthy and elevated levels of Cathepsin-L in serum (Singh, N. et al., World J Gastroenterol 2014, 20 (46), 17532-17540.). Dilution series of CTSL in PNHS accounted for the baseline 1.97 ng / mL concentration.

[0251] To further confirm enzymatic activity of Cathepsin-L, 250 pL of 0.6 mg / mL Azocasein in mPBS pH = 5.8 was mixed with 50 pL of cathepsin-L at varying concentration. A UV-vis absorption peak of the azocasein was taken and a peak was identified at 350 nm. This is consistent with similar assays with azocasein as a substrate, although the absorbance peak of azocasein does vary based on the conjugated dye (Chu, S. S.; et al. Sensors 2022, 22 (14), 5200, Klose, A. M.; et al. Anal. Chem. 2021, 93 (40), 13580-13588, Coelho, D. F.; et al. Biomed Res Int 2016, 2016, 8409183). The absorbance was then measured at 350 nm for one minute every 5 seconds. The resulting shifts in absorbance were then curve fit to a Michaelis-Menton Model using a Hyperbola Attorney Docket No. 204606-0200-00 WO function on the software OriginPro, where the maximum velocity (Vmax) is extracted. The Vmax is then plotted across concentrations of Cathepsin-L used in the assay (Figure 9). This UV-vis absorbance assay confirms that the concentrations of cathepsin-L used in these experiments exhibits enzymatic activity within a linear range.

[0252] For photonic assay serial dilution samples, the limit of blank (LOB) was determined and added to the standard deviation of low-concentration samples to derive lower limits of detection (LLOD) of human CTSL antibody capture and enzymatic activity (Klose, A. M. et al., Anal. Chem. 2021, 93 (40), 13580-13588.). Results are displayed in Figures 3 and 4. Both assays are well-behaved, with calculated LLOD of 2.0 (substrate cleavage) and 1.8 ng / mL, respectively. Both are below the required LLOD for CTSL in clinical situations of 2.2 ng / mL (Zhao, M.-M. et al., Signal Transduct. Target. Ther. 2021, 6 (1), 134.). Because this assay is label-free, its dynamic range may be tuned based on modifications to the enzyme substrate: it was observed that immobilized casein produced a lower magnitude shift in the enzymatic assay (due to both its lower mass and reduced refractive index change on cleavage) relative to the dye-tagged azocasein (Figure 20).

[0253] Dual Antibody and Enzymatic Assay using Pooled Human Serum. To assess the ability of the dual assay to report changes in CTSL concentration and activity, responses of 1 :10 diluted PNHS, PNHS + 10 ng / mL CTSL, PNHS + 10 ng / mL cathepsin- B (CTSB), and PNHS + 10 ng / mL CTSL + E-64, an irreversible inhibitor of cysteine proteases were compared (Matsumoto, K. et al., Pept. Sci. 1999, 51 (1), 99-107.). The baseline measurement of CTSL concentration and CTSL activity present in PNHS and PNHS + 10 ng / mL CTSL (Figure 5) were consistent with measurements done in the context of the serial dilution experiments. The addition of the inhibitor agent changes the enzyme activity to a point where the only observed shifts are within the noise, while the unchanged concentration results in antibody capture that is similar to the sample without an inhibitor agent. These results highlight a key advantage of this sensor, as the change in concentration and enzyme activity can be measured in a single test. T-tests were performed to ensure statistical significance. All differences were significant at 95% confidence, except the difference between the antibody capture for the spiked and spiked + inhibitor solutions. Table 1 details the statistical results (Table 1, below). Attorney Docket No. 204606-0200-00 WO

[0254] Table 1. Statistical tests comparing dual assays run on 3 different serums. A = antibody capture assay, E = enzymatic cleavage assay, U = unspiked (PNHS as- obtained), B = PNHS spiked with CTSB, S = PNHS spiked with CTSL, and I = PNHS spiked with CTSL and inhibitor solution.

[0255] While the specificity of Cathepsin-L for azocasein at acidic pH is well known, it was confirmed this was the case in this assay as well by comparing the response to samples doped with Cathepsin-B (Barrett, A. J. et al., In Methods in Enzymology; Proteolytic Enzymes, Part C; Academic Press, 1981; Vol. 80, pp 535-561.). No statistically significant difference in response was observed for PNHS doped with 10 ng / mL CTSB, vs. just the serum alone (Figure 5).

[0256] Dual Antibody and Enzymatic Assay Single Serum Samples.

[0257] Serum from various individuals was drawn throughout 2020 and beyond. An individual with severe COVID and no medication, an individual with severe COVID who was medicated with Remdesivir and Tocilizumab, an individual with asymptomatic COVID, and an individual without COVID were selected for use on this sensor. All Attorney Docket No. 204606-0200-00 WO samples were diluted 1 : 10 in mPBS (pH = 5.8) with 50 pL of fluid was then added to the micropillar cards, with 6 replicates of each assay performed. The results are shown in Figure 6 A and Figure 6B.

[0258] Finally, the performance of the dual CTSL assay in 25 single-donor human serum samples was evaluated. Baseline responses were first measured for each sample; 10 ng / mL CTSL was then doped into the sample, and it was re-measured. Figure 7 shows the differential response for all 25 single-donor serum samples after addition of 10 ng / mL CTSL relative to baseline. It was observed that the assay provided relatively consistent responses, with a coefficient of variation (CV) of 16.4% for the enzymatic cleavage and 15% for antibody capture. When not incubated at consistent times, it was observed that variation increased significantly, as would be expected for any enzyme assay.

[0259] Conclusions of the Example are now described.

[0260] A central advantage of label-free sensors is the ability to build in multiplex sensing capability without increasing complexity of the assay. Beyond this, an important but relatively unstudied area is that of multiplexing different types of assays, i.e. multimodal sensing, to increase the different types of information that can be obtained from a system under study. In this Example, a multiplex photonic sensor capable of simultaneously quantifying enzyme activity and concentration in a sample was demonstrated. Both detection modes are analytically well behaved, specific, and usable in the complex and variable background matrix of human serum. Importantly, the assay was able to distinguish inhibition of CTSL by E-64 inhibitor from a decrease in concentration of the enzyme.

[0261] It was observed that tuning both the mass and refractive index of the immobilized substrate may be used to alter the performance of the assay in subtle ways, by comparing responses with casein and azocasein. Casein has a refractive index of 1.57, whereas the dyed azocasein has a refractive index of 1.8 (Ambrose Griffin, M. C. et al., J. Colloid Interface Sci. 1985, 104 (2), 409-415., Mavrona, E. et al., Opt. Mater. Express 2018, 8 (2), 420-430.). Thus, cleavage of azocasein results in a larger change in effective refractive index than casein. Therefore, tuning both the mass and refractive index of the immobilized substrate may provide for optimizing assay performance. Attorney Docket No. 204606-0200-00 WO

[0262] In the clinical context, as already discussed, CTSL is a useful marker for severe COVID-19, and an essential enzyme in the SARS-CoV-2 infection pathway (Zhao, M - M. et al., Signal Transduct. Target. Then 2021, 6 (1), 134., Ashhurst, A. S. et al., J. Med. Chem. 2022, 65 (4), 2956-2970.). A single study noted 42 different clinical enzymes that diagnose various disorders, including cancer, liver failures, heart attacks, and more (Hemalatha, T. et al., Indian J Exp Biol 2013, 51 (10), 777-788.). Many of these are predicted to be amenable to the assay format reported herein. Moving beyond potential clinical applications, the development of enzyme inhibitors is of significant importance, and the assay format reported herein may have utility in the development of inhibitors (Levitzki, A. et al., Science 1995, 267 (5205), 1782-1788.). Other applications of the assay format reported herein include rapid screening of substrate specificity, both in biomedically relevant contexts and in biomanufacturing.

[0263] This approach may be suitable for assaying activity of any enzymes having a substrate that may be immobilized on the surface of the sensor without compromising the ability of the enzyme to react with it, and in which the enzymatic reaction causes a significant change in mass (either increase or decrease) of the sensor-immobilized substrate. Examples of enzymes and substrates fitting these criteria include collagenase (enzymatic cleavage of collagen; loss of mass) and ubiquitin ligases and artificial peptide ligases (enzymatic conjugation of peptides; increase in mass) (Liu, F. et al., Mol. Cancer 2024, 23, 148., Severin, K. et al., Nature 1997, 389 (6652), 706-709.).

[0264] An inexpensive multiplex platform capable of the detection of cathepsin-L through two different detection mechanisms, through antibody-antigen binding and through enzymatic degradation is demonstrated.

[0265] The clinical utility for Cathepsin-L is important in a variety of different circumstances. In COVID, Cathepsin-L increases in concentration for severe symptoms, and Cathepsin-L has been shown to be an important enzyme in the SARS-CoV-2 infection pathway (Zhao, M.-M.; et al. Signal Transduct. Target. Ther. 2021, 6 (1), 134, Ashhurst, A. S.; et al. J. Med. Chem. 2022, 65 (4), 2956-2970). The demonstrated measurement show that the device is capable of determining the difference in cathepsin-L concentration. Furthermore, by measuring enzymatic activity, the potential to show COVID severity by an additional metric was demonstrated. The use of an inhibitory Attorney Docket No. 204606-0200-00 WO agent demonstrates this sensor can detect the decrease in affinity as well. Paxlovid, widely used an antiviral medication against SARS-CoV-2 infection, inhibits the same pathway that Cathepsin-L utilizes. Thus, the described sensor has the capability to demonstrate the effectiveness of the Paxlovid medication. Given the increase in people who have a “rebound”, the described sensor may determine if enzymatic activity reemerges and therefore provide new insight into this phenomenon (Wang, L.; et al. medRxiv 2022). The described sensor may also help determine early on that someone should return to treatment, potentially saving at risk lives.

[0266] Outside of COVID, this sensor has clinical utility in a variety of other situations. Previous studies have shown Cathepsin-L to be key in fascioliasis confirmed by the use of an ELISA assay (Gonzales Santana, B.; et al. PloS Negl. Trop. Dis. 2013, 7 (9) e2414). Compared to ELISA, the described assay can have results ready in ten minutes, can demonstrate simultaneous enzymatic activity, and can provide insightful information about binding and enzyme kinetics. The platform can be expanded with other sensors that measure enzymatic activity of other proteases, capture other antibodies, or capture other analytes. It has been noted previously that cathepsin-L plays a role in cancer stages; initial stages show an increase in cathepsin-L concentration, but no change in activity11(Sudhan, D. R.; et al. Pharmacol Ther 2015, 155, 105-116). Later stages show an increase in activity, but no change in concentration. In this literature, Cathepsin-L is consistently confirmed by ELISA studies, with changes in optical density used to measure enzymatic activity. The described assay can put both results in a single assay.

[0267] Moving beyond Cathepsin-L, this assay is just the start of many possibilities in clinical diagnostics. A single study noted many clinical enzymes that diagnose various disorders, including cancer, liver failures, heart attacks, and more (Hemalatha, T.; et al. Indian J Exp Biol 2013, 51 (10), 777-788). All of these clinical enzymes have commercially available antibodies available from suppliers such as sinobiological and RNDSystems. All of these enzymes have substrates available for purchase from chemical suppliers such as Sigma-Aldrich and ThermoFisher. It therefore stands to reason that the developed platform can detect these many different enzymes just as it detects Cathepsin- L. Exemplary enzymes for which concentration and enzymatic activity can be detected by the device and method include alkaline phosphatase, cathepsin-D, MMP-9, Lysozyme, Attorney Docket No. 204606-0200-00 WO tartrate-resistant acid phosphatase, amylase, lipase, and lysozyme. This study goes on to cite a lack of an affordable, streamlined enzymatic assay for clinical use, and notes that this is a pressing medical need. Here, an inexpensive and accurate medical diagnostic device is presented. A device is presented that can both measure an enzyme’s specific activity and quantify its concentration via an antibody simultaneously, without the use of any secondary labels, and in a period of minutes. This technology provides real time information, including the binding kinetics and enzyme kinetics, which cannot be observed via an ELISA assay. The advantages this platform provides are limitless in providing affordable and relevant patient care throughout the clinical world.

[0268] Photonic ring resonators have been shown to demonstrate a variety of applications, and their successful use in developing a tissue chip platform was recently demonstrated (Cognetti, J. S.; et al. Lab. Chip 2021, 21 (15), 2913-2921). Given that cells and tissues are known to secrete cathepsin-L as an inflammatory response, this dual assay could be used in such a scenario (Felbor, U.; et al. EMBO J. 2000, 19 (6), 1187- 1194). The described assay can be combined with tissue chip platforms, utilizing not only cathepsin-L but a variety of secreted enzymes and inhibitors to study cellular response in vitro.

[0269] Moving beyond clinical applications, the development of drug inhibitors is a major field of research in organic chemistry (Levitzki, A.; Gazit, A. Science 1995, 267 (5205), 1782-1788). The need to quantify inhibitory features is key in research and development. In developing this assay, an additional quick and easy way for such research to occur was developed. This would remove the need to use a dyed agent, just as previous cathepsin-L studies have relied on azocasein due to its enzymatic dye. Because of the multiplexing capability, a single inhibitor could be tested against multiple targets simultaneously. The described device may be an alternative assay in laboratory settings that is quick and easy to use.

[0270] The multiplex capability of this assay may be expanded. Given the eight sensing rings, it is possible to immobilize three antibodies, three enzymatic substrates, an antibody negative control, and an enzymatic negative control on a single multiplex hub. The device can be a diagnostic device and used to detect several enzymes and quantify their enzymatic activity simultaneously. The number of ring resonators on this photonic Attorney Docket No. 204606-0200-00 WO device may be expanded so that more locations for specific antibodies and substates exist on a single assay. This creates the potential for a single device that could serve as a panel for all Cathepsin proteins.

[0271] A sensor capable of detecting enzymatic shifts and antibody binding through a single assay using a multiplex platform is demonstrated. This work is studied primarily through the lens of COVID immunity, but Cathepsin-L detecting is clinically relevant in a variety of circumstances throughout the clinical world. Moreover, the presented capability of the device for simultaneous detection of an enzyme through both its enzymatic activity and antibody binding has a litany of many applications.

Claims

Attorney Docket No. 204606-0200-00 WOCLAIMSWhat is claimed is:

1. A method for determining the concentration and enzymatic activity of one or more target molecules in a sample solution comprising the steps of: providing an apparatus comprising: a set of one or more detection elements, wherein the set comprises: one or more capture detection elements functionalized with one or more capture molecules that bind the one or more target molecules; and one or more interaction detection elements functionalized with one or more interaction molecules that are modified by the one or more target molecules; a sample addition zone; and a flowpath in fluid communication with the set of one or more detection elements and the sample addition zone; and delivering the sample solution to the sample addition zone.

2. The method of claim 1, wherein the set of one or more detection elements further comprises: one or more control detection elements functionalized with one or more control molecules having lower binding affinity to the one or more target molecules than the capture molecule or the interaction molecule.

3. The method of claim 1, wherein the set of one or more detection elements further comprises: one or more capture control detection elements functionalized with one or more capture molecules that bind a control target molecule; andAttorney Docket No. 204606-0200-00 WO one or more interaction control detection elements functionalized with one or more control molecules having lower affinity for the one or more target molecules than the interaction molecule.

4. The method of any one of claims 1-3, further comprising the step of: passing light through each of the set of one or more detection elements; detecting the light after passing through each of the set of one or more detection elements before and after each of the one or more detection elements contacts a sample solution; determining the resonance wavelength of each of the set of one or more detection elements before each of the set of one or more detection elements contacts a sample solution, wherein the determination is based on the detected light; determining the resonance wavelength of each of the set of one or more detection elements after each of the set of one or more detection elements contacts the sample solution, wherein the determination is based on the detected light; calculating the shift in resonance wavelength of each of the set of one or more detection elements before and after each of the set of one or more detection elements contacts the sample solution; determining the concentration of the one or more target molecules based on the resonance wavelength shift of the one or more capture detection elements; and determining the enzymatic activity of the one or more target molecules based on the resonance wavelength shift of the one or more interaction detection elements.

5. The method of claim 4, wherein the determination of the concentration of the one or more target molecules is further based on the difference between the resonance wavelength shift of the one or more capture detection elements and the resonance wavelength shift of the one or more capture control detection elements.Attorney Docket No. 204606-0200-00 WO6. The method of any one of claims 4-5, wherein the determination of the enzymatic activity of the one or more target molecules is further based on the difference between the resonance wavelength shift of the one or more interaction detection elements and the resonance wavelength shift of the one or more control interaction detection elements.

7. The method of any one of claims 1-6, wherein contact between the one or more target molecules and the one or more capture detection elements forms a complex comprising the one or more target molecules and the one or more capture molecules bound to the one or more capture detection elements.

8. The method of any one of claims 1-7, wherein contact between the one or more target molecules and the one or more interaction detection elements causes at least one or more interaction molecules or interaction molecule portions to be released from the one or more interaction detection elements.

9. The method of any one of claims 1-8, wherein contact between the one or more target molecules and the one or more capture detection elements causes an increase in the resonance wavelength of the one or more capture detection elements.

10. The method of any one of claims 1-9, wherein contact between the one or more target molecules and the one or more interaction detection elements causes a decrease in the resonance wavelength of the one or more interaction detection elements.Attorney Docket No. 204606-0200-00 WO11. The method of any one of claims 1-10, further comprising the step of: adding a modulator of the one or more target molecules to the sample solution.

12. The method of any one of claims 1-11, further comprising the step of: diagnosing a subject based on the determination of the concentration and enzymatic activity of the one or more target molecules in the sample solution.

13. The method of any one of claims 1-12, further comprising the step of: obtaining the sample solution from a subject.

14. The method of any one of claims 12-13, further comprising the step of: administering one or more modulators of the one or more target molecules to the subj ect.

15. The method of any one of claims 1-14, further comprising the step of: administering one or more modulators of the one or more target molecules to a subject based on the determination of the enzymatic activity of the one or more target molecules in the sample solution.

16. The method of any one of claims 1-15, wherein the one or more detection elements include at least one of a ring resonator, a double ring resonator, a cylindrical resonator, a spherical resonator, a spiral waveguide, a Vernier filter, a photonic crystal, and a Mach-Zehnder Interferometer (“MZI”).Attorney Docket No. 204606-0200-00 WO17. The method of any one of claims 1-16, wherein the one or more capture molecules is selected from one or more of the group consisting of: a protein, a peptide, an antibody, an antigen, an enzyme, a substrate, and a nucleic acid.

18. The method of any one of claims 1-17, wherein the one or more interaction molecules is selected from one or more of the group consisting of: a protein, a peptide, an enzymatic substrate, a nucleic acid, a small molecule enzymatic substrate, a phosphopeptide, and a polymer.

19. The method of any one of claims 2-18, wherein the one or more control molecules is selected from one or more of the group consisting of: a protein, a peptide, an antibody, an antigen, an enzyme, a substrate, a polymer, a brush polymer, polyethylene glycol, serum, and a nucleic acid.

20. A system for determining the concentration and enzymatic activity of one or more target molecules in a sample solution comprising: an apparatus comprising: a set of one or more detection elements comprising: one or more capture detection elements functionalized with one or more capture molecules for the one or more target molecules; and one or more interaction detection elements functionalized with one or more interaction molecules for the one or more target molecules; a sample addition zone; andAttorney Docket No. 204606-0200-00 WO a flowpath in fluid communication with the set of one or more detection elements and the sample addition zone.

21. The system of claim 20, further comprising: a light source configured to pass light through each of the set of one or more detection elements; a photodetector configured to receive the light after passing through each of the set of one or more detection elements; a display; a processor communicatively connected to the apparatus, the light source, the photodetector, and the display; and a non-transitory computer readable medium with instructions stored thereon, which when executed by a processor perform steps comprising: passing a light spectra through each of the one or more detection elements with the light source; detecting the light spectra that has passed through each of the one or more detection elements with the photodetector; analyzing the detected light spectra; and determining a resonance wavelength of each of the set of one or more detection elements based on the analysis of the detected light spectra.

22. The system of any one of claims 20-21, wherein the set of one or more detection elements further comprises: one or more control detection elements functionalized with one or control molecules having lower binding affinity to the one or more target molecules than the capture molecule or the interaction molecule.

23. The system of any one of claims 20-21, wherein the set of one or more detection elements further comprises:Attorney Docket No. 204606-0200-00 WO one or more capture control detection elements functionalized with one or more capture molecules that bind a control target molecule; and one or more interaction control detection elements functionalized with one or more control molecules having lower affinity for the one or more target molecules than the interaction molecule.

24. The system of any one of claims 21-23, further comprising the steps of: determining the resonance wavelength of each of the set of one or more detection elements before each of the set of one or more detection elements contacts a sample solution, wherein the determination is based on the detected light spectra; determining the resonance wavelength of each of the set of one or more detection elements after each of the set of one or more detection elements contacts the sample solution, wherein the determination is based on the detected light spectra; calculating the shift in resonance wavelength of each of the set of one or more detection elements before and after each of the set of one or more detection elements contacts the sample solution; determining the concentration of the one or more target molecules based on the resonance wavelength shift of the one or more capture detection elements; and determining the enzymatic activity of the one or more target molecules based on the resonance wavelength shift of the one or more interaction detection elements.

25. The system of claim 24, wherein the determination of the concentration of the one or more target molecules is further based on the difference between the resonanceAttorney Docket No. 204606-0200-00 WO wavelength shift of the one or more capture detection elements and the resonance wavelength shift of the one or more capture control detection elements.

26. The system of any one of claims 24-25, wherein the determination of the enzymatic activity of the one or more target molecules is further based on the difference between the resonance wavelength shift of the one or more interaction detection elements and the resonance wavelength shift of the one or more interaction control detection elements.

27. The system of any one of claims 20-26, wherein contact between the one or more target molecules and the one or more capture detection elements forms a complex comprising the one or more target molecules and the one or more capture molecules bound to the one or more capture detection elements.

28. The system of any one of claims 20-27, wherein contact between the one or more target molecules and the one or more interaction detection elements causes at least one or more interaction molecules or interaction molecule portions to be released from the one or more interaction detection elements.

29. The system of any one of claims 20-28, wherein contact between the one or more target molecules and the one or more capture detection elements causes an increase in the resonance wavelength of the one or more capture detection elements.

30. The system of any one of claims 20-29, wherein contact between the one or more target molecules and the one or more interaction detection elements causes aAttorney Docket No. 204606-0200-00 WO decrease in the resonance wavelength of the one or more interaction detection elements.

31. The system of any one of claims 20-30, further comprising the step of: diagnosing a subject based on the determination of the concentration and enzymatic activity of the one or more target molecules.

32. The system of any one of claims 20-31, further comprising the step of: determining a treatment for a subject based on the determination of the enzymatic activity of the one or more target molecules in the sample solution.

33. The system of any one of claims 20-32, wherein the at least one detection element includes at least one of a ring resonator, a double ring resonator, a cylindrical resonator, a spherical resonator, a spiral waveguide, a Vernier filter, a photonic crystal, and a Mach-Zehnder Interferometer (“MZI”).

34. The system of any one of claims 20-33, wherein the one or more capture molecules is selected from one or more of the group consisting of: a protein, a peptide, an antibody, an antigen, an enzyme, a substrate, and a nucleic acid.

35. The system of any one of claims 20-34, wherein the one or more interaction molecules is selected from one or more of the group consisting of: a protein, a peptide, an enzymatic substrate, a nucleic acid, a small molecule enzymatic substrate, a phosphopeptide, and a polymer.

36. The system of any one of claims 22-35, wherein the one or more control molecules is selected from one or more of the group consisting of: a protein, aAttorney Docket No. 204606-0200-00 WO peptide, an antibody, an antigen, an enzyme, a substrate, a polymer, a brush polymer, polyethylene glycol, serum, and a nucleic acid.

37. A photonic integrated circuit (PIC), comprising: at least one first grating coupler; at least two second grating couplers; at least one waveguide between the first grating coupler and the second grating couplers; at least one detection element functionalized with one or more capture molecules that bind one or more target molecule disposed within the at least one waveguide; and at least one detection element functionalized with one or more interaction molecules that are modified by the one or more target molecules disposed within the at least one waveguide.

38. The PIC of claim 37, further comprising: at least one detection element functionalized with one or more control molecules disposed within the at least one waveguide, wherein the control molecule has lower binding affinity to the one or more target molecules than the capture molecule or the interaction molecule.

39. The PIC of claim 37, further comprising: at least one detection element functionalized with one or more capture molecules that bind a control target molecule; and at least one detection element functionalized with one or more control molecules having lower affinity for the one or more target molecules than the interaction molecule.Attorney Docket No. 204606-0200-00 WO40. The PIC of any one of claims 37-39, wherein the at least one detection element includes at least one of a ring resonator, a double ring resonator, a cylindrical resonator, a spherical resonator, a spiral waveguide, a Vernier filter, a photonic crystal, and a Mach-Zehnder Interferometer (“MZI”).

41. The PIC of any one of claims 37-40, wherein the one or more capture molecules is selected from one or more of the group consisting of: a protein, a peptide, an antibody, an antigen, an enzyme, a substrate, and a nucleic acid.

42. The PIC of any one of claims 37-41, wherein the one or more interaction molecules is selected from one or more of the group consisting of: a protein, a peptide, an enzymatic substrate, a nucleic acid, a small molecule enzymatic substrate, a phosphopeptide, and a polymer.

43. The PIC of any one of claims 38-42, wherein the one or more control molecules is selected from one or more of the group consisting of: a protein, a peptide, an antibody, an antigen, an enzyme, a substrate, a polymer, a brush polymer, polyethylene glycol, serum, and a nucleic acid.

44. A substrate, comprising: a sample addition zone in fluid communication with a wicking zone and a sample detection zone, wherein the sample detection zone is between the sample addition zone and the wicking zone, and wherein the sample addition zone is upstream of the wicking zone; and at least one photonic integrated circuit (PIC) of any one of claims 37-43 positioned in the sample detection zone, disposed directly on a top or bottom surface of the substrate, optically coupled to a light source and a photodetector via a fiber bundle.

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