Reusable bioanalysis arrays

Abstract

The disclosure relates to simultaneous or near simultaneous detection of hundreds or thousands of proteins in fluids disposed on a reusable array.

Description

REUSABLE BIOANALYSIS ARRAYS PRIORITY

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No.63 / 564,428, entitled “REUSABLE BIOANALYSIS ARRAYS”, filed on March 12, 2024, which is incorporated herein by reference in its entirety. FIELD

[0002] The disclosure relates to simultaneous or near simultaneous detection of hundreds or thousands of analytes, such as proteins, in fluids disposed on a reusable array. BACKGROUND

[0003] Conventional protein chips are devices in which proteins or peptides are captured by capture reagent fixed to the surface of the chip. The specificity of separate capture reagents for individual proteins provides for the detection of protein binding. Capture reagents can include antibodies, such as those used in sandwich assays. Proteomic measurement can further include the use of chromatographic techniques in combination with mass spectrometry for detection and quantification. Preparation and storage protocols vary widely. The inability to account for variations in sample preparation and analysis means that developing consistent results for health screening, particularly for individual patients over time, is prohibitively expensive and time consuming.

[0004] The Enzyme-Linked ImmunoSorbent Assay (ELISA) uses two antibodies specific for different analyte epitopes, making it a “sandwich assay”. A first antibody is immobilized to a surface, capturing a target protein in a sample fluid. The second antibody is linked to an enzyme that catalyzes a detectable change in a specific additive. An ELISA requires several steps and expensive reagents. Antibodies in an ELISA assay cannot be renatured following application of a biological sample (e.g., blood plasma) and removal of non-covalently bound immobilized antibody by a Clean in Place (CIP) protocol. SUMMARY

[0005] The disclosure is directed to devices and methods in which binding member arrays can be used, denatured, and renatured to provide proteome-level analysis of analyte samples. Binding members are covalently bonded to an array surface. After use, the binding members can be cleaned under high stringency conditions, and renatured and reconstituted to have atleast 99.5% binding affinity. Unlike conventional ELISA formats, the reconstitution results in reusability of the arrays.

[0006] The repeatability of the binding members system drastically reduces the cost of goods, while providing the capability of frequent testing. The device and methods benefit from the denaturation-renaturation selection of SOMAmers in SELEX, but instead to provide reusable SOMAmer arrays.

[0007] The devices and methods described herein provide substantial benefits in their speed, reusability, and cost, providing the capability of macro-studies such as those described in Oh et al., Nature (624)164 (2023), incorporated herein by reference in its entirety, but in a reusable format. The reusability drastically reduces the expense in comparison to other systems. Unlike more conventional diagnostics in which large biobanks have single collection times with unique specimen sets, or smaller longitudinal biobanks that can follow conditions over time, the reused immobilized binding member panels can provide an on- going assessment of health conditions, including in a personalized medicine context, with a more fulsome understanding of highly complex biological systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The disclosure will be readily understood by the following detailed description in conjunction with the accompanying drawings, wherein like reference numerals designate like structural elements, and in which:

[0009] FIG.1 depicts a configuration in which color centers corresponding to binding members correlate to radiation emission (e.g., fluorescent radiation), according to an illustrative embodiment; and

[0010] FIG.2 depicts a configuration in which target analyte:binding member complexes form, resulting in changed measured results, according to an illustrative embodiment. DETAILED DESCRIPTION

[0011] The disclosure may be understood by reference to the following detailed description, taken in conjunction with the drawings as described below. It is noted that, for purposes of illustrative clarity, certain elements in various drawings may not be drawn to scale, may be represented schematically or conceptually, or otherwise may not correspond exactly to certain physical configurations of embodiments. 2 4899-7634-3847.1

[0012] Clinical proteomics in which proteomics in which high quality data is used for a large fraction of the human proteome are the next horizon in mass biological development. Mass clinical proteomics will include both longitudinal and repeatable assays. The positive impact on major and rare diseases should be substantial.

[0013] To achieve scale, proteomics will be repeatable, specific, have low CVs, provide speed of analysis, and at a low cost. SomaLogic has developed platforms with high specificity, low coefficient of variation (CV).

[0014] Herein is provide devices and methods in which a binding member array is covalently bonded to a surface substrate. Once the microarray is contacted with a sample, the binding members bind to their respective target analytes present in the sample and thereby enable a determination of the absence, presence, amount, and / or concentration of the target analytes in the sample. In variations, the binding member can be an aptamer, SOMAmer, or DARPin.

[0015] If capable of reuse, the cost of a covalently bonded binding member array over repeated use can be amortized over several uses. The cost combined with the specificity and less than 5% CV provides a system capable of measuring complex biological systems over a period of time.

[0016] Binding members capable of binding target analytes can be covalently bonded to the solid support surface. Such assays can involve the use of a microarray that includes one or more binding members immobilized on a solid support.

[0017] In some variations, the binding members can be denatured and washed as described in Potyrailo et al., Angew. Chem. Int. Ed.2015, 54, 2174 –2178 (incorporated herein by reference in its entirety), and then renatured to at least 99.5% binding specificity and binding affinity. The binding members described herein providing binding specificity comparable to ELISA. However, unlike antibodies used in ELISA, the binding members can be denatured and cleaned, and then renatured with binding activity at least 99.5% from a first use to a second use. The reusability of the binding members (aptamers, SOMAmers, and DARPins) is impossible with antibodies. The reusability of the binding member array reduces the cost of goods substantially.

[0018] Denaturing and cleaning conventional binders such as antibodies in an ELISA using the current cleaning methods would prevent renaturation and folding to at least 99.5% binding specificity and binding affinity. By contrast, the present disclosure provides binding members that renature with at least 99.5% binding activity. 3 4899-7634-3847.1

[0019] The binding activity can be measured by the measured property of the NK center associated with the particular binding member following kinetic challenge. The property of the NK center following denaturation of the binding members and removal of all non- covalently bound biological compounds is at least 99.5% of the property of the NK center in the previous measurement. The comparison of binding activity before and after removal of biological constituents can be measured by the KDof the binding members to their target analytes.

[0020] Taking SOMAmers as an example, unlike ELISA a SOMAmer covalently bonded to a solid surface is renaturable and therefore reusable, as evidenced by having been selected using a denaturation-renaturation cycle in every round of SELEX. Antibodies have not been selected by a denaturation / renaturation cycle, and cannot be so-selected, and thus are not re- usable in a denaturation-renaturation context. As such, a several thousandplex SOMAmer array can be generated on a surface. Binding Member Arrays

[0021] The disclosure describes devices, systems, and methods for detecting target analytes in a sample based on a change in a property of one or more binding members on the surface of the substrate upon binding a target analyte. When a target analyte binds to a binding member attached to the surface, it forms a target analyte:binding member complex. A detector can be configured to detect the change in the color center property resulting from the formation of the target analyte:binding member complex, thereby detecting the target analyte in the sample.

[0022] The binding member arrays can be reused in a manner that conventional arrays cannot. Specifically, the binding members can be renatured and reconstituted to active components following a CIP protocol. As used herein, the CIP protocol removes all biological components and denatures the binding members, while not removing binding members from the surface of the array. Following renaturation of the binding members at physiological buffer and room temperature and pressure, the binding members have at least 99.5% binding affinity to target analytes as compared to a first use before a CIP protocol and denaturation of the binding members.

[0023] A CIP protocol refers to a protocol in which all materials from blood plasma, but not the covalently bound binding members, are removed from the substrate surface after 4 4899-7634-3847.1detection and quantification of target analyte:binding member complexes. Binding members are denatured by the CIP protocol. Blood plasma includes proteins, sugars, lipids, metabolites, clotting factors, and salts, and includes target analytes. CIP protocols can include protein denaturants (e.g., urea, detergents (SDS)), proteases (e.g., trypsin), water miscible or water immiscible organic solvents (e.g., ethanol or other alcohols, chloroform, and lipid- dissolving reagents (e.g., chloroform / methanol solution), and heat, either together or separately in any combination and / or sequence. High pH (pH > 10) or low pH (pH < 4) can be used to degrade various components. Heat can also be used to degrade components. An example of a surface treatment that does not satisfy a CIP protocol includes formaldehyde, which would slowly, over many cycles, attack the nucleic acid-based binding reagents, or DNase which would attack those same DNA-based binding reagents, but not degrade DNA between uses. A CIP protocol denatures the secondary and tertiary conformation of the binding members covalently bound to the substrate, but does not degrade or break the covalent bond of the binding member with the substrate. CIP protocol can be assessed by adding plasma onto arrays of target analyte:binding member complexes and washed. Degraded biological components can be removed using any washing method known in the art. Aptamer and SOMAmer Arrays

[0024] In some variations, the binding member array is an aptamer array. Aptamers are nucleic acid ligands selected for specific binding to a target analyte (e.g., a target protein, metabolite, and or lipid), are developed and selected. Aptamers are nucleic acids that form two- and three-dimensional structures. Aptamers have sufficient chemical versatility available within their monomers to function as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as target analytes. Such an array can be, for example, as described in US Patent No.11,249,080, which is incorporated herein by reference in its entirety.

[0025] In some variations, the binding member array is a SOMAmer array. Such an array can be, for example, as described in US Patent No.11,249,080, which is incorporated herein by reference in its entirety.

[0026] Slow Off-rate Modified Aptamers (SOMAmers) are a class of binding members. SOMAmers are capable of binding to a target analyte with high specificity and affinity. SOMAmers are aptamers having improved off-rate characteristics, represented as a rate of dissociation (t1 / 2) at which 50% of the aptamer / target complex has dissociated. SomaScan, 5 4899-7634-3847.1which uses SOMAmers to detect and quantify human proteins, today can detect about 11,000 human proteins with a low coefficient of variation (CV) (under 5%). See, e.g., U.S. Pat. No. 5,475,096 entitled “Nucleic Acid Ligands” see also, e.g., U.S. Pat. Nos.6,242,246, 6,458,543, and 6,503,715, entitled “Nucleic Acid Ligand Diagnostic Biochip”, each of which is incorporated herein by reference in its entirety.

[0027] SOMAmers are aptamers having improved off-rate characteristics represented as a rate of dissociation (t1 / 2) or the point at which 50% of the aptamer / target complex has dissociated. Such rates of dissociation may vary, generally, from greater than 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220 and 240 minutes, this being the average time it takes a protein-aptamer complex to dissociate.

[0028] SOMAmers can be prepared by systematic evolution of ligands by exponential enrichment (SELEX). SOMAmers can contain modified nucleosides that provide for different built-in functionalities. These functionalities may include tags for immobilization, labels for detection, means to promote or control separation, hydrophobic sidechains to provide better affinity with proteins, etc. The modifications to improve affinity with proteins are chemical groups that are attached to the 5-position of the pyrimidine bases. By functionalizing the 5-position (e.g., with a benzyl, napthyl or indole group) the chemical diversity of the SOMAmers is expanded, allowing high affinity binding with a wider range of target analytes. Additionally, some polymerases are still able to transcribe DNA with modifications in these positions, thus allowing the amplification necessary for the SELEX process. SOMAmers, and the methods to produce them, are described in U.S. Pat. Nos. 7,964,356 and 7,947,447, both entitled “Method for generating aptamers with improved off- rates,” each of which is incorporated herein by reference.

[0029] Arrays of SOMAmers can be formed on a substrate. Conventionally, SOMAscans are performed using a surface (e.g., a glass slide) onto which short oligonucleotides are immobilized at known X-Y locations. Designing a SOMAmer array in the place of oligonucleotides at X-Y locations by covalently bonding the SOMAmers to the array provides a reusable assay. Covalently attached SOMAmers can bind specific target analyte such as a target analyte (and no others) by combining binding with a wash of the analytes with negatively charged oligonucleotides and heparin sulfate. As such, a kinetic challenge is performed directly on the SOMAmer array. The binding and kinetics are performed directly on the surface. The concentration of multiple proteins can be measured simultaneously by using SOMAmers specific to each individual protein at each separate location of an X-Y panel. 6 4899-7634-3847.1

[0030] Using a covalently binding SOMAmers in the array, the final time intensive hybridization step of the SOMAscan does not need to be performed. Instead, bound SOMAmers may be detected by any surface-based assay known in the art.

[0031] When binding member arrays are SOMAmer arrays, the SOMAmer array can be any array disclosed in the art. In various aspects, the binding member arrays can correspond to SOMAmers in the 7,000 analyte arrays, 11,000 analyte arrays, custom arrays, or disease- specific arrays. These can include any SOMAscan panel, as described in https: / / somalogic.com / somascan-panels / and all associated links, which is incorporated by reference herein in its entirety. DARPin Arrays

[0032] In some variations, the binding member is a designed ankyrin repeat protein (DARPin), also referred to herein as DARPin proteins. DARPins are derived from naturally occurring ankyrin repeat motifs, a protein class that mediates high-affinity protein-protein interactions in nature. DARPin proteins are binding members with high specificity and high binding affinity to a target protein. A DARPin protein includes at least one ankyrin repeat domain, and may comprise 2, 3, 4, 5, or more ankyrin repeat domains. The ankyrin repeat domains include a core scaffold that provides structure, and target binding residues that bind to a target. The core scaffold includes conserved amino acid residues, and the target binding surface includes amino acid residues that differ depending on the target. DARPins are as described in Stumpp et al., Drug Discovery Today 13(15-16): 695 – 701 (2008), Stumpp et al., Curr Opin Drug Discov Devel.10(2): 153-9 (2007); and Binz et al., Nature Biotech. 22(5): 575-582 (2004), each of which is incorporated by reference herein in its entirety. DARPins are a protease stable and thermodynamically stable, capable of denaturation and refolding. See Wetzel et al, J. Mol. Biol.376(1): 241-257 (2008), incorporated by reference herein in its entirety.

[0033] Any binding member known in the art that shows at least 99.5 % binding activity following cleaning in place (CIP) can be used in place of aptamers or SOMAmers. That is, any binding member configured to bind a target analyte and can have binding activity between CIP protocols can be used. Binding Member Arrays

[0034] The binding member array can include any number of binding members that correspond to a particular target analyte. In some variations, the binding member array can 7 4899-7634-3847.1include binding members on the order of 104targets. In some variations, the binding member array can include binding members on the order of 103targets. In some variations, the binding member array can include binding members on the order of 102targets. In some variations, the binding member array can include binding members only a few (e.g., on the order of ten) targets. In many aspects, the greatest benefit of the binding member arrays is the large number of binding members corresponding to a large number of targets. The assay of a large number of binding members provides for measurement of complex biological systems, not achievable on a repeated basis for other technologies. Label-Free Detection and Color Centers

[0035] The detection of bound binding members can include a detection of a color center in the substrate that changes upon binding of a specific binding member corresponding to the location on the array to its target analyte (e.g., target protein, metabolite, or lipid). The methods described herein reduce or eliminate fluids include the sample fluid, wash fluids, fluids used for regeneration, and detection agents (e.g., labels) of the binding member arrays. After a sample has been added to the surface and binding of target analytes completed, the surface is washed to remove unbound proteins. Following a kinetic challenge, target analyte:binding member complex can be measured.

[0036] In various aspects, binding members covalently bonded on surfaces can use labels or be label-free. Label-free assays can be detected using properties inherent in the assay system. Such properties include molecular charge, molecular weight, dielectric constant, or affinity for a binding member. Label applications can include a detectable tag, such as but not limited to a dye, a radioisotope, or other molecule that can be measured. Alternatively, the binding member can be labeled with a spin label without labeling the protein target. Target Analytes

[0037] Any type of target analyte can be detected, including, but not limited to proteins, metabolites, or lipids.

[0038] The target analytes can be in any samples containing target analyte may be any type of same sample. The sample can be a biological fluid, such as whole blood, blood plasma, serum, tissue extract, urine, or a different biological matrix. The sample is to the applied to the binding member array. In some variations, the sample can be processed to obtain a specific class of target analyte (e.g., a protein, metabolite, or lipid). 8 4899-7634-3847.1

[0039] In some variations, the target analyte is a protein. When the analyte is a target protein, the target protein may be naturally occurring or processed. Proteins can include folded polypeptide chains in their physiological conformation, abnormally folded polypeptide chains, denatured polypeptide chains, fragments of a polypeptide chain that may or may not be normally folded, short polypeptides, polypeptides that incorporate non-natural amino acids, and polypeptides that are post-translationally modified (e.g., phosphorylation, glycosylation, disulphide bonding, etc.) or polypeptides assembled into a protein complex. The target analytes may also include small molecules found in biological fluids such as metabolites. Alternatively, the target analyte can be a lipid. Measurement by Surface Bound binding members

[0040] FIG.1 depicts an illustrative configuration in which color centers corresponding to binding members allow optical detection of radiation emission (e.g., fluorescent radiation). In FIG.1, discrete color centers 102, 104, 106, and 108 are embedded at discrete regions in substrate 100. binding members 112, 114, 116, and 118 are covalently attached to substrate surface 120 at discrete regions. Each separate discrete binding member binding region 112, 114, 116, and 118 corresponds to each discrete color center 102, 104, 106, and 108, respectively.

[0041] When radiation 120 (e.g., fluorescent radiation) is incident on the discrete color centers 102, 104, 106, and 108, the radiation is absorbed by the color center, which then emits radiation. Properties of emitted radiation, including emission intensity, wavelength shift, or other properties, can be measured by detector 122. In other variations, electric field changes or magnetic field changes, may be detected, as described in U.S. Patent No. 11,249,080.

[0042] Substrate 100 is exposed to target analytes. The presence of target analyte:binding member complex at binding member 112, 114, 116, and 118 can be detected based on the fluorescent emission from the nitrogen-vacancy center. In one variation, the intensity of the decrease results from formation of the target analyte:binding member complex. The orientation (and resulting emission efficiency) of color centers is compensated for because misalignment will be observed for both the target analyte:binding member complex and for unbound binding member. In another variation, the wavelength spectrum of the color center can shift (e.g., wavelength shift). 9 4899-7634-3847.1

[0043] In FIG.2, binding member 114 binds target analyte 124, and binding member 118 binds target analyte 128. After binding of target analytes to the binding member to form a target analyte:binding member complex, the surface can be washed to remove unbound materials (e.g., extraneous proteins, metabolites, or lipids). This wash can be performed buffer solutions similar to the sample fluid, such as phosphate buffered saline or the same buffer used for the binding reaction used to form the target analyte:binding member complex.

[0044] The target analyte:binding member complex then undergoes a kinetic challenge by exposure to a polyanionic competitor. Because dissociation rates of cognate target analyte:binding member interactions are much slower than those of non-specific interactions, a polyanionic competitor, present in excess, rapidly occupies binding sites freed by the dissociated non-cognate complexes and prevents their rebinding.

[0045] The resulting target analyte to binding member to create a target analyte:binding member complex is then measured. With further reference to FIG.2, formation of a target analyte:binding member complex results in a change in the interaction between binding member 114 and discrete color center 104, and binding member 118 and discrete color center 108. A detectable change in properties (e.g., radiation intensity and / or spectrum) is then observed in emitted radiation 122. As described above, and as described in U.S. Patent No. 11,249,080, the presence of a target analyte:binding member complex instead of an unbound binding member can result in an electric field change and / or a magnetic field change, as described in property of the color center (e.g., a property associated with a magnetic resonance or spin of the color center). Formation of Binding Member Arrays

[0046] In the present disclosure, color centers are embedded in the surface of a substrate array, for example at a specific depth from the substrate surface. In some variations, the depth of color centers can be at least 5 nm. In other variations, the depth of color center can be less than 25 nm. The color centers can be separated by a sufficient distance to allow the access of target analytes to binding members and formation of different target analyte:binding member complexes. In some variations, the distance between color centers is at least 100 nm, 200 nm, or 300 nm. Color centers separated by hundreds of nanometers allow the signal from a single color center to be observed separately. 10 4899-7634-3847.1

[0047] In a first variation, a single binding member is associated with a single color center, which is in turn associated with a single photodetector. Binding a binding member can be detected on an individual basis. Target molecules can be counted individually, eliminating the need to calibration binding to multiple binding members. However, several of the same binding member would thereby have to be associated with a number of NV centers to measure orders of magnitude of binding.

[0048] The second variation, multiple binding members of the same type associated with multiple color centers, which are then associated with a single photodetector. Signals of multiple color enters can be averaged at the photodetector. A range of concentrations can thereby be quantified with a single photodetector. A calibration would be used to correlate the color center response to the concentration of the target protein in the fluid.

[0049] In a third variation, a collection of multiple binding members of the same type are associated with a single color center, which is associated with a single photodetector. A range of concentrations can be quantified with a single photodetector to provide an average signal due to the presence of multiple binding members. Color Centers

[0050] In some variations, the color center can be a crystalline defect in a substrate. Crystal defects have electronic properties that deviate from those of the undisturbed crystal lattice. The color center can signal the presence of binding due to light absorption and / or emission features in crystalline materials in the color center. For example, if an electrically negative ion is missing at a certain lattice position, an electron fills the vacancy and is trapped by surrounding positive ions. Alternatively, crystal defects can include the presence of interstitial ions or ion vacancies. Alternatively, a color center can be formed by administering excitation energy of laser-active ions (e.g., photodarkening of materials such as ytterbium- doped fibers).

[0051] Color centers formed from crystal defects can be created or generated. In one non- limiting example, a crystal defect can be created during crystal growth by growing crystals at a low growth temperature. Alternatively, crystalline defects can be caused by induced thermal fluctuations, resulting in different color center densities. In another alternative, color centers can be created by irradiating the crystalline lattice, for example with gamma radiation (e.g., high-energy gamma rays that transfer energy to particular ions, which then move to unusual 11 4899-7634-3847.1positions). Examples of materials with different color center densities can include nitrogen valence centers in diamond substrates.

[0052] binding members are bound to different color centers. Binding of a binding member by target analytes at a location corresponding to a substrate color center can be detected. In some variations, color center can absorb and emit radiation based on the formation of a target analyte:binding member complex. In some variations, the color center can increase intensity or change spectral properties (e.g., show a color, or show a color shift). For example, color centers with a sufficiently high density cause substantial absorption of light at optical wavelengths where there would normally be no absorption. Alternatively, completely transparent crystals can produce a color on binding due to some density of colors centers. For example, transmitted light may appear yellow if color centers lead to the absorption of blue light. Color centers can be created in any manner, for example as described in https: / / www.rp-photonics.com / color_centers.html, incorporated herein by reference in its entirety.

[0053] The surface bound binding member arrays can use label-free markers, such as those described in US Patent No.11,249,080. A label is detectable non-analyte molecule that is covalently or non-covalently attached to the analyte. The label can be detected based on any property known in the art, including molecular weight, molecular charge, dielectric constant, or affinity for an aptamer, or alternatively a property of a tag (a dye, a radio-isotope, or other measured property). Some variations of labels use spin label linked to an aptamer attached to a surface. Because this spin label is attached to a component of the detection system (the binding member) as opposed to the analyte, changes in binding can result in measurable difference in measured spin.

[0054] Alternatively, the target analytes can be detected by using binding members with magnetic spin labels, microwave emission, electrostatic interactions, or magnetic interactions, as described in U.S. Patent No.11,249,080, which is incorporated herein by reference, in its entirety. Nitrogen-Vacancy Centers

[0055] In some variations, the color center can be a nitrogen-vacancy (NV) center. NV centers can be present in diamond substrate when the substrate is a diamond surface (e.g., U.S. Patent No.11,249,080, which is incorporated herein by reference in its entirety). By creating the binding member array on a diamond substrate and containing Nitrogen Vacancy 12 4899-7634-3847.1centers just below the surface, the binding member can impact those nitrogen vacancy centers when a protein is bound providing a florescent signal.

[0056] Methods can include Optically Detected Magnetic Resonance (ODMR), such as those that make use of a nitrogen-vacancy (NV) center in a surface formed of a diamond crystal (e.g., U.S. Patent No.11,249,080, which is incorporated herein by reference in its entirety).

[0057] The NV center consists of a nitrogen substitution for a carbon atom positioned adjacent to a neighboring vacancy in a diamond lattice of the diamond substrate. The NV center is a paramagnetic color center with unique coupling between its electronic spin states and optical states. It is capable of emitting intense and stable fluorescence (i.e., large absorption coefficient combined with short lifetime of the excited state). The NV center also exhibits very long magnetic relaxation times, making it a sensitive detector of local properties such as the magnetic or electric fields. Alternatively, the NV centers can be naturally occurring.

[0058] Nitrogen-vacancy centers may be embedded in a crystalline structure, such as a diamond substrate, by introducing the nitrogen impurities and the vacancies at the desired depth, then annealing from 1000K-1300K, which allows the vacancies to collocate via diffusion to the nitrogen impurities. Nitrogen defects may be implanted either through a nitrogen pulse during chemical vapor deposition (CVD) of the diamond matrix or by ion beam implantation after the deposition has completed. Alternatively, vacancies can be implanted or created via ion beams electron, proton, or helium beams.

[0059] In NV centers, the diamond substrate is amenable to chemical modification, allowing for the covalent attachment of aptamers or binding members. Regenerating Arrays Following Use

[0060] The ability to perform routine proteomics at low consumer expense can have profound effects on science and healthcare.

[0061] Following kinetic challenge and measurement of the target analyte:binding member complex, all non-covalently bound materials, including target antigens bound in a target analyte:binding member complex. The method of removing the non-covalently bound materials can depend on the nature of the non-covalently bound materials. In some variations, the removal can depend on the components present. For example, if proteins (whether or not target antigens) are present in the sample, protein denaturants such as SDS and / or urea can be provided to denature the proteins, and proteases can be provided to degrade them. Lipids can 13 4899-7634-3847.1be removed using lipid dissolving reagents (e.g., a chloroform / methanol solution). In some variations, the method of removing non-covalently bound materials is a CIP protocol. In some variations, the binding members denature in the removal step.

[0062] Renaturation of the binding member is performed in a physiological buffer at room temperature and pressure.

[0063] The covalently bonded binding member array surface can be stripped of all target analytes (e.g., proteins) following the assay, leaving only the covalently immobilized binding members. The same binding member array can be denatured and renatured, which is part of binding member design and selection.

[0064] Denaturation and renaturation of binding members that are covalently attached to binding member arrays provides for reuse of the array in a manner not available in any other platform. In the case of SOMAmers, because the SOMAmers are selected using a denaturation-renaturation cycle in every round of SELEX, they are selected for the property of denaturation and renaturation. This provides a way to remove all target analytes, including proteins (cognate proteins (target proteins) or non-cognate proteins) after challenge.

[0065] Unlike conventional health diagnostic methods, the methods described herein provide for the detection of a full array of biomarkers can be detected instead of relying on only a few biomarkers. The resulting device results in at least 99.5% activity of a second use of the array to a first use of the array. In some variations, the resulting device results in at least 99.6% activity of a second use of the array to a first use of the array. In some variations, the resulting device results in at least 99.7% activity of a second use of the array to a first use of the array. In some variations, the resulting device results in at least 99.8% activity of a second use of the array to a first use of the array. In some variations, the resulting device results in at least 99.9% activity of a second use of the array to a first use of the array.

[0066] The assays can be performed again on regenerated binding member arrays. The method steps in the assay described herein, and included in U.S. Patent No.11,249,080, can be performed. Repeat Assays

[0067] In general, the method of identifying the presence of a a target analyte in a sample is provided. The method can be repeated any number of times. Binding members are covalently bonded to the substrate. Each binding member, or type of binding member, is associated with a discrete color center. With reference to FIG.3, a biological sample comprising a target 14 4899-7634-3847.1analyte is provided to the substrate (302). The substrate includes covalently bonded binding members. The target analyte is bound to the binding member to form a target analyte:binding member complex (304). Target analyte:binding member complexes are detected on the binding member array (306). Conditions (e.g., chemical, heat, or a combination) that denature the binding member and remove target analytes from the array while not removing the covalently bound binding members are then provided (308). Binding members on the binding member array are renatured such that each binding member has at least 99.5% binding affinity as compared to the binding member before the denaturing step (310).

[0068] In some variations, the same binding member array used for a particular individual. This binding member array can be re-used at any interval. Thus, a binding member array for a single individual encompassing large binding member panels (5,000, 7,000, 11,000 binding members, or more) can be prepared.

[0069] A full panel can be used to evaluate the full array of binding members for individuals over time. For example, the same array can be used for a healthy individual, and changes in protein abundance can be detected over any period of time. The use of the same binding member array multiple times provides for a continually decreasing cost of goods over time. Further, using the same binding member panel for a single individual over time prevents the need to re-normalize the measurements, as changes are on the same renatured panel. Likewise, binding members covalently bound to the substrate results in a non- diminishment of the binding members from the substrate.

[0070] Alternatively, the same binding member array can be used repeatedly for multiple individuals. In this case, the same reusable binding member array benefits from the consistent construction and measurement.

[0071] Having described several embodiments, it will be recognized by those skilled in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the disclosure. Additionally, a number of well-known processes and elements have not been described in order to avoid unnecessarily obscuring the embodiments disclosed herein. Accordingly, the above description should not be taken as limiting the scope of the document.

[0072] Those skilled in the art will appreciate that the presently disclosed embodiments teach by way of example and not by limitation. Therefore, the matter contained in the above description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all generic and specific 15 4899-7634-3847.1features described herein, as well as all statements of the scope of the method and system, which, as a matter of language, might be said to fall there between. 16 4899-7634-3847.1

Claims

Claims 1. A device comprising: a substrate; an array of binding members covalently bound to the substrate, each binding member configured to bind a specific target analyte to form a target analyte:binding member complex, each discrete binding member region corresponding to a discrete color center disposed in the substrate; wherein formation of said target analyte:binding member complex causes a measurable change in a property of the color center as compared to the binding member alone; wherein the binding members are configured to denature upon providing denaturing conditions; and wherein, following a clean in place (CIP) protocol that removes non-covalently bound biological components and denatures the binding members, upon providing a renaturing composition at room temperature and pressure the binding members of the binding member array are configured to have at least 99.5% binding affinity to target analytes as compared to a first use before a CIP protocol and denaturation of the binding members.

2. The device of claim 1, wherein the binding member is a SOMAmer or an aptamer.

3. The device of any one preceding claim, where in the discrete color center comprises a defect in a crystalline lattice of the substrate.

4. The device of any one preceding claim, wherein the discrete color center comprises a nitrogen vacancy (NV) in a diamond substrate.

5. The device of any one preceding claim, wherein the property of the color center intensity of emitted radiation corresponding to the color center corresponding to the target analyte:binding member complex.

6. The device of any one preceding claim, wherein the property of the color center a shift in wavelength of emitted radiation. 17 4899-7634-3847.

17. A method of identifying the presence of a target analyte in a sample comprising: providing a biological sample comprising a target analyte to the substrate of any one of claims 1 - 6; binding the target analyte to a binding members in the array of binding members to form a target analyte:binding member complex; providing conditions that denature the binding member on the binding member array and remove target analytes from the array while not removing the covalently bound binding members from the substrate; and renaturing the binding member on the binding member array such that the binding member has at least 99.5% binding affinity as compared to the binding member before the denaturing step.

8. The method of claim 7, wherein the binding member is a SOMAmer or an aptamer.

9. The method of one of claims 7 or 8, wherein, following said step of binding the target analyte to the binding member, providing a kinetic challenge to the target analyte:binding member complex.

10. The method of claim 9, wherein the kinetic challenge comprises providing a polyanionic competitor to the target analyte:binding member complex.

11. The method of one of claims 7-10, wherein, prior to said step of providing the wash composition, detecting the quantity of each target analyte:binding member complex on the binding member array.

12. The method of one of claims 7-11, wherein the step of detecting the quantity of each target analyte:binding member complex comprises determining a measurable change in a property of the color center.

13. The method of one of claims 7-12, comprising repeating the steps of binding, kinetic challenge, detecting for a second sample. 18 4899-7634-3847.1

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