CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 62/438,656, filed on December 23, 2016, and to U.S. Provisional Application Serial No. 62/439,166, filed on December 27, 2016, the content of each of which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

[0002] Devices, systems, and methods herein relate to biofluid analysis that may be used in diagnostic and/or therapeutic applications, including but not limited to protein analysis,

BACKGROUND

[0003] Analysis of biofluids from a subject may be used as a diagnostic tool for disease and to monitor subject health. For example, one approach to analysis of proteins from a biofluid sample may include the use of an enzyme-linked immunosorbent assay (ELISA) that uses antibodies and color change to identify a substance. ELISA is a "wet-lab" type analytic biochemistry assay that uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a substance, usually an antigen, in a wet (i.e., liquid) sample. An ELISA procedure may include immobilizing a protein sample having an unknown amount of antigen onto a support surface. A specific antibody (having specificity for an antigen of interest) may be applied to the surface, thereby forming a complex with the antigen of the sample. An enzyme may then be covalently linked to the antibody. Between each of these steps, the support may be washed with a mild detergent solution to remove any non-specifically bound proteins and/or antibodies. After a final wash step, the support may be developed by adding an enzymatic substrate. The subsequent reaction may produce a quantifiable signal, most commonly a color change in the substrate. The results from protein analysis may, for example, detect the presence or absence of viruses and/or bacterial infections. The requirements of conventional protein analysis techniques (e.g., purified and isolated protein) is labor intensive and requires specialized equipment that are impractical for a point-of-care setting such as a physician's office or clinic. Therefore, additional devices, systems, and methods for performing biofluid analysis may be desirable. SUMMARY OF SOME OF THE EMBODIMENTS OF THE DISCLOSURE

[0004] Described herein are inventions and embodiments of biofluid analysis systems, biosensor assemblies, methods, including the features, structure, functionality and steps thereof, for non-label analysis of analytes from a biofluid such as human plasma. That is, a label molecule is not required for detection of an analvte in a sample. These systems and methods may be used to characterize and/or quantitate a biofluid sample and permit evaluation of subject health and/or diagnosis of a condition. Conventional wet lab assay techniques such as ELISA are time consuming, labor intensive, and expensive processes. Generally, the systems and methods described herein may include a biofluid analysis system configured to spectrally analyze and quantitate a plasma sample placed on a biosensor. The biofluid analysis system may automatically process and analyze the sample on the biosensor and quantitate one or more of total analvte, a specific analvte, glycoproteins, and carbohydrates. The bioanalysis system may be compact and, for example, fit on a table. The biosensor may be configured as a dry or wet disposable sensor depending on the analvte to be measured and may use just a small volume of biofluid (e.g., about 10 μΐ). The biofluid analysis system may comprise a detector such as a spectrophotometer and controller configured to analyze the sample on the biosensor based on surface plasmon resonance transmission.

[0005] Surface plasmon resonance transmission is an optical technique for studying label- free biomolecular interactions of molecules from ions to viruses in real-time. An illumination source may be configured to output a light beam perpendicular to a surface of a biosensor. Light passes through a plurality of nanometer sized holes in the biosensor and is received by a spectrophotometer. The light may be absorbed by the free electrons on a metal layer of the biosensor to generate surface plasmons (resonating electrons). Surface plasmon polaritons are surface electromagnetic waves that propagate in a direction parallel to a metal/dielectric (or metal/vacuum) interface. Since the wave is on the boundary of the metal and the external medium (e.g., air or water), these oscillations are sensitive to any change of this boundary, such as the adsorption of molecules to the metal surface. Surface plasma resonance has a property of being proportional to the mass on the biosensor surface. The generation of surface plasmons may be measured as a loss of intensity in the light beam received by a spectrophotometer. Spectrophotometer data may be used to generate a sensorgram corresponding to molecular adsorption of the biofluid sample to the biosensor over time. A type and quantity of the analyte may be determined from the sensorgram. Other quantitative analyte characteristics that may be determined may include one or more of binding, specificity, concentration, kinetics, and/or affinity. In some embodiments, the biosensor assembly may include molecules (e.g., antibodies, aptamers, charges, ligands, etc.) that may be immobilized to a surface of the biosensor to capture one or more exosomes and/or free circulating DNA and/or minimize non-specific binding of unwanted analytes.

[0006] In some embodiments, a biofluid analysis system is provided, comprising a radiation source configured to emit a light beam and a biosensor assembly configured to hold a biosensor defining a plurality of holes. The biosensor may be configured to receive a biofluid and the light beam. A detector may be configured to receive the light beam from the plurality of holes. The biosensor assembly may be disposed between the radiation source and the detector, A controller may be coupled to the detector and comprise a processor and memory. The controller may include computer instructions for operation thereon configured to cause the processor to receive signal data corresponding to the light beam received by the detector, generate spectroscopy data using the signal data, and identify an analyte of the biofluid using the spectroscopy data.

[0007] In some embodiments, the computer instructions may further be configured to cause the processor determine at least one of a total analyte content and the identified analyte amount in the biofluid. In some embodiments, the analyte may comprise at least one of a protein, nucleic acid, lipoprotein, glycoprotein, and carbohydrate. In some embodiments, the plurality of holes may comprise openings on a first side of the biosensor. The openings may comprise a diameter of between about 100 nm and about 500 nm. In some of embodiments, the openings may comprise a diameter of between about 200 nm and about 250 nm. In some embodiments, the openings may be spaced apart by at least about 450 nm. In some embodiments, the biosensor assembly may comprise a platform configured to move with at least two degrees of freedom.

[0008] In some embodiments, the radiation source may be configured to emit the light beam comprising a wavelength of between about 100 nm and about 2500 nm. In some embodiments, the radiation source may be configured to emit the light beam comprising a diameter of between about 600 urn to about 700 μη . In some embodiments, the radiation source may comprise a first optical fiber configured to emit the light beam toward the biosensor. In some embodiments, the radiation source may comprise at least one of a collimator, a filter, and a broadband light emitting diode coupled to the first optical fiber. In some embodiments, the filter may comprise a 600 nm cut-off filter. In some embodiments, the radiation source may comprise at least one of a solid state halogen light, and broadband light emitting diode.

[0009] In some embodiments, the detector may comprise a spectrophotometer configured to measure wavelengths of the light beam between about 100 nm and about 2500 nm. In some embodiments, the detector may comprise a second optical fiber coupled to the spectrophotometer. The second optical fiber may be configured to receive the light beam from the plurality of holes. In some embodiments, the second optical fiber may be configured to move with at least one degree of freedom in a direction perpendicular to a plane of the biosensor assembly. In some embodiments, the second optical fiber may be configured to bias away from the biosensor assembly when the radiation source does not emit the light beam.

[0010] In some embodiments, the spectroscopy data may comprise wavelength data and intensity data of the received light beam. In some embodiments, the computer instructions may be configured to cause the controller to set spectroscopy data parameters comprising at least one of integration time, number of scans to average, and box car width. In some embodiments, the computer instructions may be configured to cause the controller to generate the spectroscopy data by at least one of calculating maximum and minimum intensities of the signal data and a change in wavelength.

[0011] In some embodiments, the biofluid analysis system may further comprise a calibration system configured to detect and position the biosensor relative to the radiation source using the platform. In some embodiments, the calibration system may be configured to sequentially align the radiation source to each of a plurality of wells of the biosensor. Each well may define a set of the plurality of holes. In some embodiments, the computer instructions may be configured to cause the controller to generate indexing data using the calibration system. The indexing data may correspond to locations of the wells relative to the radiation source and the detector. In some embodiments, the computer instructions may be configured to cause the controller to generate spectroscopy data for each of the plurality of wells without the biofluid and with the biofluid. In some embodiments, the calibration system may comprise at least one of a first optical sensor and a backlight provided on a first side of the biosensor assembly. A second optical sensor may be provided on a second side of the biosensor assembly. In some embodiments, the first optical sensor may comprise a microscope and the second optical sensor may comprise a camera. In some embodiments, the biosensor may comprises at least one fiducial. The second optical sensor may be configured to image at least one of the plurality of wells and at least one of the fiducials. In some embodiments, the calibration system may further comprise an input device configured to control movement of the biosensor assembly.

[0012] In some embodiments, the biosensor may comprise a plurality of wells. Each well on a first side of the biosensor may comprise a first side square of between about 50 μηι and about 150 μηι and a corresponding set of the plurality of holes within the first side square. In some embodiments, each well on a second side of the biosensor may comprise a second side square of between about 100 μηι and about 800 μηι.

[0013] Also described here are embodiments corresponding to biofluid analyzing methods. In general, these methods may include the steps of applying a biofluid to a biosensor. The biosensor may comprise a substrate and a metal layer disposed on a surface of the substrate. The biosensor may define a plurality of holes each comprising an opening of between about 100 nm and about 500 nm. A light beam may be emitted toward a first side of the biosensor. The light beam may be received through the plurality of holes at a detector from a second side of the biosensor. Spectroscopy data may be generated from the detector. An analyte of the biofluid may be identified from the spectroscopy data.

[0014] In some embodiments, the method may further comprise determining at least one of a total analyte content and the identified analyte amount in the biofluid. In some embodiments, the analyte may comprise at least one of a protein, nucleic acid, lipoprotein, glycoprotein, and carbohydrate. In some embodiments, the method may further comprise calibrating the biosensor relative to the light beam and the detector. In some embodiments, the method may further comprise sequentially aligning the light beam to a plurality of wells of the biosensor. Each well may define a set of the plurality of holes. In some embodiments, the method may further comprise generating indexing data corresponding to locations of the wells relative to the detector. In some embodiments, spectroscopy data may be generated for each of the plurality of wells without the biofluid and with the biofluid. j0015j In some embodiments, a binding agent may be deposited on the metal layer. In some embodiments, the binding agent may comprise at least one of polyethylene glycol, an amine protein group, an antibody, an aptamer, a charge, a ligand, a moiety, a zwitterion, and an amplifier. In some embodiments, the amine protein group may comprise at least one of neutravidin, streptavidin, and carboxylamine. [0016] In some embodiments, the plurality of holes may be spaced apart by at least about 450 nm. In some embodiments, the biosensor may comprise a plurality of wells. Each well on the first side of the biosensor may comprise a first side square of between about 50 μτη and about 150 μ ι and a corresponding set of the plurality of holes within the first side square. In some embodiments, each well on the second side of the biosensor may comprise a second side square of between about 100 urn and about 800 μηι and a corresponding set of the plurality of holes within the second side square.

[0017] In some embodiments, a biofluid assembly is provided, comprising a sensor comprising a substrate and a metal layer disposed on a surface of the substrate. The sensor may define a plurality of through holes. Each of the plurality of holes may comprise first openings on a first side of the substrate and a second opening on a second side of the substrate. The plurality of through holes may comprise a diameter of between about 100 nm and about 500 nm. A number of the first openings may be greater than a number of the second openings.

[0018] In some embodiments, the first openings may comprise the diameter of between about 200 nm and about 250 nm. In some embodiments, the first openings may be spaced apart by at least about 450 nm. In some embodiments, the sensor may comprise a length and a width of between about 1 cm and about 15 cm, and a thickness of between about 500 μηι and about 675 μτη.

[0019] In some embodiments, at least one of a polyethylene glycol, an amine protein, and an antibody may be disposed on the metal layer. In some embodiments, a first transparent substrate may be coupled to a first side of the substrate. In some embodiments, a second transparent substrate may be coupled to a second side of the substrate.

[0020] In some embodiments, a housing (e.g., consumable, disposable, palette, holder) may be configured to hold the sensor at a fixed position relative to the housing. In some embodiments, the housing may comprise a magnet configured to hold the sensor in contact with the housing.

[0021] In some embodiments, the sensor may be configured to bind to an analyte of a biofluid on the metal layer. The analyte may comprise a size of between about 100 nm and about 600 nm. In some embodiments, the analyte may comprise a size of between about 100 nm and about 300 nm. In some embodiments, the analyte may comprise at least one of a protein, nucleic acid, lipoprotein, glycoprotein, and carbohydrate. [0022] In some embodiments, the first openings may be spaced apart by at least about 450 nm. In some embodiments, the biosensor may comprise a plurality of wells. Each well on the first side of the biosensor may comprise a first side square of between about 50 μηι and about 150 μηι and a corresponding set of the plurality of holes within the first side square. In some embodiments, each well on the second side of the biosensor may comprise a second side square of between about 100 μιη and about 800 μηι and a corresponding set of the plurality of holes within the second side square.

[0023] Also described herein are embodiments corresponding to biosensor manufacturing methods. In general, these methods may include the steps of depositing a first layer of silicon nitride on a first side of a substrate and a second layer of silicon nitride on a second side of the substrate. A plurality of first holes may be created extending through the first layer and a plurality of second holes extending through the substrate and the second layer. A number of the first holes may be greater than a number of the second holes. The plurality of first holes may extend into respective second holes. The plurality of first holes may comprise a diameter of between about 100 nm and about 500 nm. A third layer of a metal may be deposited on the first layer.

[0024] In some embodiments, the first holes may comprise the diameter of between about 200 nm and about 250 nm. In some embodiments, the plurality of first holes may be spaced apart from each other between about 485 nm and 515 nm. The plurality of second holes may comprise a diameter of between about 700 μιη and about 750 μηι and may be spaced apart from each other between about 1.5 mm and about 2.5 mm. In some embodiments, creating the plurality of second holes may comprise a sidewall angle in the substrate of between about 45° and about 70° with respect to a plane of the second side of the substrate. In some embodiments, creating the plurality of second holes may comprise a sidewall angle in the substrate of between about 50° and about 60° with respect to a plane of the second side of the substrate.

[0025] In some embodiments, a first photoresist layer may be applied to the first layer and a second photoresist may be applied to the second layer. The plurality of second holes may be created in the second layer using the second photoresist. In some embodiments, the plurality of second holes may be created by etching the substrate using a wet etchant from the second side of the substrate. In some embodiments, the wet etchant may comprise potassium hydroxide. In some embodiments, creating the plurality of first holes may comprise maintaining an unetched portion of the first layer of between about 40 nm and about 60 nm extending from the first side of the substrate to the plurality of the first holes. In some embodiments, creating the plurality of first holes may comprise extending the plurality of first holes through the unetched portion using a dry etchant. In some embodiments, the dry etchant may comprise reactive ion etching. In some embodiments, creating the plurality of first holes may comprises the first layer of silicon nitride having a depth of between about 100 iim and about 140 nm.

[0026] In some embodiments, depositing the first and second layers of silicon nitride may each comprise a depth of between about 80 nm and about 350 nm. In some embodiments, depositing the first and second layers of silicon nitride may comprise using low pressure chemical vapor deposition. In some embodiments, creating the plurality of first holes and plurality of second holes may comprise using one or more of interference lithography, deep ultraviolet light etching, and electron beam lithography.

[0027] In some embodiments, the third layer may comprise titanium and gold. In some embodiments, depositing the third layer may comprises a pressure of less than about 3 x 10 ^"6 Torr, Depositing the titanium may comprise a thickness of about 3 nm at a deposition rate of less than about 2 A/sec. Depositing the gold may comprise a thickness of between about 90 nm and about 1 10 nm at a deposition rate of less than about 0.5 A/sec. In some embodiments, the substrate may comprise at least one of silicon, silica, and glass, having a diameter of between about 8 cm and about 20 cm,

[0028] In some embodiments, a binding agent may be deposited on the metal layer. In some embodiments, the binding agent may comprise at least one of polyethylene glycol, an amine protein group, an antibody, an aptamer, a charge, a ligand, a moiety, a zwitterion, and an amplifier. In some embodiments, the amine protein group may comprise at least one of neutravidin, streptavidin, and carboxylamine.

[0029] In some embodiments, the first holes may be spaced apart by at least about 450 nm. In some embodiments, the biosensor may comprise a plurality of wells. Each well on the first side of the substrate may comprise a first side square of between about 50 μηι and about 150 μιη and a corresponding set of the plurality of first holes within the first side square. In some embodiments, each well on the second side of the substrate may comprise a second side square of between about 100 μιη and about 800 μηι and a corresponding set of the plurality of second holes within the second side square. [0030] These and other embodiments, advantages and objects of the present disclosure will be even better understood with reference to the detailed description

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIGS. 1A-1F are illustrative views of a biofluid analysis system according to some embodiments of the disclosure. FIG 1A, and IC are perspective views of the system, FIGS. IB and I F are rear views of the system, and FIGS. ID and I E are side views of the system.

[0032] FIGS. 2A-2F are illustrative views of another biofluid analysis system according to some embodiments of the di sclosure. FIG. 2 A is a front view of the system, FIG. 2B is a plan view of the system, and FIG. 2C is a cross-sectional plan view of the system. FIGS. 2D and 2F are side views of a radiation source of the system. FIG. 2E is a side view of a radiation source and detector of the system.

[0033] FIGS. 3A and 3B are block diagrams of a biofluid analysis system according to some embodiments of the disclosure.

[0034] FIGS. 4A-4E are illustrative views of a biosensor assembly according to some embodiments of the disclosure. FIG. 4A is a perspective view of the assembly, FIG. 4B is a plan view of a biosensor, FIGS. 4C, and 4E are top views of the assembly, and FIG 4D is a bottom view of the assembly.

[0035] FIG. 5 is an illustrative flowchart of a biofluid analysis method according to some embodiments of the disclosure.

[0036] FIG. 6 is an illustrative flowchart of a method of functionalizing a biosensor according to some embodiments of the disclosure.

[0037] FIG. 7 is an illustrative flowchart of a method of manufacturing a biosensor according to some embodiments of the disclosure.

[0038] FIGS. 8A-8M are illustrative views of a biosensor manufacturing process according to some embodiments of the disclosure. FIGS, 8A-8G, 8K, and 8L are cross-sectional side views of the steps in the process. FIG, 8H is a plan view of a first side of a biosensor and FIG. 8J is a plan view of a second side of the biosensor, FIG. 81 is a scanning electron microscope image of the first side of the biosensor. FIG. 8M is a plan view of a silicon wafer. [0039] FIGS, 9A-9C are illustrative plots of spectroscopy data including wavelength and intensity collected according to some embodiments of the disclosure.

[0040] FIGS. 10A-10D are illustrative views of another a biofluid analysis system according to some embodiments of the disclosure. FIGS. 10A and 10D are perspective views of the system, FIG. 10B is an exploded schematic view of the system, and FIG. I OC is a plan view of the system.

[0041] FIGS. HA-1 1D are illustrative views of yet another a biofluid analysis system according to some embodiments of the disclosure. FIG. 11A is a perspective view of the system, FIG. 1 B is a rear view of the system, and FIGS. 11C and 1 ID are front views of the system.

DETAILED DESCRIPTION

[0042] Described herein are embodiments of systems, devices, and methods for controlling a biofluid analysis system that uses a biosensor, including methods of manufacturing and functionalizing the biosensor. In some embodiments, the biofluid analysis system provides spectral analysis of a biofluid sample placed on the biosensor in order to identify and quantitate one or more analytes. For example, a user may apply a small amount (e.g., about ten microliters) of a biofluid, as a starting material, on a surface of a biosensor (e.g., rmcrofluidic semiconductor chip). In some embodiments, the biosensor is configured to bind to an analyte of the biofluid. A radiation source (e.g., light source, illumination source) may then be used to direct a light beam at the biosensor and thereby generate surface plasmon resonance on the biosensor. A detector (e.g., optical sensor, spectrophotometer) can be used to receive the light passing through the biosensor and to measure characteristics of the light beam (e.g., intensity and wavelength). This spectroscopy data, in some embodiments, is then used to identify an analyte of the biofluid sample as a specific analyte and quantitate the amount of the analyte in the sample. The biofluid sample may comprise, for example, plasma from blood, serum from blood, cerebrospinal fluid, urine, combinations thereof, and the like.

[0043] In some embodiments, the biosensor is coupled to a portable housing (e.g., consumable, disposable, palette, holder) to aid handling, functionalization, calibration, and indexing of a biofluid sample applied to the biosensor. The housing having the biosensor may be placed by a user onto a biosensor assembly for automated processing of the biofluid sample. For example, the biosensor assembly may comprise a platform configured to hold (e.g., secure) a biosensor in place relative to the platform. The platform may further be configured to move with at least two degrees of freedom so as to position the biosensor at a predetermined location relative to the radiation source and detector. Once in position, the biosensor assembly may receive a light beam from the radiation source that may allow spectral measurement of analyte binding to a surface of the biosensor and further data procssing.

[0044] As shown in FIG. lA, a biofluid analysis system (100) may comprise a radiation source, biosensor assembly, and detector. For example, FIG. 1A illustrates a radiation source comprising a first optical fiber (110) and a light source (112). The first optical fiber (110) may be coupled to an output of the light source ( 12). A light beam having a predetermined wavelength may be generated by the light source ( 1 12) and emitted from an end of the first optical fiber (1 10) towards the biosensor assembly. The biosensor assembly may comprise a removable housing (121) that may be held on a platform (122). Similarly, a semiconductor biosensor chip (120) may be held in the housing (121). The housing (121) may allow a user (e.g., technician) to handle and/or manipulate the biosensor (120) without touching and/or damaging the biosensor (120) itself. The biosensor assembly may further comprise an XY stage (124) coupled to the platform (120) that may be configured to position the platform (122) and the biosensor (120) placed thereon) at a predetermined position relative to the first optical fiber (110) and detector. This may allow the system (100) to direct the light beam at a predetermined well of the biosensor (120).

[0045] FIG IB is a rear view of the system (100) illustrating the detector comprising a spectrophotometer (130) and a second optical fiber (132). The second optical fiber (132) may be coupled to the spectrophotometer (130) and configured to receive the light beam passed through the biosensor (120). In other words, the biosensor assembly may be disposed between the radiation source and the detector. As shown in the rear view of FIG. IB, the first optical fiber ( 110) and second optical fiber (132) may be disposed perpendicular to the housing (121). The XY stage (124) may be used to position the biosensor (120) relative to the first and second optical fibers (110, 132). The XY stage (124) may be controlled by a calibration system configured to detect and position the biosensor (120) relative to the radiation source and/or detector. For example, the calibration system may comprise a first optical sensor (140) (e.g., visualization camera, microscope) (FIGS. IB, ID) and a second optical sensor (142) (FIGS. ID and IF). During calibration of the biosensor (120) in the biofluid analysis system (100), the first and second optical sensors (140, 142) may be used to identify the alignment of the wells of the biosensor (120) with respect to the first and second optical fibers (110, 132). For example, the calibration system may generate indexing data corresponding to locations of the wells of the biosensor (120) relative to the radiation source and/or detector.

I. Devices

[0046] Described herein are devices that may be used in some embodiments of the various systems described. A biosensor as described herein may comprise an array of nanometer sized through holes (e.g., nanoholes) that extend through the biosensor. The nanoholes may be coated with an opaque gold film layer. Light illumination at predetermined wavelengths on a first side of the biosensor may generate surface plasmons on the gold surface, thereby generating surface plasmon mediated light transmission through the nanoholes. The surface plasmon mediated light may be output from a second side of the biosensor and may be measured by a spectrophotometer. One or more substances (e.g., antibodies coupled to affinity ligands) may be applied to the biosensor to increase binding of an analyte (e.g., antigen, target molecule, protein, exosome) of the biofiuid sample to the biosensor, and thus its plasma resonance. A refractive index of the biosensor surface changes when a biofiuid sample and other substances are applied (e.g., functionalized) to the biosensor. Accordingly, binding of an antigen of a biofiuid sample to a corresponding antibody shifts (e.g., red/right shift) an optical transmission profile measured by a detector. The amount of spectral shift corresponds to molecular mass density, and permits quantification of captured antigens (e.g., exosomes) on the biosensor. Since antigen binding to the biosensor creates a spectral shift, antigens may be identified in a label-free manner. The biofluids described herein may comprise any biological fluid including, but not limited to, serum, plasma, cerebrospinal fluid, ascites fluid, saliva, and cell culture media,

[0047] FIG. 4B is an illustrative example of a biosensor (410) according to some embodiments, comprising a semiconductor chip showing a first side of the biosensor (410) configured to receive a biofiuid sample. The biosensor (410) may have an outer layer of metal such as gold, and may comprise a plurality of spaced-apart wells (412) that extend through the biosensor (410). Although each well (412) depicted in FIG. 4B appears as a single hole, each well (412) may comprise a plurality of nanometer-sized openings on the first side of the biosensor (410). For each well, the number of openings on a second side of the biosensor (410) may be less than the number of openings on the first side of the biosensor (410). In some embodiments, the biosensor (410) may comprise at least one fiducial (414) that may be imaged and/or otherwise detected by an optical sensor of a calibration system and/or used to generate indexing data. For example, a fiducial (414) may be disposed in each corner of the biosensor (410). The wells (412) may be analyzed independently of each other such that the wells (412) may be functionalized independently to identify and/or quantitate different analytes of the biofluid.

[0048] In some embodiments, a sensor (410) (e.g., biosensor) may comprise a substrate having a metal layer disposed on a surface of the substrate. The sensor may define a plurality of through holes where each of the through holes may comprise first openings on a first side of the substrate and a second opening on a second side of the substrate. The first openings may comprise a diameter of between about 100 nm and about 500 nm. In particular, the openings may comprise a diameter of between about 200 nm and about 250 nm. The second openings may have a greater diameter than that of the first openings such that a number of the first openings is greater than a number of the second openings. The sensor may comprise a length and a width of between about I cm and about 15 cm, and a thickness of between about 500 μηι and about 675 μηι.

[0049] In some embodiments, one or more substances (e.g., binding agents) may be disposed on the biosensor to increase binding of an analyte (e.g., target molecule, protein, exosome) of the biofluid sample to the biosensor. For example, a binding agent may be deposited on the metal layer, where the binding agent may comprise at least one of a polyethylene glycol, an amine protein, and an antibody, an aptamer, a charge, a ligand, a moiety, a zwitteiion, and an amplifier. The amine protein group may include, for example, at least one of neutravidin, streptavidin, and carboxyl amine. The analyte to be bound to the biosensor may comprise a size of between about 100 nm and about 600 nm. In particular, the analyte may comprise a size of between about 100 nm and about 300 nm.

[0050] As described in more detail herein, the biosensor may be coupled to a housing (e.g., consumable, disposable, palette, holder) to aid in one or more of handling, sample preparation, indexing, calibration, and measurement of the biosensor for biofluid analysi s. The housing may be configured to be processed by a biofluid analysis system.

II Systems

[0051] Described herein are biofluid analysis systems that may include one or more of the components necessary to perform biofluid analysis using the devices according to various embodiments described herein. For example, the biofluid analysis systems described herein may automatically process and analyze a biofluid sample on the biosensor using surface plasmon resonance transmission to quantitate one or more of total analyte, protein, nucleic acid, lipoprotein, glycoproteins, and carbohydrates in a non-label manner.

[0052] Generally, the devices described herein for use in a biofluid analysis system may include one or more of a biosensor assembly, a radiation source, a detector, and a controller. The radiation source ma be configured to emit a light beam, A biosensor assembly may be configured to hold a biosensor and to receive the light beam. A detector may be configured to receive the light beam passed through the biosensor assembly. The biosensor assembly may be disposed between the radiation source and the detector. A controller coupled to the detector and controller may be configured to receive signal data corresponding to the light beam received by the detector and generate spectroscopy data using the signal data. An analyte of the biofluid may be identified by the controller using the spectroscopy data.

[0053] FIG. 3A is a block diagram of a biofluid analysis system (300) according to some embodiments. The system (300) may comprise a control system (320) configured to control one or more of a radiation source (310), biosensor assembly (312), and detector (314), As shown in the front view of FIG. I IC, the system (1 100) may comprise a biosensor input (1 1 10) (e.g., entry hole) configured to receive a biosensor. For example, a biosensor (not shown) may be advanced into the system (1100) using the biosensor input (1 1 10), The system (1 100) may be placed (e.g., mounted) on a table, desk, in a cart, floor, sidewall, or other suitable support surface. In some embodiments, the system (1100) may process one or more biosensors configured to receive a light beam for a detector to generate signal data corresponding to spectroscopy data useful in identifying one or more analytes from a biofluid sample.

[0054] The radiation source (310), biosensor assembly (312), and detector (314) may be coupled to the control system (320) through one or more wired or wireless communication channels. Any wired connections may be optionally built into the floor and/or walls or ceiling. The control system (320) may be coupled to one or more networks (370), databases (340), and/or servers (350). The network (370) may comprise one or more databases (340) and servers (350). In some embodiments, a remote operator (not shown) may be coupled to one or more networks (370), databases (340), and servers (350) through a user console (360). In some embodiments, one or more of the radiation source (310), biosensor assembly (312), and detector (314) may be coupled directly to any of the network (370), database (340), server (350), or each other. Indexing, calibration, data generation, processing, and analysis may be performed from any one of the devices of the sy stem (300) or distributed throughout a plurality of devices. A user (such as a technician or other operator) may use the user console (360) to control the system (300), The user console (360) may be located in the same room as the biofluid analysis system (300), in an adjacent or nearby room, or tele-operated from a remote location in a differently building, city, country, etc,

[0055J FIGS, 1 1 A-l 1 D depict an exterior of a biofluid analysis sy stem (1 100) according to some embodiments. As shown in FIGS. 11 A, 11C, and 11D, the system (1 100) may further comprise one or more optical waveguides (e.g., status indicator lights) (1120, 1 122) configured to visually communicate a status of the system (1 100). A first optical waveguide (1120) and a second optical waveguide (1122) may output color-coded light that may aid in efficiently notifying a user of a state of biofluid analysis using the system (1100). For example, a status of the system (1 100) (e.g., ON, idle, running, finished run, processing, error, user input required) may be communicated to the user using a set of light patterns emitted from respective optical waveguides. In some embodiments, the second optical waveguide (1122) may be configured to indicate the location of biosensor input ( 1 110). As shown in FIGS. 1 1 A and 1 ID, a biosensor input cover (1 130) (e.g., external light plug) may be configured to prevent external light penetration into the system (1 100) from the biosensor input (11 10). The cover (1130) may be removed from the biosensor input (11 10) when the biosensor (not shown) is advanced into or removed from the sy stem ( 11 10).

Radiation source

[0056] The biofluid analysis systems as described herein may comprise a radiation source configured to emit a light beam to generate surface plasmon resonance on a biosensor. The radiation source may be configured to generate the light beam in the UV-visible-near-IR wavelengths. For example, the radiation source may be configured to emit the light beam comprising a wavelength of between about 100 nm and about 2500 nm. In some embodiments, the light beam may be filtered and collimated. In some embodiments, the light beam may be emitted from a first optical fiber towards a well disposed on a first side of a biosensor. A second optical fiber disposed below the biosensor may receive the light beam from a second side of the biosensor and transmit the light to a spectrophotometer for data detection and analysis.

[0057] FIG. 2A is a front view of a biofluid analysis system (200) and depicts a radiation source comprising a first optical fiber (210) coupled to a first optical fiber lock (211). A light beam having a predetermined wavelength may be emitted from an end of the first optical fiber (210) towards a biosensor (not shown) held on a platform (222). The first optical fiber (210) may be coupled to a light source (not shown) that may be, for example, a solid state halogen light or a broadband light emitting diode. In some embodiments, a first optical sensor (240) (as described in more detail with respect to the calibration system) may be disposed on the first side of the biosensor.

[0058] FIGS. 2B and 2C are plan views of the biofluid analysis system (200). A light output of the first optical fiber (210) may be perpendicular to the biosensor. For example, FIG. 2C is a cross-sectional plan view of the first optical fiber (210) aligned over the wells of the biosensor (220). FIGS. 2D, 2E, and 2F are detailed side views of a first optical fiber (210) and/or second optical fiber (232) of the system (200). In some embodiments, the radiation source may comprise a collimator (214) and a filter (216) each optically coupled to the first optical fiber (210). In some embodiments, the collimator (214) may be used to focus the light emitted from the first optical fiber (210) to form a light beam of a predetermined diameter. For example, the light beam may comprise a diameter of between about 600 μιη to about 700 μηι when the light beam contacts a surface of a biosensor (not shown) disposed within a housing (221 ) of the system (200). The filter (216) may comprise a cut-off filter of a predetermined wavelength. As a non-limiting example, the filter (216) may comprise a 600 nm cut-off filter.

Biosensor assembly

[0059] A biosensor assembly may be used to aid manipulation and positioning of a biosensor relative to other components of a biofluid analysis system such as the radiation source and detector. For example, a user may place the biosensor on a retractable platform of the biosensor assembly through a biosensor input (e.g., entry hole) in a housing of the system. The biosensor assembly may hold (e.g., secure) the biosensor in place relative to the platform during biofluid analysis. The platform may then retract into the system to position the biosensor for illumination and spectral analysis. The platform may move with at least one degree of freedom (e.g., translate along the X-axis and/or Y-axis using a moveable XY stage) in response to a calibration procedure to align the radiation source to the biosensor. In some embodiments, the biosensor assembly may comprise one or more of a platform and an XY stage coupled to the system, as well as biosensor and corresponding housing. [0060] FIG. 10A is a perspective view of a biofluid analysis system (1000), according to some embodiments, including a housing (1021) having a biosensor (1020) held within a slot in a platform (1022), The housing ( 1021 ) may be advanced into the platform (1022) such that an end portion of the housing (1021) may protrude from an end of the platform (1022). When the platform (1022) is advanced toward an exterior cover (not shown) of the system (1000), the protruding portion of the housing (1021) may extend out of the system (1000) in a manner to permit a user to remove the housing (1021) and biosensor (1020) disposed thereon from the system. The platform (1022) may be coupled to an XY stage (1024) that may be configured to move the biosensor (1021) with two degrees of freedom on a plane perpendicular to a longitudinal axis of the light beam emitted from the first optical fiber (1010). Movement of the platform (1022) in this plane allows a controller of the system (1000) to direct a light beam at specific wells of the biosensor (1020), For example, the light beam emitted from the first optical fiber (1010) may be sequentially aligned to a plurality of wells of the biosensor (1020). Each well may define a set of the plurality of nanometer sized holes. In some embodiments, a first optical fiber lock (1011) may be coupled to the first optical fiber (1010) and used to hold (e.g., secure) the first optical fiber (1010) at a predetermined position relative to the other components of the system (1000).

Platform

[0061] FIG. 10B is an exploded schematic view of the biosensor assembly of the system (1000). FIG. IOC is a plan view of the biosensor assembly. In some embodiments, one or more of a controller and calibration system may use the platform (including the XY stage) to position the biosensor relative to the radiation source, detector, and/or other components of the system. The platform (1022) may comprise a size and shape to hold a housing having a biosensor disposed thereon. The platform (1022) may further define a hole having a size and shape to allow light to pass through a first side of the biosensor and out of a second side of the biosensor opposite the first side. In some embodiments, an XY stage (1024) may be coupled to an underside of the platform (1022) and configured to move the platform (1022) (and any biosensor placed thereon) along a predetermined plane. For example, the XY stage (1024) may comprise an X stage (1025) disposed on a Y stage (1026). The X and Y stages (1025, 1026) may each comprise a set of axial rails that may be driven by a motor (not shown) to move the platform (1022). In some embodiments, the X stage (1025) may have a movement range of up to about 110 mm. In some embodiments, the Y stage (1026) may have a movement range of between about 25 mm and about 55 mm. Housing

[0062] In some embodiments, the biosensor may be removably positioned within a portable housing (e.g., consumable, disposable, palette, holder) to aid in handling, functionalization, calibration, and indexing of a hiofluid sample applied to the biosensor. The housing having the biosensor may be placed by a user onto a biosensor assembly for automated processing of the biofluid sample. The housing may be particularly useful in providing physical support and protection to the biosensor. In some embodiments, the biosensor assembly may include the housing. The housing may be configured to hold the sensor at a fixed position relative to the housing.

[0063] FIG. 4A is a perspective view of a housing (400) of a biosensor assembly. FIGS, 4D and 4E are respective bottom and top views of the biosensor assembly (400), The housing (400) may comprise a handle (44) at an end that may, for example, allow a user to hold the housing (400) between a finger and thumb. The housing (400) may define a first recess and/or first hole (420) for a biosensor (410). The biosensor ( 10) may be slid into the first recess (420). The biosensor (410) may be coupled to a transparent substrate (416) (e.g., glass) such as a microscope slide. In some embodiments, the housing (400) may further define a second recess (422) that may be configured for a user to push out the slide (416) from the housing (410) such as by using a thumb. In some embodiments, the housing (400) may comprise a magnet (430) configured to attract the metal layer of the biosensor (410) and hold the biosensor (410) to the housing (400). The biosensor (410) may also be held to the housing (400) using a lock (432). In some embodiments, the biosensor (410) may comprise dimensions of between about 2 cm and about 10 cm. For example, the biosensor (410) may comprise a square of about 2.52 cm by about 2.52 cm.

[0064] In some embodiments, a housing (400) may comprise a length of between about 7.5 cm and about 25.5 cm, a width of between about 2.5 cm and about 8 cm, and a height of between about 0.25 cm and about I cm. For example, the housing (400) my comprise a length of about 15 cm, a width of about 3.8 cm, and a height of about 0,6 cm.

Biosensor

[0065] In some embodiments, the biosensor assembly may include a removable biosensor (e.g., semiconductor chip), A biosensor as described herein may be used with the housing described herein. In order to isolate a desired anaiyte from a biofluid sample, a biosensor may be formed having an array of gold-plated wells (e.g., holes) for binding to an anaiyte of interest. The biosensor may be configured to bind to an analyte of a biof!uid on the metal layer. The analyte may comprise a size of between about 100 nm and about 600 nm, and preferably between about 100 nm and about 300 nm. The analyte may comprise at least one of a protein, nucleic acid, lipoprotein, glycoprotein, and carbohydrate.

[0066] In some embodiments, a first transparent substrate (e.g., microscope slide) may be coupled to a first side of the substrate. A second transparent substrate (e.g., slide cover) may be coupled to a second side of the substrate. As described in more detail herein, the biosensor may be functionalized by applying at least one of a polyethylene glycol, an amine protein, and an antibody on the metal layer of the biosensor. This may aid binding of an analyte of interest to the biosensor.

[0067] The biosensor comprising a semiconductor chip mounted to a transparent substrate (e.g., microscope slide) may be coupled to a housing (e.g., consumable, disposable, palette, holder) and placed onto a platform of a biosensor assembly . The platform may be translated to position the biosensor at a predetermined position relative to a radiation source and detector,

[0068] As used herein, the biosensor may comprise a plurality of wells with each well comprising a set of a plurality of through holes. As shown in FIG. 4B, a biosensor (410) may define a plurality of wells (412) that extend through the biosensor. The biosensor (410) may be configured to receive a biofluid and a light beam that may pass through the wells (412). The biosensor may be formed from a substrate (e.g., silicon wafer) and a metal layer (e.g., gold) disposed on a surface of the substrate. As shown in FIGS. 8H, 81, and 8J, each of the plurality of holes may comprise first openings on a first side of the substrate and a second opening on a second side of the substrate. The first openings may comprise a diameter of between about 100 nm and about 500 nm. In some embodiments, the openings may comprise a diameter of between about 200 nm and about 250 nm. A number of the first openings is greater than a number of the second openings. The openings may be spaced apart by at least about 450 nm. The biosensor may comprise a length and a width of between about 1 era and about 15 cm, and a thickness of between about 500 μηι and about 675 μηι.

[0069] As shown in FIG. 8H, each well on a first side of the biosensor m ay comprise a fi rst side square of between about 50 μηι and about 150 μηι and a corresponding set of the plurality of holes within the first side square. As shown in FIG, 8 J, each well on a second side of the biosensor may comprise a second side square of between about 100 μτη and about 800 μηι.

Detector

[0070] Generally, the biofluid analysis systems described herein may comprise a detector used to receive light beams output from a plurality of holes in a biosensor. The received light may be used to generate signal data used for spectroscopy. The detector may be disposed on a side of the biosensor opposite that of a radiation source such that the detector receives a light beam from the radiation source that has passed through the plurality of holes of the biosensor. As shown in FIG. 2 A and 2E, the detector may comprise a spectrophotometer (230) and a second optical fiber (232) coupled to the spectrophotometer (230). The second optical fiber (232) may be configured to receive a light beam through the plurality of holes of a biosensor. The spectrophotometer (230) may receive the light beam from the second optical fiber (232) and generate signal data. In some embodiments, a spectrophotometer (230) may be configured to measure wavelengths of the light beam between about 100 nm and about 2500 nm. Spectrophotometer parameters for light acquisition such as time of integration, number of scans to average, and box car width may be set by at least one of the calibration system and control system.

[0071] In some embodiments, the second optical fiber (232) may be configured to move with at least one degree of freedom in a direction perpendicular to a plane of the biosensor assembly (e.g., biosensor). For example, the second optical fiber (232) may be configured to bias away from the biosensor assembly when the radiation source does not emit the light beam. This may prevent the detector from being damaged when spectral measurements are not being performed. For example, the Z stage (126) shown in FIG. 1C may bias the second optical fiber (132) away from the platform (122) when the platform (122) inserts and ejects the biosensor (120). The Z stage (126) may be coupled to the second optical fiber (132). The Z stage (126) may be controlled by at least one of the calibration system and control system. In some embodiments, the Z stage (126) may move the second optical fiber (132) by up to about 30 mm. In some embodiments, the second optical fiber (132) may be disposed as close as about 0.5 mm away from the biosensor (120).

Calibration system

[0072] The biofluid analysis systems described herein may comprise a calibration system coupled to a controller. The calibration system may be configured to locate and position wells of a biosensor relative to a radiation source and a detector. As shown in FIGS. 1A-1F and 10D, the calibration system may comprise a first optical sensor (140), second optical sensor (142), and backlight (1030). In some embodiments, the calibration system may be configured to determine one or more biosensor structures (e.g., fiducials, wells) and position a predetermined well at a predetermined location relative to the radiation source and detector. That is, the calibration system may move a platform to align a specific well of the biosensor to the light beam and detector. The calibration system may further set one or more parameters of a detector to calibrate the detector and optimize a spectrum.

[0073] The first optical sensor (140) and backlight (1030) may be provided on a first side of the biosensor assembly (e.g., facing the output of the radiation source), and a second optical sensor (142) may be provided on a second side of the biosensor assembly (e.g., facing the detector). For example, the first optical sensor (140) may comprise a microscope and the second optical sensor (142) may comprise a camera. The second optical sensor (142) may be configured to image at least one of the plurality of wells and at least one fiducial of the biosensor (120). For example, the first optical sensor (140) may comprise a visualization camera (e.g., microscope) and the second optical sensor (142) may comprise a feature detection camera.

|0074| As shown in FIG. 10D, the backlight (1030) may be configured to illuminate a biosensor (1020) placed beneath it such that the second optical sensor (1032) may optically image one or more structural features of the biosensor (1020) such as one or more fiducials and wells (e.g., set of nanometer sized holes). That is, the backlight (1030) may output light through the biosensor (1020) to illuminate the biosensor (1020) for the second optical sensor (1032) to image. In some embodiments, the backlight (1030) may comprise a green backlight and the first optical sensor may be used to determine a diameter of the light beam and visualize the biofluid sample. The calibration system and/or controller of a control system may process the image data to locate the biosensor (1020) and wells relative to the other system components. In some embodiments, the calibration system and/or controller may be configured to detect and position the biosensor (1020) relative to the radiation source using the platform. The calibration system may control operation of an XY stage ( 1024) of the biosensor assembly to position a well of the biosensor (1020) at a predetermined location using the image data generated by the second optical sensor (1032). The calibration system may be configured to sequentially align the radiation source to each of a plurality of wells of the biosensor (1020), with each well defining a set of the plurality of holes, [0075] In some embodiments, computer instructions may be configured to cause the controller to generate indexing data using the calibration system. The indexing data may correspond to locations of the wells relative to the radiation source and the detector. The computer instructions may be configured to cause the controller to generate spectroscopy data for each of the plurality of wells without the biofluid and with the bioiluid.

[0076] In some embodiments, the calibration system may further comprise an input device configured to control movement of the biosensor assembly. For example, a user may use a joystick (not shown) to provide coarse movements to align the platform relative to one or more of a radiation source, light beam, and detector. Additionally or alternatively, the radiation source and detector (e.g., first and second optical fibers) may be configured to move and reposition relative to a fixed biosensor. For example, the platform may be configured to move with one degree of freedom for insertion and ejection of the biosensor. Each of the first and second optical fibers may be coupled to respective XY stages to position themselves relative to each well of the biosensor.

Control system

[0077] The biofluid analysis systems as described herein may couple to one or more control systems (e.g., computer systems) and/or networks. FIG. 3B is a block diagram of the control system (320). The control system (320) may comprise a controller (322) comprising a processor (324) and a memory (326). In some embodiments, the control system (320) may further comprise a communication interface (330). The controller (322) may be coupled to the communication interface (330) to permit a user to remotely control the control system (320), radiation source (310), biosensor assembly (312), detector (314), calibration system (380), and any other component of the system (300). The communication interface (330) may comprise a network interface (332) configured to connect the control system (320) to another system (e.g., Internet, remote server, database) over a wired and/or wireless network. The communication interface (330) may further comprise a user interface (334) configured to permit a user to directly control the control system (320).

Controller

[0078] Generally, the biofluid analysis systems described herein may include a biosensor and corresponding control system coupled to a radiation source and detector. In some embodiments, a detector may be configured to generate signal data. The signal data may be received by a controller and used to generate spectroscopy data corresponding to one or more analytes of a biofluid sample. The control system may accordingly provide analyte analysis of a biofluid sample. As described in more detail herein, the controller may be coupled to one or more networks using a network interface. The controller may include a processor and memory coupled to a communication interface comprising a user interface. The controller may automatically perform one or more steps of biosensor calibration, indexing, spectroscopy analysis, and analyte analysis, and thus improve one or more of specificity, sensitivity, and speed of biofluid analysis.

[0079] The controller may include computer instructions for operation thereon to cause the processor to perform one or more of the steps described herein. In some embodiments, the computer instructions may be configured to cause the processor to determine (e.g., calculate) at least one of a total analyte content and the identified analyte amount in the biofluid. The analyte may comprise at least one of a protein, nucleic acid, lipoprotein, glycoprotein, and carbohydrate. The spectroscopy data may comprise wavelength data and intensity data of the received light beam. In some embodiments, the computer instructions may be configured to cause the controller to set spectroscopy data parameters comprising at least one of integration time, number of scans to average, and box car width. The computer instructions may be configured to cause the controller to generate the spectroscopy data by at least one of calculating maximum and minimum intensities of the signal data and a change in wavelength. Signal data and analysis may be saved for each well of the biosensor.

[0080] A control system (320), as depicted in FIG. 3B, may comprise a controller (322) in communication with the biofluid analysis system (300) (e.g., radiation source (310), biosensor assembly (312), detector (314), and calibration system (380)). The controller (322) may comprise one or more processors (324) and one or more machine-readable memories (326) in communication with the one or more processors (324). The processor (324) may incorporate data received from memory (326) and user input to control the system (300). The memory (326) may further store instructions to cause the processor (324) to execute modules, processes, and/or functions associated with the system (300). The controller (322) may be connected to and control one or more of a radiation source (310), biosensor assembly (312), detector (314), calibration system (330), communication interface (330), and the like by wired and/or wireless communication channels.

[0081] The controller (322) may be implemented consistent with numerous general purpose or special purpose computing systems or configurations. Various exemplary computing systems, environments, and/or configurations that may be suitable for use with the systems and devices disclosed herein may include, but are not limited to software or other components within or embodied on a server or server computing devices such as routing/connectivity components, multiprocessor systems, microprocessor-based systems, distributed computing networks, personal computing devices, network appliances, portable (e.g., hand-held) or laptop devices. Examples of portable computing devices include smartphones, personal digital assistants (PDAs), cell phones, tablet PCs, wearable computers taking the form of smartwatches and the like, and portable or wearable augmented reality devices that interface with the patient's environment through sensors and may use head- mounted displays for visualization, eye gaze tracking, and user input.

Processor

[0082] The processor (324) may be any suitable processing device configured to run and/or execute a set of instructions or code and may include one or more data processors, image processors, graphics processing units, physics processing units, digital signal processors, and/or central processing units. The processor (324) may be, for example, a general purpose processor, Field Programmable Gate Array (FPGA), an Application Specific Integrated Circuit (ASIC), combinations thereof, and the like. The processor (324) may be configured to run and/or execute application processes and/or other modules, processes and/or functions associated with the system and/or a network associated therewith. The underlying device technologies may be provided in a variety of component types including metal-oxide semiconductor field-effect transistor (MOSFET) technologies like complementary metal- oxide semiconductor (CMOS), bipolar technologies like emitter-coupled logic (EC!,), polymer technologies (e.g., silicon-conjugated polymer and metal -conjugated polymer-metal structures), mixed analog and digital, combinations thereof, and the like.

Memory

[0083] In some embodiments, the memory (326) may include a database (not shown) and may be, for example, a random access memory (RAM), a memory buffer, a hard drive, an erasable programmable read-only memory (EPROM), an electrically erasable read-only memory (EEPROM), a read-only memor}' (ROM), Flash memory, combinations thereof, and the like. As used herein, database refers to a data storage resource. The memory (326) may store instructions to cause the processor (324) to execute modules, processes, and/or functions associated with the control system (320), such as calibration, indexing, biosensor signal processing, spectroscopy analysis, analyte analysis, notification, communication, authentication, user settings, combinations thereof, and the like. In some embodiments, storage may be network-based and accessible for one or more authorized users. Network- based storage may be referred to as remote data storage or cloud data storage. Signal data and analysis stored in cloud data storage (e.g., database) may be accessible to authorized users via a network, such as the Internet. In some embodiments, database (340) may be a cloud-based FPGA.

[0084] Some embodiments described herein relate to a computer storage product with a non-transitory computer-readable medium (also may be referred to as a non-transitory processor-readable medium) having instructions or computer code thereon for performing various computer-implemented operations. The computer-readable medium (or processor- readable medium) is non-transitory in the sense that it does not include transitory propagating signals per se (e.g., a propagating electromagnetic wave carrying information on a transmission medium such as space or a cable). The media and computer code (also may be referred to as code or algorithm) may be those designed and constructed for a specific purpose or purposes.

[0085] Examples of non-transitory computer-readable media include, but are not limited to, magnetic storage media such as hard disks, floppy disks, and magnetic tape, optical storage media such as Compact Disc/Digital Video Discs (CD/DVDs); Compact Disc-Read Only Memories (CD-ROMs); holographic devices; magneto-optical storage media such as optical disks; solid state storage devices such as a solid state drive (SSD) and a solid state hybrid drive (SSHD); carrier wave signal processing modules; and hardware devices that are specially configured to store and execute program code, such as Application-Specific Integrated Circuits (ASICs), Programmable Logic Devices (PLDs), Read-Only Memory (ROM), and Random-Access Memory (RAM) devices. Other embodiments described herein relate to a computer program product, which may include, for example, the instructions and/or computer code disclosed herein.

[0086] The systems, devices, and methods described herein may be performed by software (executed on hardware), hardware, or a combination thereof. Hardware modules may include, for example, a general -purpose processor (or microprocessor or microcontroller), a field programmable gate array (FPGA), an application specific integrated circuit (ASIC), combinations thereof, and the like. Software modules (executed on hardware) may be expressed in a variety of software languages (e.g., computer code), including C, C++, Java®, Python, Ruby, Visual Basic®, and/or other object-oriented, procedural, or other programming language and development tools. Examples of computer code include, but are not limited to, micro-code or micro-instructions, machine instructions, such as produced by a compiler, code used to produce a web service, and files containing higher-level instructions that are executed by a computer using an interpreter. Additional examples of computer code include, but are not limited to, control signals, encrypted code, and compressed code,

C om mun i cati on interface

[0087] The communication interface (330) may permit a user to interact with and/or control the system (300) directly and/or remotely. For example, a user interface (334) of the system (300) may include an input device for a user to input commands and an output device for a user and/or other users (e.g., technicians) to receive output (e.g., view biofluid sample data on a display device) related to operation of the system (300). In some embodiments, a network interface (332) may permit the control system (320) to communicate with one or more of a network (370) (e.g., Internet), remote server (350), and database (340) as described in more detail herein.

User interface

[0088] User interface (334) may serve as a communication interface between a user (e.g., operator) and the control system (320). In some embodiments, the user interface (334) may comprise an input device and output device (e.g., touch screen and display) and be configured to receive input data and output data from one or more sensors, input device, output device, network (370), database (340), and server (350). For example, signal data generated by a detector may be processed by processor (324) and memor' (326), and output visually by one or more output devices (e.g., display). Signal data, spectroscopy data, and/or analyte data may be received by user interface (334) and output visually, audibly, and/or through haptic feedback through one or more output devices. As another example, user control of an input device (e.g., joystick, keyboard, touch screen) may be received by user interface (334) and then processed by processor (324) and memory (326) for user interface (334) to output a control signal to one or more components of the biofluid analysis system (300). In some embodiments, the user interface (334) may function as both an input and output device (e.g., a handheld controller configured to generate a control signal while also providing haptic feedback to a user). Output device

[0089] An output device of a user interface (334) may spectroscopy data and/or analvte data corresponding to a biofluid sample and/or system (300), and may comprise one or more of a display device, audio device, and haptic device. The display device may be configured to display a graphical user interface (GUI). The user console (360) may include an integrated display and/or video output that may be connected to output to one or more generic displays, including remote displays accessible via the internet or network. The output data may also be encrypted to ensure privacy and all or portions of the output data may be saved to a server or electronic healthcare record system. A display device may permit a user to view signal data, calibration data, functionalization data, spectroscopy data, analvte data, system data, biofluid data, patient data, and/or other data processed by the controller (322). In some embodiments, an output device may comprise a display device including at least one of a light emitting diode (LED), liquid crystal display (LCD), electroluminescent display (ELD), plasma display panel (PDP), thin film transistor (TFT), organic light emitting diodes (OLED), electronic paper/e-ink display, laser display, holographic display, combinations thereof, and the like.

[0090] In some embodiments, the system ( 1 10) may comprise one or more illumination sources (not shown) coupled to the optical waveguides (1 120, 1 22). The illumination source may be configured to emit light using a predetermined combination of light output parameters (e.g., wavelength, frequency, intensity, pattern, duration, etc.). Non-limiting examples of an illumination source include incandescent, electric discharge (e.g., excimer lamp, fluorescent lamp, electrical gas-discharge lamp, plasma lamp, etc.), electroluminescence (e.g., light-emitting diodes, organic light-emitting diodes, laser, etc.), induction lighting, and fiber optics. An optical waveguide may refer to a physical structure that guides electromagnetic waves such as visible light spectrum waves to passively propagate and distribute received electromagnetic waves. Non-limiting examples of optical waveguides include optical fiber, rectangular waveguides, light tubes, light pipes, combinations thereof, or the like. Any of the optical waveguides as described herein may communicate a state of any of the components of the system.

[0091] The light patterns described herein may, for example, comprise one or more of flashing light, occulting light, isophase light, etc., and/or light of any suitable light/dark pattern. For example, flashing light may correspond to rhythmic light in which a total duration of the light in each period is shorter than the total duration of darkness and in which the flashes of light are of equal duration. Occulting light may correspond to rhythmic light in which the duration of light in each period is longer than the total duration of darkness. Isophase light may correspond to light which has dark and light periods of equal length. Light pulse patterns may include one or more colors (e.g., different color output per pulse), light intensities, and frequencies.

[0092] An audio device may audibly output patient data, biofluid data, spectroscopy data, analyte data, system data, alarms and/or warnings. For example, the audio device may output an audible warning when improper insertion of the biosensor into the biosensor assembly occurs. In some embodiments, an audio device may comprise at least one of a speaker, piezoelectric audio device, magnetostrictive speaker, and/or digital speaker. In some embodiments, a user may communicate with other users using the audio device and a communication channel.

[0093] A haptic device may be incorporated into one or more of the input and output devices to provide additional sensor}' output (e.g., force feedback) to the user. For example, a haptic device may generate a tactile response (e.g., vibration) to confirm user input to an input device (e.g., joystick, keyboard, touch surface). In some embodiments, the haptic device may include a vibrational motor configured to provide haptic tactile feedback to a user. Haptic feedback may in some embodiments confirm initiation and completion of biosensor processing. Additionally or alternatively, haptic feedback may notify a user of an error such as improper placement and/or insertion of the biosensor into a biosensor assembly. This may prevent potential harm to the system.

Input device

[0094] Some embodiments of an input device may comprise at least one switch configured to generate a control signal. In some embodiments, the input device may comprise a wired and/or wireless transmitter configured to transmit a control signal to a wired and/or wireless receiver of a controller (322). For example, an input device may comprise a touch surface for a user to provide input (e.g., finger contact to the touch surface) corresponding to a control signal. An input device comprising a touch surface may be configured to detect contact and movement on the touch surface using any of a plurality of touch sensitivity technologies including capacitive, resistive, infrared, optical imaging, dispersive signal, acoustic pulse recognition, and surface acoustic wave technologies. In embodiments of an input device comprising at least one switch, a switch may comprise, for example, at least one of a button (e.g., hard key, soft key), touch surface, keyboard, analog stick (e.g., joystick), directional pad, pointing device (e.g., mouse), trackball, jog dial, step switch, rocker switch, pointer device (e.g., stylus), motion sensor, image sensor, and microphone. A motion sensor may receive user movement data from an optical sensor and classify a user gesture as a control signal. A microphone may receive audio and recognize a user voice as a control signal.

Network interface

[0095] As depicted in FIG. 3 A, a control system (320) described herein may communicate with one or more networks (370) and computer systems (350) through a network interface (332). In some embodiments, the control system (320) may be in communication with other devices via one or more wired and/or wireless networks. The network interface (332) may facilitate communication with other devices over one or more external ports (e.g., Universal Serial Bus (USB), multi-pin connector) configured to couple directly to other devices or indirectly over a network (e.g., the Internet, wireless LAN).

[0096] In some embodiments, the network interface (332) may comprise a radiofrequency receiver, transmitter, and/or optical (e.g., infrared) receiver and transmitter configured to communicate with one or more devices and/or networks. The network interface (332) may communicate by wires and/or wirelessly with one or more of the sensors, user interface (334), network (370), database (340), and server (350).

[0097] In some embodiments, the network interface (332) may comprise radiofrequency (RF) circuitry (e.g., RF transceiver) including one or more of a receiver, transmitter, and/or optical (e.g., infrared) receiver and transmitter configured to communicate with one or more devices and/or networks. RF circuitry may receive and transmit RF ^' signals (e.g., electromagnetic signals). The RF circuitry converts electrical signals to/from electromagnetic signals and communicates with communications networks and other communications devices via the electromagnetic signals. The RF circuitry may include one or more of an antenna system, an RF transceiver, one or more amplifiers, a tuner, one or more oscillators, a digital signal processor, a CODEC chipset, a subscriber identity module (SIM) card, memory, and the like. A wireless network may refer to any type of digital network that is not connected by cables of any kind.

[0098] Examples of wireless communication in a wireless network include, but are not limited to cellular, radio, satellite, and microwave communication. The wireless communication may use any of a plurality of communications standards, protocols and technologies, including but not limited to Global System for Mobile Communications (GSM), Enhanced Data GSM Environment (EDGE), high-speed downlink packet access (HSDPA), wideband code division multiple access (W-CDMA), code division multiple access (CDMA), time division multiple access (TDMA), Bluetooth, near-field communication (NFC), radio-frequency identification (RFID), Wireless Fidelity (Wi-Fi) (e.g., IEEE 802.11a, IEEE 802.1 1 b, IEEE 802.1 lg, IEEE 802.1 1η), Voice over Internet Protocol (VoIP), Wi-MAX, a protocol for email (e.g., Internet Message Access Protocol (IMAP), Post Office Protocol (POP)), instant messaging (e.g., extensible Messaging and Presence Protocol (XMPP), Session Initiation Protocol for Instant Messaging, Presence Leveraging Extensions (SIMPLE), Instant Messaging and Presence Service (IMPS)), Short Message Service (SMS), or any other suitable communication protocol. Some wireless network deployments combine networks from multiple cellular networks or use a mix of cellular, Wi-Fi, and satellite communication.

[0099] In some embodiments, a wireless network may connect to a wired network in order to interface with the Internet, other carrier voice and data networks, business networks, and personal networks. A wired network is typically carried over copper twisted pair, coaxial cable, and/or fiber optic cables. There are many different types of wired networks including wide area networks (WAN), metropolitan area networks (MAN), local area networks (LAN), Internet area networks (IAN), campus area networks (CAN), global area networks (GAN), like the Internet, and virtual private networks (VPN). As used herein, network refers to any combination of wireless, wired, public, and private data networks that are typically interconnected through the Internet, to provide a unified networking and information access system.

Other components

[00100] As shown in FIGS. 1A, IB, ID, IF, 2A and 2B, the first optical fiber (110) may be coupled to a first optical fiber lock (1 1) to fix the position of the first optical fiber ( 10) relative to the other components of the system (100). In some embodiments, the biofluid analysis system may be coupled to at least one dampener configured to reduce the transmission of vibration to the system from the surface on which the system is placed. For example, the dampener may comprise a set of six vibration isolation feet that ensures adequate dampening and isolation. FIG. 1 IB is a rear view of the biofluid analysis system (1 100) that may comprise a removable backpiate for access, a USB connector, network connector (e.g., Ethernet port), input device connector (e.g., joystick), power switch, power cable, power indicators (e.g., platform controller status indicator light, X stage status indicator light, and Y stage status indicator light.

III. Methods

[00101] Described herein are embodiments corresponding to methods for analyzing a biofluid, functionalizing a biosensor, and manufacturing a biosensor. These methods may characterize and/or quantitate a biofluid sample based on surface plasmon resonance transmission and may in some embodiments be used with the systems and devices described. For example, a biofluid analysis system may spectrally analyze and quantitate a plasma sample placed on a biosensor and identify an analyte as well as quantitate one or more of total analyte content and specific analyte content of the sample. A biosensor may be functionalized by the addition of one or more substances in order to increase binding affinity of one or more analytes. That is, the biosensor may be transformed from a "dry consumable" to a "wet consumable." A biosensor manufacturing method may process a silicon wafer substrate into a biosensor that may be used with the biofluid analysis system.

Biofluid preparation

[00102] In some embodiments, a biofluid sample may be preprocessed prior to application on a biosensor and processing by a biofluid analysis system. The biofluid sample may comprise, for example, plasma from blood, serum from blood, cerebrospinal fluid, urine, combinations thereof, and the like. The biofluid analysis system may be configured to identify and quantitate a wide range of analytes including at least one of exosomes from different origins, exosomal RNA with target sequences, cfDNA with target sequences, exosomal surface proteins, glycoproteins, and carbohydrates.

[00103] In some embodiments, a biofluid sample may be prepared by drawing blood from a patient. For example, about a 10 mL sample may be sufficient for use with the biosensors and systems described herein. Plasma may then be separated from blood to provide, for example, about 6 mL of a plasma sample. One or more analytes (e.g., exosomes) may be isolated from the plasma sample and then applied to a surface of the biosensor including a "wet" functionalized biosensor.

Biofluid analysis

[00104] Methods for analyzing a biofluid in some embodiments may use a biofluid analysis system and/or biosensor as described herein. The methods described here may quickly and easily provide a label -free method of identifying analytes from a biofluid based on surface plasmon resonance analysis techniques. Generally, the methods described here may include applying a biofluid sample to a biosensor and inserting the biosensor into a biofluid analysis system. The biosensor may be calibrated with respect to a radiation source and detector. Each of a plurality of wells of the biosensor may be located and indexed. Each well may be sequentially illuminated to generate spectroscopy data. A controller may identify one or more analytes using the spectroscopy data. In some embodiments, spectral analysis of the biosensor without the biofluid sample may be performed by the biofluid analysis system in order to generate reference spectroscopy data. A wavelength shift between the reference data and biofluid data may indicate the presence of a target analyte while differences in an intensity measurement may indicate a quantity of the target analyte. This analysis may have numerous benefits, such as label-free analysis of a sample within minutes.

[00105] FIG. 5 is a flowchart the generally described a method of analyzing a biofluid (500). The process (500) may begin in step 502 by applying a biofluid sample to the biosensor. For example, the biosensor may be a semiconductor chip as shown in FIG. 4B. In some embodiments, a binding agent may be deposited on the metal layer prior to applying the biofluid sample to the biosensor. The binding agent may comprises at least one of polyethylene glycol, an amine protein group, an antibody, an aptamer, a charge, a ligand, a moiety, a zwitterion, and an amplifier. The amine protein group may comprises at least one of neutravidin, streptavidin, and carboxylamine.

[00106] In step 504, the biosensor may be placed in a housing (e.g., consumable, disposable, palette, holder) such as depicted in FIGS. 4 A, 4D, 4E, lOA, and 10D. In step 506, the housing having the biosensor disposed thereon may be placed onto a platform and inserted into the biofluid analysis system. For example, the housing may be inserted onto a platform through a biosensor input (1110) of the biofluid analysis system (1 100). FIGS, 10A and 10D depict a housing (1021) securely held on the platform (1022), according to some embodiments. In step 508, the platform may be moved (such as by using an XY stage (1024)) to a predetermined position (e.g., under an output of a radiation source) and calibrated. The position of the biosensor and its wells may be calibrated relative to the light beam and the detector. That is, a location of a plurality of wells of the biosensor may be identified and indexed using a calibration system. The indexing data may correspond to locations of the wells relative to the detector. Indexing allows the light beam emitted from the radiation source to be aligned with each of the wells. [00107] In step 510, each well is illuminated by a radiation source sequentially after aligning the light beam to the well. Each well defines a set of the nanometer-sized holes. FIG. 4C is a measurement sequence, performed according to some embodiments. For example, sequencing may begin at a first well (450) and proceed along a first column to a tenth well (452). In some embodiments, the wells may be measured in a serpentine manner along each of the columns (or rows) until a last well (e.g., hundredth well) (454) has been illuminated and measured. Indexing data may comprise a location of each of the wells, as well as corresponding functionalization data, biofluid data, detector data (e.g., signal data), anaiyte data, and the like. In step 512, signal data may be received from a detector for each well and stored in memory. The detector may be provided opposite to an output of the radiation source. In step 514, spectroscopy data may be generated by at least one of a spectrophotometer and a controller (e.g., processor and memory) using the received signal data. For example, the spectroscopy data may be used to generate a sensorgram or a plot of wavelength and intensity. The sensorgram may correspond to molecular adsorption of the biofluid sample to the biosensor over time. As another example, FIGS. 9A-9C are illustrative plots of spectroscopy data including wavelength and intensity. In FIG. 9A, maximum intensities (910) and minimum intensities (920) of the waveform may be calculated from the spectroscopy data. In FIG. 938, shifts in wavelength between measurements within a region of interest (930) may be calculated to identify an anaiyte. For example, FIG. 9B illustrates shifts in wavelength of a local minimum within the region of interest (930) for a dry biosensor (932), a biosensor having an antibody (934), and an antibody bound to an anaiyte (e.g., exosome) (936).

[00108] In step 516, one or more analytes may be identified using the spectroscopy data. In some embodiments, at least one of a total anaiyte content and the identified anaiyte amount in the biofluid may be determined using a controller. The anaiyte may comprise at least one of a protein, nucleic acid, lipoprotein, glycoprotein, and carbohydrate. In step 518, at least one of the spectroscopy data and anaiyte analysis may be output to a user.

Functionalize a biosensor

[00109] Methods for funetionalizing biofluid in some embodiments may use a biofluid analysis system and/or biosensor as described herein. The methods described here may allow a wide range of analytes to be characterized and quantitated with high specificity. Generally, the methods described here include applying one or more substances to a metal surface of a biosensor to change binding characteristics for one or more target analytes. Accordingly, the biosensor may be transformed from a "dry sensor" to a "wet sensor" by the addition (e.g., functionalization) of one or more substances. In some embodiments, the spectral characteristics of the biosensor may be measured after applying each substance to generate reference (e.g., baseline) data. One or more wells of the biosensor may be configured as positive control for non-specific binding and a negative control comprising an antibody.

[00110] FIG. 6 is a flowchart the generally describes a method of functionalizing a biosensor (600). The process (600) may begin in step 602 by mounting a biosensor to a transparent substrate. For example, the underside of a biosensor chip (410) may be mounted (e.g., adhered) to a microscope slide. In some embodiments, the biosensor may be secured to cured polydimethylsiloxane (PDMS). In step 604, the biosensor may be placed in a solution of phosphate-buffered saline (PBS). The biosensor may then be illuminated and measured by a detector of a biofluid analysis system. In step 606, polyethylene gycol (PEG) may be applied to the biosensor and washed. In step 608, the biosensor in a solution of PBS may be illuminated and measured. In step 610, the biosensor may be incubated with neutravidin and washed. In step 6 2, the biosensor may be incubated with an antibody and washed. In some embodiments, the antibody may comprise a monoclonal antibody (e.g., absorbing monoclonal antibodies). Other substances that may be applied to the biosensor may include covalently attaching chelating moieties (e.g., charged moieties, absorbing chelating moieties). In step 614, the biosensor in a solution of PBS may be illuminated and measured. In step 616, the biosensor may be incubated with an analyte of a biofluid sample and washed. In step 618, the biosensor in a solution of PBS may be illuminated and measured. In some embodiments, one or more the antibody and biofluid may be applied to the biosensor using microfluidics. Alternatively, the biosensor may be used as a "dry consumable" that has not been functionalized.

[00111] FIG. 9C is an illustrative plot of spectroscopy data of a bare biosensor and biosensor having the biofluid sample. The change in spectra may be used to identify and quantitate an analyte of the biofluid. For example, a controller may identify a reference sentinel peak (940) and a biofluid sentinel dip (950),

Manufacturing a biosensor

[00112] Also described herein are embodiments corresponding to methods for manufacturing a biosensor that may be used in some embodiments with the biofluid analysis system embodiments as disclosed herein. The methods described here may manufacture a biosensor comprising a semiconductor chip that may be useful with surface plasmon resonance analysis techniques. This may have numerous benefits, such as label-free detection of analyte directly from a biofluid starting material. The biosensors manufactured as described herein may be processed to generate data having higher sensitivity and specificity than ELISA.

[00113] Generally, the methods described here include depositing a layer of silicon nitride on each side of a silicon wafer substrate such as by using low pressure chemical vapor deposition. A plurality of first holes may be created on one side of the substrate. A photoresist may be added to the silicon nitride layers and used to create a plurality of second holes on a second side of the substrate. The plurality of second holes may be extended through one side of the silicon nitride layer and through the substrate so as to connect the first and second plurality of holes. The connected holes form a plurality of wells for light transmission. The holes may be created using one or more techniques including interference lithography, deep ultraviolet light etching, and electron beam lithography. The number of first holes may be greater than a number of second holes such that the diameter of the first holes is smaller than the diameter of the second holes. For example, the first holes may comprise a diameter of between about 100 nm and about 500 nm while the second holes comprise a diameter of between about 700 μιη and about 750 μηι. In some embodiments, the second hole in the substrate may comprise a sidewall angle of between about 45° and about 70° with respect to a plane of the second side of the substrate. A metal layer may be deposited over the substrate and around the holes. The formed substrate may then be partitioned and cut into a plurality of chips (e.g., biosensors, dry consumables). In some embodiments, the chip may be coupled to a glass substrate (e.g., adhered to a microscope slide). The biosensor may be further coupled to a housing to form a biosensor assembly.

[00114] FIG. 7 is a flowchart the generally described a method of manufacturing a biosensor (700). Various steps of the flowchart are illustrated in corresponding FIGS, 8A-8M. A biosensor may comprise a substrate such as a silicon wafer. In some embodiments, the substrate may comprise silicon, silica, and glass having a diameter of between about 8 cm and about 20 cm. For example, a silicon wafer (800) as shown in FIG. 8A may comprise about a 10, 16 cm (4 inch) diameter and a thickness of between about 500 μιη and about 675 μιη or about a 15.24 cm (six inch) diameter and a thickness of between about 500 μηι and about 675 μηι. In some embodiments, the thickness of the substrate (800) may be between about 500 μτη and about 550 μηι. The substrate may comprise any suitable diameter such as between about 8 cm and about 20 cm.

[00115] As shown in FIGS. 8A-8B, the process (700) may begin in step 702 by depositing a layer of silicon nitride (810, 820) on opposite sides of the substrate (800). For example, a first layer of silicon nitride (810) may be disposed on a first side of a substrate (800) and a second layer of silicon nitride (820) may be disposed on a second side of the substrate (800). In some embodiments, silicon nitride may be deposited (e.g., grown) on the substrate (800) using low pressure chemical vapor deposition (LPCVD). In some embodiments, the first and second layers of silicon nitride (810, 820) may have a depth of between about 80 nm and about 350 nm. In other embodiments, the first and second layers of silicon nitride (810, 820) may have a depth of between about 200 nm and about 300 nm.

[00116] In step 704, a plurality of first holes (812) (e.g., nanotrenches), as shown in FIG. 8C, may be created in the first layer (810). In some embodiments, the first holes (812) may comprise a diameter of between about 100 nm and about 500 nm and have a hole-to-hole distance of between about 485 nm and about 515 nm (or more). In some embodiments, the first holes (812) may comprise a diameter of between about 200 nm and about 250 nm. In some embodiments, the plurality of first holes (812) may be created using interference lithography without etching of the substrate (800) and the second layer (820). In some embodiments, between about 40 nm and about 60 nm of the first layer (810) is left unetched under the first holes (812) as an unetched portion (816). In other embodiments, the plurality of first holes (812) may be created using deep UV etching, photolithography, or electron beam lithography (with or without scanning electron microscope guidance).

[00117] In step 706, a photoresist may be applied the first and second layers (810, 820) as shown in FIG. 8D. For example a first photoresist (814) may be applied on the first layer (810) and have a thickness of between about 1 μιη and about 3 μηι above a top surface of the first layer (810). In some embodiments, the first photoresist (814) may have a thickness of about 1.8 μηι above a top surface of the first layer (810), The first photoresist may protect the first layer (810) from subsequent etching performed on the second layer (820) and substrate (800). A second photoresist (not shown) may be applied on the second layer (820).

[00118] In step 708, the second photoresist may be used to create a plurality of second holes (822) in the second layer (820). FIG. 8E illustrates creation of a single second hole (822) in the second layer (820). In some embodiments, the plurality of second holes (822) in the second layer (820) may be created using lithography without etching of the substrate (800). In some embodiments, the second holes (822) in the second layer (820) may comprise a diameter of between about 700 μτη and about 750 μτη and have a hole-to-hole distance of between about 1.5 mm and about 2.5 mm. Therefore, a set of the first holes (812) may fit into an opening of each second hole (822).

[00119] A third photoresist (not shown) may be applied to the substrate (800) and used to extend the plurality of second holes (802) into the substrate (800). In some embodiments, the plurality of second holes (802) in the substrate (800) may form a sidewall angle of between about 45° and about 70° with respect to a plane of the second side of the substrate (820). In some embodiments, the plurality of second holes (802) in the substrate (800) may form a sidewall angle of about 54° or 54.74°. In some embodiments, the third photoresist may be etched using a wet etchant from the second side of the substrate without etching the first layer (810) and second layer (820) as shown in FIG. 8F. For example, the unetched portion (816) may remain after creating the second holes (822, 802). The wet etchant may include potassium hydroxide (KOH). The first and second layers (810, 820) may serve as a mask for the remaining substrate (800).

[00120] In step 710, the first holes (812) are extended into the second holes to create a plurality of through holes through the substrate (800) and silicon nitride layers (810, 820) as shown in FIG. 8G. That is, each through hole goes all the way through the biosensor. In some embodiments, the unetched portion (816) may be etched (anisotropic etch) using a dry etchant from the second side of the substrate (800), However, in some embodiments, at least about 40 nm of the first lay er (810) remains. In some embodiments, the first layer (810) may comprise a thickness of between about 100 nm and about 140 nm as result of step 710. The dry etchant may include reactive ion etching (RIE).

[00121] FIG. 8H is a plan view of the biosensor (830) from a first side of the substrate (800) with the plurality of first holes (812). FIG. 81 shows an image from a scanning electron microscope depicting the plurality of first holes (812) on the metal (e.g., gold) surface disposed on the first layer (810). In some embodiments, a set of the first holes (812) may comprise a sensor well suitable for receiving a biofluid sample. For example, a sensor well is illustrated in FIG. 8H as a 50 urn by 50 μηι area of the sensor comprising a set of first holes (812). The first holes (812) may be separated by a distance of between about 485 nm and about 515 nm. This allows one or more analytes of a biofluid having a size of between about 100 nm and about 330 nm to bind to the surface of the metal layer and generate surface plasmons. The first holes (812) may comprise a diameter of between about 200 nm and about 250 nm. In some embodiments, the openings of the first holes (812) may comprise one or more shapes including circular, square, rectangular, trapezoidal, oval or elliptical, arc-shaped, combinations thereof, and the like.

[00122] FIG. 8J is a plan view of the biosensor (830) from a second side of the substrate (800) with the plurality of second holes (822). In some embodiments, the second holes (822) may be separated by a distance of about 1.5 mm and about 2.5 mm. The second holes (822) may comprise a diameter of between about 700 nm and about 750 nm. In some embodiments, the openings of the second holes (822) may comprise one or more shapes including circular, square, rectangular, trapezoidal, oval or elliptical, arc-shaped, combinations thereof, and the like. As discussed in detail herein, a plurality of first holes (812) may extend into one of the second holes (802, 820). Accordingly, the number of first holes (812) may be greater than a number of the second holes (822) where the first holes (812) are smaller than the second holes (822). As used herein, a through hole or well may refer to all of the first holes (812) that extend into one second hole (822). For example, for a first well of the biosensor (830), light that enters into any of the first holes (812) of the first well will be output through the same second hole (822).

[00123] In step 712, a third layer of metal may be deposited on the first layer (810). For example, the metal layer (846) may comprise one or more of titanium (Ti) and gold (Au), In some embodiments, the metal layer may be deposited by vapor deposition. FIG. 8 illustrates a metal evaporator (844) configured to deposit metal particles (e.g., Ti, Au) onto the biosensor (830) generated from a metal source (840). In some embodiments, titanium may be deposited onto the biosensor (830) by evaporation and may function as an adhesive layer between the silicon nitride layers and a gold layer. In some embodiments, the deposited titanium may comprise a thickness of about 2 nm to about 3 nm and be deposited at rate of less than about 2 A/sec. The titanium may be about 99.995% pure. In some embodiments, gold may be deposited onto the biosensor (830) by evaporation and comprise a thickness of between about 90 nm and about 1 10 nm. The layer of gold may be deposited at a rate of less than about 0.5 A/sec. In some embodiments, deposition of the metal layer may be performed at a pressure of less than about 3 x 10 ^"6 Torr.

[00124] FIG. 8L is a detailed cross-sectional view of a third layer (e.g., metal) and an opening (850) of a first hole in the first layer (810). In some embodiments, the metal layer (846) may form a projection (852) (e.g., overhang) over a portion of the first hole opening (850). While the projection (852) may be preferable in some embodiments, a metal coating within the first and second holes (812, 822) is preferably minimized so as to reduce occlusions.

[00125] In some embodiments, the silicon wafer (800) may be partitioned into a predetermined size and shape. Partitioning may be performed by any suitable method including scribing, breaking, mechanical sawing, laser cutting, combinations thereof, and the like. The partitioned wafer (800) may comprise one or more shapes including rectangular, square, trapezoidal, oval or elliptical, arc-shaped (e.g., hemi-arc, hemi-spherical, hemi- cylindricai), combinations thereof, and the like. FIG. 8M illustrates a circular wafer (860) comprising a 4 inch diameter being cut into about 8 biosensor chips (862) comprising a 1 inch diameter. In FIG. 8M, the number 3 and 6 chips are candidates for failure. Similarly, a 6 inch circular wafer may be cut to form about 17 biosensor chips comprising a 1 inch diameter. For example, a 1 inch diameter biosensor chip (862) may comprise about 100 wells (e.g., sensing arrays) provided over about 10 rows and about 10 columns. This biosensor (862) may be configured for up to 23 target analytes (including 2 controls) in quadruplicate. As another example, an about 3.3 inch diameter biosensor chip (862) may comprise about 999 wells provided over about 33 rows and about 33 columns. In yet another example, a biosensor (862) comprising 16 wells may be configured for up to 2 target analytes (including 2 controls) in quadruplicate or up to 3 target analytes (including 2 controls) in triplicate,

[00126] The foregoing description, for purposes of explanation, used specific nomenclature to provide a thorough understanding of vari ous inventions and embodiments disclosed herein. However, it will be apparent to one skilled in the art that specific details are not required in order to practice the disclosed inventions and embodiments. Thus, the foregoing descriptions of specific embodiments of the inventions and corresponding embodiments thereof are presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed; obviously, many modifications and embodiments are possible in view of the above teachings. The embodiments were chosen and described in order to best explain the principles of the inventions, the corresponding embodiments thereof, and practical applications, so as to enable others skilled in the art to best utilize the invention and various implementations with various modifications as are suited to the particular use contemplated. It is intended that the following claims and their equivalents define the scope of the invention. [00127] In addition, any combination of two or more such features, structure, systems, articles, materials, kits, steps and/or methods, disclosed herein, if such features, structure, systems, articles, materials, kits, steps and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. Moreover, some embodiments of the various inventions disclosed herein may be distinguishable from the prior art for specifically lacking one or more features/elements/functionality found in a reference or combination of references (i.e., claims directed to such embodiments may include negative limitations).

[00128] Any and ail references to publications or other documents, including but not limited to, patents, patent applications, articles, webpages, books, etc., presented anywhere in the present application, are herein incorporated by reference in their entirety. Moreover, all definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.