Multiplexed surface plasmon resonance sensing of analytes in liquid sample

Abstract

To provide a hand-held or portable SPR device.SOLUTION: A hand-held or portable SPR device focuses light at two or more positions on a noble metal sensor surface, functionalized to enable detection of different SPR curves and their respective minima. The detection is accomplished substantially simultaneously by obtaining SPR signals contemporaneously from the different locations on the sensor surface, on the same sensor. The SPR device may incorporate multiple light sources or a single light source with a beam that is shifted by deflectors in seriatim to focus on different positions on along the functionalized sensor surface. To control wettability of the sensor surface (to augment multiplexing), the sensor surface may include i) the addition of surface assembled monolayers (SAMs) and ii) surface bombardment by gaseous plasmas, most notably O2.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] The present invention relates to a surface plasmon resonance (SPR)-based device and method for detecting or identifying dissolved or suspended components in a fluid and measuring their concentration. Targeted components include viruses, bacteria, endosomes, enzymes, cations, anions, lipids, pharmaceuticals, drugs, and small molecules. The present invention can be used to analyze and measure human or animal tears. Furthermore, the present invention can identify solutes or measure their concentration in other body fluids, or can be similarly used for detecting pathogens in food or the environment. [Background technology]

[0002] Patent Document 1 discloses an apparatus that uses surface plasmon resonance (SPR) technology as a sensing platform to detect analytes or chemical markers, particularly antigens, in liquid samples such as human or animal tears. Specific antibodies are immobilized or attached to the SPR sensor surface, which leads to the generation of signals specific to each chemical marker, such as an antigen.

[0003] The portable SPR device taught in the '661 patent is a one-dimensional SPR device, in other words, the entire gold-plated surface area of ​​the disposable sensor prism is functional for the detection of only one analyte in the tear sample, or has no function at all, such as when the device only uses a noble metal film to measure the osmolality of the tear fluid.

[0004] By immobilizing antibodies on the SPR sensing surface, unknown concentrations of antigenic analytes in tear samples can be detected, thereby facilitating the diagnosis of ocular diseases and conditions. The same procedure can be used to detect solutes or analytes in any fluid, for medical or other purposes.

[0005] Computer analysis of the pattern of light absorption determines, for example, the position or angle of the absorption line of maximum absorption by the sensing surface. Thus, operation of a surface plasmon resonance device may more specifically include operation of a computer or microprocessor to determine the surface resonance angle or absorption line position from each electrical signal.

[0006] The device of the '661 patent considers that different analytes or solutes in a liquid sample migrate to a sensing surface at different rates. Different SPR minima are then detected in a single sequence, providing information about the constituents of the liquid sample. Problems arise when the migration rates are not sufficiently different to facilitate easy detection of the respective SPR minima. There is also a need for reduced test time and reduced materials. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 8,249,682 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention aims to provide an improved device and / or a new and improved method utilizing SPR to facilitate efficient simultaneous detection of the presence of multiple solutes or analytes in a liquid sample. More particularly, the present invention contemplates an improved, second-generation SPR device utilizing an improved method for simultaneously measuring the concentrations of at least two components of a liquid sample with a single sensor. The device is intended for clinical use or for use in testing food samples at any point in the production chain or in trade routes, including the home. The present invention seeks to provide such a device and / or method that is simple, cost-effective, safe, and hygienic to use. [Means for solving the problem]

[0009] The present invention uses novel technology to fabricate novel handheld or portable SPR devices, such as those described in U.S. Patent Application Publication No. 2009 / 0129994, the disclosure of which is incorporated herein by reference. According to the present invention, a multidimensional or multiplexed detection and measurement device exhibits multiple distinct functionalized locations or regions on a gold-plated sensor surface, which form multiple detection channels for the simultaneous detection of multiple components dissolved or suspended in a liquid sample, such as human tear fluid. The present invention represents a significant change in the state of the art in using SPR for the detection and quantification of dissolved constituents and analytes in liquid samples, including, for example, body fluids and food samples. In the field of medicine, the present invention provides real-time point-of-care diagnostics, a long-desired goal in the industry.

[0010] Thus, the present invention contemplates focusing light at two or more locations on a noble metal sensor film or coating, particularly a coating on its underside, so that a different SPR curve (with a minimum value identifying each analyte) can be observed for each location. By generating one or more SPR curves from different locations on the sensor surface, the concentration of one or more analytes in a single liquid sample placed on the sensor surface can be analytically determined. According to the present invention, a handheld or portable SPR measurement device can detect the presence and measure the concentration of one or more constituent analytes in a liquid sample selectively attached to the sensor surface at each location along or on the sensor surface, where the sensor surface is provided on a single disposable sensor prism that can be removably attached to the SPR measurement device. Signals from different sensor locations, evoked when respective light sources are oriented to strike different locations on the sensor film or coating, can be collected substantially simultaneously in real time by multiplexing (generating) the SPR signals from the different locations on the sensor surface and directing them to a photodetector device, which can be an array of photoelectric converters, such as charge-coupled devices.

[0011] An essential technique that allows for the precise application (both location and minute layer thickness) of the adhesive binding layer, immobilization matrix, and ligands required for functionalization of the SPR surface is jetting-based bioprinting. Jetting-based bioprinting is a non-contact technique that generates 2D structures using picoliter droplets of bioink layered on a substratum. Jetting-based bioprinting methods can eject picoliter droplets with high spatial resolution.

[0012] According to the present invention, the SPR device described in U.S. Patent No. 6,277,999 can incorporate multiple light sources or a single light source whose beam is oriented to focus at different locations on or along the sensor by optical, mechanical, and / or electronic means. (For example, an electrical signal from a microprocessor may operate an electromechanical servomechanism or a piezoelectric actuator to mechanically adjust the optical focusing element.) To enable different functionalization of the sensor surface (thereby enabling multiplexing) and optionally to adjust or modify the wettability of the sensor surface, the present invention contemplates the addition of surface-assembled monolayers (SAMs). Different SAMs can be used to bind respective solute or analyte binding agents to the noble metal sensor surface, where the binding agents can include preselected antibodies to attract and retain the respective solutes or analytes, i.e., antigens, in the liquid sample.

[0013] The surface plasmon resonance (SPR)-based multiplexed analyte sensing device of the present invention comprises a sensing surface for contacting a liquid sample and configured to interact with a plurality of different solutes or analytes in the liquid sample, an optical sensing device, and a computer or microprocessor operatively coupled to the optical sensing device for receiving from the optical sensing device a plurality of electrical signals encoding a pattern of light absorption by the sensing surface. The computer or microprocessor is programmed to analyze data from the optical sensing device by analyzing the pattern to determine the concentrations of the plurality of different solutes in a single continuous or integrated sample in contact with the sensing surface. The sensing surface comprises a plurality of different molecular elements or ligands configured to bind to each of the plurality of different solutes, the plurality of different molecular elements or ligands being disposed in respective, separate, predetermined regions on the sensing surface. The sensing surface is thus spatially coded or functionally segmented (patterned) for detecting the presence and concentration of each solute or analyte. The SPR device further includes at least one light source and an optical transmission element, the optical transmission element being at least partially disposed between the light source and the sensing surface and configured to direct each beam of electromagnetic radiation toward a respective predetermined region and toward an underside of the sensing surface opposite the liquid sample, such that the beam reflected from the underside of the sensing surface impinges on the optical sensing device. (Typically, the underside of a noble metal substrate or film is attached to the prism of the sensor body via a chrome layer.) The optical transmission element may also be partially disposed between the solute sensing surface and the optical sensing device.

[0014] According to a further feature of the present invention, (a) a solute sensing surface comprises at least one noble metal film, layer, or coating, and the predetermined regions are on the at least one metal film, layer, or coating; and (b) at least one surface-assembled monolayer is provided on the sensing surface to control or adjust the wettability of the sensing surface, i.e., to regulate the degree of spreading of a liquid sample on the sensing surface. The wettability can be enhanced, for example, by the application of a surface-assembled monolayer, so that the liquid sample migrates and effectively contacts the at least one metal film, layer, or coating in one or more of the predetermined regions. The surface-assembled monolayer can include alkanethiol groups attached to the at least one metal film, layer, or coating. The alkanethiol groups can additionally function to attach different solute-binding molecular entities or ligands to the metal film, layer, or coating.

[0015] At least a portion of the alkanethiol groups may comprise a terminal hydrophobic head selected from the group consisting of an acidic hydrophobic head, a hydroxyl hydrophobic head, or an amino group, the terminal hydrophobic head being SH-(CH) n -COOH or SH-(CH2) n It could be.

[0016] Surface-assembled monolayers (SAMs) may contain both hydrophilic and hydrophobic elements in preselected amounts to vary the hydrophilicity / hydrophobicity of the surface-assembled monolayer in a given region. Differential deposition of the SAM on the sensing surface is achieved through the use of a printing device that applies pico- or nano-sized droplets of the SAM components.

[0017] Preferably, the SPR device of the present invention further comprises a hand-held housing or casing, wherein the sensing surface is disposed in the housing or casing, and wherein one or more light sources, a light sensing device, a computer or microprocessor, and a light transmission element are housed inside the housing or casing.

[0018] The computer or microprocessor is configured to efficiently simultaneously detect multiple SPR signals from analytes in the liquid sample. The electrical signal from the optical sensing device includes signals from different photoelectric elements or pixels in a predetermined sequence, allowing the computer or microprocessor to simultaneously monitor SPR optical signals from different regions of the sensor surface in a substantially real-time and interleaved manner. The real-time point-of-care diagnostics provided by the present invention involve multiple analyses on a single 1 x 3 mm sensor surface in a single test to increase the efficiency of medical professionals, which allows for earlier initiation of treatment at significantly lower medical costs.

[0019] The liquid sample may be, for example, a biological fluid, a human fluid, an animal fluid, a food, or a beverage. The solute or analyte to be detected for presence and / or concentration may be cells, bacteria, insect material, vegetable material, viruses (e.g., viruses directly or in lysates from tissues or respiratory fluids), proteins, DNA and RNA, dissolved cations or anions, endosomes, enzymes, lipids, pharmaceutical compounds, natural and synthetic drugs, small molecules, or dissolved gases. Enzymes include MMP-9; viruses include herpes simplex virus, herpes zoster virus, and adenovirus; proteins include lactoferrin, tryptase, and interleukins; and small molecules include histamine, glucose, and fructose.

[0020] A disposable sensor for analyte detection by surface plasmon resonance according to the present invention comprises a body having a predetermined geometry for attachment to an analyte detection device including a light beam generating component, a light beam transmitting or guiding component, a light detector, and a computing unit. The disposable sensor also comprises a noble metal base layer provided on a sensing surface of the body, and a plurality of different binding agents attached to the base layer on the sensing surface. The different binding agents are disposed at respective locations on the base layer that are spaced apart from one another on the sensing surface. The different binding agents are preselected to bind respective solutes or analytes.

[0021] If desired, a plurality of molecular agents may be at least indirectly attached to the sensing surface to enhance the wettability of the sensing surface, such that a liquid placed in contact with the sensing surface spreads across the sensing surface to provide contact with the sensing surface comprising a plurality of preselected locations or regions on the substrate.

[0022] Preferably, the molecular agents for enhancing surface wettability collectively constitute a surface-assembled monolayer. The surface-assembled monolayer can be configured to change the contact angle of a solvent or water droplet on the sensor surface. The surface-assembled monolayer can be configured to impart varying degrees of hydrophilicity and hydrophobicity to the sensing surface. In other words, different SAM elements can be provided and attached to different locations or regions of the sensor surface depending on the function (target solute or analyte), binder, and type of liquid sample (hydrophobic or hydrophilic).

[0023] The noble metal base layer is a film 15-100 nm thick, and the noble metal may be Au, Ag, Al, Pt, Rh, Cu, or Ni, or in some cases a mixture or alloy thereof. The sensing surface may further include an adhesion layer 0.01-50 nm thick that attaches the metal layer, film, or coating to the body of the disposable sensor. The noble metal film may have its hydrophilic or hydrophobic properties altered by plasma treatment. Plasma treatment is typically used only at the end of the manufacturing or creation of the sensing surface, if necessary.

[0024] The method of the present invention for determining the concentration of at least one analyte in a fluid sample includes measuring one or more locations of one or more SPR minima and correlating the locations of the SPR minima with locations of SPR minima of known control samples or known refractive indexes to determine the composition of at least one unknown sample or the concentration of at least one unknown analyte. Alternatively or additionally, the method may include using the measurements of one or more SPR minima to correct other SPR minima in the same measurement for environmental factors, such as air humidity, temperature, and / or air pressure. Alternatively or additionally, the method may include using the measurements of one or more SPR minima to correct other SPR minima in the same measurement for manufacturing variations or errors, including the thickness of the precious metal, the thickness of the adhesive layer, and the refractive index of the molded sensor prism.

[0025] The second generation method and apparatus of the present invention will reduce testing time, expedite medical diagnosis and treatment, and for many purposes reduce costs.

[0026] A method of manufacturing a sensor for analyte detection by surface plasmon resonance of the present invention includes the steps of providing a body having a surface formed of a precious metal coating or film, operating a pico- or nanodot printer to apply a plurality of solute or analyte binding agents to a plurality of different, spaced apart regions or locations on the precious metal coating or film, and attaching the solute or analyte binding agents to the plurality of different, spaced apart regions or locations on the precious metal coating or film, wherein the solute or analyte binding agents are configured to bind to respective constituents of a liquid sample applied to the sensing surface.

[0027] The manufacturing method may further include operating the pico- or nanodot printer or another pico- or nanodot printer to provide or print a plurality of surface-assembled monolayer elements onto a surface of the body, in particular onto a noble metal coating or film.

[0028] The step of providing a plurality of solute or analyte binding agents at different regions or locations on the precious metal coating or film may be performed after the step of providing a plurality of surface-assembled monolayer elements on the sensing surface, in which case attaching the solute or analyte binding agents to different, spaced regions or locations on the precious metal coating or film comprises attaching the solute or analyte binding agents to the precious metal coating or film via the surface-assembled monolayer elements.

[0029] The fabrication method optionally includes selecting an additional plurality of surface-assembled monolayer elements to tailor the wettability of the precious metal coating or film, and printing the additional plurality of surface-assembled monolayer elements onto the precious metal coating or film, preferably after printing or applying the solute or analyte binding agent. [Brief explanation of the drawings]

[0030] [Figure 1A] 1 is a partial perspective view of a disposable sensor component according to the present invention and a schematic enlarged elevational view of a sensing surface or film included in the sensor component. [Figure 1B] FIG. 1B is a schematic side view of the sensor components shown in FIG. 1A. [Figure 1C] FIG. 2 is a schematic side or end view of the distal end of the sensor component of FIGS. 1A and 1B. [Figure 2] FIG. 2 shows the generation of two SPR curves from two excitation spots or active regions on the noble metal surface of the sensor component of FIG. 1. [Figure 3] FIG. 2 shows the generation of three SPR curves from three excitation spots on the noble metal surface of the sensor component of FIG. 1. [Figure 4] FIG. 1 shows the generation of three SPR curves from only one excitation (light) source. [Figure 5] FIG. 2 shows the generation of three SPR curves from three unique positions on the precious metal sensor surface of the sensor prism of FIG. 1. [Figure 6]1 is a graph showing SPR data, specifically two measurements, for both air and tear fluid measured at two specific wavelengths, 855 nm and 950 nm, respectively, where the Y-axis is intensity and the X-axis is pixel position in a linear array. DETAILED DESCRIPTION OF THE INVENTION

[0031] 1A-1C show a disposable sensor 12 that can be attached to the applicator end of a handheld SPR device, such as that disclosed in U.S. Patent Application Publication No. 2009 / 022999, which is incorporated herein by reference. Sensor 12 includes a sensor body or prism 14 having a sensing surface 16 at one end that can be contacted with a target liquid. Sensing surface 16 includes a base layer 18 in the form of a 15-100 nm thick film, layer, or coating made of a noble metal such as Au, Ag, Al, Pt, Rh, Cu, and Ni. A handheld SPR device (see FIG. 7, infra) cooperates with sensor 12 to detect the presence of any of a preselected group of potential analytes or solutes in a liquid aliquot and measure the concentration of the detected analyte or solute.

[0032] 1A and 1C, the sensing surface 16, and particularly the film, layer, or coating 18, comprises a surface-assembled monolayer (SAM) 20 that includes a plurality of molecular agents 22 applied to the substrate, film, coating, or layer 18, and thus indirectly to the sensing surface 16, for adjusting or regulating the degree of wettability of the precious metal substrate 18. It is often desirable to enhance the wettability of the sensing surface 16 so that a liquid sample 13 placed on the sensing surface 16 spreads across at least a portion of the sensing surface and contacts one or more active areas or locations 24, 26, 28 (see FIG. 5) thereof. FIG. 1A shows a water droplet 13' that has beaded up without spreading on a gold or precious metal substrate 18 that has not been provided with a wettability-enhancing SAM or otherwise treated, for example, with oxygen plasma.

[0033] 5, one or more of the active areas or locations 24, 26, 28 are functionalized with respective solute or analyte binding agents 30, 32 attached to the base layer 18 at the sensing surface 16 at different active areas. The active areas or locations 24, 26, 28 and concomitantly their respective binding agents 30, 32 are separated from one another at the sensing surface 16 and are preselected to bind respective predetermined solutes or analytes.

[0034] The surface-assembled monolayer 20 may include alkanethiol groups attached to the metal film, layer, or coating of the base layer 18. The alkanethiol groups may function, in part, to attach antibodies and other preselected solute or analyte binding agents to the precious metal substrate or film 18. At least some of the alkanethiol groups may each comprise a terminal hydrophobic head group. The terminal hydrophobic head group may be an acidic hydrophobic head group, a hydroxyl hydrophobic head group, or an amino group. The terminal hydrophobic head group may be SH-(CH2) n -COOH or SH-(CH2) n It could be.

[0035] The surface-assembled monolayer 20 may contain both hydrophilic and hydrophobic elements in preselected amounts to vary the hydrophilicity / hydrophobicity of a given region. Depending on the carrier or liquid sample (e.g., oil-based or aqueous), the solute or analyte binding agents 30, 32 (e.g., antibodies), and the target solute or analyte (e.g., antigens), as well as the SAM elements 22 or other binding agents that immobilize the solute or analyte binding agents 30, 32 to the metal substrate or film 18, the wettability (hydrophilicity / hydrophobicity) of the sensing surface 16, and more particularly, the noble metal substrate or film 18, is adjusted or tuned to facilitate capture or targeting of the solute or analyte at the respective locations or regions 24, 26, 28.

[0036] Additional SAMs can be provided on the surface-assembled monolayer 20 and configured to modify the contact angle of a solvent or water droplet on the sensing surface 16. The monolayer 20 can be organized to impart varying degrees of hydrophilicity and hydrophobicity to the sensing surface 16.

[0037] 2 illustrates the incidence of reflected light energy along two lines 34, 36 of optical elements in a linear array 38 for generation by a microprocessor 40 of respective SPR curves (see FIG. 6) from two spaced-apart excitation spots or active regions 42, 44 in a noble metal substrate or film 18 of a sensing surface 16. Two illumination or incident beams 46, 48 are directed toward the underside of the noble metal substrate or film 18, pass through a prism 12, and are transmitted as reflected beams 50, 52 to the lines 34, 36 of optical elements or pixels in the linear array 38. The illumination beams 46, 48 originate from respective light sources 54, 56 and may be of the same wavelength or different wavelengths.

[0038] 3 illustrates the incidence of reflected light energy along three lines 58, 60, 62 of optical elements in the linear array 38 for the generation by the microprocessor 40 of respective SPR curves (see FIG. 6) from three spaced excitation spots or active regions 64, 66, 68 on the noble metal substrate or film 18 of the sensing surface 16. Three illumination or incident beams 70, 72, 74 are directed from respective light sources 76, 78, 80 through appropriate optical elements 75 to impinge on the underside of the noble metal substrate or film 18 at various angles (to detect SPR minima) through the prism 12. Reflected beams 82, 84, 86, having various angular directionality, are directed onto the lines 58, 60, 62 of the optical elements in the array 38. (The illustrations of FIGS. 2-4 are schematic in that the reflected light, e.g., beams 82, 84, 86, passes back through prism 14 and is directed by optical elements to light-sensitive photoelectric elements or CCDs in linear array 38.) The light in beams 70, 72, 74 may be of the same wavelength or different wavelengths.

[0039] As shown in FIG. 4, an SPR sensing device such as that disclosed in the '661 patent may be modified to include a mechanical or electromechanical beam deflector 88 and appropriate optical elements to sequentially focus light from a single light source 902 at different locations 90, 92, and 94 at various angles of impingement beneath the metal film, coating, or layer 18. FIG. 4 shows a single beam 96 being directed by the beam deflector 88 and optical elements along three separate paths 98, 100, and 102 to locations 90, 92, and 94. From these separate locations or regions functionalized to bind respective solutes or analytes as described above, respective reflected beams 104, 106, and 108 at various reflection angles travel to illuminate the linear array 38 along three lines 110, 112, and 114. A microprocessor 40 processes the output of the photosensitive elements of the linear array 38 to generate three SPR curves. This approach offers the advantage that all three SPR curves are generated essentially simultaneously, rather than sequentially, through a single sensor prism and its sensing surface. In either case, the present invention contemplates efficient, simultaneous processing of a single liquid sample 13 (FIG. 1A), allowing for the identification of multiple solutes or analytes in the sample and the measurement of the concentrations of those solutes or analytes.

[0040] As shown in Figure 5, three SPR curves can be efficiently generated simultaneously by directing light to three predetermined, spaced apart locations or active regions or areas 24, 26, 28 on the precious metal base layer 18 of the sensor prism 12. On the upper or outer side of the base layer 18, the separated regions 24, 28 each comprise a binding agent 30, 32, specifically a respective antibody for binding to a respective antigen 116, 118. A third region 26 between regions 24 and 28 can be bare metal of the gold base layer or layer 18 for osmolality or reference measurements as described in U.S. Patent No. 5,899,231. Thus, the two spatial locations or locations 24, 28 with respective analyte binding capabilities result in the detection of two unique analytes (e.g., antigens) and the measurement of their concentrations.

[0041] Figure 6 shows the SPR measurement data measured as described above, showing two measurements of both air and tear fluid, measured at two unique wavelengths, 855 nm and 950 nm, respectively. The air position was different from the tear fluid measurement position on the gold SPR film.

[0042] After applying the noble metal substrate or film 18 to the sensing surface 16 of the sensor prism 14, typically a gold substrate or film 18, an adhesion layer, e.g., chromium, a microdot printer or plotter (e.g., GeSim's Nano-Plotter™, https: / / gesim-bioinstruments-microfluidics.com / microarray-printer / ) is used to apply the molecular agents or components 22 of the surface-assembled monolayer 20 to the noble metal substrate or film 18. Depending in part on the nature of the solute or analyte binding agents 30, 32, different molecular agents 22, and concomitantly, different SAMs, can be applied or printed at different locations or regions 24, 28. If the molecular agents or components 22 are insufficient to provide adequate surface wettability, the gold or other noble metal film 18 can be further provided with additional SAMs as needed to provide the desired degree of wettability in different active regions or areas 24, 28 of the sensing surface 16.

[0043] Typically, the manufacturing process involves first printing SAM elements 22, possibly of different types, in different active regions or areas 24, 28 of the sensing surface 16. This is followed by a second printing step in which a solute or analyte binding agent 30, 32 is applied to each active region or area 24, 28 of the sensing surface 16 and binds to the active end of each SAM element 22. In a third printing step, different SAMs 22 may be applied or printed to the sensing surface 16, particularly the substrate or film 18, to adjust the hydrophilicity / hydrophobicity of the noble metal substrate or film 18 in the active regions or areas 24, 28. Finally, if desired, the sensing surface, i.e., the substrate or film 18, may be exposed to a plasma, such as a plasma of O gas, to enhance the wettability of the substrate or film.

[0044] Typically, the molecular agents or components 22 of each surface-assembled monolayer 20 serve to attach solute or analyte binding agents 30, 32 to respective regions or areas 24, 28 of the metal substrate or film 18. To that end, fabrication involves applying solute or analyte binding agents (e.g., antibodies 30, 32) to respective predetermined active areas 24, 28 ( FIG. 5 ) on the substrate or film 18 using the same or different microdot printers or plotters. The solute or analyte binding agents can be applied in any shape or configuration, such as linear, circular, or rectangular, and optionally in shapes or configurations of various sizes. Furthermore, the solute or analyte binding agents may be applied or printed with different densities or efficiencies, for example, at different locations, whether within the same or different active areas of the substrate or film 18. Different SPR efficiencies or sensitivities facilitate the collection of more information, such as information regarding solute concentration.

[0045] The printer or plotter used to create the sensing surface 16 allows for precise deposition, both in terms of location and thickness, of the various layers on the sensing surface, including the SAM layer 20 for wettability tuning, and the ligands (e.g., 30, 32) required for functionalizing the SPR surface (substrate or film 18). Such printers or plotters utilize jetting-based bioprinting technology, which involves a non-contact technique for building 2D structures using picoliter volumes of bioink that are deposited onto a substrate. This jetting-based bioprinting method can eject picoliter-volume droplets with high spatial resolution. (Generally, the terms "micro" or "micro" used thus far refer to linear dimensions, while "pico" refers to volume. Some bioprinter manufacturers use the intermediate term "nano," ostensibly referring to the area of ​​a given dot or droplet.)

[0046] As described above, a multiplexed analyte sensing device using surface plasmon resonance (SPR) has a sensing surface 16 with a noble metal film or coating 18 that is specifically and spatially functionalized (e.g., by binding agents 30, 32) to interact with multiple different solutes or analytes in a liquid sample placed in contact with the sensing surface. As further described above, the device includes a light-sensing device 38, such as a linear array or CCD camera, and a computer or microprocessor 40 operatively coupled to the light-sensing device to receive an electrical signal or signals from the light-sensing device that encode the pattern of light absorption by the sensing surface. The computer or microprocessor 40 is programmed to analyze data from the light-sensing device 38, and by analyzing the pattern of light intensity as a function of reflection angle, the computer or microprocessor 40 ascertains the concentrations of multiple different solutes in a single, continuous or integral sample 13 in contact with the sensing surface 13 and, in particular, the substrate or film 18. To specifically functionalize the sensing surface 16, the base layer or film 18 comprises a plurality of different molecular elements or ligands 30, 32 configured to bind to respective solutes or analytes, each located at a separate, predetermined area of ​​the sensing surface. Thus, the sensing surface 16 is spatially coded and functionally divided or compartmentalized for detecting the presence of each solute or analyte and their associated concentration. The SPR device further includes at least one light source 54, 56, 76, 78, 80, 90 and an optical transmission element disposed at least partially between the light source and the sensing surface 16 and configured to direct respective beams of electromagnetic radiation 46, 48, 70, 72, 74, 96 toward a respective predetermined area or active area 24, 26, 28, 42, 44, 64, 66, 68 and toward the underside of the sensing surface 16 opposite the liquid sample 13. Beams 50, 52, 82, 84, 86, 104, 106, 108 reflected from the underside of sensing surface 16 thereby impinge on optical sensing device 38. An optical transmission element (not shown) may also be positioned partially between solute sensing surface 16 and optical sensing device 38.

[0047] As described above, the surface-assembled monolayer 20 may include alkanethiol groups 22 attached to the metal substrate or film 18. The primary function of the alkanethiol groups 22 is to attach and bind antibodies to the substrate or film 18 of the sensing surface 16. However, the alkanethiol groups 22 have an additional important function: modifying, e.g., enhancing, the wettability of the sensing surface 16. If the particular alkanethiol-type groups or other binding agents 22 used to functionalize the sensing surface 16 do not exhibit the desired degree of wettability, additional SAM groups may be provided on the sensing surface 16, particularly on the substrate or film 18. These additional SAM groups, as well as the SAMs for functionalization with the respective solute or analyte binding agents 30, 32, may be of different types and provided on different active areas 24, 28 to impart different degrees of hydrophilicity / hydrophobicity. Alternatively, or in addition, the sensing surface and metal substrate or film 18 may be exposed to a plasma, such as a plasma of O2 gas. This exposure is performed only at the end of the fabrication process, after printing of the various functional groups and agents.

[0048] At least a portion of the alkanethiol groups 22 may comprise a terminal hydrophobic head selected from the group consisting of an acidic hydrophobic head, a hydroxyl hydrophobic head, or an amino group. n -COOH or SH-(CH2) n As described above, the surface-assembled monolayer 20 provided on the metal substrate or film 18 may include preselected amounts of hydrophilic and hydrophobic elements to vary the hydrophilicity / hydrophobicity of the surface-assembled monolayer in predetermined regions.

[0049] The computer or microprocessor 40 is configured to efficiently simultaneously detect multiple analytes in the liquid sample 13. The electrical signal from the optical sensing device 38 comprises signals from different photosensitive elements or pixels in a predetermined sequence, such that the computer or microprocessor simultaneously monitors SPR optical signals from different regions or active areas 24, 26, 28, 42, 44, 64, 66, 68 of the sensing surface 16 in a substantially real-time and interleaved manner.

[0050] The liquid sample 13 may be, for example, a biological fluid, a human fluid, an animal fluid, a food, or a beverage. The solutes or analytes whose presence and / or concentration are to be detected may be cells, bacteria, insect material, vegetable material, viruses (direct or in lysates), proteins, DNA and RNA, dissolved cations or anions, endosomes, enzymes, lipids, pharmaceutical compounds, natural and artificial drugs, small molecules, or dissolved gases. Enzymes may include MMP-9; viruses may include herpes simplex virus, herpes zoster virus, and adenovirus; proteins may include lactoferrin, tryptase, and interleukins; and small molecules may include histamine, glucose, and fructose.

[0051] The noble metal substrate or coating may have a thickness of 15-100 nm, and the noble metal may be Au, Ag, Al, Pt, Rh, Cu, or Ni, or possibly a mixture or alloy thereof. Sensing surface 16 also optimally includes an adhesion layer, such as a chromium adhesion layer, having a thickness of 0.01-50 nm for attaching metal layer, film, or coating 18 to prism or body 14 of disposable sensor 12. The noble metal substrate or film 18 may have its hydrophilic or hydrophobic properties altered by plasma treatment.

[0052] In a method for measuring the concentration of at least one solute, dissolved constituent, or analyte in a fluid sample, the position of one or more SPR minima is measured and the position of the SPR minima is related to the position of the SPR minima of a known control sample or a known refractive index to determine the composition of at least one unknown sample or the concentration of at least one unknown analyte. Alternatively, or in addition, the measured values ​​of one or more SPR minima are used to correct other SPR minima in the same measurement for environmental factors, such as air humidity, temperature, and / or air pressure. Alternatively, or in addition, the method of the present invention may include using the measured values ​​of one or more SPR minima to correct other SPR minima in the same measurement for manufacturing variations or errors, including the thickness of the precious metal, the thickness of the adhesive layer, and the refractive index of the molded sensor prism.

[0053] Clinical uses of the present invention include in the ophthalmic diagnosis of conjunctivitis and dry eye. 50% of corneal specialists cannot distinguish between viral, bacterial, and allergic etiologies. The sensing device of the present invention facilitates such determinations. The present invention contemplates a disposable sensor accessory or cap 12 with three distinct biofilm zones or functionalized regions 24, 26, and 28 (FIG. 5) on a sensing substrate 18, where respective light beams 70, 72, and 74 (FIG. 3) or 98, 102, and 104 (FIG. 4) from light sources 76, 78, 80, and 90 are focused at each zone or location 82, 84, and 86, or 90, 92, and 94. A single tear sample presented to the sensing substrate 18, contacting the substrate with tears, typically in vivo, yields all three SPR signals and three different diagnoses essentially or practically simultaneously in one second.

[0054] For dry eye, the sensing substrate or film 18 preferably has pure or substantially pure gold on one half of the sensing substrate 18 and MMP-9 antibody loaded via SAM 20 on the other half, allowing for two diagnoses to be achieved in one test.

[0055] In urological applications, the sensor 12 and associated sensing devices described herein can be used to detect and quantitatively assess urinary tract infections. The sensor surface 16, or more specifically, the sensing substrate or film 18, is printed with an antibody film containing Escherichia coli, Klebsiella, and Enterococcus, which account for over 90% of the bacteria causing these infections. Additionally, urethral secretions can be simultaneously tested for gonorrhea, chlamydia, and syphilis.

[0056] In veterinary medicine, the present invention allows for the simultaneous measurement of osmolality and MMP-9 for the diagnosis of dry eye, a condition commonly seen in cats.

[0057] In the meat and vegetable industries, the present invention allows for the simultaneous detection of multiple contaminants and the quantitative determination of their presence in the sample being tested.

Claims

[Claim 1] 1. A multiplexed analyte sensing device using surface plasmon resonance, the device comprising: a sensing surface for contacting a liquid sample, the sensing surface being configured to interact with a plurality of different solutes in the liquid sample; A light sensing device; a computer or microprocessor operatively coupled to the light-sensing device for receiving from the light-sensing device a plurality of electrical signals encoding a pattern of light absorption by the sensing surface; at least one light source; Optical transmission element wherein the computer or microprocessor is programmed to analyze data from the optical sensing device by analyzing the pattern to determine the concentrations of the plurality of different solutes in a single continuous or integral sample in contact with the sensing surface; the sensing surface comprises a plurality of different molecular entities or ligands configured to bind to respective ones of the plurality of different solutes, the plurality of different molecular entities or ligands being located at respective predetermined regions of the sensing surface spaced apart from one another; the optical transmission element is at least partially disposed between the at least one light source and the sensing surface and is configured to direct each beam of electromagnetic radiation toward a respective one of the predetermined regions and an underside of the sensing surface opposite the liquid sample, the beam reflected from the underside of the sensing surface impinging on the optical sensing device.

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