Devices and kits for detecting analytes of interest and methods of use thereof

Abstract

Various embodiments of devices comprising a synthetic polymer substrate having a high quality finish upper surface having at least a double layer coating comprising a first reflective layer and a second transparent layer are disclosed. Also disclosed are embodiments of the disclosed devices and kits that can detect particles. Also disclosed are various embodiments of methods of using the disclosed devices and various embodiments of methods of using the disclosed kits.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 417,965, filed Oct. 20, 2022, which is hereby incorporated by reference in its entirety. Technical Field

[0003] The subject matter described herein relates to composite material solid supports for use in bioassays to determine the presence of one or more analytes of interest. The subject matter described herein also relates to kits comprising the composite material solid supports, and methods of using the composite material solid supports and the kits. Background Art

[0004] Bioassays are used to detect the presence and / or quantity of analyte material in a biological sample. In surface - based assays, analyte substances are captured and detected on a solid support or substrate. Examples of surface - based assays include DNA or RNA microarrays (for studies of gene expression and genotyping), and arrays having one or more binding moieties such as carbohydrates, antibodies, proteins, haptens, or aptamers.

[0005] Bioassays typically capture and immobilize a sufficient amount of analyte from a test sample to provide a detectable signal when interrogated, e.g., optically (e.g., using optical tags such as fluorophores, plasmonic nanoparticles, plasmonic substrates, etc.). For advantageous application in analytical experiments, the solid supports of bioassays typically must have highly reproducible surfaces in terms of surface finish (roughness), optical properties, and / or mechanical properties (e.g., thickness, size, position). In assay formats where samples and controls must be analyzed on different carrier surfaces (e.g., different carriers or different positions on the same carrier) associated with them, a highly reproducible substrate surface is particularly desirable. When performing assays, the absence of a highly reproducible carrier can lead to significant errors due to carrier - to - carrier variations or different positions on the same carrier.

[0006] The present disclosure provides improved solid supports for use in bioassays.

[0007] In one aspect, the present disclosure provides various embodiments of polymer - based devices having one or more deposited metal and / or dielectric layers. The disclosed embodiments represent an improvement over expensive ultra - flat silicon - based chips (e.g., ultra - flat supports made of single - crystal silicon, which require more complex manufacturing methods).

[0008] In addition, embodiments that include a deposited metal and / or dielectric layer have been found to permit the detection of individual particles in a bioassay. Devices that include two or more layers as disclosed herein achieve a substantially improved signal-to-noise ratio (SNR) for individual particles in a bioassay using an optical instrument (e.g., a dark-field optical microscope or a dark-field spectrophotometer).

[0009] In some embodiments described herein, chemical or biological coatings may be applied. Those of ordinary skill in the art will recognize how to configure a coating device to be compatible with a particular choice of chemical coating. In some embodiments, a chemical overcoat is applied to one or more deposited metal and / or dielectric layers. In some embodiments, a chemical overcoat is applied to a multi-layer coating made of metal and / or dielectric layers.

[0010] The foregoing examples of the related art and the limitations related thereto are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of ordinary skill in the art upon reading the specification and studying the drawings. SUMMARY OF THE INVENTION

[0011] The following aspects and their embodiments described and illustrated below are intended to be exemplary and illustrative, not limiting in scope.

[0012] In one aspect, there is provided a device that includes a synthetic polymer substrate having an upper surface that has a coating that includes a first layer and a second layer, the first layer including a material that reflects electromagnetic radiation and the second layer including a dielectric and transparent material. In one aspect, a super-high-quality surface roughness is provided on the upper surface in the device. In some embodiments, the surface roughness of the upper surface has a surface finish quality that is comparable to, substantially equivalent to, or equivalent to the surface finish of a silicon wafer suitable for semiconductor production.

[0013] In another aspect, there is provided a device that includes (i) a composite solid carrier component that includes a synthetic polymer substrate having an upper surface and a lower surface, the upper surface having a high-quality surface finish and including a double-layer or multi-layer coating that includes at least one reflective layer deposited on the upper surface and at least one dielectric transparent layer deposited on the reflective layer, and (ii) a plurality of binding components fixed to the composite solid carrier component (e.g., fixed to the lower surface).

[0014] In another aspect, a kit is provided for detecting a bioanalyte of interest in a test sample. The kit includes: an assay comprising a detection zone that includes a composite solid support member and a plurality of binding members immobilized to the composite solid support member, the composite solid support member including a synthetic polymer substrate having an upper surface and a lower surface, the upper surface including a bilayer or multilayer coating that includes at least one reflective layer deposited on the upper surface and at least one dielectric transparent layer deposited on the reflective layer, (ii) a container including a population of detectable plasmonic particles, and (iii) instructions for use.

[0015] In another aspect, a method is provided for detecting a bioanalyte in a fluid sample. The method includes (i) contacting the device described herein with a fluid sample suspected of containing a bioanalyte of interest and with detectable particles associated with a binding substance for the analyte of interest, wherein the device includes an immobilized member that binds to the binding substance, and (ii) analyzing the device for the presence of detectable particles using an optical instrument.

[0016] In another aspect, a method is provided for detecting a bioanalyte in a fluid sample. The method includes: contacting the device described herein with a fluid sample suspected of containing a bioanalyte of interest and with detectable particles associated with a binding substance for the analyte of interest, wherein the device includes an immobilized member that binds to the binding substance; and analyzing the device using a dark-field optical microscope or a dark-field spectrophotometer that includes (i) a beam emitter that is directed over a sample via a first optical path, (ii) an array of light detectors that are arranged on an axis orthogonal to the sample surface (i.e., the sensor surface and the sample surface are parallel) and are configured to detect light reflected via a second optical path, wherein the light detectors are not in the path of the reflected light (i.e., the second optical path) and the first optical path and the second optical path do not coincide, (iii) one or more optical objectives configured to collect the light detected by the light detectors, and (iv) a processor for the beam received by the light detectors that correlates each light detector with a spatial point on the sample such that measurements are made sequentially across various wavelengths and parallel along X-Y spatial coordinates. In some embodiments, the beam emitter is a collimated beam emitter and the first optical path has at least one lens or lens array that sequentially illuminates the sample at various wavelengths. In embodiments where the beam emitter is an LED emitter, the first optical path can employ condenser optics and collimation optics. In embodiments using a LASER system, lenses are not required. Preferably, the beam is a collimated beam.

[0017] In one aspect, the photodetectors described herein include an optical objective lens that achieves an optical resolution of at least about 3 - 4 microns (i.e., the minimum optical resolution required to identify and / or classify nanoparticles). In one aspect, the second optical path cannot coincide with the first optical path. In some embodiments, the axis of the detection module is orthogonal to the surface of the sample to be detected.

[0018] In another aspect, a method for detecting a bioanalyte in a fluid sample is provided. The method includes: contacting the device described herein with a fluid sample suspected of containing a bioanalyte of interest and with a detectable particle associated with a binding substance of the analyte of interest, wherein the device includes a stationary component that binds to the binding substance; and analyzing the device using a dark field optical microscope or a dark field spectrophotometer, which includes (i) a white light beam emitter that is directed onto the sample through a first optical path having at least one lens or lens array, thereby irradiating the sample at all wavelengths, (ii) an array of photodetectors that are arranged to detect light reflected through a second optical path that can distinguish different wavelength ranges, the second optical path being defined as the path of the light beam after reflection from the sample, and (iii) one or more optical objective lenses that are configured to focus the light detected by the photodetectors, and (iv) a processor for the image received by the photodetectors, the processor correlating each photodetector with a spatial point on the sample such that measurements are made sequentially across the various wavelengths and in parallel along the X - Y spatial coordinates. In one aspect, the first optical path and the second optical path do not coincide. In some embodiments, the light beam emitter is a collimated light beam emitter, and the first optical path has at least one lens or lens array that irradiates the sample sequentially at each wavelength. In embodiments where the light beam emitter is an LED emitter, the first optical path may employ condenser optics and collimating optics. In embodiments using a LASER system, lenses are not required. Preferably, the light beam is a collimated light beam.

[0019] In another aspect, a method for detecting a bioanalyte in a fluid sample is provided. The method includes: contacting the device described herein with a fluid sample suspected of containing a bioanalyte of interest and with detectable particles associated with a binding substance for the analyte of interest, wherein the device includes a stationary component that binds to the binding substance; and analyzing the device using a dark field optical microscope or a dark field spectrophotometer, which includes (i) a plurality of light emitters or a single light emitter configured to emit a plurality of light beams, wherein the light emitter is directed onto the sample via a first optical path, thereby irradiating the sample sequentially at specific wavelengths using a collimated beam emitter, (ii) a monochromatic light detector array arranged to detect light reflected via a second optical path, which can collect light reflected at each wavelength, the second optical path being defined as the path of the light beam after reflection from the sample, and (iv) one or more optical objectives configured to focus the light detected by the light detector, and (v) a processor for the light beams received by the light detector, the processor correlating each received image with a spatial point on the sample such that measurements are made sequentially across the various wavelengths and in parallel along the X-Y spatial coordinates. In one aspect, the first optical path and the second optical path do not coincide. In some embodiments, the light beam emitter is a collimated beam emitter, and the first optical path has at least one lens or lens array that irradiates the sample sequentially at the various wavelengths. In embodiments where the light beam emitter is an LED emitter, the first optical path can employ condenser optics and collimating optics. In embodiments using a LASER system, lenses are not required. Preferably, the light beam is a collimated beam.

[0020] In some embodiments, the optical instrument can include, for example, a complementary metal oxide semiconductor (CMOS) sensor (such as an RGB CMOS sensor).

[0021] In an embodiment, the optical instrument is a microscope spectrophotometer for dark field measurements, comprising (i) a beam emitter that is directed onto a sample through a first optical path having a lens array, thereby illuminating the sample at all wavelengths, (ii) a set of filters that can select a subset of optical wavelengths, (iii) an array of light detectors that are arranged to detect light reflected through a second optical path, which can collect the reflected light, the second optical path being defined as the path of the beam after reflection from the sample, (iv) one or more optical objectives that are configured to focus the light detected by the light detectors, and (v) a processor for the beams received by the light detectors, the processor correlating each received image with a spatial point on the sample such that measurements are made sequentially across the various wavelengths and in parallel along the X-Y spatial coordinates. In one aspect, the first optical path and the second optical path do not coincide. In some embodiments, the beam emitter is a collimated beam emitter, and the first optical path has at least one lens or lens array that sequentially illuminates the sample at the various wavelengths. In embodiments where the beam emitter is an LED emitter, the first optical path can employ condenser optics and collimation optics. In embodiments using a LASER system, no lenses are required. Preferably, the beam is a collimated beam.

[0022] In addition to the exemplary aspects and embodiments described above, additional aspects and embodiments will become apparent by reference to the drawings and by study of the following description.

[0023] Additional embodiments of the apparatus, kits, methods, etc. will be apparent from the following description, drawings, examples, and claims. As can be understood from the foregoing and the following description, each and every feature described herein and every combination of two or more of such features are included within the scope of the present disclosure, provided that the features included in such combinations are not mutually inconsistent. Additionally, any feature or combination of features can be specifically excluded from any embodiment of the present disclosure. Additional aspects and advantages of the present disclosure are set forth in the following description and claims, particularly when considered in conjunction with the appended examples and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1A Showing the optical performance of an exemplary apparatus used in an AVAC 50X analyzer, which detects plasmonic nanoparticles by measuring weak scattering signals using a dark field microspectrophotometer. The AVAC technology is described, for example, in U.S. Patent Application Publication No. 2020-0319102, U.S. Patent Application Publication No. 2020-0319085, and U.S. Patent No. 10,281,330, each of which is incorporated herein by reference.

[0025] Figure 1B Showing monomers that can be determined using AVAC analysis.

[0026] Figure 2 Displays the optical properties of another coated cyclic olefin polymer (COP) substrate device.

[0027] Figures 3A - 3B Displays the signal-to-noise ratio (SNR) achieved by devices using a first reflective layer that is 100 nm ( Figure 3A ) or 50 nm thick ( Figure 3B ). As used herein, the signal-to-noise ratio (SNR) means the ratio of the scattered signal of the nanoparticles (i.e., the signal of interest) to the scattered signal from the surrounding substrate (i.e., the signal of no interest or "noise").

[0028] Figure 4A and 4B Displays that cyclic olefin polymer (COP) disc embodiments having a first aluminum layer and a second silica layer do not show damage or degradation after being cultured in water ( Figure 4A ) or carbonate ( Figure 4B ) for 20 hours, regardless of the presence or absence of a (3-glycidoxypropyl)trimethoxysilane (GPTMS) overcoat.

[0029] Figures 5A - 5E Displays the results of degradation tests conducted on various substrates coated with aluminum, copper, or gold. Figures 5A - 5E Each substrate described in

[0030] Figure 6 is coated with a 50 nm aluminum layer and a 50 nm silicon oxide coating. Figure 6 Each substrate described in

[0031] Figures 7A - 7C is coated with a 50 nm aluminum layer, however the thickness of the silicon oxide varies.

[0032] Figure 8A and 8B Describes the roughness of uncoated COP substrates obtained using high-quality molds (steel polished molds) or ultra-high-quality molds (steel polished molds with nickel inserts).

[0033] Figures 9A - 9F Displays the surface measurement results of various embodiments of COP substrates having a combination of Si and SiO2.

[0034] Figure 10 Displays the surface measurement results obtained from a reference substrate made of silicon.

[0035] Figures 11A - 11CResults obtained using substrates coated with aluminum and substrates coated with silicon having various oxide layer thicknesses are shown.

[0036] Figures 12A - 12C Results obtained for background scatter, signal, and signal-to-noise ratio using coated aluminum and coated silicon substrates having various oxide layer thicknesses compared to reference blanks made of COP or silicon are shown. DETAILED DESCRIPTION

[0037] Aspects will now be described more fully hereinafter. However, such aspects may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope thereof to those skilled in the art.

[0038] I. Definitions

[0039] When ranges of values are provided, it is intended that every intermediate value between the upper and lower limits of such range, and any other recited value or intermediate value in such range, is encompassed within the disclosure. For example, if a range of 1 μm to 8 μm is stated, it is intended that 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, and 7 μm are also expressly disclosed, as well as ranges of values greater than or equal to 1 μm and less than or equal to 8 μm.

[0040] The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polymer" includes a single polymer as well as two or more of the same or different polymers, reference to "an excipient" includes a single excipient as well as two or more of the same or different excipients, and so forth.

[0041] The disjunctive conjunction "or" is inclusive unless otherwise stated. For example, "X or Y" means "X, Y, or both X and Y", unless otherwise stated.

[0042] The term "about", when immediately preceding a numerical value, means a range of that value plus or minus 10%, e.g., "about 50" means 45 to 55, "about 25,000" means 22,500 to 27,500, etc., unless the context of the disclosure otherwise indicates or is inconsistent with such an interpretation. For example, in a list of numerical values, e.g., "about 49, about 50, about 55", "about 50" means a range that extends to less than half the interval between the previous value and the next value, e.g., greater than 49.5 to less than 52.5. Further, the phrase "less than about" a value or "greater than about" a value should be understood in view of the definition of the term "about" provided herein.

[0043] As used herein to describe a substance or material, the term "dielectric" means that the substance or material is an electrical insulator that can be polarized by an applied electric field (i.e., when the substance / material is placed in an electric field, charges do not flow through the substance / material as they do in an electrical conductor because the substance / material does not have loosely bound or free electrons). Instead, the electrons are only slightly displaced from their average equilibrium positions, thereby causing dielectric polarization. Positive charges are displaced in the direction of the field and negative charges are displaced in the direction opposite to the field.

[0044] As used herein to describe a substrate, the term "reflective" means that the substrate can reflect electromagnetic radiation with a reflectivity of at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the provided radiation. In some non-limiting embodiments, the radiation is visible light radiation or near-IR or IR radiation provided on the surface of the reflective substrate, preferably in a specular reflection manner. The radiation can be provided (i.e., irradiated) at an angle less than 90 degrees relative to the surface of the substrate, for example and without limitation. For example, the irradiation angle can be in the range of about 1-89 degrees, or about 10-80 degrees, or about 15-75 degrees, or about 20-70 degrees, or about 25-65 degrees, or about 30-60 degrees. For example and without limitation, the provided radiation can have a wavelength in the range of 300-1000 nm, or in the range of visible light (i.e., for example, about 350 nm to about 850 nm, or about 400 nm to about 825 nm, or about 450 nm to about 800 nm).

[0045] As used herein, the term "substrate" (or "solid substrate") means an object or substance having a super-high-quality surface roughness and which can be used as a carrier or base for materials (such as the coating materials and immunoassay materials described herein) that receive a biometric on one of its surfaces. In one aspect, the substrates described herein have an upper surface that is comparable in roughness to a silicon wafer used in semiconductor applications when measured using the same technique. In embodiments, a conventional atomic force microscope probe is used to measure the surface roughness, and in embodiments, the measured roughness is less than about 2 nm or less than about 1 nm. Generally, the substrate is a solid object and is not magnetic. The substrate can have any shape depending on the desired application, for example, the substrate can be provided in the form of a planar substrate, although the substrate can have any available shape or configuration.

[0046] As used herein, the term "transparent" means that the surface is non-reflective. A substrate surface is "non-reflective" if, for example, the reflectivity of the surface is less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the provided electromagnetic radiation, such as provided visible light.

[0047] The reflectance at, for example, wavelengths from 400 nm to 1000 nm means that the composite substrate has a reflectance greater than a specified amount at all wavelengths between 400 nm and 1000 nm. The reflectance of the reflective substrate can be measured using, for example, a reflectometer equipped with a multi-wavelength light source and a spectrometer at the angles described above.

[0048] With respect to a substrate or carrier, as used herein, the terms “immobilizing” or “immobilized” include covalent conjugation, non-specific association, ionic interactions, and other methods of attaching a substance (e.g., a polymer, copolymer, binding moiety) to a substrate or carrier (i.e., the surface of the substrate or carrier).

[0049] As used herein, the term “antibody” means a polypeptide ligand substantially encoded by one or more immunoglobulin genes or fragments thereof that specifically binds and recognizes an epitope (e.g., an antigen). The recognized immunoglobulin genes include the κ and λ light chain constant region genes, the α, γ, δ, ε, and μ heavy chain constant region genes, and numerous immunoglobulin variable region genes. Antibodies exist, for example, as intact immunoglobulins or as many well-characterized fragments initially produced by digestion with various peptidases. This includes, for example, Fab' and F(ab)'2 fragments. The term “antibody” as used herein also includes antibody fragments produced by modifying whole antibodies or those synthesized de novo using recombinant DNA methods. It also includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single-chain antibodies. The “Fc” portion of an antibody refers to the portion of the immunoglobulin heavy chain that contains one or more heavy chain constant domain structures, CH1, CH2, and CH3, but does not include the heavy chain variable region.

[0050] As used herein, the term “detectable response” refers to a change or appearance of a signal that can be directly or indirectly detected by observation or by an instrument, and whose presence or magnitude varies with the presence of a target analyte of interest in a test sample. Typically, the response is a plasmon-detectable response. Typically, the detectable response is an optical response from particles such as metal particles or fluorophores (e.g., plasmonic particles, such as plasmonic nanoparticles), due to a change in their position in an array or resulting in a change in the wavelength distribution pattern or reflection intensity, absorbance, or fluorescence, or a change in light scattering, fluorescence quantum yield, fluorescence lifetime, fluorescence polarization, excitation or emission wavelength shift, or a combination of the above parameters. A detectable change in a given spectral property is typically an increase or decrease and can also be a shift in a spectral measurement.

[0051] As used herein, the term "metalloid" means a chemical element recognized by one of ordinary skill in the art as having properties that are between those of metals and nonmetals or a mixture of the properties of metals and nonmetals, including alloys containing at least one such element and / or compounds containing at least one such element. In an embodiment, the metalloid employed is selenium, boron, silicon, germanium, arsenic, antimony, tellurium, and / or polonium, or one or more compounds or alloys thereof. In other embodiments, the metalloid is boron, silicon, germanium, arsenic, antimony, and / or polonium, or one or more oxides thereof.

[0052] The devices, kits, and methods of the present disclosure may comprise the disclosed components or steps, consist essentially of the disclosed components or steps, or consist of the disclosed components or steps.

[0053] All ranges disclosed herein include all subranges contained therein, as well as all specific values contained therein. Additionally, all ranges disclosed herein include their endpoints, unless otherwise indicated. For example, "X to Y" means "greater than or equal to X and less than or equal to Y", unless otherwise indicated.

[0054] When used to describe the amounts of components of a composition, all percentages, parts, and ratios are based on the total weight of the composition, unless otherwise indicated.

[0055] All measurements are made at about 25 °C, unless otherwise specified. Additionally, all measurements are made at a pressure of about 1 atm, unless otherwise specified.

[0056] By reserving the right to exclude any single member of any such group, including any subrange or combination of subranges within the group, which may be claimed according to a range or in any similar manner, less than the full measure of the present disclosure may be claimed for any reason. Additionally, by reserving the right to exclude any single substituent, analogue, compound, ligand, structure, or group thereof, or any member of a claimed group, less than the full measure of the present disclosure may be claimed for any reason.

[0057] Throughout the present disclosure, various patents, patent applications, and publications are referenced. The disclosures of these patents, patent applications, and publications are hereby incorporated by reference in their entirety into the present disclosure to more fully describe the prior art known to one of ordinary skill in the art as of the date of the present disclosure. If there is any inconsistency between the patents, patent applications, and publications cited and the present disclosure, the present disclosure shall control.

[0058] For convenience, some terms employed in the specification, examples, and claims are collected herein. Unless otherwise defined, all technical and scientific terms used in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains.

[0059] II. Composite Substrate and Device

[0060] The present disclosure provides solid supports, devices, and methods for use in bioanalytical operations. Embodiments of the supports and devices are used in assays for capturing biomolecules or analytes in test samples, including assays for nucleic acid hybridization, protein interactions, antibody binding, and other analytical assays. The solid supports, devices, and methods provide rapid, sensitive, reliable, and / or optionally multiplexed detection of biomolecules and other compounds present in biological samples. The devices and methods are intended for use, for example, in research, clinical laboratories, medical clinics, hospital outpatient departments, nursing homes, outpatient clinics, emergency rooms, point-of-care settings (e.g., doctor's offices, emergency rooms, field, etc.), and high-throughput testing applications.

[0061] The devices, methods, and kits described herein include or use a solid substrate or support (e.g., a composite solid support component) having a base layer and a coating on the base layer, wherein the coating comprises at least one layer.

[0062] In some embodiments, the devices described herein can include an inlet and / or an outlet and / or one or more chambers (e.g., mixing chambers, waste pools, etc.). In some embodiments, one or more channels can be provided to connect the ports and / or chambers provided in the device.

[0063] A. Substrate

[0064] The substrate can comprise a polymer or copolymer, consist of a polymer or copolymer, or consist essentially of a polymer or copolymer. For example, the substrate comprises a thermoplastic material. In embodiments, the substrate can comprise one or more materials selected from styrene / methyl methacrylate (SMMA) copolymers, polymethyl methacrylates (PMMAs), olefins, polyesters, polystyrenes, polyethylenes, polyamides, acrylonitrile butadiene styrene, and polyacetals. In embodiments, the substrate comprises a cyclic olefin copolymer and / or a cyclic olefin polymer (COP).

[0065] In some embodiments, the substrate is a disk, e.g., a COP disk. In other embodiments, the substrate is a glass slide. In other embodiments, the substrate can take other shapes as needed to interface with fluidics or other means of introducing a sample thereto. It can be part of a larger device and can be enclosed or attached thereto.

[0066] In other embodiments, the substrate can be silicon, or a combination of silicon and a thermoplastic material.

[0067] The substrate can have a thickness in the range of about 100 microns to about 1.2 mm, or about 300 microns to about 700 microns, or about 700 microns to about 1 mm.

[0068] In some preferred embodiments, the substrate has a thickness of less than about 1.0 mm, less than about 0.9 mm, or less than about 0.8 mm, or less than about 0.7 mm, or less than about 0.6 mm, or less than about 0.5 mm, or less than about 0.4 mm, or less than about 0.3 mm, or less than about 0.2 mm, or less than about 0.1 mm.

[0069] The substrate has a smooth or substantially smooth upper surface, exhibiting very low roughness, i.e., a very high-quality surface finish (e.g., substantially equivalent to the roughness of a silicon wafer).

[0070] Additionally, the overall shape of the substrate is preferably planar or substantially planar.

[0071] Preferably, in an embodiment, the substrate is rigid or substantially rigid.

[0072] In an embodiment, the substrate (i.e., on one or both of the upper and / or lower surfaces of the substrate) has a surface roughness equivalent to or substantially equivalent to that of a silicon wafer suitable for use in semiconductor production or as a semiconductor.

[0073] In some embodiments, the surface finish (roughness) of the substrate before applying the coating is equivalent to or substantially equivalent to the surface finish (roughness) of a silicon wafer.

[0074] In other embodiments, the surface finish (roughness) of the substrate after applying the coating is equivalent to or substantially equivalent to the surface finish of a silicon wafer.

[0075] In still other embodiments, the surface finish (roughness) of the substrate before and after applying the coating is equivalent to or substantially equivalent to the surface finish of a silicon wafer. That is, in such embodiments, applying a coating on the substrate does not affect the overall surface finish of the substrate / coating composite relative to the uncoated substrate. In these embodiments, the technique for measuring the surface roughness of the substrate is the same as the technique for measuring the surface roughness of a silicon wafer.

[0076] It has been found that when transmitted light passes through the substrate and strikes the surface below the substrate, maintaining a smooth or substantially smooth substrate surface (or coated substrate surface) minimizes the amount (i.e., intensity) of reflection generated by the imperfect (rough) top surface and the number of reflections generated by the imperfect bottom surface. The surface below the substrate may have a surface finish or roughness that is difficult to control, thus highlighting the need to maintain a smooth substrate surface to compensate for other reflections.

[0077] An ideal substrate surface with a substantially perfect surface finish (i.e., roughness) will maximize the contrast between the background and detected particles when analyzed via, for example, dark field microscopy or another analytical method. On the other hand, a rough substrate will produce a diffuse or noisy background, thereby reducing the contrast between the background and the particles. This effect is particularly relevant in dark field imaging such as dark field microscopy because the scattered light of interest (e.g., from nanoparticles) constitutes a very small fraction of the illumination. Thus, any additional source of light scattering will very likely be in the same range as the signal.

[0078] One of ordinary skill in the art will recognize that various methods can be used to measure surface finish (i.e., roughness). It is understood that the parameter Ra is a general and the most widely used roughness parameter internationally. Ra is the arithmetic mean of the deviation of the roughness curve of the surface from the mean line (i.e., the reference line representing the entire surface). Thus, a surface roughness of 10 nm for a hypothesized substrate means that such a substrate deviates from the mean (average) of 10 nm of its entire surface, as measured across the entire surface of the substrate. In one aspect, the substrates described herein have an upper surface having a surface roughness of about 0 - 100 nm, or about 0 - 75 nm, or about 0 - 50 nm, or about 0 - 25 nm, or about 0 - 10 nm. In some embodiments, the upper surface has a roughness comparable to, equivalent to, or substantially equivalent to that of a silicon wafer suitable for semiconductor production.

[0079] Additionally, the substrate is planar or substantially planar. As used herein, "flatness" is a measure or indication of the curvature or deviation of a surface from a plane (where a zero value indicates perfect flatness). For example, the surface of the substrate can have a flatness in the range of about 0 μm - about 100 μm, or about 0 μm - about 90 μm, or about 0 μm - about 80 μm, or about 0 μm - about 70 μm. For example, the flatness of the composite materials described herein can be about 0 μm, or about 5 μm, or about 10 μm, or about 15 μm, or about 20 μm, or about 25 μm, or about 30 μm, or about 35 μm, or about 40 μm, or about 45 μm, or about 50 μm, or about 55 μm, or about 60 μm, or about 65 μm, or about 70 μm, or about 75 μm, or about 80 μm, or about 85 μm, or about 90 μm, or about 95 μm, or about 100 μm. Preferably, the flatness is minimized, i.e., less than 100 μm and more preferably approximately 0 μm.

[0080] In the case of an aluminum-coated substrate (e.g., an aluminum-coated COP substrate), the flatness can be from about 0 μm to about 90 μm, or about 05 μm to about 85 μm, or about 0 μm to about 80 μm, or from about 0 μm to about 75 μm.

[0081] For example, one or both of the upper and / or lower substrate surfaces can be produced using a polished steel die or alternative techniques such as by adding nickel inserts, which cover different or varying levels of roughness and are suitable for different surface qualities (e.g., no scratching associated with polishing).

[0082] In some embodiments, the substrate comprises a ferromagnetic metal, which allows for remote surface magnetization in the substrate. In some embodiments, the ferromagnetic metal is nickel or cobalt, or an alloy comprising nickel and / or cobalt. For example, nickel vanadium can be used as the ferromagnetic metal or ferromagnetic additive.

[0083] In one aspect, the ferromagnetic metal or alloy can be provided in any layer on the coated substrate. In some embodiments, the substrate is directly coated with the ferromagnetic metal or alloy. The thickness of the ferromagnetic metal or alloy layer can be, for example, 100 nm - 200 nm thick. Subsequent layers (e.g., a reflective or substantially reflective layer, an overcoat, or other layers described herein) can be provided on the ferromagnetic metal or alloy layer.

[0084] For example and without limitation, the devices described herein can comprise a ferromagnetic layer about 100 - 200 nm thick, a reflective layer having the thickness described herein, and an optional silica layer having the thickness described herein. Aluminum or another metal can be used to implement the reflective layer, or a stacked dielectric material having alternating high and low refractive indices can be used to implement the reflective layer.

[0085] B. Coating

[0086] The coating comprises at least one layer (i.e., the first layer) that reflects or is substantially reflective of electromagnetic radiation. In an embodiment, the first layer of the coating reflects or is substantially reflective of visible light.

[0087] In an embodiment, the coating comprises at least one layer (i.e., the first layer) that comprises one or more metals or metalloids selected from aluminum, silver, gold, chromium, nickel, cobalt, and silicon, and alloys and compounds containing one or more of aluminum, silver, gold, chromium, nickel, cobalt, or silicon. In other embodiments, the first layer can comprise a stacked dielectric material having alternating high and low refractive indices. In other embodiments, the first layer comprises a metal and / or metalloid, and one or more dielectric materials.

[0088] In an embodiment, the first layer of the coating consists essentially of a metal or metalloid selected from aluminum, silver, gold, chromium, nickel, cobalt, and silicon. In other embodiments, the first layer of the coating consists of a metal or metalloid selected from aluminum, silver, gold, chromium, and silicon.

[0089] In an embodiment, the coating comprises two or more layers. In some preferred embodiments, the coating is a bilayer, i.e., a coating comprising a first reflective layer and a second transparent layer as described above or consisting of a first reflective layer and a second transparent layer as described above.

[0090] In an embodiment, the second layer of the bilayer coating is dielectric and transparent.

[0091] Preferably, the second layer of the bilayer coating can be functionalized (e.g., functionalized to be a detectable particle). More preferably, the assay is a "sandwich" type assay, where the second layer is functionalized as a capture moiety, and the detectable particle is part of the detection moiety.

[0092] The second layer of the bilayer coating can comprise a material selected from: a metalloid, an alloy and a compound containing one or more metalloids, and a polymer. In an embodiment, the polymer used as the second layer is a synthetic polymer.

[0093] When the second layer is a metalloid, an alloy of a metalloid, or a compound of a metalloid, the metalloid is selected from selenium, boron, silicon, germanium, arsenic, antimony, tellurium, and polonium.

[0094] Preferably, the metalloid is one or more of boron, silicon, germanium, arsenic, antimony, and / or polonium. When the second layer is a compound containing one or more metalloids, the compound is preferably an oxide of the metalloid. For example, in some preferred embodiments, the second layer is an oxide of silicon, preferably silica (which can be applied via any suitable application method, including but not limited to chemical vapor deposition (CVD) or remote combustion chemical vapor deposition (r-CCVD)).

[0095] In an embodiment comprising a bilayer coating, the first layer (i.e., the reflective layer) can have a thickness in the range of about 10 nm - about 1,000 nm, or in the range of about 10 nm - about 500 nm, or in the range of about 10 nm - about 250 nm, or in the range of about 10 nm - about 200 nm, or in the range of about 50 nm - about 150 nm, or in the range of about 75 nm - about 125 nm.

[0096] For example, the first layer (i.e., the reflective layer) can have a thickness in the range of about 1 nm - about 250 nm, or in the range of about 25 nm - about 250 nm.

[0097] In embodiments having a double-layer coating, the second layer (i.e., the transparent layer or the dielectric transparent layer) can have a thickness in the range of from about 10 nm to about 500 nm, or in the range of from about 20 nm to about 200 nm, or in the range of from about 20 nm to about 100 nm, or in the range of from about 50 nm to about 100 nm, or in the range of from about 75 nm to about 100 nm, or in the range of from about 70 nm to about 90 nm.

[0098] For example, the second layer (i.e., the transparent layer or the dielectric transparent layer) can have a thickness in the range of from about 1 nm to about 250 nm, or in the range of from about 25 nm to about 250 nm.

[0099] In some preferred embodiments having a double-layer coating, the first layer and the second layer have thicknesses within about 30%, within about 25%, within about 20%, within about 15%, or within about 10% of each other.

[0100] In some preferred embodiments, the first layer and the second layer are of the same thickness or approximately the same thickness (e.g., within about 5% of each other).

[0101] In other preferred embodiments, the thickness of the first layer is greater than the thickness of the second layer.

[0102] The device can optionally contain an additional layer applied to the second layer after the second layer is applied to the first layer. Preferably, an epoxy silane such as (3-glycidoxypropyl)trimethoxysilane (“GPTMS”) or a chlorosilane such as 3-chloropropyltriethoxysilane, or 3-aminopropyltrimethoxysilane (APTMS), or 3-aminopropyltriethoxysilane (APTES) is used to apply the additional layer (or “overcoating” or “overcoat layer”).

[0103] In embodiments of the composite solid support, the reflective layer (i.e., the first layer) comprises aluminum or consists essentially of aluminum and the transparent dielectric layer (i.e., the second layer) comprises silica or consists essentially of silica.

[0104] In other embodiments of the composite solid support, the reflective layer (i.e., the first layer) consists of aluminum and the transparent dielectric layer (i.e., the second layer) consists of silica.

[0105] In embodiments of the composite solid support, the coating comprises silica and APTMS. In embodiments of the composite solid support, the coating consists essentially of silica and APTMS.

[0106] In embodiments of the device described herein, the substrate is silicon, single-crystalline silicon, or a silicon wafer.

[0107] In some embodiments, the coating is a 20 nm silicon dioxide layer on a silicon wafer. In other embodiments, the coating is silicon dioxide having a thickness greater than 20 nm. In still other embodiments,

[0108] In one aspect, a device that replaces a silicon-based ultra-flat chip is achieved by manufacturing with a high-quality mold (i.e., a mold including a nickel insert) and depositing one or more layers on a polymeric substrate. The devices described herein thus represent an improvement over more expensive ultra-flat silicon chips that require more complex manufacturing methods.

[0109] In an embodiment, the devices described herein may be configured to include an inlet and / or an outlet.

[0110] In one aspect, a device is provided that includes a synthetic polymeric substrate having an upper surface with a coating thereon, the coating including a first layer and a second layer, the first layer including a material that reflects electromagnetic radiation and the second layer including a dielectric and transparent material.

[0111] In another aspect, the device includes a substrate having an upper surface on which a coating is provided. In an embodiment, the coating has at least two layers, the first layer being provided directly on the upper surface of the substrate and the second layer being provided on the first layer provided. The first layer is reflective and the second layer is transparent such that light (e.g., visible light) can pass through the second layer and be reflected back by the first layer through the second layer.

[0112] In another aspect, the device includes a substrate having an upper surface on which a coating is provided, the coating having a first layer and a second layer as described above, and an overcoat (overlayer) provided on the second layer after the second layer is provided on the first layer (which is itself provided on the substrate). In some preferred embodiments, the overcoat is APTMS or GPTMS.

[0113] As described herein, the device may further include a plurality of binding components fixed on a composite carrier member (e.g., on the second layer or on the overcoat if present).

[0114] C. Binding Component

[0115] In addition, the described device may include one or more binding components (e.g., multiple binding components) that are fixed to a composite solid support component. The composite support component includes a substrate and one or more of the layers described herein. In a preferred embodiment, the composite support component includes a substrate having a high-quality surface finish as described herein, a first reflective layer, and a second transparent layer, where the second transparent layer is optionally dielectric. In another preferred embodiment, the composite support component includes a substrate, a first reflective layer, and a second transparent layer (where the second transparent layer is optionally dielectric), and an optional overcoat (e.g., APTMS or GPTMS).

[0116] The binding components can have the same or different properties. For example, in the described device, the multiple binding components provided on the composite solid support component can consist of binding components for a single analyte or a single class of analytes. In another embodiment, the multiple binding components provided on the composite solid support component can include components that bind a first analyte and components that bind a second analyte, and optionally components that bind a third, fourth, fifth analyte or a class of analytes or more than five different analytes or multiple classes of analytes.

[0117] In a preferred embodiment, the multiple binding components include a first binding component for a first analyte and a second binding component for a second analyte.

[0118] The multiple binding components can include proteins, antibodies, or peptides.

[0119] In some non-limiting embodiments, the protein can be streptavidin.

[0120] In some non-limiting embodiments, the antibody is an anti-IL6 antibody.

[0121] The binding components provided on the composite solid support component can optionally include a binding tag. In an embodiment, the binding tag is haloalkane dehalogenase or avidin.

[0122] The multiple binding components include a ligand that specifically binds to the binding tag, and the ligand is part of a fusion protein that includes an antibody or antibody fragment that binds an analyte of interest.

[0123] In some non-limiting embodiments, in an embodiment, the binding tag can be haloalkane dehalogenase or avidin.

[0124] In some non-limiting embodiments, the ligand can be a synthetic organic compound, such as a chloroalkane linker or a crosslinker.

[0125] For example, HaloTag TM can be used to provide the binding tag.

[0126] In some non-limiting embodiments, the binding tag can be an enzyme-modified protein or peptide for attaching a single protein or peptide. For example, an enzyme such as but not limited to biotin ligase can be used to effect biotinylation of the desired protein or peptide to effect enzyme modification. For example, AviTag can be used TM to provide the binding tag.

[0127] For example, in an embodiment, there is provided a device comprising: (a) a composite solid support member comprising a synthetic polymer substrate having an upper surface and a lower surface, the upper surface having a high-quality surface finish, the upper surface comprising a bilayer coating, the bilayer coating comprising (i) a reflective layer deposited on the upper surface and (ii) a dielectric transparent layer deposited on the reflective layer; and (b) a plurality of binding members fixed to the composite solid support member.

[0128] In other embodiments, the device comprises: (a) a composite solid support member comprising a synthetic polymer substrate having an upper surface and a lower surface, the upper surface comprising a coating, the coating comprising (i) a reflective layer deposited on the upper surface and (ii) a dielectric transparent layer deposited on the reflective layer, and (iii) an overcoat (overlayer) optionally comprising GPTMS or APTMS or consisting essentially of GPTMS or APTMS; and (b) a plurality of binding members fixed to the composite solid support member via the overcoat.

[0129] In other embodiments, the device comprises: (a) a composite solid support member comprising a synthetic polymer substrate having an upper surface and a lower surface, the upper surface comprising a coating, the coating comprising (i) a reflective layer deposited on the upper surface containing a ferromagnetic material such as Ni or Co and (ii) a dielectric transparent layer deposited on the reflective layer, and (iii) an overcoat (overlayer) optionally comprising GPTMS or APTMS or consisting essentially of GPTMS or APTMS, and (b) a plurality of binding members fixed to the composite solid support member via the overcoat. The ferromagnetic material can be used to induce a surface-mediated magnetic field (i.e., remote surface magnetization) on the surface to attract magnetic particles, etc., to accelerate binding to the surface.

[0130] III. Kit

[0131] In another aspect, there is provided a kit for detecting a bioanalyte of interest in a test sample.

[0132] In an embodiment, the kit includes an analyte having a detection zone, the detection zone comprising a composite solid support member as described herein.

[0133] The composite solid support member of the detection zone can comprise, for example, a synthetic polymer substrate.

[0134] In addition, the composite solid support component may have an upper surface and a lower surface. In an embodiment, the upper surface of the composite solid support component comprises a bilayer coating, the bilayer coating comprising (i) a first reflective layer deposited on the upper surface and (ii) a second layer deposited on the reflective layer.

[0135] In an embodiment of the kits described herein, the composite solid support has a plurality of binding components immobilized thereto. The plurality of immobilized binding components are capable of binding an analyte of interest or a ligand that is specific for a binding tag, the ligand being part of a fusion protein comprising an antibody or antibody fragment that binds the analyte of interest. For example, the plurality of immobilized binding components are one or more of an antibody, an antibody fragment, or a synthetic organic compound.

[0136] In some embodiments, the ligand that is specific for the binding tag is biotin.

[0137] In an embodiment, the plurality of immobilized binding components can be a synthetic organic compound comprising a chloroalkane linker of appropriate size, and the binding tag can be a haloalkane dehalogenase.

[0138] The kits described herein also include a container comprising a population of detectable particles.

[0139] Specifically, the kit comprises at least one additional binding component that is capable of associating with the detectable particles and has specific binding for the analyte of interest. In an embodiment, the at least one additional binding component is an antibody or an antibody fragment.

[0140] In an embodiment, the binding component is an antibody conjugated to the detectable particles via a thiol (-SH) group.

[0141] In an embodiment, the antibody or antibody fragment has specific binding for a cardiac biomarker, an inflammatory biomarker (e.g., interleukin ILx, such as IL-6), a nerve cell biomarker (such as Tau and its isoforms, or other targets for Alzheimer's disease and / or Parkinson's disease), a biomarker associated with one or more infectious diseases (e.g., LAM, p24, chemokine panel, IFN panel, etc.).

[0142] For example, the cardiac biomarker is troponin, such as troponin C (TNNC1 or TNNC2), troponin I (cTnI) or troponin T (cTnT), or high-sensitivity (hs) cTnI. Alternatively, the cardiac biomarker can be B-type natriuretic peptide (BNP) or pro-BNP, or a diagnostic panel.

[0143] The detectable particles comprise a metal or consist essentially of a metal, preferably a transition metal or a noble metal.

[0144] In embodiments, the detectable particles comprise one or more metals or consist essentially of one or more metals selected from gold, silver, platinum, palladium, iridium, osmium, rhodium, ruthenium, and alloys thereof. In some embodiments, the detectable particles are nanoparticles having at least a plasmonic material (e.g., gold, aluminum, silver, or metamaterial) embedded therein.

[0145] In embodiments, the detectable particles consist of a metal selected from gold, silver, platinum, palladium, iridium, osmium, rhodium, and ruthenium.

[0146] In embodiments, the detectable particles comprise gold or consist essentially of gold. In embodiments, the detectable particles consist of gold.

[0147] The detectable particles have an average diameter in the range of about 1 nm - about 1500 nm, or about 25 nm - about 500 nm, or about 50 nm - about 250 nm, or 100 nm - 200 nm.

[0148] In embodiments, the detectable particles resonate at a wavelength in the range of about 250 nm - about 1000 nm, or about 300 nm - about 950 nm, or about 350 nm - about 900 nm, or about 400 nm - about 850 nm, or about 450 nm - about 800 nm.

[0149] In embodiments, the detectable particles have a core - shell structure, wherein the core is magnetic and the shell is a transition metal. In embodiments, the core is iron, an iron oxide, or an iron alloy. In a preferred embodiment, the core is iron or iron (II,III) oxide (i.e., Fe3O4). The shell is preferably gold.

[0150] In embodiments, the diameter of the magnetic core (i.e., the average magnetic core diameter of a plurality of detectable particles) can be in the range of about 1 nm - about 300 nm, or about 25 nm - about 250 nm, or about 50 nm - about 200 nm, or about 75 nm - about 150 nm and the thickness of the shell can be in the range of about 0.5 nm - about 50 nm, or about 1 nm - about 40 nm, or about 5 nm - about 30 nm, or about 10 nm - about 25 nm.

[0151] In other embodiments, the diameter of the magnetic core can be in the range of about 0.5 nm - about 60 nm, or about 1 nm - about 40 nm, or about 3 nm - about 30 nm, or about 5 nm - about 25 nm. The shell can have a thickness in the range of about 1 nm - about 100 nm, or about 5 nm - about 80 nm, or about 5 nm - about 60 nm, or about 10 nm - about 45 nm.

[0152] Optionally, an intermediate layer may be provided between the core and the shell of the detectable particle (i.e., the intermediate layer may serve as the first shell between the core and the outer shell). The intermediate layer may comprise silica. The diameter of the magnetic core (e.g., the average magnetic core diameter of a plurality of detectable particles) may range from about 1 nm to about 300 nm, or from about 25 nm to about 250 nm, or from about 50 nm to about 200 nm, or from about 75 nm to about 150 nm. The thickness of the intermediate layer may range from about 0.5 nm to about 50 nm, or from about 1 nm to about 40 nm, or from about 5 nm to about 30 nm, or from about 10 nm to about 25 nm. The thickness of the shell may range from about 0.5 nm to about 50 nm, or from about 1 nm to about 40 nm, or from about 5 nm to about 30 nm, or from about 10 nm to about 25 nm. The detectable particle may have a diameter (i.e., an average diameter) in the range of about 25 nm to about 500 nm, or from about 50 nm to about 450 nm, or from about 75 nm to about 350 nm, or from about 100 nm to about 300 nm.

[0153] In other embodiments, the detectable particle does not have a core - shell structure. That is, in such embodiments, the detectable particle consists essentially of a transition metal or their alloy. For example, the detectable particle may consist essentially of gold.

[0154] In an embodiment, the kit may include instructions for use.

[0155] IV. Analyte Detection Method

[0156] Embodiments of methods for detecting a bioanalyte in a fluid sample are also described herein. The method includes contacting the device described herein with (i) a fluid sample suspected of containing the bioanalyte of interest and (ii) a detectable particle associated with a binding substance for the analyte of interest.

[0157] The device to be contacted contains a fixed component that binds to the binding substance. After contacting the fluid sample and the detectable particle associated with the binding substance, the device is analyzed with an optical instrument for the presence of the detectable particle.

[0158] In an embodiment, the optical instrument is a dark - field spectrophotometer or a dark - field optical microscope having a plurality of light detectors. In one aspect, any spectrophotometer may be used. In some embodiments, the light detector is a monochromatic light detector. In other embodiments, the light detector is an RGB light detector.

[0159] In an embodiment, a microscope or a spectrophotometer is capable of performing simultaneous analysis at different points on a single sample (i.e., using a sample prepared singly on a substrate as described herein), where the analysis can be performed with high spatial resolution and without the need for a mechanical system for physically scanning the sample to be analyzed. This can be achieved, for example, by using a dark-field optical microscope or a dark-field spectrophotometer, which has means for processing the light received by two or more (i.e., multiple) light detectors and one or more optical objectives configured to collect the light detected by the light detectors, where the processing means has a correlation for each light detector and the optical objective corresponding to the same different spatial points.

[0160] In one aspect, the optical objective described herein has a resolution of about 4 nm or less.

[0161] The optical microscope or the dark-field spectrophotometer can be used for both bright-field and dark-field applications, for both reflection measurements and transmission measurements, provided that optical components suitable for each technique are used. In a preferred embodiment, the optical instrument is a spectrophotometer for dark-field measurements.

[0162] The dark-field optical microscope or the dark-field spectrophotometer can have a light source with a broad spectral band (e.g., but not limited to an LED bulb emitting white light) and a wavelength selector (e.g., but not limited to one or more monochromators, filters, prisms, etc.).

[0163] Alternatively, the spectrophotometer can have multiple light sources, each having a different wavelength. By way of example and without limitation, the multiple light sources can be multiple LEDs or multiple lasers, or a combination of one or more LEDs and one or more lasers. In such an embodiment, a wavelength selector will not be needed because they will only need to have means for selecting the LED for irradiating the sample such that wavelength scanning can be performed by changing from one LED to another.

[0164] In an embodiment, the beam source of the spectrophotometer includes a monochromator to selectively control the wavelength sent to the sample such that a beam of a certain wavelength is emitted. Thus, simultaneous analysis at different points on the same sample at the same wavelength can be performed. Thereafter, another wavelength can be selected using the monochromator such that the sample is sequentially irradiated with several wavelengths.

[0165] In an embodiment, the optical instrument is a spectrophotometer for dark-field measurement, which includes (i) a beam emitter that is guided onto a sample through a first optical path having a lens array, thereby irradiating the sample sequentially at various wavelengths, (ii) an array of light detectors that are arranged to detect light reflected through a second optical path, which is defined as the path of the beam after reflection from the sample, (iii) one or more optical objectives that are configured to collect the light detected by the light detectors, and (iv) a processor for the beam received by the light detectors, which correlates each light detector with a spatial point on the sample such that measurements are made sequentially across the various wavelengths and in parallel along the X-Y spatial coordinates.

[0166] In an embodiment, the optical instrument is a spectrophotometer for dark-field measurement, which includes (i) a white beam emitter that is guided onto a sample through a first optical path having a lens array, thereby irradiating the sample at all wavelengths, (ii) an array of light detectors that are arranged to detect light reflected through a second optical path, which can distinguish different wavelength ranges, which is defined as the path of the beam after reflection from the sample, (iii) one or more optical objectives that are configured to collect the light detected by the light detectors, and (iv) a processor for the image received by the light detectors, which correlates each light detector with a spatial point on the sample such that measurements are made sequentially across the various wavelengths and in parallel along the X-Y spatial coordinates.

[0167] In an embodiment, the array of light detectors can distinguish different wavelengths or wavelength ranges, such as the red, green, and blue portions of the visible spectrum.

[0168] In some embodiments, the optical instrument can include, for example, a complementary metal oxide semiconductor (CMOS) sensor such as an RGB CMOS sensor.

[0169] In an embodiment, the optical instrument is a spectrophotometer for dark-field measurement, which includes (i) a beam emitter that is guided onto a sample through a first optical path having a lens array, thereby irradiating the sample at all wavelengths, (ii) a set of filters that can select a subset of light frequencies, (iii) an array of light detectors that are arranged to detect light reflected through a second optical path, which can collect the reflected light, which is defined as the path of the beam after reflection from the sample, (iv) one or more optical objectives that are configured to collect the light detected by the light detectors, and (v) a processor for the beam received by the light detectors, which correlates each received image with a spatial point on the sample such that measurements are made sequentially across the various wavelengths and in parallel along the X-Y spatial coordinates.

[0170] In an embodiment, the filter may select a subset of optical frequencies in, for example, the red, green, or blue (RGB) portion of the visible spectrum.

[0171] In another aspect, a method for detecting a bioanalyte in a fluid sample is provided. The method includes: contacting the device described herein with a fluid sample suspected of containing the bioanalyte of interest and with detectable particles associated with a binding substance for the analyte of interest, wherein the device includes a stationary component that binds to the binding substance; and analyzing the device using a spectrophotometer that includes (i) a plurality of light emitters that are directed onto the sample via a first optical path, thereby sequentially irradiating the sample at specific wavelengths, (ii) a monochromatic light detector array that is arranged on a second optical path that can collect the light reflected at each wavelength, the second optical path being defined as the path of the light beam after reflection on the sample, (iii) one or more optical objectives that are configured to focus the light detected by the light detector, and (iv) a processor for the light beam received by the light detector, the processor correlating each received image with a spatial point on the sample such that measurements are made sequentially across the various wavelengths and in parallel along the X-Y spatial coordinates.

[0172] The spectrophotometer may be configured to measure cross-polarization (provided that suitable polarizers are coupled along the light beam impinging on the sample and along the light beam path directed towards the light detector array). For example, in some non-limiting embodiments, the light detector array is a CCD camera, where a series of its pixels includes light detectors. The series may be one pixel or an array of pixels.

[0173] In some embodiments, the light beam source includes a monochromator such that a light beam of a certain wavelength is emitted. In this way, parallel analysis at the same wavelength at different points on the same sample is performed.

[0174] Among different beam sources with a broad spectral band that can be used to perform spectrophotometric analysis, the beam source may be a visible light, ultraviolet, and / or infrared light source.

[0175] The spectrophotometer used in the method described herein preferably operates with high sensitivity.

[0176] In some embodiments, the detection method described herein may be configured to detect particles in the femtogram range or smaller.

[0177] In an embodiment, the spectrophotometer described in 2020-0319102, US2020-0319085, and / or US10,281,330 (incorporated herein by reference) may be used. In an embodiment, for example, the spectrophotometer operates using AVAC technology as in an AVAC analyzer (Mecwins).

[0178] In an embodiment, the spectrophotometer further includes a dark field microscope objective lens and a dark field beam splitter.

[0179] In one aspect, the methods described herein provide improved detection of individual particles in a bioassay. The methods described using the devices disclosed herein substantially achieve an improved signal-to-noise ratio (SNR) for individual particles (e.g., plasmonic particles) in a bioassay.

[0180] In one aspect, the disclosed methods detect individual particles with an SNR of at least 60, or at least 70, or at least 80, or at least 90, or at least 100.

[0181] As demonstrated by the following examples, the SNR obtained using the COP-based devices described herein in conjunction with dark field microscopy analysis allows for the detection of individual particles of interest without the need for a silicon-based ultra-flat chip / wafers.

[0182] Examples

[0183] Based on the following non-limiting examples, additional aspects of the subject matter of the present invention will be apparent to those of ordinary skill in the art.

[0184] Example 1

[0185] An exemplary bioassay was performed using the devices described herein having a COP substrate and a layer of silicon dioxide (20 nm) deposited thereon via physical vapor deposition (PVD). The limit of quantification of the device used was evaluated to be 95 fg / mL.

[0186] Figure 1A The optical performance of an exemplary device analyzed in an AVAC analyzer (Mecwins) is shown. A signal-to-noise ratio (SNR) greater than 100 was exhibited.

[0187] Figure 1B Shown is available in conjunction with Figure 1A The monomers determined using an AVAC analysis in the same device.

[0188] Figure 2 Shown is Figure 1B The optical performance of the COP substrate device, which exhibits an SNR of 80, thus allowing the detection of individual particles of interest.

[0189] Example 2

[0190] In Figures 3A - 3B it is shown that reducing the thickness of the first reflective layer from 100 nm ( Figure 3A ) to 50 nm ( Figure 3B ) while keeping the second transparent layer constant does not affect the optical performance. The two composite solid carriers of this example provide similar gold nanoparticle (GNP) detection signals.

[0191] The signal-to-noise ratio (SNR) remains substantially unchanged, where the 100 nm aluminum embodiment ( Figure 3A ) has an SNR of approximately 83 and the 50 nm aluminum embodiment ( Figure 3B ) has an SNR of approximately 81.

[0192] Example 3

[0193] Figure 4A It is shown that the COP disc embodiment with an aluminum first layer and a 50 nm silica second layer after being cultured in water for 20 hours is not damaged or degraded. Figure 4B It is shown that the COP disc embodiment with an aluminum first layer and a 50 nm silica second layer after being cultured in pH = 9 carbonate buffer for 20 hours is not damaged or degraded.

[0194] Example 4

[0195] In addition, aluminum, copper, and gold used in coating COP substrates are compared. Figures 5A to 5E The qualitative results of culturing aluminum-coated COP substrates, copper-coated COP substrates, and gold-coated COP substrates are shown.

[0196] Figure 5A It is shown that after adding only a few drops of DI water, halos and stains are observed almost immediately in the copper-coated substrate and the gold-coated substrate when in contact with water droplets.

[0197] In contrast, as Figure 5B shown, when the aluminum-coated substrate is cultured in water for 20 hours, no damage is observed. The copper-coated substrate shows halos at the edges, while the gold-coated substrate is significantly damaged overall.

[0198] Similarly, Figure 5C it is shown that the COP disc embodiment with an aluminum coating (plus GPTMS) is not damaged after being cultured in DI water for 20 hours, while visible damage is seen in the copper-coated or gold-coated embodiments.

[0199] In addition to the DI water culture test, the metal-coated substrates are cultured in carbonate. Again, as Figure 5D shown, after culturing for 20 hours, copper and gold are significantly damaged, while no visible damage is seen in the aluminum coating. Figure 5E The results of the peel test after carbonate culture are shown to verify the adhesion of the metal coatings. Aluminum shows excellent adhesion, while both copper and gold are easily peeled off.

[0200] Example 5

[0201] Evaluate two batches (Batch 5.1 and Batch 5.2) of aluminum-coated COP substrates with a second silica layer of different thicknesses (2 samples per silica layer thickness) that were reacted or not reacted with GPTMS.

[0202] In each batch, the first aluminum layer is 100 nm thick and each in Batch 5.1 and Batch 5.2 has the following second silica layer thicknesses:

[0203] Batch 5.1: Silica layer thicknesses of 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, and 90 nm.

[0204] Batch 5.2: Silica layer thicknesses of 25 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, and 90 nm.

[0205] In Batch 5.2, the samples were additionally functionalized with a GPTMS layer. As can be seen in Figure 6 the same optical properties were achieved across the two batches. Thus, the GPTMS layer does not affect the overall optical properties.

[0206] Example 6

[0207] Compare COP substrates with a double-layer coating to evaluate the effect of (1) selecting a first silicon layer 200 nm thick and a first aluminum layer 100 nm thick, and (2) selecting the second silica layer thickness. Use GNPs with a 100 nm diameter. For two samples of each of the following batches, obtain the GNP scattering signal:

[0208] Batch 6.1: A first silicon layer 200 nm thick and a second silica layer that is 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, or 90 nm.

[0209] Batch 6.2: A first aluminum layer 100 nm thick and a second silica layer that is 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, or 90 nm.

[0210] As shown in Figure 7A it was found that a silica layer between approximately 50 nm and 60 nm optimized the GNP scattering signal. Additionally, it was found that the ratio of the GNP scattering signal of the samples in Batch 6.1 (aluminum-coated) to the GNP scattering signal of the samples in Batch 6.2 (silicon-coated) was approximately 3:1.

[0211] Figure 7BShow the relationship between the background signal and the silica layer thickness for each of the samples in batch 6.1 and batch 6.2. A low background signal was obtained for each of the test substrates, and it was found that the background signal decreased as the silica layer thickness increased.

[0212] Figure 7C Show the signal-to-noise ratio (SNR) for each of the samples in batch 6.1 and batch 6.2. For the aluminum-coated substrates, it was found that by selecting a silica thickness between approximately 40 nm and approximately 60 nm, the SNR was optimized for 100 nm GNPs. For the silicon-coated substrates, it was found that by selecting a silica thickness between approximately 50 nm and approximately 80 nm, the SNR was optimized.

[0213] Thus, for 100 nm GNPs, the aluminum-coated COP substrates and the silicon-coated COP substrates analyzed in Example 6 exhibited a maximum GNP scattering signal when the layer thickness was approximately 50 nm. In the case of the current deposition method, the silica layer can be deposited precisely and reproducibly to allow for easy industrialization.

[0214] Example 7

[0215] Evaluate the effect of surface roughness on the signal and background noise. The roughness was measured using an atomic force microscope (AFM).

[0216] It was found that significantly different roughness measurements were obtained via AFM on opposite sides of the same polymer substrate sample.

[0217] The test COP substrates were prepared using a polished steel mold with a high-quality nickel insert. The nickel insert was prepared as a 1:1 replication of a silicon wafer.

[0218] Figure 8A Show the roughness of the native (i.e., uncoated) polymer (COP) substrate on the mold side of the substrate with the nickel insert.

[0219] Figure 8B Show that the native COP substrate on the polished back side (i.e., the side opposite the nickel stamper side) is significantly rougher compared to the mold with the nickel insert.

[0220] Similarly, Figure 9A 、 9B 、9C, 9D, 9E, and 9F show the COP substrates coated with 50 nm of silicon ( Figure 9A ), the COP substrates coated with 125 nm of silicon ( Figure 9B ), the COP substrates coated with 200 nm of silicon ( Figure 9C ), the COP substrates coated with 200 nm of silicon + 25 nm of silica ( Figure 9D) COP substrates coated with 200 nm silicon + 50 nm silicon dioxide( Figure 9E ) COP substrates coated with 200 nm silicon + 200 nm silicon dioxide( Figure 9F ) AFM measurement results obtained on the nickel stamper side of

[0221] Thus, significant differences in surface finish (roughness) and surface quality were found between the nickel stamper surface and the polished back side of the COP substrate, i.e., no scratches on the nickel side.

[0222] In addition, it was found that the silicon sputter coating or the silicon dioxide sputter coating had no substantial effect.

[0223] Example 8

[0224] In an additional surface roughness study using AFM, the surface finish (roughness) of the COP substrate was compared with a comparative reference substrate made of silicon, specifically a silicon wafer, having a roughness of less than 1 nm. Figure 10 The surface roughness measurement results of the reference silicon wafer are shown in

[0225] Table 1 below shows the roughness (in nm) ("Roughness") for exemplary COP substrates and the roughness of the reference silicon wafer (in nm) ("Comparative silicon wafer").

[0226] Table 1

[0227]

[0228]

[0229] Example 9

[0230] COP substrates coated differently across two batches were analyzed.

[0231] In the first batch, the COP substrates were coated with (a) 200 nm silicon, (b) 200 nm silicon and 25 nm silicon dioxide, (c) 200 nm silicon and 50 nm silicon dioxide, (d) 100 nm aluminum and 25 nm silicon dioxide, or (e) 100 nm aluminum and 50 nm silicon dioxide.

[0232] In the second batch, the COP substrates were coated with (a) 100 nm aluminum and 100 nm silicon dioxide, (b) 100 nm aluminum and 150 nm silicon dioxide, (c) 100 nm aluminum and 200 nm silicon dioxide, (d) 200 nm silicon and 100 nm silicon dioxide, (e) 200 nm silicon and 150 nm silicon dioxide, or (f) 200 nm silicon and 200 nm silicon dioxide.

[0233] It was found that in the second batch with an increased silica thickness, the background scattering increased by almost an order of magnitude. See Figure 11A .

[0234] It was also found that the GNP scattering signal obtained using the coated COP composites of the first and second batches (i.e., aluminum-coated and silicon-coated as the first layer) peaked for samples with a silica thickness of 50 nm. See Figure 11B .

[0235] In addition, it was found that using 100 nm GNP for detection, the signal-to-noise ratio (SNR) peaked for samples with 50 nm silica. See Figure 11C .

[0236] It can thus be concluded that in some embodiments the silica layer in the composite material should be between about 25 nm and about 100 nm thick.

[0237] Example 10

[0238] In addition, as described in Table 2, various embodiments of the composite material were prepared and evaluated.

[0239] Table 2

[0240] Examples Substrate Material Layer 1 Layer 2 Coated Disk Side 10.0a COP None None n / a 10.0b COP (thin) None None n / a 10.1 COP Silicon (50nm) None Front 10.2 COP Silicon (125nm) None Front 10.3 COP Silicon (200nm) None Front 10.4 COP Silicon (200nm) Silicon Dioxide (25nm) Front 10.5 COP Silicon (200nm) Silicon Dioxide (50nm) Front 10.6 COP Aluminum (100nm) Silicon Dioxide (25nm) Front 10.7 COP Aluminum (100nm) Silicon Dioxide (50nm) Front 10.8 COP Aluminum (100nm) Silicon Dioxide (25nm) Back 10.9 Silicon (Comparative Reference Disk) None None n / a

[0241] It was found that each coated substrate produced very low background scattering, except for the back-coated aluminum-based composite (Example 10.8), which showed a large increase in background scattering. Compared to other 1.0 mm thick COP disks, the thinner COP disks of about 0.6 mm thickness (Example 10.0b) showed lower background scattering. See Figure 12A .

[0242] It was also found that the produced aluminum- and silica-coated COP disks improved scattering relative to the silicon- and silica-coated COP disks and the silicon reference (Example 10.9). In addition, it was found that substrates with a thicker silicon oxide layer performed better than those with a thinner oxide layer. A reduction in scattering was shown in the silicon-coated substrates (Examples 10.1 - 10.3), about 60% of the scattering produced by the COP blanks (Examples 10.0a and 10.0b) and about 40% of the scattering produced by the silicon blank reference (Example 10.9). See Figure 12B .

[0243] In addition, it was found that the signal-to-noise ratio (SNR) of the substrates coated with aluminum and silicon oxides (Examples 10.6 - 10.8) was comparable to that of the silicon reference blank (Example 10.9). Substrates coated with silicon and silicon dioxide, particularly Example 10.5, also showed good performance. The thinner COP discs with a thickness of 0.6 mm (i.e., Example 10.0b) had better performance than the thicker COP substrates with a thickness of 1.0 mm (i.e., Example 10.0a). See Figure 12C 。

[0244] Although many exemplary aspects and embodiments have been discussed above, those skilled in the art will recognize some of their modifications, permutations, additions, and subcombinations. It is therefore intended that the following appended claims and claims hereafter introduced be construed to include all such modifications, permutations, additions, and subcombinations as fall within their true spirit and scope.

Claims

1. A device, comprising: A synthetic polymer substrate having an upper surface; A coating on at least a portion of the upper surface, wherein the coating comprises a first layer and a second layer, the first layer comprising a material that reflects electromagnetic radiation, and the second layer comprising a dielectric and transparent material.

2. The device according to claim 1, wherein the first coating layer is selected from aluminum, silver, gold, chromium, silicon, and dielectric materials.

3. The device according to claim 1 or claim 2, wherein the first coating layer has a thickness between about 10 - 1000 nm, or between 10 - 500 nm, or between 10 - 250 nm, or between 10 - 200 nm, or between 50 - 150 nm, or between 75 - 125 nm.

4. The device according to any one of claims 1 - 3, wherein the second coating layer is a metalloid, an oxide of a metalloid, or a synthetic polymer.

5. The device according to claim 4, wherein the metalloid is boron, silicon, germanium, arsenic, antimony, tellurium, or polonium.

6. The device according to claim 4 or claim 5, wherein the metalloid oxide is silicon oxide.

7. The device according to any one of claims 1 - 6, wherein the second layer has a thickness between about 10 - 500 nm, or between 20 - 200 nm, or between 20 - 100 nm, or between 50 - 100 nm, or between 75 - 100 nm, or between 70 - 90 nm.

8. The device according to any one of claims 1 - 7, wherein the second layer comprises silica.

9. The device according to any one of claims 1 - 5, wherein the first layer is at least as thick as the second layer.

10. The device according to any one of claims 1 - 5, wherein the first layer has a thickness greater than the second layer.

11. The device according to claim 9, wherein the first layer is aluminum and the second layer is silica.

12. The device according to claim 11, wherein the first layer and the second layer have thicknesses within about 30%, 25%, 20%, 15%, or 10% of each other.

13. The device according to any one of claims 1 - 12, wherein an overcoat is applied with 3 - glycidoxypropyl)trimethoxysilane (GPTMS), 3 - aminopropyltrimethoxysilane (APTMS), or 3 - aminopropyltriethoxysilane (APTES).

14. The device according to any one of claims 1 - 5, wherein the substrate has a surface roughness, before or after coating, that is substantially equivalent to a silicon wafer suitable for semiconductor production.

15. The device according to any one of claims 1 - 14, wherein the substrate has a lower surface, and one or both of the surfaces are polished to provide a surface roughness that is substantially equivalent to a silicon wafer suitable for semiconductor production.

16. The device according to any one of claims 1 - 14, wherein the substrate has a lower surface, and one or both of the surfaces are produced by metal deposition to have a surface roughness that is substantially equivalent to a silicon wafer suitable for semiconductor production.

17. The device according to any one of claims 1-16, wherein the substrate has a thickness of less than 1 mm, less than 0.5 mm, less than 0.3 mm, less than 0.25 mm, less than 0.2 mm, or less than 0.1 mm.

18. The device according to any one of claims 1-17, wherein the substrate is composed of a cyclic olefin copolymer or a cyclic olefin polymer.

19. The device according to any one of claims 1-17, wherein the substrate comprises a thermoplastic material.

20. The device according to claim 19, wherein the thermoplastic material is a styrene / methyl methacrylate copolymer, polymethyl methacrylate, an olefin, a polyester, polystyrene, polyethylene, a polyamide, acrylonitrile butadiene styrene, or a polyacetal.

21. The device according to any one of claims 1-20, wherein the substrate is rigid.

22. The device according to any one of claims 1-21, wherein the substrate is planar.

23. The device according to any one of claims 1-22, wherein the upper surface of the substrate has a flatness of less than about 100 μm.

24. The device according to any one of claims 1-23, wherein the entire surface is the bottom layer of a multi-layer structure.

25. The device according to any one of claims 1-24, wherein the functionalized surface is part of a more complex structure.

26. A device comprising: a composite solid support member comprising a synthetic polymer substrate having an upper surface and a lower surface, the upper surface comprising at least a bilayer coating, the bilayer coating comprising a reflective layer deposited on the upper surface and a dielectric transparent layer deposited on the reflective layer; and a plurality of binding members fixed to the composite solid support member.

27. The device according to claim 26, wherein the plurality of binding members comprises a first binding member for a first analyte and a second binding member for a second analyte.

28. The device according to claim 26 or 27, wherein the plurality of binding members comprises a haloalkane dehalogenase binding tag.

29. The device according to claim 26 or 27, wherein the plurality of binding members comprises a protein, an antibody, or a peptide.

30. The device according to claim 26, wherein the plurality of binding members comprises a ligand that specifically binds to a binding tag, the ligand being part of a fusion protein comprising an antibody or an antibody fragment that binds to an analyte of interest.

31. The device according to claim 30, wherein the binding tag is a haloalkane dehalogenase or a biotin ligase binding site.

32. The device according to claim 30, wherein the ligand is a synthetic organic compound.

33. The device according to claim 32, wherein the compound comprises a chloroalkane linker.

34. The device according to claim 30, wherein the ligand is biotin.

35. The device according to any one of claims 26 - 34, wherein the coating comprises (3 - glycidyloxypropyl)trimethoxysilane (GPTMS), 3 - aminopropyltrimethoxysilane (APTMS), or 3 - aminopropyltriethoxysilane (APTES).

36. The device according to any one of claims 26 - 35, wherein the reflective layer comprises one or more of silicon, aluminum, silver, gold, chromium, platinum, palladium, and their alloys, or at least two dielectric materials having different refractive indices.

37. The device according to any one of claims 26 - 36, wherein the transparent dielectric layer comprises a quasi - metal or a synthetic polymer.

38. The device according to any one of claims 26 - 37, wherein the reflective layer has a thickness between about 5 nm and about 250 nm.

39. The device according to any one of claims 26 - 38, wherein the transparent dielectric layer has a thickness between about 1 nm and about 250 nm.

40. The device according to any one of claims 25 - 39, wherein the reflective layer consists essentially of aluminum and the transparent dielectric layer consists essentially of silicon dioxide.

41. The device according to any one of claims 25 - 40, wherein the coating comprises silicon dioxide.

42. The device according to any one of the preceding claims, wherein the substrate is not silicon, single - crystal silicon, or a silicon wafer, and wherein the coating is not a 20 - nm silicon dioxide layer on a silicon wafer, or wherein the coating is silicon dioxide having a thickness greater than 20 nm.

43. The device according to any one of the preceding claims, wherein the substrate comprises a ferromagnetic metal to allow remote surface magnetization.

44. The device according to claim 43, wherein the ferromagnetic metal is nickel or cobalt.

45. The device according to any one of claims 26 - 34, wherein the device further comprises an inlet, an outlet, or both.

46. A kit for detecting a bio - analyte of interest in a test sample, comprising: An analyte comprising a detection zone, the detection zone comprising a composite solid - support member and a plurality of binding members immobilized to the composite solid - support member, the composite solid - support member comprising a synthetic - polymer substrate having an upper surface and a lower surface, the upper surface comprising a bilayer coating, the bilayer coating comprising a reflective layer deposited on the upper surface and a dielectric transparent layer deposited on the reflective layer; A container comprising a population of detectable particles; and Instructions for use.

47. The kit according to claim 46, wherein the plurality of immobilized binding members are capable of binding the analyte of interest or are ligands that are specific for a binding tag, the ligand being part of a fusion protein comprising an antibody or an antibody fragment that binds the analyte of interest.

48. The kit according to claim 46 or claim 47, wherein the plurality of immobilized binding members are antibodies, antibody fragments, or synthetic organic compounds.

49. The kit according to claim 46 or claim 47, wherein the plurality of immobilized binding components are synthetic organic compounds, the synthetic organic compounds comprising a chloroalkane linker, either directly attached to the surface or through an immobilized protein, and wherein the binding tag is a haloalkane dehalogenase.

50. The kit according to any one of claims 46-49, wherein the detectable particle comprises a magnetic core and a shell, wherein the shell consists essentially of a metal selected from gold, silver, and platinum.

51. The kit according to any one of claims 46-49, wherein the detectable particle consists essentially of a metal selected from gold, silver, and platinum, or wherein the detectable particle does not have a core-shell structure.

52. The kit according to claim 50 or claim 51, wherein the metal is selected from gold, silver, platinum, palladium, iridium, osmium, rhodium, ruthenium.

53. The kit according to claim 50 or claim 51, wherein the surface metal is gold.

54. The kit according to any one of claims 46-53, wherein the detectable particle has an average diameter in the range of about 1 nm - about 1500 nm, or about 25 nm - about 500 nm, or about 50 nm - about 250 nm, or 100 - 180 nm.

55. The kit according to claim 48 or claims 50-52, wherein the magnetic core has a diameter in the range of about 5 nm - about 150 nm and the shell has a thickness in the range of about 10 nm - about 50 nm.

56. The kit according to claim 46 or claims 52-55, wherein the core of the detectable particle consists essentially of iron oxide.

57. The kit according to claim 46 or claims 52-56, wherein the detectable particle further comprises an intermediate layer provided between the core and the shell.

58. The kit according to claim 57, wherein the intermediate layer is silica.

59. The kit according to any one of claims 46-58, wherein the detectable particle resonates at a wavelength in the range of at least about 450 nm to about 800 nm.

60. The kit according to any one of claims 34-53, further comprising an additional binding component, the additional binding component being capable of associating with the detectable particle and having specific binding to an analyte of interest.

61. The kit according to claim 58, wherein the additional binding component is an antibody or an antibody fragment.

62. The kit according to claim 59, wherein the antibody or antibody fragment has specific binding to a cardiac biomarker.

63. The device according to claim 62, wherein the cardiac biomarker is troponin.

64. The device according to claim 63, wherein the troponin is troponin C, troponin I, or troponin T.

65. The device according to claim 62, wherein the cardiac biomarker is Pro BNP, or NT-proBNP.

66. The device according to claim 62, wherein the cardiac biomarker is d-dimer.

67. A method for detecting a bioanalyte in a fluid sample, comprising: contacting a device according to any one of claims 1-45 with a fluid sample suspected of containing a bioanalyte of interest and with detectable particles associated with a binding substance for the analyte of interest, wherein the device comprises a stationary component that binds to the binding substance; and analyzing, using an optical instrument, whether the detectable particles are present in the device.

68. The method according to claim 67, wherein the optical instrument is a spectrophotometer for dark field measurement, wherein the spectrophotometer comprises (i) a multi-beam emitter having various emission wavelengths, (ii) an array of light detectors arranged to detect light reflected through a second optical path defined as the path of the beam after reflection on the sample, (iii) one or more optical objectives configured to focus the light detected by the light detectors, and (iv) a processor for the beams received by the light detectors, the processor correlating each light detector with a spatial point on the sample such that measurements are made sequentially across the various wavelengths and in parallel along X-Y spatial coordinates.

69. The method according to claim 67, wherein the optical instrument is a spectrophotometer for dark field measurement, wherein the spectrophotometer comprises: (i) a white light beam emitter that is directed onto the sample through a first optical path having a lens array, thereby irradiating the sample at all wavelengths within the visible light range, (ii) an array of light detectors arranged to detect light reflected through a second optical path that can distinguish different wavelength ranges, the second optical path being defined as the path of the beam after reflection on the sample, (iii) one or more optical objectives configured to focus the light detected by the light detectors, and (iv) a processor for the image received by the light detectors, the processor correlating each light detector with a spatial point on the sample such that measurements are made sequentially across the various wavelengths and in parallel along X-Y spatial coordinates.

70. The method according to claim 67, wherein the optical instrument is a spectrophotometer for dark field measurement, wherein the spectrophotometer comprises: (i) a beam emitter that is directed onto the sample through a first optical path having a lens array, thereby irradiating the sample at all wavelengths, (ii) a set of filters that can select a subset of optical frequencies, (iii) an array of light detectors arranged to detect light reflected through a second optical path that can collect the reflected light, the second optical path being defined as the path of the beam after reflection on the sample, and (iv) one or more optical objectives configured to focus the light detected by the light detectors, and (v) A processor for the light beam received by the light detector, the processor correlating each received image with a spatial point on the sample such that measurements are made sequentially across the various wavelengths and in parallel along the X-Y spatial coordinates.

71. The method according to any one of claims 68 - 70, wherein the one or more optical objectives have a resolution of about 4 nm or less.

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