Methods for determination of analytes

Abstract

SOLUTION: Articles and methods relating to nanosensors for determination of molecules and other features, e.g., via surface plasmonic resonance, electric resonance, magnetic resonance, color changes, or the like. For example, they may be used for sample detection. The articles include a microwell array and a nanosensor array.EFFECT: The nanosensor arrays may utilize nanoparticles positioned on nanostructures that are able to interact with a sample suspected of containing an analyte, such as a single cell. The interaction between nanoparticles and a sample can be detected by a change in applied energy, such as altered electromagnetic radiation caused by surface plasmonic resonance of incident visible light, and / or other types of resonance. Electromagnetic radiation may be applied to a microwell array and nanosensor, and the applied electromagnetic radiation may be altered as a nanosensor interacts with a sample suspected of containing an analyte.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] (Related Applications) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 556,186, filed September 8, 2017, entitled "Nanosensor Methods and Apparatuses for Determination of Analytes," inventors Quan, et al., which is incorporated herein by reference in its entirety.

[0002] (Technical field) The present invention generally relates in some aspects to articles and methods relating to nanosensors for the determination of molecules and other features, for example, via surface plasmon resonance, color change, and the like. [Background technology]

[0003] Immunoassays are widely used in disease diagnosis. Traditional immunoassays (e.g., enzyme-linked immunosorbent assays, Western blots) typically limit detection sensitivity to 100 pg / ml or greater due to bulk measurement of fluorescent or colorimetric signals derived from fluorescently labeled molecules or enzymatic reactions. Furthermore, current immunoassays can only be performed on large numbers of cells. Existing single-cell techniques include flow cytometry, which is limited by antibody selection; typical flow cytometry methods are compatible with only 10% of available antibodies. None of the currently available techniques can detect intracellular molecules at the single-cell level in a highly multiplexed format.

[0004] Therefore, improvements in the detection limits are needed for disease prognosis and drug development. Summary of the Invention [Means for solving the problem]

[0005] The present invention generally relates in some aspects to articles and methods relating to nanosensors for the determination of molecules and other structures, e.g., via surface plasmon resonance, electric dipole resonance, magnetic dipole resonance, color change, etc. The subject matter of the present invention in some cases includes interrelated products, alternative solutions to a particular problem, and / or multiple different uses of one or more systems and / or articles.

[0006] In one aspect, the invention generally relates to an article. In one set of embodiments, the article comprises a microwell array including wells containing nanoparticles positioned distally to the ends of nanostructures, where the nanoparticles interact with incident light via surface plasmon resonance, electric resonance, and / or magnetic resonance.

[0007] According to another set of embodiments, an article comprises a microwell array comprising wells and a nanosensor array comprising nanoparticles positioned distally to ends of nanostructures contained within the wells, the nanoparticles being sized to interact with and alter incident visible light via surface plasmon resonance, electric resonance and / or magnetic resonance.

[0008] In another aspect, the invention relates generally to methods. In another set of embodiments, a method of fabricating an article is described. The method includes securing a first substrate including a microwell array containing wells to a second substrate including an array of nanostructures, at least some of the nanostructures including nanoparticles positioned distal to an end of the nanostructures, the nanostructures positioned within the wells.

[0009] In one set of embodiments, the method includes applying electromagnetic radiation to nanoparticles positioned distal to the end of the nanostructure, the nanoparticles interacting with the electromagnetic radiation via surface plasmon resonance, electric resonance, and / or magnetic resonance to modify the electromagnetic radiation, and measuring the modified electromagnetic radiation.

[0010] The method, according to another set of embodiments, includes positioning cells in wells of a microwell array, the wells further including nanoparticles positioned distal to the ends of the nanostructures and a reactive entity at least partially coated on the nanoparticles. In another set of embodiments, the method further includes lysing the cells in the wells to release an analyte suspected of being capable of binding to the reactive entity, and applying electromagnetic radiation to the nanoparticles, the nanoparticles interacting with and altering the electromagnetic radiation via surface plasmon resonance, electric resonance, and / or electromagnetic resonance, and measuring the altered electromagnetic radiation to determine the analyte.

[0011] Yet another set of embodiments relates to a method that includes acquiring a first optical image of an array of nanostructures on a substrate, the nanostructures having a cross-sectional dimension orthogonal to the direction in which the first optical image is acquired that is less than 700 nm, and the nanostructures being at least partially coated with a reactive entity. In another set of embodiments, the method further includes causing an interaction between the reactive entity and the analyte, acquiring a second optical color image of the array of nanostructures, and measuring a color change between the first optical image and the second optical image, wherein the color change is caused by the interaction between the reactive entity and the analyte.

[0012] In one set of embodiments, the method includes positioning a sample in a well of a microwell array, the well further comprising nanoparticles positioned distal to an end of the nanostructure, and further includes applying electromagnetic radiation to the nanoparticles, which interact with and modify the electromagnetic radiation via surface plasmon resonance, electric resonance, and / or magnetic resonance, and measuring the modified electromagnetic radiation.

[0013] In another set of embodiments, a method includes adding a sample suspected of containing an analyte to a well of a microwell array, the well further including nanoparticles positioned distal to an end of the nanostructure, the nanoparticles being at least partially coated with a reactive entity. In another set of embodiments, the method includes applying electromagnetic radiation to the nanoparticles, the nanoparticles interacting with incident light via surface plasmon resonance, electric resonance, and / or magnetic resonance to alter the electromagnetic radiation, and measuring the altered electromagnetic radiation to determine the interaction of the reactive entity with the analyte.

[0014] In another set of embodiments, a method includes exposing a solution suspected of containing an analyte to nanoparticles positioned distal to an end of a nanostructure, the nanostructure further comprising a reactive entity capable of interacting with the analyte, and further including applying electromagnetic radiation to the nanoparticles, the nanoparticles interacting with and modifying the electromagnetic radiation via surface plasmon resonance, electric resonance, and / or magnetic resonance, and measuring the modified electromagnetic radiation.

[0015] A method according to another set of embodiments includes acquiring first and second optical color images of an array of nanostructures on a substrate, the nanostructures having cross-sectional dimensions between 400 nm and 700 nm, and measuring a change in color between the first optical color image and the second optical color image.

[0016] Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0017] Non-limiting embodiments of the present invention are described, by way of example, with reference to the accompanying drawings, which are schematic and not intended to be drawn to scale. In the drawings, each identical or nearly identical component shown is typically represented by a single reference numeral. For clarity, not every component is labeled in every figure, or every component of every embodiment of the invention is shown, and the illustrations do not necessarily enable those skilled in the art to understand the invention.

[0018] [Figure 1] 1 illustrates a description of the interaction between a nanosensor and an analyte, which alters incident electromagnetic radiation, according to some embodiments. [Figure 2] 2A and 2B show the change in visible light produced by a nanosensor upon binding to an analyte, according to some embodiments. [Figure 3] 1 shows a microwell array with a semi-permeable membrane containing nanosensors and various concentrations of analyte, which generates optical light, according to some embodiments. [Figure 4] 1 shows a representation of a microwell array, according to some embodiments. [Figure 5] An exemplary representation of the capture of HEK293 cells with a single cell capture efficiency of approximately 70% is shown. [Figure 6] 1 shows an exemplary representation of a silicon nanorod nanosensor. [Figure 7] 7A and 7B show an exemplary representation of the color of silicon nanorods under dark-field imaging. [Figure 8] 8A-8D show several forms of silicon nanosensors, including nanocones, nanoneedles, nanowires, and nanoparticles, according to some embodiments. [Figure 9] 9A-9D show exemplary representations of the color shift of the nanosensor as the concentration of BSA protein increases. [Figure 10] 10A-10C show exemplary representations of the color shift of the nanosensor as the concentration of streptavidin increases. [Figure 11]11A-11C show exemplary representations of the color shift of nanosensors with various diameters as the concentration of streptavidin increases. [Figure 12] FIG. 12 shows nanorods with diameters of ∼100 nm and spacings of ∼400 nm, according to some embodiments. [Figure 13] 13A-13C show exemplary representations of the color shift of ∼100 nm diameter nanorods at various concentrations of BSA protein as the protein is absorbed onto the metasurface. [Figure 14] 1 shows nanorods with diameters of ∼180 nm and spacings of ∼420 nm, according to some embodiments. [Figure 15] 15A-15C show exemplary representations of the color shift of ∼180 nm diameter nanorods at various concentrations of BSA protein when the protein is adsorbed onto the metasurface. [Figure 16] Figures 16A and 16B show exemplary representations of the color shift of ~180 nm nanorods at various concentrations of BSA protein when the protein is adsorbed onto a metasurface with 1.8 nm diameter gold nanoparticles as secondary labels to amplify the color shift. [Figure 17] 1 shows an exemplary representation of a dark-field image of a microwell array with a 3x3 individual nanosensor array in each well. [Figure 18] 18A-18C show a 20 microliter droplet of single cell suspension being applied onto the sensor chip of the microwell array and nanosensor array. [Figure 19] 19A-19C show exemplary representations of protein binding on nanosensors inducing a color shift that correlates with protein concentration. [Figure 20] 1 shows a 75 mm x 25 mm glass slide with a 10-microwell array, according to some embodiments. [Figure 21] 21A-21C show exemplary representations of nanosensors on silicon nano- or micro-sized needles spaced at the same period as the microwells. [Figure 22] 22A-22I show an exemplary representation of a nanosensor array for detecting protein concentrations from 0.1 to 10 pM. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention generally relates in some aspects to articles and methods relating to nanosensors for the determination of molecules and other features, e.g., via surface plasmon resonance, electric resonance, magnetic resonance, color change, etc. These articles and methods can be used, for example, for sample detection. Articles described in some aspects of the present invention include microwell arrays and nanosensor arrays. In some embodiments, the nanosensor arrays can utilize nanoparticles positioned on nanostructures that can interact with a sample suspected of containing an analyte, such as a single cell. The interaction between the nanoparticles and the sample can be detected by a change in applied energy, e.g., altered electromagnetic radiation, caused by surface plasmon resonance and / or other types of resonance of incident visible light. Electromagnetic radiation can be applied to the microwell arrays and nanosensors, and the applied electromagnetic radiation can be altered when the nanosensors interact with a sample suspected of containing an analyte. Furthermore, in some embodiments, the nanosensor arrays can utilize nanostructures, and analyte binding can be determined based on optical or color changes.

[0020] Certain aspects of the present invention generally relate to systems and methods for detecting biomolecules, e.g., proteins or nucleic acids, e.g., originating from cells or other sources. In some embodiments, the biomolecules are qualitatively and / or quantitatively determined using nanoneedles or other nanostructures used as sensors, which may be present in an array, e.g., an array of wells.

[0021] In one set of embodiments, the particle at the end of the nanoneedle has a narrowband spectrum. For example, binding of a biomolecule or other analyte to the particle via a reactive entity can affect the particle's ability to resonate with incident light, e.g., due to surface plasmon resonance, electric resonance, and / or magnetic resonance. Examples of reactive entities include antibodies, enzymes, nucleic acids, or other entities as described below. By measuring the difference in resonance, e.g., by applying light to the particle and measuring refraction and / or absorbance, the binding interaction between the particle and the biomolecule can be determined.

[0022] As a non-limiting example, FIG. 1 illustrates a system 10 in which an analyte 15 can interact with a reaction entity 20 immobilized on a nanoparticle 25. The interaction can be, for example, specific or nonspecific, covalent or noncovalent, etc. The nanoparticle 25 can be positioned on the end 30 of a nanoneedle 35 or other suitable nanostructure. In some cases, the nanoneedle can be positioned within a well 40 (e.g., an isolated well or a well of a microarray, etc.); in other cases, the nanoneedle can be positioned on a substrate, optionally on a suitable nanostructure, but not necessarily within a well. Incident light 45 from a light source 50 can interact with the nanoparticle 25 and can interact with the particle via the plasmon resonance effect. Additionally, a portion of the light 55 is directed toward a detector 60, e.g., via refraction from the particle. By measuring the difference in light reaching the detector, various interactions between the analyte 15 and the reaction entity 20 can be determined.

[0023] In another set of embodiments, binding of a biomolecule or other analyte can be determined using a change in color, e.g., visible light. Without wishing to be bound by any theory, it is believed that certain types of nanoneedles or other nanostructures can vibrate only in a fundamental mode (e.g., in response to visible light). The fundamental mode can change, e.g., due to a reaction entity, e.g., upon binding of a biomolecule to the nanoneedle. Thus, a change in the visible properties of the nanoneedle (e.g., a change in color and / or intensity) can be used to determine the binding interaction between the nanoneedle and the biomolecule.

[0024] As a non-limiting example, Figure 2A shows system 10 in which nanoneedles 15 and reactive entities 20 positioned within well 25 produce the color of visible light 30. Analyte 35 does not interact with nanoneedles 15 or reactive entities 20 and does not produce visible light. In system 40 of Figure 2B, nanoneedles 15 and reactive entities 20 positioned within well 25 bind to analyte 35. That interaction results in a change in the optical color or appearance produced by visible light 45, which may be different from visible light 30.

[0025] In some cases, biomolecules (or other analytes) originate from cells. For example, cells can be lysed in wells of a microwell plate and qualitatively and / or quantitatively determined using nanoneedles or other nanostructures used as sensors as described herein. In some cases, particles may also be present. Biomolecules can be determined, for example, using plasmon resonance effects, color changes, etc., as described herein. In some cases, cells can be introduced into particles and sealed therein, for example, using a membrane. For example, cells can be lysed individually in separate wells to prevent contamination of one well with another from cell lysate. In some cases, a semipermeable membrane is used to allow entry of a lysis reagent (e.g., lysis buffer) into the well but prevent lysate from exiting the well. Thus, the lysates of individual cells can be individually determined according to certain embodiments.

[0026] 3 shows a non-limiting example of system 10 including wells 25 and 30. Cells 15 and 20 are positioned within wells 25 and 30, respectively. Cells 15 and 20 can be lysed, for example, upon addition of a lysis buffer to wells 25 and 30. A semi-permeable membrane 55 is positioned over microwell array 35 to prevent contamination of the contents of wells 25 and 30. The semi-permeable membrane may be added, for example, after introducing the cells into the wells and before or after lysis of the cells.

[0027] Interaction of nanoparticles 45 with analyte from cells 15 produces optical light 60, and interaction of nanoparticles 45 with analyte from cells 20 (which are in higher concentration than cells 15) produces optical light 65. For example, incident light may be applied, and a portion of the light may be absorbed by the nanoneedle. Differences in analyte (e.g., concentration, type, etc.) may produce different optical light or different well or nanoneedle appearances. As another example, a well may comprise a nanosensor including nanoneedles 40 and nanoparticles 45 positioned at ends 50 of nanoneedles 35. Incident light can interact with such a system through plasmon resonance effects, which may produce different refracted light or appearances based on different interactions between the analyte and nanoparticles (e.g., via reactive entities). By measuring such light, the analyte can be determined.

[0028] The above represent various non-limiting examples of specific embodiments of the present invention. However, other embodiments are possible. Thus, more generally, various aspects of the present invention are described herein in the context of nanosensors for the determination of molecules and other structures, e.g., via surface plasmon resonance, color change, etc.

[0029] Some articles and methods of the present invention relate to sensors that include particles (e.g., nanoparticles) positioned on a nanostructure, e.g., distal to the end of the nanostructure. In some cases, the particles can interact with an analyte; e.g., as described below, the interaction between the analyte and the nanostructure and / or particle can be determined (e.g., qualitatively and / or quantitatively) by determining how light interacts with the particle. For example, light can interact with the particle via the surface plasmon resonance effect, and changes in the light can be used to determine the analyte. In some cases, other resonances, such as, e.g., electric and / or magnetic resonances, can occur.

[0030] In various embodiments of the present invention, various samples can be determined. For example, in certain aspects, the sample may include cells. Nanosensors can be used to optically interrogate or study samples, e.g., cells. In some cases, properties of cells or other samples, such as the presence or concentration of an analyte or sample, may interact with the nanosensor and / or a reactive entity on the nanosensor, which can be optically determined. Examples of optical interrogation techniques that can be used include, but are not limited to, fluorescence, phosphorescence, surface plasmon resonance, surface plasmon resonance, localized surface plasmon resonance, Raman spectroscopy, surface-enhanced Raman spectroscopy, and the like.

[0031] In certain embodiments, the nanoparticles comprise a metal, such as gold, silver, copper, etc. According to some embodiments, the nanoparticles comprise gold. In another set of embodiments, the nanoparticles comprise silver, quantum dots, or semiconductor nanoparticles.

[0032] In some embodiments, the particles are sized to interact with, e.g., alter, incident visible light via surface plasmon resonance effects. For example, upon interaction with the particles, a portion of the incident light may be absorbed, reflected, refracted, etc., resulting in a change in the light that can be measured in some way. Additionally, in some cases, the particles are sized to interact with incident visible light via electrical and / or magnetic resonance effects.

[0033] In some cases, the particles may be nanoparticles. For example, the particles may have a characteristic diameter of less than about 1 micrometer. The particles may be spherical or non-spherical. The characteristic diameter may be considered the diameter of a perfect sphere having the same volume as the non-spherical particle. According to certain embodiments, the diameter of the nanoparticles is at least 0.5 nm, at least 1.0 nm, at least 1.5 nm, at least 2.0 nm, at least 2.5 nm, at least 3 nm, at least 4 nm, at least 5 nm, at least 7 nm, at least 10 nm, at least 30 nm, at least 100 nm, at least 300 nm, etc. In some embodiments, the diameter of the nanoparticles is less than 1000 nm, less than 500 nm, less than 300 nm, less than 100 nm, less than 50 nm, less than 30 nm, less than 10 nm, less than 7 nm, less than 5 nm, less than 4 nm, less than 3.0 nm, less than 2.5 nm, less than 2.0 nm, less than 1.5 nm, or less than 1.0 nm. Furthermore, any combination of these is also possible, and for example, the characteristic diameter may be within the range of 0.5 to 3.0 nm.

[0034] In some embodiments, the particles and / or nanostructures are at least partially coated with a reactive entity. The term "reactive entity" refers to any entity that can interact with an analyte in a manner that causes a detectable change in a property of a component, such as a chemical property, an optical property, a mechanical property, a vibrational property, etc. The interaction between the reactive entity and the analyte may be specific or non-specific binding and may include a variety of interactions. The interaction of the reactive entity with the analyte may result in, for example, a change in light, which can be determined as discussed herein.

[0035] In some embodiments, the reaction entity can include a binding partner to which the analyte binds. The reaction entity can include a specific or nonspecific binding partner of the analyte. For example, the reaction entity can be a chemical or biochemical entity, such as a metal, nucleic acid, antibody, aptamer, sugar, carbohydrate, protein, polymer, oligonucleotide, catalyst, quantum dot, etc. As a non-limiting example, in certain embodiments, the reaction entity at least partially coating the nanoparticle is an antibody or a fragment thereof. The antibody can be any suitable antibody, including a monoclonal antibody, a chimeric antibody, a humanized antibody, etc. In some embodiments, the reaction entity includes an enzymatic reaction product induced by a chromogenic substrate labeled with the analyte. The chromogenic substrate can include, for example, 3,3',5,5'-tetramethylbenzidine (TMB), 3,3'-diaminobenzidine (DAB), 2,2'-azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), etc.

[0036] A binding partner may be a molecule capable of undergoing binding with a specific analyte, and includes specific, semi-specific, and non-specific binding partners, as known to those skilled in the art. The term "specifically binds," when referring to a binding partner (e.g., a protein, aptamer, nucleic acid, antibody, etc.), refers to a reaction that determines the presence and / or identity of one or other member of a binding pair (e.g., proteins and other biologics) in a mixture of heterogeneous molecules. Thus, for example, in the case of a receptor / ligand binding pair, the ligand will specifically and / or preferentially select its receptor from a complex mixture of molecules, or vice versa. An enzyme will specifically bind to its substrate, a nucleic acid will specifically bind to its complement, and an antibody will specifically bind to its antigen. Other examples include nucleic acids that specifically bind (hybridize) to their complements, antibodies that specifically bind to these antigens, etc. Binding may be by one or more of a variety of mechanisms, including, but not limited to, ionic interactions, covalent interactions, hydrophobic interactions, van der Waals interactions, and / or hydrogen bonding.

[0037] As another example, the reactive entity may include platinum, which can be used to determine hydrogen. As yet another example, the reactive entity may include a hydrogel, which can be used to determine water or humidity. Non-limiting examples of reactive entities include those disclosed in International Patent Application Publication No. WO 2015 / 175398, which is incorporated herein by reference in its entirety for all purposes.

[0038] In some cases, one or more particles may be immobilized to the nanostructure directly or indirectly, for example, via one or more linkers or spacers. For example, the particles may be attached to any suitable location on the nanostructure, such as a side or end. As an example, the nanostructure may be attached to the substrate at a first end, and the particles may be attached to a second end distal to the first end.

[0039] The nanostructures may have any suitable shape and / or size. In some cases, for example, the nanostructures may be nanoneedles, nanowires, nanorods, nanocones, etc. See, e.g., Figure 8. Other shapes are also possible, such as nanoribbons, nanofilaments, nanotubes, nanopillars, etc. In certain embodiments, the nanostructures are vertically aligned, although other angles or alignments are also possible.

[0040] In some embodiments, the nanostructures have a length determined from an edge or point of attachment to the substrate that is less than about 100 micrometers, less than about 50 micrometers, less than about 30 micrometers, less than about 20 micrometers, less than about 10 micrometers, less than about 5 micrometers, less than about 3 micrometers, less than about 2 micrometers, less than about 1 micrometer, less than about 500 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, less than about 50 nm, less than about 30 nm, less than about 20 nm, less than about 10 nm, etc. In some cases, the length may be at least about 10 nm, at least about 20 nm, at least about 30 nm, at least about 50 nm, at least about 100 nm, at least about 200 nm, at least about 300 nm, at least about 500 nm, at least about 1 micrometer, at least about 2 micrometers, at least about 3 micrometers, at least about 5 micrometers, at least about 10 micrometers, at least about 20 micrometers, at least about 30 micrometers, at least about 50 micrometers, at least about 100 micrometers, etc. Any combination of these is possible; for example, the nanostructure length may be between 0.2 and 2 micrometers.

[0041] Nanostructures may have any suitable cross-sectional shape, such as square, circular, triangular, oval, polygonal, star-shaped, irregular, etc. A nanostructure may maintain the same cross-sectional shape throughout its entire length, or different cross-sectional shapes may exist in different portions of the nanostructure. Furthermore, nanostructures may have any suitable cross-sectional diameter. The cross-sectional diameter may be constant (e.g., like a nanoneedle or nanorod) or may vary (e.g., like a nanocone). The average diameter may be, for example, less than about 1000 nm, less than about 500 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, less than about 50 nm, less than about 30 nm, less than about 20 nm, less than about 10 nm, etc. In some cases, the length may be at least about 10 nm, at least about 20 nm, at least about 30 nm, at least about 50 nm, at least about 100 nm, at least about 200 nm, at least about 300 nm, at least about 500 nm, at least about 1000 nm, etc. Combinations are also possible in various embodiments. For example, the average diameter of the nanostructures may be between 50 nm and 300 nm.

[0042] The nanostructures may be formed of any suitable material, which may be the same or different from, for example, the substrate to which they are vertically attached. In one set of embodiments, the nanostructures are formed of silicon and / or other suitable semiconductor materials (e.g., germanium). Additional non-limiting examples of materials include metals (e.g., nickel or copper), silica, glass, etc. In some cases, the nanostructures (which may be attached to the substrate) can be formed of a single material.

[0043] Any suitable method can be used to form the nanostructures. Examples include, but are not limited to, lithography techniques, such as electron beam lithography, photolithography, X-ray lithography, extreme ultraviolet lithography, ion projection lithography, etc. As another example, in some embodiments, the nanostructures may be formed of one or more materials that are susceptible to etching with an appropriate etchant. For example, the nanostructures may include materials such as silica or glass that can be etched using HF (hydrofluoric acid) or BOE (buffered oxide etch). As another example, the nanostructures may include metals such as copper, iron, nickel, and / or steel, which can be etched using acids such as HCl (hydrochloric acid), HNO3 (nitric acid), sulfuric acid (H2SO4), and / or other etching compounds, such as ferric chloride (FeCl3) or copper sulfate (CuSO4). As yet another example, the nanostructures may comprise silicon or other semiconductor materials, which can be etched using etchants such as EDP (a solution of ethylenediamine and pyrocatechol), KOH (potassium hydroxide), and / or TMAH (tetramethylammonium hydroxide). The nanostructures may, in some cases, comprise plastics or polymers, such as polymethyl methacrylate, polystyrene, polyperfluorobutenyl vinyl ether, etc., which can be etched using KOH (potassium hydroxide) and / or other acids as described herein.

[0044] Nanostructures, in some embodiments, may comprise or consist essentially of one material or more than one material, for example, in one embodiment, the nanostructures are formed from a single or solid piece of etchable material as discussed herein.

[0045] Certain embodiments of the present invention also generally relate to microwell arrays, which may contain one or more wells, which may be circular or non-circular. Any number of wells may be present, e.g., at least 1, at least 2, at least 5, at least 10, at least 25, at least 50, at least 100, at least 200, at least 500, etc. The microwells may also contain one or more sensors as described herein, e.g., one or more nanoneedles or other nanostructures. In some embodiments, the microarrays may be sized according to commercially available ANSI / SLAS standards and may comprise, for example, 6, 12, 24, 48, 96, 384, or 1536 wells. The microwell arrays may be formed from suitable materials, including plastics or polymers (e.g., polystyrene, polypropylene, polycarbonate, cycloolefins, etc.), silica, glass, metals, etc.

[0046] In certain embodiments, the wells may have diameters ranging from 10 microns to 50 microns. According to some embodiments of the invention, the wells have diameters of at least 10 microns, at least 20 microns, at least 30 microns, or at least 40 microns. In certain embodiments, the wells have diameters of less than 50 microns, less than 40 microns, less than 30 microns, or less than 20 microns. Any combination of these is also possible; for example, the wells may have diameters of 20 to 40 microns.

[0047] According to certain embodiments, the wells have a depth of at least 20 microns, at least 30 microns, at least 40 microns, or at least 50 microns. In certain aspects of the invention, the wells have a depth of less than 60 microns, less than 50 microns, less than 40 microns, or less than 30 microns. Any combination of these is also possible; for example, the wells may have a depth in the range of 20 microns to 60 microns.

[0048] In some embodiments, the wells may have a pitch (or spacing between wells) of about 1000 micrometers or less, about 700 micrometers or less, about 500 micrometers or less, about 300 micrometers or less, about 100 micrometers or less, about 50 micrometers or less, about 40 micrometers or less, about 30 micrometers or less, about 25 micrometers or less, about 20 micrometers or less, about 15 micrometers or less, about 10 micrometers or less, about 5 micrometers or less, about 3 micrometers or less, about 2 micrometers or less, about 1 micrometer or less, etc. In some cases, the pitch may be at least about 1 micrometer, at least about 3 micrometers, at least about 5 micrometers, at least about 10 micrometers, at least about 15 micrometers, at least about 20 micrometers, at least about 25 micrometers, at least about 30 micrometers, at least about 40 micrometers, at least about 50 micrometers, at least 100 micrometers, at least 300 micrometers, at least 500 micrometers, at least 700 micrometers, at least 1000 micrometers, etc. Furthermore, any combination of these is also possible, and for example, the pitch may be between about 10 micrometers and 100 micrometers.

[0049] In some cases, the microwell array may be configured to contain one or more cells. For example, one or more lysates arising from the cells may be determined in one or more wells using one or more sensors as described herein. However, it should be understood that cells (or cell lysates) are not required, and other embodiments of the invention may relate to lysates arising from other sources, including biological and non-biological specimens.

[0050] In some cases, microwell arrays may be populated with one cell per well, or two or more cells per well. Various wells may have the same or different numbers of cells present. Cells may be isolated cells, cell aggregates, or cells found in cell culture, in tissue constructs containing cells, etc. Examples of cells include, but are not limited to, bacteria or other unicellular organisms, eukaryotic cells, plant cells, or animal cells. If the cell is an animal cell, the cell may be, for example, an invertebrate cell (e.g., a cell from a fruit fly), a fish cell (e.g., a zebrafish cell), an amphibian cell (e.g., a frog cell), a reptilian cell, a bird cell, or a human or non-human mammal. If the cell is derived from a multicellular organism, the cell may be derived from any part of the organism.

[0051] In some embodiments, one or more cells are added to one or more wells and optionally sealed in place (e.g., to prevent contamination or interaction between different wells), e.g., using a membrane, such as a semi-permeable membrane. However, according to some embodiments, the cells may be lysed within the well to release one or more analytes of interest, e.g., proteins, nucleic acids, etc.

[0052] Various techniques may be used to lyse cells. For example, cells may be lysed by exposure to lysis chemicals or cell lysis buffers (e.g., detergents such as Triton-X or SDS, enzymes such as lysozyme, lysostaphin, zymolase, cellulase, mutanolysin, glycanase, protease, mannase, proteinase K, etc.), or physical conditions (e.g., ultrasound, ultraviolet light, mechanical agitation, etc.). If lysis chemicals are used, they may be added to the wells before and / or after the cells are added. In some cases, the lysis chemicals may be added before or after adding a membrane, e.g., to contain the cells within the well. In some cases, the membrane may be permeable and / or semi-permeable, which may facilitate the entry of the lysis chemicals.

[0053] Some embodiments of the present invention generally relate to semi-permeable membranes that can be attached to microwell arrays and / or nanostructured substrates, for example, to contain cells or other samples within the wells. In certain cases, the semi-permeable membranes are sized to prevent the passage of cells but allow the passage of smaller compounds.

[0054] In certain embodiments of the invention, a semi-permeable membrane can be positioned between the microwell array and the nanostructured substrate, or on top of the microwell array or the nanostructured substrate. In some cases, the semi-permeable membrane can be removed from the microwell array and / or the nanostructured substrate. In certain embodiments, the semi-permeable membrane is evaluated by microscopy, such as dark-field microscopy or other optical microscopy techniques.

[0055] For example, various semipermeable membranes having various permeabilities can be used. For example, the semipermeable membrane can be hydrophilic or hydrophobic, porous or non-porous, etc. In certain embodiments, the semipermeable membrane can comprise a polymer such as, for example, polycarbonate. Other examples of semipermeable membranes include cation exchange membranes, anion exchange membranes, etc.

[0056] As previously discussed, embodiments of the articles and methods may include a nanostructured substrate directly or indirectly associated with a microwell array. In some embodiments of the invention, the nanostructured substrate comprises a plurality of nanosensors as described herein. According to certain embodiments, the microwell array and the nanostructured substrate are separable. In some cases, separation can be achieved without the use of tools. In other embodiments of the invention, the microwell array and the nanostructured substrate are inseparable. In certain embodiments of the invention, the microwell array and the nanostructured substrate are directly attached to one another, e.g., such that a nanosensor is present in at least one well of the microwell array.

[0057] In some embodiments, light may be applied to the nanosensor, e.g., to determine an analyte as discussed herein. In some cases, the light may interact with the nanosensor (e.g., nanoparticles within the nanosensor) via surface plasmon resonance and / or other resonances, such as electric and / or magnetic resonance, and the effects of the interaction may be determined, e.g., by determining the refraction and / or absorbance of light. In one set of embodiments, the light applied to the nanosensor may be an incident beam comprising plane-polarized light from a laser, e.g., a He-Ne laser. Other lasers are commercially available.

[0058] In some cases, incident light is incident on a substrate surface, such as a silicon surface or a metal surface (e.g., gold) of a particle. In some cases, interaction of an analyte with the nanosensor (e.g., via a reaction entity) can change the refractive index or other properties of the incident light, which can be determined, for example, using a detector. For example, in certain embodiments, localized surface plasmon oscillations can cause optical changes in the nanosensor, which can produce absorption, for example, in the ultraviolet and / or visible light range. These can be determined and used to determine binding or other interactions between the analyte and a reaction entity, such as a protein or nucleic acid.

[0059] In some cases, the light can be detected using a microscope and / or spectrometer, or other optical detector. A variety of suitable optical detectors, including spectrometers, are commercially available. In some cases, other optical components may be present, for example, to facilitate interaction or detection. Examples of optical components include, but are not limited to, waveguides, optical sensors, photodetectors, optical fibers, etc.

[0060] However, it should be understood that other detection methods can be used in addition to or instead of the surface plasmon resonance effect as described herein. For example, in some embodiments, an analyte can interact with a reaction entity immobilized on a nanostructure (i.e., these may or may not comprise particles such as nanoparticles), and a change in optical appearance, e.g., a color change, can be determined to determine binding of the analyte to the reaction entity. Any suitable method, e.g., optical microscopy, fluorescence spectroscopy, etc., can be used to determine the change in optical appearance.

[0061] In certain embodiments of the present invention, an analyte can interact with the nanosensor, causing a change in optical appearance when the nanosensor interacts with incident light via electric or magnetic resonance. Without wishing to be bound by any theory, electric or magnetic resonance may arise from the interaction of light with the nanosensor or a portion thereof, such as a nanoparticle. In some cases, for example, the resonance may change upon application of an appropriate electric or magnetic field. A variety of electric and / or magnetic field generators are readily available commercially.

[0062] In some cases, the distance or pitch between nanostructures in a periodic structure can be controlled, for example, so that the nanostructures form a metasurface. For example, the pitch can be set to be smaller than the wavelength of the incident light. For example, the pitch can be less than 700 nm, less than 600 nm, less than 500 nm, and / or greater than 400 nm, greater than 500 nm, or greater than 600 nm. For example, the pitch can be between 400 nm and 500 nm. The nanostructures can have any of the dimensions provided herein. In some cases, the average cross-sectional diameter of the nanostructures is smaller than the wavelength of the incident light.

[0063] Without wishing to be bound by any theory, scattered light from individual nanostructures can interfere, and the amount of interference can be sensitive to the analyte or other entity bound to the nanostructure. Thus, changes in color or other optical properties can be used to determine changes in analyte interaction.

[0064] It should be understood that other pitches can be used, for example, when applying infrared or ultraviolet light. For example, the pitch can be less than 1000 nm, less than about 500 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, less than about 50 nm, less than about 30 nm, less than about 20 nm, less than about 10 nm, etc., and / or the pitch can be at least about 10 nm, at least about 20 nm, at least about 30 nm, at least about 50 nm, at least about 100 nm, at least about 200 nm, at least about 300 nm, at least about 500 nm, at least about 1000 nm, etc. Any combinations of these are also possible in various embodiments.

[0065] International Patent Application Publication No. 2015 / 175398 is incorporated herein by reference in its entirety. Additionally, U.S. Provisional Patent Application No. 62 / 556186, filed September 8, 2017, entitled "Nanosensor Methods and Apparatuses for Determination of Analytes," inventors "Quan et al.", is also incorporated herein by reference in its entirety.

[0066] The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.

[0067] (Example 1) Silicon nanostructures were fabricated and used as label-free nanosensors. The advantage of using silicon nanostructures is that the fabrication process is CMOS (complementary metal-oxide semiconductor) compatible, allowing for mass production. The fabricated silicon nanostructures can be in the form of nanorods, with diameters between 50 and 300 nm and heights between 0.2 and 2 microns, as shown in Figure 6. Under dark-field imaging, nanorods with different diameters display various spectral colors (see Figures 7A-7B). At diameters less than 250 nm, each nanorod has a unique color. For diameters greater than approximately 300 nm, the color spectrum becomes similar. Silicon nanostructures can also take other forms, such as cones, needles, wires, and particles, as shown in Figures 8A-8C. The dimensions used range from 50 nm to 1 micrometer.

[0068] To test the sensitivity of the silicon nanorods, chips or microwell arrays were functionalized with a 2% APTM solution in ethanol (v / v) for 20 minutes. Next, 10 mM glutaraldehyde, 10 mM sodium cyanoborohydride, and a 1 / 1000 dilution of anti-BSA were added to the microwell array and incubated for 2 hours. The microwell arrays were then rinsed. Various concentrations of BSA solution were added to the nanosensors. The microwell arrays were washed with deionized water and imaged under a dark-field microscope with a 20x objective. As shown in Figures 9A-9D, the color of each nanosensor shifted to the red (i.e., increased wavelength) with increasing BSA protein concentration.

[0069] (Example 2) In this example, streptavidin at various concentrations was tested (see Figures 10A-10C). As the streptavidin concentration increases, the color spectrum has a red shift. In the case of streptavidin, each silicon nanosensor operates independently. Therefore, nine different silicon nanorods with diameters of 80, 100, 120, 140, 160, 180, 200, 220, and 240 nm were fabricated and their color responses to streptavidin solutions of various concentrations were tested in this example, as shown in Figures 11A-11C. The spacing between each silicon nanosensor was 5 micrometers.

[0070] When the distance between nanosensors is reduced to less than a photon wavelength, the scattered light from the individual nanorods interferes and collectively forms a silicon metasurface. The metasurface can function as a sensor and be used to detect protein concentration. For example, as shown in Figure 12, if each nanorod is ~100 nm in diameter, the spacing is ~400 nm. This produces a generally green color in deionized water, and as proteins are absorbed onto the surface, the overall color is red-shifted, as shown in Figures 13A-13C.

[0071] The diameter of individual nanorods was reduced to ~180 nm (see Figure 14), with spacing of ~420 nm. The overall color was orange-green in deionized water and red-shifted upon protein absorption to the surface, as shown in Figures 15A-15C. Furthermore, 1.8 nm diameter gold particles functionalized with anti-BSA were used as secondary labels to amplify the color shift from the metasurface nanosensor. The color shift due to nanoparticle binding was an order of magnitude larger than that due to molecular analyte binding (30 nm vs. 3 nm) (Figures 16A-16B). This color shift can be quantified, for example, using an appropriate colorimetric detector.

[0072] (Example 3) To detect protein expression in single cells, in this example, silicon nanosensors were fabricated on silicon wafers using electron beam lithography and reactive ion etching. A monolayer of 2% 950K PMMA (A2) was used. The wafer was first dehydrated at 150°C for 30 minutes. The PMMA was then spun at 1600 rpm for 40 seconds to a thickness of 100 nm. The wafer was prebaked at 200°C for 2 minutes. The ELIONIX F125 was used at 125 keV and 1800 microcoulombs / cm. 2 A dose of 1000 nm was used. The resist was developed in 1:3 methyl isobutyl ketone:isopropyl alcohol (v / v) for 30 seconds and rinsed in isopropyl alcohol. The wafer was then coated with 30 nm of alumina using a thermal evaporator and lifted off in hot acetone for 3 hours. The wafer was then etched for 2 minutes using STS-RIE. As shown in Figure 17, a microwell array with 3 × 3 individual nanosensor arrays in each well was analyzed by dark-field imaging. Each nanosensor in the 3 × 3 group appeared as a different color due to its slightly different dimensions.

[0073] (Example 4) In this example, a single-cell suspension (cell concentration 20,000 cells / mL) was prepared and 20 microliters was dropped onto the microwell array. The cells diffused into the microwells with a collection efficiency that was sensitive to the microwell geometry.

[0074] Before cells were introduced into the microwell array, the array was incubated with 2% APTMS (v / v) in ethanol for 10 minutes. The microwell array was thoroughly rinsed with ethanol and blown dry with nitrogen gas. Next, 10 mM glutaraldehyde, 10 mM sodium cyanoborohydride, and a 1 / 1000 dilution of anti-beta-actin were dropped into the microwell array and incubated for 2 hours. The microwell array was then rinsed with deionized water. As shown in Figures 18A-18C, a droplet (20 microliters) of single-cell suspension was applied to a sensor chip containing both microwells and nanosensors. After seeding the cells on the chip for 10 minutes, a hydrophilic semipermeable membrane (10 nm pore size, 6 micron thick polycarbonate membrane) was attached to the microwell array. The membrane sealed the surface due to its hydrophilicity. A droplet of cell lysis buffer was then applied to the membrane and allowed to pass into each individual microwell. Cells were lysed within individual microwells, and target proteins were captured by nanosensors. Binding of proteins to the nanosensors induced a color shift that correlated with protein concentration and is shown in Figures 19A-19C.

[0075] (Example 5) In this example, multiple microwell arrays were integrated on a glass slide to detect multiple proteins from single cells. A single microwell array measured 1 cm x 1 cm and accommodated 1,000 cell traps, with nanosensors embedded in each microwell. As shown in Figure 20, a 75 mm x 25 mm glass slide accommodated 10 microwells. Each microwell was functionalized with a different antibody. Therefore, a glass slide with 10 microwells could detect 10 different proteins in 10 groups of 1,000 single cells.

[0076] To detect multiple proteins within the same group of single cells, nanosensors were fabricated on silicon nano- or micro-sized needles. The nanosensors were made of gold, silver, or silicon. The nanoneedles were spaced at the same intervals as the microwells (see Figures 21A-21C). Therefore, each nanoneedle could be aligned with each microwell. The nanosensors on each nanoneedle detected the protein concentration in the cell lysate from each microwell. To analyze multiple proteins from the same group of single cells, different nanoneedles functionalized with different antibodies were sequentially contacted with the same microwell array.

[0077] (Example 6) When the analyte in solution reaches sub-pM concentration levels, only a maximum of one analyte molecule is available to bind to each individual nanosensor, while many nanosensors in the array have no bound molecules. In this detection scheme, the threshold level of signal (e.g., color change, fluorescence) from the nanosensor can be assigned either 1 or 0. The concentration of the analyte in solution can be derived by directly counting the number of nanosensors with a signal assignment of 1, thus suppressing the noise signal inherent in traditional bulk colorimetric or fluorometric measurements at ultralow concentrations.

[0078] Because nanosensor arrays have a typical pitch in the 1-10 micrometer range, it is possible to densely pack 10,000-1,000,000 nanosensors into a small area (e.g., 1 mm x 1 mm). Figures 22A-22I illustrate the optical signal of a nanosensor array under this scheme. In Figures 22A-22I, 16 blocks of nanosensor arrays were fabricated on a silicon chip. Each block has a matrix of 32 x 32 nanosensors spaced 2 micrometers apart. Each nanosensor has a nanorod shape with a diameter of 95 nm and a length of 200 nm. Figure 22A shows the chip under dark-field imaging, with each nanosensor exhibiting a green scattering spot collected by a color camera. The nanochip was functionalized with 2% (3-aminopropyl)trimethoxysilane (APTMS) in 95% ethanol for 10 minutes, washed with ethanol, and heated at 80 °C for 2 hours.

[0079] The chip was then functionalized with 10 mM glutaraldehyde and 10 mM sodium cyanoborohydride for 1 hour. The sensor surface was coated with 1 microgram / ml of tau protein. Various concentrations of anti-tau protein were then flowed over the chip as the detection target. The anti-tau protein was prepared in a PBS buffer solution containing 0.1% gelatin and 150 mM NaCl. The anti-tau protein was labeled with horseradish peroxidase. After reacting with a chromogenic substrate, an insoluble layer of deposit formed on the nanosensor surface, causing a change in the nanosensor's optical resonance.

[0080] Figure 22B shows a dark-field image after the assay; each nanosensor changed from green to yellow upon detecting proteins captured by the antibodies functionalized on the nanosensor. Figure 22C shows the hue change extracted from aligning Figures 22A and 22B and subtracting corresponding spots. At an anti-tau protein concentration of 10 pM, all nanosensors in Figure 22C exhibited a delta hue at approximately -40. Figures 22D-22F show embodiments in which the anti-tau protein concentration was reduced to 1 pM. Figures 22G-22I show embodiments in which the anti-tau protein concentration was reduced to 0.1 pM. A subset of nanosensors had an observable delta hue in Figures 22D-22F. As shown in Figures 22G-22I, many nanosensors did not have an observable delta hue, indicating that many nanosensors did not have proteins bound to the surface.

[0081] While several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining the results, and / or achieving one or more of the advantages described herein, and each such variation and / or modification is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and / or configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application for which the teachings of the present invention are used. Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Accordingly, the foregoing embodiments are presented by way of example only, and it should be understood that, within the scope of the appended claims and their equivalents, the invention may be practiced otherwise than as specifically described and claimed. The present invention relates to each individual structure, system, article, material, kit, and / or method described herein. Furthermore, combinations of two or more such structures, systems, articles, materials, kits, and / or methods are within the scope of the present invention, provided that such structures, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0082] In the event that the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and / or inconsistent disclosure with respect to each other, the document having the later effective date shall control.

[0083] All definitions defined and used herein should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0084] The indefinite articles "a" and "an," as used in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0085] The term "and / or," as used in the specification and claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the conjoined elements. Other elements other than the elements specifically identified by the "and / or" term may optionally be present, whether related or unrelated to the elements specifically identified. Thus, as a non-limiting example, when a reference to "A and / or B" is used in conjunction with open-ended language such as "comprising," it can refer in one embodiment to A only (optionally including elements other than B); in another embodiment to B only (optionally including elements other than A); in yet another embodiment to both A and B (optionally including other elements); etc.

[0086] As used in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., the inclusion of at least one, two or more, and optionally additional unlisted items, of a number or list of elements. Only terms clearly indicating the contrary, such as "only one of" or "only one," or when "consisting of" is used in the claims, will refer to the inclusion of only one element of a plurality or list of elements. In general, the term "or" as used herein should be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "only one."

[0087] As used in the specification and claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but necessarily including at least one of all elements specifically listed in the list of elements, and not excluding combinations of elements in the list of elements. This definition allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to the elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") refers, in one embodiment, to at least one A in the absence of B, optionally including two or more A's (optionally including elements other than B); in another embodiment, to at least one B in the absence of A, optionally including two or more B's (optionally including elements other than A); in yet another embodiment, to at least one A, optionally two or more A's, and at least one B, optionally including two or more B's (optionally including other elements); etc.

[0088] When the term "about" is used herein in connection with a number, it should be understood that further embodiments of the present invention include that number unmodified by the presence of the term "about."

[0089] It should also be understood that, unless expressly stated to the contrary, in any method in the claims including more than one step or action, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are described.

[0090] In the claims, as in the specification above, all transitional phrases, e.g., "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," etc., are to be understood as open-ended, i.e., to mean inclusive without limitation. As set forth in Section 2111.03 of the U.S. Patent Office Manual of Patent Examining Procedure, only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively.

Claims

1. a microwell array including wells containing nanoparticles positioned distally to the ends of the nanostructures; The article, wherein the nanoparticles interact with incident light via surface plasmon resonance, electric resonance and / or magnetic resonance.

2. The article of claim 1 , further comprising a reactive entity immobilized to the nanoparticle.

3. The article of claim 2 , wherein binding of the analyte to the reactive entity causes a change in light refracted from the nanoparticle.

4. 4. The article of claim 2 or 3, wherein the reactive entity comprises an antibody.

5. The article of any one of claims 2 to 4, wherein the reactive entity comprises an aptamer.

6. The article of any one of claims 2 to 5, wherein the reactive entity comprises a protein.

7. The article of any one of claims 2 to 6, wherein the reactive entity comprises an oligonucleotide.

8. The article of any one of claims 1 to 7, further comprising a detector positioned to detect light refracted from the nanoparticles.

9. The article of claim 8 , wherein the detector is a spectroscopic detector.

10. The article of any one of claims 1 to 9, wherein the incident light is plane polarized light.

11. The article of any one of claims 1 to 10, wherein the incident light comprises visible light.

12. The article of any one of claims 1 to 11, further comprising a light source positioned to direct incident light onto the nanoparticles.

13. The article of claim 12 , wherein the light source comprises a laser.

14. The article of claim 13, wherein the laser is a He—Ne laser.

15. The article of any one of claims 1 to 14, wherein only one nanoparticle is attached to the nanostructure.

16. The article of any one of claims 1 to 15, wherein the nanoparticles comprise a metal.

17. The article of any one of claims 1 to 16, wherein the nanoparticles comprise gold.

18. The article of any one of claims 1 to 16, wherein the nanoparticles comprise silver.

19. The article of any one of claims 1 to 16, wherein the nanoparticles comprise quantum dots.

20. The article of any one of claims 1 to 19, wherein the nanoparticles have an average diameter of less than about 3 nm.

21. The article of any one of claims 1 to 19, wherein the nanoparticles have an average diameter of at least about 0.5 nm.

22. The article of any one of claims 1 to 21, wherein the nanostructures comprise silicon.

23. The article of any one of claims 1 to 22, wherein the nanostructures are substantially vertically aligned.

24. The article of any one of claims 1 to 23, wherein the nanostructures are nanoneedles.

25. The article of any one of claims 1 to 23, wherein the nanostructures are nanowires.

26. The article of any one of claims 1 to 23, wherein the nanostructures are nanorods.

27. The article of any one of claims 1 to 23, wherein the nanostructures are nanocones.

28. The article of any one of claims 1 to 23, wherein the nanostructures are nanopillars.

29. The article of any one of claims 1 to 28, wherein the nanostructures have a length of less than about 5 micrometers.

30. The article of any one of claims 1 to 29, wherein the nanostructures have a length greater than at least about 0.1 micrometers.

31. The article of any one of claims 1 to 30, wherein the nanostructures have an average cross-sectional diameter of at least about 50 nm.

32. The article of any one of claims 1 to 31, wherein the nanostructures have an average cross-sectional diameter of less than about 500 nm.

33. The article of any one of claims 1 to 32, wherein the microwell array comprises glass.

34. The article of any one of claims 1 to 33, wherein the microwell array comprises silicon.

35. The article of any one of claims 1 to 34, wherein the microwell array is fabricated using photolithography.

36. The article of any one of claims 1 to 35, wherein the nanostructures comprise a semiconductor.

37. The article of any one of claims 1 to 36, wherein the nanostructures comprise silicon.

38. The article of any one of claims 1 to 37, wherein the nanostructures and the microwell array have substantially the same composition.

39. The article of any one of claims 1 to 38, wherein the nanostructures and the microwell array define a single material.

40. The article of any one of claims 1 to 39, wherein the wells have a diameter of less than 50 micrometers.

41. The article of any one of claims 1 to 40, wherein the wells have a depth of at least 20 microns.

42. 42. The article of any one of claims 1-41, wherein the microwell array comprises a plurality of wells each containing a nanoparticle positioned distally to the end of the nanostructure.

43. 43. The article of any one of claims 1-42, wherein the microwell array comprises at least 10 wells, each containing a nanoparticle positioned distally at the end of the nanostructure.

44. The article of any one of claims 1 to 43, wherein the nanostructures have an average pitch of less than 100 micrometers.

45. The article of any one of claims 1 to 44, wherein the well further comprises cells.

46. The article of any one of claims 1 to 45, wherein the well further comprises a cell lysate.

47. The article of any one of claims 1 to 46, further comprising a membrane sealing the well.

48. 48. The article of claim 47, wherein the membrane is a semi-permeable membrane.

49. 49. The article of any one of claims 47 or 48, wherein the membrane is non-porous.

50. The article of any one of claims 47 to 49, wherein the membrane comprises polycarbonate.

51. applying electromagnetic radiation to nanoparticles positioned distal to the ends of the nanostructures, the nanoparticles interacting with and modifying the electromagnetic radiation via surface plasmon resonance, electric resonance, and / or magnetic resonance; and measuring the altered electromagnetic radiation.

52. 52. The method of claim 51, further comprising applying electromagnetic radiation to the nanoparticles.

53. 53. The method of claim 51 or 52, wherein the electromagnetic radiation comprises visible light.

54. 54. The method of any one of claims 51 to 53, further comprising exposing a solution suspected of containing the analyte to said nanoparticles, wherein the nanostructures further comprise reactive entities capable of interacting with the analyte.

55. 55. The method of claim 54, wherein the solution comprises lysed cells from which the specimen is derived.

56. 56. The method of any one of claims 54 or 55, further comprising determining the reactive entity by measuring the altered electromagnetic radiation.

57. 57. The method of any one of claims 51 to 56, wherein the nanoparticles are positioned within wells of a microwell array.

58. positioning cells in wells of a microwell array, the wells further comprising nanoparticles positioned distal to an end of the nanostructure and a reactive entity at least partially coated on the nanoparticles; lysing the cells in the well to release analytes suspected of being capable of binding to a reactive entity; applying electromagnetic radiation to the nanoparticles, wherein the nanoparticles interact with and modify the electromagnetic radiation via surface plasmon resonance, electric resonance, and / or magnetic resonance; and measuring the altered electromagnetic radiation to determine the analyte.

59. acquiring a first optical color image of an array of nanostructures on a substrate, the nanostructures having a cross-sectional dimension orthogonal to a direction in which the first optical image is acquired that is less than 700 nm, the nanostructures being at least partially coated with a reactive entity; allowing an interaction to occur between the reaction entity and the analyte; acquiring a second optical color image of the array of nanostructures; measuring a color change between the first optical image and the second optical image, the color change being caused by an interaction between the reaction entity and the analyte.

60. 60. The method of claim 59, wherein the array of nanostructures has an average spacing between nanostructures of less than about 3 μm.

61. 61. The method of claim 59 or 60, wherein the array of nanostructures has an average spacing between nanostructures of less than about 500 nm.

62. 62. The method of any one of claims 59 to 61, wherein the reactive entity comprises an antibody.

63. The method of any one of claims 59 to 62, wherein the reaction entity comprises an aptamer.

64. 64. The method of any one of claims 59 to 63, wherein the reactive entity comprises a protein.

65. 65. The method of any one of claims 59 to 64, wherein the reaction entity comprises an oligonucleotide.

66. 66. The method of any one of claims 59 to 65, wherein the reaction entity comprises an enzymatic reaction product induced by a chromogenic substrate labeled to the analyte.

67. 67. The method of claim 66, wherein the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine.

68. 67. The method of claim 66, wherein the chromogenic substrate is 3,3'-diaminobenzidine.

69. 67. The method of claim 66, wherein the chromogenic substrate is 2,2'-azino-di-(3-ethylbenzothiazoline-6-sulfonic acid.

70. 70. The method of any one of claims 59 to 69, wherein the nanostructures are nanoneedles.

71. 71. The method of any one of claims 59 to 70, wherein the nanostructures have a length of less than about 5 micrometers.

72. 72. The method of any one of claims 59 to 71, wherein the nanostructures have a length greater than at least about 0.1 micrometers.

73. 73. The method of any one of claims 59 to 72, wherein the nanostructures comprise a semiconductor.

74. The method of any one of claims 59 to 73, wherein the nanostructures comprise silicon.

75. 75. The method of any one of claims 59 to 74, wherein the nanostructures consist essentially of silicon.

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