WO2008021614A2 - Coded particle arrays for high throughput analyte analysis - Google Patents

Abstract

Disclosed herein are compositions and methods for analyte analysis using coded particle arrays.

Description

CODED PARTICLE ARRAYS FOR HIGH THROUGHPUT ANALYTE

ANALYSIS

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of U.S. Provisional Application No. 60/822,834, filed August 18, 2006, which is hereby incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant 0243423 awarded by NSF. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

Current protein detection systems can not analyze many different proteins with high sensitivity that have been captured in solution phase with little fear of cross contamination between capture proteins. Systems that do allow for solution phase capture of target proteins, such as DNA coded nanoparticles, either do not allow for convenient multiple protein analysis or lack decoding resolution. Moreover, DNA coded nanoparticles must be decoded using DNA chips and/or PCR, which are extensive decoding procedures that also rely on problematic spotting technology. Metallically coded nanowires limit their minimal bit widths to just the optical range, 1- 2.0 um. Color or shape variant particles are also limited to the optical ranges of a few microns and lack large coding ranges. Moreover, coded nanowires have not yet demonstrated a promising protein detection system. Protein detection systems based upon spotting machines and organized arrays suffer from strong cross contamination with neighboring protein spots, protein dehydration, and low protein density. Spotting also requires that the proteins to be held in a highly viscous solution or a gel matrix to reduce dehydration. This greatly restricts protein activity. Each spot requires at minimal 150μm² to allow for the solution drop size and adequate neighbor separation.

BRIEF SUMMARY OF THE INVENTION

Disclosed herein is a method and compositions for detecting analyte(s), comprising bringing into contact a sample and one or more coded particles, wherein at least one of the coded particles comprises a capture molecule specific for an analyte. The disclosed method makes use of coded particles with which specific target analyte interact. Once gathered onto a substrate, the disclosed particles can form a highly packed grouping of particles. For example, the coded particles can be brought into contact with analytes, such as proteins, in solution. This allows the interaction of the analyte to occur in solution (rather than on a substrate) where the analyte and capture molecule can have or be in their natural or physiological state or conformation. Once gathered onto a substrate, the coded particles can form a highly packed grouping of particles that can include a mixture of different coded particles with different analytes bound. For example, numerous different coded particles can fit within the current minimum 150 um² spot size that contains only a single type of analyte.

Also disclosed is a method and compositions for simultaneously detecting and identifying multiple analytes in a sample, comprising: coding a particle for each analyte, coating the coded particles with a capture molecule specific for the analytes, adding the coding particles to the sample, randomly gathering the particles onto a substrate, detecting the analyte, and decoding the particles to identify the analyte.

Also disclosed is a method and compositions for separating multiple analytes in a sample, comprising: coding a particle for each analyte, coating the coded particles with a capture molecule specific for the analytes, adding the coding particles to the sample, and separating the coded particles in a substrate by the electrical properties, magnetic properties, conductance properties, refractive properties, reflective properties, and/or plasmon resonance properties.

Also disclosed herein is an apparatus for detecting analytes, the apparatus comprising coded particles, wherein the particles are coded for each analyte, wherein the coded particles comprise a capture molecule specific for the analytes, a substrate for gathering the coded particles, and a scanning device for detecting the analyte and decoding the coded particles.

The coded particles can comprise a dopable semiconducting material. The dopable semiconducting material can be selected from the group consisting of silicon, polysilicon, arsenide, germanium, indium arsenide, indium tin oxide, titanium oxide, and diamond. The coded particles can be less than 100, 50, 20, 10, or 1 μm in diameter. The coded particles can have a reference feature. The reference feature can be a reference angle. The reference feature can be a variation in intensity. The coded particles can be doped. The capture molecule can be an antibody, aptamer, epitome, single stranded DNA or RNA, or molecularly imprinted polymer (MIP). The analyte can be detected using a marker. The marker can comprise a nanoparticle. The coded particles can be decoded using scanning probe microscopy (SPM). The coded particles can be decoded using local conductance measurements. The coded particles can be decoded using electrostatic measurements. The coded particles can be decoded using topographical measurements. The coded particles can be gathered onto a substrate. The coded particles can be gathered onto a substrate prior to, simultaneous with, or following bringing into contact the sample and the coded particles. The coded particles can be gathered at a density of greater than 1, 10, 20, 50, or 100 particle per 10 μm². The coded particles can be gathered onto the substrate randomly. The coded particles can be coded with different patterns. The different patterns can be detected using scanning probe microscopy (SPM). Multiple analytes can be detected. Multiple analytes can be detected simultaneously. The coded particles can be coded with different patterns. The coded particles can have different patterns each comprise a different capture molecule. The different capture molecules can each be specific for a different analyte, whereby a particular pattern is indicative of a particular analyte. The coded particles can collectively comprise 5 or more, 6, or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 200 or more, 300 or more, 400 or more, or 500 or more different capture molecules. Decoding the coded particles need not depend on the coded particles being at a predetermined position or being arranged in a pattern. Identification of the analyte need not require predetermined knowledge of the position of the analyte on a substrate. The coded particles need not be in a predetermined location during decoding. The coded particles can be in solution or in suspension after being brought into contact with the sample.

Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or can be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.

Figure 1 shows demonstration of particle packing density.

Figure 2 shows demonstration of decoding. Left: topography is flat. Right: subsurface decoding.

Figure 3 shows schematic of ESF dual pass technique.

Figure 4 shows demonstration of protein detection. Shown is a SPM image of Human Anti-IgG modified Nano Particles bound to IgG.

Figure 5 shows illustration of the data obtained from the high density particle array. Solutions are pre-prepared with coded particles comprising capture molecules (e.g. antibodies) specific for each analyte. Mixed coded particles are combined with the sample comprising the analyte(s)/ antigen(s). The particles are assembled onto a substrate for data collection. Both decoding and detection are obtained simultaneously. The dark single thick vertical stripe, code 10100, indicates the presence of the maker protein. Thus, target protein 10100 was captured in the tested solution. The redundancy of the particles increases the reliability of the system.

Figure 6 shows decoding of coded particles by topography, surface potential, and amplitude.

Figure 7 shows images of dope-coded particle assembly and handling process, (a) Scanning electron microscope image of hexagonal metal layer (25 ran Cr/20 nm Ni/35 nm Au) patterned onto 200 nm dope-coded polysilicon after reactive ion etching to remove the unprotected polysilicon and expose the underlying 100 nm sacrificial oxide layer, (b) After BHF etching of sacrificial oxide layer, dope-coded particles were stamped and transferred onto a secondary gold coated substrate with polysilicon face up. (c) After suspension phase protein binding, the particles are recollected and randomly assembled in a high density random arrangement, (d) The dope-coded particles may be decoded optically, or (e) with a surface potential scanning probe to observe the doped regions. The extracted portion of the surface potential image demonstrates subsurface decoding of the particle's doped regions while being nearly independent of the surface features. Figure 8 shows topographical AFM images of dope-coded biosensing particles after exposure to 200 ng/ml anti-human IgG conjugated with 20 nm nanoparticles. The surface was scanned in tapping mode at a rate of 0.5 Hz with a stiff 42 N/m tip. Particle density analysis is a built-in function of the DI Nanoscope software. Only particles resembling the size of the 20 nm nanoparticles were included in the particle density analysis. The overlaid lines indicate how the perpendicular and parallel codes were translated. The particle density analysis was obtained from the regions indicated by the overlain squares and is shown enlarged to the right of each AFM image. Note that the particle code is obtained regardless of the nanoparticle density measured, (a) Bar graph summarizing the average marker protein density detected from both sets of experiments. The inset depicts how the two codes were identified relative to the hexagon edge, (b) Perpendicular dope-coded particle as a control with no target protein immobilized, (c) Parallel dope-coded particle modified with complementary target protein 10 μg/ml human IgG. (d) Separate set of experiments with parallel-coded particle modified with noncomplementary target protein 10 μg/ml anti-human IgG.

Figure 9 shows analysis of μ-contact printed protein patterns by SPR-I detection.

Figure 10 shows an example of a multi-channel microfluidic device for protein separation by SPR-I. DETAILED DESCRIPTION OF THE INVENTION

The disclosed method and compositions can be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.

It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, the singular forms "a ", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a particle" includes a plurality of such particles, reference to "the particle" is a reference to one or more particles and equivalents thereof known to those skilled in the art, and so forth. "Optional" or "optionally" means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present. Ranges can be expressed herein as from "about" one particular value, and/or to

"about" another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and/or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art. Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises," means "including but not limited to," and is not intended to exclude, for example, other additives, components, integers or steps. 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 method and compositions described herein. Such equivalents are intended to be encompassed by the appended claims.

Materials Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particle is disclosed and discussed and a number of modifications that can be made to a number of molecules including the particle are discussed, each and every combination and permutation of particle and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

A. Coded Particles

Disclosed are coded particles which can be used in the detection or separation of analytes. The coded particles can be coded with a pattern that can be detected using microscale and nanoscale detection and measurement techniques (for example, using scanning probe microscopy (SPM)). Thus, for example, coded particles can be coded with patterns of, for example, microscale or nanoscale topography, electrical properties, magnetic properties, conductance properties, refractive properties, reflective properties, and/or plasmon resonance properties. The coded particles can include one or more capture molecules that can be used to associate one or more analytes with the coded particle. Generally, the coded particles can be coded with a pattern specific for each analyte target. The pattern can be embodied in changes in the materials or material properties of the coded particle. As an example, a silicon particle can comprise lines of boron. Thus a pattern could then be distinguished based on the number, thickness, spacing, and/or intensity of the lines. The pattern can also be based on a variation in coding intensity (for example, a darker or lighter line).

The pattern of a coded particle can be any shape, size, level or intensity that can be detected or measured using the microscale or nanoscale detection and measurement technique and which can be distinguished form other patterns to be used in the same assay. For example, lines of various thicknesses, orientation and/or intensity can be used. Similarly, various shapes can be used. Any combination of these or other such features can be used. Different pattern elements can be used on different coded particles. The pattern on a given coded particle can also be made up of a combination of different pattern features. For example, a coded particle can have vertical and horizontal lines (forming a cross-hatch), lines and squares, lines of different thickness, features of different concentration or intensity, square and triangles. Thus, pattern features and combinations of pattern features can be used to make up a large number or different and distinguishable codes. This allows for large scale multiplex detection and decoding of many coded particles in the same assay (and thus the separate detection and identification of many analytes). Other such patterns, designs, or marks are contemplated herein for coding the disclosed particles. The coded particle can also have a reference feature. For example, the reference feature can be a reference angle. A reference angle would allow for the distinction of, for example, horizontal, diagonal, and vertical lines or features.

The coded particles can be any suitable size, although microscale particles are particularly useful because they allow many coded particles to be gathered in a smaller space. For example, the coded particles can be less than 100, 50, 20, 10, or 1 μm in diameter/ width. The use of such small coded particles allows the coded particles to be used in solution. This allows analytes and their respective capture molecules to interact under conditions more similar to physiological conditions and/or conditions that allow the analytes and capture molecules to maintain their normal or natural conformation than is typically possible for surface immobilization.

The coded particle can be any shape amenable to an effective coding scheme. For example, the coded particles can be circular, round, triangular, square, rectangular, pentagonal, hexagonal, octagonal, and/or polygonal (regular and/or irregular). Coded particles of the same or similar general shape can be used together. Alternatively, a combination of differently shaped coded particles can be used together. It is useful if the shape(s) of the coded particles to be used together can pack together, although this is not required.

In one aspect of the disclosed method, the coded particles can be coded with different patterns, where the coded particles having different patterns each comprise a different capture molecule and where the different capture molecules are each specific for a different analyte. In this way, a particular pattern can be indicative of a particular analyte. For example, the coded particles can collectively comprise 5 or more, 6, or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 200 or more, 300 or more, 400 or more, or 500 or more different capture molecules. 1. Doping

The coded particles can be coded using any suitable material or technique that can produce patterns detectable using microscale or nanoscale techniques. For example, the coded particles can be coded by doping. As used herein, "doping" refers to the process of intentionally introducing impurities into a material such as a semiconductor. Doping is most well known for the introduction of impurities into an extremely pure (also referred to as intrinsic) semiconductor in order to change its electrical properties. Such techniques, and the semiconductors used, can be used with and for the disclosed coded particles. The disclosed coded particles need not be made from intrinsic semiconductor material if semiconductor material is used since the disclosed method does not require such levels of purity. The impurities used for doping can differ dependent upon the type of semiconductor. Thus, the coded particle can comprise a dopable semiconducting material. Non-limiting examples of dopable semiconducting material include silicon, polysilicon, arsenide, germanium, indium arsenide, indium tin oxide, titanium oxide, and diamond. Suitable doping materials for such semiconductors are known and can be used in the coded particles. Non-limiting examples of dopants include boron, arsenic, phosphorous, gallium, silicon, and indium. Doping can be done in patterns of, for example, shape and/or intensity of doping.

For the group IV semiconductors such as silicon, germanium, and silicon carbide, the useful dopants are group III or group V elements. Boron, arsenic, phosphorus and gallium can be used to dope silicon. Boron is particularly useful as a p-type dopant for silicon because it diffuses at a rate which makes doped depths easily controllable.

Phosphorus is typically used for bulk doping of silicon wafers, while arsenic is used to diffuse junctions, since it diffuses more slowly than phosphorus and is thus more controllable. By doping silicon with group V elements such as Phosphorus, extra valence electrons are added which become unbonded from individual atoms and allow the compound to be electrically conductive, n-type material. Doping with group III elements, such as Boron, which are missing the forth valence electron creates "broken bonds", or holes, in the silicon lattice that are free to move. This is electrically conductive, p-type material. In this context then, a group V element is said to behave as an electron donor, and a group III element as an acceptor. Other compound semiconductors such as gallium arsenide can be doped with group rv elements such as silicon. In most cases, a low concentration of silicon will preferentially substitute for the group III element, and function as a donor.

Conductive organic polymers can be doped by adding chemical reactants to oxidize (or sometimes reduce) the system to push electrons into the conducting orbitals within the already (potentially) conducting system. There are two primary methods of doping a conductive polymer, both through an oxidation-reduction (redox) process. These can be used to code the disclosed coded particles. The first method, chemical doping, involves exposing a polymer, such as melanin (typically a thin film), to an oxidant (typically iodine or bromine) or reductant (far less common, but typically involves alkali metals). The second is electrochemical doping in which a polymer-coated, working electrode is suspended in an electrolyte solution in which the polymer is insoluble along with separate counter and reference electrodes. An electric potential difference is created between the electrodes which causes a charge (and the appropriate counter ion from the electrolyte) to enter the polymer in the form of electron addition (n doping) or removal (p doping).

The number of dopant atoms needed to create a detectable difference in the electrical properties of a coded particle is very small. Where a comparatively small number of dopant atoms are added (of the order of 1 every 100,000,000 atoms) then the doping is said to be low, or light. Where many more are added (of the order of 1 in 10,000) then the doping is referred to as heavy, or high. This is often shown as n+ for n- type dopant or p+ for p-type doping. 2. Photolithography The disclosed coded particles can be coded or doped by photolithography.

Photolithography is a process — used in semiconductor device fabrication, for example — to transfer a pattern from a photomask (also called reticle) to the surface of a substrate. For example, crystalline silicon, glass, sapphire, and metal can be used as a substrate for photolithography. Photolithography (also referred to as "microlithography" or "nanolithography") bears a similarity to the conventional lithography used in printing and shares some of the fundamental principles of photographic processes.

Photolithography involves a combination of: substrate preparation, chemical deposition (e.g., evaporation, sputtering, plasma enhanced chemical vapor deposition), photoresist spinning, soft-baking, exposure, developing, hard-baking, etching, and various other chemical treatments (thinning agents, edge-bead removal etc.) in repeated steps on an initially flat substrate.

A part of a typical lithography procedure would begin by depositing a layer of conductive metal several nanometers thick on the substrate. A layer of photoresist — a chemical that hardens when exposed to light (often ultraviolet) — can be applied on top of the metal layer. The photoresist can be selectively "hardened" by illuminating it in specific places. For this purpose a transparent plate with patterns printed on it, called a photomask or shadowmask, can be used together with an illumination source to shine light on specific parts of the photoresist. Some photoresists work well under broadband ultraviolet light, whereas others are designed to be sensitive at specific frequencies to ultraviolet light. It is also possible to use other types of resist that are sensitive to X-Rays and others that are sensitive to electron-beam exposure.

Generally most types of photoresist will be available as either "positive" or "negative." With positive resists the area that you can see (masked) on the photomask is the area that you will see upon developing of the photoresist. With negative resists it is the inverse, so any area that is exposed will remain, whilst any areas that is not exposed will be developed. After developing, the resist can be hard-baked before subjecting to a chemical etching stage which will remove the metal underneath. Finally, the hardened photoresist can be etched using a different chemical treatment, and all that remains is a layer of metal in the same shape as the mask (or the inverse if negative resist has been used). B. Analytes

Analytes can be any molecule or portion of a molecule that is to be detected, measured, or otherwise analyzed. An analyte need not be a physically separate molecule, but can be a part of a larger molecule. Analytes include biological molecules, organic molecules, chemicals, compositions, and any other molecule or structure to which the disclosed method can be adapted. It should be understood that different forms of the disclosed method are more suitable for some types of analytes than other forms of the method. Analytes are also referred to as target molecules.

Analytes can be biological molecules. Biological molecules include but are not limited to proteins, peptides, enzymes, amino acid modifications, protein domains, protein motifs, nucleic acid molecules, nucleic acid sequences, DNA, RNA, mRNA, cDNA, metabolites, carbohydrates, and nucleic acid motifs. As used herein, "biological molecule" and "biomolecule" refer to any molecule or portion of a molecule or multi- molecular assembly or composition, that has a biological origin, is related to a molecule or portion of a molecule or multi-molecular assembly or composition that has a biological origin. Biomolecules can be completely artificial molecules that are related to molecules of biological origin. Although reference is made above and elsewhere herein to detection of a "protein" or "proteins," the disclosed method and compositions encompass proteins, peptides, and fragments of proteins or peptides. Thus, reference to a protein herein is intended to refer to proteins, peptides, and fragments of proteins or peptides unless the context clearly indicates otherwise.

Any sample from any source can be used with the disclosed method. In general, analyte samples should be samples that contain, or may contain, analytes. Examples of suitable analyte samples include cell samples, tissue samples, cell extracts, components or fractions purified from another sample, environmental samples, culture samples, tissue samples, bodily fluids, and biopsy samples. Numerous other sources of samples are known or can be developed and any can be used with the disclosed method. Preferred analyte samples for use with the disclosed method are samples of cells and tissues. Analyte samples can be complex, simple, or anywhere in between. For example, an analyte sample can include a complex mixture of biological molecules (a tissue sample, for example), an analyte sample can be a highly purified protein preparation, or a single type of molecule. C. Capture Molecules The capture molecule can be any specific binding molecule for an analyte of interest. A specific binding molecule is a molecule that interacts specifically with a particular molecule or moiety. The molecule or moiety that interacts specifically with a specific binding molecule is referred to herein as an analyte. It is to be understood that the term analyte refers to both separate molecules and to portions of such molecules, such as an epitope of a protein, that interacts specifically with a specific binding molecule. Many molecules that interact with analytes of interest are know and these can be used with the disclosed compositions and methods.

A specific binding molecule that interacts specifically with a particular analyte is said to be specific for that analyte. For example, where the specific binding molecule is an antibody that associates with a particular antigen, the specific binding molecule is said to be specific for that antigen. The antigen is the analyte. A composition containing the specific binding molecule can also be referred to as being specific for a particular analyte. Specific binding molecules include antibodies, ligands, binding proteins, receptor proteins, haptens, aptamers, carbohydrates, synthetic polyamides, peptide nucleic acids, or oligonucleotides, epitomes, and molecularly imprinted polymers (MIPs). Examples of DNA binding proteins include zinc finger motifs, leucine zipper motifs, helix-turn-helix motifs. These motifs can be combined in the same specific binding molecule. Antibodies useful as the specific binding molecules, can be obtained commercially or produced using well established methods. For example, Johnstone and Thorpe, Immunochemistry In Practice (Blackwell Scientific Publications, Oxford, England, 1987) on pages 30-85, describe general methods useful for producing both polyclonal and monoclonal antibodies. The entire book describes many general techniques and principles for the use of antibodies in assay systems.

Properties of zinc fingers, zinc finger motifs, and their interactions, are described by Nardelli et al., Zinc fmger-DNA recognition: analysis of base specificity by site- directed mutagenesis. Nucleic Acids Res, 20(16):4137-44 (1992), Jamieson et al., In vitro selection of zinc fingers with altered DNA-binding specificity. Biochemistry,

33(19):5689-95 (1994), Chandrasegaran and Smith, Chimeric restriction enzymes: what is next? Biol Chem, 380(7-8):841-8 (1999), and Smith et al., A detailed study of the substrate specificity of a chimeric restriction enzyme. Nucleic Acids Res, 27(2):674-81 (1999). One form of specific binding molecule is an oligonucleotide or oligonucleotide derivative. Such specific binding molecules are designed for and can be used to detect specific nucleic acid sequences. Thus, the analyte for oligonucleotide specific binding molecules are nucleic acid sequences. The analyte can be a nucleotide sequence within a larger nucleic acid molecule. An oligonucleotide specific binding molecule can be any length that supports specific and stable hybridization between the reporter binding probe and the analyte. Thus, the specific binding molecule can comprise an oligonucleotide with a length of, for example, 10 to 40, 16 to 25 nucleotides long. The oligonucleotide specific binding molecule can be a peptide nucleic acid. Peptide nucleic acid forms a stable hybrid with DNA. This allows a peptide nucleic acid specific binding molecule to remain firmly adhered to the target sequence.

Oligonucleotide specific binding molecules can also make use of the triple helix chemical bonding technology described by Gasparro et al., Nucleic Acids Res., 22(14):2845-2852 (1994). Briefly, the oligonucleotide specific binding molecule is designed to form a triple helix when hybridized to a target sequence. This is accomplished generally as known, preferably by selecting either a primarily homopurine or primarily homopyrimidine target sequence. The matching oligonucleotide sequence which constitutes the specific binding molecule will be complementary to the selected target sequence and thus be primarily homopyrimidine or primarily homopurine, respectively. The specific binding molecule contains a chemically linked psoralen derivative. Upon hybridization of the specific binding molecule to a target sequence, a triple helix forms. By exposing the triple helix to low wavelength ultraviolet radiation, the psoralen derivative mediates cross-linking of the probe to the target sequence. D. Substrates

The coded particles can be gathered onto a substrate prior to decoding. The substrate can include any solid material with which coded particles can be associated. This includes materials such as acrylamide, agarose, cellulose, nitrocellulose, glass, gold, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicates, polycarbonates, teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, functionalized silane, polypropylfumerate, collagen, glycosaminoglycans, and polyamino acids. Substrates can have any useful form including thin film, membrane, bottles, dishes, fibers, gels, woven fibers, shaped polymers, particles, beads, microparticles, or a combination. Substrates can be porous or non-porous. A chip is a rectangular or square small piece of material. Useful forms for substrates are sheets, films, and chips. A useful form for a substrate is a microtiter dish.

Coded particles can be associated with or gathered on a substrate at high density. For example, the coded particles can be gathered at a density of greater than 1, 10, 20, 50, or 100 particle per 10 μm². The coded particles can be gathered onto the substrate randomly. E. Markers

The presence of an analyte associated with a coded particle can be detected on the particle either directly or by using a marker. The marker generally serves to make the analyte more detectable. A marker can be linked to a binding molecule that can bind to an analyte. The binding molecule can be specific, like the capture molecule, or it can be nonspecific. Alternatively, the marker can bind directly to the analyte(s).

Generally, the marker can be a molecule capable of detection by SPM. Thus, the marker can be, for example, a nanoparticle. Any nanoparticle that can be bound, attached or coupled to a specific binding molecule can be used. For example, nanoparticles can comprise metals, alloys, polymers, or a multilayer with materials such as gold, iron, nickel, cobalt, polystyrene, polyester, or nylon, or combinations thereof. Multiple markers can be used, for example by using nanoparticles of different sizes, in order to increase discrimination of analytes.

Other methods known in the art for the detection and analysis of analytes, such as mass spectroscopy, can be used to detect and analyze the analyte on the coded particle. For example, matrix-assisted laser desorption/ionization (MALDI), surface-assisted laser desorption/ionization (SALDI), and surface-enhanced laser desorption/ionization (SELDI) can be used for detection and analysis. MALDI is a soft ionization technique used in mass spectrometry, allowing, among other things, the ionization of biomolecules (biopolymers such as proteins, peptides and sugars) which tend to be more fragile and quickly lose structure when ionized by more conventional ionization methods. SALDI is MALDI using a liquid plus particulate matrix, and SELDI is MALDI using a biochemical affinity target. Thus, as used herein, "MALDI" refers to all mass spectrometer methods using laser desorption/ionization. MALDI and mass spectrometry techniques are well known and can be applied to the detection and analysis of the disclosed analytes. Any suitable mass spectrometer can be used to detect and analyze the analytes. For example, the type of a mass spectrometer most widely used with MALDI is the TOF (time-of-flight mass spectrometer), mainly due to its large mass range. MALDI-TOF instruments are typically equipped with an "ion mirror", deflecting ions with an electric field, thereby doubling the ion flight path and increasing the resolution. MALDI and mass spectrometry can also be used to analyze and validate the capture molecule attached to the disclosed coded particles. F. Apparatus

Also disclosed herein is an apparatus for detecting analytes, the apparatus comprising coded particles, wherein the particles are coded for each analyte, wherein the coded particles comprise a capture molecule specific for the analytes, a substrate for gathering the coded particles, and a scanning device for detecting the analyte and decoding the coded particles. Optimally, the coded particle(s) can comprise a dopable semiconducting material. Thus, in one aspect of the disclosed apparatus, at least one of the coded particles is doped. The coded particles can be less than 100, 50, 20, 10, or 1 μm in diameter. Generally, the coded particles are gathered on the substrate at a density of greater than 1, 10, 20, 50, or 100 particle per 10 μm². Optimally, at least one of the coded particles can have a reference feature. For example, the reference feature can be a reference angle. The scanning device of the disclosed apparatus can use a scanning probe microscopy (SPM).

G. Kits

The materials described above as well as other materials can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the disclosed method. It is useful if the kit components in a given kit are designed and adapted for use together in the disclosed method. For example disclosed are kits for analyte detection, the kit comprising coded particles. The kits also can contain a substrate for gathering the coded particles. The kits also can contain a scanning device for detecting the analyte and decoding the coded particles. The kits also can contain a reference for matching codes to analytes. The kits also can contain a means to attach binding molecules for desired analytes to coded particles.

H. Mixtures

Disclosed are mixtures formed by performing or preparing to perform the disclosed method. For example, disclosed are mixtures comprising coded particles, analyte and binding molecules.

Whenever the method involves mixing or bringing into contact compositions or components or reagents, performing the method creates a number of different mixtures.

For example, if the method includes three mixing steps, after each one of these steps a unique mixture is formed if the steps are performed separately. In addition, a mixture is formed at the completion of all of the steps regardless of how the steps were performed.

The present disclosure contemplates these mixtures, obtained by the performance of the disclosed methods as well as mixtures containing any disclosed reagent, composition, or component, for example, disclosed herein. I. Systems

Disclosed are systems useful for performing, or aiding in the performance of, the disclosed method. Systems generally comprise combinations of articles of manufacture such as structures, machines, devices, and the like, and compositions, compounds, materials, and the like. Such combinations that are disclosed or that are apparent from the disclosure are contemplated. For example, disclosed and contemplated are systems comprising coded particles and a scanning device for decoding the coded particles and detecting a captured analyte. J. Data Structures and Computer Control

Disclosed are data structures used in, generated by, or generated from, the disclosed method. Data structures generally are any form of data, information, and/or objects collected, organized, stored, and/or embodied in a composition or medium. A list of codes associated with coded particles stored in electronic form, such as in RAM or on a storage disk, is a type of data structure.

The disclosed method, or any part thereof or preparation therefor, can be controlled, managed, or otherwise assisted by computer control. Such computer control can be accomplished by a computer controlled process or method, can use and/or generate data structures, and can use a computer program. Such computer control, computer controlled processes, data structures, and computer programs are contemplated and should be understood to be disclosed herein.

Uses

The disclosed methods and compositions are applicable to numerous areas including, but not limited to, diagnostic detection of disease markers in a bodily fluid or biopsy. For example, tumor marker levels in blood are used to screen for and diagnose certain types of cancer. Tumor markers associated with common cancers include: AFP (alpha-fetoprotein), Beta-HCG (human chorionic gonadotropin), Cancer antigens (CA 15- 3, CA 19-9, CA 27.29, CA 125), CEA (carcinoembryonic antigen), and PSA (prostate- specific antigen). The coded particles disclosed herein can also be used as research tools for multiplex evaluation of analyte expression. Other uses are disclosed, apparent from the disclosure, and/or will be understood by those in the art.

Methods

Disclosed herein is a method for detecting analytes using coded particles. Generally, the method can comprise bringing into contact a sample and one or more coded particles, wherein at least one of the coded particles comprises a capture molecule specific for an analyte. The presence of a compound on one or more of the coded particles can be detected and the coded particles can be decoded. Decoding the coded particle on which a compound is detected identifies the captured analyte. The disclosed coded particles can comprise a capture molecule for an analyte. An advantage of some forms of the disclosed method is that the captured analyte can be identified by the code specific to the particle comprising the capture molecule. An advantage of some forms of the disclosed method is that decoding the coded particles does not depend on the coded particles being at a predetermined position or being arranged in a pattern. The coded particles do not have to be in a predetermined location during decoding. Thus, identification of the analyte does not require predetermined knowledge of the position of the analyte on a substrate nor does it require specific positioning of a capture molecule on a substrate.

Also disclosed is a method for simultaneously detecting and identifying multiple analytes in a sample, comprising: coding a particle for each analyte, coating the coded particles with a capture molecule specific for the analytes, adding the coding particles to the sample, randomly gathering the particles onto a substrate, detecting the analyte, and decoding the particles to identify the analyte.

The disclosed method involves bringing into contact a sample containing (or suspected of containing) one or more analytes and one or more coded particles. At least one of the coded particles comprises a capture molecule specific for one or more of the analytes. An advantage of some forms of the disclosed method is that the target analyte can be captured while free floating in solution. Thus, the contacting step of the disclosed method can occur in a liquid solution, viscous solution, gel matrix, or any other medium suitable to the target analyte. The coded particles can be in solution or in suspension during and/or after being brought into contact with the sample.

The coded particles can be gathered on a substrate. The gathering of coded particles on a substrate can be done before or after bringing the sample into contact with the coded particles. It is preferred that the coded particles be gathered after the sample is brought into contact with the coded particles to gain the advantage of having the interaction take place in solution or suspension. The coded particles can be gathered on a substrate in any suitable manner. For example, the dope-coded biosensing particles can be designed with an embedded magnetic metal layer. As a result, these particles can be easily extracted with an external magnetic field onto a flat secondary substrate for detection & decoding analysis. Furthermore, the particles' faces can be functionalized to facilitate extraction. For example, the gold face of the particles may be modified with dithiols to facilitate it's adhesion to a secondary gold coated substrate during extraction. There are numerous other type of chemical modifications that could be used herein. In addition, other methods of high density extraction include the use of centrifugation and filters.

The coded particles can adhere to the substrate via, for example, electrostatic or other forces. For example, the coded particles can also adhere to the substrate material or to a coating on the substrate that can interact with the coded particles. Such interaction is preferable non-specific. This allows the entire substrate surface to interact with any coded particle. A. Decoding The coded particles can be decoded using any suitable microscale or nanoscale detection or measurement technique. Thus, for example, coded particles can be decoded using scanning probe microscopy (SPM). The technique used can be chosen, if necessary, based on the nature of the pattern on the coded particle. For example, the patterns can be microscale or nanoscale topography, electrical properties, magnetic properties, conductance properties, refractive properties, reflective properties, and/or plasmon resonance properties. Patterns to be detected can be patterns of, for example, shape and/or intensity of doping. SPM covers several related technologies for imaging and measuring surfaces on a fine scale, down to the level of molecules and groups of atoms. At the other end of the scale, a scan can cover a distance of at least 100 micrometers in the x and y directions and at least 4 micrometers in the z direction. SPM technologies share the concept of scanning an extremely sharp tip (for example, 3-50 ran radius of curvature) across the object surface. The tip is mounted on a flexible cantilever, allowing the tip to follow the surface profile. When the tip moves in proximity to the investigated object, forces of interaction between the tip and the surface influence the movement of the cantilever. These movements are detected by selective sensors. Various interactions can be studied depending on the mechanics of the probe. Several types of probes with different tips are used in scanning probe microscopy. Tip selection depends on the mode of operation and on the type of sample. As non-limiting examples, the coded particles can be decoded using local conductance measurements, electrostatic measurements, or topographical measurements.

The three most common scanning probe techniques are Atomic Force Microscopy (AFM), Scanning Tunneling Microscopy (STM), and Near-Field Scanning Optical Microscopy (NSOM). Atomic Force Microscopy (AFM) measures the interaction force between the tip and surface. The tip can be dragged across the surface, or can vibrate as it moves. The interaction force will depend on the nature of the sample, the probe tip and the distance between them. Scanning Tunneling Microscopy (STM) measures a weak electrical current flowing between tip and sample as they are held a very distance apart. Near-Field Scanning Optical Microscopy (NSOM) scans a very small light source very close to the sample. Detection of this light energy forms the image. NSOM can provide resolution below that of the conventional light microscope.

There are numerous variations on these techniques. For example, Surface Potential Microscopy, Electrostatic Force Microscopy (EFM), Kelvin Force Microscopy (KFM), and Scanning Capacitance Microscopy (SCM) are variations of AFM and STM where a tip/probe measures the atomic force, and also hovers above the substrate to measure the electrical characteristics of the sample. This feedback is not based upon tunneling current, but upon surface potential, potential gradient, electrostatic, and capacitive contributions. AFM can operate in several modes that differ according to the force between the tip and surface. In contact mode, the tip is usually maintained at a constant force by moving the cantilever up and down as it scans. Li non-contact mode or intermittent contact mode the tip is driven up and down by an oscillator. Especially soft materials can be imaged by a magnetically-driven cantilever. In non-contact mode, the bottom-most point of each probe cycle is in the attractive region of the force-distance curve. In intermittent contact mode the bottom-most point is in the repulsive region. Variations in the measured oscillation amplitude and phase in relation to the driver frequency are indicators of the surface-probe interaction.

To image frictional force, the probe is dragged along the surface, resulting in a torque on the cantilever. To image the magnetic field of the surface, a magnetically- susceptible probe is used. In other variations, the electric charge distribution on the surface or the surface capacitance is imaged. For thermal scanning microscopy (TSM) the thermal conductivity of the surface is probed with a resistive tip that acts as a tiny resistance thermometer. In addition to these modes, many instruments are also designed to plot the phase difference between the measured modes, for example frictional force versus contact profile. This plot is called phase mode. B. Biomolecular Amplification

The disclosed method is highly sensitive, allowing for detection of analyte at picomolar concentrations without biomolecular amplification. The direct or indirect amplification of analyte prior to detection by the methods disclosed herein can greatly improve the sensitivity of the disclosed method. For example, a number of methods have been developed for exponential amplification of nucleic acids. These include the polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), and amplification with Q replicase (Birkenmeyer and Mushahwar, J. Virological Methods, 35:117-126 (1991); Landegren, Trends Genetics 9:199-202 (1993)).

Examples 1. Example 1

Hexagonal disks were fabricated ~6μm in diameter and ~200nm thick. The particles were made of undoped polysilicon which had been encoded with doped regions. The hexagonal shape allows for reference angles from which to decoding the particles. Initial results showed that the particles could indeed form very densely packed arrays once collected (see Fig. 1). The image shows a densely packed random array of particles extracted from solution. The inset box represents about half the area required for only one protein spot using the current spotting array technology. However, within that same inset area, the disclosed system could potentially analyzed hundreds of different proteins with little fear of neighboring cross contamination. This high density, no cross contamination, and ease of decoding is a major advantage over current techniques. Furthermore, these particles can be easily scaled down while maintaining these advantages.

Coded polysilicon were decoded using an SPM technique called electrostatic force microscopy (EFM) (see Fig. 2). The image on the left shows the flat unstructured topography of the surface, and the image on the right shows the sub-surface decoding at different system settings. The bits were ~ 500nm wide with ~1.5μm spacing. In SPM- EFM, the scanning tip did a dual pass for every scan line (see Fig. 3). The first pass was simply to acquire the surface topography. In the second pass, the tip lifted off the substrate a pre-specified distance and retraces the previously measured surface topography while acquiring electrostatic information from the sample. Using SPM to decode the doped particles is of major advantage because it simultaneously acquires decoding and protein/analyte detection information, hi addition, encoding/decoding can be 3 dimensionally complex in that both 2D patterns and pattern intensity are obtained. Scaling down is possible because SPM can decode bits of widths as little as 10's of nanometers in size. The protein immobilization procedure intended for the encoded particles were applied to silicon samples (see Fig. 4). Here the target protein was directly immobilized onto the substrate, 30OpM Human IgG. The substrate was then exposed to a solution of very low concentration marker protein, ~130pM 5nm Au Nanoparticle conjugated Anti- Human IgG. Large topographical changes due to the presence of the Marker indicated the successful capture of the target protein/antigen/analyte. Figure 4 shows the detection of the Marker as indicated by the captured nanoparticles. Picomolar protein/ analyte detection is highly sensitive and sufficient for most clinical applications. It is worth noting that this picomolar detection level was obtained without biomolecular amplification, and can be greatly improved with such additional resources.

Having prior knowledge of which particular capture protein corresponds to which particular particle code allows for the successful identification of target protein captures even with only a general marker protein (Fig. 5). Figure 5 shows illustration of the data obtained from the high density particle array. Solutions are pre-prepared with coded particles comprising capture molecules (e.g. antibodies) specific for each analyte. Mixed coded particles are combined with the sample comprising the analyte(s)/ antigen(s). The particles are assembled onto a substrate for data collection. Both decoding and detection are obtained simultaneously. The single dark vertical stripe particles, code 10010, indicate the presence of marker protein capture in the topography image. Thus the corresponding target analyte, target protein 10010, is present in solution. With the current spotting technology, less than one quarter of a single protein spot could actually fit within the inset caption. Furthermore, having redundant particles increases the reliability of the system. It is important to note that with the disclosed system, the Target Proteins are captured while free floating in solution where they are most active. Current spotting techniques require that the proteins be held in a highly viscous solution or a gel matrix to reduce dehydration, but this greatly restricts protein activity. 2. Example 2

To fabricate dope-coded biosensing particles, 100 run of sacrificial oxide was first grew on a silicon wafer and then 200 nm of polysilicon deposited. The wafer was patterned with photoresist and subjected to boron ion implantation to encode the polysilicon layer. By over or under developing the resist patterns, rotating the patterns, overlaying the patterns, and/or changing the dopant and/or substrate(Sugimura, et al. 2002), a large number of codes can be generated with only one mask. The convenience of photolithography also allows for fabricating structures below the standard optical size limit, where features below 100 nm are possible using resolution enhancement techniques and extreme UV (Hsu, et al. 2004). After encoding, photoresist was used again to pattern hexagons onto the coded polysilicon; 25 run Cr, 20 Ni, and 35 run Au was sputtered onto the patterned substrate, followed by lift-off in acetone leaving metallic hexagons on the substrate. The substrate was reactive ion etched leaving hexagonal dope-coded polysilicon particles bound to the sacrificial oxide (Fig. 7A). Fabricating the coded particles in this manner leaves the sacrificial oxide/polysilicon interface nearly atomically flat, necessary for convenient protein detection.

Then, the samples were exposed to 1-10-decanedithiol chemical vapor to modify the gold layer (Hozumi, et al. 2001) and then over-exposed with buffered HF (BHF) for 40 min to remove the sacrificial oxide underlayer and slightly etch the doped regions of the polysilicon. Chemical vapor exposure was achieved by pipetting 100 pi of stock solution into a 40 ml glass flask with samples. The flask was caped and heated in an oven for 2 h at 100 °C. This slight selective etching of the doped regions leaves a 1-2 nm recession, allowing for topographical or optical decoding. The samples were then inverted and lightly pressed onto a subsequent gold coated silicon substrate sandwiching the coded particles. Upon separation of the two substrates, dithiol bonding immobilized the coded particles and transferred them onto the subsequent gold coated substrate exposing the nearly atomically flat polysilicon side of the particles (Fig. 7B). hi this new convenient arrangement, the samples were modified with 3-aminopropyltriethoxysilane through chemical vapor exposure (CVE) and afterwards with 10 mM glutaraldehyde for 30 min in order to establish the protein linker groups (Wang et al. 2004).

The samples were exposed to a solution of their corresponding target proteins in 10 mM phosphate buffer solution pH 7.5 for 2 h at room temperature and then let sit overnight in Tris buffer solution 50 mM pH 8 at 4 °C to block the remaining aldehyde ends. Afterwards, the samples were ex-posed to 0.5% Tween 20 in Tris for 30 min at room temperature in order to reduce nonspecific binding. Next, the dope-coded biosensing particles were suspended in Tris buffer soultion. The particles were suspended in Tris with a brief sonnication. In separate experiments using flat silicon substrates, sonnication retained protein binding activity levels, showed less background noise, and reduced nonspecific binding levels.

Equal concentrations from each desired solution of suspended dope-coded biosensing particles were extracted and mixed together. The marker proteins were then added to the mixture of biosensing particles and let incubate for 2 h at room temperature. With the assistance of an external magnet, a gold coated substrate was submerged into the suspension to collect the coded particles. This step can be repeated until the desired collected particle density is achieved. Particles landing on their dithiolated face were immobilized, while others were easily rinsed away. Figure 7C demonstrates a high density random arrangement of the recollected particles. The particles can be decoded optically (Fig. 7D), according to their surface potential with a surface potential AFM (Fig. 7E), or topographically. Topographical AFM based detection and decoding was demonstrated in this work to emphasize the scalability of the system (Nettikadan, et al. 2004; Ouerghi, et al. 2002). For a preliminary coding scheme, a simple mask of parallel lines were fabricated 1 μm wide and 1 μm apart. Two simple codes of lines perpendicular and parallel to the hexagonal particle's edge were chosen, which were the same mask patterns distinguished by 30° of rotation. The inset image in Fig. 8 A depicts how the two codes were identified. The proteins used to demonstrate the system were human IgG as the target proteins and anti-human IgG conjugated 20 nm nanoparticles as the marker proteins. During solid phase target protein immobilization, target protein 10 μg/ml human IgG was immobilized onto the parallel coded particles while perpendicular coded particles were selected as the control with no target proteins immobilized thereon. After suspension and mixing, the coded particles were exposed to marker protein 200 ng/ml anti-human IgG conjugated 20 nm nanoparticles for suspension phase binding. Once collected onto a substrate, an AFM was used to scan the topography, simultaneously decoding the particles and detecting the marker protein nanoparticles. Note that the particle code is obtained regardless of the nanoparticle density measured. Examples of AFM scans are shown in Figs. 8B and 8C. The results showed that perpendicular dope-coded control particles and parallel dope-coded particles exhibited an average nanoparticle density of 3.60 and 31.12/μm2, respectively (Figs. 8A-8C). The human IgG modified parallel dope-coded particles exhibited a much higher nanoparticle density count than the unmodified perpendicular dope-coded particles. This is attributed to specific complementary binding between the target human IgG proteins and marker anti-human IgG protein conjugated 20 nm nanoparticles. The low density of nanoparticles observed on the perpendicular dope-coded particles is attributed to nonspecific binding (NSB) and is nearly that of the background supporting substrate (Figs. 8B and 8C). An additional set of experiments was conducted to verify the nonspecific binding levels. During solid phase target protein immobilization, noncomplementary target protein 10 μg/ml anti-human IgG was immobilized onto the parallel coded particles while perpendicular coded particles were selected again as the control with no target proteins immobilized onto it. After suspension, mixing, and exposure to marker protein 200 ng/ml anti-human IgG conjugated 20 nm nanoparticles, only low nanoparticle density levels were observed for parallel dope-coded particles, 3.91/μm2 (Figs. 8 A and 8D). Only NSB levels were observed for these particles since the target and marker proteins were noncomplementary. 3. Example 3

Surface Plasmon Resonance-Imaging (SPR-I) can be used in combination with the disclosed coded particles for protein separation. The advantages of this system are that it is label free, real-time, and high throughput. An example of an analysis of μ-contact printed protein patterns by SPR-I detection is shown in Figure 9. Figure 10 shows an example of a multi-channel micro fluidic device for protein separation by SPR-I.

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Claims

CLAIMSWe claim:

1. A method for detecting analytes, comprising: a) bringing into contact a sample and one or more coded particles, wherein at least one of the coded particles comprises a capture molecule, wherein the capture molecule is specific for an analyte, b) detecting the presence of a compound on one or more of the coded particles, and c) prior to, simultaneous with, or following step (b), decoding at least one of the coded particles, wherein decoding the coded particle on which the compound is detected identifies the compound as the analyte.

2. The method of claim 1, wherein at least one of the coded particles comprises a dopable semiconducting material.

3. The method of claim 2, wherein the dopable semiconducting material is selected from the group consisting of silicon, polysilicon, arsenide, germanium, indium arsenide, indium tin oxide, titanium oxide, and diamond.

4. The method of claim 1, wherein at least one of the coded particles is less than 100, 50, 20, 10, or 1 μm in diameter.

5. The method of claim 1, wherein at least one of the coded particle has a reference feature.

6. The method of claim 5, wherein the reference feature is a reference angle.

7. The method of claim 5, wherein the reference feature is a variation in intensity.

8. The method of claim 1, wherein at least one of the coded particles is doped.

9. The method of claim 1, wherein the capture molecule is an antibody, aptamer, epitome, single stranded DNA or RNA, or molecularly imprinted polymer (MIP).

10. The method of claim 1, wherein the analyte is detected using a marker.

11. The method of claim 10, wherein the marker comprises a nanoparticle.

12. The method of claim 1, wherein at least one of the coded particles is decoded using scanning probe microscopy (SPM).

13. The method of claim 12, wherein at least one of the coded particles is decoded using local conductance measurements.

14. The method of claim 12, wherein at least one of the coded particles is decoded using electrostatic measurements.

15. The method of claim 12, wherein at least one of the coded particles is decoded using topographical measurements.

16. The method of claim 1, wherein, prior to decoding, the coded particles are gathered onto a substrate.

17. The method of claim 16, wherein the coded particles are gathered at a density of greater than 1, 10, 20, 50, or 100 particle per 10 μm².

18. The method of claim 16, wherein the coded particles are gathered onto the substrate randomly.

19. The method of claim 1, wherein the coded particles are coded with different patterns, wherein the different patterns can be detected using scanning probe microscopy (SPM).

20. The method of claim 1, wherein multiple analytes are detected.

21. The method of claim 20, wherein the multiple analytes are detected simultaneously.

22. The method of claim 1, wherein the coded particles are coded with different patterns, wherein the coded particles having different patterns each comprise a different capture molecule, wherein the different capture molecules are each specific for a different analyte, whereby a particular pattern is indicative of a particular analyte.

23. The method of claim 22, wherein coded particles collectively comprise 5 or more, 6, or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 200 or more, 300 or more, 400 or more, or 500 or more different capture molecules.

24. The method of claim 1, wherein decoding the coded particles does not depend on the coded particles being at a predetermined position or being arranged in a pattern.

25. The method of claim 1, wherein identification of the analyte does not require predetermined knowledge of the position of the analyte on a substrate.

26. The method of claim 1, wherein the coded particles are not in a predetermined location during decoding.

27. The method of claim 1, wherein the coded particles are in solution or in suspension after being brought into contact with the sample.

28. An apparatus for detecting analytes, the apparatus comprising a) coded particles, wherein the particles are coded for each analyte, wherein the coded particles comprise a capture molecule specific for the analytes, b) a substrate for gathering the coded particles, and c) a scanning device for detecting the analyte and decoding the coded particles.

29. The apparatus of claim 28, wherein at least one of the coded particles comprises a dopable semiconducting material.

30. The apparatus of claim 28, wherein at least one of the coded particles are less than 100, 50, 20, 10, or 1 μm in diameter.

31. The apparatus of claim 28, wherein at least one of the coded particles has a reference feature.

32. The apparatus of claim 31, wherein the reference feature is a reference angle.

33. The apparatus of claim 31, wherein the reference feature is a variation in intensity.

34. The apparatus of claim 28, wherein at least one of the coded particles are doped.

35. The apparatus of claim 28, wherein the capture molecule is an antibody, aptamer, epitome, single stranded DNA or RNA, or molecularly imprinted polymer (MIP).

36. The apparatus of claim 28, wherein the scanning device is scanning probe microscopy (SPM).

37. The apparatus of claim 28, wherein the coded particles are gathered on the substrate at a density of greater than 1, 10, 20, 50, or 100 particle per 10 μm².

38. A method for simultaneously detecting and identifying multiple analytes in a sample, comprising: a) coding a particle for each analyte, b) coating the coded particles with a capture molecule specific for the analytes, c) adding the coding particles to the sample, d) randomly gathering the particles onto a substrate, e) detecting the analyte, and f) decoding the particles to identify the analyte.