US20030231987A1

US20030231987A1 - Devices and methods for performing array based assays

Info

Publication number: US20030231987A1
Application number: US10/273,938
Authority: US
Inventors: Condie Carmack; Patrick Collins; Arthur Schleifer
Original assignee: Individual
Current assignee: Individual
Priority date: 2002-06-14
Filing date: 2002-10-17
Publication date: 2003-12-18
Also published as: EP1371980A2; EP1371980A3; US7919308B2; US20030231985A1

Abstract

Array coverslip devices and methods of using the same are provided. The subject coverslip devices are characterized by having an array coverslip and at least one wall structure having at least one orifice therein on a planar surface of the coverslip. In certain embodiments, more than one wall structure is present. The wall structures may be flexible or rigid. The subject methods include joining an array coverslip having at least one wall structure having at least one orifice on a planar surface of the coverslip with a ligand array, performing an array assay using the coverslip joined with the ligand array, and reading the ligand array to obtain a result. Also provided are systems and kits for use in the subject methods.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No. 10/172,850 filed on Jun. 14, 2002, the disclosure of which is herein incorporated by reference.[0001]

FIELD OF THE INVENTION

The field of this invention is biopolymeric arrays.

BACKGROUND OF THE INVENTION

Array assays between surface bound binding agents or probes and target molecules in solution may be used to detect the presence of particular biopolymers. The surface-bound probes may be oligonucleotides, peptides, polypeptides, proteins, antibodies or other molecules capable of binding with target molecules in solution. Such binding interactions are the basis for many of the methods and devices used in a variety of different fields, e.g., genomics (in sequencing by hybridization, SNP detection, differential gene expression analysis, identification of novel genes, gene mapping, finger printing, etc.) and proteomics.

One typical array assay method involves biopolymeric probes immobilized in an array on a substrate such as a glass substrate or the like. A solution containing analytes that bind with the attached probes is placed in contact with the substrate, covered with another substrate to form an assay area and placed in an environmentally controlled chamber such as an incubator or the like. Usually, the targets in the solution bind to the complementary probes on the substrate to form a binding complex. The pattern of binding by target molecules to biopolymer probe features or spots on the substrate produces a pattern on the surface of the substrate and provides desired information about the sample. In most instances, the target molecules are labeled with a detectable tag such as a fluorescent tag, chemiluminescent tag or radioactive tag. The resultant binding interaction or complexes of binding pairs are then detected and read or interrogated, for example by optical means, although other methods may also be used. For example, laser light may be used to excite fluorescent tags, generating a signal only in those spots on the biochip that have a target molecule and thus a fluorescent tag bound to a probe molecule. This pattern may then be digitally scanned for computer analysis.

As will be apparent, control of the assay environment and conditions contributes to increased reliability and reproducibility of the array assays. During an array assay such as a hybridization assay, the assay is often performed at elevated temperatures and care must be taken so that the array does not dry out due to evaporation or sample leakage from the array assay area. Usually, a substrate such as a coverslip is positioned over the array to help control the assay environment, as mentioned above. However, heretofore this method fails to provide complete assay control.

The simplest coverslips are planar substrates that, when positioned over an array, directly contact the array, thereby eliminating space for sample above the array. Furthermore, these coverslips do not remain in a fixed position over the array and instead float or slip around the array which can cause contamination when performing multiple array assays on a single sided array surface. More importantly, this protocol fails to provide a means to introduce sample to the array once the array and the coverslip are joined together. As such, sample must first be contacted with the array which is opened to the environment, allowing evaporation and contamination before the coverslip is joined to the array.

In light of the above disadvantages of these simple coverslips, coverslips that include spacers on two opposing sides of the coverslip have been developed such as Lifterslips™ available from Erie Scientific Co., Portsmouth N.H. The opposing spacers raise the coverslip above the array a fixed distance so as to provide a defined space between the array and the array-facing coverslip surface for sample. The spacers are positioned on opposing sides of the coverslip with two of the sides of the coverslip completely opened so that sample may be introduced to the array through the opened sides that do not have spacers. However, while effective at providing a means to introduce sample and a space for the sample between the array and the coverslip, these types of coverslips fail to prevent sample evaporation and leakage from the array assay area from the opened sides of the coverslip and thus do not solve the problem of the array drying out and uneven sample distribution over the array.

Other solutions include placing a gasket component or the like between the array substrate and the coverslip. However, while minimizing unwanted slippage or floating of the coverslip, this method too suffers from disadvantages. Typically these gaskets are separate components that need to be pealed from a backing and precisely placed either around an array before the coverslip is positioned thereover or on the coverslip before contacting the array. This procedure is time consuming and labor intensive and requires precise positioning of the gasket so as not to contact the array. After the array assay is complete, the gasket is removed from around the array. This step introduces more problems as the gaskets must be carefully removed without touching the array.

Furthermore, sample introduction into the array area is difficult because these removable gaskets do not provide a means to introduce sample to the array once the array is assembled with the coverslip as these gaskets are completely enclosed, e.g., as a ring or the like. As such, sample can only be introduced to the array before the coverslip is positioned over the array. This can cause uneven distribution of sample if the coverslip does not settle properly against the array. Furthermore, bubbles can get trapped under the coverslip when positioned over the array, also causing uneven sample distribution.

Thus, there continues to be an interest in the development of array coverslip devices and methods of using the same. Of particular interest is the development of an array coverslip device, and methods of use thereof, that provides a fluid barrier around an array assay area to prevent leakage and evaporation from the array assay area, enables fluid to be easily introduced and removed from the sealed area without removing the coverslip, is easy to use, and that may also be capable of testing multiple samples with multiple arrays without cross-contamination.

SUMMARY OF THE INVENTION

Array coverslip devices and methods of using the same are provided. The subject coverslip devices are characterized by having an array coverslip and at least one wall structure having at least one orifice therein on a planar surface of the coverslip. In certain embodiments, more than one wall structure is present. The wall structures may be flexible or rigid. The subject methods include joining an array coverslip having at least one wall structure having at least one orifice on a planar surface of the coverslip with a ligand array, performing an array assay using the coverslip joined with the ligand array, and reading the ligand array to obtain a result. Also provided are systems and kits for use in the subject methods. The subject devices and methods find use in any array based applications, including genomic and proteomic applications.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 shows an exemplary substrate carrying an array, such as may be used in the devices of the subject invention. [0012]
FIG. 2A shows an exemplary embodiment of a subject array coverslip having a single orifice. FIG. 2B shows a cross-sectional view of the coverslip device of FIG. 2A. FIG. 2C shows a cross-sectional view of the coverslip device of FIG. 2A joined with an array to provide an array assay area. [0013]
FIG. 3 shows another exemplary embodiment of a subject array coverslip having two orifices positioned in two corners that are on the same side of the rigid wall structure. [0014]
FIG. 4 shows another exemplary embodiment of a subject array coverslip having two orifices positioned on two diagonally opposed corners of the rigid wall structure. [0015]
FIG. 5 shows another exemplary embodiment of a subject array coverslip having four orifices positioned in the four corners of the rigid wall structure. [0016]
FIG. 6 shows another exemplary embodiment of a subject array coverslip having a rectangular rigid wall structure that is not commensurate with the perimeter of the surface on which it is positioned. [0017]
FIG. 7 shows another exemplary embodiment of a subject array coverslip having two rectangular rigid walls structures. [0018]
FIG. 8. illustrates the steps of the subject methods wherein a subject coverslip is joined with an array substrate having an array thereon. [0019]
FIG. 9 shows the assembled coverslip/array structure of FIG. 8 having sample retained in the array assay area provided by the assembled structure. [0020]
FIG. 10 shows an exemplary embodiment of a subject array coverslip having two rigid wall structures defining two individual bounded areas that do not have orifices.[0021]

DEFINITIONS

The term “polymer” refers to any compound that is made up of two or more monomeric units covalently bonded to each other, where the monomeric units may be the same or different, such that the polymer may be a homopolymer or a heteropolymer. Representative polymers include peptides, polysaccharides, nucleic acids and the like, where the polymers may be naturally occurring or synthetic. [0022]
The term “monomer” as used herein refers to a chemical entity that can be covalently linked to one or more other such entities to form an oligomer. Examples of monomers include nucleotides, amino acids, saccharides, peptides, and the like. In general, the monomers used in conjunction with the present invention have first and second sites (e.g., C-termini and N-termini, or 5′ and 3′ sites) suitable for binding to other like monomers by means of standard chemical reactions (e.g., condensation, nucleophilic displacement of a leaving group, or the like), and a diverse element which distinguishes a particular monomer from a different monomer of the same type (e.g., an amino acid side chain, a nucleotide base, etc.). The initial substrate-bound monomer is generally used as a building-block in a multi-step synthesis procedure to form a complete ligand, such as in the synthesis of oligonucleotides, oligopeptides, and the like. [0023]
The term “oligomer” is used herein to indicate a chemical entity that contains a plurality of monomers. As used herein, the terms “oligomer” and “polymer” are used interchangeably. Examples of oligomers and polymers include, but are not limited to: polydeoxyribonucleotides, polyribonucleotides, other polynucleotides which are B or C-glycosides of a purine or pyrimidine base, polypeptides, polysaccharides, and other chemical entities that contain repeating units of like chemical structure. [0024]
The term “ligand” as used herein refers to a moiety that is capable of covalently or otherwise chemically binding a compound of interest. The ligand may be a portion of the compound of interest. The term “ligand” in the context of the invention may or may not be an “oligomer” as defined above. The term “ligand” as used herein may also refer to a compound that is synthesized on the substrate surface as well as a compound is “pre-synthesized” or obtained commercially, and then attached to the substrate surface. [0025]
The terms “array,” “biopolymeric array” and “biomolecular array” are used herein interchangeably to refer to an arrangement of ligands or molecules of interest on a substrate surface, which can be used for analyte detection, combinatorial chemistry, or other applications wherein a two-dimensional arrangement of molecules of interest can be used. That is, the terms refer to an ordered pattern of probe molecules adherent to a substrate, i.e., wherein a plurality of molecular probes are bound to a substrate surface and arranged in a spatially defined and physically addressable manner. Such arrays may be comprised of oligonucleotides, peptides, polypeptides, proteins, antibodies, or other molecules used to detect sample molecules in a sample fluid. [0026]
The term “biomolecule” means any organic or biochemical molecule, group or species of interest that may be formed in an array on a substrate surface. Exemplary biomolecules include peptides, proteins, amino acids and nucleic acids. [0027]
The term “peptide” as used herein refers to any compound produced by amide formation between a carboxyl group of one amino acid and an amino group of another group. [0028]
The term “oligopeptide” as used herein refers to peptides with fewer than about 10 to 20 residues, i.e. amino acid monomeric units. [0029]
The term “polypeptide” as used herein refers to peptides with more than 10 to 20 residues. [0030]
The term “protein” as used herein refers to polypeptides of specific sequence of more than about 50 residues. [0031]
The term “nucleic acid” as used herein means a polymer composed of nucleotides, e.g. deoxyribonucleotides or ribonucleotides, or compounds produced synthetically (e.g. PNA as described in U.S. Pat. No. 5,948,902 and the references cited therein) which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions, Wobble interactions, etc. [0032]
The terms “ribonucleic acid” and “RNA”s used herein mean a polymer composed of ribonucleotides. [0033]
The terms “deoxyribonucleic acid” and “DNA” as used herein mean a polymer composed of deoxyribonucleotides. [0034]
The term “oligonucleotide” as used herein denotes single stranded nucleotide multimers of from about 10 to 100 nucleotides and up to 200 nucleotides in length. [0035]
The term “polynucleotide” as used herein refers to single or double stranded polymer composed of nucleotide monomers of generally greater than 100 nucleotides in length. [0036]
The term “sample” as used herein relates to a material or mixture of materials, typically, although not necessarily, in fluid form, containing one or more components of interest. [0037]
The terms “nucleoside” and “nucleotide” are intended to include those moieties which contain not only the known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. In addition, the terms “nucleoside” and “nucleotide” include those moieties that contain not only conventional ribose and deoxyribose sugars, but other sugars as well. Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, or are functionalized as ethers, amines, or the like. [0038]
The term “chemically inert” is used herein to mean substantially chemically unchanged by contact with reagents and conditions normally involved in array based assays such as hybridization reactions or any other related reactions or assays, e.g., proteomic array applications. [0039]
The term “communicating” information refers to transmitting data representing that information as electrical signals over a suitable communication channel (for example, a private or public network). [0040]
The term “forwarding” an item refers to any means of getting that item from one location to the next, whether by physically transporting that item or otherwise (where that is possible) and includes, at least in the case of data, physically transporting a medium carrying the data or communicating the data. [0041]
The term “physically inert” is used herein to mean substantially unchanged physically by contact with reagents and conditions normally involved in array based assays such as hybridization reactions or any other related assays or reactions. [0042]
The terms “target” “target molecule” and “analyte” are used herein interchangeably and refer to a known or unknown molecule in a sample, which will hybridize to a molecular probe on a substrate surface if the target molecule and the molecular probe contain complementary regions, i.e., if they are members of a specific binding pair. In general, the target molecule is a biopolymer, i.e., an oligomer or polymer such as an oligonucleotide, a peptide, a polypeptide, a protein, and antibody, or the like. [0043]
The term “hybridization” as used herein refers to binding between complementary or partially complementary molecules, for example as between the sense and anti-sense strands of double-stranded DNA. Such binding is commonly non-covalent binding, and is specific enough that such binding may be used to differentiate between highly complementary molecules and others less complementary. Examples of highly complementary molecules include complementary oligonucleotides, DNA, RNA, and the like, which comprise a region of nucleotides arranged in the nucleotide sequence that is exactly complementary to a probe; examples of less complementary oligonucleotides include ones with nucleotide sequences comprising one or more nucleotides not in the sequence exactly complementary to a probe oligonucleotide. [0044]
The term “hybridization solution” or “hybridization reagent” used herein interchangeably refers to a solution suitable for use in a hybridization reaction. [0045]
The terms “mix” and “mixing” as used herein means to cause fluids to flow within a volume so as to more uniformly distribute solution components, as after different solutions are combined or after a solution is newly introduced into a volume or after a component of the solution is locally depleted. [0046]
The term “probe” as used herein refers to a molecule of known identity adherent to a substrate. [0047]
The term “remote location” refers to a location other than the location at which the array is present and hybridization occur. As such, when one item is indicated as being “remote” from another, what is meant is that the two items are at least in different buildings, and may be at least one mile, ten miles, or at least one hundred miles apart. [0048]
The term “stringent hybridization conditions” as used herein refers to conditions that are that are compatible to produce duplexes on an array surface between complementary binding members, i.e., between probes and complementary targets in a sample, e.g., duplexes of nucleic acid probes, such as DNA probes, and their corresponding nucleic acid targets that are present in the sample, e.g., their corresponding mRNA analytes present in the sample. An example of stringent hybridization conditions is hybridization at 50° C. or higher and 0.1×SSC (15 mM sodium chloride/1.5 mM sodium citrate). Another example of stringent hybridization conditions is overnight incubation at 42° C. in a solution: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10% dextran sulfate, followed by washing the filters in 0.1×SSC at about 65° C. Stringent hybridization conditions are hybridization conditions that are at least as stringent as the above representative conditions. Other stringent hybridization conditions are known in the art and may also be employed to identify nucleic acids of this particular embodiment of the invention. [0049]

DETAILED DESCRIPTION OF THE INVENTION

Array coverslip devices and methods of using the same are provided. The subject coverslip devices are characterized by having an array coverslip and at least one wall structure having at least one orifice therein on a planar surface of the coverslip. In certain embodiments, more than one wall structure is present. The wall structure(s) may be flexible or rigid. The subject methods include joining an array coverslip having at least one wall structure having at least one orifice on a planar surface of the coverslip with a ligand array, performing an array assay using the coverslip joined with the ligand array, and reading the ligand array to obtain a result. Also provided are systems and kits for use in the subject methods. The subject devices and methods find use in any array based applications, including genomic and proteomic applications. [0050]
Before the present invention is described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, 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, since the scope of the present invention will be limited only by the appended claims. [0051]
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention. [0052]
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. [0053]
It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a surface agent” includes a plurality of such surface agents and reference to “the array” includes reference to one or more arrays and equivalents thereof known to those skilled in the art, and so forth. [0054]
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. [0055]
Introduction [0056]
As summarized above, the subject invention provides array coverslip devices and methods for using the same in array-based assays, i.e., array binding assays. By coverslip it is generally meant a device dimensioned to fit or be joined with an array to provide an array assay area between the coverslip and the array. The subject invention can be used with a number of different types of arrays in which a plurality of distinct polymeric binding agents (i.e., of differing sequence) are stably associated with at least one surface of a substrate or solid support. The polymeric binding agents may vary widely, however polymeric binding agents of particular interest include peptides, proteins, nucleic acids, polysaccharides, synthetic mimetics of such biopolymeric binding agents, etc. In many embodiments of interest, the biopolymeric arrays are arrays of nucleic acids, including oligonucleotides, polynucleotides, cDNAs, mRNAs, synthetic mimetics thereof, and the like. [0057]
While the subject devices and methods find use in array hybridization assays, the subject devices also find use in any suitable binding assay in which members of a specific binding pair interact. That is, any of a number of different binding assays may be performed using the subject invention, where typically a first member of a binding pair is stably associated with the surface of a substrate and a second member of a binding pair is free in a sample, where the binding members may be: ligands and receptors, antibodies and antigens, complementary nucleic acids, and the like. For ease of description only, the subject devices and methods described below will be described primarily in reference to hybridization assays, where such examples are not intended to limit the scope of the invention. It will be appreciated by those of skill in the art that the subject array coverslip devices and methods of using the same may be employed for use with other binding assays as well, such as immunoassays, proteomic assays, etc. [0058]
In further describing the subject invention, representative arrays that may be used with the subject invention are described to provide a proper foundation for the invention. The subject array coverslip devices are then described in greater detail, followed by a description of systems that include the subject array coverslip devices. Next, the subject methods are described, as well as kits for use in practicing the subject methods. [0059]
Representative Biopolymeric Arrays [0060]
As mentioned above, the devices of the subject invention are used with arrays and more specifically biopolymeric arrays. Such biopolymeric arrays find use in a variety of applications, including gene expression analysis, drug screening, nucleic acid sequencing, mutation analysis, and the like. These biopolymeric arrays include a plurality of ligands or molecules or probes (i.e., binding agents or members of a binding pair) deposited onto the surface of a substrate in the form of an “array” or pattern. [0061]
The subject biopolymeric arrays include at least two distinct polymers that differ by monomeric sequence attached to different and known locations on the substrate surface. Each distinct polymeric sequence of the array is typically present as a composition of multiple copies of the polymer on a substrate surface, e.g., as a spot or feature on the surface of the substrate. The number of distinct polymeric sequences, and hence spots or similar structures, present on the array may vary, where a typical array may contain more than about ten, more than about one hundred, more than about one thousand, more than about ten thousand or even more than about one hundred thousand features in an area of less than about 20 cm[0062] ²or even less than about 10 cm². For example, features may have widths (that is, diameter, for a round spot) in the range from about 10 μm to about 1.0 cm. In other embodiments, each feature may have a width in the range from about 1.0 μm to about 1.0 mm, usually from about 5.0 μm to about 500 μm and more usually from about 10 μm to about 200 μm. Non-round features may have area ranges equivalent to that of circular features with the foregoing width (diameter) ranges. At least some, or all, of the features are of different compositions (for example, when any repeats of each feature composition are excluded, the remaining features may account for at least about 5%, 10% or 20% of the total number of features). Interfeature areas will typically (but not essentially) be present which do not carry any polynucleotide (or other biopolymer or chemical moiety of a type of which the features are composed). Such interfeature areas typically will be present where the arrays are formed by processes involving drop deposition of reagents, but may not be present when, for example, photolithographic array fabrication process are used. It will be appreciated though, that the interfeature areas, when present, could be of various sizes and configurations. The spots or features of distinct polymers present on the array surface are generally present as a pattern, where the pattern may be in the form of organized rows and columns of spots, e.g. a grid of spots, across the substrate surface, a series of curvilinear rows across the substrate surface, e.g. a series of concentric circles or semi-circles of spots, and the like.
In the broadest sense, the arrays are arrays of polymeric or biopolymeric ligands or molecules, i.e., binding agents, where the polymeric binding agents may be any of: peptides, proteins, nucleic acids, polysaccharides, synthetic mimetics of such biopolymeric binding agents, etc. In many embodiments of interest, the arrays are arrays of nucleic acids, including oligonucleotides, polynucleotides, cDNAs, mRNAs, synthetic mimetics thereof, and the like. [0063]
The arrays may be produced using any convenient protocol. Various methods for forming arrays from pre-formed probes, or methods for generating the array using synthesis techniques to produce the probes in situ, are generally known in the art. See, for example, Southern, U.S. Pat. No. 5,700,637; Pirrung, et al., U.S. Pat. No. 5,143,854 and Fodor, et al. (1991) [0064] Science 251:767-777, the disclosures of which are incorporated herein by reference and PCT International Publication No. WO 92/10092. For example, probes can either be synthesized directly on the solid support or substrate to be used in the array assay or attached to the substrate after they are made. Arrays may be fabricated using drop deposition from pulse jets of either polynucleotide precursor units (such as monomers) in the case of in situ fabrication, or the previously obtained polynucleotide. Such methods are described in detail in, for example, the previously cited references including U.S. Pat. Nos.: 6,242,266, 6,232,072, 6,180,351, U.S. Pat. Nos. 6,171,797, and 6,323,043, and U.S. patent application Ser. No. 09/302,898 filed Apr. 30, 1999 by Caren et al., and the references cited therein, the disclosure of which are herein incorporated by reference. Other drop deposition methods may be used for fabrication. Also, instead of drop deposition methods, photolithographic array fabrication methods may be used such as described in U.S. Pat. Nos. 5,599,695, 5,753,788, U.S. Pat. No. 6,329,143, the disclosures of which are herein incorporated by reference. As mentioned above, interfeature areas need not be present, particularly when the arrays are made by photolithographic methods as described in those patents.
A variety of solid supports or substrates may be used, upon which an array may be positioned. In certain embodiments, a plurality of arrays may be stably associated with one substrate. For example, a plurality of arrays may be stably associated with one substrate, where the arrays are spatially separated from some or all of the other arrays associated with the substrate. [0065]
The array substrate may be selected from a wide variety of materials including, but not limited to, natural polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as fiber containing papers, e.g., filter paper, chromatographic paper, etc., synthetic or modified naturally occurring polymers, such as nitrocellulose, cellulose acetate, poly (vinyl chloride), polyamides, polyacrylamide, polyacrylate, polymethacrylate, polyesters, polyolefins, polyethylene, polytetrafluoro-ethylene, polypropylene, poly (4-methylbutene), polystyrene, poly(ethylene terephthalate), nylon, poly(vinyl butyrate), cross linked dextran, agarose, etc.; either used by themselves or in conjunction with other materials; fused silica (e.g., glass), bioglass, silicon chips, ceramics, metals, and the like. For example, substrates may include polystyrene, to which short oligophosphodiesters, e.g., oligonucleotides ranging from about 5 to about 50 nucleotides in length, may readily be covalently attached (Letsinger et al. (1975) [0066] Nucl. Acids Res. 2:773-786), as well as polyacrylamide (Gait et al. (1982) Nucl. Acids Res. 10:6243-6254), silica (Caruthers et al. (1980) Tetrahedron Letters 21:719-722), and controlled-pore glass (Sproat et al. (1983) Tetrahedron Letters 24:5771-5774). Additionally, the substrate can be hydrophilic or capable of being rendered hydrophilic.
Suitable array substrates may exist, for example, as sheets, tubing, spheres, containers, pads, slices, films, plates, slides, strips, disks, etc. The substrate is usually flat, but may take on alternative surface configurations. The substrate can be a flat glass substrate, such as a conventional microscope glass slide, a cover slip and the like. Common substrates used for the arrays of probes are surface-derivatized glass or silica, or polymer membrane surfaces, as described in Maskos, U. et al., [0067] Nucleic Acids Res, 1992, 20:1679-84 and Southern, E. M. et al., Nucleic acids Res, 1994, 22:1368-73.
Each array may cover an area of less than about 100 cm[0068] ², or even less than about 50 cm², 10 cm²or 1 cm². In many embodiments, the substrate carrying the one or more arrays will be shaped generally as a rectangular solid (although other shapes are possible), having a length of more than about 4 mm and less than about 1 m, usually more than about 4 mm and less than about 600 mm, more usually less than about 400 mm; a width of more than about 4 mm and less than about 1 m, usually less than about 500 mm and more usually less than about 400 mm; and a thickness of more than about 0.01 mm and less than about 5.0 mm, usually more than about 0.1 mm and less than about 2 mm and more usually more than about 0.2 and less than about 1 mm. With arrays that are read by detecting fluorescence, the substrate may be of a material that emits low fluorescence upon illumination with the excitation light. Additionally in this situation, the substrate may be relatively transparent to reduce the absorption of the incident illuminating laser light and subsequent heating if the focused laser beam travels too slowly over a region. For example, the substrate may transmit at least about 20%, or about 50% (or even at least about 70%, 90%, or 95%), of the illuminating light incident on the substrate as may be measured across the entire integrated spectrum of such illuminating light or alternatively at 532 nm or 633 nm.
Immobilization of the probe to a suitable substrate may be performed using conventional techniques. See, e.g., Letsinger et al. ([0069] 1975) Nucl. Acids Res. 2:773-786; Pease, A. C. et al., Proc. Nat. Acad. Sci. USA, 1994, 91:5022-5026, and AOligonucleotide Synthesis, a Practical Approach,” Gait, M. J. (ed.), Oxford, England: IRL Press (1984). The surface of a substrate may be treated with an organosilane coupling agent to functionalize the surface. See, e.g., Arkins, ASilane Coupling Agent Chemistry,” Petrarch Systems Register and Review, Eds. Anderson et al. (1987) and U.S. Pat. No. 6,258,454.
Referring first to FIG. 1, typically biopolymeric arrays use a contiguous [0070] planar substrate 110 carrying an array 112 disposed on a rear surface 111 b of substrate 110. It will be appreciated though, that more than one array (any of which are the same or different) may be present on rear surface 111 b, with or without spacing between such arrays. That is, any given substrate may carry one, two, four or more arrays disposed on a front surface of the substrate and depending on the use of the array, any or all of the arrays may be the same or different from one another and each may contain multiple spots or features. The one or more arrays 112 usually cover only a portion of the rear surface 111 b, with regions of the rear surface 111 b adjacent the opposed sides 113 c, 113 d and leading end 113 a and trailing end 113 b of slide 110, not being covered by any array 112. A front surface 111 a of the slide 110 does not carry any arrays 112. Each array 112 can be designed for testing against any type of sample, whether a trial sample, reference sample, a combination of them, or a known mixture of biopolymers such as polynucleotides. Substrate 110 may be of any shape, as mentioned above.
As mentioned above, [0071] array 112 contains multiple spots or features of biopolymers, e.g., in the form of polynucleotides. As mentioned above, all of the features may be different, or some or all could be the same. The interfeature areas, if present, could be of various sizes and configurations. Each feature carries a predetermined biopolymer such as a predetermined polynucleotide (which includes the possibility of mixtures of polynucleotides). It will be understood that there may be a linker molecule (not shown) of any known types between the rear surface 111 b and the first nucleotide.
[0072] Substrate 110 may carry on front surface 111 a an identification code (not shown), e.g., in the form of bar code or the like printed on a substrate in the form of a paper label attached by adhesive or any convenient protocol or may be a part of the array substrate such as etched, painted or printed directly on the array substrate or the like. The identification code contains information relating to array 112, where such information may include, but is not limited to, an identification of array 112, i.e., layout information relating to the array(s), etc.
Array Coverslip Devices [0073]
The subject array coverslip devices generally include a solid support or substrate having a first planar surface or planar array facing surface that has at least one wall structure thereon. The first planar surface may be regular or have irregularities, modified or unmodified, as will be described below. The coverslip substrate may be fabricated from a single material, or be a composite of two or more different materials. While the nature of the coverslip substrate may vary considerably, representative materials from which it may be selected include, but are not limited to: plastics, such as polyacrylamide, polyacrylate, polymethacrylate, polyesters, polyolefins, polyethylene, polytetrafluoro-ethylene, polypropylene, poly (4-methylbutene), polystyrene, poly(ethylene terephthalate); fused silica (e.g., glass); bioglass; silicon chips, ceramics; metals; and the like, as well as other suitable materials described in U.S. application Ser. No. 10/172,850, where in certain embodiments optically transparent substrate are employed. In many embodiments, the coverslip substrate is made from glass. [0074]
The coverslip substrates of the subject invention may assume a variety of shapes ranging from simple to complex, with the only limitation that they are suitably shaped to fit over at least on array positioned on an array substrate. In many embodiments, the coverslip substrates will assume a circular, square, or oblong, where substrates having a rectangular shape are of particular interest, although other shapes are possible as well, such as irregular or complex shapes. For example, in those embodiments where at least one array is stably associated with an array substrate that is a microscope slide, e.g., a 25 mm by 75 mm glass substrate, the coverslip may be similarly rectangularly shaped. [0075]
Similarly, the size of the coverslip substrates may vary depending on a variety of factors, including, but not limited to, the size of the array substrate, the size of the array area on the substrate, and the like. In certain embodiments of the subject devices having a substantially rectangular shape, the length of the coverslip substrates typically ranges from about 15 mm to about 40 mm, usually from about 20 mm to about 35 mm and more usually from about 20 mm to about 30 mm, the width typically ranges from about 15 mm to about 40 mm, usually from about 20 mm to about 35 mm and more usually from about 20 mm to about 30 mm and the thickness typically ranges from about 0.01 mm to about 3 mm, usually from about 0.02 mm to about 1 mm and more usually from about 0.02 mm to about 0.1 mm. However, these dimensions are exemplary only and may vary as appropriate. [0076]
The first planar surface or array surface of the coverslip substrate may or may not be a modified surface. As such, in certain embodiments the array facing surface that is contacted with the sample is unmodified glass. However, in other embodiments, the surface that is contacted with the sample may include one or more surface modification agents or components, which agents may be present in a modification layer or coating on the substrate surface. Accordingly, the surface may be physically and/or chemically modified and may include structural features. For example, the surface may include one or more agents that may promote or inhibit the interaction of sample or other fluid to the surface of the coverslip substrate or to the rigid fluid barrier of the coverslip. In other words, the coverslip substrate may include hydrophobic or hydrophilic agents, respectively, or may display a charge. In certain embodiments, a portion of the surface may be hydrophobic and a portion hydrophilic. A variety of different hydrophobic and hydrophilic agents may be employed. Representative agents include, but are not limited to: agarose, polyacrylamide, poly-L-lysine, aminosilane, etc. The surface may include agents that may be agents that make-up a surface modification layer. In certain embodiments, the surface may be modified by chemical or physical etching or the like or include one or more physical structures thereon. [0077]
A feature of the first planar surface of the subject coverslip devices is that it is bounded on all sides by a fluid barrier that forms at least one bounded area or cavity defined by the walls of the fluid barrier and the first surface of the coverslip substrate. That is, a sealing element is positioned on a first surface of the coverslip substrate to provide a rigid wall structure. [0078]
The wall structure may be flexible such as those wall structures described in U.S. application Ser. No. 10,172,850, the disclosure of which is herein incorporated by reference, or may be rigid. By rigid it is meant that the wall structure is one that does not easily or readily compress or deform and is instead one that cannot be substantially bent or folded without breaking. The invention is further described primarily with respect to wall structures that are rigid, where such description is not intended to limit the scope of the invention as it will be apparent that the description of the rigid wall structures are also applicable to, and intended to include, the wall structures described in U.S. application Ser. No. 10,172,850. [0079]
The rigid wall structures may be fabricated from a single material, or be a composite of two or more different materials with the only limitation being that the wall structure is rigid. Representative materials from which the rigid wall structure may be selected include, but are not limited to: Teflon®, polypropylene, polystyrene, metal or metal alloy, plastics, ceramics, fused silica, quartz, glass, and the like. [0080]
The rigid wall structure or fluid barrier may be at least one wall surrounding the first surface of the coverslip substrate in a manner sufficient to form a bounded area defined by the rigid wall structure and the first planar surface, as mentioned above, where the number of distinct walls surrounding the first surface will depend on the cross-sectional shape of the cavity, e.g., one wall for a cavity having a circular cross-sectional shape and 4 walls for a cavity having a rectangular or square cross-sectional shape. The bounded area may have a variety of cross-sectional shapes, including, but not limited to, circular, triangular, rectangular, square, pentagon, hexagon, etc., and irregular cross-sectional shapes, but will usually have a rectangular, circular or square cross-sectional shape. Therefore, the number of distinct walls of the rigid wall structure on the first surface of the coverslip substrate will be at least one, and may be two, three, four, five, six or more, depending on the cross-sectional shape. The height of the walls of the rigid wall structure may vary, where the height is generally at least about 0.001 mm, usually at least about 0.01 mm and more usually at least about 0.02 mm, where in many embodiments the height does not exceed about 1 mm, and typically does not exceed about 0.1 mm. The width of the walls that make-up the rigid wall structure may vary, where the width is generally at least about 0.1 mm, usually at least about 1 mm and more usually at least about 10 mm, where in many embodiments the width does not exceed about 10 mm, and typically does not exceed about 5 mm. The lengths of the rigid wall structures may be substantially the same as the dimensions of the coverslip substrate (see for example FIGS. [0081] 4-7) so as to exactly or closely approximate the dimensions of the coverslip substrate, i.e., the rigid fluid barrier is positioned along the edges of the first surface so as to be commensurate with the planar surface's perimeter, or maybe less than the dimensions of the coverslip substrate (see for example FIG. 8) so the rigid wall structure is not commensurate with the planar surface's perimeter.
Each bounded area or cavity formed by the walls of the rigid wall structure and the planar surface of the coverslip substrate generally have a volume of at least about 1 μl, usually at least about 10 μl and more usually at least about 20 μl, where the volume may range from about 1 μl to about 500 μl, and in certain embodiments the volume may range from about 500 μl to about 1000 μl or greater, but in many embodiments does not exceed about 50 μl. The bounded area is typically defined by a first surface area of at least about 1 mm[0082] ², usually at least about 100 mm²and more usually at least about 400 mm², and walls having a height as described above. Other exemplary dimensions and volumes are described in U.S. application Ser. No. 10/172,850.
The rigid wall structure may be provided on the coverslip surface using any convenient method. For example, the rigid wall structure may be attached to the coverslip substrate using adhesives or the like or may be formed on the surface of the coverslip, e.g., by painting the structure on the coverstip, etc. In certain embodiments, the rigid walls are printed on the coverslip surface using any suitable printing process such as lithographic, screen, intaglio and relief printing protocols. [0083]
As mentioned above and described in U.S. application Ser. No. 10/172,850, the invention provides for an assay chamber or assay area associated with or including a biochemical assay site. The assay area includes the above-described coverslip devices and a second substrate that is complementary to the coverslip surface and that can be placed adjacent the coverslip device to from a fluid tight assay chamber. The assay chamber further includes at least one analysis component, e.g., an array of immobilized oligonucleotides, necessary for performing a biochemical assay such as, e.g., a binding reaction between an immobilized oligonucleotide and a complementary oligonucleotide in the sample solution. In certain embodiments, a subject wall structure may have an alternate configuration that provides for the biochemical assay; for example a wall structure may be on the second substrate having the analysis component(s), i.e., on the array, rather than on the first substrate. [0084]
Accordingly, a subject coverslip device is dimensioned to fit with an array (a substrate having at least one array thereon) to produce an array assay area or assay chamber around the array having an array assay volume that is bounded on the top and bottom by the array substrate surface and the planar surface of the coverslip device, respectively, and on the sides by the wall structure. As such, in the case where both the array substrate and the coverslip are planar, the volume of the assay area provided by joining the two together would then be the array substrate surface area enclosed by the wall structure times the height of the wall structure with the coverslip in place over the array. [0085]
As mentioned above, the array facing surface of a subject coverslip is mateable with a corresponding surface of an array or a substrate having at least one array. That is, the coverslip is an array mating coverslip that is complementary to the array with which it is to be used so that the coverslip may be placed adjacent the array to form an array assay area around the array. For example, in certain embodiments the array may be rectangular in shape, e.g., having dimensions of 25 mm by 75 mm. Accordingly, the coverslip and/or one or more bounded areas of the coverslip would, in many instances, have an analogous shape, i.e., the coverslip would be rectangular in shape and/or the rigid wall structure would be rectangular, so as to mate with and the array. [0086]
In certain embodiments, more than one wall structure is present on the first surface of the coverslip substrate (see for example FIGS. 9 and 12 showing two rigid wall structures or two cavities), where the individual rigid wall structures may be formed from one or more contiguous wall or fluid barrier or may be formed from separate walls. A multiple cavity coverslip device may be used with a substrate having more than one array, where some or all of the arrays may be the same or some or all may be different, to provide separate or individual wall structures around each array such that multiple samples may be tested with multiple arrays without cross-contamination. [0087]
In order to enable transfer of fluid from one side of the wall structure to the other side of the wall structure when the coverslip is assembled adjacent a second mateable substrate, e.g., mated with a substrate having an array thereon, the subject fluid barrier may include at least one orifice in the wall structure. The at least one orifice also advantageously enables air to escape from between mated substrates, i.e., between an array and a subject coverslip, thus providing a more even distribution of fluid, i.e., a uniform distribution of fluid, over the array which minimizes variation and enables reproducible array assay results. More specifically, at least one orifice may be present in the wall structure, where each orifice is provided by a discontinuity in a location or area of the wall structure. The number of orifices present in a wall structure typically ranges from about 1 to about 10, usually from about 1 to about 5 and usually from about 1 to about 4; however in certain embodiments a greater number of orifices may be present. In certain embodiments, e.g., where the wall structures are rigid wall structures, the orifice(s) present will be positioned in a corner of the wall structure, if the wall structure defines a shape that includes corners. Having the one or more orifices in the corner(s) (or any non-linear portion of the wall structure) helps trap air bubbles in the corner orifices, thereby providing a smooth and even or uniform distribution of fluid under the coverslip which, as mentioned above, minimizes variation and enables reproducible array assay results. Such orifices may variously be referred to as ports, inlets, outlets, drains, vents, or such similar terms. [0088]
The size of a subject orifice may vary where a subject coverslip device may include orifices of different sizes. In many embodiments, an orifice present in a wall structure is sized to be small enough so that fluid from the interior of the wall structure is retained thereby due to surface tension and/or capillary forces acting upon the fluid between the coverslip and array. Accordingly, in such embodiments, the total length of a discontinuity, i.e., an orifice, may typically range from about 0.5 mm to about 4.0 mm, usually from about 1.0 to about 3.5 mm, more usually from about 1.5 mm to about 3.0 mm. For example, for a wall structure having dimensions of 26.5 mm by 22.0 mm (for example positioned on a 26.5 mm by 22.0 mm coverslip substrate), the length of the discontinuity may range from about 0.5 mm to about 2.0 mm, usually from about 0.85 mm to about 1.75 mm and more usually from about 1.25 mm to about 1.75 mm. For a wall structure having dimensions of 30.0 mm by 24.0 mm (for example positioned on a 30.0 mm by 24.0 mm coverslip substrate), the length of the discontinuity may typically range from about 1.0 mm to about 5.0 mm, usually from about 1.25 mm to about 2.25 mm and more usually from about 1.35 mm to about 1.65 mm. It is important to note that the wall structures of the subject invention are able to accommodate such small orifices therein without impairing the functionality of the wall structure or orifice. For example, in reference to the subject rigid wall structures, if mated with a second substrate and a compression force is applied thereto, e.g., if the substrates are clamped together, the rigid wall structure will continue to provide an orifice for fluid transfer and the escape of air from the interior of the rigid wall structure because the rigid material of the wall structure will not deform about the orifice thereby blocking or closing the opening. [0089]
FIGS. [0090] 2-7 and FIG. 10 show exemplary embodiments of the subject coverslips. Turning first to FIG. 10, an exemplary embodiment of array coverslip device 16 is shown having substrate 74 with first surface 74 a and second surface 74 b. Positioned on first surface 74 a are two wall structures 76 herein illustrated as rectangular wall structures, although only one rectangular wall structure may be provided or more than two rectangular wall structures may be provided. In this particular embodiment, wall structures 76 do not include orifices.
FIG. 2A shows a plan view of array coverslip [0091] 2 having substrate 4 having first planar surface 4 a and second surface 4 b, and rectangular wall structure 6 having walls 6 a, 6 b, 6 c and 6 d positioned on planar surface 4 a. As show, wall structure 6 is positioned so as to be commensurate with the perimeter of first surface 4 a; however this need not be the case. Wall structure 6 includes orifice 9, provided by a discontinuity in the wall structure. In this embodiment, orifice 9 is positioned at a corner of wall structure 6, however as described above the orifice may be positioned in any convenient location of the wall structure, e.g., the wall structure may be of a shape that does not include corners. The discontinuity may be in any corner or any side of the wall structure. FIG. 2B shows a cross-sectional view taken along lines X-X of FIG. 2A and shows the bounded area or cavity that is formed by wall structure 6 and first surface 4 a of substrate 4. FIG. 2C shows the cross-sectional view of device 2 of FIG. 2A positioned on top of a substrate having array such as substrate 110 having array(s) 112 described above to provide a substantially sealed assay area 150 having an assay volume defined by coverslip first surface 4 a, wall structure 6 and array substrate 110. The only unsealed area of the assay area is provided by the small discontinuity in the wall structure (not shown in the cross-sectional views).
As shown in the Figures and as shown particularly in the cross-sectional views of the wall structures, the walls of the wall structure prevent fluid from moving or collecting between the sides of the walls (i.e., between the array-facing [0092] side wall 6 d″ and the exterior-facing side wall 6′) of the wall structure, as described above, such that fluid is retained in the area bounded by the array facing side wall. The prevention of fluid collection in the walls themselves of the wall structure enables an even or uniform, and predictable, distribution of fluid over an array mated with a subject coverslip, which minimizes variation between array assays and provides reproducible array assay results. Furthermore, in many instances, the amount of sample available for an array assay is extremely limited and thus it is important that all available sample be utilized for the array assay and not wasted by being collected in adjacent overflow reservoirs.
Accordingly, in many embodiments, the walls are solid or non-porous such there is not a space between the array-facing side and the exterior-facing side of a wall for fluid to collect. As shown for example in FIG. 2B with regards to [0093] wall 6 d (it is to be understood that the following description is applicable to each wall that makes-up the rigid wall structure), wall 6 d is solid having array-facing side 6 d″ and exterior-facing side 6 d′ with no space therebetween for fluid to collect. However, in certain embodiments it may be desirable to have a space between the sides of the walls of the wall structure, for example for manufacturing considerations, or the like. However, even in these embodiments where the sides of the walls define a hollow or substantially hollow rigid wall structure, fluid is still prevented from collecting in the hollow space because there is no fluid communication between the interior of the wall structure and the space between the sides of the wall structure such that the array-facing side of the wall prevents fluid from penetrating into such a space between the sides of the walls. For example, the interface or contact, at least between the array-facing side of the wall and the planar surface of the coverslip substrate upon which the array-facing side wall is positioned, provides a fluid-tight seal and at least the array-facing side is non-porous, such that there is no opening or rather no fluid communication channel through which fluid may flow into a space, if present, between the sides of the walls or the wall structure. In certain embodiments, at least the array-facing side wall may be hydrophobic. The subject coverslips differ significantly in this regard from prior art coverslips which require overflow chambers of relatively large volumetric capacity (see for example U.S. Pat. No. 3,879,106 entitled “Microscope Slide Cover Slip” which describes a coverslip to be used with a microscope slide that has overflow chambers to collect and retain excess fluid placed in between the coverslip and the microscope slide).
FIG. 3 shows another exemplary embodiment of a subject coverslip device. [0094] Coverslip device 4 is analogous to coverslip 2 of FIGS. 2A-2C with the difference being the presence of two orifices 19 in wall structure 16 on first surface 14 a of substrate 14. In this embodiment, the discontinuities are positioned at two corners on the same side of wall structure 16; however they may be positioned in any convenient location of the wall structure. For example, the two orifices may be present in any two corners of wall structure 16 or one orifice may be present in a corner and another orifice in a side of wall structure 16. FIG. 4 shows array coverslip device 6 which is analogous to coverslip 4 of FIG. 3, except that the two orifices 29 are positioned in diagonally opposing corners of wall structure 26.
FIG. 5 shows another exemplary embodiment of a subject coverslip device. [0095]
[0096] Coverslip device 8 is analogous to the above described coverslips with the difference being the presence of four orifices in wall structure 39 on first surface 34 a of substrate 34. In this embodiment, the four discontinuities are positioned at the four corners on of wall structure 36; however they may be positioned in any convenient location of the wall structure. For example, one or more orifices may be present in any one or more corners of wall structure 36 and the remaining orifices may be present in one or more sides of wall structure 36.
FIG. 6 shows another exemplary embodiment of a subject coverslip device. [0097]
[0098] Coverslip device 10 is analogous to the above-described coverslips and includes two orifices 49 present in two corners of wall structure 46. However, in this particular embodiment, the wall structure is not positioned at the edges or the perimeter of substrate 44, but rather has dimensions smaller than those of substrate 46 and is positioned a distance from the edges, i.e., is not commensurate with the perimeter of the substrate.
FIG. 7 shows another exemplary embodiment of a subject coverslip device. [0099] Coverslip device 12 has two separate wall structures 56 each with orifice 59 providing two individual bounded areas or cavities, although the two bounded areas could be formed by one contiguous wall structure. In this particular embodiment, two wall structures, i.e., two bounded areas; are provided, each having one orifice. However, in certain embodiments more wall structures, i.e., more bounded areas, may be present, each having more than one orifice.
Systems [0100]
Also provided by the subject invention are array assay systems that include the subject array assay coverslips. Such systems include an array coverslip as described above and an array. In certain embodiments, the subject systems may further include reagents employed in array based assay protocols, including sample preparation reagents, e.g., labeling reagents, etc., washing fluids, buffers such as array assay buffers, etc. [0101]
Methods [0102]
The subject invention also includes methods for assaying a sample for the presence of at least one analyte using the subject array coverslip devices. The subject methods may be employed in any array based assay such as a hybridization assay or any other analogous binding interaction assay. [0103]
In the subject methods, a subject coverslip is joined to a ligand array that includes an analyte specific ligand immobilized on a surface of the ligand array substrate. In the instance where the wall structure does not include an orifice, sample is contacted with the array prior to covering the array with the coverslip. In those embodiments where the wall structure includes at least one orifice, the array may be covered by a cover slip and sample introduced to the formed assay area using at least one orifice. The subject methods will further be described primarily in reference to those embodiments where a coverslip is first joined to an array and sample is introduced into the interior of the assay area by an orifice in the coverslip, where such description is exemplary only and is in no way intended to limit the scope of the inventions. However, it will be apparent that the subject methods may be easily modified to include the above-described embodiments where sample is contacted with an array before the coverslip covers the array, following which the remainder of the methods is carried out as described below. [0104]
As shown in the cross-sectional view of FIG. 8, [0105] coverslip device 14 having substrate 64 with first surface 64a having at least one wall structure 66 (shown here as a single wall structure defining a single bounded area, but more bounded areas may be present in certain embodiments), present thereon, is placed on surface 111 b of array substrate 110. As shown, at least one orifice 69 is present in wall structure 66. Array substrate 110 includes at least one array 112 (shown here as one array 112, but more than one array may be present in certain embodiments). The coverslip and the array are brought together in the directions of the arrows 155 to form an assay area around array 112 formed by the walls of wall structure 66, surface 64 a of substrate 64 and surface 111 b of array substrate 110, wherein fluid transfer to and from the array assay area may be accomplished using orifice 69.
Once an array is covered by a subject coverslip, sample is then introduced to the array. That is, the sample is introduced into the assay area using the orifice(s) provided by the coverslip. FIG. 9 shows the assembled structure of FIG. 8 having sample S (represented here as a stippling pattern) in the assay area, where sample S has been contacted with [0106] array 112 by introducing the sample through the orifice in this particular embodiment. In those embodiments having more than one assay area, for example each covering a different array to provide multiple, individual array assay areas- each around an array, the same sample may be applied to one or more assay areas, for example when it is desirable to test the same sample with different arrays during the same assay procedure, or a different sample may be applied to one or more assay areas than is applied to one or more other assay area, for example when it is desirable to test different samples with the same array during the same array assay procedure.
The amount of sample applied to each assay area may vary depending on a variety of factors such as the type of array and the like, but generally the amount of sample applied to each assay area is at least about 1 μl, usually at least about 10 μl and more usually at least about 20 μl, where the amount of sample may be as great as 1000 μl or greater in certain embodiments, but often does not exceed about 200 μl and more often does not exceed about 50 μl. The sample may be introduced through an orifice using any convenient protocol, where in many embodiments a deposition type protocol is employed, e.g., by pipette or the like. [0107]
Following contact of the array and the sample, the resultant sample contacted array/coverslip structure is then maintained under conditions sufficient and for a sufficient period of time for any binding complexes between members of specific binding pairs to occur. In many embodiments, the duration of this step may be at least about 1 minute long, often at least about 1 hour long, and may be as long as 24 hours or longer in certain embodiments, but often does not exceed about 17 hours to about 20 hours or to about 24 hours long. The sample contacted array/coverslip structure is typically maintained at a temperature ranging from about 4° C. to about 75° C., usually from about 45° C. to about 65° C. Where desired, the sample may be agitated to ensure contact of the sample with the array. In the case of hybridization assays, the sample is contacted with the array under stringent hybridization conditions, whereby complexes are formed between target nucleic acids that are complementary to probe sequences attached to the array surface, i.e., duplex nucleic acids are formed on the surface of the substrate by the interaction of the probe nucleic acid and its complement target nucleic acid present in the sample. An example of stringent hybridization conditions is hybridization at 50° C. or higher and 0.1×SSC (15 mM sodium chloride/1.5 mM sodium citrate). Another example of stringent hybridization conditions is overnight incubation at 42° C. in a solution: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10% dextran sulfate, followed by washing in 0.1×SSC at about 65° C. Hybridization involving nucleic acids generally takes from about 30 minutes to about 24 hours, but may vary as required. Stringent hybridization conditions are hybridization conditions that are at least as stringent as the above representative conditions, where conditions are considered to be at least as stringent if they are at least about 80% as stringent, typically at least about 90% as stringent as the above specific stringent conditions. Other stringent hybridization conditions are known in the art and may also be employed, as appropriate. [0108]
Once the incubation step is complete, the array is typically washed at least one time to remove any unbound and non-specifically bound sample from the substrate, generally at least two wash cycles are used. Accordingly, the coverslip is removed from the array so that the array may be washed. Washing agents used in array assays are known in the art and, of course, may vary depending on the particular binding pair used in the particular assay. For example, in those embodiments employing nucleic acid hybridization, washing agents of interest include, but are not limited to, SSC, SDS, and the like as is known in the art, at different concentrations and may include some surfactant as well. [0109]
Following the washing procedure, as described above, the array is then interrogated or read so that the presence of the binding complexes is then detected i.e., the label is detected using calorimetric, fluorimetric, chemiluminescent or bioluminescent means. The presence of the analyte in the sample is then deduced from the detection of binding complexes on the substrate surface. [0110]
Utility [0111]
The above-described methods find use in a variety of different applications, where such applications are generally analyte detection applications in which the presence of a particular analyte in a given sample is detected at least qualitatively, if not quantitatively. [0112]
Specific analyte detection applications of interest include hybridization assays in which the nucleic acid arrays of the subject invention are employed. In these assays, a sample of target nucleic acids is first prepared, where preparation may include labeling of the target nucleic acids with a label, e.g. a member of signal producing system. Following sample preparation, a subject coverslip is positioned over an array and the prepared sample product is contacted with the array under hybridization conditions by introducing the sample through one or more orifices present on the coverslip, as described above, whereby complexes are formed between target nucleic acids that are complementary to probe sequences attached to the array surface. As mentioned above, in certain embodiments, sample is contacted with the array prior to covering the array with a coverslip. The presence of hybridized complexes is then detected. Specific hybridization assays of interest which may be practiced using the subject arrays include: gene discovery assays, differential gene expression analysis assays; nucleic acid sequencing assays, and the like. Patents describing methods of using arrays in various applications include U.S. Pat. Nos.: 5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028; 5,800,992; the disclosures of which are herein incorporated by reference. [0113]
Where the arrays are arrays of polypeptide binding agents, e.g., protein arrays, specific applications of interest include analyte detection/proteomics applications, including those described in U.S. Pat. Nos.: 4,591,570; 5,171,695; 5,436,170; 5,486,452; 5,532,128; and 6,197,599; as well as published PCT application Nos. WO 99/39210; WO 00/04832; WO 00/04389; WO 00/04390; WO 00/54046; WO 00/63701; WO 01/14425; and WO 01/40803; the disclosures of the United States priority documents of which are herein incorporated by reference. [0114]
Reading of the array may be accomplished by illuminating the array and reading the location and intensity of resulting fluorescence at each feature of the array to detect any binding complexes on the surface of the array. For example, a scanner may be used for this purpose that is similar to the AGILENT MICROARRAY SCANNER available from Agilent Technologies, Palo Alto, Calif. Other suitable apparatus and methods are described in U.S. patent applications: Ser. No. 09/846125 “Reading Multi-Featured Arrays” by Dorsel et al.; and Ser. No. 09/430214 “Interrogating Multi-Featured Arrays” by Dorsel et al.; which references are incorporated herein by reference. However, arrays may be read by any other method or apparatus than the foregoing, with other reading methods including other optical techniques (for example, detecting chemiluminescent or electroluminescent labels) or electrical techniques (where each feature is provided with an electrode to detect hybridization at that feature in a manner disclosed in U.S. Pat. No. 6,221,583 and elsewhere). Results from the reading may be raw results (such as fluorescence intensity readings for each feature in one or more color channels) or may be processed results such as obtained by rejecting a reading for a feature which is below a predetermined threshold and/or forming conclusions based on the pattern read from the array (such as whether or not a particular target sequence may have been present in the sample or an organism from which the sample was obtained has a particular condition). The results of the reading (processed or not) may be forwarded (such as by communication) to a remote location if desired, and received there for further use (such as further processing), as now described in greater detail. [0115]
In certain embodiments, the subject methods include a step of transmitting data from at least one of the detecting and deriving steps, as described above, to a remote location. By “remote location” is meant a location other than the location at which the array is present and hybridization occur. For example, a remote location could be another location (e.g. office, lab, etc.) in the same city, another location in a different city, another location in a different state, another location in a different country, etc. As such, when one item is indicated as being “remote” from another, what is meant is that the two items are at least in different buildings, and may be at least one mile, ten miles, or at least one hundred miles apart. “Communicating” information means transmitting the data representing that information as electrical signals over a suitable communication channel (for example, a private or public network). “Forwarding” an item refers to any means of getting that item from one location to the next, whether by physically transporting that item or otherwise (where that is possible) and includes, at least in the case of data, physically transporting a medium carrying the data or communicating the data. The data may be transmitted to the remote location for further evaluation and/or use. Any convenient telecommunications means may be employed for transmitting the data, e.g., facsimile, modem, Internet, etc. [0116]
Kits [0117]
Finally, kits for use in practicing the subject methods are also provided. The subject kits at least include one or more subject coverslips, as described above. The subject kits may also include one or more arrays. The kits may further include one or more additional components necessary for carrying out an analyte detection assay, such as sample preparation reagents, buffers, labels, and the like. As such, the kits may include one or more containers such as vials or bottles, with each container containing a separate component for the assay, and reagents for carrying out an array assay such as a nucleic acid hybridization assay or the like. The kits may also include a denaturation reagent for denaturing the analyte, buffers such as hybridization buffers, wash mediums, enzyme substrates, reagents for generating a labeled target sample such as a labeled target nucleic acid sample, negative and positive controls. [0118]
In addition to the above components, the subject kits also typically include written instructions for practicing the subject methods. The instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, etc. [0119]
It is evident from the above discussion that the above described invention provides devices and methods for performing array assays which are simple to use, inexpensive and can be used with a multitude of different array formats. The above described invention provides for a number of advantages, including ease of use, uniform distribution of fluid over the array, fluid transfer between locations on either side of the fluid barrier when the coverstip is assembled with an array, fluid loss prevention and the ability to test multiple samples with multiple arrays without cross-contamination. As such, the subject invention represents a significant contribution to the art. [0120]
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. [0121]
While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. [0122]

Claims

What is claimed is:

1. An array coverslip device comprising:

an array coverslip; and

at least one wall structure comprising at least one orifice on a planar surface of said coverslip.

2. The device according to claim 1, wherein said at least one orifice is a discontinuity in one or more locations of said wall structure.

3. The device according to claim 1, wherein said at least one orifice is located at a corner of said wall structure.

4. The device according to claim 1, wherein said at least one orifice is sealable.

5. The device according to claim 4, wherein said at least one orifice is self-sealing.

6. The device according to claim 1, wherein said wall structure is rigid.

7. The device according to claim 1, wherein said wall structure is flexible.

8. The device according to claim 1, wherein said array cover slip has two or more wall structures on said planar surface.

9. The device according to claim 1, wherein said at least one wall structure and said planar surface define a bounded area having a volume that ranges from about 1 μl to about 1000 μl.

10. The device according to claim 1, wherein said planar surface is hydrophobic.

11. The device according to claim 1, wherein said planar surface is hydrophilic.

12. The device according to claim 1, wherein said coverslip is dimensioned to be joined with an array to provide a defined assay area bounded by said array, said planar surface and said wall structure.

13. The device according to claim 12, wherein said wall structure is commensurate with said planar surface's perimeter.

14. The device according to claim 1, wherein said wall structure is not commensurate with said planar surface's perimeter.

15. The device according to claim 14, wherein said coverslip has a rectangular configuration.

16. The device according to claim 1, wherein said wall structure forms a bounded area.

17. An array coverslip device comprising:

an array coverslip; and

at least one rectangular rigid wall structure on a planar surface of said coverslip.

18. An array assay device comprising:

(a) an array coverslip comprising at least one wall structure having at least one orifice on a planar surface of said coverslip; and

(b) a substrate having at least one array, which substrate is joined to said array coverslip.

19. An array assay device comprising:

(a) an array coverslip comprising at least one rectangular rigid wall structure on a planar surface of said coverslip; and

20. A method of assaying a sample for the presence of at least one analyte, said method comprising:

(a) joining an array coverslip comprising at least one wall structure having at least one orifice on a planar surface of said coverslip with a ligand array,

(b) performing an array assay using said coverslip joined with said ligand array; and

(c) reading said ligand array to obtain a result.

21. The method according to claim 20, further comprising contacting said ligand array with a sample, wherein said ligand array and said coverslip are joined together prior to contacting said array with said sample.

22. The method according to claim 20, further comprising contacting said ligand array with a sample, wherein said ligand array and said coverslip are joined together subsequent to contacting said array with said sample.

23. The method according to claim 22, further comprising introducing said sample to said ligand array through said at least one orifice.

24. The method according to claim 23, further comprising removing said sample from said ligand array using said at least one orifice.

25. The method according to claim 20, wherein said ligand array is a nucleic acid array.

26. The method according to claim 25, wherein said analyte is a nucleic acid.

27. The method according to claim 20, wherein said ligand array is a polypeptide array.

28. The method according to claim 27, wherein said analyte is a polypeptide.

29. The method according to claim 20, wherein said wall structure is rigid.

30. A method comprising transmitting a result obtained by a method of claim 20 from a first location to a second location.

31. The method according to claim 30, wherein said second location is a remote location.

32. A method comprising receiving said result obtained by the method of claim 30.

33. A kit for performing an assay, said kit comprising:

(a) an array coverslip device according to claim 1; and

(b) instructions for using said array coverslip device to perform an array assay.

34. The kit according to claim 33, wherein said kit further comprises a ligand array.