WO2006055925A2 - Microarrays for analyte detection - Google Patents

Abstract

On embodiment of the present invention describes the use of microarrays to investigate the carbohydrate binding specificities of bacteria, to detect pathogens, and to screen anti-adhesion therapeutics is reported. This system is ideal for whole cell applications because microarrays present carbohydrate ligands in a manner that mimics interactions at cell-cell interfaces. Other advantages include assay miniaturization, since minimal amounts (e.g., on the order of picomoles) of a ligand (i.e., a biological recognition element) are required to observe binding, and high-throughput, since thousands of compounds can be placed on an array and analyzed in parallel. Pathogen detection experiments can be completed in complex mixtures of cells or protein using the known carbohydrate binding epitopes of the pathogens in question. The non-destructive nature of the arrays allows the pathogen to be harvested and tested for antibacterial susceptibility. These investigations allow microarray-based screening of biological samples for contaminants and combinatorial libraries for anti-adhesion therapeutics.

Description

Microarrays for Analyte Detection

Related Applications

This application claims the benefit of priority to United States Provisional Patent Application serial number 60/628,927, filed November 19, 2004; which is hereby incorporated by reference in its entirety.

Background of the Invention

Roles of Carbohydrates in Biological Processes. Carbohydrates displayed on the surface of cells play critical roles in cell-cell recognition, adhesion, signaling between cells, and as markers for disease progression. Neural cells use carbohydrates to facilitate development and regeneration; cancer cell progression is often characterized by increased carbohydrate-dependent cell adhesion and the enhanced display of carbohydrates on the cell surface; viruses recognize carbohydrates to gain entry into host cells; and bacteria bind to carbohydrates for host cell adhesion. [Kleene R, Schachner M. (2004) Glycans and neural cell interactions. Nat. Rev. Neurosci., 5, 195-208; Hakomori S, Handa K. (2002)

Glycosphingolipid-dependent cross-talk between glycosynapses interfacing tumor cells with their host cells: essential basis to define tumor malignancy. FEBS Lett., 531, 88-92; Smith AE, Helenius A. (2004) How viruses enter animal cells. Science, 304, 237-42; and Karlsson KA. (1999) Bacterium-host protein-carbohydrate interactions and pathogenicity. Biochem. Soc. Trans., 27 ^', 471-4.] Identification of the specific saccharides involved in these processes is important to better understand cell-cell recognition at the molecular level and to aid the design of therapeutics and diagnostic tools.

Many interactions at cell-cell interfaces involve multiple binding events that occur simultaneously. [Kiessling LL, Gestwicki JE, Strong LE. (2000) Synthetic multivalent ligands in the exploration of cell-surface interactions. Curr. Opin. Chem. Biol, 4, 696-703.] This "multivalent" type of binding amplifies affinities relative to interactions that involve only a single ligand. [Mammen M, Choi SK, Whitesides GM. (1998) Polyvalent interactions in biological systems: Implications for design and use of multivalent ligands and inhibitors. Angew. Chem. Inter. Ed. Engl., 37, 2755-2794.] This effect has lead to the development of multivalent anti-adhesive therapeutics against bacteria and viruses by displaying carbohydrates on flexible polymers (see below). [Autar R, Khan AS, Schad M, Hacker J, Liskamp RM, Pieters RJ. (2003) Adhesion inhibition of FlC-fimbriated Escherichia coli and Pseudomonas aeruginosa PAK and PAO by multivalent carbohydrate ligands. ChemBioChem, 4, 1317-1325; Nagahori N, Lee RT, Nishimura S, Page D, Roy R, Lee YC. (2002) Inhibition of adhesion of type 1 fimbriated Escherichia coli to highly mannosylated ligands. ChemBioChem, 3, 836-844; Reuter JD, Myc A, Hayes MM, Gan Z, Roy R, Qin D, Yin R, Piehler LT, Esfand R, Tomalia DA, Baker JR, Jr. (1999) Inhibition of viral adhesion and infection by sialic-acid-conjugated dendritic polymers. Bioconjug. Chem., 10, 271-278; Choi SK, Mammen M, Whitesides GM. (1997) Generation and in situ evaluation of libraries of poly(acrylic acid) presenting sialosides as side chains as polyvalent inhibitors of influenza-mediated hemagglutination. J. Am. Chem. Soc, 119, 4103-4111; and Sigal GB, Mammen M, Dahmann G, Whitesides GM. (1996) Polyacrylamides bearing pendant alpha-sialoside groups strongly inhibit agglutination of erythrocytes by influenza virus: The strong inhibition reflects enhanced binding through cooperative polyvalent interactions. J. Am. Chem. Soc, 1 18, 3789-3800.] Dendrimers and bovine serum albumin (BSA) have also been used as multivalent scaffolds. Additionally, devices that are responsive to the presence of a pathogen use multivalent binding for recognition. [Charych DH, Nagy JO, Spevak W, Bednarski MD. (1993) Direct Colorimetric Detection of a Receptor-Ligand Interaction By a Polymerized Bilayer Assembly. Science, 261, 585-588; Ma ZF, Li JR, Liu MH, Cao J, Zou ZY, Tu J, Jiang L. (1998) Colorimetric detection of Escherichia coli by polydiacetylene vesicles functionalized with glycolipid. J. Am. Chem. Soc, 120, 12678-12679; and Disney MD, Zhang J, Swager TM, Seeberger PH. (2004) Detection of Bacteria with Carbohydrate- Functionalized Fluorescent Polymers. J. Am. Chem. Soc, 126, 13343-13346.]

Carbohydrate Microarrays. An array is an orderly arrangement of samples. Microarrays of biological materials are comprised of a number of small discrete deposits of biological materials, such as DNA, RNA, proteins, or carbohydrates, arranged in predetermined patterns on a solid support. The deposits are generally very small (e.g., in the range 100-200 μm in diameter) which allows for fabrication of plates containing a large number of deposits for conducting a large number of separate experiments. To maximize productivity, microarrays are generally prepared using robotics. The solid support can be glass, a polymer, or metal surface. Microarray technology has proven to be very effective for the study of DNA, RNA, and proteins. Microarrays that contain carbohydrate molecules have been reported. The carbohydrate can be bound to solid support by a covalent or noncovalent interaction. The mode of attachment is limited by the necessity that the bond formed between the solid support and the carbohydrate is both durable and does not interfer with testing assays, e.g., binding affinity or biological activity. [See D. Schena et al. (1995) Science, 270, 467; and S. L. Schreiber et al. (2000) Science, 289, 176.] Carbohydrate microarrays comprise a plurality of carbohydrates (probes or targets) immobilized on a surface of a substrate, where probes or targets of known identity are located in known and hence addressable locations over the surface of the substrate. In a typical assay in which a carbohydrate microarray is used in the analysis of carbohydrates, molecules, such as antibodies directly or indirectly tagged with fluorescent markers, are interacted with the substrate, resulting in binding of targets and probes according to their identity. Following appropriate washes, the chip is scanned, typically with a laser-scanner, which excites the fluorescent tags (where present) and reads the emitted light. Depending on the application, the pattern of fluorescence over the surface of the chip provides information on the identity of the targets/probes and/or the level of their expression.

Protein-carbohydrate interactions have been studied through immobilization of carbohydrates onto glass or gold slides or onto the wells of a 96- well plate. [Adams EW, Ueberfeld J, Ratner DM, O'Keefe BR, Walt DR, Seeberger PH. (2003) Encoded fiber-optic microsphere arrays for probing protein-carbohydrate interactions. Angew Chem Int Ed Engl, 42, 5317-20; Adams EW, Ratner DM, Bokesch HR, McMahon JB, O'Keefe BR, Seeberger PH. (2004) Oligosaccharide and glycoprotein microarrays as tools in HIV glycobiology; glycan-dependent gpl20/protein interactions. Chem Biol, 11, 875-81; Fukui S, Feizi T, Galustian C, Lawson AM, Chai W. (2002) Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions. Nat Biotechnol, 20, 1011-7; Park S, Shin I. (2002) Fabrication of carbohydrate chips for studying protein-carbohydrate interactions. Angew Chem Int Ed Engl, 41, 3180-2; Ratner DM, Adams EW, Su J, O'Keefe BR, Mrksich M, Seeberger PH. (2004) Probing protein- carbohydrate interactions with microarrays of synthetic oligosaccharides. Chembiochem, 5, 379-82; Fazio F, Bryan MC, Blixt O, Paulson JC, Wong CH. (2002) Synthesis of sugar arrays in microtiter plate. J Am Chem Soc, 124, 14397-402; Bryan MC, Plettenburg O, Sears P, Rabuka D, Wacowich-Sgarbi S, Wong CH. (2002) Saccharide display on microtiter plates. Chem Biol, 9, 713-20; and Houseman BT, Mrksich M. (2002) Carbohydrate arrays for the evaluation of protein binding and enzymatic modification. Chem Biol, 9, 443-54.] Additionally, antibiotic microarrays have been used to study the interactions of these compounds to both resistance-causing enzymes and to therapeutic targets. [Disney MD, Seeberger PH. (2004) Aminoglycoside microarrays to explore interactions of antibiotics with RNAs and proteins. Chemistry, 10, 3308-14; and Disney MD, Magnet S, Blanchard JS, Seeberger PH. (2004) Aminoglycoside microarrays to study antibiotic resistance. Angew Chem Int Ed Engl, 43, 1591-4.] Each of the above examples requires a purified biomolecule to be hybridized with the array. One example where proteins did not have to be purified explored the binding of chicken hepatocytes and human T-cells to carbohydrates displayed on low density arrays. [Nimrichter L, Gargir A, Gortler M, Altstock RT, Shtevi A, Weisshaus O, Fire E, Dotan N, Schnaar RL. (2004) Intact cell adhesion to glycan microarrays. Glycobiology, 14, 197-203.]

In another example, nitrocellulose coated slides were employed for the noncovalent immobilization of microbial polysaccharides and neoglycolipid modified oligosaccharides. [S. Fukui et al. (2002) Nat. Biotechnol., 20, 1011.] Wong and coworkers used hydrophobic interactions to anchor lipid-bearing carbohydrates onto polystyrene microtiter plates. [C. H. Wong al. (2002) J. Am. Chem. Soc, 124, 14397.] This technology entailed forming triazole rings by 1,3-dipolar cycloaddition of alkynes and azides wherein the azido group was attached to the carbohydrate via an ethylene tether. The triazole ring functioned as a hydrophobic anchor to a solid support comprised of saturated hydrocarbon chains 13-15 carbons in length. However, noncovalent bonds are not as strong as a covalent bonds. In one approach to covalent attachment of a carbohydrate to a solid support, Diels- Alder- mediated covalent immobilization of cyclopentadiene-derivatized monosaccharides was achieved on a gold surface bearing benzoquinone groups. [B. Houseman and M. Mrksich (2002) Chem. Biol., 9, 443.] Another covalent immobilization technology involved reacting maleimide functionalized mono- and di-saccharide glycosylamines with a thiol-derivatized glass slide, or alternatively, thiol-functionalized carbohydrates with a self-assembled monolayer presenting maleimide groups. [See S. Park et al. (2002) Angew. Chem. Int. Ed., 41, 3180.] Cell-Surface Carbohydrate Recognition by Pathogens. A variety of pathogens use cell-surface carbohydrates present on human, animal, or plant cells as scaffolds to facilitate binding and pathogenesis. [Karlsson KA. (2001) Pathogen-host protein- carbohydrate interactions as the basis of important infections. Adv Exp Med Biol, 491, 431- 43; and Sharon N. (1987) Bacterial lectins, cell-cell recognition and infectious disease. FEBS Lett, 217, 145-57.] Extensive information on this subject has afforded a database of carbohydrate-pathogen interactions that exists in the public domain, for example, E. coli binds to mannose; influenza binds to sialic acid, etc. The interactions of pathogens with cell surface carbohydrates are often multivalent, which results in higher binding affinity compared to monovalent binding. Carbohydrate-protein interactions in a monovalent setting (i.e., one ligand-analyte interaction at a time) are very weak, often occurring with K_d 's in the millimolar range. To overcome this, carbohydrate-cell interactions are often multivalent in nature (i.e., many ligand-analyte interactions occur at a time). This multivalent binding increases affinities as the product of each interaction, minus the effect of linking the compounds together. The result is increasing the overall affinities into the micro- or nanomolar range. Figure 1 gives a schematic of multivalent interactions that occur during cell-cell recognition. While carbohydrate arrays have found use in studying protein interactions, their use to detect whole pathogens in a non-destructive manner has not been reported.

Summary of the Invention

The present invention generally relates to arrays able to participate in the pathogen- recognition process. The subject matter of the present invention involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.

In one aspect, the invention provides a method of determining the presence of a pathogen (analyte). According to one set of embodiments, the method includes an act exposing a sample suspected of containing a pathogen to an indicator and an array. If the sample contains the pathogen (analyte), then the pathogen (analyte) binds the array and the signal from the indicator (fluorescence) allows for a signal to be produced at a threshold level.

According to another set of embodiments, the method includes acts of exposing a sample suspected of containing a biological entity to an array comprising a plurality of biological recognition elements, at least some of which are able to specifically interact with the biological entity, and determining the fluorescence of the sample.

In yet another set of embodiments, the method is a method of determining a biological entity. The method includes steps of exposing a sample suspected of containing a biological entity to an array capable of multivalent binding to a biological entity, and determining the biological entity by determining fluorescence of the sample. In still another set of embodiments, the method includes a step of specifically binding an array to a biological entity. Another set of embodiments includes placing a sample into a solution that contains a cell-permeable fluorescent dye. The sample is then hybridized onto the array and the binding is detected through the fluorescence of the stained cells.

Another set of embodiments comprises capturing of a pathogen by an array; and culturing the pathogen to determine its presence and optionally the effect of therapeutics on pathogen growth. Detection can also be achieved without labeling the cells; for example, using Surface Plasmon Resonance spectroscopy (SPR)-based or other "label free" methods for determining binding of the analytes to the arrays.

In another aspect, the present invention is directed to a method of making one or more of the embodiments described herein. In yet another aspect, the present invention is directed to a method of using one or more of the embodiments described herein. In still another aspect, the present invention is directed to a method of promoting one or more of the embodiments described herein.

Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control.

Brief Descriptions of the Drawings

Figure 1 depicts a schematic diagram illustrating multivalency, in accordance with one embodiment of the invention.

Figure 2 depicts a schematic of a multivalent binding of a cell to an array. Figure 3 depicts various 2-aminoethyl pyranosides useful in the present invention.

Figure 4 depicts a schematic illustrating one embodiment of the detection scheme, involving cell staining. Figure 5 depicts an image of a carbohydrate array after incubation with ORNl 78 that were stained with SYTO 83 cell-permable nucleic acid staining dye. Each _ concentration was spotted with three rows of five spots. Each spot is the result of delivery of 1 nL of a 20 mM, 5 niM, 1.25 niM, 310 μM, 63 μM, or 15 μM, carbohydrate-containing solution. The spot diameter is about 200 μm.

Figures 6A and 6B illustrates an example of an array hybridized to E. coli in serum and erythrocytes. Figure 6A depicts both a picture of the 20 nanomole spots taken using a fluorescent slide scanner after hybridization of a solution containing ORNl 78 with various concentrations of erythrocytes (20 picomoles of mannose is the amount delivered to the surface) and brightfield microscopic images of arrays after hybridization with ORNl 78 and 10⁹ or 10⁷ erythrocytes. Figure 6B depicts plots of data after array hybridization of 10⁸ ORNl 78 in a background of erythrocytes (top) and in serum (bottom).

Figure 7 depicts a photomicrograph illustrating that bacteria can be harvested in a live form from an array and tested for antibacterial susceptibility. Bacteria were harvested from a carbohydrate array, with cells stained with SYTO 62, and tested for antibacterial susceptibility. An image of an LB plate after bacteria are harvested from an array and streaked onto the plate from mannose-containing spots and non-mannose containing spots (top). Results from anti-bacterial susceptibility testing of the harvested bacteria (bottom).

Figure 8 illustrates the use of arrays to screen bacteria for carbohydrate binding: images of mannose positions on the arrays after hybridization with mannose binding E. coli, ORNl 78, or mutant E. coli, ORN208, which has a greatly diminished mannose binding affinity; plots of the experimental data from these two slides, the errors being the standard deviation in measurement. Each spot is the result of delivering about 20 picomoles of mannose.

Figure 9 illustrates the use of arrays to screen anti-adhesion inhibitors. Images from slides after incubation with serially diluted concentrations of inhibitors are shown (top). ICt)O were determined by placing varying concentrations of inhibitors into hybridization solution containing 10 ORNl 78 and binding to mannose-containing spots. The resulting data was plotted versus the concentration of the inhibitor (for the polymer this is moles of mannose).

Figure 10 depicts the binding of ORN 178 to an array of complex mannosides. This bacterial strain has little preference for the different mannosides. Compounds that were placed on the array are shown (top). An image of an array for binding of ORNl 78; the concentration of carbohydrate in the arraying solution is 2 mM, 1 nM, 500 μM, and 250 μM from top to bottom (bottom).

Figure 11 depicts the detection limit for binding of ORNl 78 to the carbohydrate microarrays. Serially diluted ORNl 78 was incubated with the arrays after being stained with STYP 82 dye. Unbound bacteria was washed off and the slides scanned. The signal to noise ratio was determined by measuring the amount of signal from the spots from binding to mannose to the signal that is measured outside the area where mannose has been delivered.

Detailed Description Embodiments of the present invention relate to the use of carbohydrate microarrays to investigate the carbohydrate binding specificities of bacteria, to detect pathogens, and to screen anti-adhesion therapeutics. Microarrays present carbohydrates in an ideal manner to study cell-cell interactions because they can accommodate multivalent binding. Cell- surface carbohydrates are exploited by many pathogens for adherence to tissues and entry into host cells. This system is ideal for whole cell applications because microarrays present carbohydrate ligands in a manner that mimics interactions at cell-cell interfaces. As described herein, after incubation of Escherichia coli with a carbohydrate array presenting a variety of monosaccarides binding of the bacterium is observed to mannose on the array. Binding can also be observed when the bacterium is present within heterogeneous solutions of cells or protein. These results demonstrate that arrays can be used to detect pathogens.

Other advantages include assay miniaturization, since minimal amounts (e.g., picomoles) of a ligand are required to observe binding, and high-throughput, since thousands of compounds can be placed on an array and analyzed in parallel. Pathogen detection experiments can be completed in complex mixtures of cells or proteins using the known carbohydrate binding epitopes of the pathogens in question. The non-destructive nature of the arrays allows the pathogen to be harvested and tested for antibacterial susceptibility. In addition, rapid screening of potential anti-adhesion therapeutics can be facilitated with this platform. The miniaturized and high-throughput nature of microarrays makes them a promising tool to screen combinatorial libraries for discovery of anti- adhesion therapeutics, test organisms for their carbohydrate binding epitopes, and to detect pathogens. Since carbohydrate-cell interactions are ubiquitous in nature, these investigations impact a variety of important areas. Overview of a Selected Embodiment. One aspect of the present invention relates to an array consisting of a plurality of spots, each comprising a carbohydrate molecule attached to a solid support. [Seeberger PH et al., United States Patent Application 2005/0221337; hereby incorporated by reference.] In certain embodiments the carbohydrate molecule is a monosaccharide, oligosaccharide, or polysaccharide. In certain preferred embodiments, the carbohydrate comprises mannose, galactose, glucose, fucose or N-acetylglucosamine. The carbohydrate molecule may be attached to the solid support via a linker, e.g., an amine-reactive homobifuntional disuccinimidyl carbonate linker. [For additional linkers, see, for example, Seeberger PH, et al. (2003) United States Patent 6,579,725; hereby incorporated by reference.] The hydrophilic linker minimizes interactions between the array matrix and the solution phase proteins. In addition, the linker is compatible with a wide range of assay conditions, a limitation often encountered with noncovalent forms of attachment. In certain preferred embodiments, the carbohydrate molecule is attached to the linker by a sulfide bond or a nitrogen containing bond. This procedure allows carbohydrate substrates drawn from solution-phase chemistry, solid phase chemistry, and natural sources to be readily incorporated into the present method. Furthermore, the carbohydrate may be bound to the solid support at varying concentration densities to permit determination of relative binding affinities. In other preferred embodiments, the array consists of spots that are about 10 μm to about 200 μm in diameter and the distance between adjacent spots is about 300 μm. The high density of the arrays is advantageous because it requires only minute amount of carbohydrate substrate and is amenable to high-throughput technology.

Another aspect of the present invention relates to a method of preparing an array of carbohydrate molecules, comprising the steps of applying a carbohydrate compound to a support to form a localized spot that is about 10 μm to about 200 μm in diameter and a distance of about 300 μm from an adjacent spot. In certain embodiments the carbohydrate molecule is a monosaccharide, oligosaccharide, or polysaccharide. In certain preferred embodiments, the carbohydrate comprises mannose, galactose, glucose, fucose or N- acetylglucosamine. The carbohydrate molecule may be attached to the solid support via a linker, e.g., an amine-reactive homobifuntional disuccinimidyl carbonate linker. In certain preferred embodiments, the carbohydrate molecule is attached to the linker by a sulfide bond or a nitrogen-containing bond. This mode of attachment is advantageous because it is amenable to preparation of carbohydrate microarrays wherein the carbohydrate is drawn from solution-phase chemistry, solid-phase chemistry, or natural sources. In addition, the carbohydrate microarray may be prepared using precision printing robotics. The rapid construction of large arrays enables many carbohydrate binding experiments to be conducted efficiently and with little waste. Another aspect of the present invention relates to a method to detect the interaction of a carbohydrate with a pathogen of interest, comprising the steps of contacting a pathogen to a carbohydrate bound to the surface of a microsphere and detecting the presence of a complex formed between the carbohydrate and pathogen. The range of carbohydrate substrates and binding proteins discussed above for the carbohydrate microarray can be employed in the carbohydrate microsphere binding studies. In preferred embodiments, the binding event is detected by fluorescence spectroscopy. The microsphere may be composed of glass and optionally contain a signature dye to facilitate identification of the microsphere. In other preferred embodiments, the carbohydrate is bonded to the microsphere via a linker, such as an amine-reactive homobifuntional disuccinimidyl carbonate linker.

The microarray of the present invention alleviates several of the problems associated with current microarray technology. First, the arrays of the present invention are printed at a high density, requiring little material for the manufacture of many hundreds of arrays (e.g., picomoles carbohydrate/array). This characteristic has obvious implications for reducing the costs of large-scale microarray production and also means that one can generate arrays with small amounts of precious structures (e.g., difficult to isolate or synthesize). These types of experiments would not be possible with non-miniaturized assay formats. Second, the arrays of the present invention are amenable to standard technologies used in high-throughput screening applications, such as high-density precision printing robotics and fluorescence scanning instrumentation. This amenability allows researchers who routinely perform DNA and protein array experiments to adopt carbohydrate array technology with minimal difficulty. In addition, immobilization chemistry of the present invention allows for structures of interest to be drawn from solution-phase synthesis, automated solid-phase synthesis, and natural sources (e.g., glycoproteins). This technology can also be applied to the preparation of 'hybrid' arrays consisting of both carbohydrate structures and glycoproteins immobilized on a single slide. Finally, immobilization chemistry of the present invention provides structures covalently immobilized on a hydrophilic non-fouling surface. Two major advantages of this technology are: (a) the arrays are compatible with a wide range of assay conditions (e.g., wide pH range, detergent concentrations, ionic strength), whereas systems that make use of only hydrophobic or electrostatic interactions, are greatly limited in this regard; (b) non-specific interactions between solution-phase proteins and the array matrix are greatly minimized, leading to high signal/noise ratios and decreased likelihood of 'false positive' results. A simple high throughput screening system will be of utmost importance to identify important carbohydrate-protein interactions and to find small molecules that block such interactions.

Selected Microarrays. A microarray may include any one-, two- or three- dimensional arrangement of addressable regions, or features, each bearing a particular chemical moiety or moieties, such as a carbohydrate, associated with that region. Any given array substrate may carry one, two, or four or more arrays disposed on a front surface of the substrate. Depending upon the use, any or all of the arrays may be the same or different from one another and each may contain multiple spots or features. In certain embodiments, a typical array may contain more than ten, more than one hundred, more than one thousand, more ten thousand features, or even more than one hundred thousand features, in an area of less than 20 cm² or even less than 10 cm². For example, square features may have widths, or round feature may have diameters, in the range from a 10 μm to 1.0 cm. In other embodiments each feature may have a width or diameter in the range of 1.0 μm to 1.0 mm, usually 5.0 μm to 500 μm, and more usually 10 μm to 200 μm. Features other than round or square may have area ranges equivalent to that of circular features with the foregoing diameter ranges. At least some, or all, of the features may be of different compositions (for example, when any repeats of each feature composition are excluded the remaining features may account for at least 5%, 10%, or 20% of the total number of features). Interfeature areas are typically, but not necessarily, present and do not carry probe molecules. Such interfeature areas are present where the arrays are formed by processes involving drop deposition of reagents, but may not be present when the photolithographic array fabrication processes are used.

In certain embodiments, each array may cover an area of less than 100 cm², or even less than 50 cm², 10 cm² or 1 cm². In certain embodiments, the substrate carrying the one or more arrays will be shaped generally as a rectangular solid having a length of more than 4 mm and less than 1 m and a width of more than 4 mm and less than 1 m, although other shapes are possible as well. 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, a substrate may transmit at least 20%, or 50%, of the illuminating light incident on the front as may be measured across the entire integrated spectrum of such illuminating light.

Selected Methods to Make Carbohydrate Microarrays. Microarrays of biological materials are comprised of a number of small discrete deposits of biological materials, such as DNA, RNA, proteins, carbohydrates, glycoproteins, glycoconjugates, or mixtures of any of them, in predetermined patterns on a solid support. The support generally comprises glass, a polymer, or metal surface; however, glass supports can be coated with another material, e.g., gold. Gold covered surfaces would allow for direct analysis, by matrix-assisted laser desorption ionization mass spectrometry or surface plasmon resonance spectroscopy, of the material bound to the solid support. In addition, automated technologies have been developed to simplify rapid assembly of microarrays. Photolithography, mechanical microspotting, and ink jet technology have been used for the automated production of microarrays containing biomolecules. With photolithography, a glass wafer, modified with photolabile protecting groups is selectively activated by shining light through a photomask. This method has been used to prepare high- density oligonucleotide microarrays by repeated deprotection and coupling cycles. [Fodor SPA et al. (1998) United States Patent 5,744,305; hereby incorporated by reference.] Microspotting encompasses deposition technologies that enable automated microarray production by printing small quantities of pre-made biochemical substances onto solid surfaces. Printing is accomplished by direct surface contact between the printing substrate and a delivery mechanism, such as a pin or a capillary. Robotic control systems and multiplexed printheads allow automated microarray fabrication. InkJet technologies utilize piezoelectric and other forms of propulsion to transfer biochemical substances from miniature nozzles to solid surfaces. Using piezoelectricity, the sample is expelled by passing an electric current through a piezoelectric crystal which expands to expel the sample. Piezoelectric propulsion technologies include continuous and drop-on-demand devices. In addition to piezoelectric ink jets, heat may be used to form and propel drops of fluid using bubble-jet or thermal ink jet heads. However, in limited circumstances the heat associated with thermal ink jets can partially degrade biological samples. [Hayes DJ et al. (1997) United States Patent 5,658,802; hereby incorporated by reference.] Another method for making arrays of biological materials is called the "dot blot" approach. This method has been successfully employed for the production of DNA microarrays. In this method, a vacuum manifold transfers a plurality, e.g., 96, aqueous samples of DNA from 3 millimeter diameter wells to a porous membrane. The DNA is immobilized on the porous membrane by baking the membrane or exposing it to UV radiation. This manual procedure is practical for making one array at a time and usually limited to 96 samples per array. "Dot-blot" procedures are therefore inadequate for applications in which many thousand samples must be determined. Another technique employed for making ordered arrays of genomic fragments uses an array of pins dipped into the wells, e.g., the 96 wells of a microtitre plate, for transferring an array of samples to a substrate, such as a porous membrane. One array includes pins that are designed to spot a membrane in a staggered fashion, for creating an array of 9216 spots in a 22 x 22 cm area. A limitation with this approach is that the volume of DNA spotted in each pixel of each array is highly variable. In addition, the number of arrays that can be made with each dipping is usually quite small.

A variety of chemically derivatized glass slides that can be printed on and imaged using commercially available arrayers and scanners may be used as a solid support for the microarrays. In certain embodiments, glass slides that have been treated with an aldehyde- containing silane reagent are used. In one embodiment of special interest, glass slides with aldehyde moieties attached are purchased from TeleChem International (Cupertino,

California) under the trade name "Super Aldehyde Substrates". The aldehyde groups on the surface of these slides react readily with primary amines on the proteins to form a Schiff s base linkage. Since typical proteins display many lysine residues on their surface, as well as the generally more reactive α-amine at their N-terminus, they can attach to the slide in a variety of orientations, permitting different sides of the protein to interact with other proteins, small molecules, or small molecules in solution.

Selected Microarray Analysis Assay. The develop of microarrays comprising a large number of samples necessitates a detection method that is sensitive, selective, and rapid. One approach to monitoring microarray experiments employs radiometric or optical analysis. Radiometric or optical analysis produces a scanned image consisting of a two- dimensional matrix of pixels, each pixel having one or more intensity values corresponding to one or more signals. Scanned images are commonly produced electronically by optical or radiometric scanners and the resulting two-dimensional matrix of pixels is stored in computer memory or on a non-volatile storage device. Alternatively, analog methods of analysis, such as photography, can be used to produce continuous images of a microarray that can be then digitized by a scanning device and stored in computer memory or in a computer storage device. The results of microarray experiments can be detected by fluorescence spectroscopy. Fluorescence is a physical phenomenon based upon the ability of some molecules to absorb light (photons) at specified wavelengths and then emit light of a longer wavelength and at a lower energy. Substances able to fluoresce share a number of common characteristics: the ability to absorb light energy at one wavelength; reach an excited energy state; and subsequently emit light at another light wavelength. The absorption and fluorescence emission spectra are individual for each fluorophore and are often graphically represented as two separate curves that are overlapping. The same fluorescence emission spectrum is generally observed irrespective of the wavelength of the exciting light and, accordingly, the wavelength and energy of the exciting light may be varied within limits. Finally, the strength of the fluorescence signal may be measured as the quantum yield of light emitted. The fluorescence quantum yield is the ratio of the number of photons emitted in comparison to the number of photons initially absorbed by the fluorophore. [For more detailed information regarding each of these characteristics, the following references are recommended: Lakowicz, J. R., Principles of Fluorescence Spectroscopy, Plenum Press, New York, 1983; Freifelder, D., Physical Biochemistry, second edition, W. H. Freeman and Company, New York, 1982; "Molecular Luminescence Spectroscopy Methods and Applications: Part I" (S. G. Schulnan, editor) in Chemical Analysis, vol. 77, Wiley & Sons, Inc., 1985; The Theory of Luminescence, Stepanov and Gribkovskii, IHfFe Books, Ltd., London, 1968.] In certain embodiments the substance able to fluoresce is a cell-permeant cyanine nucleic acid stains (e.g., from Molecular Probes, Eugene OR), acridine homodimer (bis-(6- chloro-2-methoxy-9-acridinyl)spermine), dihydroethidium (hydroethidine), LDS 751, acridine orange, Hoechst 33258, Hoechst 33342, Hoechst 34580, or bisBenzimide H 33258. In certain embodiments, the substance able to fluoresce is selected from the group consisting of SYTO 40 blue-fluorescent nucleic acid stain, SYTO 41 blue-fluorescent nucleic acid stain, SYTO 42 blue-fluorescent nucleic acid stain, SYTO 43 blue-fluorescent nucleic acid stain, SYTO 44 blue-fluorescent nucleic acid stain, SYTO 45 blue-fluorescent nucleic acid stain, SYTO RNASelect green-fluorescent cell stain, SYTO 9 green- fluorescent nucleic acid stain, SYTO 10 green-fluorescent nucleic acid stain, SYTO BC green-fluorescent nucleic acid stain, SYTO 13 green-fluorescent nucleic acid stain, SYTO 16 green-fluorescent nucleic acid stain, SYTO 24 green-fluorescent nucleic acid stain, SYTO 21 green-fluorescent nucleic acid stain, SYTO 27 green-fluorescent nucleic acid stain, SYTO 26 green-fluorescent nucleic acid stain, SYTO 23 green-fluorescent nucleic acid stain, SYTO 12 green-fluorescent nucleic acid stain, SYTO 11 green-fluorescent nucleic acid stain, SYTO 20 green-fluorescent nucleic acid stain, SYTO 22 green- fluorescent nucleic acid stain, SYTO 15 green- fluorescent nucleic acid stain, SYTO 14 green-fluorescent nucleic acid stain, SYTO 25 green-fluorescent nucleic acid stain, SYTO 86 orange-fluorescent nucleic acid stain, SYTO 81 orange-fluorescent nucleic acid stain, SYTO 80 orange-fluorescent nucleic acid stain, SYTO 82 orange-fluorescent nucleic acid stain, SYTO 83 orange-fluorescent nucleic acid stain, SYTO 84 orange-fluorescent nucleic acid stain, SYTO 85 orange-fluorescent nucleic acid stain, SYTO 64 red-fluorescent nucleic acid stain, SYTO 61 red-fluorescent nucleic acid stain, SYTO 17 red-fluorescent nucleic acid stain, SYTO 59 red- fluorescent nucleic acid stain, SYTO 62 red-fluorescent nucleic acid stain, SYTO 60 red-fluorescent nucleic acid stain and SYTO 63 red- fluorescent nucleic acid stain.

Cell Adhesion to Carbohydrate Arrays. In one embodiment, five different monosaccharides equipped with an ethanolamine linker on their reducing ends were used to construct the carbohydrate arrays (Figure 3). Functionalized sugars were spotted onto glass slides that had been coated with the amine-reactive homobifunctional disuccinimidyl carbonate linker. In initial tests, 10 μL of a 20 mM carbohydrate solution was placed onto different positions on the surface. Slides were hybridized with 10⁹E. coli (ORN 178) cells that had been stained with a nucleic acid staining dye (Figure 4). After removing unbound bacteria by washing, slides were scanned using a fluorescent array scanner. Results show that a strongly fluorescent signal (signal to noise (S/N) greater than about 10) was observed at positions where mannose was immobilized; hybridization with unstained E. coli resulted in a weak signal (S/N of about 2). The remainder of the slide exhibited no signal above background. Next, in another embodiment, an arraying robot was used to construct high-density arrays. The robot spatially delivered 1 nL of carbohydrate-containing solutions that ranged in concentration from 20 mM to 15 μM and the resulting spots had diameters of about 200 μm. Several types of slides were tested to optimize array performance. Standard amine coated glass slides were reacted with either disuccinimidyl carbonate or disuccinimidyl tetrapolyethylenglycol linkers; alternatively CodeLink™ polymer coated slides were used. For each of these slides, ORNl 78 bound to mannose and not to the other carbohydrates. Furthermore, binding occurred with a signal to noise ration of greater than 100 despite the small size of the spots. CodeLink slides has the best performance since the gave the highest binding signal and the lowest background. Most likely the three dimentional manner in which the carbohydrates were immobilized on these slides is responsible for the enhanced performance. These types of surface are meant to illustrate possibilities. Immobilization can be done onto other surfaces such as through hydrophobic, hydrophilic, or other immobilization chemistries.

Other arrays that displayed mono- to nona-mannosides, which were constructed as described, were tested for binding to ORNl 78. [Adams EW, Ratner DM, Bokesch HR, McMahon JB, O'Keefe BR, Seeberger PH. (2004) Oligosaccharide and glycoprotein microarrays as tools in HIV glycobiology; glycan-dependent gpl20/protein interactions. Chem. Biol., 11, 875-881.] Results from these experiments show that ORNl 78 does not greatly discriminate in binding to these mannosides, despite varying lengths and linkage stereochemistry. This result likely reflects that recognition of mannose residues by this strain occurs through only a single mannose residue, and that stereochemistry of the linkage plays little role in binding. The observation of cell adhesion to arrays constructed using an arraying robot with microarray-size spots is promising. A previous report studied adhesion of chicken hepatocytes and human T-cells to carbohydrates arrays that were manually constructed. These spots were 1.7 mm in diameter and allowed for about 200 spots to be placed on a single slide. [Nimrichter L, Gargir A, Gortler M, Altstock RT, Shtevi A, Weisshaus O, Fire E, Dotan N, Schnaar RL. (2004) Intact cell adhesion to glycan microarrays. Glycobiology, 14, 197-203.] In the work of Nimrichter et al., cells were placed directly on the carbohydrate spots. In contrast, the arrays described here show that the interactions of bacteria to carbohydrates can be studied in a high-throughput manner in which the bacteria are applied to the entire slide not only to carbohydrate spots. Due to the smaller spot size used here a much larger number of interactions can be screened in parallel.

The minimal amount of carbohydrate sufficient to detect binding was determined. Analyte consumption is an important aspect for carbohydrate arrays since materials isolated from natural sources are in short supply. Several 1 nL aliquots of serially diluted solutions of carbohydrate that ranged in concentration from 20 mM to 15 μM were arrayed. A concentration dependent decrease in signal was observed and delivery of as little as 20 femtomoles to a slide was sufficient to obtain a signal above background (Figure 4). Different concentrations of bacteria were next hybridized with the arrays to determine the bacterial detection limit. As expected, a concentration dependent decrease in signal was observed. When 10 or greater ORNl 78 were incubated, signals were well above background (see Exemplification). However, hybridization of 10⁵ cells gave signal that approached background. This sensitivity rivals or exceeds that are used in methods requiring a bacterial-enrichment step prior to detection. [Willis RC (2004) Improved Molecular Techniques Help Researchers Diagnose Microbial Conditions. Modern Drug Discovery, 7, 36-42.]

Standard microscopic images were taken of ORNl 78 bound to three mannose- containing spots. Images show that ORNl 78 only adhered to these positions and they are densely covered with bacteria (Figure 4); no bacteria are observed outside of this area. This illustrates that these slides are resistant to non-specific adhesion of bacteria.

Assessing the Carbohydrate Binding Specificities of Different Bacterial Strains.

The arrays were tested for their ability to probe differences in carbohydrate binding affinities between related strains. Two E. coli strains were used, ORNl 78, and a mutant strain, ORN209, which exhibits a reduced affinity for mannose. [Harris SL, Spears PA, Havell EA, Hamrick TS, Horton JR, Orndorff PE. (2001) Characterization of Escherichia coli type 1 pilus mutants with altered binding specificities. J Bacteriol, 183, 4099-102.] As expected, incubation of ORN209 gave a much lower signal than ORNl 78 (Figure 4). A seven-fold decrease was observed at the highest spot concentration and no signal was observed at lower concentrations (Figure 4). The signal difference is not due to the uptake of the dye.

These findings demonstrate that the arrays allow for screening of mutants that have altered carbohydrate binding affinities. The ability to distinguish the different affinities of cells may have clinical applications since the virulence of many pathogens correlates with carbohydrate binding. For example, clinical isolates of E. coli that cause urinary tract infections bind to mannosides with a much higher affinity than strains that do not cause infections. [Sokurenko EV, Chesnokova V, Dykhuizen DE, Ofek I, Wu XR, Krogfelt KA, Struve C, Schembri MA, Hasty DL. (1998) Pathogenic adaptation of Escherichia coli by natural variation of the FimH adhesin. Proc Natl Acad Sci USA, 95, 8922-6; and Sokurenko EV, Chesnokova V, Doyle RJ, Hasty DL (1997) Diversity of the Escherichia coli type 1 fimbrial lectin. Differential binding to mannosides and uroepithelial cells. J Biol Chem, 272, 17880-6.] Thus, the carbohydrate binding profiles determined with the array can aid in assessing pathogenicity and the design of strain specific therapies.

Screening Inhibitors of Carbohydrate-Cell Interactions. Anti-adhesion compounds can be used as therapeutics against pathogens and other infectious agents. In certain embodiments, the inventive array-based method was used to measure the ability of compounds to inhibit binding of ORNl 78 to mannose. Inhibitors were placed in array hybridization solutions that were incubated with 10 ORN178 cells. Compounds tested included mannose, p-nitrophenyl- α-D-mannospyranoside (p-NPMan), and a water-soluble, mannose-functionalized polymer. [Gestwicki JE, Cairo CW, Strong LE, Oetjen KA, Kiessling LL. (2002) Influencing receptor-ligand binding mechanisms with multivalent ligand architecture. J. Am. Chem. Soc. 124, 14922-33.] The ICw's were measured and showed that mannose-functionalized polymer (50 μM) was significantly more effective at inhibiting bacterial adhesion than p-NPMan (1000 μM), which was a better inhibitor than mannose (50,000 μM). Figure 5 and Exemplification.

These results agree with previous reports that showed that p-NPMan is a better adhesion inhibitor than mannose. Structural information obtained for the mannose binding pocket in E. coli aided the development of p-NPMan as a tight binder to E. coli due to forming stabilizing interactions with the aromatic residues in this protein. [Sharon N. (1987) Bacterial lectins, cell-cell recognition and infectious disease. FEBS Lett., 217, 145- 57.] Also, the arrays display the carbohydrate in a multivalent manner. Multiple ligand- polymer interactions will increase the binding affinity to whole bacterial cells since there are 100 to 400 mannose binding lectins that are displayed on the surface of E. coli. [Gestwicki JE, Cairo CW, Strong LE, Oetjen KA, Kiessling LL. (2002) Influencing receptor-ligand binding mechanisms with multivalent ligand architecture. J. Am. Chem. Soc, 124, 14922-33; and Mammen M, Choi SK, Whitesides GM. (1998) Polyvalent interactions in biological systems: Implications for design and use of multivalent ligands and inhibitors. Angew. Chem. Inter. Ed. Engl., 37, 2755-2794.] Furthermore, other multivalent scaffolds, such as polymers and BSA, exhibit enhanced binding to bacteria and mammalian cells compared to monovalent ligands. [Nagahori N, Lee RT, Nishimura S, Page D, Roy R, Lee YC. (2002) Inhibition of adhesion of type 1 fimbriated Escherichia coli to highly mannosylated ligands. ChemBioChem, 3, 836-844; Disney MD, Zhang J, Swager TM, Seeberger PH. (2004) Detection of Bacteria with Carbohydrate-Functionalized Fluorescent Polymers. J. Am. Chem. Soc, 126, 13343-13346; Gestwicki JE, Cairo CW, Strong LE, Oetjen KA, Kiessling LL (2002) Influencing receptor-ligand binding mechanisms with multivalent ligand architecture. J Am Chem Soc, 124, 14922-33; and Gestwicki JE, Strong LE, Cairo CW, Boehm FJ, Kiessling LL. (2002) Cell aggregation by scaffolded receptor clusters. Chem. Biol., 9, 163-9.]

Carbohydrate Microarrays as a Means to Detect Bacteria. The carbohydrate array platform has the potential to be used as a biosensor because many different cell types bind to carbohydrates and the carbohydrate-binding "fingerprint" can be used to determine the type of bacteria present within a complex mixture. [Mammen M, Choi SK, Whitesides GM. (1998) Polyvalent interactions in biological systems: Implications for design and use of multivalent ligands and inhibitors. Angew Chem Inter Ed Engl, 37, 2755-2794.] In certain embodiments, strain ORN 178 was placed as a contaminant into solutions that included sheep erythrocytes and serum. SYTO-83 dye was added to these solutions and they were directly applied to the arrays without removal of the excess dye (Figure 2). Importantly, non cell-associated dye did not have to be removed since it did not exhibit non-specific binding to the array surface.

The results show that in both cases that the binding ORNl 78 to the arrays is observed. Bacterial detection of ORNl 78 in serum showed signals well above background that are about 2-fold lower than that observed with a homogeneous sample (Figure 6). For experiments with erythrocytes, samples that contain equal amounts of ORNl 78 and erythrocytes result in signals that are equal to that observed with a homogeneous sample (Figures 6). Further addition of erythrocytes to 10-fold excess over the bacteria decreases the signal about 6-fold (Figure 6). Despite this decrease, the signal is still well above background. Bright-field microscopic images of the mannose positions on the arrays at different erythrocyte concentrations were taken to determine if decreased signal was due to less efficient uptake of the dye or erythrocyte binding to the arrays. Images clearly show that only bacteria bind and that the decrease in signal is due to decreased cell density (Figure 6).

The observation that ORNl 78 adhesion can be detected in complex mixtures is encouraging for the use of this array-based technique as a medical diagnostic. Traditional assays for pathogen detection require selective growth of bacteria in media and such experiments may take days. [Willis RC. (2004) Improved Molecular Techniques Help Researchers Diagnose Microbial Conditions. Modern Drug Discovery, 7, 36-42.] Recently, several platforms have been developed to accelerate this process. Colorimetric detection using bacteriophages may require a few hours and other methods, such as antibody staining and PCR, require 6 to 48 h to test a sample. [Park DJ, Drobniewski FA, Meyer A, Wilson SM. (2003) Use of a phage-based assay for phenotypic detection of mycobacteria directly from sputum. J Clin Microbiol, 41, 680-688; Waites KB, Smith KR, Crum MA, Hockett RD, Wells AH, Hook EW, 3rd. (1999) Detection of Chlamydia trachomatis endocervical infections by ligase chain reaction versus ACCESS Chlamydia antigen assay. J Clin Microbiol, 37, 3072-3073; and Andreotti PE, Ludwig GV, Peruski AH, Tuite JJ, Morse SS, Peruski LF, Jr. (2003) Immunoassay of infectious agents. Biotechniques, 35, 850-85.]

In certain embodiments, bacteria captured on the arrays were harvested and tested for anti-bacterial susceptibility (Figure 7). After incubation of a homogeneous solution of ORNl 78 to a carbohydrate array and washing off the unbound cells, bound bacteria were removed from the array by placing an inoculating loop over the mannose-containing positions. These bacteria were streaked onto LB plates and incubated at 37 °C overnight. Colonies were observed on plates after samples were harvested from mannose-containing positions. The bacteria were then further tested for antibiotic susceptibility, including minimum inhibitory concentrations for a variety of different antibiotics (Figure 7). Thus, not only do the arrays allow for pathogen detection, but they can also be used to harvest the pathogens to allow for further testing. Importantly, this is not possible with destructive methods, such as those that require PCR.

One of the major impediments of using carbohydrate ligands to detect pathogens is the lack of specificity to different cell types. To circumvent this problem cross-reactive chemical sensors have been developed using ligands that have low specificity. [Cho EJ, Bright FV. (2002) Pin-printed chemical sensor arrays for simultaneous multianalyte quantification. Anal. Chem., 74, 1462-1466; and Michael KL, Taylor LC, Schultz SL, Walt DR. (1998) Randomly ordered addressable high-density optical sensor arrays. Anal. Chem., 70, 1242-1248.] The presence of a pathogen is determined through the binding ensemble from many different analytes. Such a scheme is used by the about 1000 different olfactory receptors that are present in the nose. In certain embodiments, the spatial nature and the ability to spot several thousand ligands on a single array using the techniques described here should simplify application of the cross-reactive sensing technique. Selected Methods of the Invention. One aspect of the present invention relates to a method of detecting the presence of a biological entity in a sample, comprising the steps of contacting said sample to an array of biological recognition element-containing molecules at a contacting time and contacting temperature; and detecting the presence of a complex formed between said biological recognition element-containing molecules and said biological entity; wherein said array of biological recognition element-containing molecules comprises a plurality of spots with widths in the range of about 0.1 μm to about 500 μm. In certain embodiments, each spot is within about 900 μm of an adjacent spot.

Another aspect of the present invention relates to a method of detecting the presence of a biological entity in a sample, consisting essentially of the steps of contacting said sample to an array of biological recognition element-containing molecules at a contacting time and contacting temperature; and detecting the presence of a complex formed between said carbohydrate-containing molecules and said biological entity; wherein said array of carbohydrate-containing molecules comprises a plurality of spots with widths in the range of about 0.1 μm to about 500 μm. In certain embodiments, each spot is within about 900 μm of an adjacent spot.

Another aspect of the present invention relates to a method of detecting the presence of a bacteria in a sample, comprising the steps of treating said sample with a fluorescent cell-permeable dye, thereby forming fluorescently-labeled bacteria; contacting said sample to an array of carbohydrate-containing molecules at a contacting time and contacting temperature; and detecting the presence of a complex formed between said carbohydrate- containing molecules and said fluorescently-labeled bacteria; wherein said array of carbohydrate-containing molecules comprises a plurality of spots with widths in the range of about 0.1 μm to about 500 μm. In certain embodiments, each spot is within about 900 μm of an adjacent spot.

Another aspect of the present invention relates to a method of detecting the presence of a bacteria in a sample, consisting essentially of the steps of treating said sample with a fluorescent cell-permeable dye, thereby forming fluorescently-labeled bacteria; contacting said sample to an array of carbohydrate-containing molecules at a contacting time and contacting temperature; and detecting the presence of a complex formed between said carbohydrate-containing molecules and said fluorescently-labeled bacteria; wherein said array of carbohydrate-containing molecules comprises a plurality of spots with widths in the range of about 0.1 μm to about 500 μm. In certain embodiments, each spot is within about 900 μm of an adjacent spot.

Another aspect of the present invention relates to a method of detecting the presence of a bacteria in a sample, consisting essentially of the steps of treating said sample with a fluorescent cell-permeable dye, thereby forming fluorescently-labeled bacteria; contacting said sample to an array of carbohydrate-containing molecules at a contacting time and contacting temperature; washing said array to remove unbound components of said sample; and detecting the presence of a complex formed between said carbohydrate-containing molecules and said fluorescently-labeled bacteria; wherein said array of carbohydrate- containing molecules comprises a plurality of spots with widths in the range of about 0.1 μm to about 500 μm. In certain embodiments, each spot is within about 900 μm of an adjacent spot.

Another aspect of the present invention relates to a method of detecting and identifying a biological entity in a sample, comprising the steps of contacting said sample to an array of biological recognition element-containing molecules at a contacting time and contacting temperature; detecting the presence of a complex formed between said biological recognition element-containing molecules and said biological entity; harvesting said biological entity from said array; culturing said biological entity; and identifying said biological entity; wherein said array of biological recognition element-containing molecules comprises a plurality of spots with widths in the range of about 0.1 μm to about 500 μm. In certain embodiments, each spot is within about 900 μm of an adjacent spot.

Another aspect of the present invention relates to a method of detecting and identifying a bacteria in a sample, comprising the steps of contacting said sample to an array of carbohydrate-containing molecules at a contacting time and contacting temperature; detecting the presence of a complex formed between said carbohydrate- containing molecules and said bacteria; harvesting said bacteria from said array; culturing said bacteria; and identifying said bacteria; wherein said array of carbohydrate-containing molecules comprises a plurality of spots with widths in the range of about 0.1 μm to about 500 μm. In certain embodiments, each spot is within about 900 μm of an adjacent spot. Another aspect of the present invention relates to a method for identifying a compound with anti-adhesion properties, comprising the steps of contacting a biological entity to an array of biological recognition element-containing molecules in the presence of said compound at a contacting time and contacting temperature, wherein said biological entity binds said carbohydrate-containing molecules in the absence of said compound; and detecting the presence of a complex formed between said carbohydrate-containing molecules and said biological entity; wherein said array of biological recognition element- containing molecules comprises a plurality of spots with widths in the range of about 0.1 μm to about 500 μm. In certain embodiments, each spot is within about 900 μm of an adjacent spot.

Another aspect of the present invention relates to a method for identifying a compound with anti-adhesion properties, consisting essentially of the steps of contacting a biological entity to an array of biological recognition element-containing molecules in the presence of said compound at a contacting time and contacting temperature, wherein said biological entity binds said carbohydrate-containing molecules in the absence of said compound; and detecting the presence of a complex formed between said carbohydrate- containing molecules and said biological entity; wherein said array of biological recognition element-containing molecules comprises a plurality of spots with widths in the range of about 0.1 μm to about 500 μm. In certain embodiments, each spot is within about 900 μm of an adjacent spot.

Another aspect of the present invention relates to a method for identifying a compound with anti-adhesion properties, comprising the steps of contacting a bacteria to an array of carbohydrate-containing molecules in the presence of said compound at a contacting time and contacting temperature, wherein said bacteria binds said carbohydrate- containing molecules in the absence of said compound; and detecting the presence of a complex formed between said carbohydrate-containing molecules and said bacteria; wherein said array of carbohydrate-containing molecules comprises a plurality of spots with widths in the range of about 0.1 μm to about 500 μm. In certain embodiments, each spot is within about 900 μm of an adjacent spot.

Another aspect of the present invention relates to a method for identifying a compound with anti-adhesion properties, consisting essentially of the steps of contacting a bacteria to an array of carbohydrate-containing molecules in the presence of said compound at a contacting time and contacting temperature, wherein said bacteria binds said carbohydrate-containing molecules in the absence of said compound; and detecting the presence of a complex formed between said carbohydrate-containing molecules and said bacteria; wherein said array of carbohydrate-containing molecules comprises a plurality of spots with widths in the range of about 0.1 μm to about 500 μm. In certain embodiments, each spot is within about 900 μm of an adjacent spot.

In certain embodiments, the present invention relates to the aforementioned method, wherein said compound with anti-adhesion properties is selected from the group consisting of D-mannose, p-nitrophenyl α-D-mannopyranoside, and polymer-bound α-D- mannopyranoside.

In certain embodiments, the present invention relates to the aforementioned method further comprising the step of washing said array to remove unbound components of said sample. In certain embodiments, the present invention relates to the aforementioned method further comprising the step of treating said sample with a fluorescent cell-permeable dye.

In certain embodiments, the present invention relates to the aforementioned method, wherein said fluorescent cell-permeable dye is a cell-permeant cyanine nucleic acid stain.

In certain embodiments, the present invention relates to the aforementioned method, wherein said fluorescent cell-permeable dye is SYTO 83.

In certain embodiments, the present invention relates to the aforementioned method, wherein said biological entity is capable of reproduction.

In certain embodiments, the present invention relates to the aforementioned method, wherein said biological entity is a pathogen. In certain embodiments, the present invention relates to the aforementioned method, wherein said pathogen is a mamalian pathogen.

In certain embodiments, the present invention relates to the aforementioned method, wherein said pathogen is a human pathogen.

In certain embodiments, the present invention relates to the aforementioned method, wherein said pathogen is a bacteria.

- In certain embodiments, the present invention relates to the aforementioned method, wherein said pathogen is a bacteria selected from the group consisting of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma. In certain embodiments, the present invention relates to the aforementioned method, wherein said pathogen is an E. coli.

In certain embodiments, the present invention relates to the aforementioned method, wherein said biological recognition element is selected from the group consisting of carbohydrates, saccharides, monosaccharides, oligosaccharides, polysaccarides, glycoaminoglycans, glycolipids, aminoacids, proteins, antibodies, lectins, nucleic acids, DNA, RNA, and oligonuclotides.

In certain embodiments, the present invention relates to the aforementioned method, wherein said biological recognition element comprises an antibody.

In certain embodiments, the present invention relates to the aforementioned method, wherein said biological recognition element comprises a carbohydrate.

In certain embodiments, the present invention relates to the aforementioned method, wherein said carbohydrate is an amine-presenting carbohydrate.

In certain embodiments, the present invention relates to the aforementioned method, wherein said biological recognition element is selected from the group consisting of mannose, galactose, glucose, fucose, and N-acetylglucosamine.

In certain embodiments, the present invention relates to the aforementioned method, wherein said biological recognition element is selected from those shown in Figure 10.

In certain embodiments, the present invention relates to the aforementioned method, wherein said biological recognition element is mannose. In certain embodiments, the present invention relates to the aforementioned method, wherein said sample is homogeneous.

In certain embodiments, the present invention relates to the aforementioned method, wherein said sample is heterogeneous.

In certain embodiments, the present invention relates to the aforementioned method, wherein said sample comprises a single analyte. In certain embodiments, the present invention relates to the aforementioned method, wherein said sample comprises a plurality of analytes.

In certain embodiments, the present invention relates to the aforementioned method, wherein said sample is taken from food, water, bodily fluid or the environment. In certain embodiments, the present invention relates to the aforementioned method, wherein said sample is taken from blood, saliva, urine or feces.

In certain embodiments, the present invention relates to the aforementioned method, wherein said sample is taken from blood.

In certain embodiments, the present invention relates to the aforementioned method, wherein said sample consists essentially of erythrocytes or serum.

In certain embodiments, the present invention relates to the aforementioned method, wherein said array is a glass slide, a membrane, a 96-wellplate, a surface, or a gold-coated glass, polymer or metal surface.

In certain embodiments, the present invention relates to the aforementioned method, wherein said array is a amine-reactive glass slide.

In certain embodiments, the present invention relates to the aforementioned method, wherein said array is a Corning GAPS II amine-functionalized slide which has been reacted with N,N-disuccinimidyl carbonate or a CodeLink™ polymer coated slide.

In certain embodiments, the present invention relates to the aforementioned method, wherein the width of said spots is in the range of about 10 μm to about 300 μm.

In certain embodiments, the present invention relates to the aforementioned method, wherein the width of said spots is in the range of about 50 μm to about 200 μm.

In certain embodiments, the present invention relates to the aforementioned method, wherein the width of said spots is in the range of about 80 μm to about 120 μm. In certain embodiments, the present invention relates to the aforementioned method, wherein the width of said spots is about 100 μm.

In certain embodiments, the present invention relates to the aforementioned method, wherein the width of said spots is about 125 μm.

In certain embodiments, the present invention relates to the aforementioned method, wherein the width of said spots is about 150 μm. In certain embodiments, the present invention relates to the aforementioned method, wherein the width of said spots is about 175 μm.

In certain embodiments, the present invention relates to the aforementioned method, wherein the width of said spots is about 200 μm. In certain embodiments, the present invention relates to the aforementioned method, wherein each spot is within about 700 μm of an adjacent spot.

In certain embodiments, the present invention relates to the aforementioned method, wherein each spot is within about 500 μm of an adjacent spot.

In certain embodiments, the present invention relates to the aforementioned method, wherein each spot is within about 300 μm of an adjacent spot.

In certain embodiments, the present invention relates to the aforementioned method, wherein each spot is within about 150 μm of an adjacent spot.

In certain embodiments, the present invention relates to the aforementioned method, wherein said contacting temperature is between about 15 °C and 30 °C. In certain embodiments, the present invention relates to the aforementioned method, wherein said contacting temperature is about 25 ⁰C.

In certain embodiments, the present invention relates to the aforementioned method, wherein said contacting time is between about 0.5 hours and about 2 hours.

In certain embodiments, the present invention relates to the aforementioned method, wherein said contacting time is about 1 hour.

In certain embodiments, the present invention relates to the aforementioned method, wherein said biological recognition element is attached to said array via a covalent bond.

In certain embodiments, the present invention relates to the aforementioned method, wherein said covalent bond is a glycosidic linkage. In certain embodiments, the present invention relates to the aforementioned method, wherein said covalent bond is a sulfur-containing linkage.

In certain embodiments, the present invention relates to the aforementioned method, wherein said covalent bond is a nitrogen-containing linkage. In certain embodiments, the present invention relates to the aforementioned method, wherein said biological recognition element is attached to said array via a linker.

In certain embodiments, the present invention relates to the aforementioned method, wherein said biological recognition element is attached to said array via a disuccinimidyl carbonate linker.

In certain embodiments, the present invention relates to the aforementioned method, wherein said biological recognition element is attached to said array via a disuccinimidyl tetrapolyethylenglycol or disuccinimidyl carbonate linkage.

In certain embodiments, the present invention relates to the aforementioned method, wherein said biological recognition element varies across different genotypes.

In certain embodiments, the present invention relates to the aforementioned method, wherein said biological recognition element varies across different genotypes; and said biological recognition element is comprises a carbohydrate.

Selected Kits of the Invention. In another aspect of the invention kits are provided containing an array of the invention. A "kit," as used herein, typically defines a package or an assembly including one or more of the arrays of the invention, and/or other compositions associated with the invention, for example, as described herein. Each of the compositions of the kit may be provided in liquid form (e.g., in solution), or in solid form (e.g., a dried powder). In certain cases, some of the compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species, which may or may not be provided with the kit. Examples of other compositions or components associated with the invention include, but are not limited to, solvents, surfactants, diluents, salts, buffers, emulsifiers, chelating agents, fillers, antioxidants, binding agents, bulking agents, preservatives, drying agents, antimicrobials, needles, syringes, packaging materials, tubes, bottles, flasks, beakers, dishes, frits, filters, rings, clamps, wraps, patches, containers, and the like, for example, for using, modifying, assembling, storing, packaging, preparing, mixing, diluting, and/or preserving the compositions components for a particular use, for example, to a sample.

A kit of the invention may include instructions in any form that are provided in connection with the arrays of the invention in such a manner that one of ordinary skill in the art would recognize that the instructions are to be associated with the compositions of the invention. For instance, the instructions may relate to the use, modification, mixing, diluting, preserving, assembly, storage, packaging, and/or preparation of the compositions and/or other compositions associated with the kit. In some cases, the instructions may also include instructions for the delivery of the compositions, for example, for a particular use, e.g., to a sample. The instructions may be provided in any form recognizable by a user as a suitable vehicle for containing such instructions; for example, written or published, verbal, audible (e.g., telephonic), digital, optical, visual (e.g., videotape, DVD, etc.) or electronic communications (including Internet or web-based communications), provided in any manner.

Yet another aspect of the invention provides for the promotion of any of the above- described arrays, kits, or methods of the invention. As used herein, "promoted" includes all methods of doing business including, but not limited to, methods of selling, advertising, assigning, licensing, contracting, instructing, educating, researching, importing, exporting, negotiating, financing, loaning, trading, vending, reselling, distributing, repairing, replacing, insuring, or the like that are associated with the systems, methods, compositions, kits, etc. of the invention as discussed herein. Methods of promotion can be performed by any party including, but not limited to, personal parties, businesses (public or private), partnerships, corporations, trusts, contractual or sub-contractual agencies, educational institutions such as colleges and universities, research institutions, hospitals or other clinical institutions, governmental agencies, etc. Promotional activities may include communications of any form (e.g., written, oral, and/or electronic communications, such as, but not limited to, e-mail, telephonic, Internet, Web-based, etc.) that are clearly associated with the invention.

In one set of embodiments, the method of promotion may involve one or more instructions. As used herein, "instructions" can define a component of instructional utility (e.g., directions, guides, warnings, labels, notes, FAQs or "frequently asked questions," etc.), and typically involve written instructions on or associated with the invention and/or with the packaging of the invention. Instructions can also include instructional communications in any form (e.g., oral, electronic, audible, digital, optical, visual, etc.), provided in any manner such that a user will clearly recognize that the instructions are to be associated with the invention, e.g., as discussed herein.

Selected Advantages of the Invention. There are many advantages to using the inventive carbohydrate arrays to detect pathogens and study carbohydrate-cell interactions. One such advantage is that the display of carbohydrates on the arrays allows them to interact with cells in a multivalent manner, which is similar to how these interactions occur in nature (see Figures 1 and 2). Multivalent binding is much stronger and selective relative to monovalent binding. The arrays mimic this presentation of the carbohydrates. A schematic of the interactions of the carbohydrates displayed on the array to cells is shown in Figure 2.

Another advantage is that carbohydrate arrays are durable. This is in contrast to many other detection methods that are used for bacteria, which use fluorescent antibodies, DNA probes, or the use of bacteriophage. Yu LS, Reed SA, Golden MH. (2002) Time- resolved fluorescence immunoassay (TRFIA) for the detection of Escherichia coli O157:H7 in apple cider. [J Microbiol Methods, 49, 63-81; Perry-O'Keefe H, Rigby S, Oliveira K, Sorensen D, Stender H, Coull J, Hyldig-Nielsen JJ. (2001) Identification of indicator microorganisms using a standardized PNA FISH method. J Microbiol Methods, 47, 281-92; and Goodridge L, Chen J, Griffiths M. (1999) The use of a fluorescent bacteriophage assay for detection of Escherichia coli Ol 57:H7 in inoculated ground beef and raw milk. Int J Food Microbiol, 47, 43-50.] Because these have protein components, they must be stored under conditions that do not cause the protein to mis-fold. For prolonged periods of storage, these components must be kept at 4 ⁰C. Arrays can be stored at room temperature for several months without affecting their performance.

Another advantage is that detection can be automated. This application would be particularly useful in testing many different food samples that come from a packaging plant or at a hospital. This also minimizes the potential for human error to be entered in the analysis.

Another advantage is that arrays allow for quick implementation of multiplex sensing. One disadvantage in using carbohydrates to detect pathogens is their low selectivity in binding to pathogens. For example, both E. coli and Salmonella entrica bind to mannose. This can be overcome by spatially arraying many different carbohydrates onto an array, the binding ensemble can be deconvoluted to determine what bacteria are present within a complex mixture. Such procedures have been well-documented in the chemical literature. [Albert KJ, Lewis NS, Schauer CL, Sotzing GA, Stitzel SE, Vaid TP, Walt DR. (2000) Cross-reactive chemical sensor arrays. Chem Rev, 100, 2595-626.]

Another advantage is that the inventive arrays can detect the presence of pathogens in the presence of contaminants. These results are in contrast to methods that used PCR amplification because total cellular DNA must be harvested from the samples prior to amplification.

Another advantage is that arrays require no pre-enrichment step for detection. Currently used methods for determining the presence of bacteria and their type require that the pathogens be grown in strain-specific media. This step can require as much as 24 to 48 additional hours to make a diagnosis. Detections made on the array can be completed in minutes.

Another advantage is that pathogens captured on the arrays can be isolated and further tested for antibacterial susceptibility. Bacteria bound onto the arrays can be grown in standard bacterial media (Luria-Bertani). The samples can then be placed into a 96-well plate in the presence of antibiotics and tested for which compound inhibits growth. This allows for therapeutic regimes to be tailored to the specific bacteria that are infecting a patient. In the current world where antibiotic resistance is often fatal and is becoming an increasing issue, such applications are extremely important. Another advantage is that arrays facilitate screening of anti-adhesion therapeutics.

By placing serially diluted concentrations of compounds that may disrupt the binding of bacteria to mannose and hybridization with the arrays, potency can be quickly assed.

Another advantage is that arrays can test the carbohydrate binding affinities of different strains. Often the pathogenicity of a strain correlates with it's affinity to carbohydrates. By using this method, experiments can be completed to determine if a bacterium that is infecting a patient has potential to cause an infection or not. Armed with this information, strain specific therapies can be used.

In summary, as shown herein, certain aspects of the invention relate to methods for using carbohydrate microarrays to detect the presence of pathogens within complex solutions, including blood and serum. The advantages of these technique over current methods are numerous and include, for example; (1) speed, analysis of samples can be done in a matter of minutes, which is orders of magnitude faster than current methods that require pathogen growth; (2) simplicity of design, the only information needed to detect a pathogen is a known ligand, which does not have to be a carbohydrate; (3) parallel analysis, the spatial nature of the carbohydrate arrays allows many different carbohydrates to be arrayed onto a single slide allowing the presence of any pathogen to be detected in a sample assuming there is a known ligand (e.g., carbohydrate, antibody, or protein); and (4) the non-destructive nature, pathogens harvested from the arrays can be cultured and further tested for antibacterial susceptibility.

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

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

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non- limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 211 1.03.

As used herein, "binding" can involve any hydrophobic, non-specific, or specific interaction, and the term "biological binding" refers to the interaction between a corresponding pair of molecules that exhibit mutual affinity or binding capacity, typically specific or non-specific binding or interaction. Biological binding defines a type of interaction that occurs between pairs of molecules including proteins, nucleic acids, glycoproteins, carbohydrates, hormones, and the like. Specific examples include protein/carbohydrate, antibody/antigen, antibody/hapten, biotin/streptavidin, biotin/avidin, enzyme/substrate, enzyme/inhibitor, enzyme/cofactor, protein/substrate, lectin/carbohydrate, receptor/hormone, receptor/effector, complementary strands of nucleic acid (e.g., DNA and/or RNA), protein/nucleic acid, repressor/inducer, ligand/receptor, virus/ligand, etc. Further, the term "binding partner" refers to a molecule that can undergo binding with a particular molecule. "Specific interaction" is given its ordinary meaning as used in the art, i.e., an interaction between pairs of molecules where the molecules have a higher recognition or affinity for each other than for other, dissimilar molecules. Biotin/avidin and biotin/streptavidin are examples of specific interactions. In some cases, the specific interaction involves uncharged molecules or neutral ligands.

As used herein, a "biological entity," is an entity deriving at least partially from a biological source. Non-limiting examples of biological entities include proteins, peptides, nucleic acids (e.g., oligonucleotides, which may include DNA and/or RNA), fatty acids, carbohydrates, sugars, hormones, enzymes, receptors, lipids, viruses, bacteria, cells, and the like. In some cases, the biological entity has the capability for reproduction, which can be self-reproduction, i.e., a biological entity is a cell (e.g., a bacterium) or a virus. In certain cases, the biological entity is a "pathogen," i.e., an entity capable of causing a disease when introduced into a subject, for example, a human, a dog, a cat, a horse, a cow, a pig, a sheep, a goat, a chicken, a primate, a rat, a mouse, etc.

In certain embodiments, a "biological entity" is a prokaryote, including but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma, and further including, but not limited to, a member of the species or group, Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium diptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans,

Mycobacterium leprae, Actinomyces israelii, Listeria monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli, Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, Vibrio cholera, Shigella dysenterii, Shigellaflexneri, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostridium perfringens, Clostridium tetani, Clostridium botulinum, Treponema pallidum, Rickettsia rickettsii, Helicobacter pylori and Chlamydia trachomitis.

As used herein, a "biological recognition element" is an entity able to interact with the biological entity and/or a species present on a biological entity, such as a bacterium, a cell, a virus, etc, for example, by specifically binding to the species. In some cases, the interaction may be a specific interaction. For example, the entity may interact with the species such that the entity has an affinity to the species greater than the affinity of the entity to other species present on the biological entity, or present on similar biological entities. For instance, the biological recognition element may interact with a protein expressed on the surface of a bacterium or a cell, e.g., by binding to the protein, while the biological recognition element does not interact (and/or interacts with less affinity) to other, similar proteins present on the bacterium or cell and/or other bacteria or cells.

In certain cases, the biological recognition element specifically interacts with the biological entity, i.e., the biological recognition element interacts with a particular biological entity (or biological entity type), to a significantly greater degree than to other biological entity. For example, if the biological entity is Escherichia coli, then the biological recognition element may specifically bind to Escherichia coli to a significantly greater degree than to other Escherichia species, to other bacteria, etc.

Non-limiting examples of species that may be present on a biological entity include proteins, for example, a cell surface receptor, an enzyme, a structural protein, etc. Other examples include certain receptors and lipids, for instance, phospholipids. An example of a biological recognition element are carbohydrates, for instance, which may specifically bind a protein on the surface of a bacterium or a cell. Examples of carbohydrates include monosaccharides, oligosaccharides, and polysaccharides. Other, non-limiting examples of biological recognition elements include glycosaminoglycans, glycolipids, proteins, antibodies, glycoproteins, and lectins (i.e., glycoproteins able to bind carbohydrates, in some cases, resulting in cell agglomeration). Additional, non-limiting examples of carbohydrates able to bind to biological entities include mannose (which is able to bind Escherichia coli or Salmonella entrica), fucose (which is able to bind Psuedomonas aerginosa), sialic acid (which is able to bind the influenza virus), heparin (which is able to bind herpes simplex virus), or the Lewis group antigens (which are able to bind Helicobacter pylori). In many cases, these interactions are multivalent in nature. In some cases, the carbohydrate may be specifically chosen to bind to a certain biological entity. [Non-limiting examples of such carbohydrates include those discussed in Ratner DM et al. (2004) Probing Protein-Carbohydrate Interactions with Microarrays of Synthetic Oligosaccharides. ChemBioChem, 5, 379-383.]

As used herein, a "carbohydrate" (or, equivalently, a "sugar") is a saccharide (including monosaccharides, oligosaccharides and polysaccharides) and/or a molecule (including oligomers or polymers) derived from one or more monosaccharides, e.g., by reduction of carbonyl groups, by oxidation of one or more terminal groups to carboxylic acids, by replacement of one or more hydroxy group(s) by a hydrogen atom, an amino group, a thiol group or similar heteroatomic groups, etc. The term "carbohydrate" also includes derivatives of these compounds. Non-limiting examples of carbohydrates include allose ("All"), altrose ("Alt"), arabinose ("Ara"), erythrose, erythrulose, fructose ("Fru"), fucosamine ("FucN"), fucose ("Fuc"), galactosamine ("GaIN"), galactose ("Gal"), glucosamine ("GIcN"), glucosaminitol ("GlcN-ol"), glucose ("GIc"), glyceraldehyde, 2,3- dihydroxypropanal, glycerol ("Gro"), propane- 1, 2, 3-triol, glycerone ("1,3- dihydroxyacetone"), 1,3-dihydroxypropanone, gulose ("GuI"), idose ("Ido"), lyxose ("Lyx"), mannosamine ("ManN"), mannose ("Man"), psicose ("Psi"), quinovose ("Qui"), quinovosamine, rhamnitol ("Rha-ol"), rhamnosamine ("RhaN"), rhamnose ("Rha"), ribose ("Rib"), ribulose ("RuI"), sorbose ("Sor"), tagatose ("Tag"), talose ("TaI"), tartaric acid, erythraric/threaric acid, threose, xylose ("XyI"), or xylulose ("XuI"). In some cases, the carbohydrate may be a pentose (i.e., having 5 carbons) or a hexose (i.e., having 6 carbons); and in certain instances, the carbohydrate may be an oligosaccharide comprising pentose and/or hexose units, e.g., including those described above.

A "monosaccharide," is a carbohydrate or carbohydrate derivative that includes one saccharide unit. Similarly, a "disaccharide," a "trisaccharide," a "tetrasaccharide," a "pentasaccharide," etc. respectively has 2, 3, 4, 5, etc. saccharide units. An "oligosaccharide," as used herein, has 1-20 saccharide units, and the saccharide units may be joined in any suitable configuration, for example, through alpha or beta linkages, using any suitable hydroxy moiety, etc. The oligosaccharide may be linear, or branched in certain instances. A "polysaccharide," as used herein, typically has at least 20 saccharide units. For instance, the polysaccharide may have at least 25 saccharide units, at least 50 saccharide units, at least 75 saccharide units, at least 100 saccharide units, etc. In some cases, the carbohydrate is mulitmeric, i.e., comprising more than one saccharide chain.

In some cases, the arrays comprising biological recognition elements are durable, and can be stored for extended periods of time (weeks to months or years), and/or at room temperature (about 25 ⁰C) and/or near room temperatures (i.e., between about 4 ⁰C and about 25 ⁰C), without denaturing (unlike proteins or antibodies) or decomposing. In some cases, even higher temperatures (i.e., greater than room temperature) may be used.

The term "glass" refers to a hard, brittle, non-crystalline, inorganic substance, which is usually transparent; glasses are often made by fusing silicates with soda, as described by Webster's New World Dictionary. Ed. Guralnik, DB 1984.

For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention. The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.

Exemplification

General Methods. All aqueous solutions were made with nanopure water. Solutions used for chip hybridizations were sterile filtered through a 0.2 μm syringe filter prior to use. Each array was equipped with a hybridization chamber with dimensions of 6.5 x 2.5 cm (Grace Labs). All chemicals were purchased from Sigma Aldrich or Fluka and used without purification. Sheep erythrocytes were purchased from Sigma. Amine coated glass slides were Corning GAPS II slides and were purchased from Corning Inc. CodeLink slides were purchased from Amersham Biosciences. NMR spectra were recorded on a 300 MHz Varian Inova spectrometer at room temperature. Mass spectra were recorded on an Agilent Series 1100 LC/MS. The running solvent for each measurement was acetonitrile/water (1/1) containing 5 mM ammonium formate. Arrays were constructed using a Perkin Elmer non-contact printer. Slides were scanned using a Scan Array 500 scanner from GSI Lumonics and quantified using Scan Array Express Software. All data is the average signal from at least 15 spots on a single array; errors are the standard deviations of those measurements. Bright-field microscopic images were taken using a Nikon Eclipse TSlOO inverted microscope, with images captured using an attached digital camera.

Cell Culture. Bacterial strains ORNl 78 and ORN209 were a gift from Prof. P Orndorf (University of North Carolina). [Harris SL, Spears PA, Havell EA, Hamrick TS, Horton JR, Orndorff PE. (2001) Characterization of Escherichia coli type 1 pilus mutants with altered binding specificities. J. Bacteriol., 183, 4099-102.] The ORN209 stain is a mutant derived from ORNl 78 where the FimH protein, that is responsible for mannose binding, is mutated to diminish mannose binding. All cultures were grown in LB media at 37 ⁰C with shaking to an OD₆₆₀ of ~1.0 (10⁸ cells/mL).

Chemical Synthesis. Carbohydrates equipped with an aminoethanol linker were synthesized according to published procedures. [Ni J, Singh S, Wang LX. (2003) Synthesis of maleimide-activated carbohydrates as chemoselective tags for site-specific glycosylation of peptides and proteins. Bioconjug. Chem., 14, 232-238; and Chernyak AY, Sharma GVM, Kononov LO, Krishna PR, Levinsky AB, Kochetkov NK, Rao AVR. ( 1992) 2- Azidoethyl Glycosides - Glycosides Potentially Useful For the Preparation of Neoglycoconjugates. Carbohydrate Res., 223, 303-309.] 2 '-Azidoethyl-3, 4, 5-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranoside. To a solution of /V-acetyl-D-glucosamine (3.2 g, 14 mmol) was added 2-chloroethanol (20 mL, 300 mmol), and Dowex resin 50x8 (2.0 g) and the solution was heated to 80 °C for 2 h. The reaction was filtered to remove the resin and the excess 2-cloroethanol was removed by distillation on a rotary evaporator. The crude mixture was added to pyridine (20 mL), acetic anhydride (10 mL), and a catalytic amount of 4-dimethylaminopyridine and the reaction was stirred for 2 h. Approximately 100 mL of a mixture of water and ice (~ 1 :1) was added to the reaction mixture and the aqueous solution was extracted with 3 x 100 mL dichloromethane. The organic layer was subsequently washed with 1 N HCl, brine, and dried over Na₂SO₄. The product was purified by silica gel flash column chromatography using EtOAc:hexanes (5:3). To a solution of 2'-chloroethanol-3,4,5-tri-(9-acetyl-2- acetylamino-2-deoxy-/?-D-glucopyranoside (1.7 g, 4 mmol) was added sodium azide (2.7g, 40 mmol) and sodium iodide (0.6 g, 0.4 mmol) in 18 mL DMF and the reaction heated at 80 °C overnight. The DMF was evaporated and the crude mixture resuspended in 200 mL CH₂Cl₂, washed with H₂O (150 mL), brine (3 x 150 mL), dried with Na₂SO₄ and concentrated. The product was purified by silica gel flash column chromatography using EtOAc:hexanes (7:5) to afford 1.50 g of 2'-azidoethyl-3,4,5-tri-O-acetyl-2-acetylamino-2- deoxy-/^D-glucopyranoside (27% over three steps). ¹H NMR (300 MHz, CDCl₃): δ 5.81 (d, J = 9.3 Hz, IH), 5.19 (t, J = 8.1 Hz, IH), 5.07 (d, J = 9.6 Hz, IH), 4.85 (d, J = 3.3 Hz, IH), 4.32 (td, J = 10.5, 3.3 Hz, IH), 4.19 (m, IH), 4.04 (m, IH), 3.93 - 3.83 (m, 2H), 3.61 (m, IH), 3.51 (m, IH), 3.35 (m, IH), 2.04 (s, 3H), 1.98 (s, 3H), 1.96 (s, 3H), 1.89 (s, 3H). ¹³C (75 MHz, CDCl₃): δ 171.4, 170.8, 170.3, 169.5, 97.7, 71.1, 68.2, 67.6, 62.1, 51.8, 50.5, 23.2, 20.8. ESI-MS (positive mode) = 417.3 (M + H⁺).

2 -Aminoethanyl-2-acetylamino-2-deoxy-β-D-glucopyranoside. To 2'-azidoethyl- 3,4,5-tri-O-acetyl-2-acetylamino-2-deoxy-α-D-glucopyranoside (1.5 g, 4 mmol) in 20 mL methanol was added a catalytic amount of sodium methoxide (0.05 g, 0.25 eq). The reaction was stirred for 2 h and neutralized with Dowex 50X resin. The solution was filtered and concentrated. 2'-Azidoethyl-2-acetylamino-2-deoxy-/?-D-glucopyranoside was dissolved in 30 mL methanol and stirred overnight under an atmosphere of hydrogen in the presence of 100 mg of Pd/C. The reaction mixture was filtered through a pad of celite and concentrated. The compound was isolated without purification to yield 1.2 g of product. ¹H NMR (300 MHz, H₂O): δ 4.85 (d, J= 3.3 Hz, IH), 4.80 - 3.65 (m, 6H), 3.45 (m, 2H), 2.80 (m, 2H), 2.01 (s, 3H); ¹³C (75 MHz, H₂O): 8174.44, 97.09, 71.89, 71.03, 70.27, 69.37, 60.57, 53.70, 40.08, 21.82. ESI-MS (positive mode) = 265.0 (M + H⁺).

Carbohydrate Arrays. Carbohydrate microarrays were constructed using a robotic non-contact printer. For arraying onto CodeLink slides, sugars were dissolved in 50 mM sodium phosphate buffer, pH 9.0. For the other slides, carbohydrates were dissolved in a 25% aqueous solution of DMF as described previously. After printing, slides were immediately placed in a sealed chamber that contained a slurry of sodium chloride in water to result in an atmosphere of ~70% humidity. After incubation overnight in the chamber, slides were washed several times with distilled water to remove the unreacted carbohydrates from the surface. Remaining amine groups were quenched by placing slides in a solution pre-heated to 50 °C that contained 100 mM ethanolamine in 50 mM sodium phosphate buffer, pH 9.0 for at least 30 min. Slides were removed from this solution, washed extensively with deionized water, briefly shaken in ethanol, dried by centrifugation, and stored in a dry box until use. Cell Staining and Array Hybridization. Aliquots of the bacterial cultures were centrifuged to isolate the cells and were washed twice with an equal volume of PBS buffer, pH 7.2. After each wash, bacteria were resuspended and centrifuged. Cells were fluorescently stained with SYTO 83 orange-fluorescent cell-permeable nucleic acid dye (Molecular Probes, Eugene OR). Dye was added to a 1 mL suspension of bacteria at a concentration of 50 μM in PBS buffer and shaken for 1 h. After incubation, the cell suspension was centrifuged, isolated, and washed twice with 1 mL PBS buffer. Solutions of bacteria the contained either serum or erythrocytes were suspended in PBS buffer and stained with 50 μM of SYTO 83 dye and incubated for 30 min. After staining, samples were directly applied to the arrays. Dye that was not cell-associated did not non- specifically bind to the CodeLink slides.

Each array was equipped with a hybridization chamber and then a 800 μL solution of E. coli in PBS buffer that contained 1 mM CaCl₂ and 1 mM MnCl₂ was applied to the surface. The arrays were gently shaken on a platform for 1 h at room temperature. After incubation, the hybridization chamber was removed and unbound cells washed away from the array by dipping it into a 50 mL solution of hybridization buffer. This step was repeated twice and the buffer changed between washings. A final wash with nanopure water was used to remove salts from the array. Slides were then briefly centrifuged at about 1000 rpm for 1 min to dry the arrays. The arrays were then scanned using a fluorescence slide reader.

Harvesting Bacteria from an Array and Antibiotic Testing. Strain ORNl 78 was stained with a 50 μM solution of SYTO 62 dye, as described above and incubated with the array, unbound bacteria washed off, and the slides scanned. Positions on the array where the various carbohydrates were immobilized were mapped out using the fluorescent scan of the array. An inoculating loop was scraped over each position where a carbohydrate had been delivered to harvest bound E. coli. The loop was then streaked onto LB plates and incubated at 37 ⁰C overnight. After incubation, many (hundreds to thousand) colonies were observed when the samples were taken from the mannose-containing positions and few (two) to no colonies were observed from samples taken at other positions. Bacteria from the LB plate were then transferred to LB broth and grown. The number of bacteria were then diluted to OD_66O of 0.001 and placed into the wells of a 96-well plate with or without serially diluted concentrations of antibiotics. Plates were then grown for 24 hrs at 37 ⁰C, the culture was pippeted up and down to resuspend the cells in the media, and OD'sόόo were immediately taken using a Spectra Max 250 microplate reader. Dose- response curves were then plotted as the absorbance at 660 nm versus the concentration of antibiotic.

Incorporation by Reference All of the U.S. patents and U.S. published patent applications cited herein are hereby incorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:

1. A method of detecting the presence of a biological entity in a sample, comprising the steps of: contacting said sample to an array of biological recognition element-containing molecules at a contacting time and contacting temperature; and detecting the presence of a complex formed between said biological recognition element-containing molecules and said biological entity; wherein said array of biological recognition element-containing molecules comprises a plurality of spots with widths in the range of about 0.1 μm to about 500 μm.

2. A method of detecting the identifying a biological entity in a sample, comprising the steps of: contacting said sample to an array of biological recognition element-containing molecules at a contacting time and contacting temperature; detecting the presence of a complex formed between said biological recognition element-containing molecules and said biological entity; harvesting said biological entity from said array; culturing said biological entity; and identifying said biological entity; wherein said array of biological recognition element-containing molecules comprises a plurality of spots with widths in the range of about 0.1 μm to about 500 μm.

3. A method for identifying a compound with anti-adhesion properties, comprising the steps of: contacting a biological entity to an array of biological recognition element- containing molecules in the presence of said compound at a contacting time and contacting temperature, wherein said biological entity binds said carbohydrate- containing molecules in the absence of said compound; and detecting the presence of a complex formed between said carbohydrate-containing molecules and said biological entity; wherein said array of biological recognition element-containing molecules comprises a plurality of spots with widths in the range of about 0.1 μm to about 500 μm.

4. The method of any one of claims 1-3, further comprising the step of: washing said array to remove unbound components of said sample.

5. The method of any one of claims 1-4, further comprising the step of: treating said sample with a fluorescent cell-permeable dye.

6. The method of claim 5, wherein said fluorescent cell-permeable dye is a cell- permeant cyanine nucleic acid stain.

7. The method of claim 5, wherein said fluorescent cell-permeable dye is SYTO 83.

8. The method of any one of claims 1-7, wherein said biological entity is capable of reproduction.

9. The method of any one of claims 1-7, wherein said biological entity is a pathogen.

10. The method of claim 9, wherein said pathogen is a bacteria.

11. The method of claim 9, wherein said pathogen is a bacteria selected from the group consisting of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella,

Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma.

12. The method of claim 9, wherein said pathogen is an E. coli.

13. The method of any one of claims 1-12, wherein said biological recognition element comprises is selected from the group consisting of carbohydrates, saccharides, monosaccharides, oligosaccharides, polysaccarides, glycoaminoglycans, glycolipids, aminoacids, proteins, antibodies, lectins, nucleic acids, DNA, RNA, or oligonuclotides.

14. The method of claim 13, wherein said biological recognition element comprises a carbohydrate.

15. The method of claim 13, wherein said biological recognition element is selected from the group consisting of mannose, galactose, glucose, fucose, and N- acetylglucosamine.

16. The method of claim 13, wherein said biological recognition element is mannose.

17. The method of any one of claims 1-16, wherein said sample is homogenous.

18. The method of any one of claims 1-16, wherein said sample is heterogeneous.

19. The method of any one of claims 1-16, wherein said sample is taken from food, water, bodily fluid or the environment.

20. The method of claim 19, wherein said sample is taken from blood, saliva, urine or feces.

21. The method of claim 19, wherein said sample is taken from blood.

22. The method of claim 19, wherein said sample consists essentially of erythrocytes or serum.

23. The method of any one of claims 1-22, wherein said array is a glass slide, a membrane, a 96-wellplate, a surface, or a gold-coated glass, polymer or metal surface.

24. The method of claim 23, wherein said array is a amine-reactive glass slide.

25. The method of claim 23, wherein said array is a Corning GAPS II amine- functionalized slide which has been reacted with N,N-disuccinimidyl carbonate or a CodeLink™ polymer coated slide.

26. The method of any one of claims 1-25, wherein the width of said spots is in the range of about 10 μm to about 300 μm.

27. The method of any one of claims 1-25, wherein the width of said spots is in the range of about 50 μm to about 200 μm.

28. The method of any one of claims 1-25, wherein the width of said spots is in the range of about 80 μm to about 120 μm.

29. The method of any one of claims 1-28, wherein each spot is within about 700 μm of an adjacent spot.

30. The method of any one of claims 1-28, wherein each spot is within about 500 μm of an adjacent spot.

31. The method of any one of claims 1-28, wherein each spot is within about 300 μm of an adjacent spot.

32. The method of any one of claims 1-28, wherein each spot is within about 150 μm of an adjacent spot.

33. The method of any one of claims 1-32, wherein said contacting temperature is between about 15 °C and 30 °C.

34. The method of claim 33, wherein said contacting temperature is about 25 °C.

35. The method of any one of claims 1-34, wherein said contacting time is between about 0.5 hours and about 2 hours.

36. The method of claim 35, wherein said contacting time is about 1 hour.

37. The method of any one of claims 1-36, wherein said biological recognition element is attached to said array via a linker.

38. The method of claim 37, wherein said linker is a disuccinimidyl carbonate linker.

39. The method of claim 37, wherein said linker is a disuccinimidyl tetrapolyethylenglycol or disuccinimidyl carbonate linker.