This application claims the benefit of priority of U. S. provisional application no. 60/434, 958 filed December 20,2002 entitled"Spatially Selective Deposition of a Reactive Polysaccharide Layer onto a Patterned Template, "the complete disclosure of which is incorporated herein by reference.

GOVERNMENT LICENSING CLAUSE The U. S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Grant No. BES- 01114790 awarded by the National Science Foundation.

BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates generally to methods for spatially localized deposition of polysaccharides, and optionally for coupling molecules,

including biomolecules, cellular species, and the like to the polysaccharides in deposition. This invention further relates to materials, such as films, coatings, and gels, and to devices comprising electrochemically deposited polysaccharides, alone or in combination with coupled molecules.

2. Description of Related Art The ability to create devices such as biosensors, microarrays, and microelectromechanical systems (MEMS) requires facile methods to precisely control surfaces. A variety of patterning techniques can be used to produce desired structures, while various methods have been investigated to control surface chemistries. For instance, microfabrication techniques are routinely applied to create patterned inorganic surfaces with nanometer to micrometer scale resolution. However, traditional approaches have not proven particularly successful in adequately bonding organic and biological materials to the patterned inorganic surfaces.

Two approaches have emerged to extend microfabrication techniques for the creation of patterned surfaces with organic and biological materials. The first approach is based on an extension of photolithography. Self-assembled monolayers are selectively irradiated to create a pattern of freshly exposed surface, which is then reacted with a bifunctional agent. Reactions include those between thiols and metal surfaces, or between silanes and oxidized silicon. Bain, C. D. , Whitesides, G. M. Angew. Chem. Int. Ed. Eng/. 1989, 28, 506-512 ; Whitesides, G. M.,

Laibinis, P. E. Langm. 1990,6, 87-96 ; Sagiv, J. J. Am. Chem. Soc. 102, 1980, 92-98 ; Brzoska, J. B. , Azouz, I. B. ; Rondelez, F. Langm. 1994, 10, 4367-4373 ; Allara, D. L., Parikh, A. N. , Rondelez, F. Langm. 1995, 11,2357-2360.

A first functional group of the agent attaches the agent to the freshly exposed surface, and the second functional group subsequently couples the molecules of interest. Although variations exist, lithography creates the spatial template upon which subsequent coupling occurs. This first approach has a drawback associated with the need for photo-sensitive reagents that can be expensive, hazardous and require cumbersome steps to prepare the surface. Furthermore, conventional photolithographic operations require"line-of-sight"and would be difficult to accomplish on internal surfaces in an enclosed microfluidic system. Alternatively, if the lithographic patterning and subsequent biological functionalization are carried out before the microfluidic device is covered to form a closed fluidic environment, the biofunctionality internal to the microfluidic system cannot be readily reprogrammed. Finally, since many biospecies are labile, i. e. , sensitive and delicate with respect to their environmental conditions, fabrication processes required to close the microfluidic system may degrade the biospecies.

A second approach for creating patterned surfaces with organic and biological materials is microcontact printing (pCP), in which a soft stamp (typically made of poly-dimethylsiloxane) is created with a preselected

pattern. After"inking"the stamp with a solution containing the material to be deposited, the stamp is pressed onto the surface to transfer the pattern. Drawbacks to the microcontact printing approach involve difficulties in stamping with high spatial resolution. Furthermore, the need for direct contact to the surface entails the drawbacks described above for applications to enclosed microfluidic systems. Vaeth, K. M., Jackman, R. J. , Black, A. J. Whitesides, G. M. , Jensen, K. F. , Langmuir 2000,16, 8495-8500.

Another approach to patterning biomolecules on surfaces is"dip- pen"nanolithography, in which a scanning probe microscopy (like atomic force microscopy) is used to write species onto a surface with high lateral resolution. For biomolecular species this is accomplished by transport from the writing tip through a water meniscus to the substrate. While the lateral spatial resolution of this patterning method can be very high (30nm), patterns must be written in serial fashion, entailing the throughput limitations associated with other direct-write approaches such as electron and ion beam lithographies. In addition, dip-pen nanolithography entails the drawbacks described above for applications to enclosed microfluidic systems. Piner, R. D. , Zhu, J. Z. , Xu, F. , Hong, S., Mirkin, C. A. , Science 29 Jan 1999,283, 661-663 ; Jong, S. , Mirkin, C. A., Science 9 June 2000,288, 1808-1811 ; Lyuksyutov, S. F. et. Al., Nature Materials July 2003,2, 468-474.

Electrophoretic deposition has also been used to assemble colloidal particles and proteins onto electrode surfaces. This approach has been extended to exploit an electric field to direct the spatially selective deposition of CdTe nanocrystals. Gao, M, et al, Langmuir, 18,4098-4102 (2002). In this method, a surface with patterned electrodes is first fabricated, then a combination of an applied voltage and layer-by-layer assembly is used to generate multilayers with spatial resolution in lateral directions. The drawbacks to this assembly approach are that voltages must be maintained to retain the initial layer of nanocrystals, which may not be held to the surface by strong chemical bonds or insolubility. Again, it is not clear whether these layer-by-layer approaches can be extended to enclosed microfluidic channels.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for attaining spatially and/or temporally selective deposition of a polymer film, coating, gel, or other solid or semi-solid material onto a support in response to alteration of reaction conditions.

It is another object of the present invention to provide a method for attaining spatially and/or temporally selective deposition of a polymer film, coating, gel, or other solid or semi-solid material onto a support, the polymer being capable of coupling or being capable of manipulation to permit coupling to other molecules, especially biomolecules, cellular species, and the like.

It is still another object of the present invention to provide a method for spatially and/or temporally depositing molecules, especially biomolecules, cellular species, and the like, on a polymer film coating, gel, or other solid or semi-solid material deposited in a predetermined pattern.

Another object of the present invention is to provide materials, such as films, gels, and the like, comprising an electrochemically deposited polysaccharide, alone or coupled to other molecules, especially biomolecules, cellular species, and the like.

To achieve one or more of the foregoing objects, and in accordance with the purposes of the invention as embodied and broadly described in this document, a first aspect of this invention provides a method for selectively depositing of a polymer in spatially localized regions. The method comprises providing a substrate comprising a substrate surface, the substrate surface comprising a patterned electrically conductive portion and an electrically non-conductive portion. The substrate surface is contacted with an aqueous solution comprising a selectively insolubilizable polysaccharide, and the selectively insolubilizable polysaccharide is spatially selectively deposited on the electrically conductive pattern in a spatially selective manner.

A second aspect of the invention provides a polymer material comprising a selectively insolubilizable polysaccharide electrochemically deposited in a spatially selective pattern.

According to a third aspect of the invention, a method for selectively depositing of a polymer is provided, the method comprising contacting a substrate comprising a surface having an electrically conductive pattern thereon with an aqueous solution comprising a selectively insolubilizable polysaccharide, spatially selectively depositing the selectively insolubilizable polysaccharide on the electrically conductive pattern, and modifying the selectively insolubilizable polysaccharide (before or after deposition) to facilitate its ability to conjugate with reactive groups of other compounds, such as proteins (especially enzymes, receptors, receptor ligands, and antibodies) and nucleic acids (especially DNA and RNA).

In accordance with a fourth aspect of the invention, a polymer material is provided comprising a selectively insolubilizable polysaccharide electrochemically deposited in a spatially selective pattern, the polymer having been modified to facilitate its ability to conjugate with reactive groups of other compounds, such as proteins (especially enzymes, receptors, receptor ligands, and antibodies) and nucleic acids (especially DNA and RNA).

A fifth aspect of the invention provides a method for spatially selectively depositing molecules, preferably biomolecules and/or cell species. According to this aspect, the method comprises providing a pattern of solid or semi-solid material, e. g., film, coating, gel, or the like, optionally modifying the solid or semi-solid material to make it more receptive to conjugation with reactive groups of other molecules, and

reacting the solid or semi-solid material with the other molecules. Owing to the flexibility of the invention, any of a wide variety of different compounds can be coupled (e. g. , conjugated) to the polysaccharide. Such compounds include, for example, proteins (especially enzymes, receptors, receptor ligands, and antibodies), and nucleic acid molecules (especially DNA and RNA).

According to a sixth aspect of the invention, a material is provided comprising a selectively insolubilizable polysaccharide electrochemically deposited in a spatially selective pattern, and molecules coupled to the polysaccharide in a corresponding spatially selective pattern. In an embodiment of the invention, the polymer has been modified to facilitate its ability to conjugate with reactive groups of other compounds, such as proteins (especially enzymes, receptors, receptor ligands, and antibodies) and nucleic acids (especially DNA and RNA).

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings are incorporated in and constitute a part of the specification. The drawings, together with the general description given above and the detailed description of the certain preferred embodiments and methods given below, serve to explain the principles of the invention. In such drawings : FIG. 1 shows the transformation of the selectively insolubilizable polysaccharide chitosan from a soluble phase to an insoluble phase ;

FIG. 2 shows a progression of steps of a microfabrication technique for establishing an electrically conductive, e. g. , metal, pattern on a substrate ; FIG. 3 is a simplified representation of an electrochemical deposition cell for carrying out a method according to an embodiment of the invention ; FIG. 4 is a graph showing the relationship between applied voltage (volts) and film thickness (microns) for depositing a chitosan film on a gold template according to an embodiment of the invention ; and FIG. 5 shows photomicrographs taken of Example 1 described below.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS AND PREFERRED METHODS Reference will now be made in detail to the presently preferred embodiments and methods of the invention as illustrated in the accompanying drawings. It should be noted, however, that the invention in its broader aspects is not limited to the specific details, representative assemblies and methods, and illustrative examples shown and described in this section in connection with the preferred embodiments and methods.

The invention according to its various aspects is particularly pointed out and distinctly claimed in the attached claims read in view of this specification, and appropriate equivalents.

It is to be noted that, as used in the specification and the appended claims, the singular forms"a, ""an,"and"the"include plural referents unless the context clearly dictates otherwise.

According to an embodiment of the present invention, a method is provided for spatially selectively depositing a polymer, such as a biopolymer or a natural or synthetic polymer, the method comprising contacting a substrate comprising a substrate surface having a patterned electrically conductive portion and an electrically non-conductive portion with an aqueous solution comprising a selectively insolubilizable polysaccharide, and spatially selectively depositing the selectively insolubilizable polysaccharide on the electrically conductive pattern.

As used herein and unless otherwise indicated, a"substrate"or "wafer"comprises a platform on which an electrically conductive pattern may be deposited or otherwise formed. The platform may be formed of one or more materials, may be homogeneous or heterogeneous, and may contain a surface film. The surface on which the film is formed may be flat, curved, multi-leveled, etc. , and may optionally include channels (e. g., microchannels), ridges, indentations, protuberances, and the like.

Substrates are preferably substantially electrically non-conducting or possess a substantially electrically non-conducting surface on which the electrically conductive pattern is formed. Such substrates may be made of inorganic materials such as, but not necessarily limited to, a silicon wafer

optionally having a surface oxide film. Other inorganic materials include silicon oxide, silicon nitride, and the like.

The substrate includes one or more surface portions containing a patterned electrically conductive portion and an electrically non- conductive portion. As referred to herein, a pattern refers to the spatial localization of a material, i. e. , so that the surface also contains surface portions not covered with the patterned electrically conductive portion.

The pattern may extend from one surface to another, or be localized on a single surface. A pattern may comprise a repeating arrangement of objects or shapes, a non-repeating or random arrangement of objects or shapes, a particular defined shape, array, or the like. For example, the pattern may comprise a plurality of parallel lines spaced apart from one another by uniform or non-uniform intervals. The pattern may be coplanar or offset from the principle surface of the substrate, e. g. , as in the case of microchannels. The material or materials selected for patterning are preferably those upon which the selectively insolubilizable polysaccharide may be deposited via electrochemical deposition. Suitable materials are electrically conductive, and may include but are not necessarily limited to metals (e. g. , aluminum, antimony, cadmium, chromium, cobalt, copper, gold, iron, led, magnesium, mercury, nickel, palladium, platinum, silver, steel, tin, tungsten, zinc), metal alloys, semiconductors, and conductive polymers (polypyrrole).

Deposition of the electrically conductive patterned material on the substrate may be accomplished by any known or suitable technique. For example, standard microfabrication techniques may be selected to pattern an electrically conductive material, e. g. , gold, onto an electrically insulative substrate. Referring to FIG. 2, there is shown an exemplary yet not necessarily limiting technique for patterning an electrically conductive material on a substrate. In FIG. 2, the selected substrate 10 comprises silicon wafers with a thermal oxide film. A metal layer or layers 12, for example chromium and gold in the illustrated embodiment, are sputtered (simultaneously or consecutively) or otherwise deposited onto the wafer 10 to provide a bi-layer metal structure. Next, the deposited metal is optionally covered with a primer, then a photoresist 14 is applied to the primed metal surface, e. g. , via conventional spin-coating techniques. A mask 16 is placed over the photoresist, and the photoresist is then patterned, for example, by exposure of the unmasked portions of the photoresist to UV light 18. The exposed, non-masked areas were then etched with a suitable etchant to develop the sputtered metals into a pattern. The photoresist may then be removed, such as with a solvent, e. g. , acetone, leaving the patterned sputtered metal (s) 20 over the substrate 10.

The patterned electrically conductive material serves as a platform for the electric field directed deposition of polysaccharide. Preferably, the polysaccharide is deposited on the patterned electrically conductive

portion of the substrate surface, but not the electrically non-conductive portion. Thus, the deposition of the polysaccharide is spatially selective based on the pattern of the electrically conductive portion.

The compositions of the present invention preferably comprise selectively insolubilizable polysaccharides capable of solubilizing in a liquid medium, preferably aqueous, and forming or otherwise depositing an insoluble coating, gel, or other layer onto a support in response to an alteration in reaction conditions. As used herein, the term polysaccharide includes starches and polysugars, particularly polymers containing glucosamine residues. Ionizable polysacchides include carboxymethylcellulose, chitosan and chitosan sulfate, ligninsulfonates, and synthetic polymers such as, for example, polymethacrylic acid, polyvinylsulfonic acid, polyvinylphosphonic acid and polyethyleneimine ; ionizable agar, alginate, and carrageen and similar extracts of plants may be also be used. Other suitable polysaccharides include gums from trees, pectins from fruits, starches from vegetables, and celluloses from woody fibers. Chitosan is the preferred ionizable polysaccharide of the present invention.

In preferred embodiments, the selective insolubilization of the polysaccharides of the present invention is accomplished by modifying the polysaccharide to contain one or more ionizable group (s), which may be the same or different, such that at one or more range (s) of pH the polysaccharide will be soluble in an aqueous solvent ("solubilizing pH

ranges") whereas at one or more other pH values range (s), the polysaccharide will be insoluble (or less soluble), and thus be capable of forming an insoluble coating, gel, or other layer onto a support or otherwise depositing itself onto the support. Suitable ionizable groups include those ionizable at low pH, e. g. , capable of forming a positive charge (e. g., alkyl amine groups, primary, secondary or tertiary amine groups, guanidinium groups, imidazole groups, indole groups, purine groups, pyrimidine groups, pyrrole groups, etc. ) and those that are ionizable at high pH, e. g. , capable of forming a negative charge (e. g. , alkoxide groups, carboxyl groups, , hydroxy acid groups, phenolic groups, phosphate groups, sulfhydryl groups, etc. ). Suitable groups may exhibit multiple pKs, which may be the same (e. g. polyacidic or polybasic) or different (e. g., zwitterionic). For selectively insolubilizable polysaccharides that are ionizable at low pH, amine groups are preferred ; for selectively insolubilizable polysaccharides that are ionizable at high pH, carboxyl groups are preferred.

For example, a preferred selectively insolubilizable polysaccharide is chitosan, which is an amine-rich polysaccharide derived by deacetylation of chitin. Chitin is the second most abundant polysaccharide in nature and is found in crustaceans, insects, and fungi.

Chitosan has primary amino groups that have pKa values of about 6.3. At pH's below the pKa, amino groups are protonated making chitosan a water-soluble, cationic polyelectrolyte. At pH's above the pKa of about 6.3,

chitosan's amino groups are deprotonated, and the chitosan polymer becomes insoluble. Chitosan's pH-dependent solubility allows the biopolymer to be processed in an aqueous solution, and brought out of solution and formed into various shapes (e. g. , beads, membranes, and films) by imparting a modest increase in pH, e. g. , to neutrality.

FIG. 3. shows a suitable electrochemical deposition assembly for depositing the polysaccharide onto a patterned substrate. The assembly comprises a power source 30, such as a DC source, and a positive electrode 32 (anode) and a negative electrode 34 (cathode) connected to the power source with appropriate wiring or electrical connections. The patterned electrically conductive material is polarized to serve as the negative electrode. The positive electrode may be, for example, a non-patterned metal-coated (e. g. , gold-coated) silicon wafer.

The electrodes are immersed in an aqueous solution 36 comprising the selectively insolubilizable polysaccharide, preferably in a solubilized state. A suitable pH for deposition of the polysaccharide onto a substrate is any pH below the solubility limit. For example, an aqueous solution will have a pH less than about 6.3, more preferably less than about 5 to solubilize chitosan into solution. For the chitosan solution used to deposit chitosan onto a substrate, suitable concentrations of chitosan may vary, for example, from about 0.0001 to about 0.001 (w/v) %, about 0.001 to about 0.01 (w/v) %, about 0.01 to about 0.1 (w/v) %, about 0.1 to about 1

(w/v) %, about 1 to about 10 (w/v) %, about 10 to about 20 (w/v), and about 20 to about 30 (w/v) %.

Chemical deposition of the selectively insolubilizable polysaccharide is preferably electrode selective, providing another degree of control over the process. In the case of a polysaccharide containing a group ionizable at a low pH, e. g. , capable of forming a positive charge (e. g., alkyl amine groups, primary, secondary or tertiary amine groups, guanidinium groups, imidazole groups, indole groups, purine groups, pyrimidine groups, pyrrole groups, etc. ), the solubilized polysaccharide is attracted to and deposited on the negative electrode.

Moderate increases in the pH above the pKa of the selectively insolubilizable polysaccharide stabilize the polysaccharide in an insoluble state, forming a stable coating, gel, or other solid or semi-solid layer that optionally may be removed from the negative electrode. Positively charged polysaccharides are not attracted to the positive electrode, and do not deposit on the positive electrode. In contrast, a polysaccharide containing a group ionizable at a high pH, e. g. , capable of forming a negative charge (e. g. , alkoxide groups, carboxyl groups, carboxylate groups, hydroxy acid groups, phenolic groups, phosphate groups, sulfhydryl groups, etc.), is attracted in its soluble state to the positive electrode and deposits on the positive electrode, but not the negative electrode. Hence, a moderate decrease in the pH below the pKa of such selectively insolubilizable polysaccharide will stabilize the polysaccharide

into a stable coating, gel, or other solid or semi-solid layer that may be removed from the positive electrode.

Various aspects of the electrochemical cell, reaction conditions, and process parameters may be manipulated to control the chemical deposition of the selectively insolubilizable polysaccharide on the patterned electrode and the resulting properties of the polysaccharide thin film. For example, the shape of the patterned electrically conductive material on which the polysaccharide deposits largely dictates the spatial distribution and localization of the deposited polysaccharide. Examples of conditions and parameters that affect characteristics of the deposit include the applied voltage, current, pH level, total ion concentration, polysaccharide concentration, temperature, deposition time, and the like. In a preferred embodiment of the invention, the applied current or voltage is kept constant during electrochemical deposition. The thickness and spatial resolution of the deposit can be adjusted by altering conditions. For instance, deposition thickness is increased by increases in voltage, current density, and polymer concentration in the solution.

For example, from its soluble state, the chitosan deposits onto the patterned platform, i. e. , the negative electrode, in a spatially selective manner corresponding in shape to the patterned template. The thickness of the deposited chitosan may range from tens of nanometers to micrometers. The deposition can be controlled temporally and spatially based on when and where the voltage is applied. Further, the

concentration of the chitosan solution, the voltage and the time a current is applied to deposit chitosan onto a substrate can be varied to control the extent of polysaccharide deposition. FIG. 4 is a graph illustrating the relationship between applied voltage and film thickness (microns) for chitosan deposited on a gold template. The film thickness may measure, for example, from about 0.01 to about 3 microns, from about 0.01 to about 1.5 microns, or from about 0.02 to about 0.8 microns. For the chitosan polysaccharide, deposition occurs only on the electrode which is biased negatively ; this corresponds to attracting the positively charged amine groups characteristic of the chitosan biopolymer in acidic solution.

In a preferred embodiment of the invention, the selectively insolubilizable polysaccharide deposited on the electrode (s) is stabilized (or destabilized) by pH adjustment, such as by washing the deposited polysaccharide with a liquid selected from water, a solution of neutral pH, a basic solution, and an acidic solution. In the case of a polysaccharide containing a group ionizable at a low pH, e. g. , capable of forming a positive charge (e. g. , amine groups), raising the pH will increase the insolubility of the deposited polysaccharide and improve stabilization. On the other hand, lowering the pH of the positively charged ionizable polysaccharide will lead to destablization. In contrast, in the case of a polysaccharide containing a group ionizable at a high pH, e. g. , capable of forming a negative charge (e. g. , a carboxyl group), lowering the pH will improve stabilization, whereas raising pH will lead to instability.

The electrodeposition of the polysaccharide, for example chitosan, is accomplished by application of an electrical voltage between the spatially defined deposition electrode (e. g., a patterned Au wire) and a counterelectrode. The operational electrical circuit may be controlled by using a controlled constant voltage, a controlled constant current, or a mixture of the two as the deposition proceeds. Using constant voltage there is typically a large current and high deposition rate until an initial chitosan thin film is achieved, after which the current is reduced by the series resistance of the chitosan. Using constant current, the initial voltage is typically small but then decreases rather quickly to a nearly constant value as the resistive chitosan thin film develops on the surface.

For example, under typical conditions at a constant 0.4 mA current and current density 2-5 A/mm2, the voltage rises within 1 min to slightly over 2 V and remains nearly constant over a total deposition time of 5 min.

The deposition process is more reproducible and controllable for constant current mode of electrodeposition of chitosan.

For example, washing an acidic, soluble chitosan deposit with a base neutralizes and deprotonates the chitosan, converting the chitosan into an insoluble, stable film. Suitable bases include sodium hydroxide, ammonium and organic bases. The chitosan film is stabilized by neutralization, permitting the chitosan to be retained on the electrode surface in the absence of an applied voltage. The deposited chitosan film may possess a high amine group concentration of about 1014-1015/cm2, e. g.,

101¢/cm2, preferably in a substantially homogeneous distribution. The chitosan film may include N-acetylglucosamine residues and/or blocks, preferably in a concentration of less than 40 weight percent, more preferably less than 30 weight percent. On the other hand, washing the chitosan deposits with an acid to lower the pH below the pKa will dissolve the chitosan.

In some preferred embodiments, the selectively insolubilizable polysaccharides may serve as a template for surface-controlled bonding and reaction of the molecules, such as biomolecular and cellular species (eukaryotic or prokaryotic), for example in microfluidic systems. The selectively insolubilizable polysaccharides of the present invention may be modified to facilitate their ability to stably conjugate with reactive groups of other molecules. Such modifications may include covalent cross-linking agents (e. g. , dialdehydes (such as glutaldehyde, formaldehyde, glyoxal), anhydrides (such as succinimide, carbodiimide, dicyclohexylcarbodiimide, etc. ), genipin, amino acids, etc. ) or non-covalent crosslinking agents (such as tripolyphosphate (TPP), etc. ). In one embodiment, such molecules will be nonspecifically divalent or multivalent, possessing two or more identical reactive groups that can be used to conjugate the polysaccharides of the present invention to other molecules (e. g. , glutaraldehyde, lysine, arginine, glutamate, aspartate, polysaccharides, etc. ). More preferably, however, such molecules will comprise two or more different relevant reactive groups such that an orthogonal synthetic approach may be

employed. Examples of such compounds include amino acids. The carboxyl group of such compounds can be conjugated to the amine group of, for example, chitosan, to yield a free, and more sterically accessible, amino group that can be conjugated to the carboxy group of a glutamate or aspartate residue of a protein. Likewise, the polysaccharides of the present invention can be modified to contain chloromethylbenzyl or trialkylsulfoniumbenzyl groups that can then react with the carboxyl group of other molecules.

Owing to the flexibility of the chemistry involved, any of a wide variety of different compounds can be conjugated to the polymer. Such compounds particularly include proteins (especially enzymes, receptors, receptor ligands, or antibodies) and nucleic acid molecules (especially DNA or RNA). Depending upon the particular compound selected, conjugation may occur before or after (or both) deposition of the selectively insolubilizable polysaccharide onto the substrate.

For example, chitosan possesses amino groups that confer nucleophilic properties to the polymer. Specifically, the deprotonated amino groups have an unshared electron pair that can undergo reaction with a variety of electrophiles. As a result, various chemistries can be exploited to crosslink chitosan and to graft substituents onto the polymer.

The substituent may be coupled to the chitosan and deposited from solution. Alternatively, the substituent may be coupled to the chitosan after the chitosan has been deposited onto the negative electrode. In the

examples below, the substituent selected comprises a fluorescein derivative activated with NHS to be reactive toward chitosan's amino groups. Various other molecules, including labile biomolecules, may be selected to replace the fluorescein derivative. Such biomolecules include, not necessarily by limitation, bound protein, enzyme, polynucleotide, RNA, DNA, cells, and the like. The molecules are assembled on the polysaccharide template, which acts as an interface between the molecules and the inorganic substrate.

The conjugated selectively insolubilizable polysacchides of the present invention can be used to provide a spatially and/or temporally defined two-dimensional surface or three-dimensional matrix for molecular interactions.

In one embodiment, the conjugated molecules of such surfaces or matrices will comprise one, two, three or more enzyme species, each of which will preferably be placed in a spatially and/or temporally discrete region of such surfaces or matrices. Significantly, by incubating such surfaces or matrices in contact with a fluidic layer (i. e. , a surface or matrix that contains a flowing or flowable liquid or gas capable of transporting other molecules (e. g. , nucleic acid molecules, proteins, enzymatic substrates and/or products, etc. )), multiple stepwise synthetic reactions can be made to occur, either sequentially or in parallel. Suitable enzyme species include: aminopeptidases, angiotensin converting enzymes, caspases, cathepsins, cholinesterases, collagenases, deaminases,

endonucleases, endopeptidases, esterases, exonucleases, lipases, nucleotidases, phosphatases, proteases, restriction endonucleases, etc.

In a second embodiment, the conjugated molecules of such surfaces or matrices will comprise one, two, three or more antibody species each of which will preferably be placed in a spatially and/or temporally discrete region of such surfaces or matrices. As used herein, the term"antibodies" is intended to encompass not only conventional immunoglobulins, but also single chain antibodies, humanized antibodies, monoclonal antibodies, etc.

Significantly, by incubating such surfaces or matrices in contact with a fluidic layer containing antigens, multiple immunoassays can be simultaneously or sequentially conducted. Any of a wide variety of assay formats may be used in accordance with the methods of the present invention. They may be heterogeneous or homogeneous, and they may be sequential or simultaneous. They may be competitive or non-competitive.

U. S. Patents Nos. 5,563, 036 ; 5, 627, 080 ; 5, 633, 141 ; 5,679, 525 ; 5, 691, 147 ; 5,698, 411 ; 5, 747, 352 ; 5, 811, 526 ; 5, 851,778 and 5, 976,822 illustrate several different assay formats and applications.

In a third embodiment, the conjugated molecules of such surfaces or matrices will comprise one, two, three or more bound receptor molecule species or bound ligands of receptor molecules each of which will preferably be placed in a spatially and/or temporally discrete region of such surfaces or matrices. Significantly, by incubating such surfaces or matrices in contact with a biological sample, multiple receptor'/receptor

ligand binding assays can be simultaneously or sequentially conducted.

Suitable receptor species include: 5-hydroxytryptamine receptors, acetylcholine receptors, adenosine receptors, adrenoceptor receptors, adrenomedullin receptors, amylin receptors, amyloidreceptors, angiotensin receptors, atrial natriuretic peptide (ANP) receptors, bombesin receptors, bradykinin receptors, calcium-channel receptors, cannabinoid receptors, cgrp receptors, chemokine receptors, cholecystokinin and gastrin (CCK) receptors, corticotropin releasing factor (CRF) receptors, dopamine receptors, endothelin receptors, excitatory amino acid receptors, gaba receptors, galanin receptors, gastric inhibitory peptide (GIP) receptors, GDNF receptors, glucagon receptors, glucagon- like peptide receptors, glycoprotein hormones receptors, growth hormone secretagogue receptors, GTP-binding-protein receptors, hemotopoietin receptors, histamine receptors, imidazole receptors, integrin receptors, interleukin-1 receptors, melanin-concentrating hormone receptors, melanocortin receptors, melatonin receptors, metastin receptors, motilin receptors, neuromedin receptors, neuropeptide FF receptors, neuropeptide Y receptors, neurotensin receptors, opioid receptors, orexin receptors, P2 purinoceptor receptors, parathyroid hormone (PTH) receptors, phosphodiesterase enzyme, platelet activating factor (PAF) receptors, potassium-channel receptors, prolactin receptors, prostanoid receptors, retinoid receptors, selectin receptors, somatostatin receptors, steroid receptors, tachykinin receptors, tumour necrosis factor (TNF) receptors,

tyrosine kinase receptors, urotensin II receptors, vasoactive intestinal peptide (VIP) receptors, vasopressin receptors, etc.

In a fourth embodiment, the conjugated molecules of such surfaces or matrices will comprise one, two, three or more bound nucleic acid molecule species, which may be DNA or RNA or be composed of non- naturally occurring residues (e. g. , PNA). Such nucleic acid molecules may have defined sequences (such as the sequences of genes or fragments thereof, or may be composed of random or pseudorandom oligonucleotides (i. e. , nucleic acid molecules of 3-100 nucleotides in length) or polynucleotides (i. e, nucleic acid molecules greater than 100 nucleotides in length). Significantly, by incubating such surfaces or matrices in contact with a biological sample (or an extract thereof, multiple hybridization reactions involving nucleic acid molecules present in the sample can be simultaneously or sequentially conducted. Such hybridization reactions can be used in concert with nucleic acid amplification strategies (such as the polymerase chain reaction (PCR) (e. g. , U. S. Patents Nos. 4,683, 202 ; 4,582, 788 ; US 4,683, 194,6, 642, 000, etc.)) ; ligase chain reaction (LCR), self-sustained sequence replication (3SR) (e. g. , Guatelli et al., Proc. Natl.

Acad. Sci. USA 87: 1874-1878 (1990) ; PCT Publication. WO 88/10315), nucleic acid sequence based amplification (NASBA) (e. g. , Kievits, J Virol Methods. 35: 273-86 (1991) ), strand displacement amplification (SDA) (e. g., U. S. Patent No. 5,270, 184), and amplification with Q6 replicase (Birkenmeyer et al., J. Virological Methods, 35: 117-126 (1991) ; Landegren,

Trends Genetics, 9: 199-202 (1993) ; and rolling circle amplification (e. g., U. S. Patents Nos. 5,854, 033 ; 6, 183, 960 ; 5, 354, 668 ; 5, 733,733)) to accomplish the amplification of the hybridized molecules, or their complements. The present invention permits hundreds, thousands, and tens of thousands of nucleic acid species to be deposited on to such surfaces or matrices.

Additionally, such hybridization reactions may be used to sequence the nucleic acid molecules present in the sample, or to assess the expression profile of the genes of cells present in the biological sample (or an extract thereof (see, e. g. , U. S. Patents Nos. 6,632, 606 ; 5, 002, 867 ; 5,202, 231 ; 5, 888, 819 Lipshutz et al., Biotechniques, 9 (3): 442-447 (1995) and Chee et al., Science, 274: 610-614 (1996) ; DeRisi, J. et al. (1996) "USE OF A CDNA MICROARRAY TO ANALYSE GENE EXPRESSION PATTERNS IN HUMAN CANCER"Nature Genetics 14 : 457-60 Luo, L. et al. (1999) "GENE EXPRESSION PROFILES OF LASER-CAPTURED ADJACENT NEURONAL SUBTYPES"NatureMedicine 6^117-22 ; Bonner, R. F. petal. (1997) "LASER CAPTURE MICRODISSECTION : MOLECULAR ANALYSIS OF TISSUE"Science 2781481, 1483 ; Schena, M. et al. (1995)"QUANTITATIVE MONITORING OF GENE EXPRESSION PATTERNS WITH A COMPLEMENTARY DNA MICROARRAY" Science 270, 467-70).

In a fifth embodiment, the conjugated molecules of such surfaces or matrices will comprise one, two, three or more non-ionizable polysaccharides or other polymer molecules each of which will preferably

be placed in a spatially and/or temporally discrete region of such surfaces or matrices. Significantly, this aspect of the present invention permits one to accomplish the spatial and/or temporal selective deposition of polymers that are not readily amenable to direct spatial and/or temporal deposition onto a surface or matrix. Thus, for example, the present invention permits one to accomplish the spatial and/or temporal selective deposition of polymers such as : aramids, celluloses, kevlars, nomex, nylons, poly (ether sulfone) s, poly (methyl methacrylate) s, poly (phenylene oxide) s, poly (phenylene sulfide) s, poly (vinyl acetate) s, poly (vinyl chloride) s, poly (vinyl) fluorides, poly (vinylidene chloride) s, poly (vinylidene fluoride) s, polyacrylonitriles, polybutadienes, polycarbonates, polychloroprene, polycyanoacrylates, polydicyclopentadienes, polyesters, polyethylenes, polyimides, polyisobutylenes, polyketones, polypropylenes, polystyrenes, polytetrafluoroethylenes, polyurethanes, polyvinylpyrrolidones, rayons, silicones, starches, etc.

It is contemplated that the deposited films of the present invention may be used in various settings and environments and as components for various devices, including, for example, biosensors, microarrays, micro electromechanical systems (MEMS), and complex, multi-site biomicrofluidics applications and associated multi-step biochemical reaction sequences.

The methods and materials of embodiments of the present invention provide numerous benefits and advantages when used MEMS and similar

devices. For example, the fabrication technique is relatively simple to practice compared to conventional silicon-based MEMS approaches. Also, the product cost is reduced, both in terms of material cost and processing costs. Additionally, the internal surfaces of the microfluidic MEMS environment of embodiments of the invention are polymeric, making the material surfaces considerably more biocompatible than if they included inorganic semiconductor and metallic surfaces.

The following examples serve to explain and elucidate the principles and practice of the present invention further. These examples are merely illustrative, and not exhaustive as to the scope of the present invention.

EXAMPLES Chitosan from crab shells (85% deacetylation) and phosphate- buffered saline (PES) tablets were purchased from Sigma-Aldrich Chemicals. 5- (and 6-) -Carboxyfluorescein succinimidyl ester (NHS- fluorescein, excitation maximum 495 nm and emission maximum 519 nm) was purchased from Molecular Probes and stored desiccated at-20 °C in a dark container until use. Silicon wafers with 1 llm thick thermal oxide film (four inch diameter) were obtained from MEMC Electronic Materials.

The gold and chromium used for sputtering onto the wafer were purchased from Kurt J. Lesker Co. The primer was hexamethyldisilazane (HMDS, Microelectronic Materials). The photoresist (Microposit Photoresist S1813) and developer (Microposit Developer 352) were purchased from Shipley

Co. The etchants (TFA for gold and TFD for chromium) were obtained from Transene Co.

Chitosan solutions were prepared by adding chitosan flakes to water and incrementally adding small amounts of HC1 to the solution to maintain the pH near 3. After being mixed overnight, the chitosan solutions were filtered to remove undissolved material, and the pH of solution was adjusted using NaOH (1 M). NHS-fluorescein solution was prepared by first dissolving 2.5 mg of NHS-fluorescein in 200 IL of dry dimethylformamide (DMF) and then adding 800 gL of ethanol.

Fluorescently labeled chitosan derivatives facilitate visualization, a labeled chitosan was prepared by reacting a chitosan film with NHS- fluorescein. The chitosan film was made by adding 50 mL of a 0.4% (w/v) chitosan solution (pH 3.0) to 140 mm diameter Petri dishes. The Petri dishes were oven-dried overnight at 45°C, and then the dried films were neutralized by immersion in 1 M NaOH for 3-4 h. After neutralization, the films were washed thoroughly with distilled water and equilibrated with a 0.1 M PES buffer. This buffer was prepared by dissolving PES tablets in double distilled H20 and adjusting the pH to 7.4. The labeling reaction was initiated by adding 20 uL of NHS-fluorescein solution (the DMF/ethanol solution described above) into a Petri dish containing a chitosan film in 35 mL of PES buffer. After allowing 30 min for reaction, the yellowish-green-colored chitosan films were then rinsed with distilled water and dissolved in a dilute HC1 solution (pH = 3). For purification, the

fluorescein-labeled chitosan was precipitated by adjusting the pH to about 9 using NaOH. The precipitant was then collected and rinsed with distilled water. After purification the fluorescently labeled chitosan was redissolved in a dilute HCl solution and the pH was adjusted to 5.6. To determine the polymer concentration, aliquots of known mass were oven- dried, and the residue was weighed.

The patterned surfaces were fabricated by depositing 150 A thick chromium and then 2000 A thick gold films on 4-inch diameter silicon wafers, which had previously been coated with 1 llm thick thermal oxide film. Patterning was achieved using photolithography in which a primer and then photoresist were spin-coated onto the gold surface. After soft- backing the coated wafer at 100°C for 1 min, a specially designed mask was placed over the surface and the wafer was exposed to UV light (total dosage ~ 190 mJ/cm2). After 30 seconds of development, the wafer was then hard-baked at 120°C for 10 min. The exposed areas were then etched away by gold and chromium etchants, and the photoresist was removed using acetone.

For deposition, the patterned wafers were immersed in solutions (pH = 5.6, 0.8% (w/w) polymer) containing either fluorescently labeled chitosan or unlabeled chitosan, and the patterned gold surfaces were polarized to serve as negative electrodes. The positive electrode in these experiments was an unpatterned gold-coated silicon wafer. The two electrodes were connected to a dc power supply (model 6614C, Agilent

Technologies) using alligator clips. Deposition was performed for 2 min by applying a voltage to achieve current densities of 1-2 A/m2. After deposition, the wafers were removed from the solutions, rinsed for 1 min with deionized water, disconnected from the power supply, and dried at room temperature. After drying, the wafers were immersed in 1 M NaOH for 30 min to neutralize the chitosan. After neutralization, the wafers were rinsed with distilled water and dried at room temperature overnight.

Some experiments were performed in which NHS-fluorescein was reacted with chitosan after the chitosan had been deposited onto the patterned gold surfaces of the wafers. Other experiments were performed in which NHS-fluorescein was reacted with chitosan before the chitosan was deposited onto the patterned gold surface of the wafers. For this study, chitosan was first deposited as described above and the dried wafer was placed in a 140 cm diameter Petri dish with 35 mL of PES buffer (pH = 7.4). The reaction was initiated by adding 20 gel of the DMF/ethanol solution containing NHS-fluorescein. After the reaction was allowed to proceed for 5 min, the wafer was rinsed with distilled water and dried at room temperature overnight.

The patterned wafers were examined using an optical microscope (model FS70, Mitutoyo Corp. ), and photographs were taken with this microscope using a digital camera (Nikon DXM 1200). The patterned surfaces were also examined using a fluorescence stereomicroscope (MZFLIII, Leica) using a fluorescence filter set (GFP Plus) with an

excitation filter at 480 nm (slit width of 40 nm) and an emission barrier filter at 510 nm. Photomicrographs were prepared from the fluorescence microscope using a digital camera (Spot 32, Diagnostic instruments).

Example 1 The first example examined the selective deposition of fluorescently labeled chitosan onto a patterned surface. For this example, a silicon wafer was patterned to have two independent sets of gold surfaces. The photomicrographs in the top row of FIG. 5 were obtained using an optical microscope and show the patterns of the two sets of gold surfaces, with the right upper and left upper photomicrographs showing the gold surface patterns before and after deposition, respectively. The bottom row of photomicrographs of FIG. 5 was taken with a fluorescence microscope before and after deposition. The photomicrograph on left of the bottom row of FIG. 5 shows that prior to deposition, no image could be obtained from this patterned surface when a fluorescence microscope was used.

For deposition, the wafer was immersed in a solution containing the labeled chitosan and a negative voltage was applied to the polarizable gold surfaces. After 2 min of deposition, the wafer was removed from the solution, rinsed with deionized water, and then disconnected from the power supply. After neutralization and rinsing, the wafer was dried and then examined. The photographs from the optical microscope (top row of FIG. 5) show only slight differences between the polarizable and non-

polarizable sets of gold surfaces. The photographs from the fluorescence microscope in the bottom row of FIG. 5 show dramatic differences with obvious images from the upper set of gold surfaces (which had been polarized to be negative), and no fluorescent images from the non polarized, lower set of gold surfaces. For convenience fluorescence micrographs are shown at two different magnifications (20x and 8x) in FIG. 5.

In summary, FIG. 5 shows that the patterned gold surface serves as a platform for the spatially selective deposition of the fluorescently labeled chitosan. Further, no deposition was observed on the unpolarized gold surfaces. Thus, deposition occurs only in response to an applied voltage (or current), indicating that deposition can be controlled temporally and spatially based on when and where the voltage is applied.

Example 2 In the second example, unlabeled chitosan was deposited onto a patterned surface and examined the spatial selectivity for subsequent coupling reactions. For this example, a wafer was patterned to have a variety of gold lines with different widths and different spaces between the lines. The following table lists the dimensions of the various lines and spaces and shows that the lines vary in width from 20 to 1000, um.

TABLE Line 20 50 100 500 1000 thickness (m) Space 500 500 500 1000 1000 (, um) 100 100 200 500 500 20 50 100 200 300 10 30 50 50 100 5 10 10 10 50

For deposition, this patterned wafer was immersed in a chitosan solution and the gold surface was polarized to be negative for 2 min. After deposition, the wafer was neutralized, rinsed, and dried as described above. Photomicrographs of the region of the wafer patterned with 1 mm wide gold lines spaced 1 mm apart were taken. The optical microscope showed both the lines and spaces in this region. No fluorescence was observed (through the fluorescence microscope) before and after chitosan deposition for the gold-patterned surface (GPS) and for the unlabeled chitosan.

The next step in this example was to contact the wafer with a solution containing NHS-fluorescein. This fluorescein derivative was activated to react with amine groups and should react with any chitosan that had been deposited onto the gold pattern. After the patterned wafer was allowed to react with the NHS-fluorescein solution, the wafer was rinsed with distilled water and dried. The NHS-fluorescein treatment had little effect on the patterned surface when the wafer was examined with an optical microscope. In contrast, the fluorescence microscope showed a

distinct fluorescent pattern. This photomicrograph indicated that chitosan had been deposited onto the patterned gold platform, and this "templated"chitosan layer underwent reaction with the amine-reactive fluorescein derivative.

As a control example, the patterned wafer was directly treated with NHS-fluorescein (without prior deposition of chitosan). After this treatment, the wafer was rinsed and dried. The photograph from the optical microscope revealed the distinct gold pattern while no pattern was observed using the fluorescence microscope. These observations demonstrated that there was no reaction between NHS-fluorescein and either the gold or silicon oxide surfaces of the wafer.

To further characterize the spatial selectivity of chitosan deposition and subsequent NHS-fluorescein coupling, several regions of the patterned surface were examined using the fluorescence microscope.

Photomicrographs were taken for surfaces with 500 u. m wide lines separated by spaces of different widths, i. e. , a respective photomicrograph for each of 1000 u. m, 500 u, m, 200 u. m, and 50 u. m spacing. For surfaces with 100 um, 50 u. m, and 20 u. m wide lines, respective photomicrographs were taken for each of 500 u, m and 100 pm width spacing. These photographs showed (upon magnification to 20x or 40x) that the lines were well resolved even when they were separated by only 50 um or 20 u. m.

The foregoing detailed description of the preferred embodiments of the invention has been provided for the purpose of explaining the principles of the invention and its practical application, thereby enabling others skilled in the art to understand the invention for various embodiments and with various modifications as are suited to the particular use contemplated. This description is not intended to be exhaustive or to limit the invention to the precise embodiments disclosed.

Modifications and equivalents will be apparent to practitioners skilled in this art and are encompassed within the appended claims.