IE20080934A1

IE20080934A1 - A method of immobilising biological molecules to a support and products thereof

Info

Publication number: IE20080934A1
Application number: IE20080934A
Authority: IE
Inventors: Kirk Leister; Sandeep Kumar Vashist
Original assignee: Univ Dublin City
Priority date: 2007-11-22
Filing date: 2008-11-24
Publication date: 2009-09-02
Also published as: WO2009066275A1

Abstract

A support comprising a substrate, wherein a surface of the substrate comprises functional amino or aldehyde groups, for the covalent binding of a biomolecule. The biomolecule may be a protein such as an affinity protein. The invention also provides a method for the covalent binding of a biomolecule t6 a surface of a support through its amino or carboxyl group. The method comprises the steps of: providing a support substrate; cleaning a surface of a support substrate; functionalising the support substrate by chemically creating hydroxyl groups on the cleaned surface of the support substrate; inducing amines on the surface of the support substrate; and covalently binding a biomolecule to the support substrate. The biomolecule may be protein such as an affinity protein. <Figure 1>

Description

leal molecul«^lrra^tippnrt^nd^rochicts,w*M,m“ i opens puBHcissPECTios iUKIERSWKJS 28 ABB SOLE 2S This invention relates to a method of immobilisi support and products thereof. In particular, the method relates to immobilising biological molecules to a support through covalent binding.

Many conventional laboratory techniques such as assays for the detection of biological molecules, purification of a specific biological molecule from a mixture of molecules, isolation of specific biological molecules and the like are based on exploiting the interaction between a biological molecule of interest and an affinity molecule immobilised on a support. For example, affinity chromatography exploits the specific binding of a protein such as an enzyme for its ligand such as an inhibitor of the enzyme. The ligand can be immobilised on an insoluble support and packed into a chromatography column, a mixture of biological molecules including the enzyme of interest is applied to the column and as only the enzyme has a specific binding affinity for the ligand, the enzyme becomes bound to the column while the remaining biological molecules pass through the column. The enzyme can then be eluted from the column in a highly pure fonn.

Biological molecules immobilised on a support have wide ranging applications in fields such as scientific research, diagnostics, drug discovery, clinical trials and the like. For example, antibodies immobilised on solid supports such as, microtitre plates or affinity chromatography supports are well known and companies such as BIACORE®, Sigma Aldrich® and Biomat® S.N.C produce chemical or protein functionalised generic chips for antibody immobilization that can be utilised in techniques such as high throughput immunoassays, antibody screening, immunodiagnostics and the like.

A large majority of biological molecules are immobilised on solid supports using a simple adsorption process. However, this can result in a non-homogenous layer -Ιοί absorbed biological molecules being formed on the surface of a support. A non-homogeneous layer of an absorbed biological molecule will introduce errors and non-reproducibility of results into experiments. In particular, for antibody based techniques, the simple adsorption of antibodies onto a support can result in the antibodies being immobilised in a random orientation which can result in a loss of specific binding activity of the immobilised antibodies.

Protein A, Protein G and Protein A/G are examples of Fc binding proteins. Fc binding proteins display high affinity for the constant fragment (Fc) region of antibodies and have been used to bind antibodies in an oriented and site directed manner. Fc binding proteins exhibit a strong affinity towards gold and bind to polystyrene by adsorption. Many companies including Biomat® S.N.C have exploited the simple adsorption of Fc binding proteins to polystyrene to make functionalised Fc binding protein platforms for immunoassays. But the simple adsorption based immobilisation has the disadvantage that the functional activity of tire Fc binding protein degenerates with time as it is not very stable. in an effort to overcome the problem associated with Fc binding proteins immobilised by simple adsorption, covalent immobilisation of Fc binding proteins on gold substrates has been used. For example, Briand et al., 2006 (Colloids Surf. B. Biointerfaces 53(2): 215-224) describe a method of forming monolayers of 11-mercaptoundecanoic acid on a gold substrate and covalently binding protein A to the monolayer. This strategy can only be employed on gold substrates as the first step involves fonning thiol and gold interactions. Schmid et al., 2006 (Sensors and Actuators B 113:297-303) describe a method for immobilising antibodies to a gold support via protein A which was covalently crosslinked to the support. Again this method involves forming thiol and gold interactions. The known methods of utilising an Fc binding protein as an intermediary for oriented, site directed immobilisation of antibodies onto a solid support is limited to gold supports because the methods employ thiol and gold interactions. It is not possible to crosslink Fc binding proteins to silicon or glass or polymeric supports through thiol based interactions. -3There is therefore a need for a method of immobilising biological molecules to a variety of different support materials in a reproducible manner.

Statements of Invention The invention provides a support comprising a substrate wherein a surface of the substrate comprises functional amino or aldehyde groups for covalent binding to a biomoleeule.

The substrate may be selected from one or more of: polystyrene, gold, poiylysine, silicon and silicon derivatives, porous silicon, polysilicon, quartz, Zeonex, Zeonor, glass, polymethylmethacrylate (PMMA), polycarbonate, cellulose acetate, agarose, sepharose, cellulose, polyacrylamide, trisacryl, sepacryl, ultragel AcA, azlactone, methacrylamide polymer (Eupergit) and 2hvdro-ethyl-methacrylate (HEMA).

The protein may be selected from one or more of: an Fc binding protein, an antibody, a lectin, a chaperone protein, an adhesion protein and a ligand.

The protein may be an affinity protein. The biomoleeule may be a protein The F,; binding protein may be selected from one or more of: Protein A/G, Protein A and Protein G. The support may further comprise an antibody bound to the Fc binding protein.

The antibody may be one or more selected from: anti-HBsAg, anti-troponin I, anti-troponin T, anti-myoglobin, anti-PSA (prostate specific antigen), anticreatine kinase (CK-MB), anti-insulin, anti-PAP (prostate acid phosphatase), anti-hCG (human chorionic gonadotropin), anti-leptin, anti-Legionella antigen, anti-Rotavirus, anti-verotoxin, anti-adenovirus, anti-Pf LDH, anti-digoxin, antib2-microglobulin, anti-T3, anti-T4. anti-TSH, anti-C reactive peptide (CRP) and anti-C peptide. *08093* -4The invention further provides for an antibody functionalised biochip comprising a support substrate and an antibody attached to an Fc binding protein wherein the amino or carboxyl group of the Fc binding protein is covalently attached to a surface of the substrate. The Fc binding protein may be covalently attached to a surface of the substrate through the carboxyl group.

The invention further provides a method of covalently binding a biomolecule to a support substrate comprising the steps of: (a) providing a support substrate; (b) cleaning a surface of a support substrate; (c) functionalising the support substrate by chemically creating hydroxyl groups on the cleaned surface of the support substrate; (d) inducing amines on the surface of the support substrate: (e) covalent binding of a biomolecule to the support substrate; and (f) blocking non-specific biomolecule binding sites on the support substrate.

The biomolecule may be covalently bound to the support substrate through a carboxyl group.

The method may further comprise the step of cross linking the induced amine groups with glutaraldehyde prior to step (e) to form aldehyde groups.

The method may further comprise the step of: (g) blocking unreacted aldehyde groups with glycine.

The biomolecule may be covalently bound to the support substrate through an amino group.

The substrate may be selected from one or more of: polystyrene, gold, polylysine, silicon and silicon derivatives, porous silicon, polysilicon, quartz, Zeonex, Zeonor, glass, polymethylmethacrylate (PMMA), polycarbonate, »080934 -5cellulose acetate, agarose, sepharose, cellulose, polyacrylamide, trisacryl, sepacryl, ultragel AcA, azlactone, methacrylamide polymer (Eupergit) and 2hydro-ethyl-methacrylate (HEMA).

The biomolecule may be a protein. The protein may be an affinity protein.

The protein may be selected from one or more of; an He binding protein, an antibody, a lectin, a chaperone protein, an adhesion protein and a ligand.

The Fc binding protein may be selected from one or more of: Protein A/G, Protein A and Protein G. fhe method may further comprise the step of binding an antibody to the Fe binding protein. fhe antibody may be one or more selected from: anti-HBsAg, anti-troponin 1. anti-troponin T, anti-myoglobin, anti-PSA (prostate specific antigen), anticreatine kinase (CK-MB), anti-insulin, anti-PAP (prostate acid phosphatase), anti-hCG (human chorionic gonadotropin), anti-leptin, anti-Legionella antigen, anti-Rotavirus, anti-verotoxin, anti-adenovirus, anti-Pf LDH, anti-digoxin, antib2-microglobulin, anti-T3, anti-T4, anti-TSH, anti-C reactive peptide (CRP) and anti-C peptide.

The step of creating hydroxyl groups on the support may comprise the steps of: - treating the substrate with a basic solution; - washing the substrate; and - treating the support with oxygen plasma.

The basic solution may be a solution of potassium hydroxide or sodium hydroxide.

The step of inducing amines on the support substrate may comprise the steps of: -61B0 8 0® 3 4 incubating the hydroxyl group functionalised support with an amine solution; and drying the support; The amine solution may be selected from: 3-aminopropyl triethoxy silane or 3arninopropyl trimethoxy silane.

The support may be dried in a vacuumed desiccator placed in an oven.

The step of covalently binding a biomolecule to the support substrate may comprise the steps of: forming a cross-linking solution; mixing the biomolecule with the cross-linking solution; incubating the support with the biomolecule - cross-linking solution mixture; and quenching the cross-linking solution.

The cross-linking solution may comprise 1-ethyl 3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and Sulfo-N-Hydroxysuccinmide.

The quencher may be 2-mercaptoeihanol.

The step of binding an antibody to the Fc binding protein may comprise the steps of: blocking non-specific protein binding sites on the base substrate; and incubating the support with an antibody solution.

The non-specific protein binding sites may be blocked with a bovine serum albumin solution, such as a 1% solution of bovine serum albumin.

The invention further provides isolation and purification comprising an affinity biomolecule covalently bound to a surface of the support wherein the affinity biomolecule is bound to the support through a terminal amino or carboxyl group. -Ί ΐΕΟβ®9^* The support may be selected from one or more of: agarose, sepharose, cellulose, polyacrylamide, trisacryl, sepacryl, ultragel AcA, azlactone, methacrylamide polymer (Eupergit) and 2-hydro-ethyl-methacrylate (HEMA). r□ fhe support may be in the form of a chromatography column.

The affinity biomolecule may be a protein. The protein may be selected from one or more of: an Fc binding protein, an antibody, a lectin, a chaperone protein, an adhesion protein, and a ligand.

Some advantages associated with the methods described herein include: o Covalent binding (cross-linking) of biological molecules to a surface of a substrate provides a very strong linkage of the biological molecule to the substrate; o Biological molecules can be bound to a wide range of supports including supports with an inert or a reactive surface; o Biological molecules can be bound to optically transparent supports; o Functional amino or aldehyde groups are induced on the surface of a support itself; o Antibodies are bound strongly to a surface of a substrate without any modification to the functionality of the antibody; and c Oriented and site directed attachment of antibodies to a support when using an Fc binding protein as the variable Fab region is free to bind antigens.

Brief Description of the Drawings The invention will be more clearly understood from the following description of an embodiment thereof, given by way of example only, with reference to the accompanying drawings, in which: 30 ΙΕΟ 8 0»3 4 -8Fig. 1 is a schematic of a method for immobilising antibodies to a support substrate via an Fc binding protein. This immobilisation strategy is based on the covalent crosslinking of a carboxyl group of an Fc binding protein to the solid substrate.

Fig. 2 is a schematic of an alternative method for immobilising antibodies to a support substrate via an Fc binding protein. This strategy is based on the covalent crosslinking of an amino group of an Fc binding protein to the solid substrate; Fig. 3 is a schematic of a fluorescence detection system in which SF is a spatial filter assembly, Ml-M3 are planar mirrors, CL is a collimating lens, DC is a diehroic beam splitter, and FL is a focusing lens system; Fig. 4A is a series of fluorescent images showing the decrease in fluorescent intensity when decreasing concentrations of goat anti-mouse IgG-Cy5 labelled antibody were detected by mouse IgG bound to Zeonex® substrate using the LDC-sulfo NHS and protein A based immobilisation strategy. The concentration of antibody in pg/ml is given at the top of each image. The exposure time was 10 seconds for all concentrations. Fig. 4B shows the qualitative assay curve between the captured fluorescent signal intensity and the respective concentration of goat anti-mouse IgG-Cy5 labelled antibody; Fig. 5 is the SPR response curve demonstrating the: binding of mouse IgG to protein A which has been covalently bound through carboxyl groups to a Zeonex® coated surface of a gold SPR chip; blocking excess mouse IgG binding sites on the protein A coated substrate with 1% BSA; binding of goat anti-mouse IgG to mouse IgG; and regeneration of the protein A coated surface by 30 mM HCL Fig. 6 A to F show the surface view as assessed by Atomic Force Microscopy (AFM) imaging in the non-contact mode of steps in the Fc binding protein based immobilization strategy on a Zeonex® substrate. Fig 6A is the AFM image (of view size, 5x5 pm) of the surface of a blank Zeonex® substrate after surface «080934 -9cleaning step (Step (b)); Fig 6B is the AFM image (5x5 pm) of the surface after 3-APTES treatment (Step (d)); Fig 6C is the AFM image (5x5 pm) of the surface of Protein A covalently bound to 3-APTES by EDC and sulfo NHS through carboxy groups (Step (e)); Fig 6D is the AFM image (5x5 pm) of the surface of Mouse IgG bound to Protein A; Fig 6E is the AFM image (2x2 pm) of the surface of Mouse IgG bound to Protein A; and Fig 6F is the 3-dimensional view of the mouse IgG bound surface (5x5 pm) analyzed by AFM; Fig. 7 A and B are graphs showing data for mouse IgG - anti mouse IgG assays when mouse IgG was immobilised via Fc binding proteins on a 96-well polystyrene ELISA plate. Fig. 7 A. shows the results of an assay when the Fc binding proteins have been covalent bound via their carboxyl groups and Fig. 7B shows the results of an assay when the Fc binding proteins have been covalently bound via their amino groups; and Fig. 8 is a graph showing the results of an assay when an antibody (mouse IgG) has been directly bound covalently to a surface of a substrate via its carboxyl group (EDC-SNHS) or amino group (Glutaraldehyde).

Detailed Description The invention provides a method which overcomes the need for gold coating of substrates in order to covalently attach biological molecules to a surface of a support. In particular, the invention provides a method for covalent binding of biological molecules to a surface of a support through an NH2 or COOH group of the biological molecules. In particular, the methods described herein can be used for immobilising proteins to a surface of a support. As almost all proteins have a carboxyl terminal having a COOH group and an amino terminal having an NH2 group, any of these proteins can be covalently bound to a functionalised surface of a support. In addition, many other biomolecules also have a carboxyl terminal having a COOH group and/or an amino terminal having an NH2 group, and thus they can also be covalently bound to a functionalised surface of a support. The method of the invention can be used for immobilising a variety of -10»080834 biological molecules to a support. In the case of antibodies, the antibodies can be directly or indirectly (via an Fc binding protein) bound to a support.

The method provides an immobilisation strategy for covalently binding biological molecules to a surface of commercially relevant substrates (supports) via amine or aldehyde functionalisation of the supports. A wide range of supports, including supports having an inert or reactive surface, can be functionalised using the methods described herein. Thus, biological molecules can be covalently bound to a wide range of commercial substrates via their amino or carboxyl groups. Advantageously, the immobilistation methods described herein allow for biological molecules to be covalently bound onto a surface of an optically transparent substrate.

In one aspect, the method exploits the widely known benefits of oriented and site directed immobilization of antibodies using Fc binding proteins along with the additional benefit of long-term stability provided by covalent immobilization of Fc binding proteins to a substrate.

Fc binding proteins, for example protein A/G, protein A or protein G, orientate the immobilized antibodies. The Fc binding proteins bind to the constant Fc region of antibodies thereby keeping the antigen binding sites on the variable Fab region of the antibodies free to bind to antigens. Protein A/G is a recombinant protein made commercially by gene fusion of the Fc -binding domains of protein A and protein G. Protein A/G binds with strong affinity to nearly all the antibodies being employed widely for immunodiagnostics and biosensors (such as all human IgG subclasses, IgA, IgE, IgM and to a lesser extent IgD) at pH 5- 8.

In a different aspect, affinity biomolecules such as affinity proteins for example lectins, chaperones, adhesion proteins, ligands for enzymes, antibodies, Fc binding proteins and the like can be immobilised on a support. The biomolecule immobilised support can be used for isolation or purification methods. For -11example, downstream purification of a biological molecule using various suface affinities with improved orientation or immobilisation strategies to achieve increased capacity, selectivity and specificity.

For purification and isolation applications, biomolecules can be immobilised to a variety of chromatographic supports for example: agarose, sepharose, cellulose, polyacrylamide, trisacryl, sepacryl, ultragel AcA, azlactone, methacrylamide polymer (Eupergit) and 2-hydro-ethyl-methacrylate (HEMA) and the like. For affinity chromatography and immuno-affinity chromatography applications, the biomolecule immobilised support can be packed in a chromatography column.

Fe binding proteins i.e. Protein A, protein G and protein A/G based columns have been used industrially in the biological, bioprocess, biopharmaceutical and diagnostics industries as well as in research laboratories for the purification of antibodies and proteins due to their ability to specifically select the antibody or protein of interest. There are several commercially available Fc binding protein based columns such as Nab Protein A protein columns, Nab Protein G protein columns and Nab Protein A/G protein columns (by Thermo Scientific previously Pierce); Nunc© ProPur® Antibody Purification Spin Columns based on Protein A and Protein G (by Fisher Scientific); EZview Red Protein A gel and EZview Red Protein G gel (by Sigma Aldrich); and similar products by other companies.

We have demonstrated that Fc binding proteins can be immobilised on the surface of a variety of substrate supports (including both reactive and inert supports) by inducing amine or aldehyde groups on the surface of the support. The support could be resin supports such as conventional chromatographic resins for example agarose/sepharose, cellulose, polyacrylamide, trisacryl, sepacryl, ultragel AcA, azlactone, eupergit (methacrylamide polymer), HEMA (2-hydroethyl methacrylate) being used in the form of a chromatography column. Chromatography columns made from Fc immobilised supports of this type can be used for biosensor, bioprocesses and bioanalytical applications. -12»08093* Further, the amino or carboxyl terminal of any particular IgG type antibody can be bound to the substrate supports directly or indirectly using intermediate proteins i.e. Fc binding proteins.

One of the main advantages of the biological molecule (biomolecule) immobilisation strategy described herein is that the biomolecule is covalently bound to the surface of a support. Immobilisation of biomolecules by covalent binding will result in a homogenous layer of bound biomoiecules as each functional group (EDC-SNHS or glutaraldehyde) induced on the surface of the support will form a covalent bond with one terminal NH2 or COOH group of each biomolecule therefore the induced functional groups will bind to the biomolecules in a 1:1 ratio, thereby eliminating the aggregation of biomolecules on the surface. As such, a reproducible, homogeneous layer of biomolecules are immobilised on the surface of the support. Furthermore, covalent binding of biomolecules to the support securely “tethers” the biomolecules to the support which is important as when the biomolecule immobilised supports are used, most of the experimental protocols involve numerous washing steps. Covalently bound biomolecules will remain attached to the support throughout numerous rounds of washing steps due to the chemical (covalent) bond whereas biomolecules that have been immobilised to a support by simple passive adsorption (physical interaction) leach from the support during washing steps. Thus, because of the strong covalent attachment of the biomolecule to the support, supports made in accordance with the methods described herein are stable over numerous rounds of washing. As such, these supports are particularly well suited for use in industrial scale applications, in which large volumes of solutions will be applied to the supports, as the biomolecule will remain chemically tethered to the support for the duration of the protocol. Given the strong covalent bond formed between the surface of the support and the biomolecule, the supports can be reused as the biomolecule does not leach from the surface of the support.

In brief, the method involves cleaning a bioanalytical surface of a support, chemically creating hydroxyl groups on the cleaned surface and thereafter. ΐΕΟβ0®3 * -13inducing amine groups on the surface for example by employing 3-aminopropyl triethoxy silane (3-APTES) or 3-aminopropyl trimethoxy silane.

The invention will be more clearly understood from the examples.

Examples Example 1 - Immobilising biomolecules to a surface of a substrate The invention relates to a method of covalently immobilising biomolecules such as proteins to a surface of support via their terminal amino or carboxy groups. The method comprises the following steps: (a) providing a support substrate; (b) cleaning a surface of the support substrate; (c) functionalising the surface of the support substrate by chemically creating hydroxyl groups on the surface of the support substrate; (d) inducing amine groups on the surface of the support substrate; and (e) covalently binding a biomoleeule to the surface of the support substrate.

Step (a) - providing a support substrate The support substrate can be selected from any known support suitable for use with biomolecules. The support may have a reactive or an inert surface. The support may be optically opaque. As an example, the support may be selected from: polystyrene, gold, poiylysine, silicon and silicon derivatives, porous silicon, polysilicon, quartz, Zeonex, Zeonor, glass, polymethylmethacrylate (PMMA), polycarbonate, cellulose acetate, chromatography resins such as agarose, sepharose, cellulose, polyacrylamide, trisacryl, sepacryl, ultragel AcA, -14JE08093* azlactone, methacrylamide polymer (Eupergit) and 2-hydro-ethyl-methacrylate (HEMA).

The choice of support may depend on the end use application tor the support.

Step (b) - Surface cleaning The surface cleaning procedure varies based on the substrate to be used.

Inert, and/or polymeric supports Treat support with absolute ethanol for at least 10 min and rinse five times with deionized water.

Polylysine coated supports l'he polylysine coated supports already have amine groups present on their surface. So in this case, there is no need for functionalising the surface of the support (step (c)) or for APTES treatment to induce amine groups (step (d)). For these supports, initially the surface is cleaned by treatment with absolute ethanol for about ten minutes. Thereafter, amine groups on polylvsine are activated by treatment with 0.1 M 2-[N-morpholino] ethane sulfonic acid (MES) buffer, pH 4.7 for at least 5 min. Biomolecules can then be covalently bound to the surface (step (e)).

Glass coated supports The glass substrate was treated for 1 h in boiling piranha solution (70% sulphuric acid (H2SO4): 30% hydrogen peroxide (H2O2) (at a v/v ratio of 3:1) respectively). After 1 h, the glass substrates were removed from the cleaning solution, rinsed with high purity water (Ultrapure Milli-Q Reagent Water System Millipore), then washed in ethanol three times followed by washing in high purity water five times. Finally, the cleaned glass substrates are dried in a stream of nitrogen.

Metal / metal coated supports Dip the support in a solution of 70% sulphuric acid (H2SO4): 30% hydrogen peroxide (H2O2) (at a v/v ratio of 3:1 respectively) for 1 minute. Wash with -15«08093« deionized water three times. Thereafter, treat it with absolute ethanol for at least 5 min and rinse with deionized water five times.

Silicon supports The procedure described by Ligler [1 igler, F.S. & Cass, T. (1999) Immobilized Biomolecules in Analysis: A Practical Approach. Oxford University Press, USA] was followed to remove contaminants and/or impurities from the surface of the support. The bare silicon substrate was immersed into a mixture of HC1: Methanol (1:1, v/v) at room temperature for 30 minutes and then rinsed five times with deionized water. It was immersed into concentrated sulphuric acid at room temperature for 30 minutes and then rinsed well with deionized water five times to remove all sulphuric acid residues from the substrate. The substrate was boiled for 30 minutes in boiling water and then dried in air.

Step (c) - Generation of hydroxyl groups The surface of the substrate is functionalised by treating the cleaned substrate with 1% potassium hydroxide (KOH) for 10 min and rinsed five times with deionized water. Thereafter, the support is placed in an Oxygen plasma (Harrick Scientific Corporation) for about 3 min to generate hydroxyl groups on the surface of the support.

Hydroxyl groups are generated on the surface based on the oxidation of surfaces only. Most of the substrates have got hydrocarbon chains, silicon or metals etc. on their surface, which after oxidation leads to the formation of hydroxyl groups attached to them. Thus, for the support to bear hydroxyl groups, the surface of the support must be capable of oxidation. Hydroxyl groups have even been generated on inert substrates such as Zeonor® and Zeonex® polymers using this method.

Step (d) - Induction of amine groups Exemplary example using 3-aminopropyl triethoxysilane (APTES) Put 2% 3-APTES (made in deionized water) on to hydroxyl group functionalised support placed inside a petridish in the fume hood. Thereafter, place the covered -16»0 8 0® 3 * petri dish housing the support inside a desiccator and apply a vacuum by suction. Place the desiccator in the oven at 80° C for at least 6 hours. Remove the support from the oven and place at room temp for 20 min. Finally rinse the support with deionized water five times.

Step (e) (i) Covalent binding of carboxyl groups of biomolecules to the induced amine groups on a surface of the functionalised support The carboxyl groups of biomolecules are covalently bound to the amine groups on the surface of the support using the procedure stated below. The biomolecule may be a protein such as an Fc binding protein, or an antibody a lectin, a chaperone protein, an adhesion protein, or a ligand for a protein such as an inhibitor of an enzyme.

Gross linking solution In an eppendorf tube, add 0.4 mg 1-ethyl 3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1.1 mg Sulfo-N-Hydroxysuccinmide (Sulfo NHS) and dissolve in 100 μΐ of 0.1 M MES, pH 4.7.

Biomolecule solution In a second eppendorf tube, add 10 μΐ of the above cross linking solution and add 990 μΐ of the biomolecule at a concentration of 10 μg/ml in phosphate buffered saline (PBS), pH 7.4. Incubate at room temperature for 15 min. Add 1.4 μΐ of 20 mM 2-mercaptoethanol to quench EDC.

Add the biomolecule solution to the amine coated support and incubate at room temperature for about 2 hours. Wash with PBS five times and incubate with 1% bovine serum albumin (BSA made up in PBS, pH 7.4) for 1 hour and 30 min at room temperature subsequently followed by washing with PBS five times. BSA blocks all non-specific biomolecule binding sites on the substrate.

Step (e) (ii) Covalent binding of amino groups of biomolecules to a surface of a functionalised support *08 0? 3 4 .17.

To covalently link a biomolecule to a functionalised surface of a support via amino groups, an additional step has to be performed after the amine groups have been induced on the surface of the support. Following step (d) above, the induced amine groups are cross linked to glutaraldehyde to generate functional aldehyde groups that can be covalently attached to amino groups of biomolecules. The biomolecule may be an Fc binding protein, or an antibody, lectin, a chaperone protein an adhesion protein, or a ligand for a protein such as an inhibitor of an enzyme.

Glutaraldehyde is a homobifuntional crosslinker and can therefore bind to two amino groups:- one amino group of the amine fuctionalised substrate surface and one amino group of a biomolecule thereby covalently attaching a biomolecule to the substrate. 2.5% glutaraldehyde solution made up in carbonate buffer is added to the amine functionalised support and incubated for about 2 hours at room temperature. The support is washed five times with carbonate buffer. Biomolecules can be covalently bound to the surface by adding a biomolecule solution at the desired concentration such as 10 pg/ml to the surface of the support and incubating at room temperature for about 2 hours. The surface of the support is then washed five times with PBS. Excess aldehyde groups remaining on the surface of the support are blocked by treating the surface with a solution of 0.1M glycine at room temperature for about 1 hour. The surface is then washed five times with PBS.

To block non-specific binding sites on the surface of the support, the support, is incubated with 1% BSA (in PBS, pH 7.4) for about 1 hour 30 mins at room temperature and then washed five times with PBS.

Immobilisation of antibodies to F£ binding protein functionalised support When the biomolecule in step (e) is an Fc binding protein, an antibody can be immobilised to the covalently bound Fc binding protein in a specific and site orientated manner using the following protocol. Add an antibody diluted in PBS, -18«08 0934 for example mouse IgG at a concentration of 12 pg/ml, to the functionalised support and incubate overnight at 4° C. Wash the antibody functionalised substrate five times with PBS.

These immobilisation schemes are shown in Figs. 1 and 2..

Example 2 - Characterisation of immobilised antibodies The characterisation of immobilized antibodies on a Zeonex® support was performed using a fluorescent detection system, atomic force microscopy (AFM), surface plasmon resonance (SPR) and enzyme linked immunosorbent assay (ELISA).

Fluorescence detection system A fluorescence detection system (depicted schematically in Fig. 3) was designed and built in-house using a range of commercially available components to enable the measurement of the signal emitted by the fiuorophores bound to immobilised antibodies. This system comprised of a He-Ne laser excitation source. Sharp vision charge coupled device (CCD) camera (IDT, USA) and a range of “off the shelf’ optical components supplied by Edmund Optics, USA. The system was used to examine the binding of anti-mouse IgG-Cy5 labelled antibody to the mouse IgG bound to a Zeonex® substrate via Protein A. The assay was carried out using a proprietary cone shaped substrate (fabricated in Zeonex®) having enhanced fluorescence signal collection efficiency.

Fig. 4 illustrates the successful immobilisation of anti-mouse IgG-Cy5 labelled antibody to the mouse IgG bound Zeonex® substrate. The results, as illustrated in Fig. 4, show the fluorescent immunoassay where a range of serially decreasing concentrations of goat anti-mouse IgG-Cy5 labelled were detected by the mouse IgG bound Zeonex® substrates correlating with the decrease in fluorescent intensity at each step. This experiment clearly demonstrated the successful -19»080934 employment of the antibody immobilisation strategy for fluorescence based immunoassays.

Atomic force microscopy (AFM) AFM was performed to image the surface of a support during different stages of the antibody immobilization process. Triangular silicon nitride cantilevers (type E) with nominal spring constant 0.1 N/m (Veeco, USA) having a nominal thickness of 0.6 pm were used for imaging by PicoPlus AFM system (Agilent Technologies, USA). 8 x 8 pm AFM scanner was employed. The cantilevers were washed in ethanol and deionized water and dried at 70° C for 30 minutes. They were finally cleaned by UV treatment for 20 minutes. The imaging of samples was done in non-contact mode.

AFM studies show that the immobilization strategy worked for Zeonor®, Zeonex®, Zeonex® spincoated on polylysine, Zeonex® spincoated on gold, glass and polystyrene. As an example, Fig. 6 shows the AFM based surface analysis of the uniformity of the surface at different stages in the antibody immobilization procedure on the Zeonex® substrate. Fig. 6A shows the surface view as analyzed by AFM in non-contact mode of blank Zeonex® substrate after surface cleaning step. Fig 6B shows the surface analysis after 3-APTES treatment step. Fig 6C shows the Protein A coated surface, where the carboxyl group of protein A is crosslinked to 3-APTES by EDC and sulfo NHS. Fig 6D and E show the Mouse IgG bound surface at 5 x 5 pm and 2 x 2 pm respectively and Fig 6F shows the 3dimensional view of the mouse IgG bound surface (5x5 pm). The surface analysis shows that the Zeonex® substrate was uniformly coated with 3-APTES, Protein A and Mouse IgG at various stages of the immobilization procedure without the formation of any clumps or aggregates.

Surface plasmon resonance Zeonex® (in solution form dissolved in cyclohexane) was spin coated on to a gold SPR chip and functionalized with 3-APTES. Protein A was then bound to the gold-coated surface by using EDC and sulfo-NHS crosslinking procedure. «080934 -20A Biacore 3000 instrument (BlAcore AB, Uppsala, Sweden) was used. The running buffer in all the experiments was 10 mM HEPES buffered saline (HBS). pH 7.4, which was filtered through 0.22 pm pore size filter paper and then degassed before SPR analysis. 30 mM HC1 was used for the regeneration of protein A bound gold surface. The flow rate of all the solutions employed in SPR analysis was kept constant at 5 μΐ/ιηίη and the temperature was fixed at 25 C in ail experiments.

SIA kit Au (BR-1004-05) containing the gold-coated SPR chips was purchased from BlAcore AB, UK. Prior to immobilization of biomolecules on the goldcoated SPR chips, the gold surface was thoroughly cleaned by a quick 1 minute dip in piranha solution (H2SO4 70% : 1 i.Tl· 30% = 3:1 v/v). This was followed by treatment with ethanol for 5 min and subsequent washing with deionized water three times.

Zeonex® (in solution form dissolved in cyclohexane) was spin-coated on the SPR chips by the procedure mentioned below.

Zeonex® pellets for injection moulding were initially dissolved in cyclohexane (2 g/1). The mixture was left overnight to dissolve and thereafter sonicated for 40 minutes before use to ensure complete dissolution.

Each chip was placed on the vacuum chuck of the coater. 0.25 ml of solution was dispensed on the chip, which was enough to cover the top surface area. The following spin parameters were then employed: Initial Spin: 1500 rpm for 30-60 seconds Ramp to final Spin: 5 seconds Final Spin: 3000 rpm for 5-10 seconds -21 11080934 These spin process led to uniform coatings of Zeonex® on the SPR chips.

Zeonex® coated gold SPR chips were then attached to the SPR chip supports and protein A was covalently attached io the Zeonex® surface of the coated SPR chip. The protein A coated SPR chip was then inserted in the Biaeore 3000 set up and the immobilization of mouse IgG was carried out by continuous flow of mouse IgG (0.12 mg/ml) over the protein A coated SPR chip for 30 min. The non-specific protein binding sites on the SPR chip were blocked by continuous flow of 1% blocker BSA solution in PBS, pH 7.4 for 1 min. Goat anti-mouse IgG was then detected and bound by continuous flow of goat anti-mouse IgG (120 pg/ml) for 30 min. Finally, the protein A coated SPR surface was regenerated by continuous flow of 30 mM HC1 for 7 min.

Fig. 5 shows that the various steps in the antibody immobilization procedure worked well on the protein A coated gold SPR chip, as analyzed in real time by SPR. Mouse IgG was immobilised on the protein A coated gold SPR chip by continuous flow of mouse IgG (0.12 mg,'ml) for 30 min as a flow rate of 5 pl/min. Thereafter, the unoccupied mouse IgG binding sites on the base substrate were blocked by continuous flow of 1% BSA for 1 min. Finally, goat anti-mouse IgG was detected by continuous flow of goat anti-mouse IgG (120 pg/'ml) for 30 min and the protein A coated SPR surface was effectively regenerated by continuous flow of 30 mM HC1 for 7 min.

Enzyme Linked Immunosorbent Assay (ELISA) ELISA was performed on 6 mm x 6 mm cut solid substrate chips of Zeonex®, Zeonor® and polystyrene. Mouse IgG was bound to the substrates using the immobilisation method described above. Thereafter, the immunosensing solid substrates were provided with goat anti-mouse IgG-horse radish peroxidase (HRP) labelled (1.21 pg/ml) and incubated at RT for 1 hour. The goat anti-mouse IgG-HRP bound solid substrates were then washed five times with PBS, pH 7.4 and placed in fabricated Teflon wells (diameter 10 mm) with the biomolecular functionalized side facing upwards. Thereafter, 100 μΐ of 3, 3’, 5, 5’-22«08 0934 tetramethylbenzidine (TMB) substrate was provided to the goat anti-mouse IgGHRP bound solid substrates in various Teflon wells. After 30 min, when the enzyme substrate reaction is complete (as signalled by the development of blue colour), the reaction was stopped by adding 100 μί of IN H2SO4 to each Teflon well. The coloured solution was then transferred to the standard ELISA plate and the optical density of the solution was taken at 450 nm. in a further experiment. 96-weii ELISA plates meant for fluorescent ELISA were used as supports as they had a thin layer of plastic film forming the base of the wells. These ELISA plates were initially blocked with BSA by incubation at room temperature for one and a half hours. Thereafter, the thin bottoms of the three columns of the ELISA plate (having four wells each) with a spacing of one column between them were removed using a steel punch to stamp through the well an;.! remove the base. Each column of the bottomless wells was then attached to the fiat surfaces of Zeonex®, Zeonor®, PMMA, polycarbonate, cellulose acetate, polystyrene and glass. Thereafter, immobilization procedure of Example 1 was used to compare the immobilization of antibodies on different substrates. For this assav. there was no need to use Teflon cells as the surface area of the bottom of the ELISA wells being employed was exactly the same as for the standard wells.

The ELISA performed on various solid substrates demonstrated the successful immobilization of antibodies on a surface of the substrate and therefore the applicability of the immobilisation method for immunodiagnostic and biosensor applications.

In the first set of experiments, there was variation in the amount of antibody immobilization on the Zeonex®, Zeonor® and polystyrene substrates. The variation was probably due to either the nature of the substrate material or the fact that when solutions are applied to the cut surfaces without any containment, the solution may spill over the surface unevenly. Thus, there was a possibility that the sides and bottom surfaces of the substrate were also functionalised due to -23IEO 8 Οδ 3 4 spilling of solutions from the top surface. The average O.D. at 450 nm directly corresponding to the amount of antibody immobilized on the Zeonex®, Zeonor® and polystyrene substrates (6 mm x 6 mm) were 2.6, 2.2 and 1.9 respectively. In the second set of experiments, the antibody immobilization on the Zeonex®, Zeonor®, PMMA, polycarbonate, cellulose acetate, polystyrene and glass surface was analyzed. In this case, only the upper surface of the chips were functionalized due to the containment provided by ELISA wells. The average O.D. at 450 nm directly corresponding to the amount of antibody immobilized on the Zeonex®, Zeonor®, PMMA. polycarbonate, cellulose, acetate, polystyrene and glass substrates (6 mm x 6 mm) were 1.55, 1.42, 1.30, 1.26, 1.23, 1.39 and 1.42 respectively.

Example 3 - Immunoassay using the devised immobilisation strategies on 96well polystyrene ELISA plates The standard mouse IgG and anti mouse IgG assay was performed using the devised protein immobilisation strategies on the flat bottom 96-well polystyrene ELISA plates from Nunc as described below. All experiments were done in triplicate.

A) Immobilisation strategy based on binding of carboxyl group of binding proteins Step (h) - Surface cleaning The polystyrene wells in the 96-weli ELISA plate were cleaned by treating with absolute ethanol for 10 min and then washing five times with 300 μΐ of deionized water (D1W).

Step (c) - Generation o f hydroxyl groups The surface of the 96-well ELISA plate was functionalized by treating with 100 μΐ of 1% potassium hydroxide (KOH) for 10 min and then washing five times -24ΙΕΟ 8 08 3 4 with 300 μΐ of DIW. Thereafter, the ELISA plate was placed in the Oxygen plasma for 3 minutes to generate hydroxyl groups on the surface.

Step (d) - Induction of amine groups by treatment with 3-aminopropy Itriethoxy silane (A PTES) The hydroxyl group functionalized 96-well ELISA plate was provided with 100 μΐ of 2% 3-APTES (in DIW) per well at room temperature inside the fume hood. Thereafter, the ELISA plate was placed inside the glass desiccator and a vacuum was created inside the desiccator prior to placing it in an oven at about 80° C for about 6 hours. After 6 hours, the desiccator was removed from the oven, placed at room temperature for 20 min to cool down and then washed five times with 300 plofDIW.

Step (e) - Crosslinking of induced amine groups on 96-well ELISA plate to the carboxyl group on Fp binding proteins In an eppendorf tube, 0.4 mg EDC and 1.1 mg sulfo NHS were added and dissolved in 100 μΐ of 0.1 M MES, pH 4.7. In a second eppendorf tube, 990 μΐ of an Fe binding protein such as Protein A, Protein A/G, or Protein G 110 pg/ml in PBS) was prepared and 10 μΐ of the crosslinking solution from the first eppendorf tube was added to the second eppendorf tube. The mixture was left for 15 min at room temperature. 1.4 μ) of 2-mercaptoethanol (20 mM) was then added to quench the excess unbound EDC. Finally, 100 μΐ of this crosslinking solution i.e. EDC-sulfo NHS-FC binding protein was added to each well of the amine functionalized ELISA plate wells and incubated at room temperature for 2 hours. Wells were then washed five times with 300 μΐ of PBS. immobilising antibodies to the E, binding protein functionalized 96-well ELISA plate Mouse IgG was immobilized on to Fc binding protein functionalized 96-well ELISA plate by adding 100 μΐ of mouse IgG (12.1 pg/ml in PBS) to each modified microtiter plate well and leaving it overnight at 4° C. Thereafter, the modified microtiter plate wells were washed five times with 300 μΐ of PBS. 1Ε08 0?34 -25Blocking of the base substrate in ELISA plate wells The ELISA plate wells were incubated with 1% BSA (in PBS. pH 7.4) for 1 hour and 30 min at room temperature to block non-specific protein binding sites on the base substrate and then washed five times with 300 μΐ of PBS.

Binding of goat anti-mouse IgG HRP labelled 100 μ! of goat anti-mouse IgG HRP labelled (in varying ng/'ml concentrations in PBS) was provided to each of the ELISA plate wells and thereafter, left at room temperature for 1 hour. The ELISA plate wells were then washed five times with 300 μΐ of PBS.

TMB substrate assay TMB substrate solution was made by mixing equal amounts of TMB solution (0.4 g/'L) and Peroxide solution (containing 0.02 % hydrogen peroxide in citric acid buffer) as per the instructions of the TMB substrate kit from Pierce. 100 μ! of this ΪΜΒ substrate solution was added to each ELISA plate weli used in the immunoassay. . The peroxidase enzyme, in the presence of H2O2. catalyses the oxidation of colorless TMB substrate to a blue colored product. After a fixed reaction time (30 min), the reaction was stopped with 100 μΐ of IN ffiSCfi and the absorbance of the solution was measured at 450 nm with reference at 650 nm.

B) Immobilisation strategy based on binding of amino group of Fg binding proteins 2.5 Step (b) - Surface cleaning The polystyrene wells in the 96-well ELISA plate were cleaned by treating with absolute ethanol for 10 min and then washing five times with 300 μΐ of deionized water (DIW).

Step (c) - Generation of hydroxyl groups The surface of the 96-well ELISA plate was functionalized by treating with 100 μΐ of 1% potassium hydroxide (KOH) for 10 min and then washing five times «Ο 8 Q® 3 4 -26with 300 μΐ of DIW. Thereafter, the ELISA plate was placed in the Oxygen plasma for 3 minutes to generate hydroxyl groups on the surface.

Step___tdf.......~__Induction of amine groups___by treatment____with 3aminopropyltriethoxysilane (A PTES) The hydroxy’ group functionalized 96-well ELISA plate was provided with 100 ul of 2% 3-APTES (in DIW) per well al room temperature inside the fume hood. Thereafter, the ELISA plate was placed inside the glass desiccator and a vacuum was created inside the desiccator prior to placing it in an oven at about 80° C for 2.5 about 6 hours. After 6 hours, the desiccator was removed from the oven, placed at room temperature for 20 min to cool down and then washed five times with 300 ui of DIW.

Crosslinking of induced amine groups on 96-well ELISA plate to Glutaraldehyde 2.5% glutaraldehyde solution (made in carbonate buffer) was added to the amine functionalized ELISA plate wells and incubated at room temperature tor 2 hours. Wells were then washed five times with 300 μ! of carbonate buffer. This leads to aldehyde activated surface. Glutaraldehyde is a hornob'functional crossrinker and bind to the amino groups on both ends.

Step (e) - Binding of E? binding proteins Fc binding protein solution i.e. Protein A, protein G or protein A/G (10 ug/ml) was added to the aldehyde activated ELISA plate wells and incubated at room temperature for 2 hours. Wells were then washed five times with 300 μΐ of PBS. This led to Ff binding proteins bound surface.

Blocking of excess aldehyde groups The excess aldehyde groups left on the substrate after the binding of Fc binbding proteins were blocked by treating with 0.1M glycine for 1 hour. The ELISA plate wells were then washed five times with 300 μΐ of PBS.

Blocking of the base substrate in ELISA plate wells »0 8 0934 -27The ELISA plate wells were incubated with 1% BSA (in PBS, pH 7.4) for 1 hour and 30 min at room temperature to block non-specific binding sites on the base substrate and then washed five times with 300 pi of PBS.

Immobilising antibodies to the F\. binding protein functionalized 96-well EL.1SA plate Mouse IgG was immobilized on to Fc binding protein functionalized 96-well ELISA plate by adding 100 pi of mouse IgG (12.1 pg/ml in PBS) to each modified microtiter plate well and leaving it overnight at 4° C. Thereafter, the modified microtiter plate wells were washed five times with 300 pi of PBS.

Binding of goat anti-mouse IgG HRP labelled 100 pi of goat anti-mouse IgG HRP labelled (in varying ng/ml concentrations in PBS) was provided to each of the ELISA plate wells and thereafter, left at room temperature for 1 hour. The ELISA plate wells were then washed five times with 300 pi of PBS.

TMB substrate assay TMB substrate solution was made by mixing equal amounts of TMB solution (0.4 g/L) and Peroxide solution (containing 0.02 % hydrogen peroxide in citric acid buffer) as per the instructions of the TMB substrate kit from Pierce. 100 pi of this TMB substrate solution was added to each ELISA plate well used in the immunoassay. The peroxidase enzyme, in the presence of H2O2, catalyses the oxidation of colorless TMB substrate to a blue colored product. After a fixed reaction time (30 min), the reaction was stopped with 100 pi of IN H2SO4 and the absorbance of the solution was measured at 450 nm with reference at 650 nm.

Fig. 7A and B demonstrate that Fc binding proteins can be bound in a functionally oriented manner by crosslinking their carboxyl as well as amino groups. Thus, both these strategies for binding proteins can be effectively used for binding capture antibodies in immunoassays as demonstrated here. -28(£08 0934 The strategy employing EDC-sulfoNHS for binding the carboxyl group of Fc binding proteins appears to be better compared to the strategy employing glutaraldehyde for binding the amino group of Fc binding proteins as shown in Fig. 7A and B.

Example 4 - Direct binding of biomoleeules to a surface of a substrate As an exemplary example, the immobilisation strategy of Example 1 was employed to directly immobilise a mouse IgG antibody to a surface of a substrate by covalent bonding through either amino or carboxyl groups. It will be apparent that this methodology can be used to bind any biomolecule having a terminal amino or carboxyl group to the surface of a support.

Referring to Fig. 8, mouse IgG can be directly bound to the substrate by covalent means without employing any Fc binding protein. Two immobilisation strategies were employed for direct mouse IgG binding, one based on EDC-sulfo NHS for binding the carboxyl group of mouse IgG and another based on glutaraldehyde for binding the amino group of mouse IgG,.

In the glutaraldehyde based immobilisation strategy, the direct binding of mouse IgG directly to the surface of a substrate was better than when the antibody was linked using an Fc binding protein. However, the overall assay sensitivity and the O.D. values were better for EDC-sulfo NHS based Fc binding strategy. Thus, maximum reactivity of Fc binding proteins can be achieved when their carboxyl terminal ends are used for immobilisation than their amino terminal ends.

Example 5 - Antibody functionalised biochip The immobilisation strategies described herein can be employed to create an antibody functionalised biochip. Advantageously, as the immobilisation strategies can covalently bind proteins to a wide range of support substrates, the IE 0 8 093 ί - 29 biochip can comprise any one of a number of supports. The support can be selected depending on the experimental procedures to be used with the chip.

F'e binding proteins can be covalently bound to the surface of a biochip for functionalisation with antibodies for use in drug targeting, antibody screening, immune response, immunodiagnostics, biosciences, charge transfer and nanobiotechnology applications.

The biochip can contain one or more antibody for example one or more antibody selected from anti-HBsAg, anti-troponin 1, anti-troponin T, anti-myoglobin. antiPSA. anti-creatine kinase (CK-MB), anti-insulin, anti-PAP (prostate acid phosphate), anti-hCG (human chorionic gonadotropin), anti-leptin, antiLegionella antigen, anti-Rotavirus, anti-verotoxin, anti-adenovirus. anti-Pf LDH. anti-digoxin, anti-b2-microglobulin, anti-T3, anti-T4, anti-TSll, anti-C reactive peptide (CRP) arid anti-C peptide.

As the Fc binding protein is covalently bound to the surface of the chip, the biochip is more stable and can be employed for the binding of capture antibody many times resulting in a reusable chip. Thus, many immunoassays can be performed on the same chip resulting in cost effectiveness compared to chips having passively adsorbed Fc binding proteins that can only be used only a few times. If multiple assays are performed on the passively adsorbed Fc binding proteins, the bound Fc binding proteins are washed away due to a leaching effect which will result in a decrease in assay sensibility and a decrease in the reproducibility of the assay.

The invention is not limited to the embodiment hereinbefore described, with reference to the accompanying drawings, which may be varied in construction and detail. ΙΕΟ 8 0934

Claims

1. A support comprising a substrate wherein a surface of the substrate comprises functional amino or aldehyde groups for covalent binding to a biomolecule.

2. A support as claimed in claim 1 wherein the substrate is selected from one or more of: polystyrene, gold, polvlysine, silicon and silicon derivatives, porous silicon, polysilicon, quartz. Zeonex. Zeonor, glass, polymethylmethacrylate (PMMA), polycarbonate, cellulose acetate, agarose, sepharose, cellulose, 10 polyacrylamide, trisacryl, sepacryl, ultragei AcA, azlactone, methacrylamide polymer (Eupergit) and 2-hydro-ethyl-methacrylate (HEMA).

3. A support as claimed in claim 1 or 2 wherein the biomolecule is a protein, 15

4. A support as claimed in claim 3 wherein the protein is an affinity protein.

5. A support as claimed in claim 3 or 4 wherein the protein is selected from one or more of: an F c binding protein, an antibody, a lectin, a chaperone protein, an adhesion protein and a ligand.

6. A support as claimed in claim 5 wherein the F c binding protein is selected from one or more of: Protein A/G, Protein A and Protein G.

7. A support as claimed in claim 5 or 6 further comprising an antibody bound to 25 the F c binding protein.

8. A support as claimed in claim 5 or 7 wherein the antibody is one or more seieeted from: anti-HBsAg, anti-troponin I, anti-troponin T, anti-myoglobin, anti-PSA (prostate specific antigen), anti- creatine kinase (CK-MB), anti30 insulin, anti-PAP (prostate acid phosphatase), anti-HCG (human chorionic gonadotropin), anti-leptin, anti-Legionella antigen, anti-Rotavirus, antiverotoxin, anti-adenovirus, anti-Pf LDH. anti-digoxin, anti-b2-microglobulin, anti-T3, anti-T4, anti-TSH, anti-C reactive peptide (CRP) and anti-C peptide. -319. An antibody functionalised biochip comprising a support substrate and an antibody attached to an F c binding protein wherein the amino or carboxyl group of the F c binding protein is covalently attached to a surface of the substrate.

9. 10. A biochip as claimed in claim 9 wherein the F e binding protein is covalently attached to a surface of the substrate through the carboxyl group.

10. 11. A method of covalently binding a biomoleeule to a support substrate comprising the steps of: (a) providing a support substrate; (c) cleaning a surface of a support substrate; (c) functionalising the support substrate by chemically creating hydroxyl groups on the cleaned surface of the support substrate; (d) inducing amines on the surface of the support substrate; and (e) covalent binding of a biomoleeule to the support substrate; and (f) blocking non-specific biomoleeule binding sites on the support substrate.

11. 12. A method as claimed in claim 11 wherein the biomoleeule is covalently bound to the support substrate through a carboxyl group.

12. 13. A method as claimed in claim 11 further comprising the step of cross linking the induced amine groups with glutaraldehyde prior to step (e) to form aldehyde groups.

13. 14. A method as claimed in claim 13 further comprising the step of: (g) blocking unreacted aldehyde groups with glycine.

14. 15. A method as claimed in claim 13 or 14 wherein the biomoleeule is covalently bound to the support substrate through an amino group. ΙΕΟ β 09 34 -3216. A method as claimed in any one of claims 11 to 15 wherein the substrate is selected from one or more of: polystyrene, gold, polylysine, silicon and silicon derivatives, porous silicon, polysilicon, quartz, Zeonex, Zeonor. glass, polymethylmethacrylate (PMMA), polycarbonate, cellulose acetate, agarose, 5 sepharose, cellulose, polyacrylamide, trisacryl, sepacryl, ultragel AcA, azlactone, methacrylamide polymer (Eupergit) and 2-hydro-ethylmethacrylate (HEMA).

15. 17. A method as claimed in any one of claims 11 to 15 wherein the biomolecule 10 is a protein.

16. 18. A method as claimed in claim 17 wherein the protein is an affinity protein.

17. 19. A method as claimed in claim 17 or 18 wherein the protein is selected from 15 one or more of: an F c binding protein, an antibody, a lectin, a chaperone protein, an adhesion protein and a ligand.

18. 20. A method as claimed in claim 19 wherein the F c binding protein is selected from one or more of: Protein A/G, Protein A and Protein G.

19. 21. A method as claimed in claim 19 or 20 further comprising the step of binding an antibody to the F c binding protein.

20. 22. A method as claimed in claim 19 or 21 wherein the antibody is one or more 25 selected from: anti-HBsAg, anti-troponin I, anti-troponin T, anti-myoglobin, anti-PSA (prostate specific antigen), anti-creatine kinase (CK-MB), antiinsulin, anti-PAP (prostate acid phosphatase), anti-HCG (human chorionic gonadotropin), anti-leptin, anti-Legionella antigen, anti-Rotavirus. antiverotoxin, anti-adenovirus, anti-Pf LDH, anti-digoxin, anti-b2-microglobu'in, 30 anti-T3, anti-T4, anti-TSH, anti-C reactive peptide (CRP) and anti-C peptide.

21. 23. A method as claimed in any one of claims 11 to 22 wherein the step of creating hydroxyl groups on the support comprises the steps of: -33treating the substrate with a basic solution; washing the substrate; and treating the support with oxygen plasma.

22. 24. A method as claimed in claim 23 wherein the basic solution is a solution of potassium hydroxide or sodium hydroxide.

23. 25. A method as claimed in any one of claims 11 to 24 wherein the step of inducing amines on the support substrate comprises the steps of: incubating the hydroxyl group functionalised support with an amine solution; and drying the support;

24. 26. A method as claimed in claim 25 wherein the amine solution is selected from: 3-aminopropyl triethoxy silane or 3-aminopropyl trimethoxy silane.

25. 27. A method as claimed in claim 25 or 26 wherein the support is dried in a vacuumed desiccator placed in an oven.

26. 28. A method as claimed in any one of claims 11 to 27 wherein the step of covalently binding a biomolecule to the support substrate comprises the steps of: forming a cross-linking solution; mixing the biornolecule with the cross-linking solution; incubating the support with the biomolecule - cross-linking solution mixture; and quenching the cross-linking solution.

27. 29. A method as claimed in claim 28 wherein the cross-linking solution comprises 1-ethyl 3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and Sulfo-N-Hydroxysuccinmide. »08 0934 -3430. A method as claimed in claim 28 or 29 wherein the quencher is 2mercaptoethanol.

28. 31. A method as claimed in any one of claims 19 to 30 wherein the step of binding an antibody to the F c binding protein comprises the steps of: blocking non-specific protein binding sites on the base substrate; and incubating the support with an antibody solution.

29. 32. A method as claimed in claim 31 wherein the non-specif sites are blocked with a bovine serum albumin solution. protein binding

30. 33. A method as claimed in claim 32 wherein the non specific protein binding sites are blocked with a 1% solution of bovine serum albumin.

31. 34. A support for protein isolation and purification comprising an affinity biomolecule covalently bound to a surface of the support wherein the affinity biomolecule is bound to trie support through a terminal amino or carboxyl group.

32. 35. A support as claimed in daim 34 wherein the support is selected from one or more of: agarose, sepharose, cellulose, polyacrylamide, trisacryl, sepacryl, ultragel AcA, azlactone, methacrylamide polymer (Eupergit) and 2-hydroethyl-methacrylate (HEMA).

33. 36. A support as claimed in claim 35 in the form of a chromatography column.

34. 37. A support as claimed in any of claims 34 to 36 wherein the affinity biomolecule is a protein.

35. 38. A support as claimed in claim 37 wherein the protein is selected from one or more of: an F c binding protein, an antibody, a lectin, a chaperone protein, an adhesion protein, and a ligand.