# US 9,040,309 B2 · Binding layer and method for its preparation and uses thereof

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- Number: US 9,040,309 B2
- Document ID: US9040309B2
- Status: Granted
- Kind: B2
- Application number: 11/471,716
- Filed: 2006-06-21
- Published: 2007-05-24
- Granted: 2015-05-26
- Earliest claimed: 2005-10-27
- Assignees: Bio-Rad Haifa Ltd.
- Applicants: Shai Nimri
- Inventors: Shai Nimri
- Web page: https://patentgrep.com/patent/US9040309B2

## CPC

- G01N 33/54353: {with ligand attached to the carrier via a chemical coupling agent (coatings G01N33/54393)}
- G01N 33/548: Carbohydrates, e.g. dextran
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The invention provides a binding layer comprising a polysaccharide substituted by carboxylic groups or derivatives thereof exhibiting high performance in the binding of ligand molecules and in the interaction thereof with analyte molecules. A method for the preparation of the binding layer and for the assaying of various analyte molecules is also provided.

## Claims

### Claim 1

A binding layer, comprising:
a polysaccharide substituted by a plurality of carboxylic groups, the plurality of carboxylic groups comprising a plurality of easily-activated carboxylic groups,
wherein at least one β-atom of each of the easily activated carboxylic groups is a carbon atom and remaining β-atoms are independently hydrogen or carbon atoms,
the β-carbon atom of each of the easily activated carboxylic groups is linked to a functional group of the polysaccharide through a covalent linkage, the functional group being a native or chemically introduced carboxylic group, or a derivative thereof selected from the group consisting of an ester, an amide, an anhydride, and an acetyl halide,
wherein at least a part of the plurality of the easily-activated carboxylic groups are activated carboxylic ester groups capable of binding ligand molecules, the activated carboxylic ester groups being obtained by activation of the carboxylic groups of the easily activated carboxylic groups and being selected from the group consisting of a N-hydroxysuccinimide ester and a N-hydroxysulfosuccinimide ester, and
the β-carbon atom of the easily-activated carboxylic groups has no electron withdrawing groups.

### Claim 2 (depends on 1)

The binding layer according to claim 1, wherein the polysaccharide is selected from the group consisting of agarose, alginic acid, amylopectin, amylose, carageenan, cellulose, chitin, chitosan, dextran, glycogen, heparan, heparin, hyaluronic acid, pectin, amylase, and starch.

### Claim 3 (depends on 1)

The binding layer according to claim 1, wherein the polysaccharide is selected from the group consisting of alginic acid, carboxymethyl dextran, carboxymethyl cellulose, hyaluronic acid, and pectin.

### Claim 4 (depends on 1)

The binding layer according to claim 1, wherein the ligand molecules are selected from the group consisting of nucleic acids, peptides, proteins, polypeptides, antigens, polyclonal antibodies, monoclonal antibodies, single chain antibodies, antibody fragments, glycan, small organic molecules, cells, viruses, bacteria, and biological samples.

### Claim 5 (depends on 1)

The binding layer according to claim 1, wherein the polysaccharide is immobilized onto a solid substrate having a surface.

### Claim 6 (depends on 5)

The binding layer according to claim 5, wherein the solid substrate is a sensing element of a biosensor.

### Claim 7 (depends on 6)

The binding layer according to claim 6, wherein the sensing element comprises a material selected from the group consisting of glass, plastic, and free electron metals.

### Claim 8 (depends on 7)

The binding layer according to claim 7, wherein the free electron metal is gold.

### Claim 9 (depends on 1)

The binding layer according to claim 1, wherein the plurality of carboxylic groups or derivatives thereof is activated.

### Claim 10 (depends on 1)

The binding layer according to claim 1, wherein at least 50% of the plurality of the carboxylic groups or derivatives thereof is activated.

### Claim 11

A method for preparing a binding layer comprising a polysaccharide substituted by a plurality of carboxylic groups, the plurality of carboxylic groups comprising a plurality of easily-activated carboxylic groups, each of the plurality of the easily-activated carboxylic groups having at least one β-atom being a carbon atom and remaining β-atoms are independently hydrogen or carbon atoms, the method comprising:
providing a substrate having a surface;
immobilizing a polysaccharide on the surface of the substrate wherein said polysaccharide immobilized on the substrate contains functional groups capable of chemical modification,
chemically modifying the polysaccharide by attaching a plurality of the easily-activated carboxylic groups to extend therefrom, wherein at least one β-atom of each group of the easily-activated carboxylic group is a carbon atom and remaining β-atoms are independently hydrogen or carbon atoms, wherein said attaching comprises linking the β-carbon atom of the plurality of the easily activated functional group to the functional groups of the polysaccharide through a covalent linkage, the functional groups being a native or chemically introduced carboxylic groups, or a derivative thereof selected from the group consisting of an ester, an amide, an anhydride, and an acetyl halide; and
esterifying at least part of the easily activated carboxylic groups for binding of ligand molecules with at least one ester selected from the group consisting of a N-hydroxysuccinimide ester and a N-hydroxysulfosuccinimide ester,
wherein chemically modifying and esterifying may occur, independently of each other, either before, during or after immobilizing, and a β-atom of the easily-activated carboxylic groups has no electron withdrawing groups.

### Claim 12 (depends on 11)

The method according to claim 11, wherein the polysaccharide is selected from the group consisting of alginic acid, carboxymethyl dextran, carboxymethyl cellulose, hyaluronic acid, and pectin.

### Claim 13 (depends on 11)

The method according to claim 11, wherein at least 50% of the easily activated carboxylic groups or derivatives thereof are esterified to form a plurality of ester groups selected from N-hydroxysuccinimide or N-hydroxysulfosuccinimide esters.

### Claim 14 (depends on 13)

The method according to claim 13, wherein the plurality of ester groups are reacted with ligand molecules, thereby affording ligand molecules covalently bound to the layer.

### Claim 15 (depends on 1)

The binding layer of claim 1, wherein the polysaccharide is immobilized onto a solid sensing element of a biosensor.

### Claim 16 (depends on 15)

The binding layer of claim 15, wherein the polysaccharide is selected from the group consisting of alginic acid, amylopectin, amylose, carageenan, chitin, chitosan, glycogen, heparan, heparin, hyaluronic acid, pectin, amylase, starch, carboxymethyl dextran, and carboxymethyl cellulose.