# US 2010/0167263 A1 · Novel biosensor system based on recognition induced birefringence (rib)

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- Number: US 2010/0167263 A1
- Document ID: US20100167263A1
- Status: Published application
- Kind: A1
- Application number: 12/645,005
- Filed: 2009-12-22
- Published: 2010-07-01
- Earliest claimed: 2008-12-31
- Assignees: DWI an der RWTH Aachen e.V.
- Inventors: Jürgen GROLL; Martin Möller; Matthias Eberhardt
- Web page: https://patentgrep.com/patent/US20100167263A1

## CPC

- G01N 33/54393: {Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding}
- G01N 33/54373: {involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings}
- B01J 2219/00509: Microcolumns
- B01J 2219/00605: the compounds being directly bound or immobilised to solid supports
- B01J 2219/00612: the surface being inorganic
- B01J 2219/00621: by physical means, e.g. trenches, raised areas
- B01J 2219/00623: Immobilisation or binding
- B01J 2219/00635: by reactive plasma treatment
- B01J 2219/00637: by coating it with another layer
- B01J 2219/00702: Processes involving means for analysing and characterising the products
- 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)
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS

## Abstract

The present invention relates to a label-free biosensor system, a method for manufacturing said label-free biosensor system, its use for detecting biochemical reactions and/or bindings, enzymatic reactions, nucleic acid hybridizations, protein-protein interactions and protein-ligand interactions, as well as an assay method for detecting and/or quantifying an analyte of interest in a biological sample which comprises detecting the Recognition Induced Birefringence (RIB) generated in the presence as opposed to the absence of said analyte by bringing said sample into contact with said label-free biosensor system.

## Claims

### Claim 1

Label-free biosensor system comprising:
an anisotropically microstructured elastomeric sensor substrate having elevated regions and depressions on at least one side, wherein the distance between the elevated regions is in the range from 0.5 to 100 μm and the height of the elevated regions is in the range from 1 to 500 μm, wherein either (a) said sensor substrate is made of an elastomer, preferably a polysiloxane or a fluororesin, and is further coated, at least on the top surface thereof, with a polymeric material selected from poly(ethylene oxide), poly(propylene oxide), poly(glycidol) and copolymers thereof, said polymeric material being bound to the sensor substrate either covalently or through ionic interactions, complexes or hydrogen bridges, or (b) said sensor substrate is composed in its entirety of a polymeric material selected from poly(ethylene oxide), poly(propylene oxide), poly(glycidol) or copolymers thereof or a hydrogel-forming polymer, and wherein, only on the elevated regions, biorecognition molecules are covalently bound via said polymeric material or directly to said sensor substrate, respectively.

### Claim 2 (depends on 1)

The label-free biosensor system according to claim 1, wherein the anisotropically microstructured elastomeric sensor substrate is made of PDMS.

### Claim 3 (depends on 1)

The label-free biosensor system according to claim 1, wherein the anisotropically microstructured elastomeric sensor substrate in its entirety is made of P(EO-stat-PO).

### Claim 4 (depends on 1)

The label-free biosensor system according to claim 1, wherein the polymeric material for coating the anisotropically microstructured elastomeric sensor substrate is a P(EO-stat-PO).

### Claim 5 (depends on 4)

The label-free biosensor system according to claim 4, wherein the polymeric material is made of six armed star shaped molecules with a molecular weight in the range of 1 to 3 kDa per arm and a polymer backbone of statistically copolymerized ethylene oxide (EO) and propylene oxide (PO) in a ratio 4:1, where the distal ends of the arms are functionalized with reactive isocyanate (NCO) groups.

### Claim 6 (depends on 1)

The label-free biosensor system according to claim 1, wherein the polymeric material is multifunctionalized, and wherein the biorecognition molecules are selected from the group consisting of peptides, proteins, antibodies, antibody fragments, lectins, carbohydrates, DNA, oligonucleotides, aptamers, low molecular weight ligands, complexing agents and polyionic tags, covalently bound to said multifunctionalized polymeric material on the elevated regions of the sensor substrate.

### Claim 7 (depends on 1)

The label-free biosensor system according to claim 1, wherein the elevated regions are in the form of ridges so that the depressions form channels between the ridges.

### Claim 8 (depends on 1)

A method of manufacturing the label-free biosensor system according to claim 1, comprising the steps of:
(i) providing an anisotropically microstructured sensor substrate having elevated regions and depressions on at least one side, wherein the distance between the elevated regions is in the range from 0.5 to 100 μm and the height of the elevated regions is in the range from 1 to 500 μm,
(ii) optionally functionalizing said sensor substrate to generate functional groups on the surfaces of said sensor substrate, and coating the thus functionalized sensor substrate with the polymeric material, thereby causing covalent bonding or ionic interactions, complexes or hydrogen bridges between the surface of said sensor substrate and said polymeric material, and
(iii) functionalizing said sensor substrate provided in step (i) or obtained in step (ii) only on the elevated regions of said sensor substrate with biorecognition molecules via covalent bonding to said polymeric material or to said sensor substrate, respectively.

### Claim 9 (depends on 8)

The method according to claim 8, wherein in step (i) the anisotropically microstructured elastomeric sensor substrate is made of PDMS and produced by way of soft lithography technique.

### Claim 10 (depends on 8)

The method according to claim 8, wherein in step (ii) ammonia plasma is used to generate amino functionalized surfaces.

### Claim 11 (depends on 8)

The method according to claim 8, wherein in step (ii) the functionalized polymeric material is made of six armed star shaped molecules with a molecular weight in the range of 1 to 3 kDa per arm and a polymer backbone of statistically copolymerized ethylene oxide (EO) and propylene oxide (PO) in a ratio 4:1, where the distal ends of the arms are functionalized with reactive isocyanate (NCO) groups.

### Claim 12 (depends on 8)

The method according to claim 8, wherein the biorecognition molecules are —NH2 or —OH functionalized molecules.

### Claim 13 (depends on 8)

The method according to claim 8, wherein the biorecognition molecules are applied on the elevated regions of the sensor substrate by inverse microcontact printing (μCP).

### Claim 14 (depends on 1)

Use of the label-free biosensor system according to claim 1 for detecting biochemical reactions and/or bindings, enzymatic reactions, nucleic acid hybridizations, protein-protein interactions, protein-ligand interactions and complex formation.

### Claim 15 (depends on 1)

An assay method for detecting and/or quantifying an analyte of interest in a biological sample which comprises detecting the Recognition Induced Birefringence (RIB) generated in the presence as opposed to the absence of said analyte by bringing said sample into contact with the label-free biosensor system according to claim 1.

### Claim 16 (depends on 2)

The label-free biosensor system according to claim 2, wherein the PDMS, is amino functionalized PDMS.

### Claim 17 (depends on 4)

The label-free biosensor system according to claim 4, wherein the P(EO-stat-PO) is a star shaped P(EO-stat-PO).

### Claim 18 (depends on 10)

The method according to claim 10, wherein in the amino functionalized surfaces is amino functionalized PDMS surfaces.