Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Means for analyzing liquid or solid sample
Reexamination Certificate
2002-08-29
2004-04-27
Gakh, Yelena G. (Department: 1743)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Means for analyzing liquid or solid sample
C422S082050, C422S082060, C436S072000
Reexamination Certificate
active
06726881
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a measurement chip for a surface plasmon resonance biosensor, a method for producing the chip, and the use thereof.
BACKGROUND ART
Recently, a large number of measurements using immune response are carried out in the clinical test and the like. However, since conventional methods require a complicated operation or a labeling substance, there is used an immunosensor which utilizes a surface plasmon resonance (SPR) capable of detecting change of a ligand with high sensitivity without requiring a labeling substance.
In a measurement chip commonly used for a measurement device employing such surface plasmon resonance (a surface plasmon resonance biosensor), porous materials are formed on a metal membrane coated on a glass substrate, and a physiologically active substance such as an enzyme or an antibody is deposited or immobilized on the surface of these porous materials and/or within these porous materials. Examples of these porous materials include a textile fabric, a knitted fabric and a nonwoven fabric which are composed of synthetic fibers, natural fibers, inorganic fibers etc., and also porous inorganic or organic materials (see Japanese Patent Application Laying-Open (kokai) No. 3-164195). Moreover, in a commercial product (for BIAcore 2000, Pharmacia Biosensor), carboxy methyl dextran is used as a porous material.
As a method for immobilizing a physiologically active substance on a metal membrane, LB (Langmuir-Blodgett) method may be used (see Japanese Patent Application Laying-Open (kokai) No. 5-288672), but this method has a problem in that the binding between an LB membrane and a metal membrane is so weak that the LB membrane falls off together with the physiologically active substance.
Further, Japanese Patent Application Laying-Open (kokai) No. 10-267834 discloses a method for producing an SPR sensor chip by forming an organic silicon membrane on a gold surface using a silane coupling agent, and chemically modifying the gold surface with, for example, a protein using a functional group of a silane coupling agent such as an amino group, carboxyl group, mercapto group and further using a bifunctional reagent. However, in this method, reactivity of the gold surface with the silane coupling agent is so low that an organic silicon membrane cannot be produced with good reproducibility. Moreover, Japanese Patent No. 2815120 discloses a method for introducing a functional group such as a carboxyl group or an amino group onto the surface of gold using a mercapto group.
DISCLOSURE OF THE INVENTION
An object to be achieved by the present invention is to solve the above-stated problems of the prior art. That is, an object of the present invention is to provide a measurement chip for a surface plasmon resonance biosensor which can provide good sensitivity and can be easily produced.
As a result of thorough studies directed to achieve the above object, the present inventors have found that good sensitivity can be obtained by forming an organic silicon membrane which is immobilized onto a metal membrane via a mercapto group, and then immobilizing a physiologically active substance to the organic silicon membrane via a functional group, and thereby completing the present invention.
Thus, according to the present invention, there is provided a measurement chip for a surface plasmon resonance biosensor, which comprises a transparent substrate, a metal membrane located on the transparent substrate and an organic silicon membrane immobilized on the metal membrane and in which the organic silicon membrane is immobilized on the metal membrane via a functional group capable of binding with atoms on the surface of a metal.
Preferably, the organic silicon membrane is a membrane formed by at least one or more types of silane coupling agents which contain a functional group capable of binding with atoms on the surface of a metal.
Preferably, the organic silicon membrane is a membrane formed by a mixture of at least one or more types of silane coupling agents which contain a functional group capable of binding with atoms on the surface of a metal, and at least one or more types of silane coupling agents which contain a functional group capable of binding with physiologically active substances.
Preferably, the functional group capable of binding with atoms on the surface of a metal is a disulfide group, a sulfide group, a diselenide group, a selenide group, a mercapto group, a nitrile group, an isonitrile group, a nitro group, a selenol group, a group derived from a trivalent phosphate compound, an isothiocyanate group, a xanthate group, a thiocarbamate group, a phosphine group, a thio acid group or a dithioic acid group, and particularly preferably a mercapto group.
Preferably, the organic silicon membrane is a membrane formed by a mixture of at least one or more types of silane coupling agents that contain a mercapto group, and at least one or more types of silane coupling agents that contain an amino group.
According to another aspect of the present invention, there is provided a measurement chip for a surface plasmon resonance biosensor wherein a bifunctional reagent is further bound to the organic silicon membrane.
The preferred bifunctional reagent is a disulfone compound represented by the following formula:
X
1
—SO
2
-L
2
—SO
2
—X
2
wherein X
1
and X
2
independently represent —CR
1
═CR
2
R
3
or —CHR
1
—CR
2
R
3
Y; R
1
, R
2
and R
3
independently represent an atom or a group selected from a group consisting of a hydrogen atom, an alkyl group having a carbon number of 1 to 6, an aryl group having a carbon number of 6 to 20, and an aralkyl group which contains an alkyl chain having a carbon number of 1 to 6 and has a total carbon number of 7 to 26; Y represents an atom or a group selected from a group consisting of a halogen atom, —OSO
2
R
11
, —OCOR
12
, —OSO
3
M and a quaternary pyridinium group; R
11
represents a group selected from a group consisting of an alkyl group having a carbon number of 1 to 6, an aryl group having a carbon number of 6 to 20, and an aralkyl group which contains an alkyl chain having a carbon number of 1 to 6 and has a total carbon number of 7 to 26; R
12
represents a group selected from a group consisting of an alkyl group having a carbon number of 1 to 6 and an halogenated alkyl group having a carbon number of 1 to 6; M represents an atom or a group selected from a group consisting of a hydrogen atom, an alkali metal atom and an ammonium group; and L
2
represents a linking group.
According to further another aspect of the present invention, there is provided a measurement chip for a surface plasmon resonance biosensor as mentioned above, wherein a physiologically active substance is immobilized to an organic silicon membrane directly or via a bifunctional reagent.
According to further another aspect of the present invention, there is provided a method for producing a measurement chip for a surface plasmon resonance biosensor, which comprises the step of treating a metal membrane located on a transparent substrate with a mixture of at least one or more types of silane coupling agents which contain a functional group capable of binding with atoms on the metal surface and at least one or more types of silane coupling agents which contain a functional group capable of binding with physiologically active substances, and thereby forming an organic silicon membrane on the metal membrane.
According to further another aspect of the present invention, there is provided a method for detecting and/or measuring a substance which interacts with a physiologically active substance, which comprises the steps of contacting a surface plasmon resonance biosensor having a physiologically active substance immobilized thereon according to the present invention with a sample containing a target substance; and detecting and/or measuring interaction between the physiologically active substance immobilized on the biosensor and the target substance.
DETAILED DESCRIPTION OF THE INVENTION
The embodiments of the present invention
Seshimoto Osamu
Shinoki Hiroshi
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