Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-10-27
2003-11-18
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C356S317000, C356S318000, C356S445000, C356S447000, C385S012000, C385S129000, C385S130000, C422S082010, C422S082020, C422S082050, C422S082090, C422S082110, C435S176000, C435S287100, C435S288700, C436S164000, C436S165000, C436S518000, C436S524000, C436S525000, C436S527000, C436S805000
Reexamination Certificate
active
06649361
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a surface plasmon resonance enzyme sensor and a method of measuring surface plasmon characterized by performing measurement of surface plasmon resonance with electrochemical oxidoreduction activity using an enzyme.
2. Description of the Prior Art
Biosensors using enzymes have been widely investigated as means for selectively detecting blood glucose level and a number of biomolecules such as cholesterol, urea and vitamins (cf., for example, Isao Karube, “Biosensor”, published by Kyoritsu Shuppan, May 2, 1986). In particular, combinations of an oxidase and an electrochemical detection method have been extensively studied. For example, combinations of a blood glucose level sensor and a urea glucose level sensor are commercially available from several manufacturers.
In the case of sensors using oxidases, several combinations as shown in
FIG. 3
are known, which are roughly classified into the following:
(1) Methods in which a decrease in oxygen concentration with consumption of oxygen upon enzymatic reaction is measured by an oxygen sensor;
(2) Methods in which hydrogen peroxide, which is a product of enzymatic reaction, is electrically oxidized for detection;
(3) Methods in which a redox molecule called electron mediator and an enzyme are mixed or chemically coupled and the enzyme is reacted on an electrode through the electron mediator (without oxygen consumption), etc. have been known.
As intermediate methods between (2) and (3),
(4) Methods in which hydrogen peroxide generated in an enzymatic reaction is reduced through horseradish peroxidase (HRP) electrically coupled to electron mediators (cf., for example, Vreeke, M., Maiden, R., Heller, A., Anal. Chem., 64, 3084-3090 (1992)), also have been known.
On the other hand, biosensors using optical methods have been studied. To detect the product of oxidase, chemiluminescence from the mixture of the product and chemicals such as luminol are widely used.
Recently, sensors utilizing surface plasmon resonance (SPR) have been studied as a biosensor using optical methods. The surface plasmon resonance enzyme sensors (hereinafter, referred to as SPR sensors) can detect a change in refractive index in the range of several hundreds nm from the surface of a base metal such as gold or silver. In actual measurements, the base metal is illuminated at a certain incident angle on the opposite surface of a sample and the angle at which its evanescence wave and surface plasmon resonate is measured.
The schematic of an optical system for measurement is shown in FIG.
4
(
a
). A light beam from an optical source is condensed into a wedge-form light beam, which enters a semi-cylindrical prism
7
. The sensing part
6
′ is attached to the bottom of the prism
7
using a material for matching the refractive indices (matching oil). The incidence light illuminates the sensing part
6
′ with angles of total reflection conditions. The evanescent wave and surface plasmon wave, which occur on the metal thin film side, will resonate at a certain angle of incidence (surface plasmon resonance). This phenomenon is observed as weak reflection at the resonance angle. When the reflected light is observed by a CCD camera, a valley of reflectivity is measured as shown in FIG.
4
(
b
).
Since the angle at which resonance occurs depends on the optical property (refractive index) of the surface, a molecule bound to the surface of the sensing part
6
′ changes the refractive index of the surface so that the angle at which a valley appears changes. Measurement of such a change enables high speed monitoring of the interaction between molecules on the surface.
SPR sensors are finding application to immunological sensors utilizing antigen-antibody reaction, DNA detection, receptor-protein interaction detection, etc.
However, among the conventional biosensors, electrochemical enzyme sensors, which conduct measurements using electrodes, require time and hand for a single measurement so that it is difficult for them to measure a large number of samples quickly.
Also, in the case of enzyme sensors utilizing optical methods, it is necessary to add a reagent when measuring chemiluminescence or fluorescence. This makes for a cumbersome operation and requires extra hands and/or expensive analytical apparatus.
Since, the principle of conventional SPR sensors are based on a refractive index change caused by a binding of analytes to the immobilized molecular recognition molecules on the sensing part, the refractive index change of the SPR sensor cannot be made higher than the density of molecule recognition molecule so that there is a limitation in its measurement sensitivity. Furthermore, in the conventional SPR measurement method, it is necessary that the molecule recognition molecule and the molecule to be measured bind to each other. Therefore, the method is difficult to apply to the detection of molecules to be measured having weak binding affinity or short binding lifetime for molecule recognition molecule, for example, binding of enzymes and low molecular compounds such as hydrogen peroxide, benzene derivatives, nitrogen oxides, nerve transmitter substances, amino acids, antigens, DNA oligomers, etc. and there has been the problem that its application range is limited.
SUMMARY OF THE INVENTION
The present invention has been proposed in view of the above and an object of the present invention is to provide a novel principle of measurement and an improved surface plasmon resonance enzyme sensor and a method of measuring surface plasmon resonance.
In one aspect of the surface plasmon resonance enzyme sensor of the present invention, the sensor comprises a sensing part having an optically transparent base plate, a thin metal film made of gold or silver, and a film provided on the metal thin film causing an electron transfer reaction with both the thin metal film and an enzyme.
In the surface plasmon resonance enzyme sensor, the film causing electron transfer reaction may be a polymer film.
In the surface plasmon resonance enzyme sensor, the film causing electron transfer reaction may contain an enzyme, which donates and receives charge to or from the thin metal film therethrough.
The surface plasmon resonance enzyme sensor may comprise at least one selected from the group consisting of enzymes, cells, and microorganisms which have the property of producing a molecule which causes redox reaction with the film which causes electron transfer reaction in at least one manner selected from directly, through the enzyme contained in the film, and both.
In one embodiment of the surface plasmon resonance enzyme sensor of the present invention, the sensor comprises:
an electrochemical cell (
10
) including a sensing part (
6
) comprising a glass base plate (
1
), a thin gold film (
2
) provided on one surface of the glass base plate(
1
), a working electrode (
3
a
) provided on the thin gold film (
2
), a redox polymer film (
4
) provided on the thin gold film (
2
) and an enzyme-immobilized film (
5
) provided on the polymer film (
4
),
a prism (
7
) provided on the other surface of the glass base plate (
1
),
a jig (
9
) provided in contact with the thin gold film (
2
) having a space for holding an electrolyte through a gasket (
8
) provided with a hole (
8
a
) serving as the working electrode (
3
a
); and a counter electrode (
11
) and a reference electrode (
12
) arranged remote from each other in contact with the electrolyte held in the electrochemical cell;
wherein the working electrode (
3
a
), counter electrode (
11
) and reference electrode (
12
) are connected to a potentiostat so that electrochemical measurement and surface plasmon resonance measurement can be conducted simultaneously.
In one aspect of the method of measuring surface plasmon resonance according to the present invention, the method comprises the steps of:
using the surface plasmon resonance enzyme sensor as described above,
applying predetermined potentials to respective electrodes to produce a change of state
Iwasaki Yuzuru
Niwa Osamu
Burns Doane , Swecker, Mathis LLP
Chin Christopher L.
Nippon Telegraph and Telephone Corporation
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