JPH10282104A - Measurement chip for surface plasmon resonance bio sensor and its manufacture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To immobilize nucleic acid and obtain an excellent sensitivity by forming a metal film, an organic substance layer, and avidin layer on a transparent substrate successively. SOLUTION: A measurement chip is formed by laminating a metal film 2, an organic substance layer 3, and an avidin layer 4 successively on a transparent substrate 1. Materials such as glass, polyethylenetelephtharate, and polycarbonate that are transparent to laser beams are used as the transparent substrate 1. They preferably do not show any anisotropy for polarization and have improved machining property. A material that preferably does not generate a surface plasmon resonance is used as the metal film 2 and, for example, gold, silver, copper, aluminum, and platinum can be used. The organic substance layer 3 is formed by silane coupling agent, mercapto group, and a compound with other organic functional groups. The avidin layer 4 is formed by allowing a specific amount of avidin to contact the organic substance layer 3 for a specific amount of time.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a measurement chip for a surface plasmon resonance biosensor and a method for manufacturing the same.

[0002]

2. Description of the Related Art At present, many measurements using an immunological reaction are performed in clinical examinations and the like. However, the conventional method requires complicated operations and a labeling substance. An immunosensor using surface plasmon resonance (SPR) capable of detecting a change in the sensitivity with high sensitivity is used.

In such an apparatus utilizing surface plasmon resonance (surface plasmon resonance biosensor), a physiologically active substance interacting with an object to be measured is immobilized on a measurement chip in the apparatus for measurement. The measurement chip is usually composed of a glass substrate, a metal film formed thereon, and carboxymethyl dextran bonded to the metal film (for example, a measurement chip for BIAcore 2000 manufactured by Pharmacia Biosensor Co., Ltd.). Immobilize the substance.

However, it is easy to immobilize proteins such as antibodies and enzymes on carboxymethyl dextran, but it is very difficult to immobilize acidic substances such as nucleic acids (“Protein Nucleic Acid Enzyme” Vol. 37). No.15 2997-
2984 (1992)). Further, in the layer structure of the measurement chip, since the only physiologically active substance that interacts substantially and efficiently with the measurement object is exposed on the surface of the layer made of carboxymethyl dextran, The bound biologically active substance does not function effectively, and the sensitivity is reduced accordingly.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a surface plasmon resonance biosensor capable of immobilizing a nucleic acid and having good sensitivity even when the amount of the immobilized nucleic acid is small. It is to provide a measuring chip.

[0006]

Means for Solving the Problems In view of the above problems, as a result of intensive studies, the present inventors have found that by forming an organic substance layer and an avidin layer on a metal film, the nucleic acid can be immobilized on the metal film. The present inventors have found that good sensitivity can be obtained even when a small amount of nucleic acid is used, thereby completing the present invention.

That is, the present invention comprises a transparent substrate, a metal film disposed on the transparent substrate, an organic material layer disposed on the metal film, and an avidin layer disposed on the organic material layer. A measurement chip for a surface plasmon resonance biosensor, which is characterized in that: Further, the present invention is a method for producing a measurement chip for a surface plasmon resonance biosensor, which comprises arranging a metal film, an organic material layer, and an avidin layer on a transparent substrate in this order.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The measurement chip for a surface plasmon resonance biosensor in the present invention (hereinafter, simply referred to as “measurement chip”) is a chip used for a surface plasmon resonance biosensor, and transmits and reflects light emitted from the sensor. Refers to a member including a portion and a portion for immobilizing a substance that interacts with the measurement object, and may be fixed to the main body of the sensor or detachable.

The measurement chip of the present invention comprises a transparent substrate, a metal film disposed on the transparent substrate, an organic material layer disposed on the metal film, and an avidin layer disposed on the organic material layer. ing. Here, the `` metal film disposed on the transparent substrate '' refers to not only the case where the metal film is directly in contact with the transparent substrate but also the other layer without the metal film directly contacting the transparent substrate. It is intended to include the case where they are arranged through the intermediary. The “organic substance layer disposed on the metal film” and the “avidin layer disposed on the organic substance” have the same meaning as described above.

FIG. 1 is a schematic sectional view of a measuring chip according to an example of the present invention. The measuring chip according to the present embodiment includes a transparent substrate 1, a metal film 2 formed on the transparent substrate 1, an organic material layer 3 formed on the metal film 2, and an avidin layer 4 formed on the organic material layer 3. Consisting of four layers. As the transparent substrate 1, any material may be used as long as it is generally used for a measurement chip for a surface plasmon resonance biosensor. Generally, glass, polyethylene terephthalate,
A material made of a material that is transparent to laser light such as polycarbonate can be used, and a material that does not show anisotropy with respect to polarized light and has excellent workability is desirable.
It is about 20mm.

The metal film 2 is not particularly limited as long as it can generate surface plasmon resonance. The types of metals that can be used for the metal film 2 include:
Gold, silver, copper, aluminum, platinum and the like can be mentioned, and these can be used alone or in combination. Further, in consideration of the adhesion to the transparent substrate 1, the transparent substrate 1
An intervening layer made of chromium or the like may be provided between the layer and the layer made of gold, silver, or the like.

The thickness of the metal film 2 is preferably from 100 to 2000 °, and more preferably from 200 to 600 °. 30
If it exceeds 00 °, the surface plasmon phenomenon of the medium cannot be sufficiently detected. When an intervening layer made of chromium or the like is provided, the thickness of the intervening layer is preferably 5 to 50 °. The formation of the metal film 2 may be performed by an ordinary method.
For example, it can be performed by a sputtering method, an evaporation method, an ion plating method, an electroplating method, an electroless plating method, or the like. Among these methods, it is preferable to use the sputtering method.

The organic substance layer 3 is a layer made of a substance capable of binding to metal atoms in the metal film 2 and binding to avidin molecules of the avidin layer 4. The thickness of the organic material layer 3 is preferably 10 to 200 °, more preferably 10 to 50 °.

The organic substance layer 3 comprises a silane coupling agent,
Compound having a mercapto group and another organic functional group (hereinafter, referred to as
(Hereinafter simply referred to as a “chiol compound”), and can also be formed by the LB (Langmuir-Blodgett) method. When the film is formed by the LB method, it has a disadvantage that the bonding ability to the metal film is weaker than that when the film is formed by the silane coupling agent or the thiol compound. However, it can be applied to a wide range of substances and forms an aggregated film. As a result, the number of bioactive substances to be bound per unit area can be increased.

Examples of the silane coupling agent that can be used for forming the organic material layer include 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyldiethoxymethylsilane, and 3- (2-aminoethylamino). Propyl) trimethoxysilane, 3-
(2-aminoethylaminopropyl) dimethoxymethylsilane, 3-mercaptopropyltrimethoxysilane, dimethoxy-3-mercaptopropylmethylsilane, and the like. Further, as the thiol compound,
Mercaptoaminomethane, 2-mercapto-1-aminoethane, 3-mercapto-1-aminopropane, 4-mercapto-1-aminobutane, 1,1,1-triamino-2-mercaptoethane, mercaptoacetic acid, 2-mercaptopropionic acid , 3-mercaptobutyric acid, 4-mercaptovaleric acid, 1,1,1-triamino-3-mercaptopropane, and the like. Among them, 1,1,1 which is a polyfunctional substance and has many binding sites with avidin -Triamino-2-mercaptoethane, 1,1,1-triamino-
It is preferable to use 3-mercaptopropane or the like. L
Examples of the substance applicable to the method B include aminoacetic acid, 2-aminopropionic acid, and 3-aminobutyric acid.

As a method of forming the organic material layer 3 using a silane coupling agent, a method of exposing the metal film 2 to a saturated vapor of the silane coupling agent for a predetermined time (saturated vapor method), a method of using a silane coupling agent. A method in which the metal film 2 is immersed in a solution for a certain period of time (immersion method), a method using a spin coater (spin coating method), a method using a gravure printing machine (gravure method), and the like can be used. As a method for forming the organic material layer 3, a saturated vapor method, an immersion method, a spin coating method, a gravure method, or the like can be used.

The avidin layer 4 is a layer composed of avidin molecules. The avidin layer 4 can be formed by bringing a predetermined amount of avidin into contact with the organic substance layer 3 for a predetermined time. As a specific method, there can be exemplified a method in which a transparent substrate 1 on which an organic material layer 3 is formed is placed on a flow cell type surface plasmon resonance biosensor, and a constant flow rate of avidin is flowed for a predetermined time (a predetermined amount).

As shown in FIG. 2, the measuring chip of the present invention can hybridize to the avidin layer 4 with a nucleic acid to be measured, and a complementary strand nucleic acid labeled with biotin 52 (hereinafter simply referred to as “labeled complementary nucleic acid”). Strand nucleic acid 5)). The complementary nucleic acid 51 is not particularly limited as long as it has a base sequence capable of hybridizing with the nucleic acid to be measured, and may be either DNA or RNA. Examples of the nucleic acid to be measured include DNA encoding a toxin produced by a bacterium, an oncogene, and a genetic disease gene such as phenylketonuria.

The labeled complementary nucleic acid 5 can be immobilized by binding the biotin 51 moiety to an avidin molecule.
Examples of a method for immobilizing the labeled complementary nucleic acid 5 to an avidin molecule include an ink jet method and a macro dispenser method. The ink-jet method is advantageous in that the droplet containing the labeled complementary strand nucleic acid 5 can be accurately ejected to an extremely narrow area, so that the labeled complementary strand nucleic acid 5 to be immobilized can be effectively used. The immobilization can also be performed by installing a measurement chip on a flow cell type surface plasmon resonance biosensor and flowing a labeled complementary strand nucleic acid 5 at a constant flow rate for a predetermined time (a predetermined amount). This immobilization method is advantageous in that the formation of the avidin layer 4 and the immobilization of the labeled complementary nucleic acid 5 can be performed by a series of operations. As a method for labeling the complementary strand nucleic acid 51 with biotin 52, PCR using a primer to which biotin 52 is bound is performed.
Can be exemplified.

The measurement chip of the present invention can be used, for example, for a surface plasmon resonance biosensor as shown in FIG. This surface plasmon resonance biosensor has a cartridge block 7, a light source 8, and a detector 9, and is used by mounting the measurement chip 6 of the present invention on the cartridge block 7. The measurement chip 6 is set so that the transparent substrate faces upward. A concave portion is provided on the upper surface of the cartridge block 7, and the concave portion and the measuring chip 6 constitute a measuring cell 71. Measurement cell 71
Is connected to the outside of the cartridge block 7 by the flow paths 72 and 73, and the sample flows into the measurement cell 71 through the flow path 72, and is discharged outside through the flow path 73 after being subjected to the measurement.

The light source 8 emits monochromatic light toward the transparent substrate of the measuring chip 6 (incident light 80).
The reflected light 90 reflected by the metal film provided on the back of
Light enters the detector 9. The detector 9 can detect the intensity of the reflected light 90.

With the above structure, a certain incident angle θ
, A reflected light intensity curve forming a valley is obtained. The valley in the reflected light intensity curve is due to surface plasmon resonance. That is, when light is totally reflected at the interface between the transparent substrate and the outside of the measurement chip 6, a surface wave called an evanescent wave is generated at the interface, and a surface wave called a surface plasmon is also generated on the metal film. When the wave numbers of the two surface waves match, resonance occurs, and part of the light energy is used to excite surface plasmons, and the intensity of the reflected light decreases. Here, the wave number of the surface plasmon is affected by the refractive index of the medium very close to the surface of the metal film. Is generated, the incident angle θ changes. Therefore, it is possible to detect a change in the concentration of the measurement target substance due to the shift of the valley of the reflected light intensity curve. The change amount of the incident angle θ is called a resonance signal,
A change of 10 ^-4 ° is expressed as 1 RU.

[0023]

The measurement chip of the present invention can immobilize a nucleic acid, and can measure a substance to be measured with good sensitivity even if a small amount of the nucleic acid is immobilized.

[Brief description of the drawings]

FIG. 1 is a schematic sectional view showing one embodiment of a measuring chip of the present invention.

FIG. 2 is a schematic cross-sectional view showing one embodiment of the measurement chip of the present invention on which a complementary nucleic acid is immobilized.

FIG. 3 is a conceptual diagram of a surface plasmon resonance biosensor used for the measurement chip of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Transparent substrate 2 ... Metal film 3 ... Organic material layer 4 ... Avidin layer 5 ... Labeled complementary nucleic acid 51 ... Complementary nucleic acid 52 ... Biotin 6 ... Measurement chip 7 ... Cartridge block 71 ... Measurement cell 72, 73 ... Flow path 8 ... light source 80 ... incident light 9 ... detector 90 ... reflected light

Claims (4)

[Claims] 1. A semiconductor device comprising: a transparent substrate; a metal film disposed on the transparent substrate; an organic material layer disposed on the metal film; and an avidin layer disposed on the organic material layer. Measurement chip for surface plasmon resonance biosensor. 2. The method according to claim 1, wherein the organic material layer is 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyldiethoxymethylsilane,
-(2-aminoethylaminopropyl) trimethoxysilane, 3- (2-aminoethylaminopropyl) dimethoxymethylsilane, 3-mercaptopropyltrimethoxysilane, dimethoxy-3-mercaptopropylmethylsilane, mercaptoaminomethane, 2- Mercapto-1
-Aminoethane, 3-mercapto-1-aminopropane, 4-mercapto-1-aminobutane, mercaptoacetic acid, 2-mercaptopropionic acid, 3-mercaptobutyric acid, 4-mercaptovaleric acid, 1,1,1-triamino-
2-mercaptoethane, 1,1,1-triamino-3-
Mercaptopropane, mercaptoacetaldehyde, 2
-Mercaptopropylaldehyde, 3-mercaptobutyraldehyde, 4-mercaptovaleraldehyde, dimercaptomethane, 1,2-dimercaptoethane, 1,3
The measurement chip for a surface plasmon resonance biosensor according to claim 1, wherein the measurement chip is a layer formed of dimercaptopropane, 1,4-dimercaptobutane, or 1,5-dimercaptopentane. 3. A method for manufacturing a measurement chip for a surface plasmon resonance biosensor, comprising: arranging a metal film, an organic substance layer, and an avidin layer on a transparent substrate in this order. 4. The method according to claim 1, wherein the organic material layer comprises 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyldiethoxymethylsilane,
-(2-aminoethylaminopropyl) trimethoxysilane, 3- (2-aminoethylaminopropyl) dimethoxymethylsilane, 3-mercaptopropyltrimethoxysilane, dimethoxy-3-mercaptopropylmethylsilane, mercaptoaminomethane, 2- Mercapto-1
-Aminoethane, 3-mercapto-1-aminopropane, 4-mercapto-1-aminobutane, mercaptoacetic acid, 2-mercaptopropionic acid, 3-mercaptobutyric acid, 4-mercaptovaleric acid, 1,1,1-triamino-
2-mercaptoethane, 1,1,1-triamino-3-
Mercaptopropane, mercaptoacetaldehyde, 2
-Mercaptopropylaldehyde, 3-mercaptobutyraldehyde, 4-mercaptovaleraldehyde, dimercaptomethane, 1,2-dimercaptoethane, 1,3
The method for producing a measurement chip for a surface plasmon resonance biosensor according to claim 3, wherein the measurement chip is formed using -dimercaptopropane, 1,4-dimercaptobutane, or 1,5-dimercaptopentane.