JPH11211728A - Chip for measuring hemoglobin a1 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To strongly and easily bond a hemoglobin A antibody to be fixed to a metal film by fixing the hemoglobin antibody A onto the metal film of a measurement chip for a plasmon resonance bio sensor due to hydrophobic bond or electrostatic bond. SOLUTION: A transparent substrate 1 with a metal film 2 is exposed into the saturation steam of a silane coupling agent, and an organic silicon film is formed on the metal film 2. First, 30 ml methyltriethoxysilane is loaded into a wide-mounthed petri dish as an undiluted solution and is left to stand at a room temperature for 24 hours, and the inside of the petri dish is filled with the saturation steam of propyltriethoxysilane. Then, the transparent substrate 1 is fixed to a stainless mask (support tool) so that the part of the metal film is exposed, the mask is placed on the opening of the petri dish and is left to stand for 30 minutes, an organic silicon film 3 is formed on the metal film of a cover glass, and then a 70 ml saccharification hemoglobin (HbA) antibody with 8.2×10<3> <mg> /ml concentration is dripped onto it before being left to stand still for 120 minutes. After rinsing, it is naturally dried, thus obtaining a measurement chip.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION using surface plasmon resonance biosensor (SPR sensor) for measuring a chip based on an antigen-antibody reaction, relates to a method for measuring hemoglobin A _1.

[0002]

2. Description of the Related Art It is said that diabetes accounts for more than 5% of the world population, and there are 5 million people in Japan alone. The disease is characterized by a deficiency in insulin, which metabolizes glucose and other nutrients. Therefore, in order to treat this disease, there is a method of administering a required amount of insulin from the outside of the human body, and the required amount is such that glucose in blood is measured and brought to a normal value as much as possible. Therefore, it is essential to measure the amount of glucose in the blood first, but glycated hemoglobin (HbA ₁ ) in blood, especially hemoglobin A _1C (HbA _1C )
It is known that the blood concentration of glycated hemoglobin is increased in diabetic patients, and the measurement of glycated hemoglobin is used for the diagnosis of diabetes and the measurement of the progress thereof in parallel with the measurement of blood glucose level.

[0003] Glycated hemoglobin (HbA ₁ ) is caused by non-enzymatic sugar linkages of hemoglobin. The concentration of HbA ₁ in the blood is normal about the total hemoglobin 4.5
%, But in diabetic patients this concentration rises to 2-4. The reason that glycated hemoglobin (HbA ₁ ) was selected as an indicator of diabetes is that the concentration of HbA ₁ reflects the average sugar concentration in blood in the past 1 to 3 months, and is higher than that in blood and urine. Because it is less susceptible to physiological factors, the concentration of HbA ₁ is a relatively accurate measure of the average blood glucose concentration of diabetic patients over a long period of time.

Various methods for measuring the concentration of HbA ₁ have been developed in the past. As one of them, there is one called a fluorescence measurement or a photometry. In this method, glycated hemoglobin is distinguished from non-glycated hemoglobin using haptoglobulin, and the binding of glycated hemoglobin to haptoglobulin is measured by fluorescence measurement or photometry (Japanese Patent Publication No. 1-5).
No. 3748). Another method uses high performance liquid chromatography. This is to distinguish between glycated hemoglobin and non-glycated hemoglobin using high performance liquid chromatography.
Since it is very sensitive to changes in temperature and ion concentration, it requires not only careful implementation but also has the disadvantage that the apparatus is very large and expensive.

Further, as another method, a method of measuring by ion exchange chromatography (Japanese Patent Publication No. Hei 7-6989) and a method of using column chromatography (JP-A-5-113440).
However, these methods first perform centrifugal separation of whole blood, measure Hb in blood cells, and perform column measurement, and the operation is complicated. That is, in each of the above methods, hemoglobin in a sample (extract separated from a hemolytic component) is directly bound to a solid phase, washed, and reacted with a labeled anti-hemoglobin antibody, particularly an anti-hemoglobin A _1C antibody. , Washing again and measuring the label. However, this method requires a pretreatment in which glycated hemoglobin in each sample solution is allowed to directly react with the solid phase, which takes time and makes it impossible to rapidly measure a large amount of sample. As described above, the conventional techniques have disadvantages such as complicated operation, low accuracy, need for expensive equipment, and large size that cannot be easily measured.

[0006] Therefore, there has been a demand for a method that can easily and accurately perform measurement with a small and inexpensive device. The applicant has filed a patent application as Japanese Patent Application No. 9-157226. It should be understood that use of the surface plasmon resonance biosensor, where it is characterized in that to fix the hemoglobin A ₁ antibody measuring chip therefor, which has been to find a way that allows its fixed more firmly and easily is there.

[0007]

Of the present invention 0008] problems is the measurement chip for a surface plasmon resonance biosensor, is to find a means of hemoglobin A ₁ antibodies immobilized firmly and easily bonded with a metal film .

[0008]

[Summary of a result of intense research in view of the above problems, the present inventors have immobilized hemoglobin A ₁ antibody hydrophobic bonding or electrostatic bonding by surface plasmon resonance biosensor measurement chip metal film It has been found that it is effective to solve the problem.

In the conventional method of immobilizing a protein on a metal surface through a reactive monomolecular film, a reactive group is provided at one end and an active group which is reacted and adsorbed with an oxide film on the metal surface at the other end. After forming a reactive monomolecular film on the metal surface by a chemisorption method using an organic molecule having the compound, the reactive group is chemically treated to add an -OH group, and further, using a cyanobromide method or an aldehyde method. Since it is required to fix the surface of the monolayer by reacting the amino group of the protein with the functional group that reacts with the amino group of the protein, a reagent for immobilizing the protein is required. It has the drawback that the number increases and the time required for the reaction increases. Thus, in the present invention, this disadvantage has been solved by causing a hydrophobic bond or an electrostatic bond instead of a chemical bond (covalent bond).

[0010] Namely, the present invention includes (1) a transparent substrate, are arranged in a hydrophobic bond or an electrostatic bond on the metal film and the metal film is disposed on the transparent substrate, hemoglobin A ₁ antibody (2) The measurement chip for a surface plasmon resonance biosensor according to (1), wherein the hydrophobic bond or the electrostatic bond forms an organosilicon film. (3) wherein the organosilicon film is a film formed by a silane coupling agent,
(2) the surface plasmon resonance biosensor measurement chip according to (2), (4) the metal disposed on the transparent substrate is gold, the surface plasmon resonance biosensor measurement chip according to (1), 5) The measurement chip for a surface plasmon resonance biosensor according to 1), wherein the film formed by the hydrophobic bond is mainly composed of a compound of metal and fluorine or a mixture of metal and fluorine. (6) The metal is made of gold. (5) The measurement chip for a surface plasmon resonance biosensor according to (5), wherein (7) the film formed by the hydrophobic bond is mainly composed of a compound of metal and carbon or a mixture of metal and carbon ( The measurement chip for a surface plasmon resonance biosensor according to 1), (8) the surface plasmon resonance biosensor according to (7), wherein the metal is gold. Over a measurement chip, (9) membrane by a hydrophobic bond is being formed by exposure to plasma including fluorine-based gas (1), wherein the surface plasmon resonance biosensor measuring chip, (10)
(1) The measurement chip for a surface plasmon resonance biosensor according to (1), wherein the film due to the hydrophobic bond is formed by exposing to a plasma containing carbon; (1) The measurement chip for a surface plasmon resonance biosensor according to (1), (12) the surface plasmon resonance biosensor according to (1), wherein the membrane by the hydrophobic bond contains a fluorine-containing organic compound as a main component. Measuring chip for (1,
3) The measurement chip for a surface plasmon resonance biosensor according to (1), wherein the film formed by hydrophobic bonding or electrostatic bonding is thiol; (14) the thiol film is a film formed of alkanethiol. (13) The measurement chip for a surface plasmon resonance biosensor according to (13), wherein (15) the hemoglobin A ₁ antibody is a hemoglobin A _1C antibody (1).
(16) After arranging a metal film on a transparent substrate, forming a film on the metal film by hydrophobic bonding or electrostatic bonding, and then forming hemoglobin A on the film. characterized by placing ₁ antibodies, a method of manufacturing a surface plasmon resonance biosensor measurement chip, a method of measuring hemoglobin a ₁ antibodies using the measurement chip according (17) (1), (18)
(17) The method for producing a measurement chip for a surface plasmon resonance biosensor according to (17), wherein the hemoglobin A ₁ antibody is a hemoglobin A _1C antibody; The present invention relates to the method for measuring a hemoglobin A ₁ antibody according to (17), which is characterized by administering.

In order to cause a hydrophobic bond, it is necessary to have a hydrophobic surface in the outermost layer. Therefore, a saturated alkyl chain terminal or a gold compound containing a fluorine atom or a mixture of gold and fluorine is used. In order to form a gold compound containing a fluorine atom or a mixture of gold and fluorine in a hydrophobic bond,
Conventional thin film forming technologies, such as sputtering, vacuum deposition,
Ion plating, CVD, plasma polymerization,
Dry etching, spin coating, etc. can be used. FIG. 10 shows the procedure for immobilizing hydrophobic bonds.

In order to obtain a thin film mainly containing a gold compound containing a fluorine atom or a mixture of gold and fluorine by a sputtering method, a target mainly composed of gold or a fluorinated gold of the surface is used. , Xenon, helium, nitrogen or other sputter gas alone or an atmosphere combining these sputter gases with a fluorine source gas such as fluorine, carbon tetrafluoride, sulfur hexafluoride, nitrogen trifluoride or chlorine trifluoride Inside, a sputter film is formed by direct current or high frequency discharge. At this time, if necessary, a gas serving as an oxygen source such as oxygen, nitrogen, or carbon dioxide, a nitrogen source, or a carbon source can be mixed. Here, by changing the film forming conditions such as the composition of the atmosphere gas, the pressure, and the sputtering power,
The hydrophobicity, refractive index, and extinction coefficient can be adjusted by changing the composition, structure, structure, and the like of the film.

In order to obtain a thin film mainly containing a gold compound containing a fluorine atom or a mixture of gold and fluorine by a vacuum evaporation method, a resistance heating method using a tungsten basket, a tungsten-plated coil or boat, a carbon crucible or the like is required. Or so-called EB by electron beam irradiation
Gold or gold which has been fluorinated beforehand is used as an evaporation source by an evaporation method. At this time, a fluorine source gas such as fluorine, carbon tetrafluoride and other carbon fluoride compounds, sulfur hexafluoride and other sulfur fluoride compounds, nitrogen trifluoride, chlorine trifluoride, etc. Thus, a mixed gas with an oxygen source such as oxygen, nitrogen, and carbon dioxide, a nitrogen source, and a gas serving as a carbon source can be used as the atmosphere. Also in this case, by changing the deposition conditions, the film composition, structure, structure, and the like can be changed, and the hydrophobicity, refractive index, and extinction coefficient can be adjusted.

Further, in order to obtain a thin film mainly containing a gold compound containing a fluorine atom or a mixture of gold and fluorine by an ion plating method, gold or gold which has been fluorinated beforehand is used as an evaporation source. Fluorine source gas such as fluorine, carbon tetrafluoride, sulfur hexafluoride, nitrogen trifluoride, chlorine trifluoride, etc., and, if necessary, oxygen source such as oxygen, nitrogen, carbon dioxide gas, nitrogen source, carbon The atmosphere may be a mixed gas with a source gas. Also in this case, the hydrophobicity, the refractive index, and the extinction coefficient can be adjusted depending on the film forming conditions.

In the CVD method, a siloxane monomer such as hexamethylenedisiloxane (HMDSO) is introduced with a carrier gas such as argon to form a thin film mainly composed of a siloxane film on a gold surface, and an atmosphere such as oxygen is introduced. The film is formed by a chemical vapor deposition (CVD) method in the inside. If necessary, a fluorine source gas such as a fluorine-containing siloxane monomer or carbon tetrafluoride can be mixed, or
A fluorine compound (plasma polymerized film) can also be formed using a fluorine source gas or the like without adding a siloxane monomer. Here, by changing the film formation conditions such as the composition, pressure, and power of the atmosphere gas, the composition, structure, structure, and the like of the film can be changed, and the hydrophobicity and the like can be adjusted.

In the spin coating method, for example, "CYTOP (made by Asahi glass)", which is an amorphous fluororesin, is applied on a metal film to provide an SPR substrate. Although the above description has been directed to a gold compound containing a fluorine atom or a mixture of gold and fluorine, the same applies when carbon is used instead of fluorine.

On the other hand, as one of the hydrophobic bonds, an organic silicon film is bonded to a metal atom in the metal film 2 by using a silane coupling agent to bond a hydrolyzable group in the silane coupling agent with a hemoglobin. a method of causing a binding by a ₁ antibody and physisorption. Thereby the metal film 2, the organosilicon film 3 and hemoglobin A ₁ antibody tripartite is immobilized. The organic silicon film 3 refers to a film made of a polymer containing Si—O and Si—C bonds in the molecule.

The silane coupling agent includes an organic functional group having an affinity for an organic material such as a vinyl group, an epoxy group, an amino group, and a mercapto group, a methoxy group,
It refers to an organosilicon compound having a hydrolyzable group having an affinity for an inorganic material such as an ethoxy group. The silane coupling agent that can be used in the present invention may be any silane coupling agent as long as it satisfies the above definition, and has a structural formula of CF ₃ (C
H ₂ ) nSi (OCH ₃ ) ₃ trifluoromethyltrimethoxysilane, 2,2,2-trifluoromethyltrimethoxysilane, 3,3,3-trifluoropropyltrimethoxysilane, 4,4,4-trimethylsilane A structural formula CF ₃ (C, such as fluorobutyltrimethoxysilane, 8,8,8-trifluorooctyltrimethoxysilane, 18,18,18-trifluorooctadecyltrimethoxysilane, etc.
_{H 2) nSi (OCH 2 CH} 3) 3 trifluoromethyl triethoxy silane, 4,4,4-trifluoro-butyl triethoxysilane, aminopropyl diethoxymethyl silane, 18,18,18- trifluoroacetic octadecyltriethoxysilane Silane, structural formula CH ₃ (CH ₂ ) nSi
(OCH _{3) 3} of methyltrimethoxysilane, ethyltrimethoxysilane, propyl Le trimethoxysilane, octadecyl trimethoxysilane, structural formula CH ₃ (C
_{H 2) nSi (OCH 2 CH} 3) 3 of methyltriethoxysilane, pen triethoxysilane can be used alone or in combination octadecyl trimethoxysilane. Note that n in the above structural formulas is an integer of 0 to 17.

Next, as for the electrostatic binding, a sulfur compound can be used between the two to immobilize the hemoglobin A ₁ antibody on the gold surface as a physical adsorption method.
Generally, among the sulfur compounds, thiols, especially alkanethiols, are particularly suitable in the present invention in view of operability, denseness, stability and the like. In the case of a thiol compound, a hydrophobic bond is also possible.

Alkanethiol means a methyl group, an amino group, a mercapto group,
An organic sulfur compound having an organic functional group having an affinity for an organic material such as a trifluoro group and an SH group at the other end. The alkanethiol has a structural formula of CH ₃ (CH ₂ ) nSH (n = 1 to 15) such as 1-propanethiol, 1-butanethiol, 1-hexadecanethiol in the case of a hydrophobic bond. In the case of electrostatic coupling, HOOC such as mercaptoacetic acid, 3-mercaptopropionic acid, etc.
_{(CH 2) nSH (n =} 1~15) and aminoethanethiol etc. _{H 2 N (CH 2) nSH} (n = 1~15) , and the like.

By tilting the pH toward the alkaline side, the terminal carboxyl group becomes anion-rich, which forms an electrostatic bond with a positively charged functional group of the hemoglobin A ₁ antibody (eg, an amino group in a lysine residue). Moreover, by tilting the pH to the acidic side, it becomes cation rich, which forms a capacitive coupling with the functional groups (e.g. carboxyl groups in aspartic acid residue) having a negatively charged hemoglobin A ₁ antibody.

It is also possible to form an organic silicon film by electrostatic coupling. For example, as a silane coupling agent,
Aminomethyltrimethoxysilane of structural formula H ₂ N (CH ₂ ) nSi (OCH ₃ ) ₃ , 2-aminoethyltrimethoxysilane, 3-aminopropyltrimethoxysilane, 4
-Aminobutyltrimethoxysilane, 8-aminooctyltrimethoxysilane, 18-aminooctadecyltrimethoxysilane, etc., H ₂ N (CH ₂ ) nSi (OC ₂ H ₅ )
₃ , aminoaminotriethoxysilane, 2-aminoethyltriethoxysilane, 3-aminopropyltriethoxysilane, 5-aminopentyltriethoxysilane, 8
-Aminooctyltrimethoxysilane, 18-aminooctadecyltrimethoxysilane, etc.
By tilting the pH to the acidic side, the terminal amino group becomes cation-rich. On the other hand, a functional group having a negative charge (such as a carboxyl group or a sulfonyl group) and a quaternary amino group form an electrostatic bond. Note that n in the above structural formulas is an integer of 0 to 17.

[0023]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
The measurement chip for a surface plasmon resonance biosensor (hereinafter, simply referred to as “measurement chip”) used in the present invention is a chip used for a surface plasmon resonance biosensor, and transmits and reflects light emitted from the sensor. parts, and refers to a member comprising a portion for hemoglobin a ₁ antibody and immobilized it may be intended to be secured to the body of the sensor, or may be those removable.

The measuring chip used in the present invention is a transparent substrate,
Comprises a metal film disposed on the transparent substrate, membrane bound hydrophobic bond or electrostatic is disposed on the metal film, and the hemoglobin A ₁ antibody disposed on the membrane. 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 “hydrophobically or electrostatically bonded membrane disposed on a metal film” and the “hemoglobin A ₁ antibody disposed on the film” have the same meaning as described above.

A schematic cross-sectional view of a measuring chip according to an example of the present invention is shown in FIGS.
And 3. The measuring chip used in this embodiment includes a transparent substrate 1 and a metal film 2 formed on the transparent substrate 1.
And an organic silicon film 3 formed on the metal film 2 and a hemoglobin A ₁ antibody 4 immobilized on the surface of the organic silicon film 3. The transparent substrate 1 may be any substrate as long as it is generally used for a measurement chip for a surface plasmon resonance biosensor.
It is possible to use a material such as polyethylene terephthalate or polycarbonate which is transparent to a laser beam or the like, does not exhibit anisotropy with respect to polarized light, and is excellent in workability. It is about.

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 in the range of 100 to 2000 °, more preferably 200 to 600 °. If it exceeds 3000 mm, 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 metal film 2 may be formed by a conventional method, for example, a sputtering method, a vapor deposition 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 silicon film 3 is preferably a monolayer film in which silicon atoms do not overlap in the vertical direction. By forming a monolayer film, the distance between the measurement object interacting with the hemoglobin A ₁ antibody and the surface on which the incident light is reflected can be shortened, and good sensitivity can be obtained, and the silane used can be obtained. The amount of the coupling agent can be minimized, and the cost can be reduced.

The organic silicon film 3 preferably has a close-packed structure. The close-packed structure refers to the organic silicon film 3
Means that the network structure is so dense that there is no room for other molecules to penetrate into the network structure of Si and O.
By adopting the close-packed structure, the hemoglobin A ₁ antibody can be uniformly fixed at a high density, and the measurement sensitivity can be improved. Whether or not the organic silicon film 3 has a close-packed structure can be confirmed by the following method.

An organosilicon film 3 is formed on the metal film 2 using propylethoxysilane (a compound in which the amino group of 3-aminopropyltriethoxysilane, which is a silane coupling agent, is substituted with a hydrogen atom). Since propylethoxysilane is a compound having strong hydrophobicity, the density of propylethoxysilane (that is, the degree of close packing of the organic silicon film 3) can be known from the degree of wetting of the surface of the organic silicon film 2. That is, if the surface repels water droplets uniformly when distilled water is dropped with a syringe, the organosilicon film 3 has a finely packed structure, and if the surface only partially repels water, It is presumed that voids are present in the network structure of Si and O without a filled structure.

The organic silicon film 3 can be formed by using, for example, a silane coupling agent.
Specifically, a method of exposing the metal film 2 to a saturated vapor of the silane coupling agent for a predetermined time (saturated vapor method), and a method of immersing the metal film 2 in a solution containing the silane coupling agent for a predetermined time (immersion method) ), A method using a spin coater (spin coating method), a method using a gravure printing machine (gravure method), and the like. In the present invention, any of these methods may be used, but it is preferable to use a saturated vapor method in order to form a monolayer film having a finely packed structure.

In the saturated vapor method, the temperature, humidity, and the like at the time of exposure also affect the formation of the monolayer structure and the close-packed structure, but the exposure time is the most important factor. If the exposure time is too long, a monolayer structure cannot be obtained, and if the exposure time is too short, a finely packed structure cannot be obtained. Exposure time is
Usually, it is preferably from 1 to 600 minutes, more preferably from 15 to 90 minutes.

The organosilicon film 3 of the present invention has the following advantages. Since hemoglobin A ₁ antibody can be immobilized very close to the metal film 2, it is possible to greatly improve the measurement sensitivity than with conventional measuring chip. Film formation is easy, and a large amount of film formation processing can be performed at once. By changing the type of silane coupling agent,
In addition to a film thickness suitable for physical adsorption, chemical modification such as surface modification and functional group introduction becomes possible. The measurement chip of the present invention is used by directly fixing hemoglobin A ₁ antibody to the organosilicon film 3.

Usually, the Fc fragment of the antibody 4 is immobilized only on the surface of the organic silicon film 3, and the antibody is formed in a monolayer state. However, as the Fab fragment of the antibody 4 moves away from the organosilicon film 3, the sensitivity and the reaction rate decrease, and as shown in FIG. 3, the Fab fragment (FIG. 3A)
) Or F (ab ') ₂ fragment (FIG. 3 (b)) may be directly immobilized on the organosilicon film 3 to improve sensitivity and reaction rate.

The thickness of the hemoglobin A ₁ antibody 4 depends on the size of the hemoglobin A ₁ antibody used,
It is preferably from 3,000 to 3,000, more preferably from 100 to 1,000. The hemoglobin A ₁ antibody may be immobilized by a conventional method, for example, by bringing a predetermined amount of hemoglobin A ₁ antibody into contact with the organosilicon film 3 for a predetermined time. Also be immobilized by flowing a constant flow Hemoglobin A ₁ antibody a predetermined time by installing a measuring chip to the flow cell type surface plasmon resonance biosensor (predetermined amount).

When the Fab fragment of the hemoglobin A ₁ antibody 4 is directly immobilized on the organosilicon film 3, the same treatment may be performed after the antibody 4 is partially decomposed with papain. On the other hand, when the F (ab ′) ₂ fragment of the antibody 4 is directly immobilized on the organosilicon film 3, the same treatment may be performed after the antibody 4 is partially decomposed using pepsin. By firmly immobilizing the hemoglobin A ₁ antibody directly on the silane coupling agent as described above, the immobilization of the hemoglobin A ₁ antibody can be maintained even when the measurement chip is washed, so that it can be used for repeated measurement. The advantage is obtained.

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 10 of the present invention on the cartridge block 7. The measurement chip 10 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 10 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 irradiates monochromatic light toward the transparent substrate of the measuring chip 10 (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 10, 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 in close proximity to the surface of the metal film, the refractive index of the medium by the interaction between the analyte and hemoglobin A ₁ antibody changes, surface plasmon The incident angle θ at which resonance occurs 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, and a change of 10 ⁻⁴ ° is represented as 1 RU.

[0039]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples, but the scope of the present invention is not limited to these Examples. Example 1 In this example, a measurement chip having a configuration as shown in FIG. 1 was manufactured. 13 for transparent substrates
A cover glass (manufactured by Matsunami Glass Industry Co., Ltd.) having a size of mm × 18 mm and a thickness of 0.17 mm was used. On this transparent substrate, a layer made of chromium and then a layer made of gold were formed by sputtering. Sputtering was carried out at 100 W for 30 seconds for chromium and 100 W for 150 seconds for gold. The thickness of the obtained chromium layer was 32.2 mm, and the thickness of the gold layer was 474 mm.

The transparent substrate having the above metal film was exposed to saturated vapor of a silane coupling agent to form an organosilicon film on the metal film. First, 30 ml of undiluted methyltriethoxysilane was placed in a wide-mouthed petri dish and allowed to stand at room temperature for 24 hours.
The plate was left for a period of time, and the inside of the dish was filled with saturated vapor of propyltriethoxysilane. Next, the transparent substrate prepared above was fixed to a stainless steel mask (support) so that the metal film portion was exposed, and the mask was placed on the opening of the petri dish, and allowed to stand for 30 minutes. the organic silicon film is formed on the upper, subsequently its on ^8.2 × 10 -3mg / ml concentration anti HbA ₁ antibodies of and 70ml added dropwise, and allowed to stand for 120 minutes. After rinsing, the sample was air-dried to prepare a measurement chip.

This measurement chip was set on a cartridge block of a commercially available surface plasmon resonance biosensor (BIAcore2000, manufactured by Pharmacia Biosensor). This sensor has a structure as shown in FIG. In a measurement cell with a measurement chip on which an antibody is immobilized, 0.0
The light intensity was measured under a stream of hemoglobin A1 _C, diluted to 1,0.1,1,10,100μg / ml respectively at a flow rate of 5 [mu] l / min for 10 minutes, was determined resonance signal (RU). The result is shown in FIG.
Shown in

As shown in FIG. 6, the relationship between the sample concentration and the resonance signal was found to have a similar upward slope similar to the direct proportion. This is presumed to be due to the specific reaction of the antibody. Thus, by using the measurement chip according to the present embodiment, the antigen can be quantified by measuring the value of the resonance signal. In the above, the embodiment and the test example have been described by taking the method of forming the organic silicon film as an example. Next, the embodiment of forming the metal film by another method will be described.

[Embodiment 2] As shown in FIG.
A gold compound film is formed to a thickness of about 50 ° on a glass substrate having a thickness of 0 ° by a sputtering method under the following conditions, and an anti-hemoglobin A ₁ antibody is immobilized thereon. . Film forming apparatus; Planar type DC magnetron sputtering apparatus Target: Gold gas and flow rate: Argon gas 76 sccm + sulfur hexafluoride gas 24 sccm Sputter pressure; 3 mTorr Sputter current; 6 amps In place of the target gold, metal chromium is used. It is also possible.

[0044] Example 3 as shown in FIG. 8, after 500Å deposited gold on a glass substrate, in the process as shown below in a dry etching method, to process the gold surface, anti-hemoglobin A ₁ thereon Immobilize the antibody. Film forming apparatus; RF dry etching apparatus of parallel plate type Gas and flow rate: 80 sccm of carbon tetrafluoride + 20 sccm of oxygen Processing pressure: 50 Pa Input power: 500 W Note that 100 sccm of carbon dioxide gas may be used as the gas.

[Embodiment 4] As shown in FIG. 9, after forming a film of gold on a glass substrate to a thickness of 500 °, a siloxane film was formed by the CVD method as described below, and anti-hemoglobin A ₁ was formed thereon. Immobilize the antibody. Film forming apparatus: Parallel plate type RF CVD apparatus Gas and flow rate: HMDSO 10 sccm Oxygen 30 sccm
Argon 90 sccm Deposition pressure; 200 mTorr Input power; 100 W

An embodiment of the electrostatic coupling will be described below. Example 5 As in Example 1, a cover glass was used as a transparent substrate, and a chromium layer and then a layer made of gold were formed on the transparent substrate by sputtering. The transparent substrate having the metal film was immersed in an ethanol solution of alkanethiol to form a thiol film on the metal film. First,
Dissolve alkanethiol in ethanol in a 30 ml sample bottle and adjust to 1 mM. Next, the transparent substrate prepared above is immersed in an alkanethiol ethanol solution for one hour. Thereafter, the transparent substrate is pulled up, rinsed with ethanol, dried in N ₂ or air, and a thiol film is formed on the metal film of the cover glass, and 8.2 × 1 is formed thereon.
Anti-hemoglobin A ₁ antibody at a concentration of 0 ⁻³ mg / ml
μl was added dropwise and allowed to stand for 90 minutes. After rinsing, the sample was air-dried to prepare a measurement chip. The measurement chip was set on a cartridge block of a commercially available surface plasmon resonance biosensor (Pharmacia Biosensor, BIAcore2000). This sensor has a structure as shown in FIG.

[0047]

According to the present invention, in the measuring chip for a surface plasmon resonance biosensor for fixing the hemoglobin A ₁ antibody, it can be firmly and easily bonded to the metal film.

[Brief description of the drawings]

FIG. 1 is a schematic sectional view of a measuring chip according to an example of the present invention.

FIG. 2 is a schematic sectional view of a conventional measuring chip.

FIG. 3 is a schematic sectional view of a measuring chip according to another example of the present invention.

FIG. 4 is a conceptual diagram of a surface plasmon resonance biosensor used for the measurement chip of the present invention.

FIG. 5 is a schematic sectional view of a measurement chip used in Comparative Example 1.

6 is a graph showing the relationship between hemoglobin A ₁ concentration resonance signals.

FIG. 7 is a diagram showing film formation by a sputtering method.

FIG. 8 is a diagram showing a film formation configuration by a dry etching method.

FIG. 9 is a diagram showing a film formation configuration by a CVD method.

FIG. 10 shows an antibody immobilization procedure in a physical adsorption method (hydrophobic bond).

[Explanation of symbols]

1, 1 ': transparent substrate 2: metal film 2': metal film 3: organosilicon film 4, 4 ': hemoglobin A ₁ antibody 5: porous material 6: covalent bonding film 7: cartridge block 71: measuring cell 72, 73 ... Flow path 8 ... Light source 80 ... Incident light 9 ... Detector 90 ... Reflection light 10 ... Measuring chip

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Koji Sato 1-1-1 Ichigaya-Kagacho, Shinjuku-ku, Tokyo Dai Nippon Printing Co., Ltd.

Claims (19)

[Claims] 1. A transparent substrate are arranged in a hydrophobic bond or an electrostatic bond on the metal film and the metal film is disposed on the transparent substrate, the surface plasmons, characterized in that it comprises a hemoglobin A ₁ antibody Measurement chip for resonance biosensor. 2. The measurement chip for a surface plasmon resonance biosensor according to claim 1, wherein the hydrophobic bond or the electrostatic bond forms an organic silicon film. 3. The measurement chip for a surface plasmon resonance biosensor according to claim 2, wherein the organosilicon film is a film formed by using a silane coupling agent. 4. The measurement chip for a surface plasmon resonance biosensor according to claim 1, wherein the metal disposed on the transparent substrate is gold. 5. The measurement chip for a surface plasmon resonance biosensor according to claim 1, wherein the film formed by the hydrophobic bond mainly comprises a compound of metal and fluorine or a mixture of metal and fluorine. 6. The measurement chip for a surface plasmon resonance biosensor according to claim 5, wherein the metal is gold. 7. The measurement chip for a surface plasmon resonance biosensor according to claim 1, wherein the film formed by the hydrophobic bond mainly comprises a compound of metal and carbon or a mixture of metal and carbon. 8. The measurement chip for a surface plasmon resonance biosensor according to claim 7, wherein the metal is gold. 9. The measurement chip for a surface plasmon resonance biosensor according to claim 1, wherein the film by the hydrophobic bond is formed by exposing to a plasma containing a fluorine-based gas. 10. The measurement chip for a surface plasmon resonance biosensor according to claim 1, wherein the film by the hydrophobic bond is formed by exposing to a plasma containing carbon. 11. The measurement chip for a surface plasmon resonance biosensor according to claim 1, wherein the film formed by the hydrophobic bond contains siloxane as a main component. 12. The measurement chip for a surface plasmon resonance biosensor according to claim 1, wherein the membrane formed by the hydrophobic bond contains a fluorine-containing organic compound as a main component. 13. The measurement chip for a surface plasmon resonance biosensor according to claim 1, wherein the membrane formed by hydrophobic bonding or electrostatic bonding is thiol. 14. The thiol film is a film formed of alkanethiol.
The measurement chip for a surface plasmon resonance biosensor according to the above. 15. The measurement chip for a surface plasmon resonance biosensor according to claim _1, wherein the hemoglobin A ₁ antibody is a hemoglobin A _1C antibody. 16. After placing the metal film on a transparent substrate, and characterized in that film is formed by hydrophobic bonding or electrostatic binding on the metal film, then placing the hemoglobin A ₁ antibody on the membrane To manufacture a measurement chip for a surface plasmon resonance biosensor. 17. A method for measuring hemoglobin A ₁ antibody using the measurement chip according to claim 1. 18. The method for producing a measurement chip for a surface plasmon resonance biosensor according to claim 17, wherein the hemoglobin A ₁ antibody is a hemoglobin A _1C antibody. 19. The method for measuring hemoglobin A ₁ antibody according to claim 17, wherein whole blood is directly administered to a measurement chip for a surface plasmon resonance biosensor.