JPH112632A - Method for measuring hemoglobin A1 antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To make it possible to measure scarified hemoglobin A1 c antibody simply and quickly by providing an organic silicon film on the metal film on a transparent substrate, and using a surface plasmon resonance biosensor having a hemoglobin A1 antibody on the film. SOLUTION: A metal film 2 of gold, silver, copper or the like is formed on a transparent substrate 1, which is transparent for laser light such ad glass polyethylene terephthalate and polycarbonate, by sputtering, evaporation and the like. On the metal film 2, an organic silicon film 3 containing Si-O and SiC coupling in molecules is formed by using a silane coupling agent and the like. On the organic silicon film 3, HbA1 c 4 is fixed. The hydrolysis group in the silane coupling agent is coupled with the metal atoms in the metal film 2, and the organic functional group is coupled with the metal atoms in the metal film 2, and the organic functional group is coupled with the HbA1 c 4. Therefore, the three members of the metal film 2, the organic silicon film 3 and the HbA1 c 4 are rigidly coupled. Thus, the HbA1 c 4 can be made to be fixed very close to the metal film 2, and the measuring sensitivity can be improved by a large extent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION Surface plasmon resonance-based biosensor antigen-antibody reaction using (SPR sensor) for measurement chip, relates to a method for measuring hemoglobin A ₁ c antibody.

[0002]

2. Description of the Related Art It is said that diabetes accounts for more than 5% of the world population, and there are 6 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.

[0003] Therefore, although it is important to first measure the amount of glucose in the blood, glycated hemoglobin in the blood (HbA _1), in particular hemoglobin A ₁ c (HbA ₁ c)
It is known that the blood concentration of glycated hemoglobin is increased in diabetic patients, and the measurement of glycated hemoglobin is used in combination with the measurement of blood sugar level for the diagnosis and measurement of the progress of diabetes. Glycated hemoglobin (HbA ₁ ) is caused by non-enzymatic sugar linkages of hemoglobin. Normally Hb in the blood
The concentration of A ₁ is within about 4.5% of the total hemoglobin, this concentration the case of diabetic patients increased to 2-4 times.

[0004] The reason that glycated hemoglobin (HbA ₁ ) was selected as an indicator of diabetes was that the concentration of HbA ₁ was in the past 1 to 2
Reflects the average sugar concentration in the blood of months, that it hardly depends on physiological factors compared to the blood sugar value or urine sugar value, blood average glucose of diabetic patients concentration of HbA ₁ includes long term This is because it represents the concentration relatively accurately. Various methods for measuring the concentration of HbA ₁ have been developed in the past.

As one of them, there is a method 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. 1-37484). .

As another method, there is a method using 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 ion concentration, it requires not only careful implementation, but also has the disadvantage that the device is very large and expensive.

Further, as another method, a method of measuring by ion exchange chromatography (Japanese Patent Publication No. Hei 7-6989) or a method of using column chromatography (Japanese Patent Application Laid-Open No. Hei 5-113440).
However, in these methods, the whole blood is first centrifuged, Hb in the blood cells is lysed, and the column is measured, and the operation is complicated.

Namely, any of the above methods, the analyte is bound directly to the solid phase hemoglobin (extract was separated from the hemolysis component) in, washed, labeled anti-hemoglobin antibody, especially an anti-hemoglobin A ₁ c It is performed by reacting the 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. Therefore, there has been a demand for a method that can easily and accurately perform measurement with a small and inexpensive device.

[0009]

[SUMMARY OF THE INVENTION Diabetes index a is glycated hemoglobin, especially glycated hemoglobin A ₁ c antibody,
It is an object of the present invention to provide a method for measuring glycated hemoglobin which can be simply and rapidly measured.

[0010]

Means for Solving the Problems As a means for solving the above problems, as a result of the present inventors' intensive efforts, the present applicant has previously filed a surface plasmon resonance biosensor (SPR).
It has been found that it is extremely effective to use a measurement chip for sensors. As described in a prior application filed by the present applicant (Japanese Patent Application No. 9-73646), a measurement chip for an SPR sensor used in the present invention is a transparent substrate, a metal film disposed on the transparent substrate. , An organosilicon film disposed on the metal film, and Hb disposed on the organosilicon film
A ₁ c is provided.

In a conventional measurement method, whole blood is centrifuged,
After removing the blood cell component, the extract containing hemolyzed and glycated hemoglobin had to be collected.By using this chip, whole blood can be directly lysed and administered directly to the sensor chip. . This is because the antigen forms a specific bond with the antibody based on the antigen-antibody reaction, but other than the antigen can be removed by rinsing.
This is because only the information on the surface of the PR sensor chip that is very close to gold or the antibody is faithfully reflected as a resonance signal.

[0012]

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 ₁ c and immobilize it HbA, 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,
A metal film disposed on the transparent substrate; an organic silicon film disposed on the metal film; and HbA ₁ c disposed on the organic silicon film. 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. "Organic silicon film placed on metal film"
And "HbA ₁ c disposed on the organosilicon film" has 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 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 HbA ₁ c4 fixed 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, and is generally transparent to laser light such as glass, polyethylene terephthalate, and polycarbonate. A material made of a material can be used, and a material which does not show anisotropy with respect to polarized light and has excellent workability is desirable, and its thickness is about 0.1 to 20 mm.

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 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 includes Si—O and Si
A film made of a polymer containing a -C bond in a molecule. The organic silicon film 3 can be formed using, for example, a silane coupling agent. A silane coupling agent is an organic functional group that has an affinity for organic materials such as vinyl group, epoxy group, amino group, and mercapto group in its molecule, and an inorganic material such as methoxy group and ethoxy group that has affinity. Refers to an organosilicon compound having a certain hydrolyzable group. The hydrolyzable groups in the silane coupling agent are
The organic functional group binds to HbA ₁ c4 with the metal atom therein. Thereby, the metal film 2, the organic silicon film 3, and Hb
The three members of A ₁ c4 are firmly immobilized. 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, such as 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyldiethoxymethylsilane, 3
Using-(2-aminoethylaminopropyl) trimethoxysilane, 3- (2-aminoethylaminopropyl) dimethoxymethylsilane, 3-mercaptopropyltrimethoxysilane, dimethoxy-3-mercaptopropylmethylsilane or the like alone or in combination can do.

The organic silicon film 3 is preferably a monolayer film in which silicon atoms do not overlap vertically. By using a monolayer film, the distance between the measurement object interacting with HbA ₁ c and the surface on which the incident light is reflected can be shortened, and good sensitivity can be obtained. The amount of the ring agent can be minimized, and the cost can be reduced.

It is preferable that the organic silicon film 3 has a close-packed structure. The close-packed structure means that 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 taking a close-packed structure, HbA ₁ c can be fixed uniformly 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 in the present invention comprises:
It has the following advantages.

Since HbA ₁ c4 can be immobilized at a position very close to the metal film 2, the measurement sensitivity can be greatly improved as compared with the case where a conventional measurement chip is used. 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 film thickness, chemical modification such as surface modification and functional group introduction becomes possible.

Usually, as shown in FIG. 1, the Fc fragment of the antibody is immobilized only on the surface of the organosilicon film 3,
Antibodies are formed in a monolayer state. However, the Fab of the antibody
Since the sensitivity and the reaction rate decrease as the fragment moves away from the organosilicon film 3, the Fab fragment (FIG. 2 (a)) or the F (ab ') ₂ fragment (FIG. 2 (b)) as shown in FIG. The sensitivity and the reaction rate may be improved by directly immobilizing the organic silicon film 3.

The thickness of HbA ₁ c4 depends on the HbA ₁ c used.
Although it depends on the size of itself, it is preferably from 100 to 3000 °, particularly preferably from 100 to 1000 °. HbA ₁
The method of immobilizing c may be a conventional method. For example, the immobilization can be performed by bringing a predetermined amount of HbA ₁ c4 into contact with the organosilicon film 3 for a predetermined time. Further, a transparent substrate 1 on which an organosilicon film 3 is formed is set on a flow cell type surface plasmon resonance biosensor, and H is supplied at a constant flow rate.
It can also be fixed by flowing bA ₁ c4 for a predetermined time (a predetermined amount).

When the Fab fragment of HbA ₁ c is directly immobilized on the organosilicon film 3, the same treatment may be performed after partially decomposing HbA ₁ c using papain. On the other hand, when the F (ab ') ₂ fragment of HbA ₁ c is directly immobilized on the organosilicon film 3, the same treatment may be performed after partially decomposing HbA ₁ c using pepsin.

FIG. 3 is a schematic sectional view of a measuring chip according to another example of the present invention. The measuring chip according to the present embodiment has substantially the same configuration as the above-described measuring chip, but is formed on the organosilicon film 3 with a water-soluble divalent reagent (this is referred to as a “covalent bond film”). Is provided, and HbA ₁ c 4 is fixed to the organosilicon film 3 via the covalent bond film 6.

The water-soluble bivalent reagent forming the covalent bond film 6 is not particularly limited as long as it can firmly immobilize HbA ₁ c4 covalently. Examples of such a water-soluble divalent reagent include glutaraldehyde, periodic acid, N, N′-o-phenylenedimaleimide, N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, N-succinimidyl maleimide acetic acid, N-succinimidyl-4-maleimidobutyric acid, N-succinimidyl-6-maleimidohexanoic acid,
N-sulfosuccinimidyl-4-maleimidomethylcyclohexane-1-carboxylic acid, N-sulfosuccinimidyl-3-maleimidobenzoic acid, N- (4-maleimidobutyryloxy) sulfosuccinimide sodium salt, N
-(6-maleimidocaproyloxy) sulfosuccinimide sodium salt, N- (8-maleimidocapryloxy) sulfosuccinimide sodium salt, N- (11-
Maleimide undecanoyloxy) sulfosuccinimide sodium salt, N [2- (1-piperazinyl) ethyl] maleimide dihydrochloride and the like can be used alone or in combination. Among these, glutaraldehyde which is highly versatile and easy to handle is preferable.

By providing such a covalent bonding film 6, HbA ₁
By firmly immobilizing c4 by a covalent bond, HbA ₁ c4 can be maintained immobilized even when the measurement chip is washed, so that there is an advantage that it can be used repeatedly for measurement. The thickness of the covalent bonding film 6 is preferably from 10 to 100 °, more preferably from 10 to 20 °. The covalent bonding film 6 can be formed by bringing a water-soluble bivalent reagent into contact with the organosilicon film 3. The method of immobilizing HbA ₁ c4 on the covalent bond film 6 can be performed in the same manner as the method of immobilizing HbA ₁ c4 on the organosilicon film 3. 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 measuring 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. The measurement cell 71 communicates with the outside of the cartridge block 7 through the flow paths 72 and 73. The sample flows into the measurement cell 71 through the flow path 72, and is discharged to the outside through the flow path 73 after being used for measurement. .

The light source 8 irradiates monochromatic light toward the transparent substrate of the measuring chip 10 (incident light 80), and the measuring chip 10
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 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.

[0032]

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. As the transparent substrate, a cover glass (manufactured by Matsunami Glass Industry Co., Ltd.) having a size of 18 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 is 10 for chrome
The test was performed at 0 W for 30 seconds and for gold at 100 W for 150 seconds. 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 a saturated vapor of a silane coupling agent to form an organic silicon film on the metal film. First, an undiluted solution of γ-aminopropylethoxysilane (H ₂
N- (CH ₂ ) ₃ Si (OEt) ₃ , TSL 8331, Toshiba Silicone Co., Ltd.
30 ml at room temperature for 24 hours.
-Filled with saturated vapor of aminopropylethoxysilane. Next, the transparent substrate prepared above was fixed to an aluminum mask (support) so that the metal film portion was exposed, and the mask was placed on the opening of a petri dish and left for 60 minutes to form a cover glass metal film. An organic silicon film was formed thereon 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. It poured into a measuring cell of the sensor 5% glutaraldehyde at a flow rate of 1 [mu] l / min for 20 minutes, then poured 10 hours 1mg / ml anti-human hemoglobin A ₁ c antibody at a flow rate of 1 [mu] l / min, anti on the surface of the organic silicon film It was immobilized human hemoglobin a ₁ c antibody. Thereafter, BSA was used to block the immobilized antibody according to a conventional method. Next, 5 μl of 0.1 N hydrochloric acid was poured into the measurement cell at a flow rate of 5 μl / min in order to wash away the unfixed excess antibody.

The measuring cell was installed measurement chip immobilized with antibody, 0.01, 0.1, 1, 10, or while 100ppm of human Hemoglobin A ₁ c a (HbA ₁ c) flowing each at a flow rate of 5 [mu] l / min for 10 minutes light The intensity was measured and the resonance signal (RU) was determined. The result is shown in FIG. In the figure, the open circles indicate the case where HbA ₁ c was measured with the chip according to the present invention (Example 1). (2) shows a case where HbA ₁ c was measured with a commercially available measurement chip (Comparative Example 1).

As shown in FIG. 6, the relationship between the sample concentration and the resonance signal showed 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.

Comparative Example 1 A commercially available measuring chip (unfixed antibody) for the biosensor used in Example 1 was set in a cartridge block. This measuring chip has a structure as shown in FIG. To activate the porous material (carboxymethyldextran) of the measurement chip has, 1-ethyl-2,3-dimethylaminopropyl carbodiimide (400 mM / H ₂ O) and N
35 μl of a mixture with -hydroxysuccinimide (100 mM / H ₂ O) was poured into the measuring cell at a flow rate of 5 μl / min. Then, 50 [mu] g / ml of anti-human hemoglobin A ₁ c antibody 35μl
Was poured into a measuring cell at a flow rate of 5 [mu] l / min, it was immobilized anti-human hemoglobin A ₁ c antibody carboxymethyldextran. Thereafter, 35 μl of ethanolamine was poured into the measuring cell at a flow rate of 5 μl / min to block the immobilized antibody, and then 5 μl of 0.1 N hydrochloric acid at a flow rate of 5 μl / mi to wash away the unimmobilized excess antibody.
It poured into the measurement cell at n.

The measuring cell was installed measurement chip immobilized antibody, the light intensity was measured while flowing HbA ₁ c in the same manner as in Example 1 to obtain resonance signals (RU). The result is shown in FIG. An almost directly proportional relationship was observed between the sample concentration and the resonance signal. This is presumed to be due to the specific reaction of the antibody.

FIG. 6 shows a comparison of the resonance signal between the case where the measurement chip of Comparative Example 1 was used and the case where the measurement chip of Example 1 was used. A resonance signal (RU) almost twice as large as that obtained when one measurement chip was used was measured. Therefore,
By using the measurement chip of Example 1, it is possible to quantify antigens and the like with about twice the sensitivity.

Test Example 1 The intensity of the reflected light corresponding to the incident angle θ was measured for the measurement chip manufactured in Example 1 and the measurement chip used in the comparative example. The result is shown in FIG. In the figure, で indicates the reflected light intensity curve of the measurement chip manufactured in Example 1, and... Indicate the reflected light intensity curve of the measurement chip used in Comparative Example 1. As shown in FIG. 7, it can be seen that a sufficient degree of surface plasmon resonance occurs in either case.

[0042]

According to the present invention, a compact with good sensitivity, without the whole blood is centrifuged, i.e. easy to operate and with high accuracy, hemoglobin A ₁ c (H is an indicator substance for diabetes
bA ₁ c) can be measured without a label.

[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 measuring chip according to another embodiment of the present invention. (a) shows an example in which an antibody Fab fragment is immobilized, and (b) shows an example in which an antibody F (ab ') ₂ fragment is immobilized.

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.

FIG. 6 shows HbA ₁ c obtained in Example 1 and Comparative Example 1.
It is a graph which shows the relationship between a concentration and a resonance signal.

FIG. 7 is a graph showing a reflected light intensity curve of a measurement chip.

[Explanation of symbols]

1, 1 '... transparent substrate 2 ... metal film 2' ... metal film 3 ... organosilicon film 4 ... hemoglobin A ₁ antibody 6 ... covalent bonding film 7 ... cartridge block 71 ... measurement cell 72, 73 ... flow path 8 ... light source 80 … Incident light 9… detector 90… reflected light 10… measurement chip

Claims (13)

[Claims] 1. A transparent substrate, it is provided with a transparent metal film placed on a substrate, hemoglobin A ₁ antibodies which are disposed in the organic silicon film, and the organosilicon film is disposed on the metal film A measurement chip for a surface plasmon resonance biosensor characterized by the following. 2. The measurement chip for a surface plasmon resonance biosensor according to claim 1, wherein the organosilicon film is a film formed by a silane coupling agent. 3. The method according to claim 1, wherein the silane coupling agent is 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyldiethoxymethylsilane, 3- (2-aminoethylaminopropyl) trimethoxysilane, It is at least one selected from the group consisting of 3- (2-aminoethylaminopropyl) dimethoxymethylsilane, 3-mercaptopropyltrimethoxysilane, and dimethoxy-3-mercaptopropylmethylsilane. 2. The measurement chip for a surface plasmon resonance biosensor according to 2. 4. The organic silicon film and the hemoglobin A
_{2. The} measurement chip for a surface plasmon resonance biosensor according to claim 1, wherein a layer formed with a water-soluble bivalent reagent is further provided between the antibody and _one antibody. 5. The method according to claim 1, wherein the water-soluble divalent reagent is glutaraldehyde, periodic acid, N, N′-o-phenylenedimaleimide, N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate. , N-succinimidyl maleimide acetic acid, N-succinimidyl-4
-Maleimidobutyric acid, N-succinimidyl-6-maleimidohexanoic acid, N-sulfosuccinimidyl-4-maleimidomethylcyclohexane-1-carboxylic acid, N-sulfosuccinimidyl-3-maleimidobenzoic acid, N- ( 4
-Maleimidobutyryloxy) sulfosuccinimide sodium salt, N- (6-maleimidocaproyloxy) sulfosuccinimide sodium salt, N- (8-maleimidocapryloxy) sulfosuccinimide sodium salt, N- (11-maleimidoundecanoy) The surface plasmon resonance bio according to claim 4, characterized in that it is at least one member selected from the group consisting of roxy) sulfosuccinimide sodium salt and N [2- (1-piperazinyl) ethyl] maleimide dihydrochloride. Measurement chip for sensors. 6. A surface, comprising: arranging a metal film on a transparent substrate, arranging an organic silicon film on the metal film, and then arranging hemoglobin A ₁ antibody on the organic silicon film. A method for manufacturing a measurement chip for a plasmon resonance biosensor. 7. A method of manufacturing a surface plasmon resonance biosensor measurement chip, wherein the hemoglobin A ₁ antibodies described is hemoglobin A ₁ c antibodies to claim 6. 8. The method of claim 6, wherein the organosilicon film is formed using a silane coupling agent. 9. A silane coupling agent comprising 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyldiethoxymethylsilane, 3- (2-aminoethylaminopropyl) trimethoxysilane, It is at least one selected from the group consisting of 3- (2-aminoethylaminopropyl) dimethoxymethylsilane, 3-mercaptopropyltrimethoxysilane, and dimethoxy-3-mercaptopropylmethylsilane. 8. The method for producing a measurement chip for a surface plasmon resonance biosensor according to 7 above. 10. A method for measuring hemoglobin A ₁ antibody using the measurement chip according to claim 1. 11. The hemoglobin A _{1 according} to claim 10,
Measurement wherein the antibody is a hemoglobin A ₁ c antibody. 12. The method for measuring hemoglobin A ₁ antibody according to claim 10, wherein whole blood is directly lysed and directly administered to a measurement chip for a surface plasmon resonance biosensor. 13. The method of claim 1 or a surface plasmon resonance biosensor measurement chip, wherein the hemoglobin A ₁ antibodies described is hemoglobin A ₁ c antibody 4.