JP2002228661A - Measuring chip for surface plasmon resonance biosensor and method for producing the same - Google Patents

Abstract

(57) [Summary] [Problem] To provide a measurement chip for a biosensor capable of detecting and measuring a microorganism, a virus, or an environmental hormone in real time with high sensitivity without complicated operation or using a labeling substance. An optically transparent substrate, a metal thin film formed on the substrate, an aminosilane film formed on the metal thin film, and a physiologically active substance immobilized on the aminosilane film are provided. In a measurement chip for a surface plasmon resonance biosensor, at least one substance selected from the group consisting of concanavalin A, sialic acid-containing sialo-glycan, estrogen receptor, testosterone receptor and cyclodextrin is used as a physiologically active substance. A measurement chip for a surface plasmon resonance biosensor and a method for producing the same.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a surface capable of easily detecting microorganisms such as Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, viruses such as influenza virus, or exogenous secretory disrupting chemicals, ie, environmental hormones, which have recently become a problem. The present invention relates to a measurement chip for a plasmon resonance biosensor and a method for manufacturing the same.

[0002]

2. Description of the Related Art At present, a method utilizing an immunological reaction such as an antigen-antibody reaction, for example, an ELISA method is used as a method for detecting and measuring a substance to be measured in a sample in a clinical test or the like. However, conventional methods such as the ELISA method require complicated operations and labeling substances. Therefore, as a method of detecting and measuring a target substance in a sample without complicated operations or a labeling substance, a so-called surface plasmon resonance that occurs when surface plasmon is excited at a metal / liquid interface is used. A method utilizing the method is developed, a transparent substrate as a measurement chip for a surface plasmon resonance measurement device (hereinafter, referred to as “surface plasmon resonance biosensor”) used in the method, a metal film disposed on the transparent substrate, An organosilicon film disposed on the metal film and a measurement chip including a physiologically active substance disposed on the organosilicon film have been proposed and used for detection and measurement of human serum albumin and the herbicide atrazine. (JP-A-10-267930). Further, as a measurement chip for a plasmon resonance biosensor, a transparent substrate, a metal film formed on the transparent substrate, a vapor-deposited polymer film formed on the metal film, and immobilized on the surface of the vapor-deposited polymer film A measurement chip in which a layer comprising a physiologically active substance is provided on an optical portion has been proposed and used for detecting a base sequence complementary to probe DNA (Japanese Patent Application Laid-Open No. H11-281569).

[0003] Conventionally, detection of microorganisms such as Escherichia coli and Staphylococcus aureus, and viruses such as influenza virus has been carried out by culturing them on an appropriate medium for a long time and growing them. In addition, in recent years, exogenous endocrine disrupting chemicals (hereinafter, referred to as “exogenous endocrine disrupting chemicals”, which are widely found in the outside world and are taken into the body along with the life activities of living organisms and affect various physiological endocrine phenomena including their reproduction, development, behavior, etc. "Environmental hormone"), for example, 17β-estradiol, bisphenol A, nonylphenol, DD
T, dioxins, vinfurozolin, DDE, TCDD, PCB
The detection and measurement of these substances are highly accurate gas chromatography mass spectrometers (GC-M
S) has been done using. Therefore, there is a demand for the development of a technique capable of easily detecting and measuring microorganisms, viruses and environmental hormones in a short time and without the need for complicated operations or labeling substances.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a measuring chip for a surface plasmon resonance biosensor, which can detect and measure microorganisms, viruses or environmental hormones without complicated operations or a labeling substance. And

[0005]

Means for Solving the Problems In view of the above objects, the present inventors have conducted intensive studies focusing on mannose sugar chains on the cell surface of microorganisms and viruses, and as a result, can recognize the ends of mannose sugar chains. By selecting concanavalin A or a sialic acid-containing sugar chain as a substance, and immobilizing the concanavalin A or the sialic acid-containing sugar chain on the surface of an aminosilane film formed on the surface of a metal thin film on an optically transparent substrate. Using the prepared surface plasmon resonance measurement chip, it was unexpectedly found that Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae or influenza virus A can be detected with high sensitivity in real time. Or surface plasmid prepared by immobilizing cyclodextrin With emissions resonance measurement chip surprisingly found that can detect environmental hormones, and have completed the present invention based on these findings.

That is, according to the first gist of the present invention,
A surface plasmon resonance bioassay comprising an optically transparent substrate, a metal thin film formed on the substrate, an aminosilane film formed on the metal thin film, and a physiologically active substance immobilized on the aminosilane film. In a sensor chip for a sensor, at least one substance selected from the group consisting of concanavalin A, sialic acid-containing sialo-glycan, estrogen receptor, testosterone receptor and cyclodextrin is used as a physiologically active substance. A measurement chip for a surface plasmon resonance biosensor is provided.

According to a second aspect of the present invention, there is provided an optically transparent substrate, a metal thin film formed on the substrate,
An aminosilane film formed on the metal thin film, wherein concanavalin A, a sialic acid-containing sialo-glycan, an estrogen receptor or a testosterone receptor is immobilized on the aminosilane film via a glutaraldehyde polymerized film. The present invention provides a measurement chip for a surface plasmon resonance biosensor.

Further, according to a third aspect of the present invention, a metal thin film is formed on an optically transparent substrate, an aminosilane film is formed on the metal thin film, and then a surface of the aminosilane film is formed. A measurement chip for a surface plasmon resonance biosensor, comprising immobilizing at least one substance selected from the group consisting of concanavalin A, sialic acid-containing sialo-glycan, estrogen receptor, testosterone receptor and cyclodextrin. A manufacturing method is provided.

Further, according to a fourth aspect of the present invention, a metal thin film is formed on an optically transparent substrate, an aminosilane film is formed on the metal thin film, and then a surface of the aminosilane film is formed. A glutaraldehyde polymerized film is formed, and at least one substance selected from the group consisting of concanavalin A, sialic acid-containing sialo-glycan, estrogen receptor and testosterone receptor is immobilized on the surface of the glutaraldehyde polymerized film. A method for producing a measurement chip for a surface plasmon resonance biosensor is provided.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION A measuring chip for a surface plasmon resonance biosensor of the present invention (hereinafter simply referred to as a “measuring chip”) comprises an optically transparent substrate, a metal thin film formed on the substrate, An aminosilane film formed on a metal thin film, concanavalin A immobilized on the aminosilane film, a sialic acid-containing sialo-glycan, an estrogen receptor (a natural product or a synthetic product if it has physiological activity), a testosterone receptor and Cyclodextrin (α-,
and at least one substance selected from the group consisting of β- or γ-cyclodextrin).

The optically transparent substrate constituting the measuring chip of the present invention may be any substrate as long as it is used for a measuring chip for a surface plasmon resonance biosensor. It is made of a material that is transparent to laser light, such as polyethylene terephthalate and polycarbonate, and has a thickness of about 0.1 to 20 mm.

The metal thin film constituting the measuring chip of the present invention is not particularly limited as long as it produces surface plasmon resonance. Examples of metals usable for this metal thin film include gold, silver, copper, and the like. Examples thereof include aluminum and platinum, which may be used alone or in combination. In addition, an intervening layer made of chromium or the like may be provided between the transparent substrate and the metal thin film in consideration of the adhesion to the transparent substrate. The thickness of the metal thin film is preferably 100 to 2000 mm, and 200 to 600 mm.
Is more preferred. If the thickness of the metal thin film exceeds 3000 °, the surface plasmon phenomenon of the medium cannot be sufficiently detected, which is not preferable. When an intervening layer made of chromium or the like is provided, the thickness of the intervening layer is 30 to 50.
Å is preferred.

The above-mentioned metal thin film can be formed on the above-mentioned optically transparent substrate by a conventional method, for example, by a sputtering method, a vapor deposition method, an ion plating method, an electroplating method, an electroless plating method or the like. Can be formed.

The aminosilane film constituting the measurement chip of the present invention is a film made of a polymer containing Si—O and Si—C bonds and NH ₂ groups in the molecule. Can be formed. The silane coupling agent is an organic silicon compound having an organic functional group having an affinity for an organic material in a molecule, for example, an amino group, and a hydrolyzable group having an affinity for an inorganic material, for example, a methoxy group or an ethoxy group. Yes, the hydrolyzable group binds to a metal atom in the metal thin film, and the organic functional group binds to concanavalin A, sialic acid-containing sialo-glycan, estrogen receptor, testosterone receptor or cyclodextrin. As a result, the above-mentioned metal thin film, aminosilane film, and concanavalin A, sialic acid-containing sialo-glycan, estrogen receptor, testosterone receptor or cyclodextrin are firmly immobilized. Examples of the silane coupling agent that can be used in the present invention include 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyldiethoxymethylsilane, and 3- (2-aminoethylaminopropyl) trimethoxy Silane, 3- (2-aminoethylaminopropyl) dimethoxymethylsilane, 3-mercaptopropyltrimethoxysilane, dimethoxy-3-mercaptopropylmethylsilane and the like, which may be used alone or in combination May be used.

The aminosilane film is obtained by exposing a metal thin film formed on an optically transparent substrate as described above to, for example, saturated vapor of a silane coupling agent for a predetermined time (saturated vapor method). A film can be formed by a method of dipping in a solution containing a silane coupling agent for a certain period of time (dipping method), a method using a spin coater (spin coating method), a method using a gravure printing machine (gravure method), or the like. .

A measurement chip or cuvette for a surface plasmon resonance biosensor having an aminosilane film formed on a metal thin film thus formed on an optically transparent substrate is commercially available from Biacore and Affinity Sensors. Therefore, these may be used.

Immobilization of concanavalin A, sialic acid-containing sialo-glycan, estrogen receptor, testosterone receptor or cyclodextrin on the surface of the aminosilane film formed as described above can be carried out according to a conventional method. To a predetermined amount of concanavalin A, sialic acid-containing sialo-glycan, estrogen receptor, testosterone receptor or cyclodextrin for a predetermined time. For this immobilization, for example, a transparent substrate on which a metal thin film and an aminosilane film are formed as described above is placed on a cartridge block of a flow cell type or batch type surface plasmon resonance biosensor, and concanavalin A, It can also be carried out by contacting a sialic acid-containing sialo-glycan, an estrogen receptor, a testosterone receptor or a cyclodextrin for a predetermined time.

Concanavalin A, sialic acid-containing sialo-glycan immobilized on the aminosilane film as described above,
The thickness of the estrogen receptor, testosterone receptor or cyclodextrin layer is preferably from 100 to 3000 mm, depending on the size of the substance used, and is particularly preferably 1 to 3000 mm.
It is preferably between 100 and 1000 °.

Further, a measuring chip according to another aspect of the present invention comprises:
Concanavalin A, a sialic acid-containing sialo-glycan, an estrogen receptor or a testosterone receptor immobilized on an aminosilane film via a glutaraldehyde polymerized film, comprising an optically transparent substrate and a substrate formed on the substrate. Metal thin film, an aminosilane film formed on the metal thin film, and a glutaraldehyde polymer film on the aminosilane film, and concanavalin A, sialic acid-containing sialo-glycan, estrogen receptor through the polymer film. Alternatively, the testosterone receptor is immobilized.

As described above, when a glutaraldehyde polymerized film is provided and concanavalin A, sialic acid-containing sialo-glycan, estrogen receptor or testosterone receptor are firmly immobilized by covalent bonds, the measurement of the substance can be performed even when the measurement chip is washed. It has the advantage that the immobilization can be maintained and used for repeated measurements. In this case, the thickness of the glutaraldehyde polymerized film is preferably from 10 to 100 °, and particularly preferably from 10 to 20 °.

The formation of the above-mentioned glutaraldehyde polymerized film can be carried out according to a conventional method. For example, as described above, glutaraldehyde is polymerized on an aminosilane film on a transparent substrate on which a metal thin film and an aminosilane film have been formed in advance. It can be done by doing. Immobilization of concanavalin A, sialic acid-containing sialo-glycan, estrogen receptor or testosterone receptor onto the glutaraldehyde polymerized membrane can be performed in the same manner as when these substances are immobilized on an aminosilane membrane.

The thus prepared measurement chip of the present invention can be used for detecting and measuring microorganisms, viruses or environmental hormones in real time with high sensitivity in a surface plasmon resonance biosensor. For example, when a measurement chip on which concanavalin A is immobilized as a physiologically active substance is used, a virus such as influenza virus A can be detected with high sensitivity, and a measurement chip on which a sialic acid-containing sialo-glycan is immobilized as a physiologically active substance is used. When used, microorganisms such as Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae can be detected with high sensitivity, and when a measurement chip on which estrogen receptor, testosterone receptor or cyclodextrin is immobilized as a physiologically active substance is used,
Environmental hormones such as 17β-estradiol, bisphenol A, DDT, dioxin, PCB, and phthalate can be detected in real time. Further, since many environmental hormones are hydrophobic, when detecting and measuring them, detection and measurement can be performed with higher sensitivity by adding β-cyclodextrin to the solution to be measured and measuring.

[0023]

The present invention will be described in more detail with reference to the following examples.

Example 1: Preparation of a measurement chip on which a sialic acid-containing sialo-glycan is immobilized 1.1 Preparation of a metal thin film As an optically transparent substrate, a width of 18 mm, a length of 18 mm, and a thickness of 0.1 mm is used.
A 15 mm glass plate was used. A thin film made of chromium was formed on the transparent substrate by sputtering, and then a thin film made of gold was formed on the surface thereof. The sputtering conditions are 100 W for 30 seconds for chromium and 100 for gold.
W was performed for 150 seconds. The thickness of the obtained chromium thin film is 3
0 °, and the thickness of the gold thin film was 470 °.

1.2 Preparation of Aminosilane Film A glass plate having a metal thin film prepared as described above was prepared by:
The aminosilane film was formed on the metal thin film by exposing to the saturated vapor of the silane coupling agent. That is, the capacity
500 μl of γ-aminopropylethoxysilane [(H ₂ N— (CH ₂ ) ₃ Si (OEt) ₃ , TSL 8331 manufactured by Toshiba Silicone Co., Ltd.) is placed in a 10 ml sample bottle, and left at room temperature for 24 hours.
The interior of the sample bottle was filled with saturated vapor of γ-aminopropylethoxysilane. Next, a glass plate having a metal thin film prepared as described in 1.1 above is fixed to the center of a PET mask (support) so that the metal thin film portion is exposed, and this mask is placed in a sample bottle. And left for 15 minutes or 90 minutes to form an aminosilane film on the metal thin film of the glass plate.

1.3 Immobilization of sialic acid-containing sialo-glycan on aminosilane film A measurement cuvette for a surface plasmon resonance sensor having an aminosilane film on a metal thin film on a transparent substrate, which is commercially available from Affinity Sensors for a surface plasmon resonance sensor. The test was performed using a 200 μl aminosilane cuvette as described. The aminosilane cuvette having a volume of 200 μl was placed on a cartridge block of a surface plasmon resonance sensor IAsys manufactured by Affinity Sensors. Then, distilled water was injected into the aminosilane cuvette to wash it, and the baseline of the sensorgram displayed on the personal computer display connected to Iasys was stabilized. After the baseline was stabilized, 1 mM of sialic acid containing sialo-glycan (abbreviated as Sialo-OH), 1 mM of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 1 mM of N-hydroxysuccinimide were distilled water. 100 μl of the sialic acid complex saccharide solution prepared by dissolving in water was poured into the cuvette to sufficiently contact the aminosilane film, and then the cuvette was washed by pouring distilled water into the cuvette. The injection of the sialic acid complex saccharide solution and the washing with distilled water were repeated again. Next, 100 ml of a 1M acetic anhydride / acetic acid mixed solution was added to the cuvette.
μl was injected to block the amino groups of the unreacted aminosilane film on the cuvette surface, and then washed with distilled water. In this manner, a measurement chip for a surface plasmon resonance biosensor in which a sialic acid complex sugar chain (that is, a sialic acid-containing sialo sugar chain) was immobilized on the aminosilane film of the aminosilane cuvette was prepared.

Example 2: Preparation of a measurement chip in which concanavalin A is immobilized on an aminosilane film via a glutaraldehyde polymerized film 2.1 Preparation of metal thin film and aminosilane film An optically transparent substrate having a width of 18 mm, a length of 18 mm and a thickness of 18 nm 0.
Using a 15 mm glass plate, a thin metal film is formed on this glass plate as described in 1.1 of Example 1 and then an aminosilane film is formed on the thin metal film as described in 1.1 of Example 1. Formed.

2.2 Immobilization of Concanavalin A to Aminosilane Film This is a measurement cuvette for a surface plasmon resonance sensor having an aminosilane film on a metal thin film on a transparent substrate, and 200 μl commercially available from Affinity Sensors for the surface plasmon resonance sensor. Performed using a volumetric aminosilane cuvette. The 200 μl aminosilane cuvette was placed on a cartridge block of a surface plasmon resonance sensor IAsys manufactured by Affinity Sensors. Next, phosphate buffer solution (hereinafter, `` PBS '') is added to the aminosilane cuvette.
(PH 7.2) and waited for the baseline of the sensorgram displayed on the computer display connected to the IAsys to stabilize. After the baseline has stabilized, 5 ml of 5% glutaraldehyde and 0.1 ml
Mix 500 μl of M Na0H and let stand for 30 minutes.
A 4.2% glutaraldehyde polymer in which 500 μl of HCl was added to polymerize glutaraldehyde was placed in an aminosilane cuvette and left for 30 minutes. Next, the cuvette was injected with PBS (pH 7.2) and washed for 5 minutes. next,
Concanavalin A (concentration: 300 μ / ml) was injected into the cuvette, allowed to stand for 30 minutes, and then washed with PBS (pH 7.2) for 5 minutes. Next, a regeneration buffer (1 M formic acid) is put in the cuvette, left for 2 minutes, and washed with PBS (pH 7.2) for 5 to 10 minutes.
Next, a blocking agent (1 M ethanolamine) was injected and left for 5 to 10 minutes, and then PBS (pH 7.2) was injected and washed for 5 minutes. The regeneration buffer is then placed in the cuvette.
(1M formic acid) and left for 2 minutes, then PBS (pH 7.2)
For 5 to 10 minutes. Thus, a measurement chip for a surface plasmon resonance biosensor in which concanavalin A was immobilized on the aminosilane film of the aminosilane cuvette via the glutaraldehyde polymerized film was produced.

Example 3 Detection of Influenza Virus A A measurement chip having a sialic acid-containing sialo-glycan immobilized on the aminosilane membrane prepared in Example 1 was mounted on a cartridge block of a surface plasmon resonance sensor IAsys manufactured by Affinity Sensors. It was installed in a measurement cell. An acetate buffer solution (pH 5.3) was put into the measurement cell for washing, and the baseline of the sensorgram displayed on the personal computer display connected to IAsys was stabilized. After draining the acetate buffer solution in the measurement cell, the concentration of 32 HAU / 0.025 ml (about 10
A solution obtained by diluting influenza virus A ( ⁴ TCID ₅₀ / ml) with an acetate buffer solution was injected, and the sensorgram was measured. Dilution concentration is 0.1, 0.2, 0.4, 0.5, 0 to stock solution.
There were seven levels of 7, 0.8 and 1.0. On a personal computer display connected to the surface plasmon resonance sensor Iasys,
The change in the amount of influenza virus A associated with concanavalin A is represented in real time by a sensorgram (curve). This is defined as an association curve. When the association curve reached an equilibrium state, the sample solution of influenza virus A in the measurement cell was discharged, and an acetate buffer solution was injected. The curve of the sensorgram on the PC display has dropped. This indicates that influenza virus A dissociates from concanavalin A. This is defined as a dissociation curve. When the dissociation curve reached an equilibrium state, the acetate buffer solution in the measurement cell was drained, and a 20 mM hydrochloric acid aqueous solution was injected. Thereby, the chemical bond between the sialic acid-containing sialo-glycan and the influenza virus A is released, and the sample can be regenerated to the state of the measurement chip before the sample injection. As a result of the numerical processing, the concentration of the stock solution containing influenza virus A was 3
The association constant (Ka) when 2 HAU / 0.025 ml is 1 is 10.3
M ^-1 , association rate constant (ka) is 0.18 s ^-1 , dissociation rate constant (kd)
Was 0.018 s ^-1 . Influenza A can be identified by collating with these parameters.

Example 4 Detection of Influenza Virus A Concanavalin A was applied to the aminosilane film prepared in Example 2.
The measurement chip on which is immobilized was placed in a measurement cell on a cartridge block of a surface plasmon resonance sensor IAsys manufactured by Affinity Sensors. Acetate buffer solution (p
H 5.3) was added and washed, and the baseline of the sensorgram displayed on the personal computer display connected to IAsys was stabilized. After draining the acetate buffer solution in the measurement cell, the concentration of 32 HAU / 0.025 ml (about 10 ⁴ TCID
₅₀ / ml) of influenza virus A was diluted with an acetate buffer solution, and a sensorgram was measured.
Dilution concentration is 0.1, 0.2, 0.4, 0.5, 0.7, 0.8
And 1.0. Surface plasmon resonance sensor
The change in the amount of influenza virus A associated with concanavalin A is represented by a sensorgram (curve) in real time on a personal computer display connected to IAsys. This is defined as an association curve. When the association curve reached an equilibrium state, the sample solution of influenza virus A in the measurement cell was discharged, and an acetate buffer solution was injected. The curve of the sensorgram on the PC display has dropped. This shows that influenza virus A dissociates from concanavalin A. This is defined as a dissociation curve. When the dissociation curve reached an equilibrium state, the acetate buffer solution in the measurement cell was drained, and a 20 mM hydrochloric acid aqueous solution was injected. This allows concanavalin A and influenza virus A
The chemical bond of the sample can be broken and the sample can be regenerated to the state of the measurement chip before the sample is injected. As a result of numerical processing, the concentration of the stock solution containing influenza virus A was changed to 32 HAU / 0.025 ml.
, The association constant is 5.3 M ^-1 and the association rate constant is 0.017
s ^-1, the dissociation rate constant was 0.0032s ^-1. Influenza A can be identified by collating with these parameters. Further, detection could be performed with high sensitivity even if PBS (pH 8.15) was used instead of the acetate buffer solution.

Example 5: Detection of Escherichia coli The measurement chip on which concanavalin A prepared in Example 2 was immobilized was placed in a measurement cell on a cartridge block of a surface plasmon resonance sensor IAsys manufactured by Affinity Sensors. The measurement cell was washed with an acetate buffer solution (pH 5.3), and the baseline of the sensorgram displayed on a personal computer display connected to IAsys was stabilized. After draining the acetate buffer solution in the measurement cell, a solution prepared by diluting 70 mg / 3 ml E. coli with an acetate buffer solution was injected into the measurement cell, and the sensorgram was measured. The dilution concentration is 0.01, 0.025, 0.04, 0.05, 0.07, 0.0
There were seven levels of 8 and 0.1. Surface plasmon resonance sensor
The change in the amount of Escherichia coli associated with concanavalin A is represented by a sensorgram (curve) in real time on a personal computer display connected to IAsys. This is defined as an association curve. When the association curve reached an equilibrium state, the sample solution of E. coli in the measurement cell was discharged, and an acetate buffer solution was injected. The curve of the sensorgram on the PC display has dropped. This shows that E. coli dissociates from Concanavalin A. This is defined as a dissociation curve.
When the dissociation curve reached an equilibrium state, the acetate buffer solution in the measurement cell was drained, and a 20 mM hydrochloric acid aqueous solution was injected. This breaks the chemical bond between Concanavalin A and E. coli,
It is possible to reproduce the state of the measurement chip before the sample injection. As in Example 3, the association constant when the concentration of the stock solution containing E. coli was 1 was 12.4 M ^-1 and the association rate constant was 0.
The dissociation rate constant was 90 s ^-1 and the dissociation rate constant was 0.0073 s ^-1 . Escherichia coli can be identified by collation with these parameters.

Example 6: Detection of Klebsiella pneumoniae The measurement chip on which concanavalin A prepared in Example 2 was immobilized was placed in a measurement cell on a cartridge block of a surface plasmon resonance sensor IAsys manufactured by Affinity Sensors. An acetate buffer solution (pH 5.3) was put into the measurement cell for washing, and the baseline of the sensorgram displayed on the personal computer display connected to IAsys was stabilized. After draining the acetate buffer solution in the measurement cell, a solution obtained by diluting 70 mg / 3 ml of Klebsiella pneumoniae with the acetate buffer solution was injected into the measurement cell, and the sensorgram was measured. Dilution concentrations were 0.005, 0.01, 0.03, 0.05, 0.07, and 0.
There were seven levels of 08 and 0.1. The change in the amount of Klebsiella pneumoniae associated with Concanavalin A is shown in real time by a sensorgram (curve) on a personal computer display connected to the surface plasmon resonance sensor IAsys. This is defined as an association curve. When the association curve reached an equilibrium state, the sample solution of Klebsiella pneumoniae in the measurement cell was discharged, and an acetate buffer solution was injected into the measurement cell. The curve of the sensorgram on the PC display has dropped. This indicates that Klebsiella pneumoniae dissociates from Concanavalin A. This is defined as a dissociation curve. When the dissociation curve reached an equilibrium state, the acetate buffer solution in the measurement cell was drained, and a 20 mM hydrochloric acid aqueous solution was injected. As a result, the chemical bond between concanavalin A and Klebsiella pneumoniae can be broken and the measurement chip can be regenerated to a state before the sample injection. As in Example 3, the association constant when the concentration of the stock solution containing Klebsiella pneumoniae was 1 was 26.6.
M ⁻¹ , association rate constant is 0.15 s ⁻¹ , dissociation rate constant is 0.0058
s ^-1 . Escherichia coli can be identified by collation with these parameters.

Example 7: Detection of Staphylococcus aureus The measurement chip on which concanavalin A prepared in Example 2 was immobilized was placed in a measurement cell on a cartridge block of a surface plasmon resonance sensor IAsys manufactured by Affinity Sensors. The measurement cell was washed with an acetate buffer solution (pH 5.3), and the baseline of the sensorgram displayed on a personal computer display connected to IAsys was stabilized. After draining the acetate buffer solution in the measurement cell, a solution obtained by diluting Staphylococcus aureus at a concentration of 10 mg / 3 ml with an acetate buffer solution was injected into the measurement cell, and a sensorgram was measured. The dilution concentration is 0.01, 0.02, 0.04, 0.07,
There were six levels of 0.08 and 0.1. The change in the amount of Staphylococcus aureus associated with Concanavalin A is represented in real time by a sensorgram (curve) on a personal computer display connected to a surface plasmon resonance sensor IAsys.
This is defined as an association curve. When the association curve reached an equilibrium state, the sample solution of Staphylococcus aureus in the measurement cell was discharged, and an acetate buffer solution was injected into the measurement cell. The curve of the sensorgram on the PC display has dropped. This shows that Staphylococcus aureus dissociates from Concanavalin A. This is defined as a dissociation curve. When the dissociation curve reached an equilibrium state, the acetate buffer solution in the measurement cell was drained, and a 20 mM hydrochloric acid aqueous solution was injected. As a result, the chemical bond between concanavalin A and Staphylococcus aureus can be broken and the measurement chip can be regenerated to a state before the sample injection. As in Example 3, when the concentration of the stock solution containing Staphylococcus aureus was 1, the association constant was 8.6 M ^-1 and the association rate constant was 0.0
8s ^-1, dissociation rate constant was 0.009s ^-1. Escherichia coli can be identified by collation with these parameters.

Example 8: Detection of 17β-estradiol and bisphenol A Estrogen was applied to the aminosilane film of a commercially available aminosilane cuvette in the same manner as described in 2.2 of Example 2 except that the estrogen receptor was used instead of concanavalin A. A measurement chip on which a receptor was immobilized was prepared. Using this measurement chip, 17β-
Estradiol 4.81 × 10 ^-5 mol and β-cyclodextrin 4.02 × 10 ^-4 mol, or bisphenol A 4.81 × 10
^The association constant, association rate constant and dissociation rate constant of 17β-estradiol and bisphenol A were determined in the same manner as in Example 5 except that ^-5 mol was used. As a result, for 17β-estradiol, the association constant was 4.10 × 10 ⁵ ± 0.080 M ⁻¹ and the association rate constant was 74964.50.
± 6651.82 M ⁻¹ s ⁻¹ and the dissociation rate constant were 0.18 ± 0.020 s ⁻¹ . For bisphenol A, the association constant is 1.90 × 10 ² ± 1.28 M ⁻¹ and the association rate constant is 142.16 ± 101.98.
M ⁻¹ s ⁻¹ and the dissociation rate constant were 0.71 ± 0.060 s ⁻¹ .

Example 9: Detection of 17β-estradiol and bisphenol A A commercially available aminosilane cuvette was prepared in the same manner as described in 1.3 of Example 1 except that a cyclodextrin solution was used instead of the sialic acid complex saccharide solution. A measurement chip in which cyclodextrin was immobilized on an aminosilane film was prepared. Except for using this measurement chip, the association constant, association rate constant and dissociation rate constant of 17β-estradiol and bisphenol A were determined in the same manner as in the method described in Example 8. As a result, for 17β-estradiol, the association constant was 3.83 × 10 ⁴ ± 0.026 M ⁻¹ , the association rate constant was 21407.50 ± 2164.23 M ⁻¹ s ⁻¹ , and the dissociation rate constant was
It was 0.56 ± 0.060 s ⁻¹ . For bisphenol A, the association constant is 2.59 × 10 ² ± 0.76 M ⁻¹ , the association rate constant is 82.14 ± 26.54 M ⁻¹ s ⁻¹ , and the dissociation rate constant is 0.31 ±
It was 0.0097 s ^-1 .

[0036]

The measurement chip of the present invention is easy to prepare, and can detect and measure microorganisms such as Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae, and viruses such as influenza virus A in real time with good sensitivity. By immobilizing hormone receptors such as steroids and testosterone, it is possible to detect and measure environmental hormones, ie, exogenous endocrine disrupting chemicals, and at the same time to examine the effects of other ecosystems on hormones or enzymes.

Continued on front page F-term (reference) 2G059 AA05 EE02 4B029 AA07 BB15 BB20 CC03 FA03 4B063 QA01 QA18 QQ06 QQ10 QQ79 QR43 QR48 QR82 QS39 QX04

Claims (8)

[Claims] 1. An optically transparent substrate, a metal thin film formed on the substrate, an aminosilane film formed on the metal thin film, and a physiologically active substance immobilized on the aminosilane film. In a measurement chip for a surface plasmon resonance biosensor, at least one substance selected from the group consisting of concanavalin A, sialic acid-containing sialo-glycan, estrogen receptor, testosterone receptor and cyclodextrin is used as a physiologically active substance. A measurement chip for a surface plasmon resonance biosensor. 2. The measurement chip according to claim 1, wherein the physiologically active substance is concanavalin A or a sialic acid-containing sialo-glycan. 3. The measurement chip according to claim 1, wherein the physiologically active substance is an estrogen receptor, a testosterone receptor, or a cyclodextrin. 4. Concanavalin A on an aminosilane film
The measurement chip according to claim 1, wherein the sialic acid-containing sialo-glycan, estrogen receptor or testosterone receptor is immobilized via a glutaraldehyde polymerized membrane. 5. A metal thin film is formed on an optically transparent substrate, an aminosilane film is formed on the metal thin film, and then concanavalin A, sialic acid-containing sialo-glycan, estrogen receptor are formed on the surface of the aminosilane film. The method for producing a measurement chip for a surface plasmon resonance biosensor according to claim 1, wherein at least one substance selected from the group consisting of testosterone receptor and cyclodextrin is immobilized. 6. The production method according to claim 5, wherein concanavalin A or sialic acid-containing sialo-glycan is immobilized on the surface of the aminosilane film. 7. The production method according to claim 5, wherein an estrogen receptor, a testosterone receptor or a cyclodextrin is immobilized on the surface of the aminosilane film. 8. A metal thin film is formed on an optically transparent substrate, an aminosilane film is formed on the metal thin film, and a glutaraldehyde polymer film is formed on the surface of the aminosilane film. 5. The surface plasmon according to claim 4, wherein at least one substance selected from the group consisting of concanavalin A, sialic acid-containing sialo-glycan, estrogen receptor and testosterone receptor is immobilized on the surface of the polymer film. A method for producing a measurement chip for a resonance biosensor.