JP4248154B2 - Method for measuring 17β-estradiol or bisphenol A using a measuring chip for surface plasmon resonance biosensor - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention is directed to a method of measurement of 17β- estradiol or bisphenol A with measurement chip front surface plasmon resonance biosensor.
[0002]
[Prior art]
Currently, a method using an immune reaction such as an antigen-antibody reaction, such as an ELISA method, is used as a method for detecting and measuring a measurement target substance 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 for detecting and measuring a substance to be measured in a sample without requiring a complicated operation or a labeling substance, so-called surface plasmon resonance that occurs when surface plasmon is excited at a metal / liquid interface is used. As a measurement chip for a surface plasmon resonance measuring apparatus (hereinafter referred to as “surface plasmon resonance biosensor”) used in this method, a transparent substrate, a metal film disposed on the transparent substrate, A measuring chip comprising an organosilicon film disposed on the metal film and a physiologically active substance disposed on the organosilicon film has been proposed and used for detection and measurement of human serum albumin and herbicide atrazine. (Japanese Patent Laid-Open No. 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 deposition polymer film formed on the metal film, and a surface of the vapor deposition polymer film are immobilized. A measuring chip in which a layer made of a physiologically active substance is provided in the optical part has been proposed and used for detection of a base sequence complementary to probe DNA (Japanese Patent Laid-Open No. 11-28169).
[0003]
Conventionally, microorganisms such as Escherichia coli and Staphylococcus aureus, and viruses such as influenza virus have been detected by culturing them on a suitable medium for a long time and growing them. Furthermore, in recent years, exogenous endocrine disrupting chemicals (hereinafter referred to as “exogenous endocrine disrupting chemicals”) that are widely present in the outside world and are taken into the body along with living activities of living organisms and affect physiological endocrine phenomena including reproduction, development, and behavior. "Environmental hormones"), such as 17β-estradiol, bisphenol A, nonylphenol, DDT, dioxins, binfurozoline, DDE, TCDD, PCB, etc. are problematic. This is done using a graphy mass spectrometer (GC-MS). Therefore, it is desired to develop a technique capable of easily detecting and measuring microorganisms, viruses, and environmental hormones in a short time without requiring complicated operations and labeling substances.
[0004]
[Problems to be solved by the invention]
The present invention detects 17β- estradiol or bisphenol with measurable surface plasmon resonance biosensor measuring chip without the 17β- estradiol or bisphenol A, which is a kind of environmental hormones, require complicated operations or labeling substances An object is to provide a measuring method of A.
[0005]
[Means for Solving the Problems]
In view of the above-mentioned object, the present inventors have made extensive studies by paying attention to mannose sugar chains on the cell surfaces of microorganisms and viruses. As a result, aminosilane formed on the surface of a metal thin film on an optically transparent substrate. When using a surface plasmon resonance measuring chip prepared by immobilizing cyclodextrin on the membrane surface, it was surprisingly found that 17β-estradiol or bisphenol A, which are environmental hormones , can be detected, and based on these findings, the present invention It came to complete.
[0006]
That is, the first gist of the present invention is an optically transparent substrate, a metal thin film formed on the substrate, an aminosilane film formed on the metal thin film, and immobilized on the aminosilane film. by using the physiologically active substance to the front surface plasmon resonance biosensor measurement chip using cyclo dextrin, and measuring the 17β- estradiol or bisphenol a.
[0007]
The second aspect of the present invention is an optically transparent substrate, a metal thin film formed on the substrate, an aminosilane film formed on the metal thin film, and immobilized on the aminosilane film. A method for measuring 17β-estradiol or bisphenol A using a measurement chip for a surface plasmon resonance biosensor using cyclodextrin as a physiologically active substance , and the measurement target when measuring 17β-estradiol or bisphenol A It is characterized by measuring 17β-estradiol or bisphenol A after preparing a solution to be measured by adding β-cyclodextrin to the solution .
[0008]
DETAILED DESCRIPTION OF THE INVENTION
The measurement chip for surface plasmon resonance biosensor of the present invention (hereinafter simply referred to as “measurement chip”) is formed on an optically transparent substrate, a metal thin film formed on the substrate, and the metal thin film. It has an aminosilane film and a cyclodextrin (α-, β-, or γ-cyclodextrin, etc.) immobilized on the aminosilane film, and is used for detection of environmental hormones.
[0009]
The optically transparent substrate constituting the measurement chip of the present invention may be any substrate as long as it is used in a measurement chip for a surface plasmon resonance biosensor, but generally glass, polyethylene terephthalate, It is made of a material that is transparent to laser light such as polycarbonate, and its thickness is about 0.1 to 20 mm.
[0010]
The metal thin film constituting the measurement chip of the present invention is not particularly limited as long as it produces surface plasmon resonance. Examples of metals that can be used for the metal thin film include gold, silver, copper, aluminum, and platinum. Etc., and these may be used alone or in combination. In consideration of adhesion to the transparent substrate, an intervening layer made of chromium or the like may be provided between the transparent substrate and the metal thin film. The thickness of the metal thin film is preferably 100 to 2000 mm, and more preferably 200 to 600 mm. If the thickness of the metal thin film exceeds 3000 mm, the surface plasmon phenomenon of the medium cannot be sufficiently detected, which is not preferable. Moreover, when providing the intervening layer which consists of chromium etc., it is preferable that the thickness of the intervening layer is 30-50 mm.
[0011]
The metal thin film can be formed on the optically transparent substrate according to a conventional method. For example, the metal thin film is formed by sputtering, vapor deposition, ion plating, electroplating, electroless plating, or the like. Can do.
[0012]
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, and is formed using, for example, a silane coupling agent. Can do.
A silane coupling agent is an organosilicon compound having in its molecule an organic functional group having an affinity for an organic material, such as an amino group, and a hydrolyzable group having an affinity for an inorganic material, such as a methoxy group or an ethoxy group. The hydrolyzable group is bonded to a metal atom in the metal thin film, and the organic functional group is bonded to a cyclodextrin. As a result, the metal thin film, aminosilane film, and cyclodextrin are firmly fixed.
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. Examples include silane, 3- (2-aminoethylaminopropyl) dimethoxymethylsilane, 3-mercaptopropyltrimethoxysilane, dimethoxy-3-mercaptopropylmethylsilane, and the like. These may be used alone or in combination. May be used.
[0013]
The aminosilane film is formed by exposing the metal thin film formed on the optically transparent substrate as described above to, for example, saturated vapor of a silane coupling agent (saturated vapor method) or silane coupling. The film can be formed by a method of immersing in a solution containing an agent (immersion method), a method using a spin coater (spin coating method), a method using a gravure printer (gravure method), or the like.
[0014]
A measurement chip or cuvette for a surface plasmon resonance biosensor having an aminosilane film formed on a metal thin film formed on an optically transparent substrate in this way is commercially available from Biacore and Affinity Sensors. These may be used.
[0015]
Immobilization of cyclodextrin on the surface of the aminosilane film formed as described above can be performed according to a conventional method, for example, by bringing a predetermined amount of cyclodextrin into contact with the aminosilane film 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 cyclodextrin is added thereto. It can also be performed by contacting for a predetermined time.
[0016]
The thickness of the cyclodextrin layer immobilized on the aminosilane film as described above is preferably from 100 to 3000 mm, particularly preferably from 100 to 1000 mm, depending on the size of the substance used.
[0017]
The measurement chip of the present invention thus produced can be used for detecting and measuring environmental hormones with high sensitivity in real time in a surface plasmon resonance biosensor. For example, when a measurement chip with cyclodextrin immobilized as a physiologically active substance is used, 17β-estradiol and bisphenol A can be detected in real time. In addition, since 17β-estradiol and bisphenol A are hydrophobic, when they are detected and measured, β-cyclodextrin is added to the solution to be measured, and measurement can be performed with higher sensitivity. it can.
[0018]
【Example】
The invention is further illustrated by the following examples.
[0019]
Example:
1) Preparation of measurement chip on which cyclodextrin was immobilized 1.1 Preparation of metal thin film A glass plate having a width of 18 mm, a length of 18 mm and a thickness of 0.15 mm was used as an optically transparent substrate. 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 were 100 W for 30 seconds for chromium and 100 W for 150 seconds for gold. The resulting chromium thin film had a thickness of 30 mm, and the gold thin film had a thickness of 470 mm.
[0020]
1.2 Preparation of Aminosilane Film A glass plate having a metal thin film prepared as described above was exposed to saturated vapor of a silane coupling agent to form an aminosilane film on the metal thin film. That is, 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 sample bottle was filled with a saturated vapor of γ-aminopropylethoxysilane.
Next, the glass plate having the 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 attached to the glass plate. The aminosilane film was formed on the metal thin film of the glass plate by placing it on the opening of the sample bottle and leaving it for 15 minutes or 90 minutes.
[0021]
1.3 Immobilization of cyclodextrin 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 has a volume of 200 μl commercially available from Affinity Sensors for surface plasmon resonance sensor An aminosilane cuvette was used. The 200 μl volume of aminosilane cuvette was placed on the cartridge block of the surface plasmon resonance sensor IAsys manufactured by Affinity Sensors.
The aminosilane cuvette was then rinsed with distilled water and waited for stabilization of the baseline of the sensorgram displayed on the personal computer display connected to Iasis. After the baseline was stabilized, the cyclodextrin solution was poured into the cuvette to bring it into full contact with the aminosilane membrane and then washed with distilled water injected into the cuvette.
The above cyclodextrin solution injection and distilled water washing were repeated again.
Next, 100 μl of a 1M acetic anhydride / acetic acid mixed solution was injected into the cuvette to block the amino groups of the unreacted aminosilane film on the cuvette surface, and then washed with distilled water.
Thus, a measurement chip for a surface plasmon resonance biosensor in which cyclodextrin was immobilized on the aminosilane film of the aminosilane cuvette was prepared.
[0022]
2) Detection of 17β-estradiol and bisphenol A Using this measurement chip, the association constant, association rate constant and dissociation rate constant were determined for 17β-estradiol and bisphenol A.
That is, using the above measurement chip, using 17β-estradiol 4.81 × 10 ⁻⁵ mol and β-cyclodextrin 4.02 × 10 ⁻⁴ mol, or bisphenol A 4.81 × 10 ⁻⁵ mol, The association constant, association rate constant and dissociation rate constant were determined for 17β-estradiol and bisphenol A.
First, a measurement chip 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 placed in the measurement cell and washed to stabilize the baseline of the sensorgram displayed on the personal computer display connected to the IAsys.
After the acetic acid buffer solution in the measurement cell was discarded, 17β-estradiol 4.81 × 10 ⁻⁵ mol and β-cyclodextrin 4.02 × 10 ⁻⁴ mol, or bisphenol A 4.81 × 10 ⁻⁵ was added to the measurement cell. Sensorgrams were measured by injecting a solution of moles diluted with acetate buffer solution.
Dilution concentrations were set to seven levels of 0.01, 0.025, 0.04, 0.05, 0.07, 0.08 and 0.1 with respect to the stock solution.
Surface Plasmon Resonance Sensor A change in the amount of association with 17β-estradiol or bisphenol A is represented by a sensorgram (curve) in real time on a personal computer display connected to IAsys. This is the association curve.
When this association curve reached an equilibrium state, the sample solution in the measurement cell was discharged, and an acetate buffer solution was injected. The sensorgram curve on the computer display went down. This represents a state in which 17β-estradiol and β-cyclodextrin 4.02 × 10 ⁻⁴ mol or bisphenol A is dissociated from the cyclodextrin. This is the dissociation curve.
When the dissociation curve reached an equilibrium state, the acetate buffer solution in the measurement cell was discarded, and a 20 mM hydrochloric acid aqueous solution was injected. Thereby, the chemical bond between cyclodextrin and 17β-estradiol or bisphenol A can be released and regenerated to the state of the measurement chip before sample injection.
As a result, when the concentration of the stock solution containing 17β-estradiol or bisphenol A is ¹ , the association constant is 17.83 × 104 ± 0.026 M ⁻¹ for 17β-estradiol and the association rate constant is 21407.50. ± 216.23 M ⁻¹ s ⁻¹ and the dissociation rate constant were 0.56 ± 0.060 s ⁻¹ . As 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 ±. 0.0097 s ⁻¹ .
By comparison with these parameters, 17β-estradiol or bisphenol A can be identified.
[0023]
【The invention's effect】
The measurement chip of the present invention is easy to prepare, and can detect and measure 17β-estradiol or bisphenol A in real time with good sensitivity, and at the same time, can examine the influence on other hormones or enzymes in the ecosystem.

Claims (2)

An optically transparent substrate, a thin metal film formed on the substrate, and an aminosilane film formed on the metal thin film, a cyclo dextrin as an immobilized physiologically active substance on the aminosilane film front surface plasmon resonance using a biosensor measuring chip, 17.beta.-estradiol or 17.beta.-estradiol or measuring method of bisphenol a, which comprises measuring the bisphenol a used. An optically transparent substrate, a metal thin film formed on the substrate, an aminosilane film formed on the metal thin film, and a surface using cyclodextrin as a physiologically active substance immobilized on the aminosilane film A method for measuring 17β-estradiol or bisphenol A using a measurement chip for a plasmon resonance biosensor,
When measuring 17.beta.-estradiol or bisphenol A, measured after the preparation of the test liquid added β- cyclodextrin solution, 17.beta.-estradiol or and measuring the bisphenol A 17.beta.-estradiol or bisphenol A measuring method of A.