KR20110060583A - Sensor chips for detecting estradiol and the methods thereof - Google Patents

Abstract

PURPOSE: A sensor chip for detecting estradiol is provided to simultaneously detect various substances and a smell amount of substance such as estrogen. CONSTITUTION: A sensor chip for detecting estradiol comprises: a substrate having a gold layer; a self-assembled monolayer(SAM) of thiol-acrylate polymers formed on the gold layer; and a molecular polymer layer which is formed by polymerization of the acrylate monomers and cross-linking agent. The sensor chip is applied to surface Plasmon resonance or quartz crystal microbalance. The thiol-acrylate polymer is prepared by mixing a compound with acrylate at both ends with a compound containing multiple thiol groups, and performing photopolymerization.

Description

Sensor chip for detecting estradiol and its manufacturing method {Sensor chips for detecting estradiol and the methods}

The present invention relates to a sensor chip for detecting estradiol, more specifically, a substrate on which a gold layer is formed; A self-assembled monolayer (SAM) of thiol-acrylate polymer formed on the gold layer; And a molecular imprinted polymer layer formed by polymerizing an acrylate monomer and a crosslinking agent on the SAM layer and including a binding site to which estradiol or a structural analog thereof is bonded.

Estradiol (β-estradiol) is one of the highest levels of estrogen (estrogen), one of the endocrine female hormones. As the use of various chemicals increases in modern society, substances that exhibit in vivo activity such as estrogen have been detected one after another even though they are not highly similar in structure, and are classified as endocrine barriers or environmental hormones. The in vivo activity of estradiol is mainly measured by ER binding assay, which measures the degree of binding to the estrogen receptor, and E-SCREEN assay using a cell line that specifically reacts with estrogen. These assays are complex procedures and expensive assays, and there is a high interest in a method of measuring hormone levels more easily.

In the present invention, by preparing a detection site capable of recognizing estradiol using the molecular imprinting method, it is possible to solve the inconvenience caused by the use of cells or antibodies in the conventional method. In addition, by improving the conventional method for producing a molecular stamping polymer, the thiol-acrylate (thiol-acrylate) can be formed on the surface of the gold by a self-assembled monolayer method, and then the molecular stamping polymer on the It provides a two-step method for generating it. In addition, the sensor chip is to provide a method for detecting the endocrine obstacle having structural similarity in the position and arrangement of estradiol and other functional groups.

Accordingly, a main object of the present invention is to provide a sensor chip for estradiol detection, including a molecular imprinting polymer for detecting estradiol and its derivatives using molecular inscription, and a method of manufacturing the same.

According to one aspect of the invention, the invention is a substrate with a gold layer; A self-assembled monolayer (SAM) of thiol-acrylate polymer formed on the gold layer; And it provides a sensor chip for estradiol detection is composed of a polymer layer of a molecular stamp formed by polymerizing an acrylate monomer and a crosslinking agent on the SAM layer and including a binding site to which estradiol or a structural analog thereof is bonded.

In the present invention, the term "molecular immersed polymer layer" is used interchangeably with the term 'molecular imprinted polymer (MIP)' in the present specification, which is used with a suitable template (Estradiol in the present invention) It refers to a polymer having the same space as the template material by synthesizing the polymer using a monomer (or unit) (adding a crosslinking agent if necessary) and then removing the template material. In addition, the control group as a control for the molecular imprinted polymer is a non-printed polymer is represented as a non imprinted polymer (NIP).

In the present invention, the sensor chip can be applied to a method or apparatus for recognizing a change in molecular binding, for example, surface plasmon for analyzing the binding affinity of a biomaterial such as a protein by measuring a change in refractive index of a sample on the chip surface. Surface plasmon resonance, or crystals, can be applied to quartz crystal microbalance, which precisely measures the weight of a substance adsorbed on the surface of the sensor chip using a change in vibration frequency. In Experimental Example 4 of the present invention, it was demonstrated that the estradiol can be detected using the surface plasmon resonance.

In the sensor chip for detecting estradiol of the present invention, the thiol-acrylate polymer is photopolymerized by mixing a compound having a multi thiol group and a compound having an acrylate group at both ends thereof, whereby the thiol group is bonded to the gold surface and the acrylate is It may be prepared in an externally exposed form, and preferably, may be prepared by mixing and polymerizing PETMP (Pentaerithritol tetrakis (3-mercaptopropionate)) and EGDMA (Ethylene glycol dimethacrylate).

The 'photopolymerization' refers to a polymerization reaction in which a monomer absorbs light and is activated when light is irradiated to a compound (unit) having a relatively low molecular weight, which is the basis of a repeating unit in a polymer compound structure. Photopolymerization reaction of EGDMA with is preferably performed for 20-40 minutes in the ultraviolet (UV).

In the sensor chip for detecting estradiol of the present invention, the thiol group of the thiol-acrylate polymer in the SAM layer is bonded to a substrate, and the acrylate group is characterized in that it is bonded to a polymer layer having a molecular angle. As a result of the photopolymerization reaction, two thiols are immobilized on the gold layer formed on the substrate, and two acrylates are used for the synthesis of highly molecularly compounded molecules.

In the sensor chip for detecting estradiol of the present invention, the acrylate monomer is selected from the group consisting of methacrylate, acrylic acid, 4-vinylpyridine and acrylamide. Can be. Preferably it is methacrylate.

In the sensor chip for detecting estradiol of the present invention, the crosslinking agent is composed of ethylene glycol dimethacrylate (EGD), ethylene glycol dimethacrylate (EGD), triethyleneglycol dimethacrylate (TGD), polyethylene glycol dimethacrylate (PEG), and acrylamide (acrylamide). Can be selected from.

In the sensor chip for estradiol detection of the present invention, the polymerization of the acrylate monomer and the crosslinking agent is characterized in that for 6-18 hours at 60-100 ℃ by adding an initiator (initiator). Preferably, the initiator is AIBN (2-2'-azobisisobutylonitrile) as.

According to another aspect of the present invention, the present invention provides a method of manufacturing a sensor chip for detecting estradiol, comprising the following steps:

a) mixing a compound having a multi-thiol group and a compound having an acrylate at both ends, and then forming a self-assembled monolayer having an acrylate group on the sensor chip using thiol-acrylate photopolymerization;

b) injecting a mixture of an acrylate monomer, a crosslinking agent and an estradiol onto the monolayer and then applying a radical initiator to form a molecular imprinted polymer layer; And

c) eluting with acetone and methanol / acetic acid mixed solvent in the molecular imprinting polymer layer to remove estradiol molecules.

In the production method of the present invention, by modifying the gold surface by using a thiol-acrylate photopolymerization in the step a), there is an advantage that can fix the molecular imprinted on the gold surface. Existing estradiol molecular imprinting polymer is mainly produced in the form of particles has a disadvantage that takes a long time, such as using column chromatography to detect the amount of binding of estradiol molecules.

In the production method of the present invention, the photopolymerization in step a) may be preferably performed for 20-40 minutes in UV, more preferably for 30 minutes in UV.

In the preparation method of the present invention, AIBN (2,2'-Azobisisobutyronitrile) is applied as an initiator in step b) for 6-18 hours at 60-100 ° C to polymerize a mixture of monomer, crosslinking agent and estradiol. It is characterized by.

By the reaction, as shown in FIG. 1, the crosslinking agent forms a nest-like structure surrounding the estradiol on the self-assembled single layer, and is filled in the engraved portion of the estradiol, thereby resulting in total estradiol molecular engraving.

According to another aspect of the present invention, the present invention comprises dropping an analytical sample on the sensor chip for detecting estradiol and then detecting estradiol or a structural analog thereof by surface plasmon resonance or crystal oscillator microbalance. Or a method for detecting a structural analog thereof.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1 Fabrication of Estradiol Molecular Engraving Sensor Chip

The method for manufacturing a molecular imprinting sensor chip is divided into a gold surface treatment step using thiol-acrylate photopolymerization and a molecular imprinting step for forming an estradiol detection site. Reagents and the amount used in each step are shown in Table 1 below.

TABLE 1 Materials Used and Usage

Step Component Compound Quantity Molecular ratio Thiol-acrylate photopolymerization Thiol Pentaerithritol tetrakis (3-mercaptopropionate) (PETMP) 1.2 mmole One Acrylate Ethylene glycol dimethacrylate (EGDMA) 1.2 mmole One Solvent Methylene chloride 7.6 ml - Molecular imprinting Template Estradiol (E2) 100 μmole One Monomer Methacrylate (MA) 400 μmole 4 Crosslinker Ethylene glycol dimethacrylate (EGDMA) 2.5 mmole 25 Initiator 2,2’-Azobisisobutyronitrile (AIBN) 30 μmole 0.3 Solvent Acetonitrile 20 ml -

Specifically, the specific manufacturing process of the present invention will be described with reference to FIG.

To 7.6 ml of methylene chloride solution, PETMP (Pentaerithritol tetrakis (3-mercaptopropionate) and EGDMA (Ethylene glycol dimethacrylate) were added with 1.2 mmole, respectively, and the gold layer formed substrate (gold sensor chip) was deposited and UV After 30 minutes of application, thiol and EGDMA acrylates were combined by photopolymerization to allow the thiol-acrylate material to be formed on the gold surface by self-assembly. Two of the four thiols are immobilized on the gold surface and the two acrylates bind to the polymers that are the molecular molecules to be synthesized later.Then, after washing the sensor chip three times or more with acetonitrile, the solvent is acetonitrile ( Acetonitrile) 20 ml and 400 μmole of methacrylate as monomer, 2.5 mmole of EGDMA as crosslinker and 100 μl of estradiol as template Mole was added, and 30 μmole of AIBN (2,2'-Azobisisobutyronitrile) was added as an initiator of the radical polymerization, and polymerization was carried out at 80 ° C. for 12 hours to prepare a molecular imprinted polymer of estradiol on the sensor chip. The prepared sensor chip was eluted with estradiol as a substrate in acetone and methanol: acetic acid (4: 1) solvent, and thus was reusable.

Example 2 Reflection Angle Shift of Molecular Imprinted Sensor Chips

The amount of estradiol binding to the molecular imprinting sensor chip was measured using an SPRi (Kamac) instrument measuring surface plasmon resonance. In other words, while injecting acetonitrile at a rate of 20 ul / min and injecting a sample in which 2 mmole of estradiol was dissolved in 500 μl of acetonitrile, the incident angle showing the lowest reflection on the surface of the gold sensor chip was introduced. The degree of shift of was measured.

2 shows how the incident angle of the sensor chip, which is the molecular angle, is shifted compared to the surface of the gold which is not treated with the molecular angle. As a result, it was confirmed that the surface incident angle due to molecular imprint was shifted by 0.8 degrees and was modified with a polymer on the gold surface.

Example 3 Comparison of Molecular Imprinting Effects of Molecular Imprinted Chips (MIP) and Non-Molecular Imprinted Chips (NIP)

Molecular engraving effects were estimated based on the degree of angle shift indicating the amount of estradiol binding to MIP and NIP. The degree of reflection shift is shown in Table 2 below. The degree of shift is proportional to the amount of coupling, and a 1 degree (°) reflection angle shift corresponds to approximately 1 ng coupling. An increase in the shift of the MIP chip compared to the NIP chip was calculated as an imprinting factor.

TABLE 2 Molecular Imprinting Index of Sensor Chip

Type Estradiol binding response
(angle shift, °) Imprinting factor * MIP 0.370 4.3 NIP 0.070

* Imprinting factor = (MIP-NIP) / NIP

As a result of the calculation, the molecular imprinting index was 4.3, which is higher than previously reported estradiol molecular imprinting index value (Noir, M. L., et al., Water Res. 41, 2825-2831 (2007)).

Experimental Example 4 MIP binding capacity of estradiol, its derivatives, and other endocrine disruptors

Figure 3 shows the binding amount of compounds known as endocrine disruptors such as estradiol, estriol, estrone, and bisphenol A, bisphenol A, nonylphenol, tamoxifen to MIP of the present invention Surface plasmon resonators (KMA, SPRi) were used to detect shifts in reflection. In addition, it is shown in Table 3 to compare the binding force and the ER-binding measurement and E-SCREEN results of the molecular weight of the materials analyzed in these examples.

TABLE 3 Comparison of Molecular Engraving Adhesion and Other Methods

Name CAS number ER binding * E-SCREEN * MIP binding
(angle shift) 4-Hydroxytamoxifen 68047-06-3 2.24 0.58 3.08 Estradiol 00050-28-2 2 2 0.37 Estriol 00050-27-1 0.9 1.2 0.052 Estron 00053-16-7 0.86 0.32 0.081 4-Nonylphenol 25154-52-3 -1.53 -3.03 0.087 Bisphenol a 00080-05-7 -1.86 -2.79 0.38

Liu, H., et al., Chemosphere 70, 1889-1897 (2008).

In FIG. 3, it was confirmed that only estradiol, which was used as a template, remained undissolved over time in combination with the sensor chip. In addition, tamoxifen, which was reported to be the most active in the estrogen receptor binding test (ER-binding assay), had the greatest instantaneous binding force to the sensor chip, but was eluted by acetonitrile solvent. In Table 3, bisphenol A showed a low value in ER-biding assay or E_SCREEN assay, but is actually known to cause endocrine disorders even at low concentrations. In the case of a substance that binds estradiol to a sensor chip with a molecular imprint, it is predicted that the position or shape of a functional group capable of specific binding may be similar to that of a circular template. Thus, although bisphenol A shows low activity in ER-binding or E-SCREEN, high activity in vivo shows that it is likely to react with other receptors other than the ER receptor due to its structural similarity. As such, the molecular imprinting sensor chip can be used as a tool for screening how similar the activity of estradiol and the active site is, while showing specificity with continual binding to estradiol. Showed.

As described above, according to the present invention, a substance present in a small amount, such as estrogen, can be measured by using a sensor chip, enabling immediate measurement of a sample without pretreatment, and having a higher molecular imprinting effect than a conventional molecular imprinted polymer. Indicates. In addition, although a sensor using a molecular imprinting technique uses a polymer-based bead or a modified surface, the present invention is manufactured on a gold surface chip so that it can be measured by surface plasmon resonance measurement to simultaneously recognize various materials on the gold surface in the future. It is possible to develop an array chip.

1 is a schematic diagram illustrating a manufacturing process of a sensor chip for estradiol detection according to the present invention.

Figure 2 shows the change in the shift compared to the gold surface without the treatment, the reflection angle of the estradiol detection sensor chip according to the present invention.

3 is a surface plasmon resonator for measuring the amount of binding of estrogen derivatives such as estradiol, estriol, estrone, bisphenol A, nonylphenol, and tamoxifen to MIP according to the present invention. It shows the reflection angle shift through.

Claims (13)

A substrate on which a gold layer is formed; A self-assembled monolayer (SAM) of thiol-acrylate polymer formed on the gold layer; And A sensor chip for estradiol detection, comprising a polymer layer having a molecular angle formed by polymerizing an acrylate monomer and a crosslinking agent on the SAM layer and including a binding site to which an estradiol or a structural analog thereof is bonded. The sensor chip of claim 1, wherein the sensor chip is applied to surface plasmon resonance or quartz crystal microbalance. The thiol-acrylate polymer is prepared by mixing a compound having a multi thiol group and a compound having an acrylate at both ends thereof to photopolymerize to form a thiol group bonded to the gold surface and the acrylate exposed to the outside. Sensor chip for estradiol detection, characterized in that. The estradiol detection according to claim 3, wherein the compound having a multi-thiol group is PETMP (Pentaerithritol tetrakis (3-mercaptopropionate)), and the compound having acrylates at both ends is ethylene glycol dimethacrylate (EGDMA). Sensor chip. 4. The sensor chip for estradiol detection according to claim 3, wherein the photopolymerization is performed for 20 to 40 minutes under UV light. The sensor chip of claim 1, wherein the thiol group of the thiol-acrylate polymer in the SAM layer is bonded to a substrate, and the acrylate group is bonded to a polymer layer having a molecular angle. The method of claim 1, wherein the acrylate monomer is estradiol, characterized in that selected from the group consisting of methacrylate (acrylic acid), acrylic acid (acrylic acid), 4-vinylpyridine and acrylamide (acrylamide) Sensor chip for detection. The method of claim 1, wherein the crosslinking agent is selected from the group consisting of ethylene glycol dimethacrylate (EGD), ethylene glycol dimethacrylate (EGD), triethyleneglycol dimethacrylate (TGD), polyethylene glycol dimethacrylate (PEG) and acrylamide (acrylamide). Sensor chip for estradiol detection. The method of claim 1, wherein the polymerization of the acrylate monomer and the crosslinking agent is reacted with estradiol for 6-18 hours at 60-100 ℃ by applying AIBN (2-2'-azobisisobutylonitrile) as an initiator. chip. Method for manufacturing a sensor chip for estradiol detection comprising the following steps: a) mixing a compound having a multi-thiol group and a compound having an acrylate at both ends, and then forming a self-assembled monolayer having an acrylate group on the sensor chip using thiol-acrylate photopolymerization; b) injecting a mixture of an acrylate monomer, a crosslinking agent and an estradiol onto the monolayer and then applying a radical initiator to form a molecular imprinted polymer layer; And c) eluting with acetone and methanol / acetic acid mixed solvent in the molecular imprinting polymer layer to remove estradiol molecules. The method according to claim 10, wherein the photopolymerization in step a) is performed for 30 minutes in UV. The method of claim 10, wherein in step b), AIBN (2-2'-azobisisobutylonitrile) is applied as an initiator to react at 60-100 ° C. for 6-18 hours to polymerize the mixture of monomer, crosslinking agent and estradiol. How to feature. An estradiol comprising dropping an analytical sample on the sensor chip for detecting estradiol according to any one of claims 1 to 9 and then detecting estradiol or a structural analog thereof by plasmon resonance or crystal oscillator microbalance. Or a method for detecting a structural analog thereof.

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