阅读说明：本技术 分子印迹智能凝胶光栅及其制备方法与凝血酶检测方法 (Molecular imprinting intelligent gel grating and preparation method thereof and thrombin detection method ) 是由 褚良银 赵佳佳 汪伟 蔡泉威 谢锐 巨晓洁 刘壮 于 2021-08-08 设计创作，主要内容包括：本发明提供了一种分子印迹智能凝胶光栅及其制备方法,所述该凝胶光栅由基底和基底上的相互平行的凝胶条组成,凝胶条之间的间隙为凝胶光栅的狭缝,所述凝胶条由凝血酶响应型分子印迹凝胶构成；所述凝血酶响应型分子印迹凝胶是由含凝血酶印迹复合物的凝胶在洗脱掉凝胶网络中的凝血酶后形成的。本发明还提供了基于分子印迹智能凝胶光栅的凝血酶检测方法。本发明可提高凝血酶检测的灵敏度和便捷性,所述凝血酶检测方法的检出限低至10～(-12)mol/L,能实现对10～(-12)～10～(-7)mol/L浓度级别的凝血酶的检测,特别适用于超灵敏定量检测尿液中凝血酶。(The invention provides a molecular imprinting intelligent gel grating and a preparation method thereof, wherein the gel grating consists of a substrate and gel strips which are parallel to each other on the substrate, a gap between the gel strips is a slit of the gel grating, and the gel strips consist of thrombin response type molecular imprinting gel; the thrombin response type molecularly imprinted gel is formed by eluting thrombin in a gel containing a thrombin imprinted complex. The invention also provides a thrombin detection method based on the molecular imprinting intelligent gel grating. The invention can improve the sensitivity and convenience of thrombin detection, and the detection limit of the thrombin detection method is as low as 10 ‑12 mol/L, can realize the ratio of 10 ‑12 ～10 ‑7 The detection of thrombin with mol/L concentration level is particularly suitable for the ultra-sensitive quantitative detection of thrombin in urine.)

1. The intelligent molecularly imprinted gel grating is characterized by consisting of a substrate and gel strips which are parallel to each other on the substrate, wherein gaps among the gel strips are slits of the gel grating, and the gel strips are formed by thrombin-responsive molecularly imprinted gel;

the thrombin response type molecularly imprinted gel is formed by eluting thrombin in a gel network from a gel containing a thrombin imprinted complex; the thrombin imprinted complex is formed by combining thrombin specific aptamer A1, thrombin specific aptamer A2 and thrombin at different sites, wherein the nucleotide sequence of the thrombin specific aptamer A1 is 5'-/5Acryd/GGT TGG TGT GGT TGG-3', and the nucleotide sequence of the thrombin specific aptamer A2 is 5'-/5Acryd/AGA CCG TGG TAG GGC AGG TTG GGG TGA CT-3';

the gel containing the thrombin imprinted complex is formed by cross-linking reaction of N-isopropylacrylamide, the thrombin imprinted complex and a cross-linking agent, and in the cross-linking reaction process, the carbon-carbon double bond of the N-isopropylacrylamide, the 5 'end of thrombin specific aptamer A1 in the thrombin imprinted complex and the carbon-carbon double bond on the 5' end of thrombin specific aptamer A2 and the carbon-carbon double bond on the cross-linking agent are subjected to addition reaction to form a gel network; in the crosslinking reaction process, the molar ratio of the N-isopropylacrylamide to the crosslinking agent is controlled to be 1 (0.01-0.05), and the molar ratio of the thrombin imprinted complex to the crosslinking agent is controlled to be 1 (1000-5000).

2. The molecularly imprinted smart gel grating of claim 1, wherein the cross-linking agent is a four-arm-polyethylene glycol acrylamide, an eight-arm-polyethylene glycol acrylamide, or a two-arm-polyethylene glycol acrylamide.

3. The molecularly imprinted intelligent gel grating of claim 1, wherein the ratio of the period of the gel grating to the grating height is 1 (0.01-0.1).

4. The molecularly imprinted smart gel grating of claim 1, wherein the substrate is a silanized quartz glass plate, and the gel strips are bonded to the substrate through covalent bonds.

5. The method for preparing the molecularly imprinted intelligent gel grating of any one of claims 1 to 4, comprising the steps of:

(1) preparing gel pre-polymerization liquid

Adding the thrombin specific aptamer A2 into a thrombin solution, stirring for 20-40 min, then adding the thrombin specific aptamer A1, and stirring for 20-40 min to obtain a solution containing a thrombin imprinted complex; the molar ratio of the thrombin-specific aptamer A1 to the thrombin-specific aptamer A2 to the thrombin is 1 (0.02-1);

adding N-isopropylacrylamide, a cross-linking agent, an initiator and an accelerator into a solution containing the thrombin imprinted complex, and sufficiently shaking and dissolving under an ice bath condition to obtain a gel pre-polymerization solution; in the gel pre-polymerization liquid, the concentration of N-isopropylacrylamide is 0.5-2.5 mol/L, the molar ratio of the N-isopropylacrylamide to the crosslinking agent is 1 (0.01-0.05), the molar ratio of the N-isopropylacrylamide to the initiator is 1 (0.005-0.05), the molar ratio of the N-isopropylacrylamide to the accelerator is 1 (0.02-0.2), and the molar ratio of the thrombin imprinted complex to the crosslinking agent is 1 (1000-5000);

(2) preparation of thrombin-containing gel gratings

Dripping the gel prepolymer onto a substrate, pressing a gel grating template on the gel prepolymer, reacting for 4-12 h at 4-10 ℃, and stripping the gel grating template to obtain a gel grating containing thrombin;

(3) washing to remove thrombin in gel network

And (2) placing the gel grating containing thrombin in a PBS buffer solution for soaking and washing to remove incompletely polymerized reactants, then taking out the gel grating and placing the gel grating in a mixed solution of guanidine hydrochloride and sodium chloride for fully soaking to remove thrombin in a gel network structure of the gel grating containing thrombin, replacing the mixed solution of guanidine hydrochloride and sodium chloride every 6-12 hours in the soaking process of the mixed solution of guanidine hydrochloride and sodium chloride, taking out the gel grating and placing the gel grating in the PBS buffer solution for fully soaking and washing after soaking is completed, and thus obtaining the molecular imprinting intelligent gel grating.

6. The method for preparing a molecularly imprinted intelligent gel grating according to claim 5, wherein in the mixed solution of guanidine hydrochloride and sodium chloride in the step (2), the concentration of guanidine hydrochloride is 3-6 mol/L, and the concentration of sodium chloride is 1-2 mol/L.

7. The method for preparing a molecularly imprinted intelligent gel grating according to claim 5, wherein the PBS buffer solution in the step (2) has a pH value of 7.4 and a concentration of 1-3 mmol/L.

8. The method for preparing a molecularly imprinted smart gel grating as claimed in any one of claims 5 to 7, wherein the substrate of step (2) is a silanized quartz glass plate.

9. The method for preparing the molecularly imprinted intelligent gel grating according to claim 8, wherein the silanization treatment method of the quartz glass sheet is as follows:

immersing the cleaned quartz glass sheet in an acetic acid-sodium acetate buffer solution containing a silane coupling agent for 10-30 min, then keeping for 10-20 min in an oven at 50-60 ℃, washing with deionized water, and drying to finish the silanization treatment of the quartz glass sheet; in the acetic acid-sodium acetate buffer solution containing the silane coupling agent, the volume percentage concentration of the silane coupling agent is 0.5-2%, the pH value of the acetic acid-sodium acetate buffer solution is 4.5-5.5, and the concentration is 0.05-0.2 mol/L.

10. A thrombin detection method based on a molecularly imprinted intelligent gel grating, which is characterized in that the method uses an optical detection device comprising the molecularly imprinted intelligent gel grating of any one of claims 1 to 4 for detection, and comprises the following steps:

(1) determination of the conversion equation for the Thrombin concentration

Firstly, adding a blank sample into a quartz sample pool of an optical detection device to immerse a gel grating, and reading first-order diffraction light intensity I of the blank sample after the diffraction light intensity of the gel grating is stable₁And intensity of zero-order diffracted light I₀Calculating the diffraction efficiency of the blank sample;

sequentially replacing blank samples in the step I with standard samples with known thrombin concentration according to the sequence of thrombin concentration in the standard samples from low to high, and respectively measuring the diffraction efficiency corresponding to each standard sample to obtain the diffraction efficiency corresponding to a series of standard samples;

calculating the change rate of the diffraction efficiency corresponding to each standard sample relative to the diffraction efficiency of a blank sample, recording the change rate as the relative diffraction efficiency to obtain a series of relative diffraction efficiencies, drawing a working curve by taking the relative diffraction efficiency as an abscissa and taking the thrombin concentration as an ordinate, and determining a conversion relation between the thrombin concentration and the relative diffraction efficiency;

(2) measuring the concentration of thrombin in a test sample

Replacing the original gel grating with the gel grating in the step (1), adding a blank sample into a quartz sample pool of the optical detection device to immerse the gel grating, and reading the first-order diffraction light intensity I of the blank sample after the diffraction light intensity of the gel grating is stable₁And intensity of zero-order diffracted light I₀Calculating the diffraction efficiency of the blank sample;

replacing the blank sample in the step (2) with a sample to be tested with unknown thrombin concentration, and measuring the diffraction efficiency of the sample to be tested;

calculating the relative diffraction efficiency of the sample to be measured relative to the diffraction efficiency of the hollow white sample in the step (2), and calculating the concentration of the thrombin in the sample to be measured according to the conversion relation between the concentration of the thrombin and the relative diffraction efficiency;

and (3) controlling the constant temperature of the step (1) and the step (2) which is the same as the testing temperature and is within the range of 20-35 ℃.

Technical Field

The invention belongs to the field of biomarker detection, and relates to a molecular imprinting intelligent gel grating, a preparation method thereof and a thrombin detection method based on the molecular imprinting intelligent gel grating.

Background

Thrombin is a multifunctional serine protease, which is used as a main effect protein in blood coagulation waterfall, and can promote the conversion of soluble fibrinogen in blood plasma into insoluble fibrin to realize quick-acting hemostasis. At the same time, thrombin also acts as a hormone regulating platelet aggregation, endothelial cell activation and other important responses in biological blood vessels. More importantly, recent reports indicate that thrombin can also be used as a biomarker in urine for detecting disease signals such as glomerulonephritis, hepatitis, lupus, diabetes and cancer. Therefore, it is of great importance to realize sensitive detection of thrombin.

For thrombin detection, although instruments such as a blood coagulation analyzer have high accuracy, the method needs to depend on large and expensive analysis equipment and operation of professionals, and cannot meet the requirement of convenient detection, so that most of the researches are mainly biosensor detection methods at present. The gel diffraction grating with functionalized aptamer was prepared by copolymerizing a thrombin-specific aptamer and its complementary sequence into the gel network. When the gel grating interacts with thrombin-specific aptamers, the interaction between the aptamers and the complementary sequence is disrupted, resulting in changes in the geometric parameters and refractive index of the grating, and thus detection of thrombin is achieved. However, in their experiments, only one aptamer capable of recognizing thrombin was used, and the aptamer was directly copolymerized into the gel network, resulting in insufficient binding sites of thrombin to the aptamer, and thus the detection limit of thrombin was only 0.1. mu.M. Normally, the concentration of thrombin in human blood varies between nM and lower μ M during coagulation. In order to realize the detection of thrombin, the current biosensor detection method for thrombin detection needs to carry out fluorescence labeling or adopt an additional signal enhancement mode, has the problems of complex detection operation process, expensive detection equipment, high detection limit and the like, and cannot meet the actual requirement. Therefore, it is necessary to develop a method for sensitive and convenient detection of thrombin.

In recent years, with the rapid development of molecular imprinting technology, molecular imprinting materials have been applied to the construction of biosensors. The molecular imprinting technology is also called as molecular template technology, when a template molecule contacts with a polymer monomer, multiple action points exist, the combination action can be memorized in the monomer polymerization process, and when the template molecule is removed, a cavity which is matched with the spatial structure of the template molecule and has a spatial site can be formed in the polymer. The molecular imprinting polymer formed by the molecular imprinting technology has good space three-dimensional shape, so that the removal of the template molecules does not influence the recombination capability of the action sites. The molecular imprinting technology has been applied to the detection and identification of biological macromolecules such as protein, DNA, cells, viruses and the like due to the characteristics of specific structure-effect prearrangement, specific identification, practicability and the like. The Spivak topic group utilizes thrombin as a template, prepares aptamer functionalized hydrogel by using a molecular imprinting technology, and utilizes the specificity recognition capability of an aptamer on the thrombin to directly measure the volume change of the hydrogel so as to realize the detection of the thrombin.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a molecular imprinting intelligent gel grating, a preparation method thereof and a thrombin detection method based on the molecular imprinting intelligent gel grating so as to improve the sensitivity and convenience of thrombin detection.

The main technical concept of the invention is as follows: the method comprises the steps of combining a molecular imprinting technology with an intelligent gel grating sensing technology, polymerizing a gel network with a cross-linking agent by using thrombin as an imprinting template, two thrombin specific aptamers A1 and a thrombin specific aptamer A2 which can be combined with different sites of the thrombin and N-isopropylacrylamide as comonomers, removing the imprinting template in the gel network to obtain a molecular imprinting intelligent gel capable of specifically recognizing the thrombin, combining the molecular imprinting intelligent gel with the intelligent gel grating sensing technology to prepare the molecular imprinting intelligent gel grating capable of specifically recognizing the thrombin to cause the change of diffraction light intensity, and thus realizing sensitive sensing detection of the thrombin.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

the invention provides a molecular imprinting intelligent gel grating, which consists of a substrate and gel strips which are parallel to each other on the substrate, wherein gaps among the gel strips are slits of the gel grating, and the gel strips are formed by thrombin response type molecular imprinting gel;

the thrombin response type molecularly imprinted gel is formed by eluting thrombin in a gel network from a gel containing a thrombin imprinted complex; the thrombin imprinted complex is formed by combining thrombin specific aptamer A1, thrombin specific aptamer A2 and thrombin at different sites, wherein the nucleotide sequence of the thrombin specific aptamer A1 is 5'-/5Acryd/GGT TGG TGT GGT TGG-3', and the nucleotide sequence of the thrombin specific aptamer A2 is 5'-/5Acryd/AGA CCG TGG TAG GGC AGG TTG GGG TGA CT-3';

the gel containing the thrombin imprinted complex is formed by cross-linking reaction of N-isopropylacrylamide, the thrombin imprinted complex and a cross-linking agent, and in the cross-linking reaction process, the carbon-carbon double bond of the N-isopropylacrylamide, the 5 'end of thrombin specific aptamer A1 in the thrombin imprinted complex and the carbon-carbon double bond on the 5' end of thrombin specific aptamer A2 and the carbon-carbon double bond on the cross-linking agent are subjected to addition reaction to form a gel network; in the crosslinking reaction process, the molar ratio of the N-isopropylacrylamide to the crosslinking agent is controlled to be 1 (0.01-0.05), and the molar ratio of the thrombin imprinted complex to the crosslinking agent is controlled to be 1 (1000-5000).

In the technical scheme of the molecular imprinting intelligent gel grating, the cross-linking agent is four-arm-polyethylene glycol acrylamide, eight-arm-polyethylene glycol acrylamide or two-arm-polyethylene glycol acrylamide.

In the technical scheme of the molecular imprinting intelligent gel grating, the ratio of the period of the gel grating to the grating height is 1 (0.01-0.1), and the ratio of the period of the gel grating to the grating height in a dry state or a wet state is generally within the ratio range. Generally, the period of the gel grating is 1700-1800 nm, and the grating height is 30-200 nm.

In the technical scheme of the above molecular imprinting intelligent gel grating, the substrate is a silica glass sheet subjected to silanization treatment, the gel strip is combined with the substrate through a covalent bond, specifically, after the silica glass sheet is subjected to silanization treatment, a silane coupling agent is combined on the surface of the silica glass sheet through a Si-O bond generated by a reaction of the silane coupling agent and hydroxyl on the surface of the silica glass sheet, and meanwhile, the silane coupling agent has polymerizable double bonds and can participate in a crosslinking reaction process of N-isopropylacrylamide, a thrombin imprinting compound and a crosslinking agent, so that hydrogel formed after polymerization is stably fixed on the surface of the silica glass sheet. The gel strip is combined with the substrate through a covalent bond, so that the period stability of the gel grating can be ensured, the fixation of the position of a diffraction light spot in the diffraction efficiency test process is further ensured, the volume change of the gel strip is converted into the change of the height of the grating after the gel grating identifies thrombin, the change of the diffraction light intensity is further caused, and the volume change of the gel strip is stably converted into the change of the diffraction light intensity.

The invention also provides a preparation method of the molecular imprinting intelligent gel grating, which comprises the following steps:

(1) preparing gel pre-polymerization liquid

Adding the thrombin specific aptamer A2 into a thrombin solution, stirring for 20-40 min, then adding the thrombin specific aptamer A1, and stirring for 20-40 min to obtain a solution containing a thrombin imprinted complex; the molar ratio of the thrombin-specific aptamer A1 to the thrombin-specific aptamer A2 to the thrombin is 1 (0.02-1);

adding N-isopropylacrylamide, a cross-linking agent, an initiator and an accelerator into a solution containing the thrombin imprinted complex, and sufficiently shaking and dissolving under an ice bath condition to obtain a gel pre-polymerization solution; in the gel pre-polymerization liquid, the concentration of N-isopropylacrylamide is 0.5-2.5 mol/L, the molar ratio of the N-isopropylacrylamide to the crosslinking agent is 1 (0.01-0.05), the molar ratio of the N-isopropylacrylamide to the initiator is 1 (0.005-0.05), the molar ratio of the N-isopropylacrylamide to the accelerator is 1 (0.02-0.2), and the molar ratio of the thrombin imprinted complex to the crosslinking agent is 1 (1000-5000);

(2) preparation of thrombin-containing gel gratings

Dripping the gel prepolymer onto a substrate, pressing a gel grating template on the gel prepolymer, reacting for 4-12 h at 4-10 ℃, and stripping the gel grating template to obtain a gel grating containing thrombin;

(3) washing to remove thrombin in gel network

And (2) placing the gel grating containing thrombin in a PBS buffer solution for soaking and washing to remove incompletely polymerized reactants, then taking out the gel grating and placing the gel grating in a mixed solution of guanidine hydrochloride and sodium chloride for fully soaking to remove thrombin in a gel network structure of the gel grating containing thrombin, replacing the mixed solution of guanidine hydrochloride and sodium chloride every 6-12 hours in the soaking process of the mixed solution of guanidine hydrochloride and sodium chloride, taking out the gel grating and placing the gel grating in the PBS buffer solution for fully soaking and washing after soaking is completed, and thus obtaining the molecular imprinting intelligent gel grating.

In the step (3) of the preparation method of the molecular imprinting intelligent gel grating, in the mixed solution of guanidine hydrochloride and sodium chloride, the concentration of guanidine hydrochloride is 3-6 mol/L, and the concentration of sodium chloride is 1-2 mol/L.

In the step (3) of the preparation method of the molecular imprinting intelligent gel grating, the pH value of the PBS buffer solution is 7.4, and the concentration of the PBS buffer solution is 1-3 mmol/L.

In the step (3) of the preparation method of the molecular imprinting intelligent gel grating, the mixed solution of guanidine hydrochloride and sodium chloride is replaced every 6-12 hours, and the thrombin in the gel network structure of the thrombin-containing gel grating can be completely removed after the mixed solution of guanidine hydrochloride and sodium chloride is replaced for 4-6 times.

In the step (2) of the preparation method of the molecular imprinting intelligent gel grating, the substrate is a silanized quartz glass sheet. Further, the silanization treatment method of the quartz glass sheet is as follows:

immersing the cleaned quartz glass sheet in an acetic acid-sodium acetate buffer solution containing a silane coupling agent for 10-30 min, then keeping for 10-20 min in an oven at 50-60 ℃, washing with deionized water, and drying to finish the silanization treatment of the quartz glass sheet; in the acetic acid-sodium acetate buffer solution containing the silane coupling agent, the volume percentage concentration of the silane coupling agent is 0.5-2%, the pH value of the acetic acid-sodium acetate buffer solution is 4.5-5.5, and the concentration is 0.05-0.2 mol/L. The silane coupling agent is 3- (acryloyloxy) propyl trimethoxy silane, vinyl trimethoxy silane or vinyl trichlorosilane.

Based on the molecular imprinting intelligent gel grating, the invention also provides a thrombin detection method based on the molecular imprinting intelligent gel grating, which uses an optical detection device comprising the molecular imprinting intelligent gel grating for detection, and comprises the following steps:

(1) determination of the conversion equation for the Thrombin concentration

Firstly, adding a blank sample into a quartz sample pool of an optical detection device to immerse a gel grating, and reading first-order diffraction light intensity I of the blank sample after the diffraction light intensity of the gel grating is stable₁And intensity of zero-order diffracted light I₀Calculating the diffraction efficiency of the blank sample;

sequentially replacing blank samples in the step I with standard samples with known thrombin concentration according to the sequence of thrombin concentration in the standard samples from low to high, and respectively measuring the diffraction efficiency corresponding to each standard sample to obtain the diffraction efficiency corresponding to a series of standard samples;

calculating the change rate of the diffraction efficiency corresponding to each standard sample relative to the diffraction efficiency of a blank sample, recording the change rate as the relative diffraction efficiency to obtain a series of relative diffraction efficiencies, drawing a working curve by taking the relative diffraction efficiency as an abscissa and taking the thrombin concentration as an ordinate, and determining a conversion relation between the thrombin concentration and the relative diffraction efficiency;

(2) measuring the concentration of thrombin in a test sample

Replacing the original gel grating with the gel grating in the step (1), adding a blank sample into a quartz sample pool of the optical detection device to immerse the gel grating, and reading the first-order diffraction light intensity I of the blank sample after the diffraction light intensity of the gel grating is stable₁And intensity of zero-order diffracted light I₀Calculating the diffraction efficiency of the blank sample;

replacing the blank sample in the step (2) with a sample to be tested with unknown thrombin concentration, and measuring the diffraction efficiency of the sample to be tested;

calculating the relative diffraction efficiency of the sample to be measured relative to the diffraction efficiency of the hollow white sample in the step (2), and calculating the concentration of the thrombin in the sample to be measured according to the conversion relation between the concentration of the thrombin and the relative diffraction efficiency;

and (3) controlling the constant temperature of the step (1) and the step (2) which is the same as the testing temperature and is within the range of 20-35 ℃.

Experiments prove that the detection limit of the thrombin detection method provided by the invention is as low as 10^-12mol/L, can realize the ratio of 10^-12～10^-7The detection of thrombin with mol/L concentration level is particularly suitable for the ultra-sensitive quantitative detection of thrombin in urine.

In the thrombin detection method, the adopted optical detection device comprises a laser light source, a quartz sample cell provided with the molecular imprinting intelligent gel grating, a first silicon photoelectric detector for detecting the intensity of zero-order diffraction light, a second silicon photoelectric detector for detecting the intensity of first-order diffraction light, a data acquisition system and a computer processing system; the substrate of the molecular imprinting intelligent gel grating is fixed on the inner wall of a quartz sample cell, a slit of the molecular imprinting intelligent gel grating is perpendicular to the horizontal plane, the quartz sample cell is preferably arranged on a heating platform on a temperature control heat table to regulate and control the test temperature, the quartz sample cell is positioned between a laser and a first silicon photoelectric detector and a second silicon photoelectric detector, a laser beam emitted by the laser vertically irradiates the molecular imprinting intelligent gel grating and generates diffraction to penetrate out of the quartz sample cell, the first silicon photoelectric detector and the second silicon photoelectric detector are respectively aligned to light spots of zero-order diffraction and first-order diffraction, the silicon photoelectric detector is connected with a data acquisition system, and the data acquisition system is connected with a computer processing system. The laser, first and second silicon photodetectors, data acquisition system, and computer processing system are preferably mounted on a damped vibration isolation optical platform to reduce interference of external environmental vibrations with test results.

In the thrombin detection method, the wavelength of a laser beam emitted by a laser of the adopted optical detection device is 510-635 nm, and the detection wavelengths and the powers of the first silicon photoelectric detector and the second silicon photoelectric detector are matched with the wavelength and the power emitted by the laser.

In the thrombin detection method, the diffraction efficiency refers to the intensity I of first-order diffraction light₁Intensity of zero-order diffracted light I₀The relative diffraction efficiency is the change rate of the diffraction efficiency of the standard sample or the sample to be detected relative to the diffraction efficiency of the blank sample, and the calculation formula is shown as formula (1):

in the formula (1), R_DEFor relative diffraction efficiency, DE_T,0The diffraction efficiency of the blank at a temperature T, DE_TThe diffraction efficiency of a standard sample or a sample to be measured at the temperature T.

The principle of the invention for detecting the thrombin in the sample to be detected is as follows:

the invention provides a molecular imprinting intelligent gel grating which consists of a substrate and mutually parallel gel strips on the substrate, wherein the gel strips consist of thrombin response type molecular imprinting gel, and the thrombin response type molecular imprinting gel is formed by eluting imprinting template thrombin in a gel network from gel containing a thrombin imprinting compound. When the molecular imprinting intelligent gel grating provided by the invention is in a solution to be detected containing thrombin, the thrombin in the solution to be detected is combined with thrombin specific aptamers A1 and A2 in a gel network of the gel grating, and the combination of the thrombin in the solution to be detected and the thrombin specific aptamers A1 and A2 can increase non-covalent crosslinking points in the gel network and increase crosslinking density in the gel network, so that the volume of a gel strip is shrunk, and the intensity of diffracted light penetrating through the gel grating is changed. The light diffraction signal is detected by a photoelectric probe, so that the thrombin can be sensitively and conveniently detected. Because a stable cavity which is matched with the space structure of thrombin molecules and has space sites is formed in the gel network of the gel strip, the recombination capability of the thrombin and thrombin specific aptamers A1 and A2 in the gel network is greatly improved, the detection sensitivity of the gel grating to the thrombin is greatly enhanced, and the high-sensitivity sensing detection to the thrombin is realized.

Compared with the prior art, the technical scheme of the invention has the following beneficial technical effects:

1. the invention combines a molecular imprinting technology and an intelligent gel grating sensing technology, takes thrombin as an imprinting template and two thrombin specific aptamers which can be respectively combined with different sites of thrombin as comonomers, and prepares the thrombin imprinting type intelligent gel grating sensor with functionalized aptamers. The gel grating can specifically identify thrombin, when the molecularly imprinted intelligent gel grating provided by the invention is in a solution to be detected containing thrombin, the thrombin in the solution to be detected is combined with thrombin specific aptamers A1 and A2 in a gel network of the gel grating, and the combination of the thrombin in the solution to be detected and the thrombin specific aptamers A1 and A2 can increase non-covalent crosslinking points in the gel network and increase crosslinking density in the gel network, so that the volume of a gel strip is reduced, and the intensity of diffracted light penetrating through the gel grating is changed. By detecting the diffraction light signal, the sensitive and convenient detection of the thrombin can be realized.

2. Because a stable cavity which is matched with the space structure of thrombin molecules and has a space site is formed in the gel network of the gel strip on the gel grating, the recombination capability of the thrombin and the thrombin specific aptamer in the gel network is greatly improved, the detection sensitivity of the gel grating to the thrombin is greatly enhanced, and the high-sensitivity sensing detection of the thrombin is realized. Furthermore, the gel strip on the gel grating is fixed on the substrate through a covalent bond, so that the period of the gel grating can be basically kept fixed in the process of gel volume contraction, and only the fluctuation height of the gel grating can be obviously changed, so that the change of the diffraction light intensity is caused, and the Bragg diffraction angle is unchanged, which is favorable for improving the detection sensitivity of thrombin.

3. Because the invention adopts the use of macromolecule cross-linking agent in the process of preparing the gel grating, the cross-linked PNIPAM macromolecule network structure can keep good transparency in the process of volume change, which ensures that a high-intensity diffraction light signal is obtained in the test process. Meanwhile, the chain length of the macromolecular cross-linking agent enables the gel to have more excellent swelling performance in the molecular recognition process. The above factors also contribute to the improvement of the detection sensitivity for thrombin.

4. The invention also provides a thrombin detection method based on the molecular imprinting intelligent gel grating, and experiments show that the detection limit of the method to thrombin is as low as 10^-12mol/L, can realize the ratio of 10^-12～10^-7The detection of thrombin with the mol/L concentration level can be used for detecting the concentration of the biomarker thrombin in urine with ultra-sensitivity. Compared with the prior art, the detection method has higher detection sensitivity, does not need to depend on an additional signal enhancement mode, and provides a new way for in vitro gel grating sensing detection of the biomarker.

Drawings

FIG. 1 is a schematic diagram of the preparation process and thrombin response principle of the molecularly imprinted intelligent gel grating, wherein in FIG. 1, h₂And h₁The heights of the gel grating before and after the gel grating recognizes thrombin are respectively shown.

FIG. 2 is a schematic view of a silanization process of a quartz glass plate.

FIG. 3 is the result of chemical composition analysis of the gel grating prepared in example 1, in which a) is a DNA standard curve and b) is an ultraviolet absorbance curve of the gel pre-polymerization solution and the eluent.

FIG. 4 shows the optical photograph (a) and SEM photograph (b) of the gel grating prepared in example 1.

Fig. 5 is an AFM photograph of the gel grating prepared in example 1, wherein a and c are two-dimensional and three-dimensional shape pictures of the gel grating in a dry state, and b and d are two-dimensional and three-dimensional shape pictures of the gel grating in a wet state after the gel grating reaches a swelling equilibrium in a buffer solution.

Figure 6 is a plot of diffraction efficiency versus temperature for the gel grating prepared in example 1.

FIG. 7 shows the transmittance at different temperatures of the gel grating prepared in example 1.

FIG. 8 shows the R values of the gel gratings prepared in example 1 in different protein buffers_DE。

FIG. 9 is an AFM picture of the gel grating prepared in example 1 in different concentrations of thrombin solution.

FIG. 10 shows the variation of the structural parameters of the gel grating prepared in example 1 with thrombin concentration, wherein a) b) are the curves of the grating height and the grating period with thrombin concentration.

Fig. 11 is a schematic structural diagram of an optical detection device constructed based on the gel grating provided by the present invention, in which 1-a laser light source, 2-a gel grating, 3-a quartz sample cell, 4-1-a first silicon photodetector, 4-2-a second silicon photodetector, 5-a data acquisition system, and 6-a computer processing system 6.

FIG. 12 is a graph of the diffraction efficiency of a gel grating as a function of thrombin concentration at different temperatures.

FIG. 13 is a graph of thrombin concentration versus R_DEA linear quantitative relationship between them.

Detailed Description

The molecularly imprinted intelligent gel grating, the preparation method thereof and the thrombin detection method are further described by the following embodiments. It should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make certain insubstantial modifications and adaptations of the present invention based on the above disclosure and still fall within the scope of the present invention.

In the following examples, the nucleotide sequences of thrombin-specific aptamer a1 (abbreviated as a1) and thrombin-specific aptamer a2 (abbreviated as a2) were 5' -/5Acryd/GGT TGG TGT GGT TGG-3' (see SEQ ID No.1) and 5' -/5Acryd/AGA CCG TGG TAG GGC AGG TTG GGG TGA CT-3' (see SEQ ID No.2), respectively, and Acryd in the nucleotide sequences represents a polymerizable methacrylamide group, which group was attached to the base at the 5' end by modification. A1 and A2 are nucleotide sequences which are available and can be synthesized by professional companies.

In the following embodiments, based on the gel grating provided by the present invention, an optical detection device as shown in fig. 11 is set up, where the optical detection device includes a laser light source 1, a quartz sample cell 3 in which the gel grating 2 of the present invention is installed, a first silicon photodetector 4-1 for detecting the intensity of zero-order diffracted light, a second silicon photodetector 4-2 for detecting the intensity of first-order diffracted light, a data acquisition system 5, and a computer processing system 6.

The substrate of the gel grating is fixed on the inner wall of the quartz sample cell, the slit of the gel grating is vertical to the horizontal plane, the quartz sample cell is arranged on a heating platform on a temperature control heat table to regulate and control the test temperature, the quartz sample cell is positioned between the laser and the first and second silicon photoelectric detectors, the laser beam emitted by the laser vertically irradiates the gel grating and generates diffraction to penetrate out of the quartz sample cell, the first silicon photoelectric detector and the second silicon photoelectric detector are respectively aligned to light spots of zero-order diffraction and first-order diffraction, the silicon photoelectric detectors are connected with a data acquisition system, and the data acquisition system is connected with a computer processing system. The laser, the first and second silicon photoelectric detectors, the data acquisition system and the computer processing system are arranged on the damping vibration isolation optical platform so as to reduce the interference of the vibration of the external environment on the test result. The first and second silicon photodetectors are photoelectric probes (DSi200, Zolix).

Before the optical detection device is used to start detection, the heights and positions of the first and second silicon photodetectors positioned behind the sample cell are adjusted so that light spots of zero-order diffraction light and first-order diffraction light generated after the gel grating is vertically irradiated by the laser beam are just aligned with the central positions of the receiving screens of the first and second silicon photodetectors. The laser used in the test was a He-Ne laser with a wavelength of 635 nm.

Example 1

In this embodiment, a schematic diagram of a preparation process and a thrombin response principle of the molecularly imprinted intelligent gel grating is shown in fig. 1, and the steps are as follows:

(1) silanization of substrates

And (3) putting the quartz glass sheet into a concentrated sulfuric acid solution, performing ultrasonic treatment for 15min to remove inorganic impurities on the surface of the quartz glass sheet, then putting the quartz glass sheet into deionized water, performing ultrasonic treatment for 15min, and cleaning the quartz glass sheet by using the deionized water. And putting the cleaned glass sheet into acetone for ultrasonic treatment for 15min to remove organic impurities on the surface of the glass sheet, then putting the glass sheet into deionized water for ultrasonic treatment for 15min and cleaning the glass sheet with the deionized water, and putting the cleaned quartz glass sheet into the deionized water for later use. The purpose of this step is to keep hydrocarbon contamination to a minimum while ensuring that the quartz glass sheet remains fully wettable when immersed in a solution containing a silane coupling agent.

As shown in fig. 2, the silanization treatment was performed on the quartz glass plate by dip coating: preparing an acetic acid-sodium acetate buffer solution with the pH value of 5 and the concentration of 0.1mol/L, adding a silane coupling agent 3- (acryloyloxy) propyl trimethoxy silane (3-MAPTMS) into the acetic acid-sodium acetate buffer solution according to the volume ratio of the silane coupling agent to the acetic acid-sodium acetate buffer solution of 1:99, stirring for 30min to fully perform hydrolysis reaction, then adding a washed quartz glass sheet, immersing for 10min, taking out the quartz glass sheet, placing the quartz glass sheet in a constant-temperature oven at 60 ℃ for 10min, and washing with deionized water to remove silane coupling agent molecules physically adhered to the surface of the quartz glass sheet. The silanized quartz glass plate is dried under negative pressure and placed in a drying cabinet for standby.

(2) Preparing gel pre-polymerization liquid

Adding A2 into thrombin (thrombin) solution, stirring for 30min, then adding A1, and stirring for 30min to obtain a solution containing thrombin imprinted complex A1-thrombin-A2. The molar ratio of A2 to thrombin was 1:1, and the molar ratio of A1 to thrombin was 1: 0.94.

Adding N-isopropylacrylamide (NIPAM) and a cross-linking agent quadri-arm-polyethylene glycol acrylamide (tetra-arm PEGAAm) into a solution containing a thrombin imprinted complex, fully shaking for dissolution under an ice bath condition, then adding an ammonium persulfate solution with the concentration of 10 wt.%, then adding an accelerator N, N, N ', N' -Tetramethylethylenediamine (TEMED), fully shaking for dissolution under the ice bath condition to obtain a gel pre-polymerization solution, and placing the gel pre-polymerization solution into an ice bath for later use.

In the gel pre-polymerization liquid, the concentration of NIPAM is 1.5mol/L, the molar ratio of NIPAM to tetra-arm PEGAAm is 1:0.03, the molar ratio of NIPAM to initiator is 1:0.02, the molar ratio of NIPAM to accelerator is 1:0.04, and the molar ratio of thrombin imprinted complex to tetra-arm PEGAAm is 1: 3800.

(3) Method for preparing gel grating containing thrombin by micro-contact printing method

And (2) taking 8 mu L of gel pre-polymerization liquid by using a micro-contact printing method and a micro-pipette, quickly dripping the gel pre-polymerization liquid on the surface of the silanized quartz glass sheet, quickly and lightly impressing a gel grating template on the gel pre-polymerization liquid, ensuring that the grating template and the silanized quartz glass sheet are tightly combined, transferring the gel grating template to an environment at 5 ℃, initiating a crosslinking reaction through free radicals, and stripping the gel grating template after crosslinking for 6 hours to obtain the gel grating containing thrombin. The size of the gel grating template is 18mm multiplied by 12mm, the material is polyethylene terephthalate (PET), a grating pattern is arranged on the gel grating template, the period of the gel grating template is 1728nm, and the grating height is 39 nm.

After the reaction of the step, the gel prepolymerization solution is converted into a gel state, which indicates that each component in the gel prepolymerization solution realizes successful polymerization.

(4) Washing to remove thrombin in gel network

Placing the gel grating containing thrombin in PBS buffer solution with pH 7.4 and concentration of 2mmol/L, soaking and washing to remove incompletely polymerized reactants, then taking out and placing in a mixed solution of guanidine hydrochloride and sodium chloride, soaking to denature thrombin to remove thrombin in the gel network structure of the gel grating containing thrombin, replacing the mixed solution of guanidine hydrochloride and sodium chloride every 6h in the process of soaking in the mixed solution of guanidine hydrochloride and sodium chloride, replacing washing solution for 5 times, taking out and placing in PBS buffer solution with pH 7.4 and concentration of 2mmol/L, fully soaking and washing, and balancing to obtain the molecular imprinting intelligent gel grating. In the mixed solution of the guanidine hydrochloride and the sodium chloride, the concentration of the guanidine hydrochloride is 4.3mol/L, and the concentration of the sodium chloride is 1.4 mol/L.

Example 2

In this embodiment, the chemical components of the molecularly imprinted intelligent gel grating are tested by the following steps:

(1) the absorbance values of the DNA (A1 and A2) standard solutions were measured using an ultraviolet-visible spectrophotometer, and the resulting DNA concentration-absorbance standard curve was plotted based on the DNA content in the standard solution and its absorbance value at 257nm wavelength of ultraviolet light, as shown in a) of FIG. 3, in which the abscissa DNA concentration represents the ratio of the DNA content tested to the theoretical amount added. The linear regression equation of the standard curve is that y is 0.0021x-0.0005, and the correlation coefficient R²The content of the DNA is 0.9984, which shows that the proportion of the DNA content to the theoretical addition value is in the range of 0-14 percent and follows the beer law, namely the concentration of the light absorbing substance DNA is in direct proportion to the absorbance of the light absorbing substance DNA.

Therefore, by measuring the absorbance value of the eluate obtained by washing the thrombin-containing gel grating in the step (4) of example 1, the ratio of the DNA content in the eluate to the theoretical addition amount can be calculated by using the standard curve, and the DNA ratio polymerized into the intelligent gel network can be calculated.

(2) After the eluate obtained by washing the thrombin-containing gel grating in step (4) of example 1 was dialyzed several times to remove thrombin and other small molecule impurities which interfere with the ultraviolet absorbance value, the absorbance curve of the resulting dialysate was measured, as shown in the gel eluate in b) of FIG. 3, from which it was seen that the absorbance at 257nm was 0.01. And (3) calculating according to the standard curve in the step (1) to obtain that the DNA concentration of the dialysate is 5%, the total volume of the dialysate is 5mL, and 3mL of dialysate is measured when an absorbance test is carried out, so that the total DNA content of the dialysate is 8.3%. The DNA content in the dialyzate was only 8.3% of the theoretical amount of DNA added to the gel pre-polymerization solution, i.e., the remaining about 91.7% of DNA was successfully polymerized into the gel network.

Example 3

In this example, the morphology characteristics of the molecularly imprinted intelligent gel grating prepared in example 1 were characterized.

1. An optical photograph of the gel grating prepared in example 1, as shown in a of fig. 4, after peeling the grating template, obtained a gel grating having the same size as the gel grating template, the size of the gel grating being 18mm × 12 mm. It can be seen from the optical picture that under natural illumination, the gel grating surface has a very obvious diffraction phenomenon, i.e. stripes with different colors can be observed on the gel grating surface.

The Scanning Electron Microscope (SEM) photograph of the gel grating of example 1 is shown in b of fig. 4, and it can be seen that the gel grating has a large-area regular microstructure, and the large-area regular microstructure ensures the stability and repeatability of the test result. Meanwhile, the gel grating is provided with a microstructure which is complementary with the gel grating template.

2. The microscopic appearances of the gel grating prepared in the step (4) of example 1 in the dry state and the wet state were observed by using an Atomic Force Microscope (AFM), as shown in fig. 5, wherein the two pictures a and c are two-dimensional and three-dimensional appearance pictures of the gel grating in the dry state, respectively, and the two pictures b and d are two-dimensional and three-dimensional appearance pictures of the gel grating in the wet state after the gel grating reaches swelling equilibrium in the buffer solution. The cross-sectional analysis of the AFM image revealed that: the period of the gel grating in a dry state is 1728nm, and the grating height is 39 nm; the period of the gel grating in the wet state is 1740nm, and the grating height is 154 nm.

The microscopic appearances of the gel grating in the dry state and the wet state are contrasted, and the gel grating has a very regular grating structure before and after swelling. From specific structural parameters, the height of the gel grating is increased by about 4 times after the gel grating absorbs water from a dry state and undergoes volume swelling, but the period is basically kept unchanged.

Compared with the volume change of the gel grating from a dry state to water absorption and swelling, the volume change of the gel grating is mainly shown as the great increase of the height of the gel grating, and the gel grating is kept stable in the whole process in a period. This is mainly because the silica glass sheet is silanized before the gel grating is produced, and the silane coupling agent is bonded to the surface of the silica glass sheet by reacting with hydroxyl groups on the surface of the silica glass sheet to form Si — O bonds, and on the other hand, since the silane coupling agent itself has polymerizable double bonds, the silane coupling agent participates in the radical polymerization reaction of the gel pre-polymerization solution, so that the hydrogel formed after polymerization is stably fixed on the surface of the silica glass sheet.

The stability of the period of the gel grating ensures that the position of a diffraction light spot is fixed in the diffraction efficiency test process, and the change of the grating height can cause great change of diffraction light intensity, so that the volume change of the gel grating is stably converted into the change of the diffraction light intensity.

Example 4

In this example, an optical detection device as shown in fig. 11 was constructed based on the gel grating prepared in example 1, and the temperature response performance of the gel grating was examined using the optical detection device.

5mL of PBS buffer (pH 7.4, concentration 2mmol/L) was added to the quartz sample cell, the temperature of the solution in the quartz sample cell was adjusted by a temperature-controlled hot stage, and a temperature-measurable thermistor was inserted into the solution in the quartz sample cell to monitor the temperature of the solution in real time. Respectively testing the first-order diffraction light intensity I of the gel grating at the temperature points of 20-50 ℃ every 5 DEG C₁And intensity of zero-order diffracted light I₀When the solution in the quartz sample cell reaches the set temperature during the test, the solution needs to be balanced for 30min and then the test is carried out, and data is recorded.

The curve of the diffraction efficiency of the gel grating with temperature is shown in fig. 6, and it can be seen that the diffraction efficiency of the gel grating increases with the increase of temperature, and the increase is relatively obvious at 35 ℃. This is because at a low temperature, the amide group of the PNIPAM reacts with water molecules to form hydrogen bonds, so that the water molecules in the solvent form a highly ordered solvent shell layer composed of hydrogen bonds around the polymer chain of the PNIPAM, and the hydrogel at this time is in a highly swollen hydrophilic state. With the increase of the temperature, the hydrogen bond interaction between the amide group and the water molecule is destroyed, the interaction between the hydrophobic groups of the PNIPAM macromolecule is enhanced, a hydrophobic layer is formed, the water molecule in the gel network is discharged, and the contraction of the macromolecule and the volume reduction are caused. Since the gel grating is fixed on the surface of the quartz glass sheet by the silane coupling agent, the volume change of the gel grating is only reflected by the change of the grating height and the consequent change of the refractive index of the gel grating. During diffraction, these two changes occur simultaneously, eventually manifesting as an increase in the diffraction efficiency of the gel grating with increasing temperature.

Example 5

In this example, the change of the light transmittance of the molecularly imprinted intelligent gel grating prepared in example 1 with temperature was tested.

The detection principle of the gel grating is based on the change of diffracted light intensity, and the light transmittance of the gel material on the gel grating has a great influence on the light intensity after the gel material is transmitted. Because the invention adopts the PNIPAM high molecular material sensitive to the temperature, the determination of the light transmittance of the gel grating at different temperatures is very necessary for the measurement of the intensity of the diffracted light. The transmittance of the gel grating at every 5 ℃ temperature point between 20 ℃ and 50 ℃ was measured at a wavelength of 635nm and the results are shown in fig. 7.

As can be seen from fig. 7, the light transmittance of the gel grating at each of the above temperature points is greater than 99%, which indicates that the gel grating has excellent light transmittance in the test temperature range, i.e., the gel grating has almost no absorption of light with a wavelength of 635 nm. This is mainly due to the use of the macromolecular cross-linker tetra-arm PEGAAm in the preparation of gel gratings. On the one hand, since the macromolecular crosslinking agent has a very uniform structure, the internal structure of the hydrogel molecular network formed by crosslinking the macromolecular crosslinking agent is also very uniform. On the other hand, the hydrophilic polyethylene glycol chain is directly used as the main chain of the hydrogel network, so that the hydrophobic aggregation of the PNIPAM long chain can be prevented to a certain extent, and the internal structure of the hydrogel is always kept in a homogeneous phase. Therefore, even if the volume of the hydrogel changes in the temperature rise process, the difference of the light transmittance is very small, the influence on the diffraction light intensity is basically negligible, and the very high light transmittance can be kept at different temperatures.

Example 6

In this example, an optical detection device as shown in fig. 11 was constructed based on the gel grating prepared in example 1, and the specific recognition ability of the gel grating for thrombin was examined using the optical detection device.

In order to study whether the gel grating has the specific recognition capability on Thrombin, human immunoglobulin (IgG), Bovine Serum Albumin (BSA) and Lysozyme (Lysozyme) are selected as a control group, Thrombin (Thrombin) is selected as an experimental group, and the recognition performance of the gel grating on Thrombin is respectively tested. Before the detection, PBS with pH 7.4 and concentration of 2mmol/L is used to dissolve each protein in the control group and the experimental group, and the concentration of each protein in each group is 10^-7mol/L。

5mL of each control protein solution was added to the quartz sample cell and the temperature of the solution in the quartz sample cell was maintained at 30 ℃ by adjusting the temperature of the temperature-controlled hotplate. Testing first order diffraction light intensity I of gel grating₁And intensity of zero-order diffracted light I₀When the solution in the quartz sample cell reaches the set temperature during the test, the solution is balanced for 30min, the test is carried out again, data are recorded, and the relative diffraction efficiency (R) is calculated_DE)。

As a result, as shown in FIG. 8, it was found that the R of the gel grating was observed in the solution of IgG, BSA and Lysozyme_DEAre all less than 0.01, and when thrombin is added to the sample fluid at the same concentration, the R of the gel grating_DEThen up to 0.11, indicating that the gel grating has high selectivity for thrombin.

Example 7

In this example, the gel grating prepared in example 1 was tested for its ability to detect thrombin.

1. Testing structural parameters of gel grating in thrombin buffer solutions with different concentrations

Placing the gel grating at thrombin concentration of 10^-12、10^-11、10^-10、10^-9、10^-8、10^-7After sufficiently soaking in mol/L PBS buffer (pH 7.4, concentration 2mmol/L), AFM pictures were tested, and the results are shown in fig. 9.

FIGS. 9 a-f are views of a gel grating 10^-12、10^-11、10^-10、10^-9、10^-8、10^-7AFM pictures after well soaking in mol/L PBS buffer. As can be seen from FIG. 9, the gel grating has a very regular grating morphology in thrombin solutions of different concentrations. Analysis of the cross section of the AFM image shows that the grating heights of the gel grating are different in thrombin buffers with different concentrations, and the grating heights in the a-f graphs of fig. 9 are respectively: 145. 136, 128, 116, 108 and 99nm, and the grating periods are respectively as follows: 1742. 1758, 1760, 1739 and 1756 nm.

The curve of the structural parameters of the gel grating with the thrombin concentration is shown in FIG. 10, in which a) b) are the curves of the grating height and the grating period with the thrombin concentration. As can be seen, the height of the gel grating decreased significantly with increasing thrombin concentration, but the grating period remained essentially constant at different thrombin concentrations.

This is because when thrombin in solution contacts the gel grating, thrombin specific aptamers a1 and a2 in the gel network specifically recognize thrombin, and the binding of thrombin and thrombin specific aptamers results in an increase in non-covalent crosslinking points within the gel network, resulting in an increase in the crosslink density of the gel network, causing the gel strip to undergo volume shrinkage. Because the bottom surface of the gel strip on the gel grating is fixed on the surface of the quartz glass sheet by the silane coupling agent, the volume shrinkage of the gel strip is mainly embodied as the reduction of the grating height, and the grating period is basically kept unchanged. Meanwhile, compared with thrombin-specific aptamers which are randomly arranged in a conventional gel grating network, thrombin is coupled with A1 and A2 to form a compound A1-thrombomin-A2 before polymerization reaction, and the structure of the compound A1-thrombomin-A2 is integrally polymerized into the space network of the gel grating during the polymerization reaction. Therefore, after the thrombin template in the gel network is removed, the A1 and the A2 are regularly arranged in the gel network of the gel grating by taking the thrombin as the template. This reduces the mass transfer resistance of re-recognition to a certain extent, making the detection sensitivity for thrombin superior to conventional gel gratings that do not use molecular imprinting techniques. The experimental result also proves that the gel grating can realize the detection of low-concentration thrombin.

2. An optical detection device shown in fig. 11 was constructed based on the gel grating prepared in example 1, and the detection ability of the gel grating for thrombin was examined using the optical detection device.

The thrombin concentrations were 10 each prepared using a PBS buffer solution at a pH of 7.4 and a concentration of 2mmol/L as a solvent^-12、10^-11、10^-10、10^-9、10^-8、10^-7mol/L of the solution to be tested. Since the temperature of urine is similar to body temperature, usually 36-37 ℃, and after being discharged from the body, the temperature will rapidly drop until the temperature drops to room temperature (about 23-25 ℃). Therefore, in the present embodiment, the first-order diffraction light intensity I of the gel grating is used as a temperature test point every 5 ℃ in each solution to be tested at 20-35 DEG C₁And intensity of zero-order diffracted light I₀Equilibration at each set temperature point was required for 20min prior to testing. PBS buffer (pH 7.4, 2mmol/L) was used as a blank. The results are shown in FIG. 12.

As can be seen from FIG. 12, the DE of the gel grating was found to be within the temperature range tested_T/DE_T,0Increasing linearly with increasing thrombin concentration. This is mainly due to the binding of thrombin to thrombin specific aptamers a1, a2, causing the gel grating to contract volumetrically, resulting in a reduction in the height of the grating.

Example 8

In this embodiment, an optical detection device as shown in fig. 11 is built based on the gel grating prepared in embodiment 1, and the optical detection device is used to detect thrombin standard solutions with different concentrations, so as to determine a conversion relation of thrombin concentration, which includes the following steps:

(1) the thrombin concentrations were 10 each prepared using a PBS buffer solution at a pH of 7.4 and a concentration of 2mmol/L as a solvent^-12、10^-11、10^-10、10^-9、10^-8、10^-7mol/L standard sample.

(2) At a pH of 7.4 and a concentration ofAdding 2mmol/L PBS buffer solution as blank sample into quartz sample pool of optical detection device, immersing gel grating, and reading first-order diffraction light intensity I of the blank sample after the diffraction light intensity of the gel grating is stable₁And intensity of zero-order diffracted light I₀Calculating the diffraction efficiency of the blank sample;

(3) sequentially replacing the blank samples in the step I with standard samples with known thrombin concentration according to the sequence of thrombin concentration in the standard samples from low to high, and respectively measuring the diffraction efficiency corresponding to each standard sample to obtain the diffraction efficiency corresponding to a series of standard samples;

(4) calculating the change rate of the diffraction efficiency corresponding to each standard sample relative to the diffraction efficiency of the blank sample, recording the change rate as the relative diffraction efficiency to obtain a series of relative diffraction efficiencies, drawing a working curve by taking the relative diffraction efficiency as an abscissa and taking the thrombin concentration as an ordinate, and determining a conversion relation between the thrombin concentration and the relative diffraction efficiency.

The test temperatures in the steps (2) - (4) are controlled to be the same, in this example, the test temperatures are controlled to be 20, 25, 30 and 35 ℃ respectively for testing, and the thrombin concentration and the R are determined_DEThe conversion relationship therebetween is shown in fig. 13.

Based on the above experimental results, Thrombin concentration ([ Thrombin ] can be obtained]) And R_DEThe linear quantitative relationship between:coefficients a, b and related coefficient R at different temperatures²As shown in table 1.

TABLE 1 Thrombin concentration and R_DELinear quantitative relationship between coefficients and related coefficient list

Example 9

In this example, an optical detection device as shown in fig. 11 was constructed based on the gel grating prepared in example 1, and the detection capability of the method of the present invention on thrombin in artificial urine was tested.

(1) Solution preparation

The artificial urine was prepared according to literature methods and the components and contents of the artificial urine are shown in table 2. Then dissolving thrombin with artificial urine to prepare thrombin with concentration of 10^-7、10^-8、10^-9、10^-10、10^-11、10^-12A standard sample of artificial urine at mol/L.

(2) Determination of the conversion equation for the Thrombin concentration

Taking an artificial urine standard solution without thrombin as a blank sample, adding the blank sample into a quartz sample pool of an optical detection device, immersing a gel grating in the quartz sample pool, and reading the first-order diffraction light intensity I of the blank sample after the diffraction light intensity of the gel grating is stable₁And intensity of zero-order diffracted light I₀Calculating the diffraction efficiency of the blank sample;

sequentially replacing blank samples in the step I with standard samples with known thrombin concentration according to the sequence of thrombin concentration in the standard samples from low to high, and respectively measuring the diffraction efficiency corresponding to each standard sample to obtain the diffraction efficiency corresponding to a series of standard samples;

calculating the change rate of the diffraction efficiency corresponding to each standard sample relative to the diffraction efficiency of the blank sample, recording the change rate as the relative diffraction efficiency to obtain a series of relative diffraction efficiencies, drawing a working curve by taking the relative diffraction efficiency as an abscissa and taking the thrombin concentration as an ordinate, and determining a conversion relation between the thrombin concentration and the relative diffraction efficiency.

(3) Measuring the concentration of thrombin in a test sample

Replacing the original gel grating with the same gel grating in the step (2), adding a blank sample into a quartz sample pool of the optical detection device to immerse the gel grating, and reading the first-order diffraction light intensity I of the blank sample after the diffraction light intensity of the gel grating is stable₁And intensity of zero-order diffracted light I₀Calculating the diffraction efficiency of the blank sample;

replacing the blank sample in the step (3) with a sample to be tested with unknown thrombin concentration, and measuring the diffraction efficiency of the sample to be tested; the sample to be tested is prepared from artificial urine and thrombin;

and thirdly, calculating the relative diffraction efficiency of the sample to be measured relative to the diffraction efficiency of the hollow white sample in the step (3), and calculating the concentration of the thrombin in the sample to be measured according to the conversion relation between the concentration of the thrombin and the relative diffraction efficiency.

The test temperature controlled in the steps (2) and (3) was controlled to 30 ℃.

The detection results are shown in table 3, and the detection results show that the Relative Standard Deviation (RSD) of the method for monitoring the thrombin in the artificial urine is within-6.26% -5.05%, which shows that the method provided by the invention has excellent detection performance on the thrombin in the artificial urine.

TABLE 2 composition of artificial urine

TABLE 3 actual value of thrombin concentration in artificial urine compared to the test value

Example 10

In this example, the operation process of preparing the molecularly imprinted intelligent gel grating is basically the same as that of example 1, and the difference is only that: in the gel pre-polymerization solution prepared in the step (2), the concentration of NIPAM is 2.5mol/L, the molar ratio of NIPAM to tetra-arm PEGAAm is 1:0.05, the molar ratio of NIPAM to initiator is 1:0.05, the molar ratio of NIPAM to accelerator is 1:0.2, and the molar ratio of thrombin imprinted complex to tetra-arm PEGAAm is 1: 5000. In the preparation of a solution containing thrombin blot complex A1-thrombin-A2, the molar ratio of A2 to thrombin was controlled to be 1:0.9 and the molar ratio of A1 to thrombin was controlled to be 1: 1.

Example 11

In this example, the operation process of preparing the molecularly imprinted intelligent gel grating is basically the same as that of example 1, and the difference is only that: in the gel pre-polymerization solution prepared in the step (2), the concentration of NIPAM is 0.5mol/L, the molar ratio of NIPAM to tetra-arm PEGAAm is 1:0.01, the molar ratio of NIPAM to initiator is 1:0.005, the molar ratio of NIPAM to accelerator is 1:0.02, and the molar ratio of thrombin imprinted complex to tetra-arm PEGAAm is 1: 1000. In the preparation of a solution containing thrombin blot complex A1-thrombin-A2, the molar ratio of A2 to thrombin was controlled to be 1:1 and the molar ratio of A1 to thrombin was controlled to be 1: 1.

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