CN109908982B - Preparation method and application of three-dimensional chip for screening of traditional Chinese medicine components - Google Patents
Preparation method and application of three-dimensional chip for screening of traditional Chinese medicine components Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a three-dimensional chip for screening traditional Chinese medicine components. The chip comprises a supporting layer, an initiator layer and a polymer brush layer; the initiator layer is covalently anchored to the support layer by a metal-sulfur bond; the polymeric brush layer is covalently bonded to the initiator layer. The traditional Chinese medicine component screening three-dimensional chip can fix medicine small molecules or traditional Chinese medicine components with different structures; in addition, the chip is of a three-dimensional structure, and a strong nonspecific monomer is introduced into a macromolecule layer, so that the three-dimensional chip for screening the traditional Chinese medicine components not only greatly improves the fixed quantity of the small medicine molecules or the traditional Chinese medicine components, obviously enhances the sensing signals of the chip, but also solves the problem of poor specificity of the small molecule array chip.
Description
Technical Field
The invention relates to the field of biochip preparation, in particular to a preparation method and application of a three-dimensional chip for screening traditional Chinese medicine components.
Background
At present, the effective components of the medicine need to be determined, and the modern medicine screening technology is necessary. Drug screening is the detection of a possible pharmacological activity of a natural product or an artificially synthesized compound that may be used as a drug by applying appropriate methods and techniques. The latest technology of modern drug screening is applied to the research of Chinese pharmacology, and plays a great role in promoting the modern research of Chinese medicines, such as high-throughput drug screening technology. Nowadays, high throughput screening technology has become a core technology for theoretical research of research structure and drug development of pharmaceutical enterprises, and the application of the chip based on small molecule array is the most extensive. However, for such chips, the problems currently encountered are:
(1) small molecules with different structures are difficult to uniformly fix on one chip; (2) after the micromolecules with different structures are fixed, the surface of the chip is changed, so that the specificity of the chip is greatly reduced, and further false positive signals are generated, and the occurrence of the false signals seriously reduces the reliability of a test result.
In the prior art, patent CN102866132B discloses a technique for fixing small molecules, i.e. fixing small molecules by using photo-crosslinking method, which solves the problem of uniformly fixing small molecules with different structures on a chip. However, since this technology fixes small molecules on the basis of a two-dimensional chip, the fixed amount of the small molecules is low, and the signal is weak. Therefore, when the small molecule array chip technology is used for high-throughput screening of traditional Chinese medicine components containing different small molecule structures, the lower solid-carrying amount of the small molecules of the medicine and the poorer specificity of the small molecule array chip become the technical bottlenecks of the technology.
Disclosure of Invention
The invention aims to provide a preparation method and application of a three-dimensional chip for screening traditional Chinese medicine components. The chip has the advantages of high immobilization amount and strong specificity, and is very suitable for immobilization and high-throughput screening of traditional Chinese medicine components.
The technical scheme of the invention is as follows: a three-dimensional chip for screening Chinese medicinal components comprises a support layer, an initiator layer and a polymer brush layer; the initiator layer is covalently anchored to the support layer by a metal-sulfur bond; the polymeric brush layer is covalently bonded to the initiator layer.
A preparation method of a three-dimensional chip for screening traditional Chinese medicine components comprises the following steps:
(1) adding deionized water, ammonia water and hydrogen peroxide into a chip cleaner, then placing a glass substrate with a gold film plated on the surface, heating to clean the substrate, washing with deionized water and ethanol, drying with nitrogen, and cleaning;
(2) soaking the cleaned substrate obtained in the step (1) in a 1-5 mM 2-mercaptoethylamine ethanol solution, placing at normal temperature, self-assembling a monomolecular layer on the surface of the substrate, soaking the substrate with the 2-mercaptoethylamine monomolecular layer assembled on the surface in an N, N-dimethylformamide solution containing triethylamine and 2-bromopropionyl bromide, reacting, and then cleaning the chip to obtain the substrate with the surface connected with an initiator;
(3) and (3) soaking the substrate with the surface connected with the initiator obtained in the step (2) in a solution containing carboxylic acid betaine acrylate, o-azido propenyl benzene, a catalyst and a solvent for reaction to obtain the traditional Chinese medicine component screening three-dimensional chip containing the polymer brush layer.
In the step (1), the content of the ammonia water is 25-28%, the mass fraction of the hydrogen peroxide is 28-32%, and the volume ratio of the ionized water to the ammonia water to the hydrogen peroxide is 5:1: 1.
In the preparation method of the three-dimensional chip for screening the traditional Chinese medicine components, in the step (1), the temperature is raised to 50-70 ℃; the cleaning time is 10-30 minutes.
The preparation method of the three-dimensional chip for screening the traditional Chinese medicine components comprises the following steps of (2), standing at the normal temperature for 10-15 hours; the reaction mode is that the reaction is carried out for 2 to 3 hours at minus 5 to 5 ℃, and then the reaction is carried out for 5 to 8 hours at normal temperature.
The preparation method of the three-dimensional chip for screening the traditional Chinese medicine components comprises the following steps of (3), wherein the reaction temperature is 20-50 ℃; the time is 2-5 hours.
In the step (3), the catalyst is cuprous halide and 1,4,8, 11-tetramethyl-1, 4,8, 11-tetraazacyclotetradecane in a molar ratio of 1: 1.
In the preparation method of the three-dimensional chip for screening traditional Chinese medicine components, in the step (3), the solvent is methanol or ethanol.
In the step (3), the ratio of the carboxylic acid betaine acrylate, the o-azido propenyl benzene, the catalyst and the solvent in molar parts is (1-2): (1-2): (0.01-0.1): (25-100).
An application of a three-dimensional chip for screening traditional Chinese medicine components comprises the following steps:
(1) dissolving the traditional Chinese medicine components in dimethyl sulfoxide, designing a sample application mode, and applying a sample on a traditional Chinese medicine component screening three-dimensional chip in a microarray mode through an automatic sample application machine;
(2) placing the spotted chip in the step (1) under 280-300 nm ultraviolet light for irradiating for 2-10 minutes, and then cleaning the chip to obtain a three-dimensional chip immobilized with traditional Chinese medicine components;
(3) fixing the three-dimensional chip immobilized with the traditional Chinese medicine components in the step (2) in an SPR instrument according to the operation flow of the SPR instrument, detecting the interaction between the immobilized traditional Chinese medicine components and biomolecules according to the related operation flow, and identifying and screening the related traditional Chinese medicine components according to the obtained specific signal.
Compared with the prior art, the invention has the following beneficial effects:
(1) the three-dimensional chip for screening the traditional Chinese medicine components comprises a supporting layer, an initiator layer and a high-molecular brush layer, and the introduction of the photo-crosslinking group enables the chip to fix the traditional Chinese medicine components with different molecular structures on a chip sensing layer, so that the problems of various traditional Chinese medicine component structures and difficult fixing are solved;
(2) the traditional Chinese medicine component screening three-dimensional chip prepared by the invention has three-dimensional characteristics, can greatly improve the fixed quantity of the traditional Chinese medicine components, and has low component content for the traditional Chinese medicine components, so that the improvement of the fixed quantity of the chip is crucial to the specific binding of the screened components and the feedback of sensing signals;
(3) the carboxylic acid betaine acrylate monomer is introduced into the traditional Chinese medicine component screening three-dimensional chip prepared by the invention, and the monomer has extremely strong non-specific adsorption resistance, so that the problem of poor specificity of a small molecule array chip is solved;
(4) the three-dimensional chip for screening the traditional Chinese medicine components has the characteristic of simple preparation method, and the uniformity of the quality of the chip is effectively ensured.
Drawings
FIG. 1 is a schematic structural diagram of a biosensing three-dimensional chip;
FIG. 2 is a comparison graph of SPR signals of the three-dimensional chip for fixing the Chinese medicinal component M1 and the two-dimensional chip for fixing the Chinese medicinal component M1 in experiment 1;
FIG. 3 is a graph showing the non-specific resistance of the three-dimensional chip on which the Chinese medicinal component M1 was immobilized and the two-dimensional chip on which the Chinese medicinal component M1 was immobilized to Fibrinogen (Fibrinogen, Fg) in test 2.
Description of reference numerals: 1-support layer, 2-initiator layer, 3-macromolecule brush layer, 4-drug small molecule or traditional Chinese medicine component, 5-enhancing specificity, A-target protein P, B-three-dimensional chip for fixing traditional Chinese medicine component M1 in example 1, C-two-dimensional chip for fixing traditional Chinese medicine component M1 in comparative example 1, a-fiber protein, B-three-dimensional chip for example 2, C-two-dimensional chip for comparative example 2, d-nonspecific adsorption,
Detailed Description
The present invention is further illustrated with reference to the following figures and examples, but not by way of limitation, and reference may be made to conventional techniques for processing parameters not specifically identified.
Example 1.
(1) Adding deionized water, 25% ammonia water and 30% hydrogen peroxide by mass into a chip cleaner, wherein the volume ratio of the deionized water to the ammonia water to the hydrogen peroxide is 5:1:1, then placing the chip cleaner on a glass substrate with a gold film plated on the surface, heating to 50 ℃, cleaning the substrate for 30 minutes, washing with the deionized water and ethanol, drying by using nitrogen, and cleaning;
(2) soaking the cleaned substrate obtained in the step (1) in a 1mM 2-mercaptoethylamine ethanol solution, standing at normal temperature for 15 hours, self-assembling a monomolecular layer on the surface of the substrate, soaking the substrate with the 2-mercaptoethylamine monomolecular layer assembled on the surface in a N, N-Dimethylformamide (DMF) solution containing triethylamine and 2-bromopropionyl bromide, reacting at-5 ℃ for 3 hours, reacting at normal temperature for 8 hours, and then cleaning the chip to obtain the substrate with the surface connected with an initiator;
(3) and (3) placing the substrate with the surface connected with the initiator obtained in the step (2) into a chip reactor, adding 1 mol part of carboxylic acid betaine acrylate monomer, 1 mol part of o-azido propenyl benzene monomer, 0.01 mol part of cuprous bromide, 0.01 mol part of 1,4,8, 11-tetramethyl-1, 4,8, 11-tetraazacyclotetradecane (Me4 Cyclame) and 25 mol parts of methanol solvent, and reacting for 5 hours at 20 ℃ to obtain the three-dimensional chip for screening the traditional Chinese medicine components containing the polymer brush layer.
Example 2.
(1) Adding deionized water, 25% ammonia water and 30% hydrogen peroxide by mass into a chip cleaner, wherein the volume ratio of the deionized water to the ammonia water to the hydrogen peroxide is 5:1:1, then placing the chip cleaner on a glass substrate with a gold film plated on the surface, heating to 70 ℃, cleaning the substrate for 10 minutes, washing with the deionized water and ethanol, drying by using nitrogen, and cleaning;
(2) soaking the cleaned substrate obtained in the step (1) in a 5mM 2-mercaptoethylamine ethanol solution, standing at normal temperature for 10 hours, self-assembling a monomolecular layer on the surface of the substrate, soaking the substrate with the 2-mercaptoethylamine monomolecular layer assembled on the surface in a N, N-Dimethylformamide (DMF) solution containing triethylamine and 2-bromopropionyl bromide, reacting at 5 ℃ for 2 hours, reacting at normal temperature for 5 hours, and then cleaning the chip to obtain the substrate with the surface connected with an initiator;
(3) and (3) placing the substrate with the surface connected with the initiator obtained in the step (2) into a chip reactor, adding 2 molar parts of carboxylic acid betaine acrylate monomer, 2 molar parts of o-azido propenyl benzene monomer, 0.1 molar part of cuprous bromide, 0.1 molar part of 1,4,8, 11-tetramethyl-1, 4,8, 11-tetraazacyclotetradecane (Me4 Cyclame) and 100 molar parts of ethanol solvent, and reacting at 50 ℃ for 2 hours to obtain the three-dimensional chip for screening the traditional Chinese medicine components containing the polymer brush layer.
Example 3.
(1) Adding deionized water, 25% ammonia water and 30% hydrogen peroxide by mass into a chip cleaner, wherein the volume ratio of the deionized water to the ammonia water to the hydrogen peroxide is 5:1:1, then placing the chip cleaner on a glass substrate with a gold film plated on the surface, heating to 60 ℃, cleaning the substrate for 20 minutes, washing with the deionized water and ethanol, drying with nitrogen, and cleaning;
(2) soaking the cleaned substrate obtained in the step (1) in 2mM 2-mercaptoethylamine ethanol solution, standing at normal temperature for 12 hours, self-assembling a monomolecular layer on the surface of the substrate, soaking the substrate with the 2-mercaptoethylamine monomolecular layer assembled on the surface in N, N-Dimethylformamide (DMF) solution containing triethylamine and 2-bromopropionyl bromide, reacting at 0 ℃ for 2.5 hours, reacting at normal temperature for 6 hours, and then cleaning the chip to obtain the substrate with the surface connected with an initiator;
(3) and (3) placing the substrate with the surface connected with the initiator obtained in the step (2) into a chip reactor, adding 1.5 molar parts of carboxylic acid betaine acrylate monomer, 2 molar parts of o-azido propenyl benzene monomer, 0.05 molar parts of cuprous bromide, 0.1 molar parts of 1,4,8, 11-tetramethyl-1, 4,8, 11-tetraazacyclotetradecane (Me4 cycloam) and 50 molar parts of methanol solvent, and reacting at 30 ℃ for 3 hours to obtain the three-dimensional chip for screening the traditional Chinese medicine components containing the polymer brush layer.
To demonstrate the effect of the present invention, the following comparative examples and experiments were made:
comparative example 1
Adding deionized water, 25% ammonia water and 30% hydrogen peroxide (wherein the volume ratio of the deionized water to the ammonia water to the hydrogen peroxide is 5:1:1) into a chip cleaner, placing a glass substrate with a gold film plated on the surface, heating to 50 ℃, cleaning for 30 minutes, washing with the deionized water and ethanol, drying with nitrogen, and cleaning; the cleaned substrate is soaked in 1mM 2-mercaptoethylamine ethanol solution and placed at normal temperature for 15 hours, and a monomolecular layer is self-assembled on the surface of the substrate. And then, soaking the substrate with the 2-mercaptoethylamine monomolecular layer assembled on the surface in N, N-Dimethylformamide (DMF) solution containing 4-azidobenzoyl hydrazine, diisopropylethylamine and N, N' -disuccinimidyl carbonate, reacting for 24 hours at room temperature, and cleaning the chip to obtain the two-dimensional chip with the surface connected with the photocrosslinking group.
Comparative example 2
Adding deionized water, 25% ammonia water and 30% hydrogen peroxide (wherein the volume ratio of the deionized water to the ammonia water to the hydrogen peroxide is 5:1:1) into a chip cleaner, placing a glass substrate with a gold film plated on the surface, heating to 70 ℃, cleaning for 10 minutes, washing with the deionized water and ethanol, drying with nitrogen, and cleaning; soaking the cleaned substrate in 5mM 2-mercaptoethylamine ethanol solution, standing at normal temperature for 10 hours, self-assembling a monomolecular layer on the surface of the substrate, soaking the substrate with the 2-mercaptoethylamine monomolecular layer assembled on the surface in N, N-Dimethylformamide (DMF) solution containing triethylamine and 2-bromopropionyl bromide, reacting at 5 ℃ for 2 hours, reacting at normal temperature for 5 hours, and cleaning the chip to obtain the substrate with the initiator connected on the surface.
Experiment 1
The known traditional Chinese medicine component M1 of the active ingredient target protein P is dissolved in dimethyl sulfoxide (DMSO), a sample application mode is designed, and the sample is applied to the traditional Chinese medicine component screening three-dimensional chip in the example 1 and the photo-crosslinking two-dimensional chip in the comparative example 1 in a microarray mode through an automatic sample application machine.
Irradiating the spotted chip for 10 minutes under 300nm ultraviolet light, and cleaning the chip to obtain a three-dimensional chip immobilized with traditional Chinese medicine components;
fixing the three-dimensional chip and the two-dimensional chip loaded with the traditional Chinese medicine components in an SPR instrument according to the operation flow of the SPR instrument, respectively, taking phosphate buffer solution (PBS buffer,10mM, pH 7.4) as a mobile phase, setting the flow rate to be 2 mu L/s, introducing target protein and PBS with known effective components in sequence after a base line is stable, and identifying and screening the related traditional Chinese medicine components according to the obtained specific signals. As shown in FIG. 2, since the three-dimensional chip screened by the Chinese medicinal components in example 1 has a high sample loading amount, the signal of the specific action with the target protein is obviously stronger than that of the two-dimensional chip photo-crosslinked in comparative example 1.
Experiment 2
The chinese medicinal component M2, which is known to have no specific effect with Fibrinogen (Fibrinogen, Fg), was dissolved in dimethyl sulfoxide (DMSO), and the spotting method was designed to be spotted on the chinese medicinal component screening three-dimensional chip of example 2 and the photo-crosslinked two-dimensional chip of comparative example 2 in a microarray manner by an automatic spotting machine.
Irradiating the spotted chip for 5 minutes under 280nm ultraviolet light, and cleaning the chip to obtain a three-dimensional chip immobilized with traditional Chinese medicine components;
the three-dimensional chip of example 2 and the two-dimensional chip of comparative example 2 were fixed to the SPR apparatus according to the SPR apparatus protocol, and after a flow rate of 2 μ L/s was stabilized using phosphate buffer solution (PBS buffer,10mM, pH 7.4) as a mobile phase, Fibrinogen (Fg) and PBS (1 mg/mL) were sequentially introduced. As shown in FIG. 3, since the three-dimensional chip of example 2 introduced a monomer strongly resistant to nonspecific adsorption, which had strong nonspecific resistance, the specificity of the three-dimensional chip was significantly stronger than that of the two-dimensional chip of comparative example 2, which did not incorporate a monomer having nonspecific function.
Claims (9)
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