CN102590185B - Colloidal crystal gel label-free visual detection method with aptamer as identification unit - Google Patents

Abstract

The invention discloses a colloidal crystal gel non-marking visual detection method using an aptamer as a recognition unit. The highly specific and sensitive combination between the target detection substances is used to detect the target molecules. After the aptamer binds the target molecule, the conformation change of the aptamer leads to the change of the volume of the colloidal crystal gel, which is directly reflected in the reflection peak of the colloidal crystal, that is, the change of the color of the colloidal crystal, that is, the presence or amount of the substance to be tested It is reflected by the reflection peak shift or color of the colloidal crystal gel film. This aptamer-based non-labeled visual detection method for colloidal crystal hydrogel films has the advantages of high sensitivity, good specificity, simple operation, and low detection cost.

Description

Colloidal crystal gel non-labeled visual detection method using aptamer as recognition unit

technical field

The invention relates to a non-label visual detection method, in particular to a non-label visual detection method using a colloidal crystal gel film as a recognition unit with an aptamer as a carrier.

Background technique

Biosensors are simple, fast and cheap important detection tools. Among the molecular recognition components of biosensors, the most mature application is antibodies. However, the application of antibodies is limited due to the disadvantages of cumbersome preparation, high cost, long time-consuming, harsh storage conditions, and easy inactivation after immobilization. Exploring new high-specificity and high-stable molecular recognition substances and establishing high-selectivity and high-sensitivity biosensing technology have become one of the frontier topics of modern analytical chemistry research. With the completion of the Human Genome Project and the development of nucleic acid research, especially in 1990, American scientists Tuerk and Ellington screened out the first nucleic acid aptamer, followed by the rapid development of nucleic acid aptamer and SELEX technology, antibody technology has been challenged unprecedentedly. Due to its unique advantages, the aptamer obtained by in vitro screening technology has become a new generation of ideal sensor recognition elements, and the research on aptamer sensors has attracted great attention from scientists.

Compared with antibodies currently widely used in the biomedical field, nucleic acid aptamers are a new molecular recognition method. Nucleic acid aptamers can not only bind to target molecules efficiently and specifically like antibodies, but also have many advantages that antibodies cannot match. : Short screening cycle, low cost, wide range of target molecules, high specificity and high affinity, flexible modification, good stability, etc. Due to the above advantages, aptamers have become a molecular recognition probe with great application potential, which can be applied to the construction of various small molecules, proteins, and cell detection systems, providing a new way for the research of biosensors. platform.

Colloidal crystals are a type of substance with a two-dimensional or three-dimensional ordered structure formed by the assembly of one or more monodisperse colloidal particles (inorganic or organic, with a scale of micron or submicron), and its most basic feature is the photon Bandgap. The colloidal crystal diffraction peak position follows the Bragg diffraction formula, that is, mλ=2ndsinθ, where m is the diffraction order, λ is the diffraction wavelength, n is the average refractive index of the colloidal crystal, d is the lattice spacing of the colloidal crystal, and θ is the incident light angle . It can be seen that the central wavelength of diffracted light (ie, the characteristic reflection peak) is related to the average refractive index and lattice constant. Responsive colloidal crystals refer to colloidal crystals whose photonic bandgap responds to changes in the external environment. Since Asher et al. developed a new type of colloidal crystal chemical intelligent sensing material in 1997, colloidal crystals that respond to different external environments have gradually become a hot spot in the field of colloidal crystal research. The colloidal crystal sensor mainly utilizes the property that colloidal crystals can produce Bragg diffraction, and the average refractive index or lattice constant is changed by the excitation of the external environment, which will cause the change of the diffraction peak. In some cases, the change of the characteristic reflection peak caused by the response of colloidal crystals to external stimuli can be directly observed by the naked eye. Therefore, colloidal crystals are used as a new visual sensing material in biological and chemical sensing. has emerged. Through the design of colloidal crystal structures, many sensors based on colloidal crystals have been prepared.

However, the colloidal crystal sensors that have been developed so far still have the disadvantages of simple sensor function, easy inactivation of the molecular recognition unit antibody, and single detection method, which seriously restrict the performance of the colloidal crystal sensor such as sensitivity, selectivity, reusability, and rapid response capability. index.

Contents of the invention

In order to solve the disadvantages of poor selectivity and high cost of existing non-labeled visual detection, the present invention provides a colloidal crystal gel non-labeled visual detection method using aptamers as molecular recognition units.

In order to solve these problems in the prior art, the technical solution provided by the invention is:

A colloidal crystal gel non-labeled visual detection method using an aptamer as a recognition unit, characterized in that the method comprises the following steps:

(1) Preparation of colloidal crystal hydrogel film: Add monodisperse colloidal nanoparticles into the hydrogel prepolymer solution, adjust the mass percentage of colloidal nanoparticles in the colloidal nanoparticle solution to 10% to 80%, and ultrasonically disperse , until the colloidal nanoparticle solution produces structural color; then add the initiator, pour it into the mold, let it stand for 0.1 to 2 hours, and cure the structural color colloidal solution by ultraviolet light irradiation or thermal curing in an oven to obtain colloidal crystals Hydrogel film, the obtained colloidal crystal hydrogel film is peeled off from the mold, and placed in pure water after washing to preserve;

(2) Preparation of aptamer-modified colloidal crystal hydrogel film:

Coupling the aptamer on the colloidal crystal hydrogel film washed in step (1) by a chemical bond coupling method to make an aptamer-modified colloidal crystal hydrogel film, and washing the prepared film with phosphate buffer Afterwards, the colloidal crystal hydrogel film used for detection is obtained, and the reflectance spectrum of the colloidal crystal hydrogel film used for detection is measured;

(3) Detection of target analytes:

The colloidal crystal hydrogel film obtained in step (2) is mixed with the sample to be tested in the buffer solution to carry out the specific binding reaction between the aptamer and the sample. After the reaction is completed, the colloidal crystal hydrogel film after the reaction is detected. The reflectance spectrum of the gel film is determined by comparing the color of the colloid crystal hydrogel film before and after the reaction or by measuring the reflectance spectrum of the colloid crystal hydrogel film to determine the content of the substance to be tested.

Preferably, the aptamer used in step (2) of the method is a molecular recognition unit, which is one or more of nucleic acid aptamers or RNA aptamers.

Preferably, the method step (1) uses colloidal nanoparticle materials selected from one or more of silicon dioxide, polystyrene, polymethyl methacrylate, titanium dioxide, iron oxide, gold, and silver material; the hydrogel precursor is acrylamide, methyl methacrylate, polyhydroxyethyl methacrylate, cellulose acetate butyrate, silicone methacrylate, fluorosilicon methacrylate , perfluoroether, N-vinylpyrrolidone, polyvinyl alcohol, glycidyl methacrylate or ethylene glycol dimethacrylate, or any mixture of two or more.

Preferably, in the method step (1), the particle size of the colloidal nanoparticles is between 10 nm and 500 nm; the concentration of the colloidal nanoparticles is between 10% and 80%.

Preferably, the aptamer-modified chemical bond coupling method in step (2) of the method is selected from one or any combination of two or more of the reaction between amino groups, amino groups and carboxyl groups, or carboxyl groups and hydroxyl groups.

Preferably, the detection objects in the sample to be tested in step (3) of the method are ions, drug molecules, drug molecules, additives, small molecules, neurotransmitters, hormones, proteins, enzymes, antigen antibodies, cytokines, growth factors, A tumor marker or one of various biomolecules.

Preferably, the detection object in the sample to be tested in step (3) of the method is one of mercury ions, lead ions, potassium ions, cocaine, and thrombin.

The present invention starts from the construction of the molecular recognition element of the colloidal crystal sensor, takes the nucleic acid aptamer as the entry point, and develops a new type of colloidal crystal non-labeled visual detection method with the aptamer as the recognition unit. The detection steps are:

The first step: preparation of colloidal crystal hydrogel film:

Add monodisperse colloidal nanoparticles into hydrogel prepolymer solution, adjust the mass percentage of colloidal nanoparticles in the colloidal nanoparticle solution to 10%-80%, and ultrasonically disperse until the colloidal nanoparticle solution produces structural color. After adding the initiator, pour it into the mold, let it stand for 0.1-2 hours, and cure the structural color colloidal solution by ultraviolet light irradiation polymerization or thermal curing method in an oven to obtain a colloidal crystal hydrogel film. The obtained colloidal crystal The hydrogel film was peeled off from the mold, washed and stored in pure water.

The second step is to prepare aptamer-modified colloidal crystal hydrogel film:

Coupling the aptamer on the colloidal crystal hydrogel film prepared in step (1) by a chemical bond coupling method to make an aptamer-modified colloidal crystal hydrogel film, and washing the prepared film with a buffer , to measure the reflectance spectra of colloidal crystal hydrogel films.

The third step, detection:

Mix the colloidal crystal hydrogel film obtained in step (2) with the sample to be tested in the buffer solution to carry out the specific binding reaction between the aptamer and the sample. After the reaction is completed, compare the colloidal crystal hydrogel before and after the reaction after washing. The color of the film or by measuring the reflectance spectrum of the colloidal crystal hydrogel film determines the content of the substance to be tested. The sample to be tested can be any one of ions, drug molecules, drug molecules, additives, small molecules, neurotransmitters, hormones, proteins, enzymes, antigen antibodies, cytokines, growth factors, tumor markers or various biomolecules .

The colloidal crystal gel non-marking visual detection method using the aptamer as the recognition unit of the present invention adopts a colloidal crystal hydrogel thin film as a carrier. The molecular recognition unit in the colloidal crystal gel film is one or more of nucleic acid aptamers or RNA aptamers.

The colloidal nanoparticle material described in the method is selected from one or more materials in silicon dioxide, polystyrene, polymethyl methacrylate, titanium dioxide, iron oxide, gold, silver; Hydrogel precursors are acrylamide, methyl methacrylate, polyhydroxyethyl methacrylate, cellulose acetate butyrate, silicone methacrylate, fluorosilicon methacrylate, perfluoroether, N - One or any mixture of two or more of vinylpyrrolidone, polyvinyl alcohol, glycidyl methacrylate or ethylene glycol dimethacrylate. The particle diameter of the colloidal nanoparticles is between 10 nanometers and 500 nanometers; the concentration of the colloidal nanoparticles is between 10% and 80%.

The aptamer-modified chemical bond coupling method described in the method can be one or any combination of two or more of the reaction between amino groups, amino groups and carboxyl groups, or carboxyl groups and hydroxyl groups. The detection objects are ions (such as mercury ions, lead ions, potassium ions, etc.), drug molecules, drug molecules (such as cocaine), additives, small molecules, neurotransmitters, hormones, proteins, enzymes (such as thrombin), Antigens and antibodies, cytokines, growth factors, tumor markers or any of various biomolecules.

Compared with the existing non-labeled visual detection method, the colloidal crystal gel non-labeled visual detection method using aptamer as the molecular recognition unit of the present invention has the following advantages:

(1) The present invention uses aptamers as molecular recognition units, and the advantages of aptamer molecules such as designability, high selectivity, high sensitivity, high stability, modifiability, and a wide range of detection objects make the stability of colloidal crystal membranes Greatly improved, side reaction interference is small, and it can also meet the needs of different detection samples.

(2) In the present invention, the aptamer is combined with the colloidal crystal gel film, and detection is performed by utilizing the change of the colloidal crystal gel photonic band gap caused by the conformational change after the aptamer binds to the target molecule. The change of the color of the colloidal crystal film brought about by the change of the photonic band gap can be directly identified by the human eye, which greatly simplifies the detection tools and greatly reduces the detection cost.

(3) In the present invention, the target molecule to be detected does not need to be labeled or other pretreatments, the detection steps can be simplified, and the inactivation caused by the target molecule label can be reduced.

(4) The high hydrophilicity, high water content, excellent biocompatibility and stimuli responsiveness of the colloidal crystal gel film can greatly improve the detection sensitivity.

Description of drawings

The present invention will be further described below in conjunction with accompanying drawing and embodiment:

Figure 1 is a schematic diagram of the colloidal crystal gel detection process using aptamers as recognition units.

Detailed ways

The above solution will be further described below in conjunction with specific embodiments. It should be understood that these examples are used to illustrate the present invention and not to limit the scope of the present invention. The implementation conditions used in the examples can be further adjusted according to the conditions of specific manufacturers, and the implementation conditions not indicated are usually the conditions in routine experiments.

Example 1 Aptamer-modified colloidal crystal gel film detection of heavy metal ions Hg ²⁺ in environmental water samples

1. Preparation of colloidal crystal hydrogel film

Add monodisperse silica colloidal nanoparticles with a diameter of 200 nm into a mixed solution of acrylamide and methylene bisacrylamide (29:1, molar ratio), adjust the mass percentage of silica to 30%, and ultrasonically disperse, Until the colloidal nanoparticle solution produces a bright and bright structural color; add photoinitiator 1173 (0.1%, mass volume ratio) to this solution, after fully mixing, pass through nitrogen for 5min, get 500 microliters of volume colloidal solution into the mold Stand still for 20 minutes, and irradiate with a high-pressure mercury lamp at 4°C for 15 minutes to obtain a colloidal crystal hydrogel film.

2. Preparation of aptamer-modified colloidal crystal hydrogel films

The colloidal crystal gel film prepared in the first step was reduced in 0.1M NaOH (10% N, N, N', N'-tetramethylethylenediamine) solution for 1 h, and then treated with 2-(N-morphine (Phenyl)ethanesulfonic acid (MES) buffer solution washing, add 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide in MES buffer After amine (NHS) activates the reduction membrane, add an aptamer coupling with amino-modified mercury ions at both ends to make an aptamer-modified colloidal crystal hydrogel film, wash the prepared film with phosphate buffer, and measure Reflectance spectra of colloidal crystal hydrogel films.

3. Hg ²⁺ detection

The prepared gel film was washed with Tris buffer solution, mixed with the water sample to be tested in 0.5ml Tris buffer solution, and reacted at 35°C for 1h. After the reaction is finished, wash with Tris buffer solution, and determine the content of mercury ions in the environmental water sample according to the color of the colloidal crystal hydrogel film before and after the reaction or by measuring the reflectance spectrum of the colloidal crystal hydrogel film.

Example 2 Detection of drug cocaine using aptamer-modified colloidal crystal gel film

1. Preparation of colloidal crystal hydrogel film

Add monodisperse polystyrene colloidal nanoparticles with a diameter of 150 nm into a mixed solution of polyethylene glycol diacrylate and acrylamide (1:10, molar ratio), adjust the mass percentage of polystyrene to 40%, and ultrasonically Disperse until the colloidal nanoparticle solution produces a bright and bright structural color; add photoinitiator 907 (0.1%, mass volume ratio) to this solution, after fully mixing, pass through nitrogen for 10min, and get 500 microliters of colloidal solution Add it into the mold, let it stand for 10 minutes, and irradiate it with a high-pressure mercury lamp at 4°C for 20 minutes to obtain a colloidal crystal hydrogel film.

2. Preparation of aptamer-modified colloidal crystal hydrogel films

After reducing the colloidal crystal gel film prepared in the first step in 0.3M NaOH solution for 30 min, wash it with phosphate (PBS) buffer solution, add EDC/NHS reagent to the PBS buffer solution to activate the reduction film, and then add two Aptamer-modified colloidal crystal hydrogel film was prepared by aptamer coupling with amino-modified cocaine, and the prepared film was washed with PBS buffer solution to measure the reflectance spectrum of the colloidal crystal hydrogel film.

3. Detection of cocaine

After the prepared gel film was washed with PBS buffer solution, it was mixed with the sample to be tested in 0.5 ml of PBS buffer solution, and reacted at 35° C. for 1 h. After the reaction was completed, it was washed with PBS buffer solution, and the content of cocaine in the sample was determined according to the reflection spectra of the colloidal crystal hydrogel film before and after the reaction.

Example 3 Detection of platelet-derived growth factor using aptamer-modified colloidal crystal gel film

1. Preparation of colloidal crystal hydrogel film

Add the monodisperse polymethyl methacrylate colloidal nanoparticles with a diameter of 100 nanometers in the PEGDA solution of 10% (mass volume ratio), adjust the mass percentage of polymethyl methacrylate to 50%, and ultrasonically disperse until the colloidal solution Produce translucent bright-coloured structural color; Add initiator ammonium persulfate (0.1%, mass volume ratio) and accelerator TEMED (0.1%, mass volume ratio) in this solution, after fully mixing, pass through nitrogen 30min, take 500 A microliter volume of colloidal solution was added into the mold, left to stand for 30 minutes, and polymerized in an oven at 40°C for 40 minutes to obtain a colloidal crystal hydrogel film.

2. Preparation of aptamer-modified colloidal crystal hydrogel films

After reducing the colloidal crystal gel film prepared in the first step in 0.2M KOH solution for 50 minutes, wash it with Tris buffer solution, add EDC/NHS reagent to the Tris buffer solution to activate the reduced film, and then add The colloidal crystal hydrogel film modified by the aptamer was prepared by the aptamer coupling of the platelet-derived growth factor, and the prepared film was washed with Tris buffer solution, and the reflectance spectrum of the colloidal crystal hydrogel film was measured.

3. Detection of platelet-derived growth factor

After the prepared gel film was washed with Tris buffer solution, it was mixed with the sample to be tested in 1.0 ml Tris buffer solution, and reacted at 4° C. for 2 h. After the reaction is completed, it is washed with Tris buffer solution, and the content of the platelet-derived growth factor in the sample is determined according to the reflection spectrum of the colloidal crystal hydrogel film before and after the reaction.

Example 4 Detection of thrombin using an aptamer-modified colloidal crystal gel film

1 Preparation of colloidal crystal hydrogel film

Add the monodispersed silica colloidal nano-particles with a diameter of 250 nanometers to the solution in the mixed solution of polyethylene glycol diacrylate and hydroxyethyl methacrylate (mass volume ratio 1: 5), adjust the concentration of silica Mass percent is 60%, ultrasonic dispersion, until colloidal solution produces bright and bright structural color; Add photoinitiator 1173 (0.1%, mass volume ratio) and photoinitiator 907 (0.1%, mass volume ratio) in this solution, After fully mixing, pass nitrogen gas for 15 minutes, take 500 microliters of colloidal solution into the mold, let it stand for 150 minutes, and irradiate with a high-pressure mercury lamp at 4°C for 20 minutes to obtain a colloidal crystal hydrogel film.

2 Preparation of aptamer-modified colloidal crystal hydrogel films

After reducing the colloidal crystal gel film prepared in the first step in 0.2M NaOH (containing 5% TEMED) solution for 1 h, wash with Tris buffer solution, add EDC/NHS reagent to the Tris buffer solution to activate the reduced film, An aptamer-modified colloidal crystal hydrogel film is prepared by adding an aptamer coupling with amino-modified thrombin at both ends, and after the prepared film is washed with Tris buffer, the reflectance spectrum of the colloidal crystal hydrogel film is measured.

3 Detection of thrombin

After the prepared gel film was washed with Tris buffer solution, it was mixed with the sample to be tested in 1.0 ml of Tris buffer solution, and reacted at 4° C. for 1 h. After the reaction was completed, it was washed with Tris buffer, and the content of thrombin in the sample was determined according to the reflection spectra of the colloidal crystal hydrogel film before and after the reaction.

The above examples are only to illustrate the technical conception and characteristics of the present invention, and its purpose is to allow people familiar with this technology to understand the content of the present invention and implement it accordingly, and cannot limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention shall fall within the protection scope of the present invention.

Claims (6)

1. A colloidal crystal gel non-labeled visual detection method with an aptamer as a recognition unit, characterized in that the method comprises the following steps: (1) Preparation of colloidal crystal hydrogel film: Add monodisperse colloidal nanoparticles into the hydrogel prepolymer solution, adjust the mass percentage of colloidal nanoparticles in the colloidal nanoparticle solution to 10% to 80%, and ultrasonically disperse , until the colloidal nanoparticle solution produces structural color; then add the initiator, pour it into the mold, let it stand for 0.1 to 2 hours, and cure the structural color colloidal solution by ultraviolet light irradiation or thermal curing in an oven to obtain colloidal crystals Hydrogel film, the obtained colloidal crystal hydrogel film is peeled off from the mold, and placed in pure water after washing to preserve; (2) Preparation of aptamer-modified colloidal crystal hydrogel film: Coupling the aptamer on the colloidal crystal hydrogel film washed in step (1) by chemical bond coupling method to make an aptamer-modified colloidal crystal hydrogel film, and washing the prepared film with phosphate buffer Afterwards, the colloidal crystal hydrogel film used for detection is obtained, and the reflectance spectrum of the colloidal crystal hydrogel film used for detection is measured; (3) Detection of target analytes: Mix the colloidal crystal hydrogel film obtained in step (2) with the sample to be tested in the buffer solution to carry out the specific binding reaction between the aptamer and the sample. After the reaction is completed, the colloidal crystal hydrogel film after the reaction is detected. The reflectance spectrum of the gel film is determined by comparing the color of the colloid crystal hydrogel film before and after the reaction or by measuring the reflectance spectrum of the colloid crystal hydrogel film to determine the content of the substance to be tested. 2. The method according to claim 1, characterized in that the aptamer used in the method step (2) is one or more of nucleic acid aptamers as a molecular recognition unit. 3. The method according to claim 1, characterized in that the method step (1) uses colloidal nanoparticle materials selected from silicon dioxide, polystyrene, polymethyl methacrylate, titanium dioxide, iron oxide, One or more materials of gold and silver; the hydrogel prepolymer is acrylamide, methyl methacrylate, polyhydroxyethyl methacrylate, cellulose acetate butyrate, siloxane One or more of methacrylate, fluorosilicon methacrylate, perfluoroether, N-vinylpyrrolidone, polyvinyl alcohol, glycidyl methacrylate or ethylene glycol dimethacrylate mix. 4. The method according to claim 1, characterized in that the particle size of the colloidal nanoparticles in the method step (1) is between 10 nanometers and 500 nanometers. 5. The method according to claim 1, characterized in that the chemical bond coupling method modified by the aptamer in the method step (2) is selected from the reaction between amino and amino, the reaction between amino and carboxyl or the reaction between carboxyl and hydroxyl One or any combination of two or more. 6. The method according to claim 1, characterized in that the detection object in the sample to be tested in the method step (3) is one of mercury ions, lead ions, potassium ions, cocaine, and thrombin.

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