阅读说明：本技术 一种同时可视化检测两种病毒的分子印迹荧光传感器的制备方法及应用 (Preparation method and application of molecular imprinting fluorescent sensor for simultaneously and visually detecting two viruses ) 是由 蔡昌群 陈思宇 罗谅晖 陈小明 于 2019-09-30 设计创作，主要内容包括：本发明提供了一种同时可视化检测两种病毒的分子印迹荧光传感器的制备方法及应用。在本发明中,以两种量子点为荧光信号源,同时加入功能单体和亲水单体,以HAV和HBV为模板分别印迹在不同的载体上,洗脱模板后将得到的印迹聚合物混合,得到可同时检测这两种病毒的荧光传感器。该传感器同时结合亲水单体N-异丙基丙烯酰胺(NIPAAm)及功能单体丙烯酸锌的优点,实现了对HAV、HBV两种病毒的高特异性识别。由于量子点在结合不同浓度目标物后荧光猝灭程度不同,从而实现可视化检测。本发明提供的策略为病毒分子印迹同时检测多种类似病毒奠定了很好的基础,同时也具有临床治疗与诊断病毒性疾病的潜在能力。(The invention provides a preparation method and application of molecular imprinting fluorescence sensors for simultaneously and visually detecting two viruses.)

The preparation method of molecular imprinting fluorescence sensors for simultaneously and visually detecting two viruses is characterized in that two different quantum dots are used as fluorescence signal sources, silicon dioxide is coated on the surfaces of the two different quantum dots to serve as carrier materials, a metal chelating agent is used as a functional monomer, N-isopropylacrylamide (NIPAAm) is added to enhance the specific recognition capability, different viruses are respectively imprinted on different carriers, and the imprinted polymers obtained after elution are mixed to construct the fluorescence sensors capable of simultaneously detecting two similar viruses.

2. And (3) preparing the virus molecular imprinting fluorescence sensor.

3. The virus molecular imprinting fluorescence sensor is applied.

4. The preparation of imprinted polymer according to claim 1, comprising the steps of:

1) synthesizing G-CdTe QDs/R-CdTe QDs, and coating layers of SiO on the surface₂As a carrier material in imprinting;

2) vinylating the G-CdTe QDs/R-CdTe QDs obtained in the step 1);

3) adding functional monomer, hydrophilic monomer, cross-linking agent and template viruses HAV and HBV on the basis of 2), eluting the template after polymerization is completed, and obtaining HAV and HBV imprinted polymers respectively.

5. The method of claim 1, wherein the specific recognition of the template molecule by the imprinted polymer is enhanced by metal chelation and hydrophilicity.

6. The method for producing a viral molecularly imprinted polymer according to claim 1, characterized in that: the carrier materials used, template viruses were: SiO coated on fluorescent quantum dots₂Microparticles, HAV and HBV mixed solution.

7. The method according to claim 1, wherein the functional monomer and the hydrophilic monomer are respectively: zinc acrylate, NIPAAm.

8. The preparation of the molecularly imprinted fluorescent sensor of claim 2, characterized in that: the optimal experimental conditions are as follows: zinc acrylate, N-isopropyl acryloyl (NIPAAm), Tetraethoxysilane (TEOS), Ethylene Glycol Dimethacrylate (EGDMA) dosage is 40mg, 20mg, 300 μ L, 150 μ L respectively; the pH was 7.5; the mass ratio of the G-MIP to the R-MIP is 1:1.5, and the incubation time is 20 min.

9. The preparation method of the molecularly imprinted fluorescent sensor according to claim 2, wherein two viruses are used as templates, and two imprinted polymers are obtained by imprinting on different carriers respectively, mixing the eluted imprinted polymers at ratio, adding an appropriate amount of virus mixture, adsorbing under optimized adsorption conditions, then, amounts of the above mixture is put into a cuvette, and fluorescence intensity is measured by RF-5301PC fluorescence spectrophotometer to construct fluorescent sensors for simultaneously detecting two viruses, wherein the detection conditions are that excitation wavelength is 370nm, emission wavelength is 555nm (detection of HAV), 655nm (detection of HBV), and the width of excitation and emission slits are both 5.0 nm.

10. The use of the viral molecularly imprinted fluorescent sensor of claim 3, wherein: analyzing HAV and HBV mixed solutions with different concentrations by using the virus molecular imprinting fluorescence sensor to evaluate the detection range and detection limit of the virus on the template; detecting different virus molecules with the same concentration by using the virus molecular imprinting fluorescence sensor to evaluate the selective recognition and detection capability of the prepared molecular imprinting fluorescence sensor on template molecules; the intelligent virus molecularly imprinted fluorescent sensor is applied to the labeling recovery of HAV and HBV mixed solution in human serum, and is used for evaluating the actual analysis capability of the molecularly imprinted fluorescent sensor on similar viruses HAB and HBV.

Technical Field

The invention belongs to the technical field of analytical chemistry detection, and particularly relates to a preparation method and application of molecular imprinting fluorescent sensors for simultaneously and visually detecting two viruses.

Background

Molecular imprinting technology has gained more and more attention and application in the field of virus detection because of its easy preparation, and its advantages such as higher stability, sensitivity and selectivity. However, as the larger the size of the virus, the more fragile the structure formed by its self-assembly, the more difficult it is to blot; meanwhile, the molecular imprinting of viruses generally relies on the specific recognition of the coat structure on the surface of the viruses, and since the structural units of the coats of similar viruses are also similar [ Cumbo, a., Lorber, b., Corvini, p.f.x., Meier, w., Shahgaldian, p.nat. commun.2013,4, 1925-.

In recent years, the development of molecular imprinting technology in the field of virus detection provides powerful means for breaking through the problem, for example, by introducing hydrophilic groups or monomers on the polymer surface to increase the hydrophilicity of imprinted particles, reduce non-specific binding [ Yang, y., Niu, h., Zhang, h., ACS appl.mater.interface, 2016,8, 15741. 15749 ], Ma, y, Pan, g., Zhang, y., Guo, x., Zhang, h., angelw.chem., int.ed.2013,52, 1511. 1514 ], and achieve higher template-specific recognition capability by metal-assisted molecular imprinting [ Liu, j., Yang, k., Deng, q., Li, Qi., Zhang, l., Liang, z., Zhang, y, chem, 2011mu, 2011, k., 2011, 9, 3, k.

As a functional monomer, zinc acrylate is widely applied to the construction of molecular imprinting polymers by because of the high recognition capability of a template due to the fact that zinc acrylate can be chelated with a target to form a six-membered ring structure [ Yan, Y.J., He, X.W., Li, W.Y., Zhang, Y.K., biosens, Bioelectron.2017,91,253 261.; Qin, Y.P., Wang, H.Y., He, X.W., Li, W.Y., Zhang, Y.K., Talanta 2018,185,620 and 627.], and N-isopropylacrylamide (NIPAAm) has the advantages of good thermal properties and good hydrophilicity at a temperature lower than a critical solution, and is widely applied to the construction of intelligent imprinting molecules by [ Li, C., Ma, Y., Niu, H.J., Zhang, H.63, Appl. 2015, 27340, and 2752 ] because zinc acrylate has the advantages of being simultaneously detected by NIPAAm, MAT, 272015, 350, and a plurality of important detection molecules for the simultaneous detection of zinc acrylate.

HAV and HBV are two viruses which cause hepatitis most frequently, cause substantially the same clinical symptoms, are infected with each other and coexist in patients, and thus, the simultaneous detection of the two viruses is of great significance in clinical treatment. The invention takes the two viruses as target objects for research, takes two quantum dots as different fluorescence signal sources, respectively imprints different viruses on different carriers, mixes the obtained imprinted polymers after elution, and constructs the fluorescence sensor which can be used for simultaneously detecting the two viruses. According to the invention, NIPAAm and zinc acrylate are added at the same time, the rapid elution of the template is promoted by using the NIPAAm thermal sensitive characteristic, and the selective recognition of a target object is enhanced by using the hydrophilic characteristic and the metal coordination action of the zinc acrylate. Meanwhile, the quantum dots can generate fluorescence quenching with different degrees after being combined with target objects with different concentrations, so that the obtained sensor can realize visual detection.

As shown in FIG. 2, the results show that the sensor constructed by the invention hardly interferes in the actual measurement, can realize the separate or simultaneous detection of HAV and HBV, and has the advantages of high selectivity, high sensitivity, low detection limit and the like. The strategy provided by the invention lays a good foundation for simultaneous detection of virus molecular imprinting, and has potential capability of clinically treating and diagnosing viral diseases.

Disclosure of Invention

The invention aims to provide a preparation method of molecular imprinting fluorescent sensors for simultaneously detecting two viruses in a visual manner, and the sensors are applied to specific recognition and detection of similar virus molecules.

The purpose of the invention is realized by the following technical scheme.

molecular engram fluorescence sensor for detecting two viruses simultaneously is prepared and applied, which is characterized in that the method comprises the following steps:

(1) obtaining red and green fluorescent quantum dots R-CdTe QDs and G-CdTe QDs under different experimental conditions, coating layers of silicon dioxide around the quantum dots by hydrolyzing Tetraethylorthosilicate (TEOS), grafting C-C to connect zinc acrylate, adsorbing virus by the hydrophilic action of a hydrophilic monomer NIPAAm, forming a zinc-containing six-membered ring coordination structure between the zinc acrylate and the virus by metal chelation to fix the template virus, and eluting the template molecule after polymerization is completed;

(2) non-specific binding is reduced by metal chelation and hydrophilic monomers: after synthesizing the imprinting carrier material, adding a functional monomer zinc acrylate and a hydrophilic monomer NIPAAm to assemble the imprinting material surface, wherein the NIPAAm is in a hydrophilic swelling state at a low critical dissolving temperature, so that a hydrophobic matrix is excluded to reduce nonspecific adsorption; zinc acrylate and the template virus form a zinc-containing six-membered ring coordination structure through coordination, so that the specific recognition capability of the template virus is improved;

(3) the preparation and application of the virus molecular imprinting fluorescence sensor are that two imprinting polymers G-MIPs and R-MIPs are mixed according to a determined proportion, template viruses HAV and HBV are added, after the mixture is absorbed for periods of time under optimized experimental conditions, the mixture is taken and placed in a cuvette, the excitation wavelength is 370nm, the emission wavelength is 555nm (for HAV detection) and 655nm (for HBV detection), the slit width is 5.0nm, the fluorescence intensity is measured by adopting RF-5301PC fluorescence spectrophotometry, and novel molecular imprinting fluorescence sensors for simultaneously detecting the two viruses are constructed.

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

(1) the zinc acrylate is used as a functional monomer, and a six-membered ring is formed between the zinc acrylate and virus molecules through metal chelation, so that the fixation of template molecules can be facilitated, and the elution of the template molecules is not hindered;

(2) NIPAAm is used as a hydrophilic monomer, and nonspecific adsorption is reduced through the repulsion of hydrophilic groups on the surface of the NIPAAm to hydrophobic substances;

(3) the experimental result shows that the virus molecular imprinting fluorescent sensor has high specific recognition capability on similar viruses HAV and HBV, can realize simultaneous detection, and has high selectivity and sensitivity and satisfactory imprinting effect;

(4) the quantum dots are used as a fluorescence signal source, and visual detection can be realized under the irradiation of ultraviolet light based on fluorescence quenching after combination with a target object;

(5) the sensor has the potential possibility of being applied to simultaneous detection of a plurality of similar targets, has low professional requirements on operators in the detection process, and has the potential capability of clinically treating and diagnosing viral diseases.

Drawings

FIG. 1 (A) is a flow chart for preparing the virus molecularly imprinted fluorescent sensor; (B) schematic representation of metal coordination.

FIG. 2 Mixed G-MIPs/R-MIPs (A) without added virus; (B) HAV is added; (C) adding HBV; (D) fluorescence intensity profiles of HAV and HBV were added.

[ FIG. 3 ]](A)G-CdTe(a)，[email protected]₂(b)，[email protected]₂C＝C(c)，G-MIP(d) G-NIP；(B)R-CdTe(a)，[email protected]₂(b)，[email protected]₂C ═ C (C), R-mip (d), and fourier transform infrared spectra of R-NIP particles.

[ FIG. 4 ]](A)[email protected]₂；(B)G-MIPs；(C)G-NIPs；(D)[email protected]₂(ii) a (E) R-MIPs; (F) scanning electron micrographs of R-NIPs particles.

[ FIG. 5 ]](A)[email protected]₂，(B)G-MIPs，(C)G-NIPs，(D)[email protected]₂(E) particle size distribution of R-MIPs, (F) particles of R-NIPs.

[ FIG. 6] (A) fluorescence intensity detection of specified concentrations of HAV and HBV in G-MIPs/R-MIPs; (B) visible fluorescence images of HAV and HBV with specified concentrations in MIPs mixed solution under the irradiation of a 365nm ultraviolet lamp; (C) fluorescence intensity detection of specified concentrations of HAV and HBV in G-NIPs/R-NIPs; (D) a plot of Δ F versus HAV concentration for G-MIPs; plot of Δ F versus HBV concentration for R-MIPs (difference in fluorescence intensity with Δ F in the presence or absence of template virus).

FIG. 7 is a diagram of selective and competitive investigation of G-MIPs/R-MIPs.

FIG. 8 is a graph showing reproducibility and stability of G-MIPs/R-MIPs.

FIG. 9 results of HAV (HBV-free) and HBV (HAV-free) detection in human serum.

FIG. 10 shows the results of simultaneous detection of HAV and HBV in human serum.

Detailed description of the preferred embodiments

The invention will now be described in further detail by with reference to the accompanying drawings and examples.