阅读说明：本技术 一种检测赭曲霉毒素a的荧光生物传感器的制备方法 (Preparation method of fluorescence biosensor for detecting ochratoxin A ) 是由 由天艳 毕晓雅 李丽波 刘晓红 陈柏年 于 2019-09-17 设计创作，主要内容包括：本发明属于生物传感检测技术领域,公开了一种检测赭曲霉毒素A的荧光生物传感器的制备方法。本发明属于一种基于能量共振转移作用的生物传感器,通过利用适配体与CoOOH纳米片的相互作用以及与apt耦合的g-CNQDs(g-CNQDs-apt)和CoOOH纳米片之间的能量共振转移作用,同时引入适配体作为生物识别元件,运用其可与目标物OTA特异性结合的特性,实现实际样品中OTA的快速、低成本、特异性检测。本发明构建的荧光适配体传感器对OTA的响应范围为1.00×10<Sup>-9</Sup>-1.40×10<Sup>-7</Sup>mol·L-1,检出限为3.3×10<Sup>-</Sup><Sup>10</Sup>mol·L<Sup>-1</Sup>,同时具有选择性好、快速、成本低的优点,为测定实际样品中的OTA提供了一种新型适配体传感平台。(The invention belongs to the technical field of biosensor detection, and discloses a preparation method of a fluorescence biosensor for detecting ochratoxin A. The invention belongs to a biosensor based on energy resonance transfer effect, which realizes rapid, low-cost and specific detection of OTA in an actual sample by utilizing the interaction of an aptamer and a CoOOH nano sheet and the energy resonance transfer effect between g-CNQDs (g-CNQDs-apt) coupled with apt and the CoOOH nano sheet, and simultaneously introducing the aptamer as a biological recognition element by utilizing the characteristic that the aptamer can be specifically combined with a target object OTA. The response range of the fluorescent aptamer sensor constructed by the invention to OTA is 1.00 multiplied by 10 ‑9 ‑1.40×10 ‑7 mol.L-1, detection limit of 3.3X 10 ‑ 10 mol·L ‑1 Meanwhile, the method has the advantages of good selectivity, rapidness and low cost, and provides a novel aptamer sensing platform for measuring OTA in an actual sample.)

1. A preparation method of a fluorescence biosensor for detecting ochratoxin A is characterized by comprising the following steps:

(1) synthesizing g-CNQDs;

(2) the carboxyl of the g-CNQDs and the amino modified by the OTA aptamer are subjected to an amide reaction to synthesize g-CNQDs-apt:

mixing and stirring the g-CNQDs prepared in the step (1) and the PBS solution according to a certain proportion, slowly adding the OTA aptamer solution under stirring, continuously stirring the mixture solution for a certain time to prepare g-CNQDs-apt, and storing at a certain temperature for later use;

(3) synthesizing a CoOOH nano sheet;

(4) mixing g-CNQDs-apt with CoOOH nanosheets:

and (3) mixing the g-CNQDs-apt prepared in the step (2) and the CoOOH nanosheet solution prepared in the step (3) according to a proportion, adjusting the pH value with a PBS solution, and acting for a period of time to obtain the fluorescence biosensor for detecting ochratoxin A.

2. The method of preparing a fluorescence biosensor for detecting ochratoxin a according to claim 1, wherein: in the step (1), the specific method for synthesizing g-CNQDs is as follows: 1.68mmol trisodium citrate and 10.08mmol urea were mixed and ground to a fine powder in an agate mortar; pouring the powder mixture into a 20mL polytetrafluoroethylene-lined autoclave and heating at 180 ℃ for 1h and then cooling to room temperature; dispersing the solid product in 30mL of water and centrifuging at 10000rpm for 10min to remove insoluble impurities; subsequently, the resulting upper solution was dialyzed with a 100Da dialysis bag in secondary water for 24h to remove unreacted precursor; oven dried, weighed, dissolved in secondary water and stored at 4 ℃.

3. The method of preparing a fluorescence biosensor for detecting ochratoxin a according to claim 1, wherein: in the step (2), the volume ratio of the g-CNQDs to the PBS is 2:1, the mixing and stirring time is 15-30min, wherein the concentration of the g-CNQDs is 4 mg/mL^-1PBS concentration 100mM, pH 7.4, containing 10mM EDC and 5mM NHS;

the volume ratio of the g-CNQDs to the OTA aptamer solution is 1: 1; wherein, the concentration of the OTA aptamer solution is 1 mu M; the mixture solution is continuously stirred for 2 to 4 hours; the storage temperature of the prepared g-CNQDs-apt is 4 ℃.

4. The method of preparing a fluorescence biosensor for detecting ochratoxin a according to claim 2, wherein: in the step (3), the preparation method of the CoOOH nanosheet specifically comprises the following steps: 125 μ L of 1.0M NaOH solution was mixed with 500 μ L of 10.0mM CoCl₂·6H₂Mixing the O solution in a microcentrifuge tube, carrying out ultrasonic treatment for 1min, adding 25 mu L of 0.9M NaClO solution, and continuing ultrasonic treatment for 10 min; subsequently, the CoOOH nanosheets were collected by centrifugation at 12000rpm for 15min, washed with two times of water, centrifuged three times, and redispersed in water after drying.

5. The method of preparing a fluorescence biosensor for detecting ochratoxin a according to claim 2, wherein: in the step (4), the volume ratio of the g-CNQDs-apt solution to the CoOOH nanosheet solution is 3: 2; adjusting pH to 7.4 with PBS solution, and allowing the solution to act for 1-25 min;

in the prepared fluorescence biosensor for detecting ochratoxin A, the final concentration of g-CNQDs-apt is 0.05-0.25 mg/mL^-1The final concentration of the CoOOH nanosheet is 0-30 [ mu ] g & mL^-1。

6. Use of the fluorescence biosensor for detecting ochratoxin A prepared by the preparation method of any one of claims 1-5 in detection of ochratoxin A.

7. Use according to claim 6, characterized in that: the detection method comprises the following steps:

s1: adding 100 mu L of 1-140nM OTA standard solution into 900 mu L of fluorescence biosensor for detecting ochratoxin A, reacting for 3-25min, and detecting the fluorescence intensity of the solution at 469nM with a fluorescence spectrophotometer at room temperature to obtain a standard curve of fluorescence intensity and OTA concentration; the excitation wavelength of the fluorescence spectrophotometer is set to be 390nm, the width of an excitation slit is 3nm, and the width of an emission slit is 3 nm;

s2: adding 100 mu L of solution to be detected into 900 mu L of fluorescence biosensor for detecting ochratoxin A, reacting for 3-25min, respectively detecting fluorescence intensity of the solution at 469nm at room temperature by using a fluorescence spectrophotometer, and substituting into the standard curve in step S1 to obtain the concentration of ochratoxin A in the solution to be detected.

Technical Field

The invention belongs to the technical field of biosensing detection, and particularly relates to a preparation method of a fluorescence biosensor for detecting ochratoxin A, which is used for selective detection of ochratoxin A (OTA).

Background

The grains are easy to rot and deteriorate due to fungal infection in the growth, harvesting and storage links of the grains, and a compound with strong toxicity, namely mycotoxin, can be generated under the appropriate temperature and humidity. Mycotoxins are one of the major pollutants of agricultural products around the world, and more than 400 species of mycotoxins are found at present. Ochratoxin a (ota) is a compound produced by ochratoxin a or penicillium, and is widely distributed in grains such as corn, barley, wheat, etc. OTA is classified as a potential carcinogen (group 2B) by the International cancer research institute due to its nephrotoxic, teratogenic and immunotoxic effects, and China national standards require that the OTA content in cereals and their products not exceed 5 μ g Kg^-1. Therefore, there is a need to develop an accurate and reliable analysis method to realize the OTA analysis in the grain, so as to ensure the health of human body. At present, the existing OTA detection methods have the advantages and the disadvantages. Such as high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and Thin Layer Chromatography (TLC) have high accuracy and reliability, but are relatively costly and time consuming, and must be performed in specialized laboratories by specialized personnel. Enzyme-linked immunosorbent assay (ELISA) can achieve rapid detection of OTA, but has the disadvantages that expensive enzyme-labeled reagents must be used, and environmental conditions such as temperature, pH, etc. have a significant effect on the antibody or antigen. In contrast, fluorescence assays are of great interest in biosensor applications because of their advantages of convenient, rapid, and easy automation.

Energy resonance transfer is a commonly used strategy for constructing a fluorescence sensing method and is realized by the existence of spectral overlap between a fluorescence donor and an acceptor within a distance of 10 nm. In the receptor substances reported at present, cobalt oxyhydroxide (CoOOH) nanosheets have the advantages of high synthesis speed and good stability, have wide ultraviolet absorption peaks (340nm-800nm), and can perform energy resonance transfer with various fluorescent probes (such as gold nanoclusters and fluorescent dyes), but the fluorescent probes used in the sensing technology constructed based on the CoOOH nanosheets at present still need to be improved in the aspects of stability, simplification of a synthesis route and the like.

The carbon nitride quantum dots (g-CNQDs) as a novel fluorescent probe have unique physical and chemical properties, such as good water solubility, stability and nontoxicity, and are rich in functional groups such as hydroxyl, carboxyl, amino and the like, thereby being beneficial to further modification. At present, a plurality of fluorescent sensors using g-CNQDs as donors are developed and applied, but a sensing system using the g-CNQDs as CoOOH nanosheet donors is not reported yet.

Disclosure of Invention

Aiming at the problems in the prior art, the invention introduces OTA aptamer (OTA-apt) on the basis of combining the advantages of g-CNQDs and CoOOH nano sheets to construct a novel fluorescent aptamer sensor for rapid and specific detection and analysis of OTA in an actual sample. Wherein, the sequence of the OTA aptamer is as follows: 5' -GAT CGG GTG TGG GTG GCG TAA AGG GAGCAT CGG ACA-C₆H₁₂-NH₂-3'。

The invention aims to couple a fluorescence method and an aptamer sensing technology and construct an OTA aptamer sensor based on a g-CNQDs-CoOOH system. The method is realized by the following technical scheme: the rapid, low-cost and specific detection of the OTA in the actual sample is realized by utilizing the interaction of the aptamer and the CoOOH nanosheet and the energy resonance transfer effect between g-CNQDs (g-CNQDs-apt) coupled with apt and the CoOOH nanosheet and combining the characteristic of aptamer specific recognition of the OTA.

A preparation method of a fluorescence biosensor for detecting ochratoxin A comprises the following steps:

(1) synthesizing g-CNQDs;

(2) the carboxyl of the g-CNQDs and the amino modified by the OTA aptamer are subjected to an amide reaction to synthesize g-CNQDs-apt:

mixing and stirring the g-CNQDs prepared in the step (1) and the PBS solution according to a certain proportion, slowly adding the OTA aptamer solution under stirring, continuously stirring the mixture solution for a certain time to prepare g-CNQDs-apt, and storing at a certain temperature for later use;

(3) synthesizing a CoOOH nano sheet;

(4) mixing g-CNQDs-apt with CoOOH nanosheets:

and (3) mixing the g-CNQDs-apt prepared in the step (2) and the CoOOH nanosheet solution prepared in the step (3) according to a proportion, adjusting the pH value with a PBS solution, and acting for a period of time to obtain the fluorescence biosensor for detecting ochratoxin A.

Wherein, in the step (1), the specific method for synthesizing g-CNQDs comprises the following steps: 1.68mmol trisodium citrate and 10.08mmol urea were mixed and ground to a fine powder in an agate mortar; pouring the powder mixture into a 20mL polytetrafluoroethylene-lined autoclave and heating at 180 ℃ for 1h and then cooling to room temperature; dispersing the solid product in 30mL of water and centrifuging at 10000rpm for 10min to remove insoluble impurities; subsequently, the resulting upper solution was dialyzed with a 100Da dialysis bag in secondary water for 24h to remove unreacted precursor; oven dried, weighed, dissolved in secondary water and stored at 4 ℃.

In the step (2), the volume ratio of the g-CNQDs to the PBS is 2:1, the mixing and stirring time is 15-30min, wherein the concentration of the g-CNQDs is 4 mg/mL^-1PBS concentration 100mM (pH 7.4, containing 10mM EDC and 5mM NHS); the volume ratio of the g-CNQDs to the OTA aptamer solution is 1: 1; wherein, the concentration of the OTA aptamer solution is 1 mu M; the mixture solution is continuously stirred for 2 to 4 hours; the storage temperature of the prepared g-CNQDs-apt is 4 ℃.

In the step (3), the preparation method of the CoOOH nanosheet specifically comprises the following steps: 125 μ L of 1.0M NaOH solution was mixed with 500 μ L of 10.0mM CoCl₂·6H₂Mixing the O solution in a microcentrifuge tube, carrying out ultrasonic treatment for 1min, adding 25 mu L of 0.9M NaClO solution, and continuing ultrasonic treatment for 10 min; subsequently, the CoOOH nanosheets were collected by centrifugation at 12000rpm for 15min, washed with two times of water, centrifuged three times, and redispersed in water after drying.

In the step (4), the volume ratio of the g-CNQDs-apt solution to the CoOOH nanosheet solution is 3: 2; adjusting pH to 7.4 with PBS solution, and acting for 1-25 min.

In the prepared fluorescence biosensor for detecting ochratoxin A, the final concentration of g-CNQDs-apt is 0.05-0.25 mg/mL^-1The final concentration of the CoOOH nanosheet is 0-30 [ mu ] g & mL^-1。

The fluorescent biosensor for detecting ochratoxin A prepared by the invention is used for detecting ochratoxin A.

The detection method comprises the following steps:

s1: adding 100 mu L of 1-140nM OTA standard solution into 900 mu L of fluorescence biosensor for detecting ochratoxin A, reacting for 3-25min, and detecting the fluorescence intensity of the solution at 469nM with a fluorescence spectrophotometer at room temperature to obtain a standard curve of fluorescence intensity and OTA concentration; the excitation wavelength of the fluorescence spectrophotometer is set to be 390nm, the width of the excitation slit is 3nm, and the width of the emission slit is 3 nm.

S2: adding 100 mu L of solution to be detected into 900 mu L of fluorescence biosensor for detecting ochratoxin A, reacting for 3-25min, respectively detecting fluorescence intensity of the solution at 469nm at room temperature by using a fluorescence spectrophotometer, and substituting into the standard curve in step S1 to obtain the concentration of ochratoxin A in the solution to be detected.

The invention has the beneficial effects that:

(1) combining the advantages of the g-CNQDs and the CoOOH nano-sheets, and quantitatively detecting the OTA based on the energy resonance transfer effect between the g-CNQDs and the CoOOH nano-sheets;

(2) introducing an aptamer of a specific recognition element OTA to realize specific analysis of the target object OTA;

(3) the fluorescence aptamer sensor constructed by the invention is used for OTA detection, has good selectivity, is rapid (can be completed within 10 min), is low in cost, and has a linear range of 1-140 nM.

Drawings

FIG. 1 is a schematic diagram of the fluorescent aptamer sensor for detecting OTA.

FIG. 2 is a graph of fluorescence spectra of different solutions in a feasibility assay of the fluorescence aptamer sensor.

FIG. 3 is a TEM image of g-CNQDs.

FIG. 4 is a gel electrophoresis image of apt and g-CNQDs-apt.

Fig. 5 is an SEM image of CoOOH nanosheets.

FIG. 6A is a graph showing the relationship between g-CNQDs-apt concentration and fluorescence recovery intensity △ F, B is a graph showing the relationship between CoOOH concentration and fluorescence recovery intensity △ F, C is a graph showing the relationship between g-CNQDs-apt and CoOOH nanosheet different reaction time and fluorescence intensity, and D is a graph showing the relationship between OTA and aptamer different combination time and fluorescence recovery intensity △ F.

FIG. 7A is a graph of the fluorescence spectra of solutions in the presence of different concentrations of OTA standard solution; b is a linear relationship graph between OTA concentration and fluorescence intensity.

FIG. 8 is a graph showing changes in fluorescence of solutions in the presence of various interferents.

Detailed Description

The invention is described in detail below with reference to the drawings and examples of the specification:

a preparation method of a fluorescence biosensor for detecting ochratoxin A is specifically realized by the following technical scheme: the rapid, low-cost and specific detection of the OTA in the actual sample is realized by utilizing the interaction between the aptamer and the CoOOH nanosheet and the energy resonance transfer effect between g-CNQDs (g-CNQDs-apt) coupled with the aptamer and the CoOOH nanosheet and combining the characteristic of specificity recognition of the aptamer to the OTA.

The feasibility analysis of the detection method is as follows:

CoOOH nanoplates bind ssDNA more strongly than G-quadruplexes and can act as acceptors to quench the fluorescent signal of apt end-coupled G-CNQDs. Aptamers are single-stranded DNA or RNA molecules that bind to a target with high specificity and high affinity. Therefore, interference of OTA structural analogs and other mycotoxins can be avoided, and interference signals can be effectively shielded. The fluorescence sensing strategy for OTA specific detection is designed based on the quenching characteristic of the CoOOH nanosheet and the specific recognition and binding effect of the aptamer on OTA.

The detection process of the sensor is shown in fig. 1, and the basic principle is as follows:

the aptamer is a section of oligochain ssDNA sequence, and after a CoOOH nanosheet is added into g-CNQDs coupled with the aptamer, the g-CNQDs-apt and the CoOOH nanosheet are adsorbed together due to the strong van der Waals force effect between the aptamer and the CoOOH nanosheet, so that the distance between the g-CNQDs and the CoOOH nanosheet is shortened, the energy resonance transfer between the g-CNQDs and the CoOOH nanosheet is effectively promoted, and the fluorescence signal of the g-CNQDs is quenched. When a target object OTA exists, the OTA and apt are specifically combined to form a G-quadruplex, and the acting force of the G-quadruplex and the CoOOH nanosheet is weaker than that of an aptamer single chain on the CoOOH nanosheet, so that G-CNQDs are far away from the CoOOH nanosheet, the quenching efficiency of the CoOOH nanosheet on the G-CNQDs is reduced, and the fluorescence of the G-CNQDs is recovered. Therefore, OTA specific detection can be realized by monitoring the change of the fluorescence signal of g-CNQDs.

In order to further verify the feasibility of the scheme, the fluorescence change conditions before and after a certain amount of CoOOH nanosheets and OTA are considered. As shown in FIG. 2, aptamer-coupled g-CNQDs have a strong fluorescence signal at 469nm (curve a). When the CoOOH nano-sheet is added into the solution, the basic group of the aptamer reacts with the CoOOH nano-sheet through Van der Waals force, so that g-CNQDs-apt and the CoOOH nano-sheet are adsorbed together, and further energy resonance transfer between the g-CNQDs and the CoOOH nano-sheet is initiated, so that the fluorescence signal of the g-CNQDs is quenched (curve b). After the target object OTA solution is added, G-CNQDs-apt is pulled away from the CoOOH nanosheets due to the formation of G-quadruplexes by the specific combination of the OTA and the aptamers, so that the occurrence of energy resonance transfer is hindered, and thus the fluorescence signals of the G-CNQDs are recovered (curve c). Therefore, when OTA is present in the solution, the aptamer can be specifically bound with OTA to form an aptamer complex, and the binding capacity of the aptamer complex to CoOOH nanosheets can be obviously weaker than that of free aptamers, so that the fluorescence signal of the system is enhanced. The experiment can effectively prove that the constructed aptamer sensor can be used for measuring the content of the target object OTA. In FIG. 2, a is a fluorescence spectrum of the g-CNQDs-apt solution; b is a fluorescence spectrum chart of the solution after g-CNQDs-apt is mixed with the CoOOH nano-sheet; c is a fluorescence spectrogram of a solution obtained by mixing g-CNQDs-apt with a CoOOH nano sheet and then with OTA.

The present invention will be described in more detail with reference to the following embodiments: