阅读说明：本技术 一种黄曲霉毒素产毒基因nor-1的荧光传感器制备和检测方法 (Preparation and detection method of fluorescence sensor of aflatoxin toxin-producing gene nor-1 ) 是由 李雅琪 李继洋 余茜 肖琪 孔德昭 盛建国 于 2019-10-17 设计创作，主要内容包括：本发明公开了一种黄曲霉毒素产毒基因nor-1的荧光传感器的制备方法和检测方法,荧光传感器为基于特异性识别黄曲霉毒素产毒基因nor-1的长臂发夹探针,在所述长臂发夹探针的两端分别带有NH<Sub>2</Sub>和SH,NH<Sub>2</Sub>修饰上核壳结构的CdTe/CdS量子点,SH修饰上金纳米粒子AuNPs；制备步骤包括：制备CdTe/CdS量子点溶液备用；制备AuNPs备用；长臂发夹探针的修饰：检测包括荧光传感器的构建,当目标物nor-1出现时,nor-1与发夹探针特异性互补配对,使发夹探针伸直,拉大了CdTe/CdS量子点和AuNPs的距离,阻断了荧光共振能量转移,从而使CdTe/CdS量子点的荧光恢复,通过荧光恢复强度测定nor-1的浓度,从而实现nor-1的快速检测。本发明制备荧光传感器具有灵敏、简单、快速等优点,源头检测产毒基因,成本低廉,检测迅速等优点。(The invention discloses a preparation method and a detection method of a fluorescence sensor of an aflatoxin toxin-producing gene nor-1, wherein the fluorescence sensor is a long-armed hairpin probe based on specificity recognition of the aflatoxin toxin-producing gene nor-1, and both ends of the long-armed hairpin probe are respectively provided with NH (NH) 2 And SH, NH 2 Modifying CdTe/CdS quantum dots with an upper core-shell structure, and modifying gold nanoparticles AuNPs by SH; the preparation method comprises the following steps: preparing CdTe/CdS quantum dot solution for later use; preparing AuNPs for later use; modification of long-arm hairpin probes: the detection comprises the construction of a fluorescence sensor, when a target object nor-1 appears, the nor-1 and the hairpin probe are subjected to specific complementary pairing, the hairpin probe is straightened, the distance between the CdTe/CdS quantum dot and AuNPs is enlarged, the fluorescence resonance energy transfer is blocked, the fluorescence of the CdTe/CdS quantum dot is recovered, the concentration of the nor-1 is measured through the fluorescence recovery intensity, and the quick detection of the nor-1 is realized. Hair brushThe fluorescent sensor has the advantages of sensitivity, simplicity, rapidness and the like, and has the advantages of low cost, rapidness in detection and the like when the toxin producing gene is detected from the source.)

1. A preparation method of a fluorescence sensor of an aflatoxin toxin-producing gene nor-1 is characterized by comprising the following steps: the fluorescence sensor is a long-arm hairpin probe based on specificity recognition of aflatoxin toxin-producing gene nor-1, and both ends of the long-arm hairpin probe are respectively provided with NH₂And SH, NH₂CdTe/CdS quantum dot modified with core-shell structure, gold nanoparticle AuNPs modified with SH, and CdTe/CdS quantum dot and A when no target substance appearsThe method comprises the following steps that the uNPs are close in space distance and generate fluorescence resonance energy transfer, so that fluorescence quenching of CdTe/CdS quantum dots is caused, when a target object nor-1 appears, the nor-1 is in specific complementary pairing with a hairpin probe, the hairpin probe is straightened, the distance between the CdTe/CdS quantum dots and AuNPs is enlarged, the fluorescence resonance energy transfer is blocked, so that fluorescence of the CdTe/CdS quantum dots is recovered, the concentration of nor-1 is measured through fluorescence recovery intensity, and a fluorescence sensor is established by adopting a method constructed by a fluorescence recovery mechanism; the steps for preparing the fluorescence sensor are as follows:

1) preparing CdTe/CdS quantum dot solution for later use;

2) preparing AuNPs for later use;

3) modification of long-arm hairpin probes: a. ultrasonically dispersing the CdTe/CdS quantum dot solution prepared in the step 1, adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution, uniformly oscillating, adding an N-hydroxysuccinimide (NHS) solution, uniformly oscillating, adding a hairpin probe stock solution, reacting at room temperature, oscillating overnight to obtain a CdTe/CdS-hairpin probe, washing with ethanol, centrifuging, and finally dispersing the CdTe/CdS-hairpin probe in a Tris-HCl buffer solution for later use; b. and (b) transferring a proper amount of prepared AuNPs into the CdTe/CdS-hairpin probe dispersion liquid prepared in the step (a), adding a NaCl solution to adjust the concentration of sodium ions to a certain value, carrying out oscillation reaction for 24 hours at normal temperature, and then washing and centrifuging to obtain the CdTe/CdS-hairpin probe-AuNPs.

2. The method for preparing the fluorescence sensor of the aflatoxin toxin-producing gene nor-1 as claimed in claim 1, wherein the method comprises the following steps: in the step a of the step 3, the volume ratio of the CdTe/CdS quantum dots, the EDC and the NHS to the hairpin probe is 50:2.2:2.2:1, the concentration of the EDC solution is 380 mM, the concentration of the NHS solution is 190 mM, and the concentration of the hairpin probe is 100 mu M.

3. The method for preparing the fluorescence sensor of the aflatoxin toxin-producing gene nor-1 as claimed in claim 2, wherein the method comprises the following steps: and (b) in the step (a) of the step (3), the volume ratio of the Tris-HCl buffer solution to the CdTe/CdS quantum dots dispersed in the Tris-HCl buffer solution is 52:42, the concentration of the Tris-HCl buffer solution is 10 mM, and the pH value is 7.4.

4. The method for preparing the fluorescence sensor of the aflatoxin toxin-producing gene nor-1 as claimed in claim 3, wherein the method comprises the following steps: and (c) in the step (b) of the step (3), the concentration of the NaCl solution is 6M, and the concentration of sodium ions is adjusted to be 2M.

5. The method for preparing the fluorescence sensor of the aflatoxin toxin-producing gene nor-1 as claimed in claim 4, wherein the method comprises the following steps: and the volume ratio of the CdTe/CdS-hairpin probe to the AuNPs in the step b of the step 3 is 30: 1.

6. A detection method of a fluorescence sensor of an aflatoxin toxin-producing gene nor-1 is characterized by comprising the following steps: adding an array of 20 mu L of standard solution of the toxigenic gene nor-1 with different concentrations into 0.1 mL of the prepared CdTe/CdS-hairpin probe-AuNPs solution, fixing the volume to 2 mL by adopting Tris-HCl buffer solution with the pH of 7.4, oscillating and incubating at constant temperature to form a CdTe/CdS-hairpin probe-AuNPs/nor-1 compound, measuring the fluorescence intensity of the solution at 380nm by adopting a fluorescence spectrophotometer, and establishing a standard curve of the fluorescence recovery intensity and nor-1.

7. The method for detecting a fluorescence sensor for aflatoxin toxin-producing gene nor-1 according to claim 6, which is characterized in that: the standard solution of the toxin-producing gene nor-1 with different concentrations is 10 groups, and the concentration of each group is 0.05, 0.5, 5, 10, 20, 40, 50, 100, 150 and 200nM respectively.

8. The method for detecting a fluorescence sensor for aflatoxin toxin-producing gene nor-1 according to claim 6, which is characterized in that: the incubation time is 40-60 min.

Technical Field

The invention relates to the field of fluorescence sensing, in particular to a preparation method of a fluorescence sensor for detecting aflatoxin toxin-producing gene nor-1 by using a fluorescence recovery mechanism of signal quenching and target object specific identification of fluorescence resonance energy transfer and a rapid detection method of the nor-1 by using the fluorescence sensor.

Background

Aflatoxin was identified as a carcinogen earlier than 1961, especially aflatoxin B1 has 68 times toxicity of arsenic and 10 times toxicity of potassium cyanide, and is the most toxic and carcinogenic natural pollutant discovered so far, and according to the reports of world food and agriculture organization, about 25% of grains in the world generate aflatoxin due to mildew, and aflatoxin pollution in grains has become a global problem. If the aflatoxin toxigenic bacteria can be detected from the source and effective monitoring is carried out from the source, the problems of safety and economic benefit caused by the generation of aflatoxin can be reduced.

The toxigenic bacteria of aflatoxin mainly belong to aspergillus flavus and aspergillus parasiticus, belong to saprophytic fungi, the common detection of the toxigenic bacteria at home and abroad at present mainly focuses on traditional morphological identification, identification of a culture medium, multiple PCR detection, immunosensing detection and the like, and the methods are complex and complicated, have long detection period, poor sensitivity and high error rate, and cannot meet the daily real-time, quick and accurate monitoring requirements. The DNA sensor has the advantages of simple operation, high sensitivity, strong specificity and high detection speed, and has become a research hotspot for detecting microbial pathogenic bacteria, the gene nor-1 in the biological synthesis process of aflatoxin is also called as aflD gene, and can encode a catalytic enzyme with the molecular weight of 29 kDa, thereby playing an important role in AFT synthesis. At present, the electrochemical DNA sensor for detecting the specific gene nor-1 of AFT toxigenic bacteria is reported, but the electrochemical DNA detection method has the defects of large difference between electrodes, tedious and time-consuming electrode modification process, existence of a detection interface between a solid phase and a liquid phase and the like, so that a simple, rapid and stable AFT toxigenic bacteria nor-1 gene detection method based on DNA detection is constructed, a new thought can be provided for AFT toxigenic bacteria detection, and technical support is provided for the DNA sensor in the field of detection and monitoring of microbial pathogenic bacteria. Meanwhile, the DNA fluorescent sensor has the advantages of homogeneous phase, simple steps, high sensitivity, strong specificity and the like, and is applied to the detection of the nor-1 gene, thereby being an effective way for solving the existing scientific problems in the detection of AFT toxigenic bacteria.

The semiconductor nano material quantum dots have the characteristics of adjustable emission wavelength, relatively stable fluorescence performance and the like, and are currently used as signal markers of fluorescent biological probes, but when single quantum dots are directly used as markers, the defects of difficult dispersion, easy agglomeration, easy influence of particle size and dispersion state on fluorescence intensity and the like exist, and the preparation of the core-shell type quantum dots can effectively improve the defects, so that the method is an effective method for a fluorescent biosensor. When the ultraviolet spectrum of the metal nano particles and the fluorescence emission spectrum of the quantum dots are highly overlapped, when the ultraviolet spectrum of the metal nano particles and the fluorescence emission spectrum of the quantum dots are close enough, Fluorescence Resonance Energy Transfer (FRET) can occur to quench fluorescence, so that a proper fluorescence energy donor and an acceptor are screened, and the detection effect of the fluorescence sensor is also decisive, so that the fluorescence energy donor-acceptor pair is reasonably used, and the reasonable space distance is designed, and is an important factor for preparing the fluorescence biosensor.

Disclosure of Invention

In order to solve the problems, the invention provides a method for detecting a source of aflatoxin toxigenic gold, which comprises the steps of preparing a fluorescence sensor of aflatoxin toxigenic gene nor-1 based on a fluorescence mechanism of fluorescence resonance energy transfer and target object specific identification, selecting CdTe/CdS-gold nanoparticles (AuNPs) as an energy supply-receptor pair, drawing the distance between the CdTe/CdS-gold nanoparticles and the AuNPs by utilizing hairpin probe loop design to promote the generation of fluorescence resonance energy transfer, specifically identifying and drawing the hairpin probe by a DNA complementary pairing principle of the nor-1 to the hairpin probe, increasing the distance between the energy supply-receptor pair, preventing the fluorescence resonance energy transfer from causing fluorescence recovery, and realizing the rapid and sensitive detection of nor-1 by the change of fluorescence recovery intensity.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the invention relates to a preparation method of a fluorescence sensor of an aflatoxin toxin-producing gene nor-1, wherein the fluorescence sensor is a long-arm hairpin probe based on specificity recognition of the aflatoxin toxin-producing gene nor-1, and both ends of the long-arm hairpin probe are respectively provided with NH₂And SH, NH₂Modifying CdTe/CdS quantum dots with a top core-shell structure, modifying gold nano-particles AuNPs with SH, when no target object appears, enabling the CdTe/CdS quantum dots to be close to the AuNPs in space distance, and generating fluorescence resonance energy transfer, so that the fluorescence of the CdTe/CdS quantum dots is quenched, when the target object nor-1 appears, enabling the nor-1 to be in specific complementary pairing with the hairpin probe, straightening the hairpin probe, enlarging the distance between the CdTe/CdS quantum dots and the AuNPs, and blocking the fluorescence resonance energy transfer, so that the fluorescence of the CdTe/CdS quantum dots is recovered, measuring the concentration of the nor-1 through the fluorescence recovery intensity, and establishing a fluorescence sensor by adopting a method constructed by a fluorescence recovery mechanism; the steps for preparing the fluorescence sensor are as follows:

1) preparing CdTe/CdS quantum dot solution for later use;

2) preparing AuNPs for later use;

3) modification of long-arm hairpin probes: a. ultrasonically dispersing the CdTe/CdS quantum dot solution prepared in the step 1, adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution, uniformly oscillating, adding an N-hydroxysuccinimide (NHS) solution, uniformly oscillating, adding a hairpin probe stock solution, reacting at room temperature, oscillating overnight to obtain a CdTe/CdS-hairpin probe, washing with ethanol, centrifuging, and finally dispersing the CdTe/CdS-hairpin probe in a Tris-HCl buffer solution for later use; b. and (b) transferring a proper amount of prepared AuNPs into the CdTe/CdS-hairpin probe dispersion liquid prepared in the step (a), adding a NaCl solution to adjust the concentration of sodium ions to a certain value, carrying out oscillation reaction for 24 hours at normal temperature, and then washing and centrifuging to obtain the CdTe/CdS-hairpin probe-AuNPs.

The invention is further improved in that: in the step a of the step 3, the volume ratio of the CdTe/CdS quantum dots, the EDC and the NHS to the hairpin probe is 50:2.2:2.2:1, the concentration of the EDC solution is 380 mM, the concentration of the NHS solution is 190 mM, and the concentration of the hairpin probe is 100 mu M.

The invention is further improved in that: and (b) in the step (a) of the step (3), the volume ratio of the Tris-HCl buffer solution to the CdTe/CdS quantum dots dispersed in the Tris-HCl buffer solution is 52:42, the concentration of the Tris-HCl buffer solution is 10 mM, and the pH value is 7.4.

The invention is further improved in that: and (c) in the step (b) of the step (3), the concentration of the NaCl solution is 6M, and the concentration of sodium ions is adjusted to be 2M.

The invention is further improved in that: and the volume ratio of the CdTe/CdS-hairpin probe to the AuNPs in the step b of the step 3 is 30: 1.

The invention relates to a method for detecting a fluorescence sensor of an aflatoxin toxin-producing gene nor-1, which comprises the steps of adding an array of 20 mu L of standard solution of toxin-producing gene nor-1 with different concentrations into prepared 0.1 mL of CdTe/CdS-hairpin probe-AuNPs solution, fixing the volume to 2 mL by adopting Tris-HCl buffer solution with pH of 7.4, oscillating and incubating at constant temperature to form a CdTe/CdS-hairpin probe-AuNPs/nor-1 compound, measuring the fluorescence intensity of the solution at 380nm by adopting a fluorescence spectrophotometer, and establishing a standard curve of the fluorescence recovery intensity and nor-1.

The invention is further improved in that: the standard solution of the toxin-producing gene nor-1 with different concentrations is 10 groups, and the concentration of each group is 0.05, 0.5, 5, 10, 20, 40, 50, 100, 150 and 200nM respectively.

The invention is further improved in that: the incubation time is 40-60 min.

The invention has the beneficial effects that: CdTe/CdS is used as an energy donor, gold nanoparticles (AuNPs) are used as energy acceptors, and the CdTe/CdS and the AuNPs are respectively modified to two ends of the hairpin probe to prepare the DNA fluorescent sensor. By applying the fluorescence resonance energy transfer principle and the DNA complementary pairing principle, the DNA fluorescent sensor for rapidly detecting nor-1 of a fluorescence recovery mechanism is established, a new method is provided for detecting aflatoxin, and compared with other aflatoxin detection methods, the core-shell type quantum dot CdTe/CdS required by the sensor has stable fluorescence performance and good dispersibility; the sensor is constructed based on a fluorescence resonance energy transfer principle and a DNA complementary pairing principle, the theoretical basis is mature, and the reliability is strong; the hairpin probe is a medium for drawing in the space distance between the energy donor and the energy acceptor, the distance between the energy donor and the energy acceptor is enlarged through specific recognition with a target object nor-1, so that fluorescence recovery is caused, and the detection of nor-1 is realized through the measurement of the fluorescence recovery degree; the method is simple to operate, rapid in detection and suitable for rapid detection and analysis. In addition, the method detects the toxin-producing gene of the aflatoxin from the source, and is expected to provide a basis for inhibiting the amplification of aflatoxin toxin-producing bacteria.

Drawings

FIG. 1 is a schematic diagram of the overlap of CdTe/CdS fluorescence emission spectrum and AuNPs ultraviolet spectrum.

FIG. 2 is a schematic diagram showing the preparation of a fluorescence sensor and its detection principle for nor-1;

FIG. 3 is a standard graph of fluorescence intensity for detecting different concentrations of nor-1.

Detailed Description

For the purpose of enhancing an understanding of the present invention, the present invention will be further described with reference to the accompanying drawings and examples, which are included to further illustrate the invention and are not to be construed as limiting the invention, and modifications and substitutions of the method, steps or conditions of the invention may be made without departing from the spirit of the invention.

The invention relates to a preparation method of a fluorescence sensor of an aflatoxin toxin-producing gene nor-1, wherein the fluorescence sensor is a long-arm hairpin probe based on specificity recognition of the aflatoxin toxin-producing gene nor-1, and both ends of the long-arm hairpin probe are respectively provided with NH₂And SH, NH₂When no target object appears, the CdTe/CdS quantum dots are close to the AuNPs in space distance, fluorescence resonance energy transfer occurs, so that fluorescence quenching of the CdTe/CdS quantum dots is caused, when the target object nor-1 appears, the nor-1 is in specific complementary pairing with the hairpin probe, the hairpin probe is straightened, the distance between the CdTe/CdS quantum dots and the AuNPs is enlarged, the fluorescence resonance energy transfer is blocked, so that the fluorescence of the CdTe/CdS quantum dots is recovered, and the intensity is recovered through fluorescenceMeasuring the concentration of nor-1, and establishing a fluorescence sensor by adopting a method of constructing a fluorescence recovery mechanism; the steps for preparing the fluorescence sensor are as follows:

1) preparing CdTe/CdS quantum dot solution for later use;

the preparation process comprises the following steps: 0.0673 g NaBH₄Adding 0.0957 g of Te powder into a 10 mL colorimetric tube, adding 4 mL of secondary water, introducing nitrogen to remove oxygen for 15 min, and reacting in a refrigerator at 4 ℃ to obtain a purple transparent NaHTe solution as a supernatant; in a 100 mL three-necked flask, 50 mL of secondary water and 0.1142 g of CdCl were added₂And 75 mu L MPA, introducing nitrogen for protection under the stirring condition, and adding NaOH to adjust the pH of the solution to 8.5; and quickly adding 2 mL of the freshly prepared NaHTe solution, fully stirring, and refluxing at 100 ℃ for 0.5h to obtain the CdTe quantum dot. Weighing cadmium chloride according to stoichiometric ratio, adding into the obtained CdTe quantum dots, reacting for 0.5h, and adding CN₂H₄And continuously refluxing the S to obtain the core-shell CdTe/CdS quantum dots.

2) Preparing AuNPs for later use;

the preparation process comprises the following steps: putting a certain amount of chloroauric acid solution with the concentration of 200 mu M into a flask, heating to be vigorously boiled under stirring, adding sodium citrate solution with the concentration of 0.4M L and the concentration of 350 mM into the boiled chloroauric acid solution under vigorous stirring, and continuously heating and boiling for 15 min under stirring after the solution changes from light yellow to wine red. The synthesized gold nanoparticles are then slowly cooled to room temperature under stirring, and the synthesized solution is then filtered through a 0.8 μm microporous membrane to obtain the desired AuNPs.

3) Modification of long-arm hairpin probes: a. ultrasonically dispersing the CdTe/CdS quantum dot solution prepared in the step 1, adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution, uniformly oscillating, adding an N-hydroxysuccinimide (NHS) solution, uniformly oscillating, adding a hairpin probe stock solution, reacting at room temperature, oscillating overnight to obtain a CdTe/CdS-hairpin probe, washing with ethanol, centrifuging, and finally dispersing the CdTe/CdS-hairpin probe in a Tris-HCl buffer solution for later use; b. and (b) transferring a proper amount of prepared AuNPs into the CdTe/CdS-hairpin probe dispersion liquid prepared in the step (a), adding a NaCl solution to adjust the concentration of sodium ions to a certain value, carrying out oscillation reaction for 24 hours at normal temperature, and then washing and centrifuging to obtain the CdTe/CdS-hairpin probe-AuNPs.

A method for detecting a fluorescence sensor of aflatoxin toxin-producing gene nor-1 comprises the steps of dividing standard liquid of the toxin-producing gene nor-1 into ten groups before detection, wherein each group is 20 mu L, the concentration is 0.05, 0.5, 5, 10, 20, 40, 50, 100, 150, 200nM, the spare CdTe/CdS-hairpin probe-AuNPs solution is divided into ten groups, each group is 0.1 mL, the photometer standard liquid of the ten groups of toxin-producing gene nor-1 is respectively added into the ten groups of CdTe/CdS-hairpin probe-AuNPs solution, Tris-HCl buffer solution with the pH value of 7.4 is adopted to fix the volume to 2 mL, the constant temperature shaking incubation is carried out for 40-60 min, preferably 50min, a compound of the CdTe/CdS-hairpin probe-AuNPs/nor-1 is formed, the fluorescence intensity of the solution is measured at 380nM by adopting fluorescence spectroscopy, and a standard curve of the fluorescence recovery intensity with nor-1 was established.

And detecting the actual sample of the mold produced by the aflatoxin by using the prepared fluorescent sensor, and determining whether the toxin producing strain contains a toxin producing gene of the aflatoxin. The detection process is as follows: adding 100 mu L of PCR amplification product containing 1%, 5% and 10% Aspergillus flavus toxigenic bacteria DNA extract into 400 mu L of prepared CdTe/CdS-hairpin probe-AuNPs dispersion, adding NaCl to adjust the sodium ion concentration to 2M, continuously incubating for 50min, measuring the fluorescence recovery intensity of a fluorescence sensor by using a fluorescence spectrophotometer, and determining whether the toxigenic strain contains the toxigenic gene of the aflatoxin through regression calculation of a standard curve. The method can be used for detecting the toxin-producing gene of the aflatoxin from the source, is rapid in detection and is suitable for rapid detection and analysis.