CN109852389B

CN109852389B - Preparation method and application of fluorescence sensor for detecting ochratoxin A

Info

Publication number: CN109852389B
Application number: CN201910053705.2A
Authority: CN
Inventors: 吴永江; 刘丽; 蒋可秋; 栾连军; 刘雪松
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2019-01-21
Filing date: 2019-01-21
Publication date: 2020-11-13
Anticipated expiration: 2039-01-21
Also published as: CN109852389A

Abstract

The invention provides a preparation method and application of a fluorescence sensor for detecting ochratoxin A. The method adopts water-soluble ZnCdSe quantum dots as fluorescent probes, and self-assembled nano-porphyrin prepared from a tetra- (4-pyridyl) zinc porphyrin N, N-dimethylformamide solution and dodecyl dimethyl betaine as a quantum dot fluorescence quencher to form the quantum dot-nano-porphyrin fluorescence 'on-off' sensor. The OTA is added to combine with the nano porphyrin to release the quantum dots, so that the fluorescence of the quantum dots is recovered, and the quantum dot-nano porphyrin fluorescence 'on-off-on' sensor is formed. The invention realizes the high-selectivity detection of OTA, effectively amplifies signals and obtains a detection result with high sensitivity and strong specificity. The method does not need to rely on expensive large-scale instruments, and has simple pretreatment and quick detection.

Description

Preparation method and application of fluorescence sensor for detecting ochratoxin A

Technical Field

The invention belongs to the technical field of nano material preparation and food safety detection, and particularly relates to a preparation method and application of a fluorescence sensor for detecting ochratoxin A.

Background

Ochratoxins belong to the class of mycotoxins, which are secondary metabolites of aspergillus and penicillium. The ochratoxin types comprise ochratoxin A (OTA), ochratoxin B, ochratoxin C and ochratoxin D, wherein the ochratoxin A has the characteristics of highest toxicity, widest distribution, highest toxicity yield, highest pollution to agricultural products and closest relationship with human health. OTA consists of a dihydroisocoumarin chloride moiety linked to an L- β -phenylalanine molecule through seven carboxyl groups via an amide bond. OTA has strong nephrotoxicity, hepatotoxicity, immunotoxicity, and potentially carcinogenic, teratogenic, and mutagenic properties. OTA-producing molds are widely distributed in nature, resulting in widespread distribution of OTA in various foods and feeds. Furthermore, its high chemical stability to hydrolysis and heat treatment during food processing makes it the most dangerous toxin for humans. Therefore, it is of utmost importance that OTA is rapidly and sensitively quantitatively analyzed and detected in food safety.

Traditional methods use different techniques such as thin layer chromatography, enzyme linked immunosorbent, high performance liquid chromatography, ultra high performance liquid chromatography and gas chromatography to detect OTA. Although they have high sensitivity and accuracy, these techniques are laborious, expensive, time consuming and require high precision instruments or sample preparation, limiting their practical application. The novel detection mode based on quantum dot-porphyrin fluorescence 'on-off-on' has been successfully applied to detection of cyanide, melamine, amino acid, biological thiol and DNA due to excellent selectivity and high sensitivity.

Therefore, it is urgently needed to develop a simple and rapid analysis method to realize the detection of the OTA with high sensitivity and strong specificity.

Disclosure of Invention

The invention aims to provide a preparation method of a fluorescence sensor for detecting ochratoxin A, which is realized by the following steps:

(1) synthesis of ZnCdSe quantum dot fluorescent probe

Weighing zinc dichloride and N-acetyl-L-cysteine, dissolving in ultrapure water, stirring for 10-20 minutes at low temperature and normal pressure, and adjusting the pH to 9-10 by using a sodium hydroxide solution. And (3) after the pH value is adjusted, filling nitrogen into the cadmium dichloride at normal pressure and low temperature, and reacting for 10-20 minutes. And injecting sodium hydrogen selenide, performing ice bath, and filling nitrogen to react for 10-20 minutes. And after the reaction is finished, transferring the solution into a reaction kettle, putting the reaction kettle into an oven, and reacting for 50-70 minutes at 200 ℃. After the reaction is finished, taking out the reaction product and cooling the reaction product to room temperature to obtain a green light ZnCdSe quantum dot fluorescent probe with the emission wavelength of 510-520 nm;

(2) synthesis of tetra- (4-pyridyl) zinc porphyrin nanorod self-assembly solution

Tetra- (4-pyridyl) zinc porphyrin was dissolved in N, N-Dimethylformamide (DMF) to prepare a solution having a concentration of 3.3X 10^-4～7.3×10^-6mol/L stock solution. Adding 1-3 mL of DMF solution of tetra- (4-pyridyl) zinc porphyrin into 30-50 mL of ultrapure water, and finally adding the DMF solution with the concentration of 1.5 multiplied by 10^-2～2.6×10^-2Stirring the dodecyl dimethyl betaine with mol/L for 10-20 minutes at normal temperature and normal pressure to form a very stable green transparent colloidal solution. When the DMF solution of porphyrin is dissolved in water, porphyrin molecules cannot be dissolved in a water phase and are separated out from the DMF phase, and under the strong pi-pi stacking effect and the hydrophobic effect among porphin rings, the porphyrin molecules spontaneously form J-aggregates and H-aggregates in the micelle structure of dodecyl dimethyl betaine, so that the ordered and stable nanorod structure is formed. Preparing the tetra- (4-pyridyl) zinc porphyrin nanorod self-assembly solution.

(3) Synthesis of quantum dot-nano porphyrin 'on-off' fluorescent probe

Adding the self-assembly solution of the tetra- (4-pyridyl) zinc porphyrin nanorod into a ZnCdSe quantum dot fluorescent probe, adding a Tris-HCl buffer solution with the pH value of 8.0, and quenching the fluorescence of the quantum dot by the self-assembly solution of the tetra- (4-pyridyl) zinc porphyrin nanorod through the action of electron transfer and fluorescence resonance energy transfer.

(4) OTA with different concentrations is added into the quantum dot fluorescence quenching compound obtained in the step (3), the fluorescence of the quantum dots is recovered to different degrees, and the identification and quantification of the OTA are realized; thereby obtaining the quantum dot-nano porphyrin fluorescence 'on-off-on' sensor.

Or directly combining the steps (3) and (4), mixing OTA with different concentrations, the tetra- (4-pyridyl) zinc porphyrin nanorod self-assembly solution synthesized in the step (2) and a Tris-HCl buffer solution with the pH value of 8.0, and standing for 3-10 minutes; and (2) adding the ZnCdSe quantum dots synthesized in the step (1), and performing fluorescence spectrum measurement at 470-550 nm to obtain a fluorescence spectrum after the reaction is performed for 3-10 minutes.

Furthermore, the mass ratio of the substances of zinc dichloride, N-acetyl-L-cysteine and sodium hydroselenide in the quantum dot probe in the step (1) is 1:2: 0.1-1: 5:0.1, and the emission wavelength of the ZnCdSe quantum dot fluorescent probe in the step (1) is preferably 515 nm.

Furthermore, in the invention, the mass ratio of the tetra- (4-pyridyl) zinc porphyrin and the dodecyl dimethyl betaine in the mixed solution in the step (2) is 3: 50-4: 50.

Furthermore, the mass ratio of the tetra- (4-pyridyl) zinc porphyrin to the ZnCdSe quantum dot fluorescent probe in the tetra- (4-pyridyl) zinc porphyrin nanorod in the step (3) is 25: 1-28: 1.

Furthermore, the concentration of the tetra- (4-pyridyl) zinc porphyrin nanorod in the step (3) of the invention is 7.9 multiplied by 10^-8～4.75×10^-7The concentration of the mol/L and ZnCdSe quantum dots is 3.52 multiplied by 10^-9～2.27×10^-8At mol/L, the fluorescence intensity of the tetra- (4-pyridyl) zinc porphyrin nanorod and the ZnCdSe quantum dot form a good linear relationship.

Furthermore, the quantum dot-nano porphyrin fluorescence sensor is a compound obtained by specific combination of nano porphyrin and quantum dots. The fluorescence intensity is reduced from 870-890 nm to 380-400 nm.

It is another object of the present invention to provide use of the fluorescence sensor for detecting ochratoxin a. The fluorescent sensor provided by the invention has strong OTA detection capability. After OTA (0.5-80 ng/mL) is combined with the quantum dot-nano porphyrin fluorescence 'on-off-on' sensor, the fluorescence intensity of the quantum dot is enhanced along with the increase of the OTA concentration, and a good linear relation (R) is formed²0.995). The reason is that the binding capacity of OTA and nano porphyrin is stronger than the weak electrostatic interaction between quantum dots and nano porphyrin, so that the fluorescence of the quantum dots is released.

The invention provides a novel method for quantitatively detecting OTA by using a fluorescent sensor with high speed, strong specificity and high sensitivity, namely a fluorescent sensor for detecting ochratoxin A and a preparation method thereofThe method is carried out. The fluorescent sensor based on the fluorescent 'on-off-on' mode method provided by the invention can be used for identifying quantitative OTA (over the air technology) in milk and coffee matrixes with high sensitivity and strong specificity. The invention is characterized in that: (1) the novel fluorescence sensor is high in sensitivity, the high-concentration tetra- (4-pyridyl) zinc porphyrin nanorod can effectively quench ZnCdSe quantum dot fluorescence, and when the added concentration is large enough, the quantum dot fluorescence can even be completely quenched. The invention optimizes the concentration of the tetra- (4-pyridyl) zinc porphyrin nano rod, and the concentration is selected to be 3.96 multiplied by 10^- ⁷And the mol/L nano porphyrin is used as a quencher, so that trace detection of OTA is realized. (2) The novel fluorescence sensor has strong specificity. The fluorescence intensity of the combined sensor is unchanged after the Zearalenone (ZEN) with the concentration of 60ng/mL, the Deoxynivalenol (DON) and the trichothecene toxoid (T-2 and HT-2) are combined with the sensor. (3) The novel fluorescent sensor has good stability. Adding 1X 10 concentration into the system^-4mol/mL metallic ion Mg²⁺,Zn²⁺,Na⁺,Ca²⁺,K⁺Then, the fluorescence recovery intensity of the OTA to the quantum dot-nano porphyrin is almost unchanged. (4) The novel fluorescent sensor has high response speed to OTA, and can finish detection within five minutes. (5) The invention uses the novel fluorescent sensor to carry out OTA detection, and can realize high sensitivity and strong specificity detection of OTA in milk and coffee.

Drawings

FIG. 1 is a schematic view of a method for preparing a novel fluorescence sensor for detecting ochratoxin A according to the present invention.

Fig. 2 is an ultraviolet-visible spectrum of a tetra- (4-pyridyl) zinc porphyrin self-assembly solution in the novel fluorescence sensor for detecting ochratoxin a of the present invention, with the abscissa as wavelength and the ordinate as absorbance.

FIG. 3 is a transmission electron microscope photograph of a tetra- (4-pyridyl) zinc porphyrin self-assembly solution in the novel fluorescent sensor for detecting ochratoxin A of the present invention, which is a nanorod.

FIG. 4 is a scanning electron microscope photograph of a tetra- (4-pyridyl) zinc porphyrin self-assembly solution in the novel fluorescent sensor for detecting ochratoxin A of the present invention, which is a nanorod.

FIG. 5 is a transmission electron microscope image of a complex formed by a tetra- (4-pyridyl) zinc porphyrin self-assembly solution and ZnCdSe quantum dots in the novel fluorescence sensor for detecting ochratoxin A of the present invention.

FIG. 6 is a fluorescence spectrum before and after the ZnCdSe quantum dot and the tetra- (4-pyridyl) zinc porphyrin self-assembly solution are specifically combined, wherein the abscissa is wavelength and the ordinate is fluorescence intensity.

FIG. 7 is a fluorescence recovery spectrogram of the novel fluorescence sensor for detecting ochratoxin A after the novel fluorescence sensor is acted with OTAs (0.5-80 ng/mL) with different concentrations, wherein the abscissa is wavelength and the ordinate is fluorescence intensity.

FIG. 8 is a linear correlation diagram of the novel fluorescence sensor for detecting ochratoxin A of the invention after the novel fluorescence sensor and ochratoxin A (0.5-80 ng/mL) with different concentrations act, wherein the abscissa is the concentration of ochratoxin A, and the ordinate is fluorescence recovery intensity (F)₂) And original fluorescence intensity (F) of ZnCdSe quantum dot₀) The ratio of (a) to (b).

FIG. 9 shows the stability of the novel fluorescence sensor for detecting ochratoxin A of the present invention. At the addition concentration of 1 × 10^-4mol/mL metallic ion Mg²⁺,Zn²⁺,Na⁺,Ca²⁺,K⁺Stability after the action in the case of (3). The abscissa is the added interfering substance and the ordinate is the fluorescence recovery intensity (F)₂) And original fluorescence intensity (F) of ZnCdSe quantum dot₀) The ratio of (a) to (b).

FIG. 10 shows the specificity of the novel fluorescence sensor for detecting ochratoxin A of the present invention. The abscissa is OTA, Zearalenone (ZEN), Deoxynivalenol (DON), and trichothecene toxoid (T-2, HT-2), and the ordinate is fluorescence recovery intensity (F)₂) And original fluorescence intensity (F) of ZnCdSe quantum dot₀) The ratio of (a) to (b).

Detailed Description

The invention will be further described with reference to the following figures and examples, but the scope of the invention is not limited thereto.

Material sources for the following examples:

tetra- (4-pyridyl) porphyrin was purchased from carbofuran technologies ltd, purity 97%;

the dodecyl dimethyl betaine is purchased from Shandong Yousio chemical engineering Co., Ltd, and the content is 35%;

n-acetyl-cysteine was purchased from Shanghai leaf Biotech limited and has a purity of 99%;

n, N-dimethylformamide and acetonitrile are purchased from Yongda chemical reagent limited company of Tianjin and are analytically pure;

zinc chloride was purchased from Shirong scientific Co., Ltd with a purity of 98%;

the selenium powder is purchased from Shanghai Aladdin Biotechnology, Inc., and has a purity of 99.99%;

the cadmium chloride is purchased from Shanghai Aladdin Biotechnology, Inc., and has a purity of 98%;

HT-2 toxin (HT-2), T-2 toxin (T-2), Zearalenone (ZEN), OTA, and Deoxynivalenol (DON) standards were purchased from Romer International trade, Beijing, Inc.;

milk was purchased from the Hangzhou century Lianhua supermarket, and coffee was purchased from the Singapore supermarket;

all fluorescence experiments were performed on a Hitachi F-2700 fluorometer.

Example 1

A fluorescence sensor for detecting ochratoxin A is mainly prepared by the following four steps (see figure 1):

(1) first, synthesis of ZnCdSe quantum dots

Zinc chloride (0.035g, 0.25mmol) and N-acetyl-L-cysteine (0.1253g,0.76mmol) were dissolved in 40mL of ultrapure water and stirred at normal pressure and low temperature (5 ℃ C.) for 20 minutes. After stirring, the pH was adjusted to 9.7 with sodium hydroxide solution (0.1 mol/L). After the pH was adjusted, CdCdTe (0.58mg,0.0025mmol) was added, followed by stirring in a nitrogen ice bath for 15 minutes. Sodium hydroselenide (0.0026g, 0.025mmol) was injected and stirred in a nitrogen bath for 15 minutes. After the reaction was completed, the solution was transferred to a reaction vessel and oven-reacted at 200 ℃ for 65 minutes. The concentration obtained was 4.2X 10^-7mol/L ZnCdSe quantum dot fluorescent probeNeedle, emission wavelength 515 nm.

(2) Second step of synthesizing tetra- (4-pyridyl) zinc porphyrin nano-rod self-assembly solution

Tetra- (4-pyridyl) zinc porphyrin was dissolved in N, N-Dimethylformamide (DMF) to prepare a solution having a concentration of 7.3X 10^- ⁵mol/L stock solution. To 42mL of ultrapure water, 1mL of a DMF solution of tetrakis- (4-pyridyl) zinc porphyrin was added, and the resulting solution was added to a concentration of 2.6X 10^-2The dodecyl dimethyl betaine in mol/L is stirred for 10 minutes at normal temperature and normal pressure to form a very stable green transparent colloidal solution. When the DMF solution of porphyrin is dissolved in water, porphyrin molecules cannot be dissolved in a water phase and are separated out from the DMF phase, and under the strong pi-pi stacking effect and the hydrophobic effect among porphin rings, the porphyrin molecules spontaneously form J-aggregates and H-aggregates in the micelle structure of dodecyl dimethyl betaine to form an ordered and stable nanorod structure. As shown in FIG. 2, the porphyrin self-assembly occurs, and the porphyrin monomer has a strong absorption peak at 424nm, called soret peak (B band), and a weak peak at 557nm (Q band). For the nano-porphyrins, the B band split from a single peak at 424nm to broad peaks at 425nm and 470nm, the intensity of the Q-band was greatly enhanced compared to the monomer, and there was a significant red shift, indicating successful assembly of the porphyrins into nano-porphyrins by the surfactant. As shown in fig. 3 and 4, the synthesized nano porphyrin is in an ordered nano rod-like structure.

(3) And thirdly, preparing the quantum dot-nano porphyrin fluorescence 'on-off' sensor, namely adding 70uL of ZnCdSe quantum dots synthesized in the step (1), the tetra- (4-pyridyl) zinc porphyrin self-assembly solution synthesized in the step of 250uL and a Tris-HCl buffer solution with the pH value of 680uLpH being 8 into a 1.4mL cuvette, and standing for 5 minutes. Fluorescence emission spectrum measurement was performed at 470-550 nm, and the emission spectrum after 5 minutes was measured. As shown in FIGS. 5 and 6, the transmission electron microscope images show that the quantum dots are combined on the surface of the nano porphyrin, and the tetra- (4-pyridyl) zinc porphyrin nanorod self-assembly solution quenches the fluorescence of the quantum dots through the electron transfer and fluorescence resonance energy transfer effects.

(4) Fourthly, establishing a standard curve for restoring the fluorescence of the quantum dots by the interaction of the OTA and the nano porphyrin, which comprises the following steps: adding 100uL of OTA aqueous solution with different concentrations, the tetra- (4-pyridyl) zinc porphyrin self-assembly solution synthesized in the 250uL step and the Tris-HCl buffer solution with the pH value of 580 uL8 into a 1.4mL cuvette, standing for 5 minutes, adding 70uL of ZnCdSe quantum dots synthesized in the step (1), performing fluorescence emission spectrum measurement at 470-550 nm, measuring the emission spectrum after 5 minutes, and performing parallel measurement for three times at each concentration.

As a result:

(1) as shown in FIG. 7, after OTA (0.5-80 ng/mL) is combined with nano porphyrin, the fluorescence intensity of the quantum dot is enhanced along with the increase of the concentration of the OTA. As shown in figure 8, the sensor has good linear relation (R) to OTA in the range of OTA concentration of 0.5-80 ng/mL²＝0.995)。

(2) Anti-interference evaluation of the sensor detection OTA: the sensor prepared by the invention has good anti-interference capability, and the concentration is 1 multiplied by 10 as shown in figure 9^-4mol/mL metallic ion Mg²⁺,Zn²⁺,Na⁺,Ca²⁺And K⁺The method has no obvious influence on OTA spectral signals and no obvious interference in detection.

(3) Selective evaluation of the sensors: in order to examine the selectivity of the novel fluorescence sensor, as shown in fig. 10, Zearalenone (ZEN), Deoxynivalenol (DON) and trichothecene toxins (T-2 and HT-2) with the concentration of 60ng/mL were added to the system, the fluorescence of the quantum dot-nano porphyrin did not change significantly, and after OTA was added, the fluorescence of the system was recovered significantly, indicating that the fluorescence sensor has good selectivity.

Example 2A novel fluorescence sensor for determining OTA in milk

Preparation of sample to be tested

1.5mL of milk was taken, OTA standard was added, and acetonitrile was further added to make the total volume 5mL, to prepare a sample having a concentration of (2,6,30, 60 ng/mL).

Synthesis of (di) ZnCdSe Quantum dot fluorescent Probe, same as example 1

Synthesis of self-assembled solution of (tri) tetrakis- (4-pyridyl) zinc porphyrin nanorod, as in example 1

(IV) detection of OTA in milk samples

Adding 100uL of milk samples containing OTA with different concentrations, a tetra- (4-pyridyl) zinc porphyrin self-assembly solution synthesized in the 250uL step and a Tris-HCl buffer solution with the pH value of 580uLpH being 8 into a 1.4mL cuvette, standing for 5 minutes, adding 70uL of ZnCdSe quantum dots synthesized in the step (1), performing fluorescence emission spectrum measurement at 470-550 nm, measuring the emission spectrum after 5 minutes, and performing parallel measurement for three times at each concentration.

And (V) verifying the recovery rate of the detection method: and (3) inspecting the recovery rate of the method by adopting a matrix standard adding method, taking a blank milk sample, and respectively adding four OTA standard substances with concentration levels (2,6,30 and 60ng/mL), wherein the recovery rate result is 99.4-106.6%. The fluorescence sensor prepared by the invention has high accuracy and can be used for detecting and analyzing OTA in milk samples.

TABLE 1 detection of OTA in milk samples according to the invention

Sample (I)	Addition concentration (ng/mL)	Detection (ng/mL)	Recovery (%)	RSD(％)
					Milk	2	2.06	102.8	2.75
	6	5.96	99.4	1.57
						30	31.98	106.6	1.59
	60	60.89	101.5	2.86

Example 3A novel fluorescence sensor for determining OTA in coffee

Preparation of sample to be tested

Coffee 0.5g was weighed, mycotoxin standard added, and acetonitrile added to make the total volume 5mL, to prepare a (2,6,30, 60ng/mL) sample.

Synthesis of (di) ZnCdSe Quantum dot fluorescent Probe, same as example 1

(IV) detection of OTA in coffee samples

100uL of coffee samples containing OTA with different concentrations, a 250uL of the tetra- (4-pyridyl) zinc porphyrin self-assembly solution synthesized in the step and a 580 uLTris-HCl buffer solution with the pH value of 8 are added into a 1.4mL cuvette, the cuvette is kept still for 5 minutes, 70uL of the ZnCdSe quantum dots synthesized in the step (1) are added, fluorescence emission spectrum measurement is carried out at 470-550 nm, the emission spectrum after 5 minutes is measured, and each concentration is measured in parallel three times.

And (V) verifying the recovery rate of the detection method: and (3) inspecting the recovery rate of the method by adopting a matrix standard adding method, taking a blank coffee sample, and respectively adding four OTA standard substances with concentration levels (2,6,30 and 60ng/mL), wherein the recovery rate result is between 98.5 and 102.5 percent. The fluorescent sensor prepared by the invention has high accuracy and can be used for detecting and analyzing OTA in coffee samples.

Table 2 shows detection of OTA in coffee samples of the invention

Sample (I)	Addition concentration (ng/mL)	Detection (ng/mL)	Recovery (%)	RSD(％)
					Coffee	2	2.05	102.5	2.47
	6	6.14	102.3	4.83
						30	29.6	98.8	1.44
	60	57.92	98.5	2.54

In conclusion, the fluorescence sensor for detecting ochratoxin A, which is prepared by the invention, can effectively amplify signals and can realize detection of trace mycotoxins in foods such as milk and coffee. And the preparation method is simple, the detection time is short, the anti-interference capability is strong, the sensitivity is high, and the selectivity is good. Compared with the existing detection method, the method greatly saves cost, is quick in detection and improves detection efficiency.

The scope of protection of the invention is not limited to the description of the embodiments, and modifications that do not depart from the center of the invention are intended to be included within the scope of the invention.

Claims

1. The application of a fluorescent sensor in detection of ochratoxin A is characterized in that the fluorescent sensor is prepared by the following steps:

(1) synthesis of ZnCdSe quantum dot fluorescent probe

Weighing zinc dichloride and N-acetyl-L-cysteine, dissolving in ultrapure water, stirring at low temperature and normal pressure, adjusting the pH value to 9-10 by using a sodium hydroxide solution, adding cadmium dichloride, filling nitrogen at normal pressure and low temperature for reaction for 10-20 minutes, then injecting sodium selenhydride, carrying out ice bath, filling nitrogen for reaction for 10-20 minutes, transferring the solution into a reaction kettle after the reaction is finished, putting the reaction kettle into an oven, reacting at 200 ℃ for 50-70 minutes, taking out and cooling to room temperature after the reaction is finished, and obtaining a green light ZnCdSe quantum dot fluorescent probe with the emission wavelength of 515 nm; the mass ratio of the zinc dichloride, the N-acetyl-L-cysteine and the sodium hydroselenide in the quantum dot probe is 1 (2-5) to 0.1;

Dissolving tetra- (4-pyridyl) zinc porphyrin in N, N-dimethylformamide to obtain a solution with a concentration of 3.3 × 10^-4~7.3×10^- ⁶Adding 1-3 mL of N, N-dimethylformamide solution of tetra- (4-pyridyl) zinc porphyrin into 30-50 mL of ultrapure water, and finally adding 1.5 multiplied by 10^-2~2.6×10^-2Stirring the dodecyl dimethyl betaine at normal temperature and pressure for 10-20 minutes to form a very stable green transparent colloidal solution, wherein when the N, N-dimethylformamide solution of porphyrin is dissolved in water, porphyrin molecules cannot be dissolved in a water phase and are separated out from a DMF (dimethyl formamide) phase, and under the strong pi-pi stacking effect and hydrophobic effect among porphin rings, the porphyrin molecules spontaneously form J-aggregates and H-aggregates in the micelle structure of the dodecyl dimethyl betaine to form an ordered and stable nanorod structure, so that the tetra- (4-pyridyl) zinc porphyrin nanorod self-assembly solution is prepared; wherein the mass ratio of substances in the mixed solution of tetra- (4-pyridyl) zinc porphyrin and dodecyl dimethyl betaine is 3: 50-4: 50;

(3) synthesis of quantum dot-nano porphyrin 'on-off' fluorescent probe

Adding the self-assembly solution of the tetra- (4-pyridyl) zinc porphyrin nanorod into a ZnCdSe quantum dot fluorescent probe, adding a Tris-HCl buffer solution with the pH =8.0, and quenching the fluorescence of the quantum dot by the self-assembly solution of the tetra- (4-pyridyl) zinc porphyrin nanorod under the action of electron transfer and fluorescence resonance energy transfer; wherein the mass ratio of the tetra- (4-pyridyl) zinc porphyrin to the ZnCdSe quantum dot fluorescent probe in the tetra- (4-pyridyl) zinc porphyrin nanorod is 25: 1-28: 1; when the concentration of the tetra- (4-pyridyl) zinc porphyrin nano rod is 7.9 multiplied by 10^-8~ 4.75×10^-7The concentration of the mol/L and ZnCdSe quantum dot fluorescent probe is 3.52 multiplied by 10^-9~2.27×10^-8At mol/L, the fluorescence intensity of the tetra- (4-pyridyl) zinc porphyrin nanorod and the ZnCdSe quantum dot form a good linear relationship.

2. The use according to claim 1, wherein after the preparation steps (1) and (2), ochratoxin A with different concentrations, the self-assembly solution of tetra- (4-pyridyl) zinc porphyrin nanorods synthesized in the step (2), and Tris-HCl buffer solution with pH =8.0 are mixed and left to stand for 3-10 minutes; and (2) adding the ZnCdSe quantum dots synthesized in the step (1), and performing fluorescence spectrum measurement at 470-550 nm to obtain a fluorescence spectrum after the reaction is performed for 3-10 minutes.