Detection method and application of OTA by a ratiometric fluorescence sensor based on CHA

Abstract

The present invention provides a detection method and application of a ratio-type fluorescence sensor for detecting OTA based on CHA. A ratio-type fluorescence sensor is constructed by using CDs and FAM as dual-emission fluorescence probes in combination with the CHA signal amplification technology. Finally, the ratio F FAM / F CDs is used to achieve the quantitative detection of ochratoxin A, with a linear range of 5.0 pg / mL - 3.0 ng / mL and a detection limit of 1.5 pg / mL. The ratio-type fluorescence sensor for detecting ochratoxin A in the present invention not only has a self-calibration function, can eliminate the interference of various variable or difficult-to-quantify factors such as probe concentration, polarity, temperature, and excitation intensity, broaden the response range, and effectively improve the accuracy and reliability of the detection results, but also does not involve enzymes such as proteases in the whole operation process, is less affected by the external environment, has high sensitivity, low requirements for operating conditions and operating costs, can achieve the accurate detection of ultra-trace OTA, and has the value of industrial application and popularization.

Description

Technical Field

[0001] The present invention relates to the field of biosensing technologies, and in particular to a detection method and application of detecting OTA by a ratio-type fluorescence sensor based on CHA. Background Art

[0002] Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium with immunotoxicity, teratogenicity and carcinogenic effects. It can contaminate various foods such as grains, rice, licorice and wine, and the threat to human health cannot be underestimated. Therefore, it is necessary to establish an accurate and sensitive analytical method for the detection of OTA in foods.

[0003] Fluorescence spectrometry has been widely used in the detection industry due to its advantages such as simplicity, sensitivity and convenient simultaneous detection. However, due to the limitations of low abundance of target substances, matrix effects and method sensitivity, conventional fluorescence biosensors are difficult to achieve accurate detection of ultra-trace components. Therefore, using signal amplification technology to improve the detection sensitivity is an important way to break through the application bottleneck of fluorescence biosensors. Currently, the reported enzyme-free signal amplification technologies mainly include hybridization chain reaction (HCR), catalytic hairpin assembly signal amplification technology (CHA), and DNA walkers, etc. Among them, CHA is an enzyme-free signal amplification technology established through the cyclic assembly of hairpin DNA. The primer strand cyclically catalyzes the assembly of hairpin substrates and is released after each cascade reaction for initiating the next cycle reaction. Therefore, fluorescence biosensors based on CHA signal amplification technology have been widely used in the detection of DNA, RNA, proteins, metal ions and small biological molecules.

[0004] Although signal amplification technology can improve the sensitivity of fluorescence sensors, the matrix interference and false positive problems of complex samples still limit the application of fluorescence sensors, and the accuracy of fluorescence sensors is restricted. For example, the patent "Patent No. CN 106370638 B" discloses a method for Hg 2+The detected colorimetric and fluorescence dual-signal biosensor and detection method construct a colorimetric-fluorescence dual-signal biosensor based on the catalytic hairpin assembly enzyme-free amplification technology. Although it has the advantages of the convenience of colorimetry and the high sensitivity of fluorescence method, there are also matrix interference of complex samples and false positive problems, and the detection results are prone to errors. The patent "Patent No. CN 114113264 A" discloses a dual-amplification detection method and application of tobramycin based on EXOⅢ-assisted strand circulation and CHA reaction. However, the protease used in inducing the CHA reaction in this sensor is susceptible to the environment, which greatly increases the operation condition requirements and costs of this method, and also has an adverse impact on the accuracy of the detection results, restricting the application of this method.

[0005] Meanwhile, current research on constructing biosensors for OTA detection is still in development. The patent "Patent No. CN109884009 A" discloses a detection method for detecting ochratoxin A by a target-mediated fluorescence ratio-type sensor, but its nucleic acid signal is weak and the sensitivity of the fluorescence sensor is limited. The patent "Patent No. CN 111424072 A" discloses an electrochemical biosensor for detecting ochratoxin A and its preparation method, but the detection limit can only reach 100 ng / mL, and it is difficult to achieve ultra-trace detection of ochratoxin A. Therefore, overcoming the defects of the above-mentioned existing technologies and providing a simple, convenient, fast and highly sensitive method for detecting OTA is an urgent problem to be solved in the current OTA detection application. Summary of the Invention

[0006] Aiming at the technical problems of poor sensitivity, poor precision and difficulty in ultra-trace detection when detecting OTA by existing detection methods, the present invention proposes a detection method and application of a ratio-type fluorescence sensor for detecting OTA based on CHA.

[0007] For the above purpose, the technical solution of the present invention is realized as follows:

[0008] The detection method of a ratio-type fluorescence sensor for detecting OTA based on CHA is as follows:

[0009] (1) Take 20 mL of chloroauric acid solution (HAuCl4·4H2O) with a concentration of 1 mmol / L in a 50 mL two-necked round-bottom flask, heat it to 120 °C, then quickly add 2 mL of sodium citrate solution with a concentration of 38.8 mmol / L, continue heating and stirring for 15 min, then cool and reflux to room temperature, and finally filter and collect the filtrate with a 0.45 μm nylon filter membrane to obtain an AuNP solution with an average particle size of 13 nm and a concentration of 11.5 nM of gold nanoparticles (AuNPs), which is stored in a refrigerator at 4 °C for later use. All glass instruments used in the preparation process need to be treated with aqua regia (V HCl :VHNO3 = 3: 1) Soak for 30 min and then rinse thoroughly with ultrapure water.

[0010] (2) Take 4 mL of the AuNPs solution from step (1), add 60 μL of the acetate buffer solution (pH: 5) of hairpin probe H1 (concentration 10 μM) thereto, and then add 10 μL of TCEP with a concentration of 20 mM (purchased from Sigma-Aldrich Co., Ltd. (Shanghai, China)). Incubate at room temperature for 16 h, and then slowly add a 1 M NaCl solution to the mixed solution within the next 44 h until the concentration of NaCl in the final solution is 0.1 M. Centrifuge the resulting solution three times at a speed of 15000 rpm, with each centrifugation time being 30 min, to remove the DNA that has not been modified onto the surface of AuNPs. Collect the centrifuged red sol to obtain the hairpin probe H1-modified AuNPs probe, i.e., H1-AuNPs.

[0011] (3) Mix 100 μL each of EDC and NHS with concentrations of 100 μM, add 5 mL of the CDs solution with a concentration of 1 mg / L thereto, stir at room temperature for 30 min to activate the carboxyl group, and then add 20 μL of the buffer solution of hairpin probe H2. Continue to stir at room temperature for 24 h to obtain the hairpin probe H2-modified CDs, i.e., H2-CDs, with a concentration of 100 μM, and store it in a refrigerator at 4 °C for later use.

[0012] (4) Take 10 μL each of the aptamer AP and the auxiliary probe HP with concentrations of 100 μM, hybridize at room temperature to obtain the initial reaction solution, then add the test sample thereto and incubate at room temperature for 40 min, then add 100 μL of the H1-AuNPs from step (2), react at room temperature for 45 min, then add 10 μL of the H2-CDs from step (3) and react at room temperature for 60 min. Finally, centrifuge the mixture to remove the unreacted solution, and dilute it to 500 μL to obtain the mixture to be detected. Detect the fluorescence of the mixture to be detected at an excitation wavelength of 360 nm.

[0013] (5) Substitute the ratio of the FAM fluorescence intensity and the CDs fluorescence intensity measured in step (4) into the linear equation F FAM / F CDs = 4.43 c + 0.46, and calculate to obtain the c value, that is, the concentration of OTA in the test sample. The c unit is ng / mL.

[0014] Further, the nucleotide sequence of the hairpin probe H1 in step (2) is as shown in SEQ ID NO: 1, with a thiol group modified at its 5' end and a FAM group modified at its 3' end.

[0015] Further, the nucleotide sequence of the hairpin probe H2 in step (3) is as shown in SEQ ID NO: 2, with an amino group modified at its 5' end.

[0016] Further, the nucleotide sequence of the aptamer AP in step (4) is as shown in SEQ ID NO: 3; the nucleotide sequence of the auxiliary probe HP is as shown in SEQ ID NO: 4.

[0017] Application of the above detection method of the catalytic hairpin assembly (CHA)-based ratiometric fluorescence sensor for detecting OTA in the detection of OTA. The linear range for detecting OTA is 5.0 pg / mL - 3.0 ng / mL, and the detection limit is 1.5 pg / mL.

[0018] Beneficial effects of the present invention:

[0019] The present invention provides a detection method and application of a CHA-based ratiometric fluorescence sensor for detecting OTA. By using the CHA signal amplification technology, the sensitivity of the sensor is improved. Meanwhile, enzymes such as proteases are not required, and the influence of the external environment is smaller, and the experimental conditions are less demanding. High accuracy of the sensor can be achieved through simple operations. At the same time, a ratiometric fluorescence sensor is constructed. By establishing an internal standard and obtaining the intensity ratio of the signal through dual-signal ratio processing for self-calibration, compared with single fluorescence probes and sensors, various variable or difficult-to-quantify factors such as the probe concentration, polarity, temperature, and excitation intensity of single fluorescence probes and sensors are effectively eliminated, false positive interference is avoided, the response range is wider, and the accuracy and reliability of the detection results are also significantly improved. The present invention constructs a CHA-based ratiometric fluorescence sensor for detecting ochratoxin A, with a linear range of 5.0 pg / mL - 3.0 ng / mL and a detection limit of up to 1.5 pg / mL, enabling accurate detection of ultra-trace OTA, and having excellent accuracy and sensitivity, simple operation, low cost, and good industrial application and promotion value. Description of the drawings

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0021] Figure 1Schematic diagram of the principle for detecting OTA by the ratio fluorescence sensor of the present invention.

[0022] Figure 2 Transmission electron micrograph (a) and hydrated radius distribution diagram (b) of AuNPs in Example 1.

[0023] Figure 3 Transmission electron micrograph (a) and hydrated radius distribution diagram (b) of CDs in Example 1.

[0024] Figure 4 UV-visible absorption spectra of H2 (a), CDs (b) and H2-CDs (c) in Example 1.

[0025] Figure 5 X-ray photoelectron spectrum of H2-CDs in Example 1.

[0026] Figure 6 For the influence analysis results of different time conditions on F FAM / F CDs in Example 1; wherein, a, the influence of the incubation time of OTA and the aptamer AP on F FAM / F CDs ; b, the influence of the hybridization time of H1 and the auxiliary probe HP on F FAM / F CDs ; c, the influence of the hybridization time of H1 and H2 on F FAM / F CDs ; the error bars are the average values of three measurements.

[0027] Figure 7 Agarose gel electrophoresis analysis results under different conditions in Example 2; wherein, lane a, DNA molecular weight standard; lane b, 2.0 μM H1; lane c, 2.0 μM H1 + 2.0 μM HP; lane d, 2.0 μM H1 + 2.0 μM HP + 2.0 μM H2.

[0028] Figure 8 Fluorescence spectra of the system under different conditions in Example 2; wherein, a, blank control; b, fluorescence spectrum of H1-FAM modified AuNPs and H2-CDs DNA probe after adding 300 pg / mL OTA; c, fluorescence spectrum of H1-FAM modified AuNPs and H2-CDs DNA probe after adding 1.0 ng / mL OTA.

[0029] Figure 9 Fluorescence emission spectra of different concentrations of OTA (a) and fluorescence emission spectra of different concentrations of OTA and F FAM / F CDsLinear relationship analysis (b); where the error bar is the average of three measurements.

[0030] Figure 10 For the selectivity analysis of the ratio-type fluorescence sensor in Example 1 of the implementation effect; where a is 0.02 ng / mL, b is 0.4 ng / mL, and c is 1 ng / mL.

[0031] Figure 11 For the practicality analysis of the ratio-type fluorescence sensor in Example 2 of the implementation effect; where the error bar is the average of three measurements. Specific implementation manners

[0032] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0033] The experimental methods used in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0034] In the embodiments of the present invention, the nucleotide sequence of the hairpin probe H1 is shown in SEQ ID NO: 1, and its 5' end is modified with a mercapto group and its 3' end is modified with a FAM group; the nucleotide sequence of the hairpin probe H2 is shown in SEQ ID NO: 2, and its 5' end is modified with an amino group; the nucleotide sequence of the aptamer AP is shown in SEQ ID NO: 3; the nucleotide sequence of the auxiliary probe HP is shown in SEQ ID NO: 4.

[0035] The synthesis and purification of the hairpin probe H1, hairpin probe H2, aptamer AP and auxiliary probe HP are all completed by Sangon Biotech Co., Ltd. (Shanghai, China), and are respectively diluted to a fixed concentration with a buffer solution: the hairpin probe H1 is diluted to 10 μM with an acetate buffer solution with a pH of 5.0, and the hairpin probe H2, aptamer AP and auxiliary probe HP are all diluted to 100 μM with Tris-HCl Buffer respectively.

[0036] The detection principle of the present invention is as Figure 1As shown in the figure, carbon quantum dots (CDs) and 6-carboxyfluorescein (FAM) are used as dual-emission fluorescent probes, and combined with catalytic hairpin assembly technology (CHA) to construct a ratiometric fluorescence sensor for the detection of ochratoxin A (OTA). When OTA is absent in the test sample, the catalytic hairpin assembly reaction cannot occur, and the fluorescence of the FAM (6-carboxyfluorescein)-labeled hairpin probe H1 (H1-FAM) is quenched by gold nanoparticles (AuNPs). The fluorescence signal of FAM in the system is very weak, while the signal of carbon quantum dots CDs modified with hairpin probe H2 (H2-CDs) is very strong. When OTA is present in the test sample, the aptamer AP specifically recognizes the target OTA and forms a complex, and then releases the auxiliary probe HP. The auxiliary probe HP initiates the CHA reaction. The auxiliary probe HP hybridizes with the hairpin probe H1, opening the hairpin structure of H1-FAM. Subsequently, the hairpin probe H2 binds to the exposed part of the hairpin probe H1 through a strand displacement reaction initiated by the fulcrum to form a double strand, and at the same time releases the auxiliary probe HP. The released auxiliary probe HP triggers the next round of reaction between the hairpin probe H1 and the hairpin probe H2, thus playing a role in signal amplification. As the reaction progresses, the hybridization of the hairpin probe H1 and the hairpin probe H2 causes fluorescence resonance energy transfer (FRET) to occur after the distance between FAM and CDs approaches, the fluorescence of FAM is significantly enhanced, and the fluorescence of CDs is weakened. Therefore, the quantitative detection of OTA can be achieved by the ratio (F FAM / F CDs ) of the fluorescence intensity of FAM and the fluorescence intensity of CDs.

[0037] Example 1

[0038] (1) Preparation of AuNPs

[0039] Take 20 mL of chloroauric acid solution (HAuCl4·4H2O) with a concentration of 1 mmol / L in a 50 mL two-neck round-bottom flask, heat it to 120 °C, and then quickly add 2 mL of sodium citrate solution with a concentration of 38.8 mmol / L. Continue heating and stirring for 15 min, then cool and reflux to room temperature. Finally, filter and collect the filtrate with a 0.45 μm nylon filter membrane to obtain an AuNPs solution with an average particle size of 13 nm and a concentration of 11.5 nM, which is stored in a refrigerator at 4 °C for later use.

[0040] All glass instruments used in the preparation process need to be soaked in aqua regia (V HCl : V HNO3 = 3:1) for 30 min, and then rinsed thoroughly with ultrapure water. The chloroauric acid and sodium citrate used are both purchased from Sigma-Aldrich Co., Ltd. (Shanghai, China).

[0041] The typical morphology of AuNPs was characterized by TEM (transmission electron microscopy), and the average particle size was 13 nm ( Figure 2 a); the hydrodynamic diameter of AuNPs was measured by DLS (dynamic light scattering), and the average hydrodynamic diameter was 15 nm ( Figure 2 b).

[0042] (2)Modification of AuNPs

[0043] Take 4 mL of the AuNPs solution in step (1), add 60 μL of the buffer solution of hairpin probe H1 and 10 μL of TCEP with a concentration of 20 mM (purchased from Sigma-Aldrich Co., Ltd. (Shanghai, China)) thereto, incubate at room temperature for 16 h, and then slowly add a 1 M NaCl solution to the mixed solution within the next 44 h until the concentration of NaCl in the final solution is 0.1 M. Centrifuge the obtained solution three times at a speed of 15000 rpm for 30 min each time to remove the DNA that has not been modified onto the surface of AuNPs. Collect the centrifuged red sol to obtain the hairpin probe H1-modified AuNPs probe, namely H1-AuNPs.

[0044] (3)Preparation of hairpin probe H2-modified CDs

[0045] Take 100 μL each of EDC and NHS with a concentration of 100 μM and mix them. Add 5 mL of a CDs solution with a concentration of 1 mg / L (purchased from Sigma-Aldrich Co., Ltd. (Shanghai, China)) thereto, stir at room temperature for 30 min to activate the carboxyl group, and then add 20 μL of the buffer solution of hairpin probe H2. Continue to stir at room temperature for 24 h to obtain the hairpin probe H2-modified CDs, namely H2-CDs, with a concentration of 100 μM, and store them in a refrigerator at 4℃ for later use.

[0046] As Figure 3 a, it can be observed by TEM (transmission electron microscopy) that the average radius of CDs is 4 nm, the particle size is uniform, and the dispersibility is good. As Figure 3 b, the hydrodynamic radius of CDs can be measured by DLS to be 8 nm.

[0047] The ultraviolet absorption properties of H2-CDs were studied by ultraviolet-visible absorption spectroscopy (UV-Vis): Due to the π→π * transition of the C=C bond of CDs, CDs have a characteristic ultraviolet-visible absorption peak at 340 nm ( Figure 4 , curve b); compared with the ultraviolet-visible absorption spectrum of CDs, in addition to the absorption peak at 340 nm, H2-CDs also show a characteristic absorption peak of DNA at 260 nm ( Figure 4, curve c), indicating that the hairpin probe H2 has been successfully self-assembled on the surface of CDs. Figure 5 The X-ray photoelectron spectroscopy of H2-CDs shows obvious absorption peaks of C1s, N1s, O1s, P2s and P2p at 284.8 eV, 400.8 eV, 541.3 eV, 200.8 eV and 168.8 eV, respectively. The results further verify that the hairpin probe H2 has been successfully attached to the surface of CDs.

[0048] (4) Fluorescence detection of OTA

[0049] a. Condition optimization:

[0050] The incubation time of OTA with the aptamer AP, the hybridization time of the hairpin probe H1 and the auxiliary probe HP, and the hybridization time of the hairpin probe H1 and the hairpin probe H2 will directly affect the ratio of the FAM fluorescence intensity to the CDs fluorescence intensity (F FAM / F CDs ). Therefore, they were investigated separately. As Figure 6 a, the effect of the incubation time of OTA with the aptamer AP on F FAM / F CDs was studied. When the incubation time varied in the range of 0 - 40 min, F FAM / F CDs gradually increased with the increase of the reaction time. When the reaction time was 40 min, F FAM / F CDs reached the maximum and remained unchanged in the range of 40 - 75 min. As Figure 6 b, the effect of the hybridization time of the hairpin probe H1 and the auxiliary probe HP on F FAM / F CDs was studied. F FAM / F CDs gradually increased with the increase of the hybridization time in the range of 0 - 45 min. Continuing to increase the reaction time, F FAM / F CDs remained unchanged. As Figure 6 c, the effect of the hybridization time of the hairpin probe H1 and the hairpin probe H2 on F FAM / F CDs was studied. When the hybridization time of the hairpin probe H1 and the hairpin probe H2 was in the range of 0 - 60 min, F FAM / F CDs increased with the extension of the hybridization time and reached the highest value at 60 min. In summary, the final optimal conditions were selected as follows: incubating OTA with the aptamer AP for 40 min, hybridizing the hairpin probe H1 and the hairpin probe H2 for 60 min, and hybridizing the hairpin probe H1 and the auxiliary probe HP for 45 min.

[0051] b. Detection method:

[0052] Take 10 μL of aptamer AP and helper probe HP with a concentration of 100 μM each, hybridize them at room temperature to obtain the initial reaction solution, then add the sample to be tested and incubate at room temperature for 40 min. Then add 100 μL of H1-AuNPs from step (2) and react at room temperature for 45 min. Next, add 10 μL of H2-CDs from step (3) and react at room temperature for 60 min. Finally, centrifuge the mixture to remove the unreacted solution, and dilute it to 500 μL to obtain the mixture to be detected. Detect the fluorescence of the mixture to be detected at an excitation wavelength of 360 nm.

[0053] Feasibility verification of Example 2

[0054] To verify the feasibility of the detection method of the present invention, agarose gel electrophoresis of the system under different conditions was carried out. As Figure 7 shown, the number of bases of H1 is 33 nt (band b). When the hairpin probe H1 is incubated with the helper probe HP and hybridizes to form a double strand, the migration speed of the double strand is slower than that of the hairpin probe H1 (band c). However, adding the hairpin probe H2 triggers a toehold-mediated strand displacement reaction, and the number of bases of the formed H1:H2 double strand increases, and the migration speed is slower. Therefore, a band appears at the position of 66 bp (band d), indicating that the hybridization between the hairpin probe H1 and the hairpin probe H2 is completed.

[0055] To further verify the feasibility of the detection method of the present invention, the fluorescence spectra of the system under different conditions were studied. As Figure 1 shown, the maximum emission wavelength of CDs is 440 nm, the maximum excitation wavelength of FAM is 485 nm, and the emission spectrum of CDs and the absorption spectrum of FAM partially overlap. When the two are close, fluorescence energy transfer will occur, in the FRET-ON state, the fluorescence of CDs decreases, and the fluorescence of FAM increases. As Figure 8 shown, when there is no OTA, the catalytic hairpin self-assembly reaction cannot occur, in the FRET off state (curve a), and only the characteristic fluorescence absorption peak of CDs appears at 340 nm. However, when 0.5 ng / mL OTA is added, the aptamer AP specifically recognizes OTA and releases the helper probe HP. The helper probe HP triggers the CHA reaction, and the hybridization of the hairpin probe H1 and the hairpin probe H2 causes fluorescence energy transfer when FAM and CDs are close, in the FRET on state, and obvious fluorescence absorption peaks can be observed at 520 nm and 340 nm (curve b). Continuing to increase the OTA concentration to 1.0 ng / mL, the fluorescence at 340 nm weakens, and the fluorescence at 520 nm increases (Curve c). It shows that the fluorescence energy transfer is concentration-dependent on OTA, providing an effective premise for the quantitative detection of OTA.

[0056] Example 3 Fluorescence Detection Performance Analysis

[0057] Under the optimal experimental conditions, the fluorescence intensity changes of FAM and CDs in OTA solutions with different concentrations were measured respectively. As Figure 9 shown in Fig. a, in the range of OTA concentration from 5.0 pg / mL to 5.0 ng / mL, the fluorescence intensity of FAM gradually increased, while the fluorescence intensity of CDs gradually decreased. As Figure 9 shown in Fig. b, the ratio of F FAM / F CDs showed a good linear relationship with the OTA concentration in the range of 5.0 pg / mL - 3.0 ng / mL. The linear equation was F FAM / F CDs = 4.43 c + 0.46( c : ng / mL), and the linear correlation coefficient R 2 = 0.9975. The lowest detection limit was 1.5 pg / mL, which was much lower than the limit index of OTA in the national food safety standard. Therefore, the ratiometric fluorescence sensor proposed in this patent fully meets the detection requirements of OTA.

[0058] Example of Implementation Effect 1 Selective Detection Experiment of OTA

[0059] To explore the selectivity of the ratiometric fluorescence sensor, a reaction system was constructed for detection according to the operation method of Example 1. Except for the different substances to be detected added to each reaction system, other experimental operations were the same. The substances to be detected were ultrapure water (Blank), aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), fumonisin B1 (FB1), and ochratoxin A (OTA). Three concentration levels of 0.02 ng / mL, 0.4 ng / mL, and 1 ng / mL were set for each substance to be detected to construct the reaction system, and then the changes in fluorescence signals were measured in the same way. The selective detection results of the ratiometric fluorescence sensor are shown in Figure 10 Fig. It can be seen that only the addition of OTA would cause a change in the signal intensity, while the addition of interfering substances such as AFB1, AFB2, AFG1, and FB1 had basically no effect on the signal intensity of the system, indicating that the ratiometric fluorescence sensor had good selectivity.

[0060] Among them, aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), fumonisin B1 (FB1), and ochratoxin A (OTA) used were all purchased from Aladdin Biotechnology Co., Ltd. (Beijing, China).

[0061] Example of Implementation Effect 2 Analysis of Actual Samples

[0062] To verify the practicability of the ratiometric fluorescence sensor of the present invention in OTA detection, rice purchased from a local supermarket was used as a sample. A certain amount of OTA was added to the rice sample. Methanol and water were mixed at a volume ratio of 75:25. An aliquot of the rice sample was extracted and vigorously stirred at room temperature for 30 min. After standing for stratification, the supernatant of the sample was collected. The operations of stirring, standing, and collecting the supernatant were repeated until the OTA was completely dissolved. All the supernatants were combined, and then the OTA detection was carried out according to the detection method of the present invention. At the same time, enzyme-linked immunosorbent assay (ELISA) was used as a control to detect the same concentration of OTA. As Figure 11 shown, the detection results of OTA measured by the experimental method of the present invention are consistent with those of the ELISA method, indicating that the ratiometric fluorescence sensor of the present invention has a certain accuracy and can be applied to the analysis of actual samples.

[0063] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Detection method of OTA by CHA-based ratiometric fluorescence sensor, characterized in that, The steps are as follows: (1) Prepare an AuNPs solution by reducing a chloroauric acid solution with a sodium citrate solution; (2) Dissolve the hairpin probe H1 in an acetate buffer solution, then add TCEP. The resulting solution is added to the AuNPs solution in step (1), incubated at room temperature, and then a NaCl solution is added in batches. The supernatant is removed by centrifugation to obtain an AuNPs probe modified with the hairpin probe H1, namely H1-AuNPs; (3) Add a mixture of EDC and NHS to the CDs solution, stir at room temperature, and then add the hairpin probe H2 and continue to stir at room temperature to obtain CDs modified with the hairpin probe H2, namely H2-CDs; (4) Hybridize the aptamer AP and the auxiliary probe HP at room temperature to obtain an initial reaction solution, then add the sample to be tested. After incubation at room temperature, add the H1-AuNPs in step (2), react for a period of time, then add the H2-CDs in step (3), and remove the reaction solution by centrifugation. The resulting precipitate is diluted and then subjected to fluorescence detection; (5) Substitute the ratio of the FAM fluorescence intensity and the CDs fluorescence intensity measured in step (4) into the linear equation F FAM / F CDs = 4.43 c + 0.46 to obtain the c value, and thus obtain the concentration of OTA in the sample to be measured.

2. The detection method of detecting OTA by the CHA-based ratiometric fluorescence sensor according to claim 1, wherein: In the AuNPs solution in step (1), the diameter of the AuNPs is less than 0.45 μm, and the concentration of the AuNPs is 11.5 nM.

3. The detection method according to claim 1 or 2, characterized in that: In step (2), the nucleotide sequence of the hairpin probe H1 is as shown in SEQ ID NO: 1, and its 5' end is modified with a thiol group and its 3' end is modified with a FAM group.

4. The detection method of OTA by the CHA-based ratiometric fluorescence sensor according to claim 3, characterized in that: In the acetate buffer solution, the concentration of the hairpin probe H1 is 10 μM, the pH of the acetate buffer solution is 5, the volume is 60 μL; the concentration of TCEP is 20 mM, the volume is 10 μL; the volume of the AuNPs solution is 4 mL; the incubation time is 16 h; the final concentration of NaCl after adding the NaCl solution is 0.1 M.

5. The detection method of OTA by the CHA-based ratiometric fluorescence sensor according to claim 4, characterized in that: In step (3), the nucleotide sequence of the hairpin probe H2 is as shown in SEQ ID NO: 2, and its 5' end is modified with an amino group.

6. The detection method of detecting OTA by the CHA-based ratiometric fluorescence sensor according to claim 5, characterized in that: The concentrations of both EDC and NHS are 100 μM, and the volumes are both 100 μL; the concentration of CDs in the CDs solution is 1 mg / L, the volume is 5 mL; the concentration of the hairpin probe H2 is 100 μM, the volume is 20 μL; the concentration of the resulting H2-CDs is 100 μM.

7. The detection method of detecting OTA by the CHA-based ratiometric fluorescence sensor according to claim 6, wherein: In step (4), the nucleotide sequence of the aptamer AP is as shown in SEQ ID NO: 3; the nucleotide sequence of the auxiliary probe HP is as shown in SEQ ID NO:

4.

8. The detection method of detecting OTA by the CHA-based ratiometric fluorescence sensor according to claim 7, characterized in that: The concentrations of both the aptamer AP and the auxiliary probe HP in the initial reaction solution are 100 μM; the incubation time is 40 min; the reaction time after adding the hairpin probe H1 is 45 min; the reaction time after adding H2-CDs is 60 min; the excitation wavelength for fluorescence detection is 360 nm.

9. The detection method of detecting OTA by the CHA-based ratiometric fluorescence sensor according to claim 8, characterized in that: In the linear equation of step (5) c the unit is ng / mL.

10. Use of the method according to any one of claims 1, 2 or 4-9 in detecting OTA, characterized in that: The linear range for ochratoxin A detection is 5.0 pg / mL - 3.0 ng / mL, and the detection limit is 1.5 pg / mL.

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