Ota analysis method based on efficient homogeneous electrochemiluminescence aptamer sensor

Abstract

Disclosed is an OTA analysis method based on a high-efficiency homogeneous electrochemiluminescence aptamer sensor, which comprises the following steps: preparing a mixed solution of L / S*-Ru, IA / B, hairpin probe H1, hairpin probe H2 and exonuclease Exo-III; putting OTA into the mixed solution for reaction to obtain a reaction solution; pouring the reaction solution into a solution containing 0.02M TPrA to obtain a test solution; and immersing an electrode in the test solution for ECL detection and reading the detection result. The analysis method has the advantages of high stability, high sensitivity and good selectivity, and can be used for detecting other toxins, proteins and other biomolecules by changing the aptamer sequence and blocking chain.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular detection, and particularly relates to an OTA analysis method based on a high-efficiency homogeneous electrochemiluminescence aptamer sensor. BACKGROUND

[0002] Ochratoxin A (OTA) is a highly toxic member of the ochratoxin family, which is a secondary metabolite produced by various Aspergillus and Penicillium fungi. It can be produced in food that is improperly stored and exposed to high humidity and high temperature, and mainly contaminates food such as grain crops, grape wine, coffee and its products. Ochratoxin A is stable in nature, and its activity cannot be destroyed by general food processing methods. OTA has nephrotoxicity, carcinogenicity, teratogenicity and immunotoxicity, and can cause great harm to the human body when mis-eaten in excess of the limit. In China, according to the national food safety standard GB 2761-2017, the allowable amount of OTA in cereals, legumes and their products shall not exceed 5 μg / kg.

[0003] The conventional analysis methods for determining OTA mainly include enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) methods. However, these methods usually require tedious operations, time-consuming and expensive devices, and their application in OTA detection is limited. In recent years, other methods such as surface-enhanced Raman scattering, spectrophotometry and electrochemical determination have been developed for the detection of OTA. However, they are still plagued by tedious modification processes and low recognition efficiency. Due to the high specificity, programmability and stability of DNA, some DNA-based amplification methods have become a research hotspot in the field of food safety, such as catalytic nucleic acid (DNAzyme) mediated amplification reaction, exonuclease mediated amplification reaction, catalytic hairpin self-assembly reaction (CDA) and hybridization chain reaction (CHR). In order to improve the signal amplification ability, by integrating different isothermal nucleic acid amplification technologies, two or more technologies are combined to develop various cascade signal amplifiers, which greatly improve the output intensity of the signal. However, due to the increase in the number of stacked layers of the cascade, the analysis efficiency is also limited. Chinese patent CN111424072B discloses an electrochemical biosensor for detecting ochratoxin A and a preparation method thereof, which realizes two-step amplification of the target signal by using catalytic hairpin self-assembly reaction and strand displacement isothermal amplification technology, thereby realizing high-specificity detection of the target OTA. However, this method requires a tedious electrode modification process and is relatively complex to operate.

[0004] In summary, it is of great significance to establish a high-efficiency and high-sensitivity method for detecting OTA in food in the field of food safety. SUMMARY

[0005] In view of the problems in the prior art, the present application provides an OTA analysis method based on a high-efficiency homogeneous electrochemiluminescence aptamer sensor. The method ingeniously combines CDA and Exo-III catalytic hydrolysis, and significantly amplifies the detection signal. When OTA exists, the CDA technology can continuously generate stable H1 / H2 duplex structures, resulting in the continuous accumulation of amplicon S. This accumulation further activates the continuous cycle of Exo-III catalytic hydrolysis reaction, thereby accelerating the entire reaction process and greatly enhancing the ECL output signal until all reactants are consumed. The method not only has excellent stability, but also exhibits extremely high sensitivity and good selectivity, enabling it to realize high-sensitivity and accurate determination of OTA in complex food samples.

[0006] According to an aspect of the present application, a high-efficiency homogeneous electrochemiluminescence aptamer sensor is provided, comprising: an initiation chain IA and a blocking chain B, a hairpin probe H1, a hairpin probe H2, a DNA double strand L / S*-Ru and an exonuclease Exo-III; the initiation chain IA comprises an I initiation sequence and an OTA aptamer sequence; the I initiation sequence comprises three parts d, a and b; the hairpin probe H1 comprises six parts a, b, c*, b*, a* and d*, wherein a-b and a*-b* are complementary to form a double strand as the H1 stem, c* is the loop part of the hairpin structure, d* is the 3' sticky end extending from the stem of the hairpin structure, and the 3' end of the H1 is connected with the d* fragment in the split amplicon S fragment; the H2 comprises five parts c*, a, b, c and b*, wherein c and c* are complementary to form a double strand as the stem of the H2, a-b is the loop part of the hairpin structure of the H2, b* is the 3' sticky end extending from the stem of the hairpin structure, and the c* fragment is the split unit of the amplicon S; the amplicon S comprises two parts c* and d*; the DNA double strand L / S*-Ru comprises a blocking chain L and a S*-Ru chain; the 5' end of the S*-Ru chain is modified by Ru(bpy)3 2+ The sensor ingeniously combines CDA and Exo-III catalytic hydrolysis, and can significantly amplify the detection signal.

[0007] The sequence of the initiation chain IA is shown in SEQ ID NO. 1, specifically: 5'-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACAGAGTGT CGGTCTCTATCATTATCTTTTTT-3'.

[0008] The sequence of the blocking chain B is shown in SEQ ID NO. 2, specifically: 5'-CCGACACTCTGTCCTTTT-3'.

[0009] The sequence of the hairpin probe H1 is shown in SEQ ID NO. 3, specifically: 5'-GTCTCTATCATTATCTTGCTTCATCTTCATCAAGATAATGATAGAGACCGACACTCTTTTT-3'.

[0010] The sequence of the hairpin probe H2 is shown in SEQ ID NO. 4, specifically: 5'-GCTTCATCTTCATCTTCTCTATCATTATCTTGATGAAGATGAAGCAAGATAATTTTT-3'.

[0011] The sequence of the blocking chain L is shown in SEQ ID NO. 5, specifically: 5'-CATCTTCATCTCCGTTTT-3'.

[0012] The sequence of the S*-Ru chain is shown in SEQ ID NO. 6, specifically: 5'-Ru-AGTGTCGGAGATGAAGATGAAGC-3'.

[0013] According to a second aspect of the present application, a preparation method of an efficient homogeneous electrochemiluminescence aptamer sensor is provided, comprising the following steps:

[0014] A1, the S*-Ru chain and the blocking chain L are put into a buffer solution for homogeneous reaction to obtain a hybridization product L / S*-Ru;

[0015] A2, the initiation chain IA and the blocking chain B are put into a buffer solution for homogeneous reaction to obtain a hybridization product IA / B;

[0016] A3, the L / S*-Ru, IA / B, hairpin probe H1, hairpin probe H2 and exonuclease Exo-III are mixed to complete the preparation of the aptamer sensor.

[0017] Further, the buffer solution contains 10mM Tris, 100mM KCl and 2mM MgCl2, and the pH of the buffer solution is 7.2. This buffer solution can provide a safe and stable reaction environment.

[0018] Further, the homogeneous reaction condition is 35℃, 30min. Temperature has a greater impact on the pH value of the buffer solution, and this temperature can stabilize the pH value of the buffer solution at about 7.2, thereby ensuring the stable progress of the reaction.

[0019] According to a third aspect of the present application, an OTA analysis method based on the efficient homogeneous electrochemiluminescence aptamer sensor is provided, comprising the following steps:

[0020] S1, preparing a mixture of the L / S*-Ru, IA / B, hairpin probe H1, hairpin probe H2 and exonuclease Exo-III;

[0021] S2, reacting the OTA with the mixture to obtain a reaction solution;

[0022] S3, pouring the reaction solution into a solution containing 0.02M TPrA to obtain a test solution, immersing an electrode in the test solution for ECL detection and reading the detection result.

[0023] Further, in the S2, the reaction temperature of the OTA with the mixture is 35°C, and the reaction time is 1h.

[0024] Further, in the S3, when performing the ECL detection, scanning is performed by cyclic voltammetry, wherein the potential range is set to 0.6-1.6V, and the scanning rate is 100mV / s.

[0025] The working principle of the method is as follows:

[0026] When the target OTA exists, the aptamer sequence in IA immediately binds to the target and causes the exposure of the initiator strand I sequence, triggering the CDA reaction, and d, a, b of the I sequence can hybridize with d*, a*, b* in the hairpin probe H1 to open H1, generating the intermediate product IA-H1; after H1 is opened, c*, b is released, which can hybridize with c, b* in the hairpin probe H2 to open c*, a, b of H2, and a, b continue to hybridize with a*, b* in H1 to generate a stable H1 / H2 duplex structure, and the IA sequence is released to trigger the next round of CDA reaction, promoting the generation of a large number of H1 / H2 duplex structures; the H1 / H2 duplex structure brings the splitting units of the S fragment close to each other, and assembles into a complete S sequence; then, c*, d* in the S sequence hybridizes with c, d in the S*-Ru of the L / S*-Ru hybrid to generate S-S* dsDNA with a 3' flat end, which can be hydrolyzed and digested by Exo-III; then, the S sequence is released and hybridizes with the next S*-Ru sequence, triggering the cycle of the Exo-III activated digestion process, until all S*-Ru chains are digested, generating a large number of Ru molecules, thereby significantly amplifying the ECL signal. When the OTA does not exist, the initiator strand IA, the hairpin probes H1 and H2 can maintain their own stability, the CDA reaction cannot occur, and the Exo-III hydrolysis reaction cannot be activated, and the amplified ECL signal cannot be detected. In the synergistic amplification reaction of CDA and Exo-III, CDA can continuously produce H1 / H2 duplex structures, produce a large number of S fragments to accelerate the Exo-III hydrolysis reaction, and the S fragments can be reused, thereby achieving the purpose of significantly amplifying the ECL signal. The ECL signal is positively correlated with the concentration of OTA, and the purpose of judging the size of the OTA concentration is achieved through the ECL signal intensity.

[0027] Further, the application further discloses an OTA analysis method based on the high-efficiency homogeneous electrochemiluminescence aptamer sensor, and the specific steps are as follows: mixing L / S*-Ru, IA / B, the hairpin probe H1, the hairpin probe H2 and exonuclease Exo-III to obtain a mixed solution; putting a food sample into the mixed solution to react for 1 h to obtain a reaction solution, and the reaction temperature is 35 DEG C; pouring the reaction solution into a solution containing 0.02M TPrA to obtain a test solution, immersing an electrode in the test solution to perform ECL detection and reading a detection result, wherein the ECL detection is performed by cyclic voltammetry, the potential range is set to 0.6-1.6V, and the scanning rate is 100 mV / s. The analysis method has universality and can also be used for detecting other toxins, proteins and other biomolecules.

[0028] According to a fourth aspect of the present application, an OTA detection kit based on the high-efficiency homogeneous electrochemiluminescence aptamer sensor is provided, comprising: an initiation strand IA and a blocking strand B, a hairpin probe H1, a hairpin probe H2, a DNA double strand L / S*-Ru, and an exonuclease Exo-III.

[0029] Compared with the prior art, the present application has the following beneficial effects:

[0030] The present application improves the detection sensitivity of the target OTA by designing a high-efficiency homogeneous electrochemiluminescence aptamer sensor, wherein the synergistic amplification reaction of CDA and Exo-III can significantly amplify the detection signal, and the ECL signal intensity value of the system is positively correlated with the concentration of the target OTA, which makes the analysis method have the advantages of high stability, high sensitivity and good selectivity. In addition, the analysis method can also be used for detecting other toxins, proteins and other biological molecules, only by introducing new aptamer sequences and blocking strand B, and the sequences of hairpin probes H1 and H2 remain unchanged. When the target analyte can bind to the added DNA strand and release the initiation strand I triggering CDA, the synergistic amplification reaction of CDA and Exo-III can be realized, and the purpose of detecting the target analyte can be achieved. BRIEF DESCRIPTION OF DRAWINGS

[0031] The accompanying drawings are included to provide a further understanding of the embodiments and are incorporated in and constitute a part of this specification. The drawings illustrate the embodiments and, together with the description, serve to explain the principles of the present application. Other embodiments and many of the intended advantages of the present application will be readily appreciated as the same becomes better understood by reference to the following detailed description. The elements of the drawings are not necessarily to scale relative to each other. Like reference numerals designate corresponding similar parts.

[0032] Figure 1 The preparation flowchart of the present application based on the high-efficiency homogeneous electrochemiluminescence aptamer sensor is shown.

[0033] Figure 2 The operation flowchart of the OTA analysis method of the present application based on the high-efficiency homogeneous electrochemiluminescence aptamer sensor is shown.

[0034] Figure 3 The working principle diagram of the synergistic amplification reaction of CDA and Exo-III for OTA detection is shown.

[0035] Figure 4 The detection result diagram of Example 1 is shown.

[0036] Figure 5 The detection result diagram of Example 2 is shown.

[0037] Figure 6 The detection result diagram of Example 3 is shown. Detailed Implementation

[0038] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0039] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0040] All hairpin probe powders were first dissolved in phosphate buffer, and their absorbance was measured using a UV spectrophotometer to calculate the accurate concentration. Then, all hairpin probes were prepared to 4 μM using reaction buffer (10 mM Tris, 100 mM KCl, 2 mM MgCl2, pH 7.2) and subjected to PCR at 95°C for 5 min and 25°C for 2 h to allow them to form stable hairpins.

[0041] Figure 3 A schematic diagram of the synergistic amplification reaction of CDA and Exo-III for OTA detection is shown.

[0042] Example 1

[0043] An OTA analysis method based on a high-efficiency homogeneous electrochemiluminescence aptamer sensor is described below:

[0044] S101: Mix the S*-Ru chain (675 nM) and the blocking chain L (844 nM) in a buffer solution and react at 35 °C for 30 min to obtain the hybridization product L / S*-Ru; mix the initiating chain IA (100 nM) and the blocking chain B (150 nM) in a buffer solution and react at 35 °C for 30 min to obtain the hybridization product IA / B; mix L / S*-Ru, IA / B, hairpin probe H1 (150 nM), hairpin probe H2 (150 nM) and exonuclease Exo-III (675 nM) at a concentration of 0.4 U / μL to obtain a mixed solution;

[0045] S102: Add the target compound OTA (100 nM) to the mixture and react at 35°C for 1 h to obtain the reaction solution;

[0046] S103: Pour the reaction solution into a solution containing 0.02M TPrA to obtain the test solution. Immerse the electrode in the test solution for ECL detection. Use cyclic voltammetry to scan the potential range of 0.6-1.6V and the scan rate of 100mV / s.

[0047] Test results as followsFigure 4 As shown in Figure 4 (A) and Figure 4 (B) can be seen, in the absence of target OTA (curve b), the ECL response generated by the ECL sensor is negligible, indicating that all DNA probes are stable and there is no signal leakage. In contrast, in the presence of target OTA (curve a), OTA activates the circuit and shows a significantly enhanced ECL signal after 1 h, indicating that the synergistic amplification reaction of CDA and Exo-III is activated, thereby enhancing the ECL signal. And when the target OTA exists without the addition of Exo-III, a significant decrease in the ECL signal is observed (curve c), although OTA can activate the CDA circuit and produce H1-H2-S*, but in the absence of Exo-III, the digestion process of the S*-Ru strand is hindered.

[0048] To further confirm the successful construction of the sensor of the present method, the sensor was further characterized by gel electrophoresis, which was performed as follows: the reaction solution obtained in S102 above was mixed with 2 μL loading buffer uniformly; then added to a 9% acrylamide gel to obtain a detection gel; the detection gel was placed in an electrophoresis tank, the voltage of the electrophoresis instrument was set to 100 V, and after 1 h the gel was taken out and stained with GelRed, and the DNA was visualized under ultraviolet light by a chemiluminescence imaging system. The electrophoresis results are shown in Figure 4 (C) shows that lanes c, d and h indicate that all probes can coexist stably; while in lane e, an H1-H2 hybrid band is generated, indicating that OTA has activated the CDA circuit; in lane f, a new H1-H2-S* hybrid band is found, while in lane g, only an H1-H2 hybrid band is found without the H1-H2-S* hybrid band, indicating that the S* sequence in the H1-H2-S* hybrid is completely digested by Exo-III. These results confirm the successful construction of the proposed OTA sensing ECL sensor.

[0049] Example 2

[0050] An OTA analysis method based on a high-efficiency homogeneous electrochemiluminescence aptamer sensor, which is performed as follows:

[0051] S201: Put S*-Ru chain (675 nM) and blocking chain L (844 nM) into buffer solution, react at 35℃ for 30 min to obtain hybridization product L / S*-Ru; put initiating chain IA (100 nM) and blocking chain B (150 nM) into buffer solution, react at 35℃ for 30 min to obtain hybridization product IA / B; mix L / S*-Ru, IA / B, hairpin probe H1 (150 nM), hairpin probe H2 (150 nM) and exonuclease Exo-III (675 nM) with a concentration of 0.4 U / μL to obtain a mixed solution;

[0052] S202: Put target OTA (0 nM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 5 nM, 10 nM, 25 nM, 50 nM, 100 nM and 200 nM) with different concentrations into the mixed solution respectively, and react at 35℃ for 1 h to obtain a reaction solution;

[0053] S203: Pour the reaction solution into a solution containing 0.02 M TPrA to obtain a test solution, immerse the electrode in the test solution for ECL detection, and scan by cyclic voltammetry with a potential range of 0.6-1.6 V and a scan rate of 100 mV / s.

[0054] The detection results are shown in Figure 5 . As can be seen from Figure 5 (A) and 5(B), when there is no target OTA in the system, each DNA probe can maintain its own stability, and the ECL intensity only changes slightly (curve 0 nM in Figure 5 (A)), when different concentrations of OTA are added, the ECL intensity value is positively correlated with the concentration of OTA, and thus OTA can be detected. As can be seen from Figure 5 (C), with the increase of the concentration of OTA, the ECL signal has a linear relationship with the concentration of OTA (0.01-1 nM), and the detection limit is 0.2 pM, realizing rapid and high-sensitivity detection of OTA. As can be seen from Figure 5 (D), through 8 cycles of continuous cyclic scanning, the RSD is calculated to be 3.6%, and the ECL intensity has no obvious fluctuation, proving that the sensor has high stability.

[0055] To further confirm the selectivity of the method for OTA detection, ochratoxin B (OTB), aflatoxin (AFB1), zearalenone (ZEN) and T-2 mycotoxin (T-2) were selected as interference toxins for testing. As can be seen from Figure 6(A, B, C) It can be seen that only when OTA and the mixture containing OTA are used, the ECL signal of the system will change significantly, and the ECL signal changes caused by OTB, AFB1, ZEN and T-2 are very small. Therefore, it can be shown that the method has good selectivity for OTA detection.

[0056] Example 3

[0057] An OTA analysis method based on a high-efficiency homogeneous electrochemiluminescence aptamer sensor is applied to the detection of wheat and grape wine samples, and the specific operation process is as follows:

[0058] S301: Put S*-Ru chain (675 nM) and blocking chain L (844 nM) into a buffer solution and mix, react at 35°C for 30 min to obtain hybridization product L / S*-Ru; Put initiation chain IA (100 nM) and blocking chain B (150 nM) into a buffer solution and mix, react at 35°C for 30 min to obtain hybridization product IA / B; Mix L / S*-Ru, IA / B, hairpin probe H1 (150 nM), hairpin probe H2 (150 nM) and exonuclease Exo-III (675 nM) with a concentration of 0.4 U / μL to obtain a mixed solution;

[0059] S302: Add OTA to wheat and grape wine samples, and the addition value is 1.6, 8 and 16 μg / kg, respectively, and store for 24 h;

[0060] S303: Prepare wheat sample reaction solution: mix 1 g of wheat sample with 2 mL of extraction solvent (V 甲醇 :V 水 = 7:3) and incubate for 15 min, then centrifuge at 7200 x g for 15 min to obtain supernatant, add 5 μL of supernatant to 195 μL of the mixed solution prepared in S301 above, and react at 35°C for 1 h to obtain the wheat sample reaction solution; Prepare grape wine sample reaction solution: dilute the grape wine sample 5 times with Tris buffer solution, then filter with a 0.22 μm filter membrane to obtain a filtrate, add 12.5 μL of the filtrate to 187.5 μL of the mixed solution prepared in S301 above, and react at 35°C for 1 h to obtain the wheat sample reaction solution;

[0061] S304: Pour the reaction solution into a solution containing 0.02 M TPrA to obtain a test solution, immerse the electrode in the test solution for ECL detection, and use cyclic voltammetry for scanning, with a potential range of 0.6-1.6 V and a scanning rate of 100 mV / s.

[0062] The wheat and grape wine samples in S302 above are detected using an OTA-ELISA commercial kit.

[0063] The results are shown in Table 1.

[0064] Table 1 Results of the OTA recovery experiment by the high-efficiency homogeneous electrochemiluminescence aptamer sensor and the ELISA commercial kit

[0065]

[0066]

[0067] As shown in Table 1, the recovery rate from wheat in the present work was between 99.67-100.16%, and the recovery rate from grape wine was between 97.50%-105.00%, both of which had RSDs less than 5%, and the detection results were close to those of the ELISA commercial kit. This indicates that the OTA analysis method based on the high-efficiency homogeneous electrochemiluminescence aptamer sensor proposed in the present application can reliably determine OTA in actual samples, and the flexibility and programmability of the high-efficiency homogeneous electrochemiluminescence aptamer sensor are conducive to the extensive exploration of various biomolecules or proteins in actual samples, and have great potential in food analysis.

[0068] The specific embodiments of the present application are described above, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical range disclosed in the present application, which should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

[0069] In the description of the present application, it should be understood that the wording ‘comprising’ does not exclude the presence of elements or steps not listed in the claims. The wording ‘a’ or ‘an’ in front of an element does not exclude the presence of multiple such elements. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be interpreted as limiting the scope.

Claims

1. A highly efficient homogeneous electrochemiluminescence based aptamer sensor, characterized in that, The aptamer sensor includes an initiating strand IA, a blocking strand B, a hairpin probe H1, a hairpin probe H2, and a double-stranded DNA. and exonuclease Exo-III; the priming strand IA includes an I priming sequence and an OTA aptamer sequence; the I priming sequence includes three parts: d, a, and b; the hairpin probe H1 includes a, b, , , and Six parts, of which ab and The complementary double strands form the stem of the hairpin probe H1. The ring part is a hairpin structure. The hairpin probe H1 has a 3' sticky end extending from the stem of the hairpin structure, and the 3' end of the hairpin probe H1 is attached to a fragment of the split amplicon S. Fragment; the hairpin probe H2 includes a, b, c and Five parts, of which c and The complementary double chains form the stem of the hairpin probe H2, and ab is the loop portion of the hairpin structure of the hairpin probe H2. It is the 3' sticky end extending from the stem of the hairpin structure. A fragment is a splitting unit of amplicon S; the amplicon S includes and Two parts; the DNA double strand Including blocking chains = Chain; the The 5' end of the chain is Ru(bpy)3 2+ Modification; The priming strand IA sequence is shown as SEQ ID NO. 1, specifically: 5'-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACAGAGTGTCGGTCTCTATCATTATCTTTTTT-3'; The blocking strand B sequence is shown as SEQ ID NO. 2, specifically: 5'-CCGACACTCTGTCCTTTT-3'; The hairpin probe H1 sequence is shown as SEQ ID NO. 3, specifically: 5'-GTCTCTATCATTATCTTGCTTCATCTTCATCAAGATAATGATAGAGACCGACACTCTTTTT-3'; The hairpin probe H2 sequence is shown as SEQ ID NO. 4, specifically: 5'-GCTTCATCTTCATCTTCTCTATCATTATCTTGATGAAGATGAAGCAAGATAATTTTT-3'; The blocking strand L sequence is shown as SEQ ID NO. 5, specifically: 5'-CATCTTCATCTCCGTTTT-3'; The The chain sequence is shown as SEQ ID NO. 6, specifically: 5'-Ru-AGTGTCGGAGATGAAGATGAAGC-3'.

2. A preparation method of the high-efficiency homogeneous electrochemiluminescence aptamer sensor-based sensor according to claim 1, comprising the following steps: A1, the blocking chain L is added to the reaction mixture and the blocking chain L is added to the reaction mixture A1, the blocking chain L is added to the reaction mixture and the blocking chain L is added to the reaction mixture A1, the blocking chain L is added to the reaction mixture A2, the priming strand IA and the blocking strand B are put into a buffer solution for reaction to obtain a hybrid product IA / B; A3. The mixture is heated to 95°C for 5 min, then cooled to 4°C for 5 min. IA / B, the hairpin probe H1, the hairpin probe H2 and the exonuclease Exo-III are mixed, completing the preparation of the aptamer sensor.

3. The method for preparing a high efficiency homogeneous electrochemiluminescence aptamer sensor according to claim 2, characterized in that, The buffer solution contains 10 mM Tris, 100 mM KCl and 2 mM MgCl2, and the buffer solution has a pH of 7.

2.

4. The method for preparing a high efficiency homogeneous electrochemiluminescence aptamer sensor according to claim 2, characterized in that, The homogeneous reaction condition is 35°C for 30 min.

5. The OTA assay method based on the high-efficiency homogeneous electrochemiluminescence aptamer sensor according to claim 1, characterized in that, Comprising the following steps: S1, preparing the , IA / B, mixture of hairpin probe H1, hairpin probe H2 and exonuclease Exo-III; S2, the OTA is put into the mixed solution for reaction to obtain a reaction solution; S3, the reaction solution is poured into a solution containing 0.02 M TPrA to obtain a test solution, the electrode is immersed in the test solution for ECL detection, and the detection result is read. 6.The OTA analysis method based on the high-efficiency homogeneous electrochemiluminescence aptamer sensor of claim 5, wherein, In the S3, when the ECL detection is performed, the scanning is performed by cyclic voltammetry, wherein the potential range is set to 0.6-1.6 V, and the scanning rate is 100 mV / s.

7. The method of OTA analysis based on efficient homogeneous electrochemiluminescence aptamer sensor according to any one of claims 5-6, characterized in that, Application in food detection: the food sample is put into the mixed solution to react for 1 h to obtain a reaction solution, and the reaction temperature is 35 DEG C; the reaction solution is poured into a solution containing 0.02 M TPrA to obtain a test solution, and the electrode is immersed in the test solution for ECL detection and reading of the detection result, wherein the ECL detection is scanned by cyclic voltammetry, the potential range is set to 0.6-1.6 V, and the scanning rate is 100 mV / s. , IA / B, hairpin probe H1, hairpin probe H2 and exonuclease Exo-III are mixed to obtain a mixed solution; the food sample is put into the mixed solution to react for 1 h to obtain a reaction solution, and the reaction temperature is 35 DEG C; the reaction solution is poured into a solution containing 0.02 M TPrA to obtain a test solution, and the electrode is immersed in the test solution for ECL detection and reading of the detection result, wherein the ECL detection is scanned by cyclic voltammetry, the potential range is set to 0.6-1.6 V, and the scanning rate is 100 mV / s.

8. An OTA detection kit based on the high efficiency homogeneous electrochemiluminescence aptamer sensor according to claim 1, characterized in that, The kit comprises the priming strand IA and the blocking strand B, the hairpin probe H1, the hairpin probe H2, the DNA duplex and the exonuclease Exo-III.

Images