Quenching-Competition Fluorescent Biosensor Based on ARGET-ATRP Signal Amplification Strategy and Detection Method

Abstract

The present invention discloses a quenching-competitive fluorescence biosensor and a detection method based on the ARGET-ATRP signal amplification strategy. The sensor is mainly prepared from the following raw materials: carboxylated Fe3O4 magnetic beads, EDC, NHS, PBS buffer solution, Apt, BPAA, cDNA, Me6TREN, CuBr2, FA, AA. In the present invention, the Apt sequence is immobilized on the surface of carboxylated magnetic beads through an amide reaction. Meanwhile, BPAA is linked to cDNA through an amide reaction. Then, Apt and cDNA are combined through base complementary pairing. Subsequently, an ARGET-ATRP reaction occurs, enabling a large number of FA monomers to graft and polymerize at the active sites of BPAA, thereby significantly amplifying the fluorescence signal. Finally, the target substance OTA competes with cDNA for Apt, causing the cDNA with fluorescence signal to fall off. The sensor of the present invention has the advantages of high sensitivity, good selectivity, simple operation, low cost, environmental friendliness, etc., and the detection limit can be as low as 7.6 fg / mL.

Description

Technical Field

[0001] The present invention relates to a quenching-competitive fluorescence biosensor and a detection method based on an ARGET-ATRP signal amplification strategy, belonging to the technical field of bioanalysis. Background Art

[0002] Ochratoxin A (OTA) is one of the most toxic foodborne mycotoxins. It is the most abundant, widespread, and toxic among the known ochratoxins and is classified as a Group 2B carcinogen by the International Agency for Research on Cancer. OTA widely exists in crops, feeds, and various medicinal plants, and has hazards such as renal toxicity, intestinal toxicity, immunosuppression, and carcinogenic, teratogenic, and mutagenic effects, which can cause various kidney, liver, and brain diseases in humans and animals. Therefore, the detection of OTA is of great significance for improving industrial quality and maintaining human health.

[0003] Currently, a variety of methods for detecting OTA have been developed, including thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), and biosensing methods, etc. Among them, the biosensing method has been widely used in the fields of food and drug analysis, environmental monitoring, life science, etc. due to its advantages such as high sensitivity, fast response speed, low cost, and simple operation.

[0004] The present invention uses electron transfer-activated regenerable catalyst atom transfer radical polymerization (ARGET-ATRP) as a signal amplification strategy. It is a living / controlled radical technology that can polymerize a large number of fluorescein monomers at an active site, thus effectively improving the sensitivity of the sensor. Moreover, it has the advantages of less catalyst consumption, improved oxygen tolerance of compounds, and mild reaction conditions, and has been widely favored by researchers. The purpose of the present invention is to develop a biosensor for highly sensitive detection of OTA based on the ARGET-ATRP signal amplification strategy, making it have the characteristics of high sensitivity, good selectivity, simple operation, low cost, and environmental friendliness, and can be used for trace detection of OTA. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a quenching-competitive fluorescence biosensor and a detection method based on the ARGET-ATRP signal amplification strategy. This sensor has the advantages of high sensitivity, good selectivity, simple operation, low cost, and environmental friendliness.

[0006] To achieve the above purpose, one of the technical solutions of the present invention is:

[0007] A quenching competition-based fluorescent biosensor based on the ARGET-ATRP signal amplification strategy is mainly prepared from the following raw materials: carboxylated Fe3O4 magnetic beads, EDC, NHS, PBS buffer solution, Apt, BPAA, cDNA, Me6TREN, CuBr2, FA, AA.

[0008] Further,

[0009] Apt sequence: 5’-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACACGCCACCCACACA-3’;

[0010] cDNA sequence: 5’-CCTTTACGCCACCCACACCCGATC-3’.

[0011] One of the technical solutions of the present invention is: a preparation method of a quenching competition-based fluorescent biosensor based on the ARGET-ATRP signal amplification strategy, comprising the following steps:

[0012] (1) Modification of carboxyl magnetic beads

[0013] ① Activation of carboxyl magnetic beads: Mix carboxylated Fe3O4 magnetic beads with EDC solution, NHS solution and PBS buffer solution evenly and react; after the reaction is completed, separate and wash the magnetic beads;

[0014] ② Modification of magnetic beads: Add Apt solution and PBS buffer solution to the magnetic beads in step ①, mix evenly and react;

[0015] ③ Activation of BPAA carboxyl: Mix BPAA solution, EDC solution and NHS solution evenly and react;

[0016] ④ Modification of cDNA: Add cDNA solution to the reaction solution in step ③ and react;

[0017] ⑤ Fixation of cDNA: Separate and wash the magnetic beads in step ②, add them to the reaction solution in step ④ and react; after the reaction is completed, separate and wash the magnetic beads;

[0018] (2) ARGET-ATRP reaction

[0019] Sequentially add CuBr2 / Me6TREN solution, FA solution, AA solution and PBS buffer solution to the magnetic beads in step ⑤, mix evenly and react; after the reaction is completed, separate and wash the magnetic beads, and the separated magnetic beads are the quenching competition-based fluorescent biosensor.

[0020] Furthermore, the concentration of the EDC solution is 200 mM, the concentration of the NHS solution is 50 mM, the concentration of the BPAA solution is 10 mM, the concentration of the cDNA solution is 1 μM / mL, the concentration of the Apt solution is 1 μM / mL, the concentration of the FA solution is 20 mM, the concentration of Me6TREN in the CuBr2 / Me6TREN solution is 12 mM, the concentration of CuBr2 is 10 mM, the concentration of the AA solution is 2 mM, and the concentration of the PBS buffer solution is 0.1 M, with pH = 7.4.

[0021] Furthermore, in steps (1) ③④, the volume ratio of the BPAA solution, the EDC solution, the NHS solution, and the cDNA solution is 2:1:1:1; in step (2), the volume ratio of the CuBr2 / Me6TREN solution, the FA solution, the AA solution, and the PBS buffer solution is 1:1:1:7.

[0022] Furthermore, in step (1): the reaction temperatures of ①②③④⑤ are all 35 - 40 °C, the reaction times of ①②③④ are all 1.0 - 1.5 h, and the reaction time of ⑤ is 0.5 - 1.0 h; in step (2), the reaction temperature is 35 - 40 °C, and the reaction time is 80 - 100 min.

[0023] Furthermore, in step (1), the washing in ①⑤ is performed with the PBS buffer solution and repeated twice; in step (2), the washing steps are to first wash with DMSO and repeat twice, and then wash the magnetic beads with the PBS buffer solution and repeat twice.

[0024] Furthermore, the carboxylated Fe3O4 magnetic beads are pretreated before activation: the carboxylated Fe3O4 magnetic beads are placed in a centrifuge tube, added with the PBS buffer solution for thorough washing, and then the magnetic beads are separated and repeated twice.

[0025] One of the technical solutions of the present invention is: a method for detecting ochratoxin A using ARGET-ATRP as a signal amplification strategy, comprising the following steps:

[0026] (1) First fluorescence detection

[0027] Measure the fluorescence response value of the magnetic beads obtained in claim 1;

[0028] (2) Add ochratoxin A

[0029] Mix the sample solution to be detected and the mixed buffer solution containing Tris-HCl, NaCl, CaCl2, and Tween-20 evenly with the magnetic beads in step (1) and react; after the reaction, separate and wash the magnetic beads;

[0030] (3) Second fluorescence detection

[0031] Measure the fluorescence response value of the magnetic beads obtained in step (2) for the second time; subtract the intensity of the first fluorescence signal from the intensity of the second fluorescence signal to calculate the concentration of ochratoxin A.

[0032] Furthermore, the reaction temperature in step (2) is 35 - 40 °C, and the reaction time is 1.0 - 1.5 h.

[0033] One of the technical solutions of the present invention is: an application of the above-mentioned biosensor in detecting ochratoxin A.

[0034] The schematic diagram of the detection principle of the present invention is as Figure 1 shown.

[0035] Advantages of the present invention:

[0036] 1. The present invention uses ARGET-ATRP as a signal amplification strategy, avoiding the use of nanomaterials and bioenzymes (which are susceptible to external environments such as pH and temperature). Compared with traditional ATRP reactions, this reaction has a wide range of available monomers, mild reaction conditions, and can reduce the use of heavy metal ion catalysts. And the used initiator is commercially available, convenient and easy to obtain.

[0037] 2. The present invention immobilizes the Apt sequence on the surface of carboxylated magnetic beads through an amide reaction. At the same time, BPAA is connected to cDNA through an amide reaction. Then, Apt and cDNA are combined through base complementary pairing. Subsequently, an ARGET-ATRP reaction occurs, enabling a large number of FA monomers to graft and polymerize at the active sites of BPAA, thus significantly amplifying the fluorescence signal. Finally, the target substance OTA competes with cDNA for Apt, causing the cDNA with fluorescence signal to fall off. In addition, the quenching competition-type fluorescence biosensor can greatly avoid false positive results caused by non-specific adsorption. Under optimized conditions, the detection limit of the constructed sensor can be as low as 7.6 fg / mL.

[0038] 3. The present invention has high selectivity for OTA detection and is suitable for detection in complex medicinal plant sample matrices. It also has the advantages of high sensitivity, simple operation, low cost, and environmental friendliness, and is expected to be applied to the detection of other mycotoxin contaminations in traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is the schematic diagram of the detection principle of the present invention.

[0040] Figure 2 is the feasibility study of the sensor of the present invention.

[0041] Figure 3 is the fluorescence confocal characterization. A is without OTA, and B is with OTA added.

[0042] Figure 4 Characterization by scanning electron microscope. A is without OTA, and B is with OTA.

[0043] Figure 5 Study on condition optimization. A is the optimization of FA monomer concentration, and B is the optimization of ARGET-ATRP reaction time.

[0044] Figure 6 Study on analytical performance.

[0045] Figure 7 Study on selectivity. Specific embodiments

[0046] The following further elaborates on the specific embodiments of the present invention in conjunction with examples.

[0047] The aptamer sequence and cDNA sequence were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0048] Aptamer sequence: 5’-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACACGCCACCCACACA-3’ (SEQ ID NO.1).

[0049] cDNA sequence: 5’-CCTTTACGCCACCCACACCCGATC-3’ (SEQ ID NO.2).

[0050] Carboxylated Fe3O4 magnetic beads were purchased from Suzhou Beaver Biomedical Engineering Co., Ltd.

[0051] Example 1: Quenching competition-type fluorescence biosensor based on ARGET-ATRP signal amplification strategy

[0052] The quenching competition-type fluorescence biosensor based on the ARGET-ATRP signal amplification strategy is mainly prepared from the following raw materials: carboxylated Fe3O4 magnetic beads (MBs), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), PBS buffer, aptamer (Apt), α-bromophenylacetic acid (BPAA), cDNA, tris(2-dimethylaminoethyl)amine (Me6TREN), copper bromide (CuBr2), fluorescein-o-acrylate (FA), ascorbic acid (AA).

[0053] Its preparation method is as follows:

[0054] (1) Modification of carboxyl magnetic beads

[0055] ① Pretreatment of carboxyl magnetic beads: First, aspirate 20 μL of carboxylated Fe3O4 magnetic beads (MBs) (d = 300 nm, 10 mg / mL) into a centrifuge tube, then add 20 μL of PBS buffer and wash thoroughly. Place the centrifuge tube on a magnetic stand to separate the MBs. Repeat the above steps 2 times.

[0056] ② Activation of carboxyl magnetic beads: Add 10 μL of EDC solution, 10 μL of NHS solution, and 160 μL of PBS buffer to the centrifuge tube in step ①, and react at 37 °C for 1.0 h; After the reaction, place the centrifuge tube on a magnetic stand to separate the MBs, add 200 μL of PBS buffer and wash thoroughly, place the centrifuge tube on a magnetic stand to separate the MBs, and repeat the above steps 2 times;

[0057] ③ Modification of magnetic beads: Add 10 μL of Apt solution and 90 μL of PBS buffer to the MBs in step ②, mix well, and react in a constant temperature shaker at 37 °C for 1.0 h;

[0058] ④ Activation of BPAA carboxyl: Take another centrifuge tube and add 20 μL of BPAA solution, 10 μL of EDC solution, 10 μL of NHS solution, and react in a constant temperature shaker at 37 °C for 1.0 h;

[0059] ⑤ Modification of cDNA: Add 10 μL of cDNA solution to the reaction solution in step ④, and react in a constant temperature shaker at 37 °C for 1.0 h;

[0060] ⑥ Fixation of cDNA: Place the centrifuge tube in step ③ on a magnetic stand to separate the MBs, add 200 μL of PBS buffer and wash thoroughly, place the centrifuge tube on a magnetic stand to separate the MBs, and repeat the above steps 2 times; Add the separated MBs to the reaction solution in step ⑤, and react in a constant temperature shaker at 37 °C for 0.5 h; After the reaction, place the centrifuge tube on a magnetic stand to separate the MBs, add 200 μL of PBS buffer and wash thoroughly, place the centrifuge tube on a magnetic stand to separate the MBs, and repeat the above steps 2 times;

[0061] (2) ARGET-ATRP reaction

[0062] Add 20 μL of CuBr2 / Me6TREN solution, 20 μL of FA solution, 20 μL of AA solution, and 140 μL of PBS buffer to the MBs in step ⑥ in sequence, and react in a constant temperature shaker at 37 °C for 80 min; After the reaction, place the centrifuge tube on a magnetic stand to separate the MBs, add 200 μL of DMSO and wash thoroughly, place it on the magnetic stand to separate again, and repeat the washing 2 times. Then, wash thoroughly with 200 μL of PBS buffer, place the centrifuge tube on a magnetic stand to separate the MBs, and repeat the above steps 2 times. The separated MBs are the biosensor of the present invention.

[0063] Among them, the concentration of the EDC solution is 200 mM, the concentration of the NHS solution is 50 mM, the concentration of the BPAA solution is 10 mM, the concentration of the cDNA solution is 1 μM / mL, the concentration of the Apt solution is 1 μM / mL, the concentration of the FA solution is 20 mM, the concentration of Me6TREN in the CuBr2 / Me6TREN solution is 12 mM, the concentration of CuBr2 is 10 mM, the concentration of the AA solution is 2 mM, and the concentration of the PBS buffer solution is 0.1 M, pH = 7.4.

[0064] Example 2: Method for detecting ochratoxin A using ARGET-ATRP as a signal amplification strategy

[0065] (1) First fluorescence detection

[0066] The MBs prepared in Example 1 above were diluted to 3 mL with PBS buffer solution, and the fluorescence signal F1 was detected using a transient / steady-state fluorescence spectrophotometer under the excitation wavelength (Ex) of 489 nm and the slit width of 2 nm, and the dilution solution was collected.

[0067] (2) Addition of ochratoxin A

[0068] The MBs in the above dilution solution were collected, 10 μL of the sample solution to be detected and 90 μL of a mixed buffer solution containing Tris-HCl, NaCl, CaCl2, and Tween-20 (after mixing equal volumes of 10 mmol / L Tris-HCl solution, 120 mmol / L NaCl solution, 20 mmol / L CaCl2 solution, and 0.02% (v / v) Tween-20 solution, 90 μL of the mixed solution was used as the mixed buffer solution) were added, and the reaction was carried out in a constant temperature shaker at 37 °C for 1.0 h; after the reaction, it was placed on a magnetic stand to separate the MBs, 200 μL of PBS buffer solution was added for thorough washing, and then placed on a magnetic stand to separate the MBs, and the above steps were repeated 2 times.

[0069] (3) Second fluorescence detection

[0070] After separating the above MBs, they were thoroughly washed with 200 μL of PBS buffer solution, placed on a magnetic stand for separation, and the washing steps were repeated 2 times. Then, they were diluted to 3 mL with PBS buffer solution, and the fluorescence signal F2 was detected using a transient / steady-state fluorescence spectrophotometer under the excitation wavelength (Ex) of 489 nm and the slit width of 2 nm. The fluorescence signal difference was obtained by subtracting the intensity of the first fluorescence signal F1 from the intensity of the second fluorescence signal F2, and the relationship between the fluorescence signal difference and the added OTA concentration was obtained.

[0071] Example 3: Feasibility verification

[0072] To study the feasibility of highly sensitive fluorescence detection of OTA using ARGET-ATRP as a signal amplification strategy, the present invention compared the fluorescence intensities of MBs under different modification conditions. The results are as Figure 2 shown. A strong fluorescence signal peak was observed at 489 nm for MBs modified with Apt / cDNA / BPAA / FA (curve d), and fluorescence quenching occurred at 489 nm for MBs modified with Apt / cDNA / BPAA / FA / OTA (curve f). Without the modification of Apt (curve a), cDNA (curve b), BPAA (curve c), or FA (curve e), only a very weak fluorescence signal could be detected. This is because without the modification of Apt and cDNA, the polymers generated by the ARGET-ATRP reaction cannot be connected to the MBs; without the modification of the initiator BPAA or the monomer FA, the polymerization cannot be initiated, and polymers cannot be grafted onto the MBs. Without the modification of OTA, there is no competitor in the reaction system, and no fluorescence quenching signal can be generated. The above results indicate that the sensor is feasible for the detection of OTA.

[0073] Example 4: Fluorescence confocal and scanning electron microscope characterization

[0074] In this study, fluorescence confocal microscopy and scanning electron microscopy were used to characterize the MBs modified with OTA. First, fluorescence confocal characterization was carried out. As Figure 3 shown in A, when there was no OTA modification on the MBs, obvious green fluorescent spots could be observed in the fluorescence confocal image, indicating that a large amount of FA monomers were polymerized on the surface of the MBs at this time. On the contrary, in Figure 3 B, for the MBs modified with OTA, only a small number of green fluorescent spots were observed in the fluorescence confocal image, indicating that OTA participated in the competition at this time, causing the cDNA fragments with a large amount of FA monomers to fall off. Second, scanning electron microscope characterization was carried out. As Figure 4 shown in A - B, when there was no OTA modification, the FA monomer polymers could not be competed off, so the surface of the MBs was relatively rough. On the contrary, when OTA was modified, OTA competed with cDNA for Apt, causing the cDNA with fluorescent molecules to fall off, and the surface of the MBs became relatively smooth. The results of fluorescence confocal and scanning electron microscopy proved that OTA could compete for cDNA fragments and specifically bind to Apt to achieve the detection of OTA, indicating that the sensor was successful in the detection of OTA.

[0075] Example 5: Optimization of experimental conditions

[0076] To optimize the analytical performance of the sensor, several key parameters affecting the fluorescence intensity during the construction of the sensor were optimized in this study, including the concentration of FA monomers and the reaction time of ARGET-ATRP.

[0077] First, the effect of the concentration of monomer FA in the ARGET-ATRP reaction on the fluorescence intensity of the sensor was studied. Figure 5 Figure A shows that in the range of FA concentration from 5 to 20 mM, as the FA concentration increases, the fluorescence signal gradually increases. When the concentration reaches 20 mM, the fluorescence intensity of the sensor reaches the maximum value, and when the FA concentration continues to increase, the fluorescence intensity remains basically unchanged. This indicates that the polymerization density of FA monomers on the surface of MBs is closely related to the signal amplification of the sensor. Therefore, the optimal concentration of FA is 20 mM.

[0078] Secondly, the effect of the reaction time of ARGET-ATRP on the fluorescence intensity of the sensor was studied. Figure 5 Figure B shows that in the range of 20 - 120 min, the fluorescence intensity continuously increases with the increase of the ARGET-ATRP reaction time. When the reaction time is 80 min, the fluorescence intensity reaches the maximum, and with the prolongation of time, the fluorescence intensity does not change significantly. This indicates that the ARGET-ATRP reaction has reached the optimal state at this time. Therefore, the optimal reaction time of ARGET-ATRP is 80 min.

[0079] Example 6: Analytical performance

[0080] To study the performance of the present invention in detecting OTA, a series of OTA with different concentrations were detected under the optimal conditions. As Figure 6 shown, when the concentration range of OTA is 2.00×10 -3 ~2.00×10 3 ng / mL, the fluorescence intensity gradually increases with the increase of the OTA concentration. In addition, the intensity of the fluorescence signal and the logarithm of the OTA concentration show a good linear relationship in the range of 2.00×10 -3 ~2.00×10 3 ng / mL. The linear regression equation is y = 35515x + 248196, R 2 = 0.998 (x is lgC OTA , unit: ng / mL; y is the fluorescence intensity, unit: a.u.), and the detection limit is calculated to be 7.6 fg / mL (S / N = 3).

[0081] As shown in Table 1, compared with several existing methods for detecting OTA, the present invention has a wider detection range and a lower LOD.

[0082] Table 1

[0083]

[0084] Example 7: Selectivity experiment

[0085] To verify the selectivity of the present invention, under the same conditions, the fluorescence signal response values of a mixture of 20 ng / mL OTA and aflatoxin B1 (AFB1), ochratoxin B (OTB), ochratoxin C (OTC), zearalenone (ZEN) and five substances were studied. As Figure 7 shown, compared with the AFB1, OTB, OTC, ZEN groups, significant fluorescence signal response values can be observed in OTA and the mixture. The reason for this result may be that the Apt modified on the MBs can specifically recognize OTA and bind to it specifically. However, Apt cannot recognize AFB1, OTB, OTC and ZEN, so it cannot participate in the competition and weaken the fluorescence signal intensity. The above experimental results show that the fluorescence sensor based on the ARGET-ATRP signal amplification strategy has good selectivity for OTA.

[0086] Example 8: Reproducibility and stability study

[0087] The reproducibility of the present invention was studied through within-batch and between-batch experiments. The coefficients of variation of the fluorescence signal intensities within-batch and between-batch (n = 5) were 1.8% and 4.3% respectively. The results show that the OTA detection method established by the present invention has good reproducibility.

[0088] To evaluate the stability of the constructed magnetic beads, a storage experiment was carried out in the present invention. Specifically, two groups of modified magnetic beads (n = 5) were prepared under the same conditions. One group was immediately subjected to fluorescence measurement after preparation, and the other group was measured after being stored in an environment at 4 °C for two weeks. The average values of the fluorescence intensities of the two groups were compared. The results showed that the modified magnetic beads after being stored for two weeks could still retain up to 96.0% of the fluorescence signal compared with the newly prepared magnetic beads. It shows that the sensor constructed by the present invention has good stability.

[0089] Example 9: Detection of OTA in traditional Chinese medicine samples

[0090] To further evaluate the accuracy and potential application ability of the sensor in the analysis of traditional Chinese medicine samples, three different concentrations of OTA standards (20 ng / mL, 2.0 ng / mL, 0.2 ng / mL) were added to four traditional Chinese medicine samples (forsythia, astragalus membranaceus, liquorice, xanthium sibiricum) for fluorescence detection, and the recoveries were calculated and analyzed. The results are shown in Table 2. The recoveries of this method in actual samples were 93.19 - 108.39%; these results show that the sensor can be used for the detection of OTA in traditional Chinese medicine samples and can be used to evaluate the exposure level of mycotoxins in traditional Chinese medicine.

[0091] Table 2

[0092] Sequence Listing <110> Henan University of Chinese Medicine <120> Quenching Competitive Fluorescent Biosensor and Detection Method Based on ARGET-ATRP Signal Amplification Strategy <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 49 <212> DNA <213> Artificial Sequence <400> 1 gatcgggtgt gggtggcgta aagggagcat cggacacgcc acccacaca 49 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <400> 2 cctttacgcc acccacaccc gatc 24

Claims

1. A quenching competition type fluorescence biosensor for ochratoxin A detection based on the ARGET-ATRP signal amplification strategy, characterized in that, It is mainly prepared from the following raw materials: carboxylated Fe3O4 magnetic beads, EDC, NHS, PBS buffer solution, Apt, BPAA, cDNA, Me6TREN, CuBr2, FA and AA; Apt sequence: 5’-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACACGCCACCCACACA-3’; cDNA sequence: 5’-CCTTTACGCCACCCACACCCGATC-3’; The preparation method of the biosensor includes the following steps: (1) Modification of carboxyl magnetic beads Activated carboxyl magnetic beads: Mix carboxylated Fe3O4 magnetic beads evenly with EDC solution, NHS solution and PBS buffer solution, and react; after the reaction, separate and wash the magnetic beads; Modification of magnetic beads: Add Apt solution and PBS buffer to the magnetic beads in step , mix well, and react; Activating the carboxyl group of BPAA: Mix the BPAA solution, EDC solution, and NHS solution evenly and react; Modification of cDNA: Add the cDNA solution to the reaction solution in step and react; Fixation of cDNA: Separate and wash the magnetic beads in step , add them to the reaction solution in step , and react; after the reaction is completed, separate and wash the magnetic beads; (2) ARGET-ATRP reaction Add CuBr2 / Me6TREN solution, FA solution, AA solution, and PBS buffer to the magnetic beads in step in sequence, mix well, and react; after the reaction is completed, separate and wash the magnetic beads, and the separated magnetic beads are the quenched competitive fluorescence biosensor.

2. A method for preparing the biosensor according to claim 1, characterized in that, It includes the following steps: (1) Modification of carboxyl magnetic beads Activated carboxyl magnetic beads: Mix carboxylated Fe3O4 magnetic beads with EDC solution, NHS solution and PBS buffer evenly and react; after the reaction, separate and wash the magnetic beads; Modification of magnetic beads: Add Apt solution and PBS buffer to the magnetic beads in step , mix well, and react; Activating the carboxyl group of BPAA: Mix the BPAA solution, EDC solution, and NHS solution evenly and react; Modification of cDNA: Add the cDNA solution to the reaction solution of step and react; Fixation of cDNA: Separate and wash the magnetic beads in step , add them to the reaction solution in step , and react; after the reaction is completed, separate and wash the magnetic beads; (2) ARGET-ATRP reaction Add CuBr2 / Me6TREN solution, FA solution, AA solution, and PBS buffer to the magnetic beads in step in sequence, mix well, and react; after the reaction is completed, separate and wash the magnetic beads, and the separated magnetic beads are the quenching competitive fluorescence biosensor.

3. The preparation method according to claim 2, wherein The concentration of the EDC solution is 200 mM, the concentration of the NHS solution is 50 mM, the concentration of the BPAA solution is 10 mM, the concentration of the cDNA solution is 1 μm / mL, the concentration of the Apt solution is 1 μm / mL, the concentration of the FA solution is 20 mM, the concentration of Me6TREN in the CuBr2 / Me6TREN solution is 12 mM, the concentration of CuBr2 is 10 mM, the concentration of the AA solution is 2 mM, and the concentration of the PBS buffer solution is 0.1 M, pH = 7.4; Step (1) In step (1), the volume ratio of the BPAA solution, the EDC solution, the NHS solution and the cDNA solution is 2:1:1:1; in step (2), the volume ratio of the CuBr2 / Me6TREN solution, the FA solution, the AA solution and the PBS buffer solution is 1:1:1:

7.

4. The preparation method according to claim 2, wherein In step (1): The reaction temperature is all 35 - 40 °C, The reaction time is all 1.0 - 1.5 h, The reaction time is 0.5 - 1.0 h; in step (2), the reaction temperature is 35 - 40 °C and the reaction time is 80 - 100 min.

5. The preparation method according to claim 2, characterized in that, In step (1) The washing is performed by washing with PBS buffer twice; in step (2), the washing step is to first wash with DMSO twice, and then wash the magnetic beads with PBS buffer twice.

6. The preparation method according to claim 2, characterized in that, Before activation, the carboxylated Fe3O4 magnetic beads are pretreated: the carboxylated Fe3O4 magnetic beads are placed in a centrifuge tube, added with PBS buffer solution and washed thoroughly, and then the magnetic beads are separated and repeated twice.

7. A method for detecting ochratoxin A with ARGET-ATRP as a signal amplification strategy, characterized in that, It includes the following steps: (1) First fluorescence detection Measure the fluorescence response value of the biosensor obtained in Claim 1; (2) Add ochratoxin A Mix the sample solution to be detected and the mixed buffer solution containing Tris-HCl, NaCl, CaCl2 and Tween-20 evenly with the biosensor in step (1) and react; after the reaction, separate and wash the biosensor; (3) Second fluorescence detection Measure the second fluorescence response value of the biosensor obtained in step (2); subtract the first fluorescence signal intensity from the second fluorescence signal intensity to calculate the concentration of ochratoxin A.

8. The method according to claim 7, characterized in that The reaction temperature in step (2) is 35 - 40 °C, and the reaction time is 1.0 - 1.5 h.

9. Application of a biosensor as described in Claim 1 in the detection of ochratoxin A.

Images