An electrochemical biosensor for detecting ochratoxin a based on DNA tetrahedron

Abstract

The application belongs to the technical field of biosensors and provides an electrochemical biosensor for detecting ochratoxin A based on DNA tetrahedron, which comprises an A-P probe, a gold electrode with a modified capture probe CP, a circular template CT, phi 29 polymerase, dNTPs, Mg 2+ , hemoglobin, K + , H2O2; the A-P probe is obtained by hybridization of an aptamer Apt and a primer P; the nucleotide sequences of the aptamer Apt, the primer P, the capture probe CP and the circular template CT are shown as SEQ ID NO: 1-4; the 5' end of the capture probe CP is modified with SH. The sensor has a simple preparation method, stable performance and is suitable for the detection of OTA in food and environment; the process cost of the preparation process is low, the performance is stable, the electrode has good repeatability and is suitable for the requirements of cheapness in industrialization.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology and relates to an electrochemical biosensor for detecting ochratoxin A based on DNA tetrahedrons. Background Technology

[0002] Ochratoxins are a class of fungal secondary metabolites and are typical biotoxins. Ochratoxin A (OTA), in particular, is highly toxic, widely polluting, and difficult to degrade and remove. It can damage the human liver and kidneys and cause cancer and liver and kidney poisoning in most mammals. OTA is widely produced during food and feed processing, storage, and transportation, and small amounts are also present in water bodies. The International Agency for Research on Cancer (IARC) has classified ochratoxin A as a potential human carcinogen. In my country, the standard limit for OTA in legumes, cereals, and their products is 5 μg / kg, and the standard limit for OTA in alcoholic beverages (wine) is 2 μg / kg.

[0003] Currently, there are many methods for detecting OTA, such as optical analysis, electrophoresis, and chromatography. However, these analytical methods have drawbacks such as cumbersome procedures, expensive instruments, and complex equipment, making them unsuitable for on-site detection. Therefore, it is essential to develop rapid, trace-level, highly sensitive, and selective analytical methods for detecting OTA. Summary of the Invention

[0004] To achieve more sensitive and specific detection of ochratoxin A (OTA), this invention proposes an electrochemical biosensor constructed based on the specific recognition of OTA and its aptamers, the RCA reaction, and the DNAzyme catalytic cleavage reaction. It has the advantages of fast detection speed, high sensitivity, and high specificity, and can make up for the defects and deficiencies of existing OTA detection methods, so as to achieve convenient and accurate quantitative detection of OTA.

[0005] To achieve the above objectives, the present invention adopts the following technical solution.

[0006] An electrochemical biosensor for detecting ochratoxin A based on DNA tetrahedrons, comprising:

[0007] AP probe, gold electrode modified to capture CP probe, circular template CT, phi 29 polymerase, dNTPs, Mg 2+ Heme, K + H2O2;

[0008] The AP probe was obtained by hybridization of aptamer Apt and primer P;

[0009] The nucleotide sequences of the aptamer Apt, primer P, capture probe CP, and circular template CT are shown in SEQ ID NO:1-4;

[0010] The 5' end of the capture probe CP is modified with -SH;

[0011] The method for preparing the modified gold electrode for capturing the CP includes the following steps:

[0012] (i) The gold electrode was immersed in a mixture of H2O2 and concentrated H2SO4 in a volume ratio of 7:3, then sonicated and washed with double-distilled water; then polished in 0.3 µm and 0.05 µm alumina slurry until it was mirror-like, and then rinsed repeatedly with PBS buffer and double-distilled water to obtain the pretreated gold electrode.

[0013] (ii) The capture probe CP was denatured in a buffer solution at 95°C and then subjected to an ice-water bath to obtain the hairpin-structured capture probe CP;

[0014] (iii) The solution of the capture probe CP was added to the pretreated gold electrode and incubated at 37°C, and then washed with PBS buffer and water in sequence; the gold electrode was immersed in 6-mercapto-1-hexanol (MCH) and stored at 0-4°C.

[0015] The method for preparing the ring-shaped template CT includes the following steps:

[0016] (a) The padlock probe PP and the ligation probe LP, whose nucleotide sequences are shown in SEQ ID NO: 4-5, are denatured and annealed in a buffer solution to complete hybridization and obtain a hybridization chain solution;

[0017] (b) Add T4 ligase to (a) to inactivate the enzyme, then add Exo I and Exo III to inactivate the enzyme and obtain a circular template CT solution.

[0018] A kit comprising the above-mentioned ochratoxin A electrochemical biosensor.

[0019] The kit may also include ochratoxin A and a buffer solution.

[0020] A method for detecting ochratoxin A using the above-mentioned electrochemical biosensor or kit includes the following steps:

[0021] (1) The sample to be tested was mixed with AP probe, circular template CT, phi 29 polymerase, dNTPs, and Mg. 2+ Heme in K + The mixture was mixed in a buffer solution and then dropped onto the gold electrode modified with the capture probe CP. The electrode was washed three times with buffer and ultrapure water respectively, and dried under a nitrogen stream to obtain the modified gold electrode.

[0022] (2) Using Ag / AgCl as the reference electrode, Pt electrode as the counter electrode, and modified gold electrode as the working electrode, the electrode was placed in an electrolyte containing H2O2, and the change in electrical signal was read by differential pulse voltammetry.

[0023] In step (1), the reaction temperature is 37℃ and the reaction time is 2h.

[0024] In step (2), the parameters of the differential pulse voltammetry are set as follows: the potential is set to 0 to -1 V, the amplitude is 0.05 V, and the pulse width is 0.2 s.

[0025] The detection principle of this invention is as follows: Figure 1 As shown, the sequences of each nucleic acid element and intermediate product are as follows:

[0026] Aptamer Apt:

[0027] 5'-GATC GGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3';

[0028] Primer P:

[0029] 5'-TGTCCGATGTTTTTACACCCGATC-3';

[0030] Padlock probe PP:

[0031] 5'-p-GGTAGGGTGTcACTATTTTCGACCGGCTCGGAGAAGAGAGGTAGGGTGTcACTATTTTCGACCGGCTCGG AGAAGAGA-3';

[0032] Connect probe LP:

[0033] 5'-CTTCGGTGCTCG CTTGCCTGCTCG-3';

[0034] Capture probe CP:

[0035] 5'-HS-AAAAAAGTGGGTAGGGCGGGTTGGGCACTATrAG GAAGAGA GCCCTACC-3'

[0036] Mg 2+ DNAzyme:

[0037] 5'-TCTCTTCTCCGAGCCGGTCGAAAA TAGTG-3';

[0038] G-quadruplex DNA zyme:

[0039] 5'-GTGGGTAGGGCGGGTTGGG-3';

[0040] In the presence of the target OTA, OTA specifically binds to the aptamer in AP, thereby releasing the RCA primers. The primers hybridize with the circular template at a concentration ratio of 2:1. The addition of phi 29 and dNTPs initiates bidirectional RCA, and the reaction produces a large amount of Mg. 2+ Metal DNAzyme. The electrode sensing part is first composed of CP modified on the surface of a bare gold electrode via Au-S interaction through the 5' end thiol group. CP contains a G-quadruplex DNAzyme sequence and Mg. 2+ The base sequence of the metal DNA enzyme. After the homogeneous reaction is complete, the detection process is performed on the electrode surface in Mg. 2+ Under the influence of K, the DNAzyme cleavage function in the rolling circle amplification reaction product is activated, cleaving CP to release G-quadruplex DNAzyme, and K is added. + The aptamer forms a G-quadruplex DNAzyme with heme, which then catalyzes the H2O2 reaction. Simultaneously, it captures another portion of the probe, which serves as a primer and can be recycled into the next bidirectional rolling circle amplification reaction. When OTA is absent from the system, its aptamer is not carried away, the primer is not released, the bidirectional rolling circle amplification reaction does not occur, the DNAzyme is not generated, the G-quadruplex DNAzyme does not form, and no electron transfer occurs on the electrode surface; therefore, no strong electrochemical signal is detected.

[0041] The present invention has the following advantages:

[0042] The sensor provided by this invention utilizes the specific binding characteristic between OTA and its aptamers for detection; it leverages bidirectional rolling circle amplification and DNAzyme catalytic cleavage reactions to amplify the signal, thereby improving detection sensitivity. The sensor operates under mild reaction conditions and has a fast reaction rate; the main processes of the detection principle are implemented on the electrodes, increasing reaction speed, reducing operational complexity, and achieving rapid, simple, and sensitive detection of the target analyte. The sensor's fabrication method is simple, its performance is stable, and it is suitable for the detection of OTA in food and the environment; the fabrication process has low cost, stable performance, and good electrode repeatability, making it suitable for the cost-effective requirements of industrialization. Attached Figure Description

[0043] Figure 1 This is a schematic diagram illustrating the principle of the invention;

[0044] Figure 2 The image shows the results of the optimized detection of phi 29 DNA polymerase concentration.

[0045] Figure 3 Optimize the detection results graph for heme concentration;

[0046] Figure 4 The graph shows the optimized detection results for H2O2 concentration.

[0047] Figure 5 The working curve for OTA detection by the sensor. Detailed Implementation

[0048] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0049] Example 1: Construction of an electrochemical sensor

[0050] (1) Preparation and synthesis of components

[0051] The aptamer Apt, primer P, capture probe CP, padlock probe PP and ligation probe LP were synthesized according to the nucleotide sequences shown in SEQ ID NO:1-5, wherein the 5' end of the capture probe CP was modified with -SH and the 5' end of the padlock probe PP was phosphorylated.

[0052] (a) Preparation of AP probe:

[0053] Dissolve aptamer Apt and primer P in PBS buffer to an appropriate concentration, then incubate equal volumes of aptamer Apt solution and primer P solution together at 95°C for 5 min, and allow to cool naturally to room temperature to form AP probe (10 μM), which is then stored at 4°C for later use.

[0054] (b) Fabrication of a gold electrode (CP-AuE) modified with the trapping probe CP:

[0055] (i) Soak in a mixture of 30% H2O2 and H2SO4 (concentrated) (volume ratio 7:3), then sonicate and wash with double-distilled water to remove surface residue; then polish in 0.3 µm and 0.05 µm alumina slurry to a mirror finish, and then rinse three times with PBS buffer (pH 7.4) and double-distilled water to obtain the pretreated gold electrode;

[0056] (ii) 100 μM of the capture probe CP was denatured in PBS buffer (pH 7.4) at 95°C for 5 min, and then rapidly in an ice-water bath for 30 min to obtain a solution of 10 μM of the capture probe CP.

[0057] (iii) The solution of the capture probe CP (final concentration of 1 μM) was added dropwise to the pretreated gold electrode. After incubation at 37 °C for 4 h, the electrode was washed three times with PBS buffer (pH 7.4), then immersed in 6-mercapto-1-hexanol (MCH), and the modified electrode was rinsed multiple times with PBS and stored at 4 °C.

[0058] (c) Preparation of ring-shaped template CT:

[0059] Mix 35 μL of sterile water, 6 μL of 100 μM PP and 6 μL of 100 μM LP in T4 DNA polymerase buffer, denature at 95℃ for 5 min, and then gradually cool to room temperature to complete the hybridization process of ligating the probe and the padlock probe.

[0060] Then, 3 μL of 400 U / μL T4 ligase was added to the mixed solution, and the reaction was carried out overnight at 16°C to ligate the padlock probe into a loop. Subsequently, the T4 ligase was inactivated in a 65°C water bath.

[0061] Finally, 2 μL of 20 U / μL Exo Ⅰ and 2 μL of 100 U / μL Exo Ⅲ were added, and the mixture was reacted at 37℃ for 8 hours to remove excess single strands and hybrid double strands. Then, Exo Ⅰ and Exo Ⅲ were inactivated in an 85℃ water bath. Finally, the obtained 10 μM circular template CT was stored at 4℃.

[0062] (2) Working electrode reaction

[0063] (a) Prepare the ochratoxin A detection system according to the following proportions: 8 μL 25 μM AP probe, 20 μL 100 ng / mL OTA, 2 μL 1 M MgCl2, 2 μL 50 μM circular template CT, 1.25 μL 10 U / μL phi 29 DNA polymerase, 2 μL 10 mM dNTPs, and 2 μL 10 mM heme;

[0064] (b) Take 10 μL of the above solution and add it to CP-AuE. Incubate at 37°C for 2 h. Then wash three times each with PBS buffer (pH 7.4) and ultrapure water. Dry under nitrogen flow to obtain the modified gold electrode (working electrode).

[0065] (3) Target object detection

[0066] (a) Preparation of electrolyte: Take 4 mL of 5×PBS buffer (pH 7.4) into 16 mL of sterile water, add 200 µL 2 M H2O2 solution, mix thoroughly and use immediately;

[0067] (b) Detection of the target analyte: The modified gold electrode was placed in the electrolyte, and DPV testing was performed using an electrochemical workstation. Ag / AgCl was used as the reference electrode, and a Pt electrode as the counter electrode. The potential was set from 0 to -1 V, the amplitude was 0.05 V, and the pulse width was 0.2 s. The change in the electrical signal was read using differential pulse voltammetry to detect the target analyte. Compared with the control, the current in the detection system containing OTA increased significantly, indicating that the constructed electrochemical sensor can detect OTA.

[0068] Example 2: Effect of phi 29 DNA polymerase concentration on electrical signals

[0069] Take the components of each electrochemical sensor prepared in Example 1, and then screen the optimal concentration of phi 29 DNA polymerase according to the following steps:

[0070] Prepare the ochratoxin A detection system according to the following proportions: 8 μL 25 μM AP probe, 20 μL 100 ng / mL OTA, 2 μL 1 M MgCl2, 2 μL 50 μM circular template CT, 1.25 μL phi 29 DNA polymerase (final concentrations of 0.25, 0.5, 1.0, 1.25, 1.5, and 2.0 U / μL, respectively), 2 μL 10 mM dNTPs, and 2 μL 10 mM heme;

[0071] 10 μL of the above solution was added to CP-AuE and incubated at 37°C for 2 h. Then, the electrode was washed three times each with PBS buffer (pH 7.4) and ultrapure water, and dried under nitrogen flow to obtain the modified gold electrode (working electrode).

[0072] The current signal was detected according to the method in step (3) of Example 1, and the result is as follows. Figure 2 As shown, before 1.25 U / μL, the current signal increases with the increase of its concentration, but after the concentration reaches 1.25 U / μL, the current signal remains almost unchanged. Therefore, the optimal condition for phi 29 is 1.25 U / μL.

[0073] Example 3: The effect of heme concentration on electrical signals

[0074] Take the elements of each electrochemical sensor prepared in Example 1, and then screen the optimal heme concentration according to the following steps:

[0075] Prepare the ochratoxin A detection system according to the following ratio: 8 μL 25 μM AP probe, 20 μL 100 ng / mL OTA, 2 μL 1 M MgCl2, 2 μL 50 μM circular template CT, 1.25 μL 10 U / μL phi 29 DNA polymerase, 2 μL 10 mM dNTPs, and 2 μL double-distilled water or different concentrations of heme (5 mM, 10 mM, 15 mM, 20 mM).

[0076] 10 μL of the above solution was added to CP-AuE and incubated at 37°C for 2 h. Then, the electrode was washed three times each with PBS buffer (pH 7.4) and ultrapure water, and dried under nitrogen flow to obtain the modified gold electrode (working electrode).

[0077] The current signal was detected according to the method in step (3) of Example 1, and the result is as follows. Figure 3 As shown: when the heme concentration reaches 10 mM, its corresponding current signal is the strongest, so the optimal concentration of heme is 10 mM.

[0078] Example 4: Effect of H2O2 concentration on electrical signals

[0079] Take the elements of each electrochemical sensor prepared in Example 1, and then screen the optimal H2O2 concentration according to the following steps:

[0080] Prepare the ochratoxin A detection system according to the following ratio: 8 μL 25 μM AP probe, 20 μL 100 ng / mL OTA, 2 μL 1 M MgCl2, 2 μL 50 μM circular template CT, 1.25 μL 10 U / μL phi 29 DNA polymerase, 2 μL 10 mM dNTPs, and 2 μL 10 mM heme.

[0081] 10 μL of the above solution was added to cDNA-AuE and incubated at 37°C for 2 h. Then, the cells were washed three times each with PBS buffer (pH 7.4) and ultrapure water, and dried under a nitrogen stream to obtain the modified gold electrode (working electrode).

[0082] Take 4 mL of 5×PBS buffer (pH 7.4) into 16 mL of sterile water, and add 200 µL of H2O2 solution (final concentration 5, 10, 15, 20, 25 mM). After mixing thoroughly, detect the current signal according to the method in step (3) of Example 1.

[0083] The results are as follows Figure 4As shown, within the H2O2 concentration range of 0-25 mM, the current signal increases with the increase of its concentration. When the concentration reaches 20 mM, the current reaches its maximum. After the concentration exceeds 20 mM, the current decreases slightly. Therefore, the optimal concentration of H2O2 is 20 mM.

[0084] Example 5: Sensor detection limit for OTA

[0085] Take the elements of each electrochemical sensor prepared in Example 1, and then determine the detection limit of the sensor according to the following steps:

[0086] Prepare the ochratoxin A detection system according to the following ratio: 8 μL 25 μM AP probe, 20 μL double-distilled water (control) or 100 ng / mL OTA, 2 μL 1 M MgCl2, 2 μL 50 μM circular template CT, 1.25 μL 10 U / μL phi 29 DNA polymerase, 2 μL 10 mM dNTPs, and 2 μL 10 mM heme;

[0087] 10 μL of the above solution was added to CP-AuE and incubated at 37°C for 2 h. Then, the electrode was washed three times each with PBS buffer (pH 7.4) and ultrapure water, and dried under nitrogen flow to obtain the modified gold electrode (working electrode).

[0088] The current signal was detected according to the method in step (3) of Example 1, and the result is as follows. Figure 5 As shown: DPV response corresponding to different concentrations of OTA. The higher the concentration, the stronger the DPV response. Figure 5 A). A logarithmic linear fit between the peak current of the DPV response and the OTA concentration yields the equation: I = 2.089 + 0.5312lgC OTA (R) 2 =0.995)( Figure 5 B). Calculations show that the detection limit of this electrochemical sensor for OTA is 1.91 ng / mL.

Claims

1. An electrochemical biosensor for detecting ochratoxin A based on DNA tetrahedrons, characterized in that, include: AP probe, gold electrode modified to capture CP probe, circular template CT, phi 29 polymerase, dNTPs, Mg 2+ Heme, K + H2O2; The AP probe was obtained by hybridization of aptamer Apt and primer P. The nucleotide sequences of the aptamer Apt, primer P, capture probe CP, and circular template CT are shown in SEQ ID NO:1-4; The 5' end of the capture probe CP is modified with -SH.

2. The electrochemical biosensor according to claim 1, characterized in that, The method for preparing a gold electrode modified to capture the CP probe includes the following steps: (i) The gold electrode was immersed in a mixture of H2O2 and concentrated H2SO4 in a volume ratio of 7:3, then sonicated and washed with double-distilled water; then polished in 0.3 µm and 0.05 µm alumina slurry until it was mirror-like, and then rinsed repeatedly with PBS buffer and double-distilled water to obtain the pretreated gold electrode. (ii) The capture probe CP was denatured in a buffer solution at 95°C and then subjected to an ice-water bath to obtain the hairpin-structured capture probe CP; (iii) The solution of the capture probe CP was added dropwise to the pretreated gold electrode and incubated at 37°C. Then it was washed with PBS buffer and water in sequence. The gold electrode was then immersed in 6-mercapto-1-hexanol.

3. The electrochemical biosensor according to claim 1, characterized in that, The method for preparing the ring-shaped template CT includes the following steps: (a) The padlock probe PP and the ligation probe LP, whose nucleotide sequences are shown in SEQ ID NO: 4-5, are denatured and annealed in a buffer solution to obtain a hybridization chain solution; (b) Add T4 ligase to the hybridization chain solution to react, inactivate the enzyme, then add Exo I and Exo III to react, inactivate the enzyme, and obtain the circular template CT solution.

4. A kit comprising the electrochemical biosensor as described in any one of claims 1-3.

5. The reagent kit according to claim 4, characterized in that, It also includes ochratoxin A and buffer solution.

6. A method for detecting ochratoxin A using an electrochemical biosensor as described in any one of claims 1-3 or a kit as described in claim 4 or 5, characterized in that, Includes the following steps: (1) The sample to be tested was mixed with AP probe, circular template CT, phi 29 polymerase, dNTPs, and Mg. 2+ Heme in K + The mixture was mixed in a buffer solution and then dropped onto the gold electrode modified with the capture probe CP. The electrode was washed three times with buffer and ultrapure water respectively, and dried under a nitrogen stream to obtain the modified gold electrode. (2) Using Ag / AgCl as the reference electrode, Pt electrode as the counter electrode, and modified gold electrode as the working electrode, the electrode was placed in an electrolyte containing H2O2, and the change in electrical signal was read by differential pulse voltammetry.

7. The method according to claim 6, characterized in that, In step (1), the reaction temperature is 37℃ and the reaction time is 2h.

8. The method according to claim 6, characterized in that, In step (2), the parameters of the differential pulse voltammetry are set as follows: the potential is set to 0 to -1V, the amplitude is 0.05V, and the pulse width is 0.2s.

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