Preparation method and application of an ochratoxin A aptasensor strip based on PAMAM sensitization

Abstract

The present invention discloses a preparation method and application of an ochratoxin A aptamer test strip based on PAMAM sensitization, belonging to the field of nano-biological detection. The preparation method includes the following steps: (1) preparation of PAMAM-AuNPs conjugate, (2) preparation of PAMAM-AuNPs-aptamer probe, (3) preparation of conjugate pad, (4) preparation of the complex of aptamer complementary strands DNA1 and DNA2 and streptavidin, (5) preparation of T line and C line, and (6) assembly of the test strip. The present invention utilizes the fact that the ends of PAMAM dendrimers have multiple active groups to connect multiple gold nanoparticles, improving the sensitivity of the aptamer test strip, and reads the optical density value of the color development band on the test strip through Image J software to achieve semi-quantitative analysis of the test strip.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano-biological detection, and particularly relates to a preparation method and application of an Ochratoxin A aptamer test strip based on PAMAM sensitization. Background Art

[0002] Ochratoxin A (OTA) is a secondary metabolite produced by certain toxigenic strains of Aspergillus and Penicillium genera, widely present in coffee, beer, red wine, grape juice, corn, wheat, vegetables, meat and meat products. It has high chemical and thermal stability, is easily absorbed in the human body but metabolizes very slowly, with a half-life exceeding 30 days. Research shows that it has nephrotoxicity, hepatotoxicity, teratogenicity, carcinogenicity and immunosuppressive effects on humans and animals. To better monitor the OTA content in food, there is an urgent need to establish a method for quickly, accurately and sensitively detecting OTA in food. Currently, the instrumental analysis methods for detecting OTA mainly include high performance liquid chromatography, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, etc. Although these methods have high sensitivity and accuracy, they require professional personnel to operate expensive large-scale instruments during detection, and the sample preparation process is complex. Enzyme-linked immunosorbent assay and immunochromatography do not require large-scale instruments, but rely on antibodies. The antibody synthesis cycle is long, the price is high and the antibody activity is unstable.

[0003] Compared with antibodies, aptamers have the advantages of a wide range of target substances recognition, strong specificity, easy modification, good stability, can be transported at room temperature, easy to obtain and low cost, and have been widely used in aptamer lateral flow chromatography technology. It is reported [1] that the qualitative detection limit of the gold-labeled OTA aptamer lateral flow test strip can reach 1 ng / mL, and the semi-quantitative detection limit is as low as 0.18 ng / mL. However, the weak optical properties and low biomolecule binding rate of colloidal gold result in low sensitivity of traditional gold-labeled lateral flow test strips, making it difficult to meet the rapid analysis of trace hazards in food media. PAMAM dendrimers are composed of alkyl diamines and tertiary amine branches, with high structural flexibility, hydrophilicity, good biocompatibility, high mechanical and chemical stability, and a large number of amine groups at their molecular terminals, which can immobilize multiple nanoparticles. Currently, there is no research on PAMAM-sensitized aptamer lateral flow test strips. Therefore, this patent intends to construct a gold-labeled aptamer lateral flow test strip based on PAMAM sensitization. Summary of the Invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a preparation method and application of an Ochratoxin A aptamer test strip based on PAMAM sensitization.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] A preparation method of an ochratoxin A aptamer test strip based on PAMAM sensitization is as follows:

[0007] (1) Preparation of PAMAM-AuNPs conjugate

[0008] Measure the concentrated AuNPs solution, add 0.1 mol / L K 2 CO 3 solution to adjust the pH to 8.0 - 9.0, then add a PAMAM solution with a concentration of 50 μM, react at room temperature for 5 h, centrifuge at 12000 rpm for 20 min, resuspend with ultrapure water, and store at 4°C for later use;

[0009] (2) Preparation of PAMAM-AuNPs-aptamer probe

[0010] Add the aptamer solution with a concentration of 100 μM to the PAMAM-AuNPs conjugate, add an 80 mM NaCl solution to a final concentration of 50 mM, incubate in the dark at room temperature for 48 h, centrifuge at 10000 rpm for 18 min, wash twice with PB buffer solution at pH 8.0 - 9.0, add the conjugate complex solution to resuspend, and finally store at 4°C.

[0011] (3) Preparation of conjugate pad

[0012] Spray the prepared PAMAM-AuNPs-aptamer probe onto a polyester cellulose membrane using a membrane scribing and gold spraying instrument, dry at 37°C and store in the dark for later use; the polyester cellulose membrane is pre-treated with a conjugate pad treatment solution;

[0013] (4) Preparation of the complex of aptamer complementary strands DNA1 and DNA2 and streptavidin

[0014] Dissolve streptavidin in a pH 7.4, 0.01 mol / L PBS buffer solution. Take 40 μL, 1 mg / mL streptavidin and add them to 40 μL of aptamer complementary strands DNA1 and DNA2 respectively, then add 20 μL, 0.01 mol / L PBS buffer solution, and incubate at 4°C for 2 h;

[0015] (5) Preparation of T line and C line

[0016] Use a membrane scribing and gold spraying instrument to spray the complex of complementary strands DNA1 and DNA2 and streptavidin onto a nitrocellulose membrane as the T line and C line respectively;

[0017] (6)Assembly of the test strip

[0018] Fix the sample pad, conjugate pad, nitrocellulose membrane and absorbent paper on the polyethylene base plate in sequence, then cut into strips 3.5 mm wide, seal and store in dry condition; wherein the sample pad is pre-treated with the sample pad treatment solution.

[0019] Further, the aptamer nucleotide sequence described in step (2) is as shown in SEQ ID NO:1, specifically: 5’-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACAAAAAAAAAAAAAAAAAA-SH-3’.

[0020] Further, the conjugate reconstitution solution described in step (2) is: 0.01 mol / L PB buffer solution containing 0.5% polyethylene glycol, 5% sucrose, 1% OVA, and 0.25% Tween-20.

[0021] Further, the conjugate pad treatment solution described in step (3) is: 0.1 mol / L PBS buffer solution containing 4% sucrose, 1% OVA, and 0.25% Tween 20.

[0022] Further, the nucleotide sequence of the complementary strand DNA1 described in step (4) is as shown in SEQ ID NO:2,

[0023] specifically: 5’-Biotin-TGTCCGATGCTCCCTTTACGCCACCCACACCCGATC-3’.

[0024] Further, the nucleotide sequence of the complementary strand DNA2 described in step (4) is as shown in SEQ ID NO:3,

[0025] specifically: 5’-Biotin-TTTTTTTTTTTTTTTTTT-3’.

[0026] Further, the sample pad treatment solution described in step (6) is: 0.01 mol / L PBS buffer solution containing 2% sucrose and 0.25% Tween-20.

[0027] The present invention also discloses a test strip prepared by using the preparation method described in the present invention and the application of the test strip in the detection of OTA.

[0028] The present invention utilizes the special structure of PAMAM to fix multiple AuNPs around it to form gold nanoclusters. When the PAMAM-AuNPs-aptamer nanoprobe migrates to the T line, more AuNPs captured by the complementary strand DNA1 on the T line than using a single AuNP, which improves the color development intensity on the T line, reduces the detection limit, and improves the sensitivity of the test strip.

[0029] Beneficial effects: The present invention provides a preparation method and application of an ochratoxin A aptamer test strip based on PAMAM sensitization. The test strip can effectively reduce the detection limit, improve the detection sensitivity, and achieve semi-quantitative detection through Image J software. In the present invention, the aptamer is applied to the test strip as an identification element. At the same time, the special structure of PAMAM is used to aggregate single AuNPs into gold nanoclusters, so that the detection limit of the gold-labeled aptamer test strip is lower and the sensitivity is higher. Description of the Drawings

[0030] Figure 1 It is the UV characterization diagram of AuNPs, PAMAM-AuNPs and PAMAM-AuNPs-aptamer;

[0031] Figure 2 It is the transmission electron microscope (TEM) characterization diagram of AuNPs and PAMAM-AuNPs;

[0032] Figure 3 It is the detection result diagram of the test strip for OTA standard solutions with different concentrations; (A) is the detection result diagram of the AuNPs-labeled aptamer test strip for OTA standard solutions with different concentrations; (B) is the detection result diagram of the PAMAM-AuNPs-based aptamer test strip for OTA standard solutions with different concentrations.

[0033] Figure 4 It is the standard curve diagram of the test strip for detecting OTA; (A) is the standard curve diagram of the AuNPs-labeled aptamer test strip for detecting OTA; (B) is the detection result diagram of the PAMAM-AuNPs-based aptamer test strip for OTA standard solutions with different concentrations

[0034] Figure 5 It is the specific analysis diagram of the test strip;

[0035] Figure 6 It is the schematic diagram of the test strip. Detailed Embodiments

[0036] The present invention will be further described below in conjunction with specific embodiments. However, the embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

[0037] Example 1 Detection of OTA standard solution using an ochratoxin A aptamer test strip based on PAMAM sensitization

[0038] (1) First, use 0.1 mol / L K 2 CO3 The solution was used to adjust the pH of the newly prepared AuNPs solution (10 nm, 5 mL) to 8.0 - 9.0, then a PAMAM solution with a concentration of 50 μM was added, and the reaction was carried out at room temperature for 5 h. After centrifugation (12000 rpm, 20 min), it was resuspended in ultrapure water to 1 mL and stored at 4 °C for later use.

[0039] (2) Then, 2 μL of the OTA aptamer (100 μM) activated by tris(2-chloroethyl) phosphate (1 mg / mL) was added to 1 mL of the PAMAM-AuNPs solution. After incubation at 4 °C for 12 h, a NaCl solution with a final concentration of 50 mM was added, and the reaction was continued for 48 h. After centrifugation (10000 rpm, 18 min), it was washed twice with PB buffer solution (pH adjusted to 8.0 - 9.0), and then resuspended in the conjugate complex solution (0.01 mol / L PB, 0.5% PEG, 5% sucrose, 1% OVA, 0.25% Tween 20) to 1 mL and stored at 4 °C.

[0040] The nucleotide sequence of the aptamer is as follows:

[0041] 5-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACAAAAAAAAAAAAAAAAAA-SH-3’.

[0042] (3) Streptavidin was dissolved in 0.01 mol / L PBS buffer (pH = 7.4). 40 μL of streptavidin (1 mg / mL) was respectively added to 40 μL of the aptamer complementary strand DNA1 and aptamer complementary strand DNA2, and then 20 μL of PBS buffer solution (0.01 mol / L) was added. Incubation was carried out at 4 °C for 2 h.

[0043] The nucleotide sequence of the complementary strand DNA1 is as follows:

[0044] 5’-Biotin-TGTCCGATGCTCCCTTTACGCCACCCACACCCGATC-3’.

[0045] The nucleotide sequence of the complementary strand DNA2 is: 5’-Biotin-TTTTTTTTTTTTTTTTTT-3’.

[0046] (4) The prepared PAMAM-AuNPs-aptamer probe was sprayed onto a polyester cellulose membrane using a membrane scribing and gold spraying instrument, dried at 37 °C, and stored for later use under light-protected conditions. Then, the complementary strand DNA1 of the aptamer and the complex of the complementary strand DNA2 of the aptamer and streptavidin were respectively sprayed onto a nitrocellulose membrane as the T line and the C line using the membrane scribing and gold spraying instrument. Finally, the sample pad, conjugate pad, nitrocellulose membrane, and absorbent paper were sequentially fixed onto a polyethylene base plate, and then cut into strips 3.5 mm wide and stored in a sealed and dry manner.

[0047] The conjugate pad treatment solution described above is: a 0.1 mol / L PBS buffer solution containing 4% sucrose, 1% OVA, and 0.25% Tween-20.

[0048] The sample pad treatment solution described above is: a 0.01 mol / L PBS buffer solution containing 2% sucrose and 0.25% Tween-20.

[0049] (5) First, the OTA standard was dissolved in a methanol solution to form a standard solution with a concentration of 1 mg / mL, and then diluted with a sample diluent to 0.05, 0.1, 0.2, 0.4, 0.8, 1.0 ng / mL. 100 μL of each was added to the wells of an enzyme-linked immunosorbent assay (ELISA) plate. After 15 minutes, the color development of the T line and the C line was observed, and then the optical density value on the T line was read using software Image J for semi-quantitative analysis of OTA. The results obtained with the aptasensor strip labeled with AuNPs are as Figure 3 shown in (A), and the results obtained with the aptasensor strip labeled with AuNPs-PAMAM are as Figure 3As shown in (B). It can be seen from the figure that as the OTA concentration increases, the color development degree of the T line becomes weaker. When the OTA concentration is 0.4 ng / mL, the T line hardly shows color. When the OTA concentration reaches 0.8 ng / mL, the T line does not show color. According to the definition of visual detection limit (LOD), it can be obtained from the figure that 2 ng / mL is used as the visual detection limit of the aptamer strip labeled with AuNPs, and 0.4 ng / mL is used as the visual detection limit of the aptamer strip labeled with AuNPs-PAMAM. Therefore, the visual detection limit (also called the cut-off value) of the aptamer strip based on AuNPs-PAMAM labeling is 4 times higher than that of the traditional one. The software Image J is used to measure the optical density value of the T line of the test strip, and then the optical density ratio when OTA exists and does not exist in the solution is calculated to obtain the relative optical density value (Peak-ROD) on the T line. Then the Origin software is used to fit to obtain a linear equation. According to the linear equation, the semi-quantitative detection limit of the aptamer strip labeled with AuNPs is 0.25 ng / mL, and the semi-quantitative detection limit of the aptamer strip labeled with AuNPs-PAMAM is 0.04 ng / mL. Therefore, the semi-quantitative detection limit of the aptamer strip labeled with AuNPs-PAMAM is about 6 times lower than that of the aptamer strip labeled with AuNPs.

[0050] (6) To detect the specificity of the test strip, 4 common mycotoxins with similar structures to OTA were selected for detection. Under the optimal conditions, the test strips were respectively inserted into the enzyme-linked immunosorbent assay (ELISA) plate wells containing 100 μL of OTA, aflatoxin B 1 (aflatoxin B 1 , AFB 1 ), zearalenone (ZEN), fumonisin B 1 (fumonisin B 1 , FB 1 ), and deoxynivalenol (DON), where the OTA concentration was 2 ng / mL and the other mycotoxins were all 10 ng / mL. The detection results are as shown in Figure 5 . It can be seen from the figure that when the test sample is OTA, the T line does not show color. Thus, it can be seen that the test strip has good specificity for OTA.

[0051] (7) To verify the stability of the test strip, the prepared test strips of the same batch were stored in a sealed bag containing desiccant and stored at 4 °C for 7 days, 14 days, 21 days and 30 days respectively, and then OTA with a concentration of 0.1 ng / mL was detected. Compared with the newly prepared test strips, the T line showed the same color development for the test strips stored for 7 days, 14 days and 21 days. The color development of the test strips stored for 30 days was slightly weaker, but the influence of color loss during the detection process was negligible. Therefore, the test strips still worked well after being stored sealed at 4 °C for 30 d, showing good stability.

[0052] Example 2 Detection of OTA in red wine samples using an ochratoxin A aptamer test strip based on PAMAM sensitization.

[0053] To evaluate the practicability of the test strip, red wine purchased from a supermarket was selected for the experiment in this study. Exactly 2.97 mL of red wine sample was accurately transferred into a 50 mL centrifuge tube, 30 μL of 0.01 mg / mL OTA standard solution was added to make the final concentration of OTA contained in the red wine 100 ng / mL, then 20 mL of methanol, 0.5 g of sodium acetate and 2 g of anhydrous magnesium sulfate were added, and then it was shaken for 3 min to mix evenly, centrifuged at 8000 r for 10 min, and the supernatant was taken out. The following steps were repeated once for the lower layer substance obtained after centrifugation, and the supernatants obtained from the two centrifugations were combined, rotary evaporated to near dryness at 40 °C, then re-dissolved with an aqueous solution of methanol, filtered through a 0.22 μm microporous filter membrane, and made up to 3 mL with an aqueous solution of methanol. Then, a certain volume of the sample solution was taken and diluted with a sample diluent to 0.05, 0.1, 0., 0.5, 1 ng / mL for detection, and the spike recovery rate was between 93% - 105.8%.

[0054] The sample diluent is a 5 × SSC solution containing 1% OVA, 0.2% Tween - 20 and 0.01% SDS.

[0055] Table 1. Detection results of OTA in red wine samples by the test strip (n = 3)

[0056] 。

Claims

1. Preparation method of Ochratoxin A aptamer test strip based on PAMAM sensitization, characterized in that, the preparation method is as follows: (1)Preparation of PAMAM-AuNPs conjugate Measure the concentrated AuNPs solution and add 0.1 mol / L K 2 CO 3 solution to adjust the pH to 8.0 - 9.

0. Then add the PAMAM solution with a concentration of 50 μM, react at room temperature for 5 h, centrifuge at 12000 rpm for 20 min, resuspend with ultrapure water, and store at 4 °C for later use; (2)Preparation of PAMAM-AuNPs-aptamer probe Add the aptamer solution with a concentration of 100 μM to the PAMAM-AuNPs conjugate, add 80 mM NaCl solution to a final concentration of 50 mM, incubate in the dark at room temperature for 48 h, centrifuge at 10000 rpm for 18 min, wash twice with PB buffer solution with pH 8.0 - 9.0, resuspend with the conjugate complex solution, and finally store at 4℃; Preparation of conjugate pad Spray the prepared PAMAM-AuNPs-aptamer probe onto the polyester cellulose membrane through a membrane scribing and gold spraying instrument, dry at 37℃ and store for later use in the dark; the polyester cellulose membrane is pre-treated with the conjugate pad treatment solution; Preparation of aptamer complementary strand DNA1 and DNA2 and streptavidin complex Dissolve streptavidin in PBS buffer solution with pH 7.4 and 0.01 mol / L, take 40 μL of 1 mg / mL streptavidin and add it to 40 μL of aptamer complementary strand DNA1 and DNA2 respectively, then add 20 μL of 0.01 mol / L PBS buffer solution, and incubate at 4℃ for 2 h; Preparation of T line and C line Use a membrane scribing and gold spraying instrument to spray the complementary strand DNA1 and DNA2 and streptavidin complex onto the nitrocellulose membrane as the T line and C line respectively; Assembly of test strip Fix the sample pad, conjugate pad, nitrocellulose membrane and absorbent paper on the polyethylene bottom plate in sequence, then cut into 3.5 mm wide, and store in a sealed and dry manner; wherein the sample pad is pre-treated with the sample pad treatment solution.

2. The preparation method according to claim 1, characterized in that, the nucleotide sequence of the aptamer described in step (2) is as shown in SEQ ID NO:1, specifically: 5’-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACAAAAAAAAAAAAAAAAAA-SH-3’.

3. The preparation method according to claim 1, characterized in that, the conjugate complex solution described in step (2) is: 0.01 mol / L PB buffer solution containing 0.5% polyethylene, 5% sucrose, 1% OVA, and 0.25% Tween-20.

4. The preparation method according to claim 1, characterized in that, the conjugate pad treatment solution described in step (3) is: 0.1 mol / L PBS buffer solution containing 4% sucrose, 1% OVA, and 0.25% Tween 20.

5. The preparation method according to claim 1, characterized in that, the nucleotide sequence of the complementary strand DNA1 described in step (4) is as shown in SEQ ID NO:2, Specifically: 5’-Biotin-TGTCCGATGCTCCCTTTACGCCACCCACACCCGATC-3’.

6. The preparation method according to claim 1, characterized in that the nucleotide sequence of the complementary strand DNA2 described in step (4) is as shown in SEQ ID NO:3, specifically: 5’-Biotin-TTTTTTTTTTTTTTTTTT-3’.

7. The preparation method according to claim 1, characterized in that: the sample pad treatment solution described in step (6) is: 0.01 mol / L PBS buffer solution containing 2% sucrose and 0.25% Tween-20.

8. A test strip prepared by the preparation method according to any one of claims 1-7.

9. Use of the test strip according to claim 8 in the detection of OTA.

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