Detection method of AFB1 by Au-NaCl gel and DSAI-labeled nucleic acid aptamer fluorescence sensor

Abstract

The present invention belongs to the technical field of the detection of AFB1 in biochemistry, environmental detection and food safety, and discloses a detection method of an AFB1 fluorescence sensor based on Au-NaCl gel and DSAI-labeled nucleic acid aptamer. The binding of the ammonium cation of DSAI itself to the phosphate anion in the nucleic acid aptamer enables the biosensing platform to carry stable fluorescence. Based on the large surface area and three-dimensional porous structure of the Au-NaCl gel, the Au on the surface of the Au-NaCl gel is covalently bound to the thiol group on the nucleic acid aptamer through "Au-S", which solves the false positive signals that may occur due to physical adsorption, and according to the FRET principle, the fluorescence is reduced to a certain extent. After adding the target AFB1 to be detected, specific binding occurs between AFB1 and the nucleic acid aptamer and conformational changes occur, and the fluorescence is restored. The detection method of the present invention effectively reduces the detection limit of AFB1 and improves the detection sensitivity of AFB1.

Description

Technical Field

[0001] The present invention belongs to the field of detection methods for aflatoxin B1 (AFB1) in biochemistry, environmental detection, food safety, etc., and is based on the detection of AFB1 by an Au-NaCl gel and a fluorescence sensor of a nucleic acid aptamer labeled with DSAI. Background Art

[0002] Aflatoxin (AFT) is a mycotoxin with high toxicity and carcinogenicity, which is extremely easy to contaminate various foods and agricultural products. It can accumulate in feeds and foods contaminated by mycotoxins during agricultural production, processing, transportation, and storage. Among them, AFB1 is the most common and the most toxic and carcinogenic mycotoxin. It can hinder the synthesis of intracellular RNA, and it is a pathogen of hepatocellular carcinoma (HCC), as well as growth inhibition, immune system regulation, and malnutrition. Research shows that AFB1 can be transferred from poultry feed to eggs, meat, and other edible parts, seriously contaminating the animal food supply. It not only causes huge economic losses in livestock and poultry production but also poses a risk to human health. Since AFB1 is inevitable before and after crop production and is difficult to be decomposed at quite high temperatures, it is very easy to enter the food chain and be absorbed by the human body. In order to avoid humans and animals from ingesting AFB1, a highly sensitive, fast, and convenient detection method for low-concentration AFB1 in agricultural products is needed.

[0003] A nucleic acid aptamer is an oligonucleotide sequence that can bind to various target substances with high specificity and high selectivity. Due to its advantages such as simple preparation, strong specificity, and good stability, aptamers have been widely used in the field of biosensors. Noble metal gel is a new type of functional material, which combines the unique physical and chemical properties of noble metals and the characteristics of a whole porous material. It has been widely studied due to its catalytic performance, large specific surface area, self-supporting structure, and three-dimensional porous structure.

[0004] Although traditional instrumental analysis methods show excellent performance, they are expensive, require high technical skills for detection personnel, and have a long determination time. They are not suitable for on-site rapid detection and are not friendly to underdeveloped areas. And some other detection methods will show false positives, resulting in low sensitivity. Therefore, developing a simple, fast, highly sensitive, and highly selective AFB1 detection method is an essential process. Summary of the Invention

[0005] In order to reduce the detection limit of AFB1 and improve the sensitivity of AFB1. The present invention is based on a biosensor in which an Au-NaCl gel is combined with an aptamer of another AIE fluorophore (DSAI) with aggregation-induced emission effect to detect AFB1. The binding of the ammonium cation of DSAI itself to the phosphate anion in the aptamer makes the biosensing platform carry stable fluorescence. Based on the large surface area and three-dimensional porous structure of the Au-NaCl gel, the Au on the surface of the Au-NaCl gel and the thiol group on the aptamer are covalently bonded through "Au-S", solving the false positive signals that may occur due to physical adsorption, and according to the fluorescence resonance energy transfer (FRET) principle, the fluorescence decreases to a certain extent. After adding the target AFB1 to be detected, specific binding occurs between AFB1 and the aptamer and conformational changes occur, and the fluorescence is restored. Compared with the Au gel, adding the initiator NaCl to the Au gel to adjust its nanowire diameter improves the sensitivity and accuracy of AFB1 detection.

[0006] A method for detecting AFB1 using an Au-NaCl gel and a DSAI-labeled aptamer fluorescence sensor includes the following steps:

[0007] (1) Design a DNA sequence DNA-SH with a thiol group at the end, and dissolve DNA-SH in a Tris-HCl solution;

[0008] In step (1), in the Tris-HCl solution, the concentration of DNA-SH is 10 μM; the concentration of the Tris-HCl solution is 10 mM;

[0009] The sequence of DNA-SH is: 5’-SH-GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCTTCG CTA GGC CC-3’;

[0010] (2) Preparation of the Au-NaCl gel material:

[0011] Add the sodium citrate solution and the HAuCl4 solution to H2O and stir for the first time for 15 min; quickly inject the newly prepared reducing agent aqueous NaBH4 solution and stir for the second time for 30 min; then add the initiator NaCl solution and stir for the third time for 10 - 20 s, and let it stand for 6 - 12 h to obtain a hydrogel;

[0012] Wash the prepared hydrogel several times with a large amount of water, with the total washing time lasting for 2 - 3 days, exchange it with tert-butanol several times, and then put it into a freeze dryer for freeze-drying for 12 - 24 h to obtain a solid Au-NaCl gel;

[0013] Weigh a certain amount of Au-NaCl gel solid, dissolve it in water, wash it, and remove tert-butanol in the solution to obtain an Au-NaCl gel solution with a concentration of 500 μg / mL;

[0014] In step (2), the dosage ratio of sodium citrate solution, HAuCl4 solution, H2O, NaBH4 aqueous solution, and NaCl solution is 2.5 mL: 3.08 mL: 493 mL: 2 mL: 555 μL; among them, the concentration of the sodium citrate solution is 400 mM, the concentration of the HAuCl4 solution is 32.5 mM, the concentration of the NaBH4 aqueous solution is 200 mM, and the concentration of the NaCl solution is 1 M;

[0015] (3) Quenching:

[0016] Mix the Tris-HCl solution of DNA-SH in step (1), the Au-NaCl gel solution prepared in step (2), and the Tris-HCl buffer solution. After the mixture reacts for 12 h, centrifuge and wash it to obtain a sensing system based on Au-NaCl gel, and measure its fluorescence intensity F0;

[0017] In the mixture, the concentration of DNA-SH is 10 - 50 nM; the concentration of Au-NaCl gel is 0.4 - 1.2 μg / mL; the concentration of the Tris-HCl buffer solution is 10 mM, and pH = 7.5.

[0018] (4) Detection:

[0019] Add AFB1 with a certain gradient concentration to the sensing system of the Au-NaCl gel obtained in step (3). The obtained reaction system reacts at room temperature, measure its fluorescence intensity F, and compare it with the quenched fluorescence value F0 to obtain the fluorescence recovery rate F / F0 - 1. The concentration of AFB1 added has a certain proportional relationship with the fluorescence recovery rate.

[0020] In step (4), in the reaction system, the concentration of AFB1 is 0.01 - 10 pg / mL, and the reaction time is 40 min. The present invention also prepares Au gel for comparison;

[0021] Compare the detection of AFB1 by the Au-NaCl gel added with the initiator with the Au gel without the initiator. It is found that the detection limit of the Au-NaCl gel added with the initiator in the present invention for detecting AFB1 is much lower than that of the Au gel without the initiator, indicating that regulating the nanowire diameter of the Au gel by adding the initiator NaCl will improve the detection sensitivity.

[0022] The present invention has the following advantages

[0023] (1) The principle of the present invention is simple, the operation is convenient, time-saving and labor-saving, which brings great convenience to the detection of AFB1 for food safety.

[0024] (2) The gel material in the present invention is easy to obtain, the production method is simple, the cost is low, and the property is stable.

[0025] (3) The present invention utilizes the physical and chemical properties of Au in the gel material to form a "Au-S" covalent bond with the thiol group on the nucleic acid aptamer, avoiding false positive signals caused by physical adsorption.

[0026] (4) The present invention studies the detection of AFB1 by adjusting the nanowire diameter of Au gel by adding the initiator "NaCl", which has high sensitivity and provides a certain basis for optimizing the detection of AFB1 in the future. Brief Description of the Drawings

[0027] Figure 1 It is the experimental schematic diagram of the detection of AFB1 by the Au-NaCl gel and the fluorescence sensor of the nucleic acid aptamer labeled with DSAI;

[0028] Figure 2 It is the DNA-SH concentration selection diagram in the Au gel sensing system (A1) and the Au-NaCl gel sensing system (B2);

[0029] Figure 3 It is the concentration selection diagram of Au gel (B1) and Au-NaCl gel (B2);

[0030] Figure 4 It is the reaction kinetics diagram after adding AFB1 to the Au gel sensing system (C1) and the Au-NaCl gel sensing system (C2);

[0031] Figure 5 It is the sensitivity diagram, the fluorescence recovery rate diagram of the Au gel added with different concentrations of AFB1;

[0032] Figure 6 It is the sensitivity diagram, the fluorescence recovery rate diagram of the Au-NaCl gel added with different concentrations of AFB1;

[0033] Figure 7 It is the selectivity diagram, the fluorescence recovery rate diagram after adding different types of toxins to the gel sensing system. Detailed Embodiments

[0034] The following further illustrates the present invention in conjunction with embodiments. The embodiments are used to illustrate the present invention rather than to limit the scope of the present invention.

[0035] Based on the experimental schematic diagram of the detection of AFB1 by the Au-NaCl gel and the fluorescence sensor of the nucleic acid aptamer labeled with DSAI, asFigure 1 As shown, the steps are as follows:

[0036] (1) Design a DNA sequence with a thiol group at the end according to the detection principle, and dissolve the modified DNA sequence in a 10 mM Tris-HCl solution.

[0037] DNA-SH: 5’-SH-GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTAGGC CC-3’;

[0038] (2) Preparation of Au hydrogel material

[0039] Nanoparticle solution: Add ligand sodium citrate (400 mM, 2.5 mL) and metal salt HAuCl4 (32.5 mM, 3.08 mL) solutions to H2O (482 mL), stir for about 1 min, quickly inject freshly prepared reducing agent aqueous NaBH4 solution (200 mM, 2 mL), and stir for 2 min to obtain a nanoparticle solution; the molar ratio of the metal salt, ligand, and reducing agent is 1:10:4;

[0040] Hydrogel: Further gelify the nanoparticle solution within 15 min. Add aqueous NaBH4 solution (1 M, 100 ul) to the above nanoparticle solution, stir for 10 - 20 s, and let it stand for 6 - 12 h to obtain a gold hydrogel solution;

[0041] Wash the prepared hydrogel 4 - 5 times with a large amount of water for a total of 2 - 3 days, exchange it with tert-butanol several times, and freeze it in a freeze dryer for 12 - 24 h to obtain a Au hydrogel solid;

[0042] Weigh a certain amount of Au hydrogel solid, dissolve it in water, wash it two to three times to remove tert-butanol in the solution, and prepare a Au hydrogel solution with a concentration of 500 μg / mL.

[0043] (3) Preparation of Au-NaCl hydrogel material

[0044] Add sodium citrate (400 mM, 2.5 mL) and HAuCl4 (32.5 mM, 3.08 mL) solutions to H2O (493 mL) and stir for about 15 min; quickly inject freshly prepared reducing agent aqueous NaBH4 solution (200 mM, 2 mL) and stir for about 30 min; add initiator NaCl solution (1 M, 555 ul) and stir for about 10 - 20 s, then let it stand for 6 - 12 h to obtain a hydrogel;

[0045] The prepared hydrogel was washed 4 - 5 times with a large amount of water for a total duration of 2 - 3 days, exchanged with tert-butanol several times, and then placed in a freeze dryer and frozen for 12 - 24 h to obtain the Au-NaCl gel solid;

[0046] Weigh a certain amount of the Au-NaCl gel solid, dissolve it in water, wash it two to three times to remove the tert-butanol in the solution, and prepare an Au-NaCl gel solution with a concentration of 500 μg / mL.

[0047] (4) Add Au-NaCl gel (B2) or Au gel (B1) with gradient concentrations to the 20 nM DNA-SH system, and select the gel concentration with the best quenching rate. The selection of gel concentration is shown in Figure 3 .

[0048] Figure 3 In B1, different concentrations of Au gel (0.4 - 1.2 μg / mL) were added to the Au gel sensing system containing 20 nM DNA. When the concentration of Au gel was 0.6 μg / mL, F / F0-1 reached the maximum, and it would gradually decrease as the concentration increased.

[0049] Figure 3 In B2, different concentrations of Au-NaCl gel (0.4 - 1.2 μg / mL) were added to the Au-NaCl gel sensing system containing 20 nM DNA. When the concentration of Au-NaCl gel was 1 μg / mL, F / F0-1 reached the maximum, and it would gradually decrease as the concentration increased.

[0050] Therefore, the optimal concentration of Au-NaCl gel (B2) was selected as 1 μg / mL, and the Au gel (B1) was selected as 0.6 μg / mL.

[0051] (5) Add different concentrations of DNA-SH (10 nM, 20 nM, 30 nM, 40 nM, 50 nM) to the above systems containing the optimal concentration of Au-NaCl gel (1 μg / mL) or Au gel (0.6 μg / mL) respectively, and then add the same concentration of AFB1, observe the change rate of its fluorescence intensity, and select the DNA concentration with the best quenching rate. The selection diagram of the optimal concentration of DNA-SH is shown in Figure 2 . Figure 2 In A2, in the Au-NaCl gel system, the fluorescence change rate caused by a DNA-SH solution concentration of 20 nM was the largest;

[0052] Figure 2 In A1, in the Au gel system, the fluorescence change rate caused by adding a DNA-SH solution concentration of 20 nM was the largest;

[0053] Therefore, the DNA-SH concentration selected in both systems was 20 nM.

[0054] (6) Different concentrations of AFB1 (0.1 pg / mL, 0.5 pg / mL, 1 pg / mL) were added to the Au-NaCl gel sensing system containing DNA-SH (20 nM) and Au-NaCl gel (1 μg / mL);

[0055] Different concentrations of AFB1 (0.5 ng / mL, 1 ng / mL) were added to the Au gel sensing system containing DNA-SH (20 nM) and Au gel (0.6 μg / mL);

[0056] The change in fluorescence intensity at different reaction times was observed. The kinetic diagram is shown in Figure 4 .

[0057] Figure 4 In C1 of

[0058] Figure 4 Based on the Au gel sensing system with the addition of AFB1 (0.5 ng / mL, 1 ng / mL), the fluorescence intensity reached relative equilibrium at about 40 min of reaction time.

[0058] Figure 4 In C2 of

[0059] Based on the Au-NaCl gel sensing system with the addition of AFB1 (0.1 pg / mL, 0.5 pg / mL, 1 pg / mL), the fluorescence intensity reached relative equilibrium at about 40 min of reaction time. Therefore, the reaction time after adding AFB1 in both gel systems was 40 min.

[0059] Therefore, the optimal reaction time selected in both sensing systems was 40 min. (7) Based on the Au gel sensing system, different concentrations of AFB1 (0.01 ng / mL - 10 ng / mL) were added to the reaction system containing 0.6 μg / mL of Au gel and 20 nM of DNA-SH. 200 μL was taken to measure its fluorescence intensity and record it.

[0060] Figure 5 In a of

[0061] Figure 5 Based on the Au gel sensing system, as the concentration of AFB1 increased, the fluorescence recovery rate (F / F0 - 1) also gradually increased.

[0061] Figure 5 In b of 2 There was an obvious linear relationship between the fluorescence recovery rate caused by the AFB1 concentration in the range of 0 - 5 ng / mL and its concentration. The linear regression equation was expressed as: Y = 0.17341x + 0.13119, R 2 = 0.988. Based on 3S / N, the detection limit was 8.743 pg / mL.

[0062] (8) In the Au-NaCl gel sensing system, different concentrations of AFB1 (0.01 pg / mL - 10 pg / mL) were added to the reaction system containing 1 μg / mL of Au-NaCl gel and 20 nM of DNA-SH. 200 μL was taken to measure its fluorescence intensity and record it.

[0063] Figure 6 In a of, based on the Au-NaCl gel sensing system, as the concentration of AFB1 increases, the fluorescence recovery rate (F / F0 - 1) also gradually increases.

[0064] Figure 6 In b of, the fluorescence recovery rate caused by AFB1 of 0 - 0.8 pg / mL has an obvious linear relationship with its concentration. The linear regression equation is expressed as: Y = 0.20645x + 0.1731, R 2 = 0.97596. Based on 3S / N, the detection limit is 5.356 fg / mL.

[0065] From Figure 5 and Figure 6 By comparison, comparing the detection results of AFB1 by Au gel and Au-NaCl gel shows that the Au-NaCl gel with the initiator NaCl added to regulate the diameter of the gel nanowires will reduce its detection limit and improve the detection sensitivity.

[0066] (9) Several substances similar to AFB1 (AFB2, STE, ZEA, FOM, OCH) were detected and compared with the target AFB1 to be detected under the same conditions, so as to analyze the selectivity of the sensor.

[0067] In the sensing system containing 0.6 μg / mL of Au gel and 20 nM of DNA-SH, 1.5 μL of 100 ng / mL of AFB2, STE, ZEA, FOM, OCH were added respectively, so that their final concentrations were all 0.5 ng / mL. Based on the change of fluorescence intensity after adding different kinds of toxins in the Au gel sensing system, see Figure 7 in D1 of, where the final concentration of each toxin in D1 is 0.5 ng / mL and the volume is 1.5 μL.

[0068] In the sensing system containing 1 μg / mL of Au-NaCl gel and 20 nM of DNA-SH, 1.5 μL of 100 pg / mL of AFB2, STE, ZEA, FOM, OCH were added respectively, so that their final concentrations were all 0.5 pg / mL. Based on the change of fluorescence intensity after adding different kinds of toxins in the Au-NaCl gel sensing system, see Figure 7 in D2 of, the final concentration of each toxin in D2 is 0.5 pg / mL and the volume is 1.5 μL.

[0069] From Figure 7 D1 and D2, it can be seen that the fluorescence recovery rate of AFB1 is the highest, indicating that based on these two sensors, both have good selective specificity for AFB1.

Claims

1. Detection method of AFB1 by Au-NaCl gel and DSAI-labeled nucleic acid aptamer fluorescence sensor, comprising the following steps: (1) Design a DNA sequence DNA-SH with a thiol group at the end, and dissolve DNA-SH in Tris-HCl solution; (2) Preparation of Au-NaCl gel material: Add sodium citrate solution and HAuCl4 solution to H2O and stir for the first time; quickly inject the newly prepared reducing agent NaBH4 aqueous solution and stir for the second time; then add the initiator NaCl solution and stir for the third time, and let it stand to obtain a hydrogel; Wash the prepared hydrogel with a large amount of water several times, with the total washing time lasting 2-3 days, exchange it with tert-butanol several times, and then put it into a freeze dryer for freeze drying to obtain Au-NaCl gel solid; Weigh a certain amount of Au-NaCl gel solid, dissolve it in water, wash it to remove tert-butanol in the solution to obtain Au-NaCl gel solution; (3) Quenching: Mix the Tris-HCl solution of DNA-SH in step (1), the Au-NaCl gel solution prepared in step (2) and Tris-HCl buffer solution. After the mixture reacts, centrifuge and wash to obtain a sensing system based on Au-NaCl gel, and measure its fluorescence intensity F0; (4) Detection: Add AFB1 with a certain gradient concentration to the sensing system of Au-NaCl gel obtained in step (3). The obtained reaction system reacts at room temperature, measure its fluorescence intensity F, and compare it with the quenched fluorescence value F0 to obtain the fluorescence recovery rate F / F0 - 1. The concentration of added AFB1 has a certain proportional relationship with the fluorescence recovery rate, and a linear equation is obtained.

2. The detection method according to claim 1, characterized in that In step (1), in the Tris-HCl solution, the concentration of DNA-SH is 10 μM; the concentration of Tris-HCl solution is 10 mM.

3. The detection method according to claim 1, characterized in that, In step (1), the sequence of DNA-SH is: 5’-SH-GTTGGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CC-3’.

4. The detection method according to claim 1, characterized in that, In step (2), the dosage ratio of sodium citrate solution, HAuCl4 solution, H2O, NaBH4 aqueous solution, and NaCl solution is 2.5 mL: 3.08 mL: 493 mL: 2 mL: 555 μl; among them, the concentration of sodium citrate solution is 400 mM, the concentration of HAuCl4 solution is 32.5 mM, the concentration of NaBH4 aqueous solution is 200 mM, and the concentration of NaCl solution is 1 M.

5. The detection method according to claim 1, characterized in that, In step (2), stir for the first time for 15 min; stir for the second time for 30 min; stir for the third time for 10 - 20 s; the standing time is 6 - 12 h; the freeze drying time is 12 - 24 h.

6. The detection method according to claim 1, wherein In step (2), the concentration of Au-NaCl gel solution is 500 μg / mL.

7. The detection method according to claim 1, wherein In step (3), in the mixed solution, the concentration of DNA-SH is 10 - 50 nM; the concentration of Au-NaCl gel is 0.4 - 1.2 μg / mL; the reaction time is 12 h; the concentration of the Tris-HCl buffer solution is 10 mM, and pH = 7.

5.

8. The detection method according to claim 1, characterized in that In step (4), in the reaction system, the concentration of AFB1 is 0.01 - 10 pg / mL, and the reaction time is 40 min.

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