Preparation method of electrochemical sensor based on RecJf exonuclease and gold and silver ZIF-8 composite material

Abstract

The invention designs a preparation method of an electrochemical sensor based on RecJf exonuclease and a gold and silver ZIF-8 composite material. And mixing the complementary strand with the aptamer strand to form the double-stranded DNA through complementary base pairing. An electrode modification material gold and silver ZIF-8 is dropwise added to the surface of the pretreated electrode, so that the material is physically adsorbed to the surface of the electrode; and when the beta-lactoglobulin and the RecJf exonuclease exist, the beta-lactoglobulin is combined with the aptamer chain to cause the falling of the complementary chain, and more complementary chains are cyclically fallen under the action of the RecJf exonuclease. The fallen complementary chain is fixed on the modification material through a gold-sulfur bond and a silver-sulfur bond. DNA hairpins HP1 and HP2 are added for a hybridization chain reaction, so that methylene blue is embedded into double strands; and when the beta-lactoglobulin does not exist, the complementary chain cannot fall off, and a hybridization chain reaction cannot occur to embed methylene blue. Detection of beta-lactoglobulin is realized through signal change of methylene blue, and the kit has the advantages of simplicity in operation, good stability, high specificity and the like.

Description

Technical Field

[0001] The invention relates to the technical field of food safety detection, and in particular to a method for preparing an electrochemical sensor based on RecJf exonuclease and a gold-silver ZIF-8 composite material. Background Art

[0002] Food allergy refers to the hypersensitivity reaction of the immune system to specific food ingredients (allergens), which can even be life-threatening in severe cases. It is a major global public health issue. Most allergic reactions are caused by milk and dairy products, eggs and egg products, fish and shellfish. As one of the most widely consumed foods, milk is rich in nutrients such as protein and minerals, and consuming milk is very beneficial to the growth and development of infants and children. However, milk has been listed as one of the eight major food allergens in the world by the Food and Agriculture Organization of the United Nations (FAO), and about one-third of allergic events are caused by milk. β-Lactoglobulin (β-LG) is the main milk protein in milk that causes allergies. It is composed of 162 amino acid residues, has a molecular weight of 18.4 kDa, and accounts for 10% of milk protein. Hypoallergenic formula milk is suitable for patients with milk allergies, but low concentrations of β-LG remaining during the production and processing of the product may also cause allergic reactions. Since milk allergy is incurable, patients with milk allergy should completely avoid contact with or ingestion of allergens. Therefore, in order to meet current regulations while protecting public health, it is necessary to develop a convenient, rapid, accurate, and sensitive β-LG detection method.

[0003] At present, analytical methods used to detect β-LG include high performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), fluorescence analysis, molecular imprinting, etc. Although these methods have high sensitivity and specificity, they also have disadvantages such as high cost, poor stability, complex sample pretreatment procedures, false positives and false negatives, and the need for professional experimental personnel, which makes it difficult to meet the requirements of accurate on-site detection. Biosensors have good stability, sensitivity, low cost and portability. They are an analytical tool with broad application prospects and have received widespread attention. Electrochemical sensors have the advantages of low cost, low detection limit and good selectivity, and are an important method for analytical detection. In order to further improve the sensitivity of electrochemical sensors, this method introduces RecJf exonuclease and gold-silver ZIF-8 composite materials. The use of gold-silver ZIF-8 composite materials gives electrochemical sensors a larger surface area and higher conductivity, and the introduction of metal nanoparticles can facilitate the fixation of DNA. The use of RecJf exonuclease better expands the recognition and response of the sensor to the detection target. The electrochemical sensor based on the present invention has the characteristics of good selectivity, high sensitivity, strong stability, convenience and economy, which is conducive to the promotion and application of the invention. Summary of the invention

[0004] A method for preparing an electrochemical sensor based on RecJf exonuclease and gold-silver ZIF-8 composite material is carried out according to the following steps: (1) Preparation of zeolite imidazole framework: zinc nitrate hexahydrate and 2-methylimidazole were dissolved in equal volumes of methanol, mixed and stirred vigorously, allowed to stand for aging, and then centrifuged at high speed to collect the product, which was then washed with methanol to remove unreacted solvent, and dried at a constant temperature in vacuum to constant weight to obtain zeolite imidazole framework ZIF-8; (2) Preparation of gold-silver-zeolite imidazole framework composite material: ZIF-8 was ultrasonically dispersed in water, and equal volumes of tetrachloroauric acid solution and silver nitrate solution were added under stirring with a magnetic stirrer. After stirring and mixing, a reducing agent was slowly added with continuous stirring, and the product was collected by high-speed centrifugation and washed with ultrapure water to remove the unreacted solvent. The product was dried at a constant temperature in vacuum to constant weight to obtain a gold-silver-zeolite imidazole framework composite material, gold-silver ZIF-8; (3) Preparation of double-stranded DNA: The complementary chain cDNA treated with reduced disulfide bonds is mixed with equal volumes of aptamer chain Apt of equal concentration, and then incubated in a constant temperature oscillator to form double-stranded dsDNA through base complementary pairing, and then placed at 4°C for use; (4) Construction of electrochemical sensor: gold-silver ZIF-8 is added to the pretreated electrode surface and incubated in a constant temperature and humidity environment to allow the material to be physically adsorbed on the electrode surface. When β-LG and RecJf exonuclease are present in the dsDNA system, β-LG binds to Apt, causing cDNA to fall off. Under the action of RecJf exonuclease, Apt bound to β-LG is cleaved, releasing β-LG and binding to other Apt. Since the 5' end of cDNA is modified with a protective thiol group, cDNA will not be cleaved. After multiple cycles, the RecJf exonuclease in the system is inactivated to end the cycle. The above system is added to the electrode surface to fix the uncut cDNA on the modified material through gold-sulfur bonds and silver-sulfur bonds. Then, a mixed solution of DNA hairpins HP1 and HP2 is added to generate a large number of double-stranded structures through a hybridization chain reaction, so that the signal molecule is embedded in the double-stranded structure. Finally, the electrode is placed in a buffer solution, and the electrochemical signal of the signal molecule is detected by square wave voltammetry.

[0005] It is further defined that in the step (1), the mass ratio of zinc nitrate hexahydrate to 2-methylimidazole is 1:4 to 1:6.

[0006] It is further defined that in step (2), the mass of ZIF-8 dispersed in water is 2 to 5 mg; the volume of tetrachloroauric acid solution and silver nitrate solution is 150 μL to 250 μL; and the reducing agent is sodium borohydride.

[0007] It is further defined that in step (3), the concentrations of the complementary chain cDNA and the adapter chain Apt are 50-100 μM.

[0008] It is further defined that in step (4), the volume added to the electrode surface is 2 to 8 μL; the concentration of the DNA hairpins HP1 and HP2 is 1 to 2 μM; and the signal molecule is methylene blue.

[0009] It is further defined that in steps (3) and (4), the incubation time is 0.5 to 2.5 h; the incubation temperature is 20 to 50°C; the sequence of the aptamer chain Apt is 5'- CGA CGA TCG GAC CGC AGT ACC CAC CCA CCA GCC CCA ACATCA TGC CCA TCC GTG TGT G -3'; the sequence of the complementary chain cDNA is 5'- TGC GGT CCG ATC GTC GAGAGA GAC -3', wherein the 5' end is modified with a thiol group; the sequence of the DNA hairpin HP1 is 5'- GAG AGA CTG TAT GTG GTCTCT CTC GAC GA -3'; and the sequence of the DNA hairpin HP2 is 5'- ACA TAC AGT CTC TCT CTC GTC GAG AGAGAC -3'. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Schematic diagram of an electrochemical sensor based on RecJf nuclease and gold-silver ZIF-8 composite material.

[0011] Figure 2 Square wave voltammetry curves of the sensor constructed in Example 1 of the present invention before (solid line) and after (dashed line) the addition of 50 ng / mL β-LG.

[0012] Figure 3 This is the standard curve of the sensor constructed in Example 1 of the present invention for detecting β-LG.

[0013] Figure 4 The specificity of the sensor constructed in Example 1 of the present invention to β-LG in the presence of other interfering factors. DETAILED DESCRIPTION

[0014] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments, but it should be understood that the protection scope of the present invention is not limited by the specific implementation. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified. Example 1

[0015] A method for preparing an electrochemical sensor based on RecJf exonuclease and gold-silver ZIF-8 composite material, wherein the method for implementing the electrochemical sensor is as follows: Figure 1 shown.

[0016] A method for preparing an electrochemical sensor based on RecJf exonuclease and gold-silver ZIF-8 composite material comprises the following steps: (1) Preparation of zeolite imidazole framework ZIF-8: 0.6375 g zinc nitrate hexahydrate and 3.1875 g 2-methylimidazole were dissolved in 15 mL methanol, and ultrasonicated for 20 min and 40 min, respectively. The solutions were mixed and stirred with a magnetic stirrer at 1000 rpm for 30 min, and then aged at room temperature for 24 h. The resulting solid precipitate was collected by centrifugation at 8000 rpm for 5 min, and then rinsed with methanol to remove the unreacted solvent. Finally, it was dried in a vacuum drying oven at 60 °C for 12 h to obtain zeolite imidazole framework ZIF-8.

[0017] (2) Preparation of gold-silver-zeolite imidazole framework composite material gold-silver ZIF-8: 3 mg ZIF-8 was ultrasonically dispersed in 1 mL ultrapure water, 200 μL 25 mM tetrachloroauric acid solution and 200 μL 25 mM silver nitrate solution were added and stirred for 20 min, then 2 mL 0.1 M sodium borohydride solution was added and continued to stir for 30 min. The product was collected by centrifugation at 8000 rpm for 10 min, rinsed with ultrapure water to remove the unreacted solvent, and dried in a vacuum drying oven at 60 °C for 12 h to obtain gold-silver ZIF-8.

[0018] (3) Preparation of double-stranded dsDNA: 2 μL of 100 μM complementary chain cDNA and 10 μL of 10 mM TCEP were added to a centrifuge tube and mixed. The mixture was reacted at 37°C in the dark for 1 h to reduce the disulfide bonds. Then, 2 μL of 100 μM aptamer chain Apt and 86 μL Tris buffer were added. After mixing, the mixture was oscillated in a constant temperature oscillator at 37°C for 2 h to form double-stranded dsDNA through base complementary pairing. The mixture was placed at 4°C for use.

[0019] (4) Construction of electrochemical sensor and identification of target: 5 μL of gold-silver ZIF-8 composite material was added to the pretreated electrode surface and incubated in a constant temperature and humidity incubator at 37°C for 1 h to allow the material to be physically adsorbed on the electrode surface. The electrode was then washed with Tris buffer to remove the gold-silver ZIF-8 that was not bound to the electrode surface. 20 μL of dsDNA was placed in a centrifuge tube, and 16 μL of β-LG of different concentrations, 0.5 μL of 30U / μL RecJf exonuclease, and 4 μL of enzyme reaction buffer solution were added. After mixing, the mixture was shaken at 37°C for 1 h. h; when β-LG and RecJf exonuclease are present, β-LG binds to Apt and causes cDNA to fall off. Under the action of RecJf exonuclease, Apt bound to β-LG is cleaved to release β-LG and bind to other Apts. Since the 5' end of cDNA is modified with a protective thiol group, cDNA will not be cleaved; after multiple cycles, the system is placed at a constant temperature of 65°C for 20 min to inactivate RecJf exonuclease, and the cycle is terminated. 5 μL of the above solution is added to the electrode surface and incubated at 37°C for 1.5 h to fix the uncut cDNA on the modified material through gold-sulfur bonds and silver-sulfur bonds. The electrode is then washed with Tris buffer to remove cDNA that is not bound to the electrode surface. 5 μL of 0.1 mM MCH blocking agent is added and incubated at 37°C for 1 h. The electrode is then washed with Tris buffer to remove MCH that is not bound to the electrode surface. Add 5 μL of 1 μM DNA hairpin HP1 and HP2 mixture to generate a large number of double-stranded structures by hybridization chain reaction. Wash the electrode with Tris buffer to remove HP1 and HP2 that are not bound to the electrode surface. Soak the working surface of the electrode in 8 mM methylene blue solution at 37°C for 30 min to allow the methylene blue molecules to embed into the double-stranded structure. Wash the electrode with Tris buffer to remove the methylene blue that is not embedded in the double-stranded structure. Finally, place the electrode in the buffer solution, and use square wave voltammetry to detect the electrochemical signal of methylene blue. The change in the response signal before and after the addition of β-LG can be used to quantitatively analyze β-LG.

[0020] (5) Establishment of standard curve: Add β-LG standard solution of different concentrations into step (4), obtain electrochemical signal through subsequent reaction, and perform linear fitting with the logarithm of β-LG concentration as the horizontal axis and the current signal as the vertical axis to establish the standard curve of the sensor for β-LG.

[0021] like Figure 2 As shown, the square wave voltammetric curve results of the sensor constructed in Example 1 of the present invention before (solid line) and after (dashed line) the addition of 50 ng / mL β-LG.

[0022] like Figure 3As shown, it is the standard curve of the sensor constructed in Example 1 of the present invention for detecting β-LG. Example 2

[0023] A preparation method of an electrochemical sensor based on RecJf exonuclease and gold-silver ZIF-8 composite material, and its practical application, comprises the following steps: (1) In order to verify that the prepared electrochemical sensor based on RecJf exonuclease and gold-silver ZIF-8 composite material has specific recognition of β-LG, β-LG standard was added to ultrapure water to make the concentration of β-LG in the sample 125 ng / mL; ultrapure water or Tris-HCl buffer solution were used to prepare other interference factor standard solutions, and the concentration was 125 ng / mL. The above-mentioned several different interference factor standard solutions were tested according to the detection system constructed in Example 1, and the test results are as follows: Figure 4 As shown, it is shown that the method of the present invention has good specificity for β-LG. Example 3

[0024] A preparation method of an electrochemical sensor based on RecJf exonuclease and gold-silver ZIF-8 composite material, and its practical application, is carried out according to the following steps: (1) Food sample processing: For the liquid sample milk, take 1 μL of milk sample and mix it with 2999 μL ultrapure water, then take 1 mL of the above solution and mix it with 3 mL of ultrapure water, and finally take 100 μL of the above mixed solution and mix it with 100 μL of β-LG standard samples of different concentrations to obtain milk spiked samples; for the solid sample amino acid formula milk powder, accurately weigh 1 mg and dissolve it in 1 mL of ultrapure water, then take 1 μL and mix it with 999 μL ultrapure water, finally take 125 μL from the mixed solution and mix it with 875 μL ultrapure water, and take 100 μL of the above mixed solution and mix it with 100 μL of β-LG standard samples of different concentrations to obtain milk powder spiked samples.

[0025] (2) Sample detection: The electrical signal was measured according to the steps of Example 1 and the concentration of β-LG in the sample was obtained by inserting the signal into the standard curve.

[0026] (3) When milk and milk powder are used as actual samples for determination, different concentrations of β-LG standard are added to the samples. 16 μL of sample solution is taken, and the electrical signal is measured according to steps (1) to (4) of Example 1. The β-LG concentration in the sample is obtained by inserting the standard curve detected in Example 1. Each sample is measured three times and the average value is taken. The average recovery rate is calculated to be 97.3%-104.3%.

[0027] The electrochemical sensor prepared has the advantages of simple operation, good stability, high specificity and wide detection range for the detection of β-LG. The detection of actual samples shows that the prepared sensor has good practical application value.

[0028] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention and do not limit the present invention in any way. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention, and these changes and improvements shall fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing an electrochemical sensor based on RecJf exonuclease and gold-silver ZIF-8 composite material, characterized in that: The following steps are involved: (1) Preparation of gold-silver ZIF-8: zinc nitrate hexahydrate and 2-methylimidazole were dissolved in equal volumes of methanol, mixed and stirred vigorously after being fully dissolved, and aged to obtain zeolite imidazole framework ZIF-8. ZIF-8 was dissolved in ultrapure water and tetrachloroauric acid solution, silver nitrate solution and a reducing agent were added to obtain a metal nanocomposite material, gold-silver ZIF-8. Two kinds of precious metal nanoparticles were simultaneously grown in situ on the surface of ZIF-8 by a one-step reduction method and used as electrode modification materials. The good biocompatibility and conductivity made it a DNA connection carrier and also improved the electron transfer rate on the electrode surface. (2) Identification of the target β-lactoglobulin: The complementary chain cDNA treated with reduced disulfide bonds and the aptamer chain Apt with the same concentration are added to a centrifuge tube and mixed and incubated. Double-stranded dsDNA is formed through base complementary pairing. The complementary chain and the aptamer chain in the double-stranded structure are designed to be closed at the 5' end to prevent enzyme shearing in the absence of the target. When β-lactoglobulin and RecJf nuclease are present in the double-stranded dsDNA system, β-lactoglobulin binds to the aptamer chain Apt to cause the complementary chain cDNA to fall off, and a cycle occurs under the action of RecJf nuclease to cause more complementary chain cDNA to fall off. (3) Construction of electrochemical aptamer sensor: Gold-silver ZIF-8 was added dropwise to the pretreated electrode surface to allow the material to be physically adsorbed on the electrode surface. The detached complementary chain cDNA was added and fixed on the modified material through gold-sulfur bonds and silver-sulfur bonds. A mixed solution of DNA hairpins HP1 and HP2 was added to undergo a hybridization chain reaction to produce a large number of double-stranded structures, so that a large number of signal molecules were embedded in the double-stranded structures. Finally, the electrochemical signal of the signal molecule was detected by square wave voltammetry to achieve quantitative detection of β-lactoglobulin. Compared with the common electrochemical sensor construction strategy, the zeolite imidazole framework metal nanocomposite material and the RecJf exonuclease and HCR double-cycle strategy were introduced to cause signal amplification.

2. The method for preparing an electrochemical sensor based on RecJf exonuclease and gold-silver ZIF-8 composite material according to claim 1, characterized in that: In step (1), the mass ratio of zinc nitrate hexahydrate to 2-methylimidazole is 1:4 to 1:6; the mass of ZIF-8 dissolved in ultrapure water is 2 to 5 mg; the volume of tetrachloroauric acid solution and silver nitrate solution is 150 μL to 250 μL; and the reducing agent is sodium borohydride.

3. The method for preparing an electrochemical sensor based on RecJf exonuclease and gold-silver ZIF-8 composite material according to claim 1, characterized in that: In step (2), the concentrations of the complementary chain cDNA and the adapter chain Apt are 50-100 μM.

4. The method for preparing an electrochemical sensor based on RecJf exonuclease and gold-silver ZIF-8 composite material according to claim 1, characterized in that: In step (3), the volume of the gold-silver ZIF-8 added to the electrode surface is 2 ~ 8 μL; the concentration of the DNA hairpins HP1 and HP2 is 1 ~ 2 μM; and the signal molecule is methylene blue.

5. The method for preparing an electrochemical sensor based on RecJf exonuclease and gold-silver ZIF-8 composite material according to claim 1, characterized in that: In steps (2) and (3), the incubation time is 0.5 to 2.5 h; the incubation temperature is 20 to 50°C.

6. The method for preparing an electrochemical sensor based on RecJf exonuclease and gold-silver ZIF-8 composite material according to claim 1, characterized in that: Further defined, in step (2), the aptamer chain Apt is as shown in SEQ ID NO.1, and the specific sequence is 5'- CGA CGA TCG GAC CGC AGT ACC CAC CCA CCA GCC CCA ACA TCATGC CCA TCC GTG TGT G -3'; the complementary chain cDNA is as shown in SEQ ID NO.2, and the specific sequence is 5'- TGCGGT CCG ATC GTC GAG AGA GAC -3', wherein the 5' end is modified with a thiol group.

7. The method for preparing an electrochemical sensor based on RecJf exonuclease and gold-silver ZIF-8 composite material according to claim 1, characterized in that: Further defined, in step (3), the DNA hairpin HP1 is as shown in SEQ ID NO.3, and the specific sequence is 5'-GAG AGA CTG TAT GTG GTC TCT CTC GAC GA -3'; the DNA hairpin HP2 is as shown in SEQ ID NO.4, and the specific sequence is 5'-ACA TAC AGT CTC TCT CTC GTC GAG AGA GAC -3'.

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