A preparation method of a ZIF-8 / Thi / Au-based ratio type electrochemical aptamer sensor

Abstract

The application relates to a preparation method and application of a ZIF-8 / Thi / Au-based ratio type electrochemical aptamer sensor, and ZIF-8 / Thi / Au with a signal label function is combined with a catalytic hairpin assembly strategy for the first time to detect mycotoxins, signal amplification is effectively realized, and the detection sensitivity is improved. ZIF-8 / Thi / Au composite materials are synthesized first, and a signal label is prepared by combining a hairpin chain 2. When aflatoxin B1 exists, aflatoxin B1 is combined with an aptamer chain, a complementary chain that is dropped off opens a hairpin chain 1, catalytic hairpin assembly is carried out after a signal label is added, the signal label is combined on the electrode surface, the electrode surface has a high resistance value, and a buffer solution containing [Fe(CN)6] 3‑ / 4‑ is used to carry out electrochemical measurement by using a square wave voltammetry method, a higher ZIF-8 / Thi / Au signal and a lower [Fe(CN)6] 3‑ / 4 signal can be obtained. When no aflatoxin B1 exists, the changes of the two electrochemical signals are opposite. Finally, an electrochemical aptamer sensor for detecting aflatoxin B1 is obtained by the change of the ZIF-8 / Thi / Au and [Fe(CN)6] 3‑ / 4 signal ratio.

Description

Technical Field

[0001] This invention belongs to the field of hazardous substance detection technology, specifically relating to a method for preparing a ratiometric electrochemical aptamer sensor based on ZIF-8 / Thi / Au. Background Technology

[0002] Among aflatoxins, aflatoxin B1 is the most toxic and is classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC). Furthermore, aflatoxin B1 is heat-resistant and can be present during normal cooking. To prevent such food safety incidents, many countries and regions strictly control the content of aflatoxin B1 in various foods.

[0003] Traditional methods for detecting aflatoxin B1 include high-performance liquid chromatography (HPLC), spectrophotometry, and liquid chromatography-mass spectrometry (LC-MS). These methods generally require complex sample pretreatment procedures, skilled operators, and expensive equipment, making them unsuitable for accurate on-site detection. Furthermore, enzyme-linked immunosorbent assay (ELISA) is widely used for aflatoxin B1 detection, but the preparation conditions for monoclonal antibodies are demanding, purification is difficult, and they cannot be stored for extended periods. ELISA can also produce false positives and has low sensitivity. Therefore, it is necessary to establish a simple, economical, and effective technique for detecting trace amounts of aflatoxin B1.

[0004] In recent years, electrochemical sensors have been widely used in the detection of aflatoxin B1 due to their advantages such as simple instrumentation and short detection cycles. As a novel specific recognition element, nucleic acid aptamers are structured single-stranded oligonucleotide sequences (DNA or RNA) screened using the Systematic Evolution of Ligands Exponential Enrichment (SELEX) technique. Their ease of simple, mass production has led to their widespread application in the fabrication of electrochemical sensors. Conventional single-signal electrochemical aptamer sensors typically generate an electrochemical signal by immobilizing a nucleic acid aptamer labeled with methylene blue or thionine onto a gold electrode surface, providing a rapid detection method for aflatoxin. However, single-signal output is easily affected by environmental factors, leading to insufficient accuracy and precision.

[0005] This invention addresses the problems of existing detection technologies by providing a method for preparing ZIF-8 / Thi / Au nanocomposite materials. The aim is to overcome the drawbacks of commonly used detection methods, such as high cost and insufficient sensitivity. This method uses a novel nanocomposite material as a signal probe, effectively reducing the sensor's fabrication cost and improving detection sensitivity. Furthermore, [Fe(CN)6] is introduced into the buffer solution. 3- / 4-As an internal reference probe, it makes the detection results more accurate and reliable. This invention constructs a ratiometric electrochemical aptamer sensor that can achieve accurate and rapid detection of aflatoxin B1 at low cost, which is conducive to the promotion and application of the invention. Summary of the Invention

[0006] A method for fabricating a ratiometric electrochemical aptamer sensor based on ZIF-8 / Thi / Au includes the following steps:

[0007] (1) Synthesis of signal tag: A methanol solution containing zinc nitrate and a methanol solution containing 2-methylimidazole were rapidly mixed and stirred at room temperature to obtain ZIF-8. Polyvinylpyrrolidone, thionine and chloroauric acid were added in sequence and stirred under heating conditions to obtain ZIF-8 / Thi / Au composite material. Then hairpin strand 2 was added and mixed and incubated to allow hairpin strand 2 to bind with ZIF-8 / Thi / Au composite material as a signal tag in subsequent experiments. ZIF-8 / Thi / Au can not only serve as a carrier of DNA strands, but also has good electrical properties and can generate electrochemical signals on its own.

[0008] (2) Construction of a ratiometric electrochemical aptamer sensor: Hairpin chain 1 was modified onto the electrode surface, and unbound active sites were blocked with 6-mercapto-1-ethanol. The aptamer chain and the complementary chain were bound together to form a double chain through base complementarity. After the target substance was added, the target substance bound to the aptamer chain, causing the complementary chain to fall off. The fallen complementary chain was dropped onto the modified electrode surface, and then the signal tag prepared in step (1) was added. After incubation, the electrode was rinsed, and the electrode was tested using an electrochemical workstation containing [Fe(CN)6]. 3- / 4- The electrochemical response values ​​were determined in a buffer solution to obtain ZIF-8 / Thi / Au and [Fe(CN)6]. 3- / 4- Compared to single-signal electrochemical sensors, this device cleverly utilizes two unlabeled signal tags as ratiometric signal outputs, shielding the detection environment from the influence of the signal generation and effectively improving the reliability of the detection.

[0009] Further specifying, in step (1), the heating conditions are 50~80 ℃.

[0010] Further specifying, the hairpin chain 1 sequence mentioned in steps (1) and (2) is 5'-AAT AGAGAA CAC GGT TGG GCA CGT GTT CTC T-3', and the hairpin chain 2 sequence is 5'-AAT AAT GAA CACGTC CCC AAC CGT GTT CTC T-3'.

[0011] Further specifying, in step (1), one end of the hairpin chain 2 is modified with a group that combines with the ZIF-8 / Thi / Au nanocomposite material, which is one or more of biotin, amino, carboxyl, and thiol, with thiol being preferred.

[0012] Further specifying, in step (2), one end of the hairpin chain 1 is modified with a group that binds to the electrode surface, which is one or more of biotin, amino, carboxyl, and thiol, with thiol being preferred.

[0013] Further specifying, in step (2), the concentration of the DNA strand is 2~5 μmol / L, the volume used is 5~10 μL, and the incubation time is 0.5~2 h.

[0014] Further specifying, in step (2), the volume of the signal tag is 5~10 μL and the incubation time is 1~3 h.

[0015] Further specifying, in step (2), the buffer solution is one or more of Tris buffer, PBS buffer, and PB buffer.

[0016] Compared with the prior art, the present invention has the following significant advantages:

[0017] 1. In this invention, chloroauric acid and thionine are first reacted with ZIF-8 to synthesize a ZIF-8 / Thi / Au nanocomposite material with good electrical properties and capable of generating current signals on its own, which serves as a signal tag with higher stability.

[0018] 2. This invention introduces a catalytic hairpin assembly reaction driven by the target fungal toxin, which effectively amplifies the signal and improves the detection sensitivity.

[0019] 3. This invention obtains an electrochemical aptamer sensor for detecting aflatoxin B1 by changing the signal ratio, which shields the influence of the detection environment on the signal and effectively improves the reliability of detection.

[0020] 4. The electrochemical sensing strategy based on the present invention has the characteristics of low cost, high sensitivity and good stability, which is conducive to the promotion and application of the invention. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the fabrication of a ratiometric electrochemical aptamer sensor based on ZIF-8 / Thi / Au.

[0022] Figure 2 The images shown are scanning electron microscope (SEM) images (A) and (B) of ZIF-8 and ZIF-8 / Thi / Au prepared in Example 1 of this invention.

[0023] Figure 3This is the electrochemical detection standard curve of aflatoxin B1 in Example 1 of the present invention.

[0024] Figure 4 This is a comparative graph showing the selectivity of the electrochemical aptamer sensor constructed in Example 2 of the present invention for aflatoxin B1 in the presence of other interfering toxins. Implementation

[0025] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Example

[0026] A method for fabricating a ratiometric electrochemical aptamer sensor based on ZIF-8 / Thi / Au is described below. Figure 1 As shown.

[0027] A method for fabricating a ratiometric electrochemical aptamer sensor based on ZIF-8 / Thi / Au includes the following steps:

[0028] (1) Preparation of ZIF-8: First, methanol (30 mL) containing zinc nitrate (1.0 g) was rapidly mixed with another type of methanol (30 mL) containing 2-methylimidazole (3.0 g) to obtain a milky white suspension. The prepared suspension was stirred at room temperature for 3 hours to synthesize white ZIF-8 powder.

[0029] (2) Preparation of the signal tag: 7 mg of ZIF-8 was ultrasonically dissolved in 21 mL of ultrapure water, and polyvinylpyrrolidone (5 wt% 2.1 mL), thionine (0.5 mM 21 mL), and chloroauric acid (1 wt% 175 μL) were added sequentially. The mixture was vigorously shaken and heated at 60 °C for 5 minutes to obtain the ZIF-8 / Thi / Au composite material. The obtained ZIF-8 / Thi / Au composite material was redissolved in 1 mL of ultrapure water, and hairpin chain 2 (4 mM 200 μL) was added. The mixture was gently shaken at 25 °C for 12 hours to allow hairpin chain 2 to bind with ZIF-8 / Thi / Au, thus obtaining the signal tag. Figure 2 A and Figure 2 Figure B shows scanning electron microscope images of ZIF-8 and ZIF-8 / Thi / Au.

[0030] (3) Construction of the electrochemical aptamer sensor: Hairpin chain 1 (2 μM 5 μL) was dropped onto the gold electrode surface and incubated at 37°C for 2 hours. Then, 5 μL of 6-mercapto-1-ethanol was added to block the unbound active sites and incubated at 37°C for 1 hour. Streptavidin-coated magnetic beads (10 mg / mL 3 μL), aptamer chain (2 μM 10 μL), and complementary chain (2 μM 10 μL) were mixed and incubated at 37°C for 1 hour. 10 μL of aflatoxin B1 standard solution was added and mixed and incubated at 37°C for another 1 hour. Then, magnetic separation was performed, and 5 μL of the supernatant was dropped onto the modified electrode surface and incubated at 37°C for 1 hour. Then, 5 μL of the signal tag prepared in step (2) was dropped onto the electrode surface and incubated at 37°C for 1 hour, thus completing the construction of the electrochemical aptamer sensor.

[0031] (4) Electrochemical detection of aflatoxin B1: The electrochemical aptamer sensor uses a saturated silver chloride electrode as the reference electrode and a platinum wire electrode as the counter electrode. The detection is achieved by detecting aflatoxin B1 in a substance containing [Fe(CN)6]. 3- / 4- The detection of aflatoxin B1 was performed based on the response of ZIF-8 / Thi / Au to ZIF-8 / Thi / Au in a buffer solution. The determination of the relationship between ZIF-8 / Thi / Au and [Fe(CN)6] was also performed. 3- / 4- The relationship between the signal ratio and the concentration of added aflatoxin B1 was analyzed, and a corresponding linear relationship curve was plotted.

[0032] like Figure 2 As shown, ZIF-8 / Thi / Au and [Fe(CN)6] 3- / 4- The signal ratio increases with increasing aflatoxin B1 concentration in the solution, and the linear regression equation is y = 0.733 + 0.165lgC(R). 2 =0.992), and the limit of detection (LOD) is 8.9 × 10⁻⁶. -5 ng / mL, where y represents ZIF-8 / Thi / Au and [Fe(CN)6] 3- / 4- The signal ratio, where C represents the concentration of aflatoxin B1 in the solution (ng / mL). Example

[0033] A method for fabricating a ratiometric electrochemical aptamer sensor based on ZIF-8 / Thi / Au includes the following steps:

[0034] To verify the specific recognition of aflatoxin B1 by the prepared ZIF-8 / Thi / Au-based electrochemical aptamer sensor, aflatoxin B1 standard was added to the buffer solution to a concentration of 10 ng / mL. Other interfering toxins (AFB2, AFM1, AFG1, DON, OTA, ZEN) standard solutions were prepared with buffer solution at a concentration of 100 ng / mL. The detection system constructed in Example 1 was used to detect these different interfering toxin standards, and the detection results are as follows: Figure 4 As shown, this demonstrates that the method of the present invention has high selectivity for aflatoxin B1. Example

[0035] A method for fabricating a ratiometric electrochemical aptamer sensor based on ZIF-8 / Thi / Au includes the following steps:

[0036] (1) Actual sample processing: Take 0.5 g of peanut spiked sample and add it to 10 mL of methanol / water solution (ratio 7:3). Place it in a water bath constant temperature shaker and shake for 30 minutes. Then take it out and place it in a centrifuge at 10000 rpm for 10 minutes. After centrifugation, take out the supernatant to obtain the spiked sample extract.

[0037] (2) Sample detection: ZIF-8 / Thi / Au and [Fe(CN)6] were obtained according to steps (1) to (4) of Example 1. 3- / 4- The signal ratio was used to calculate the concentration of aflatoxin B1 in the sample by substituting it into the standard curve. Each sample was measured three times, and the average value was taken. The relative standard deviation (RSD) and average recovery rate were calculated, as shown in the table below.

[0038]

[0039] The prepared electrochemical aptamer sensor has been verified to exhibit high accuracy, good sensitivity, and good stability in the detection of aflatoxin B1. Furthermore, detection results on actual samples (such as peanuts) demonstrate that the prepared electrochemical aptamer sensor has significant practical application value.

[0040] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0041] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing a ZIF-8 / Thi / Au-based ratio-type electrochemical aptamer sensor, characterized by, Includes the following steps (1) Synthesis of signal tag: A methanol solution containing zinc nitrate and a methanol solution containing 2-methylimidazole were rapidly mixed and stirred at room temperature to obtain ZIF-8. Polyvinylpyrrolidone, thionine and chloroauric acid were added in sequence and stirred under heating conditions to obtain ZIF-8 / Thi / Au composite material. Then hairpin strand 2 was added and mixed and incubated to allow hairpin strand 2 to bind with ZIF-8 / Thi / Au composite material as a signal tag in subsequent experiments. ZIF-8 / Thi / Au can not only serve as a carrier of DNA strands, but also has good electrical properties and can generate electrochemical signals on its own. (2) Construction of a ratiometric electrochemical aptamer sensor: Hairpin chain 1 was modified onto the electrode surface, and unbound active sites were blocked with 6-mercapto-1-ethanol. The aptamer chain and the complementary chain were bound together to form a double chain through base complementarity. After the target substance was added, the target substance bound to the aptamer chain, causing the complementary chain to fall off. The fallen complementary chain was dropped onto the modified electrode surface, and then the signal tag prepared in step (1) was added. After incubation, the electrode was rinsed, and the electrode was tested using an electrochemical workstation containing [Fe(CN)6]. 3- / 4- The electrochemical response values ​​were determined in a buffer solution to obtain ZIF-8 / Thi / Au and [Fe(CN)6]. 3- / 4- Compared to single-signal electrochemical sensors, this device cleverly utilizes two unlabeled signal tags as ratiometric signal outputs, shielding the detection environment from the influence of the signal generation and effectively improving the reliability of the detection. (3) In steps (1) and (2) above, the sequence of the hairpin chain 1 is 5'-AATAGAGAACACGGTTGGGCACGTGTT CTCT-3', the hairpin chain 2 has a sequence of 5'-AATAATGAACACGTCCCCAACCGTGTTCTCT-3', and the complementary chain has a sequence of 5'-CAG AGA GAC AAC ACG TGC CCAAC-3'.

2. The preparation method of a ZIF-8 / Thi / Au-based ratio-type electrochemical aptamer sensor according to claim 1, characterized in that, In step (1), the heating conditions are 50~80 ℃.

3. The preparation method of a ZIF-8 / Thi / Au-based ratio-type electrochemical aptamer sensor according to claim 1, characterized in that, In step (1), one end of the hairpin chain 2 is modified with a group that is combined with the ZIF-8 / Thi / Au nanocomposite material. This group is one or more of biotin, amino, carboxyl, and thiol groups.

4. The preparation method of a ZIF-8 / Thi / Au-based ratio-type electrochemical aptamer sensor according to claim 1, characterized in that, In step (2), one end of the hairpin chain 1 is modified with a group that binds to the electrode surface, which is one or more of biotin, amino, carboxyl, and thiol groups.

5. The method for preparing a ZIF-8 / Thi / Au-based ratio-type electrochemical aptamer sensor according to claim 1, characterized in that, In step (2), the concentrations of the hairpin chain 1, aptamer chain, and complementary chain are all 2~5 μmol / L, the volume used is 5~10 μL, and the incubation time is 0.5~2 h.

6. The method for preparing a ZIF-8 / Thi / Au-based ratio-type electrochemical aptamer sensor according to claim 1, characterized in that, In step (2), the volume of the signal tag is 5~10 μL and the incubation time is 1~3 h.

7. The method for preparing a ZIF-8 / Thi / Au-based ratio-type electrochemical aptamer sensor according to claim 1, characterized in that, In step (2), the buffer solution is one or more of Tris buffer, PBS buffer, and PB buffer.

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