Aptamer photoelectrochemical sensor for detecting toxins in food, preparation method and application thereof

Abstract

The present invention discloses a preparation method of an aptamer photoelectrochemical sensor for detecting toxins in food, comprising the steps of: dripping a TiO2 dispersion on the surface of an ITO conductive glass electrode, adding a chloroauric acid solution for electrodeposition after drying; modifying the electrode surface after deposition with a toxin aptamer in food for incubation, drying with nitrogen to obtain an apt / Au / TiO2 / ITO electrode, incubating the apt / Au / TiO2 / ITO electrode surface with 6-mercaptohexanol as a blocking agent, and drying to obtain a working electrode; using a saturated TiO2 solution as an electrolyte to obtain an aptamer photoelectrochemical sensor for detecting toxins in food. The preparation method can accurately detect the toxin concentration in food. The present invention also provides an aptamer photoelectrochemical sensor prepared by the method, and an application of the aptamer photoelectrochemical sensor in mycin analysis and detection.

Description

Technical Field

[0001] The present invention belongs to the field of sensor detection technology, and specifically relates to an aptamer photoelectrochemical sensor and its preparation method and application Background Art

[0002] Aflatoxin B1 (AFB1) is more toxic than cyanide, arsenide, and organic pesticides, causing serious damage to human organs, particularly the liver. Commonly used methods for detecting AFB1 residues include high-performance liquid chromatography (HPLC) and high-performance liquid chromatography-mass spectrometry (HPLC-MS). However, chromatographic methods are expensive, require high sample preparation, and require specialized operators. Therefore, a new method for rapid detection of aflatoxins in jujubes is urgently needed.

[0003] Photoelectrochemical (PEC) sensing uses two different forms of energy as the excitation and detection signals, resulting in low background signals and noise. However, under conditions of light excitation, the holes generated by PEC sensing technology have strong oxidizing power and can undergo redox reactions with organic matter, but cannot specifically oxidize the target. Nucleic acid aptamers, also known as aptamers, are oligonucleotide sequences or short polypeptides, generally single-stranded DNA or RNA sequences, selected from random single-stranded nucleic acid sequences through systematic evolution of ligands by exponential enrichment (SELEX). They have high specificity and high affinity for the target substance and have functional properties similar to antibodies, with the property of specifically binding to the target molecule. Due to their high specificity, high affinity, wide detection range, easy synthesis, and good stability, nucleic acid aptamers have been widely used in the fields of medicine, environmental testing, food safety testing, and other fields.

[0004] At the same time, the core of PEC sensing is the choice of photoelectric conversion materials. TiO2 is the most commonly used photoelectric conversion material due to its low preparation cost and non-toxicity. Yan et al. prepared anatase / rutile mixed phase titanium dioxide deposited with Au nanoparticles using a simple hydrothermal method and found that after 10,800 seconds of continuous illumination, the photocurrent exhibited significant attenuation. Therefore, effectively addressing the stability issues of the sensor during detection is urgent.

[0005] By combining the low background current and high sensitivity of photoelectrochemical detection technology with the excellent biocompatibility of gold nanoparticles, a nucleic acid aptamer is used as the sensitive recognition element of the sensor. Utilizing the aptamer's high specificity, a thiol-modified AFB1 aptamer is immobilized on a modified electrode via Au-S. This enables rapid detection and accurate quantification of AFB1 in food, overcoming a series of issues with traditional mass spectrometry and chromatography detection technologies, such as expensive instrumentation, time-consuming operation, complex operation, and the need for specialized personnel. Furthermore, by changing the active ingredient of the electrolyte, the stability of the TiO2 is effectively improved, thereby enhancing the stability of the sensor. Summary of the Invention

[0006] The present invention provides a method for preparing an aptamer photoelectrochemical sensor for detecting toxins in food. The preparation method can accurately detect the toxin concentration in food.

[0007] A method for preparing an aptamer photoelectrochemical sensor for detecting toxins in food, comprising:

[0008] Step 1: drop-coating a TiO2 dispersion on the surface of an ITO conductive glass electrode and drying the TiO2 / ITO electrode, and then adding the TiO2 / ITO electrode to a chloroauric acid solution for electrodeposition to obtain an Au / TiO2 / ITO electrode;

[0009] Step 2: Modify the food toxin aptamer on the surface of the Au / TiO2 / ITO electrode and incubate it. After drying with nitrogen, obtain the apt / Au / TiO2 / ITO electrode. Wherein, apt is the food toxin aptamer. 6-mercaptohexanol is used as a blocking agent to incubate it on the surface of the apt / Au / TiO2 / ITO electrode. After drying, obtain the MCH / apt / Au / TiO2 / ITO electrode.

[0010] Step 3: Use the MCH / apt / Au / TiO2 / ITO electrode as the working electrode and the TiO2 saturated solution as the electrolyte to obtain an aptamer photoelectrochemical sensor for detecting toxins in food.

[0011] The method for preparing the TiO2 dispersion comprises: adding TiO2 into ultrapure water to form a TiO2 solution, and ultrasonically dispersing the TiO2 solution to obtain the TiO2 dispersion.

[0012] The concentration of the TiO2 dispersion is 0.2-2 mg / mL.

[0013] The ultrasonic time is 30-60 min.

[0014] Before applying TiO2 dispersion on the surface of ITO conductive glass electrode, pre-treat the ITO conductive glass electrode, including:

[0015] The ITO electrode was placed in a 0.5-1M NaOH ethanol solution and ultrasonically cleaned. After rinsing with deionized water, the ITO electrode was immersed in acetone and ultrasonically cleaned, and then rinsed with deionized water. The cleaned ITO electrode was then immersed in ethanol, ultrasonically cleaned, and rinsed with deionized water. The electrode was immersed in deionized water and ultrasonically cleaned, rinsed with deionized water, blown dry with N2, and fixed.

[0016] The ultrasonic time in the NaOH ethanol solution is 30-60 h.

[0017] The modified area of the ITO electrode is 1.0 cm×0.5 cm.

[0018] In step 1, the parameters of the electrodeposition are: scanning range: -0.3 to 0 V; scanning rate: 30-40 mV / s; and number of scanning cycles: 2 to 5 cycles.

[0019] In step 2, the food toxin aptamer is an aflatoxin B1 aptamer, and the concentration of the aflatoxin B1 aptamer is 2-5 μM.

[0020] In step 2, the incubation parameters of the aflatoxin B1 aptamer are: incubation temperature of 25-37° C., and incubation time of 30-60 min.

[0021] In step 2, the incubation parameters of 6-mercaptohexanol are: incubation temperature is room temperature, and incubation time is 30-45 minutes.

[0022] In step 3, the preparation method of the electrolyte includes: dispersing TiO2 in a PBS buffer solution, ultrasonicating for 20-30 minutes, stirring at room temperature to obtain a pre-electrolyte, and centrifuging and filtering the pre-electrolyte to obtain an electrolyte, wherein the mass / volume ratio of TiO2 to PBS buffer solution is 1:10-20 g / mL.

[0023] The TiO2 concentration is 0.5-2 mg mL -1 .

[0024] The PBS buffer solution is 0.1-0.5M.

[0025] The stirring time is 2-3h.

[0026] The centrifugal speed is 5000-10000 rpm.

[0027] The present invention also provides an aptamer photoelectrochemical sensor prepared by using the preparation method of the aptamer photoelectrochemical sensor for detecting toxins in food.

[0028] The present invention also provides an application of the aptamer photoelectrochemical sensor in the analysis and detection of mycin, including:

[0029] Step 1: Apply aflatoxin B1 solution to the surface of the MCH / apt / Au / TiO2 / ITO electrode for incubation;

[0030] Step 2: Immerse the MCH / apt / Au / TiO2 / ITO electrode obtained in step 1 in the electrolyte of the aptamer photoelectrochemical sensor to perform current-time scanning to obtain a photocurrent signal, and detect the concentration of aflatoxin B1 based on the photocurrent signal.

[0031] The incubation parameters are: incubation time of 30-60 minutes, incubation temperature of room temperature, and a certain incubation time is given to fully identify and adsorb aflatoxin B1.

[0032] The aflatoxin B1 absorption amount is 35-50 μL.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention adds a sufficient amount of TiO2 to the electrolyte so that the concentration of TiO2 in the electrolyte reaches saturation, thereby avoiding the reaction of TiO2 in the MCH / apt / Au / TiO2 / ITO electrode in the electrolyte and avoiding interference of the photocurrent signal due to the reaction of TiO2 in the electrode, thereby obtaining an aptamer photoelectrochemical sensor capable of detecting toxin concentrations in food with high stability, accuracy and reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 A schematic diagram of a method for preparing an aptamer photoelectrochemical sensor for detecting toxins in food provided by an embodiment of the present invention;

[0036] Figure 2 The photocurrent linearity diagram of aflatoxin B1 under different concentration conditions provided in Application Example 1 of the present invention;

[0037] Figure 3 This is a comparison chart of the reproducibility results of the aptamer photoelectrochemical sensor prepared in Example 1 of the present invention in detecting aflatoxin B1;

[0038] Figure 4 This is a comparison chart of the photocurrent stability of the aptamer photoelectrochemical sensors prepared in Example 1 of the present invention and Comparative Example 1. DETAILED DESCRIPTION

[0039] The following specific examples further illustrate the present invention. The following examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The various reagents, reaction conditions, detection methods, etc. used in the following examples are, unless otherwise stated, considered to be conventional reagents, reaction conditions, and detection methods used in the art.

[0040] This invention uses Au / TiO2 as a photoelectric conversion material and combines it with aptamer technology to successfully fabricate an aptamer photoelectrochemical sensor. This sensor, which uses an Au / TiO2 composite material as the photoelectric conversion layer, achieves specific detection of aflatoxin B1 by surface-modifying an aptamer containing an aflatoxin B1 recognition site. The sensor exhibits a wide detection range, high sensitivity, stable response, and good reproducibility.

[0041] Example 1

[0042] A method for preparing an aptamer photoelectrochemical sensor for detecting toxins in food, such as Figure 1 Shown, including:

[0043] (1) 1 mg of TiO2 was dispersed in 1 mL of ultrapure water and ultrasonicated for 40 min to obtain a uniform TiO2 dispersion. 40 μL of the dispersion was then applied to the surface of an ITO electrode and dried under an infrared lamp for 30 min to form a TiO2 / ITO electrode (modified area 0.5 cm). 2 ); TiO2 / ITO was placed in a 1% HAuCl4 solution and cyclic voltammetry was performed in the potential range of -0.3V to 0V for 5 cycles. The electrode was removed and carefully rinsed with ultrapure water and dried with nitrogen to obtain an Au / TiO2 / ITO electrode.

[0044] (2) 40 μL of aflatoxin B1 (AFB1) aptamer solution was added dropwise onto the surface of the Au / TiO2 / ITO electrode and incubated at room temperature for 40 min. The electrode was then rinsed with ultrapure water to remove the unbound aflatoxin B1 (AFB1) aptamer and dried with nitrogen to obtain a modified electrode Apt / Au / TiO2 / ITO electrode. 40 μL of 6-mercaptohexanol (MCH) was added dropwise onto the modified electrode to block the sites on the modified electrode that were not bound to the aptamer. The electrode was incubated at room temperature for 1 h to obtain an MCH / Apt / Au / TiO2 / ITO electrode. The construction of the aptamer photoelectrochemical sensing interface was completed.

[0045] (3) 1 g of TiO2 was dispersed in 20 mL of PBS buffer solution and ultrasonically stirred at room temperature for 20 min to obtain a pre-electrolyte. The pre-electrolyte was centrifuged and filtered to obtain a Ti solution as the electrolyte for the prepared sensor. The MCH / Apt / Au / TiO2 / ITO electrode was placed in the electrolyte to obtain an aptamer photoelectrochemical sensor for detecting toxins in food, such as Figure 1 As shown in a.

[0046] Application Example 1

[0047] The present invention provides an application of an aptamer photoelectrochemical sensor in the analysis and detection of mycin, including

[0048] Jujube samples (from a local supermarket) were selected to evaluate the practical application performance of the aptamer photoelectrochemical sensor. A certain amount of AFB1 standard solution was added to the jujube sample. After sample pretreatment, an aflatoxin B1 solution was prepared. The aflatoxin B1 solution was applied to the surface of the MCH / apt / Au / TiO2 / ITO electrode prepared in Example 1 and incubated for 1 hour at room temperature. The aflatoxin B1 absorption amount was 45 μL. Figure 1 As shown in Figure b, when aflatoxin B1 solution (AFB1) is added to the aptamer photoelectrochemical sensor for detecting toxins in food, the photocurrent signal decreases significantly, and the concentration of aflatoxin B1 can be detected based on the decrease ratio.

[0049] The incubated MCH / apt / Au / TiO2 / ITO electrode was immersed in 4 mL of the electrolyte of the aptamer photoelectrochemical sensor prepared in Example 1, and then a current-time scan was performed to obtain a photocurrent signal, and the concentration of the mycin was detected based on the photocurrent signal. Figure 2 As shown in (a), the concentration of aflatoxin B1 is 0.0001 μg·mL -1 , 0.001 μg·mL -1 , 0.01 μg·mL -1 , 0.1 μg·mL -1 , 1 μg·mL -1 and 10 μg·mL -1 , the photocurrent signal formed is relatively stable; Figure 2 As shown in (b), the photocurrent response decreases with the increase of aflatoxin B1 concentration, and the related linear relationship is ΔI = 0.66 logC (ng mL -1 )+0.09(R 2 =0.992).

[0050] Example 2

[0051] (1) 1 mg of TiO2 was dispersed in 1 mL of ultrapure water and ultrasonicated for 40 min to obtain a uniform TiO2 dispersion. 40 μL of the dispersion was then applied to the surface of an ITO electrode and dried under an infrared lamp for 30 min to form a TiO2 / ITO electrode (modified area 0.5 cm). 2 ); TiO2 / ITO was placed in a 1% HAuCl4 solution and cyclic voltammetry was performed in the potential range of -0.3V to 0V for 5 cycles. The electrode was removed and carefully rinsed with ultrapure water and dried with nitrogen to obtain an Au / TiO2 / ITO electrode.

[0052] (2) 40 μL of aptamer solution was added dropwise to the surface of the Au / TiO2 / ITO electrode and incubated at room temperature for 40 min. The electrode was then rinsed with ultrapure water to remove unbound aptamers and dried with nitrogen to obtain a modified electrode, Apt / Au / TiO2 / ITO. 40 μL of 6-mercaptohexanol (MCH) was added dropwise to block the sites on the modified electrode that were not bound to the aptamer. The electrode was incubated at room temperature for 1 h to obtain an MCH / Apt / Au / TiO2 / ITO electrode. The construction of the aptamer photoelectrochemical sensing interface was completed.

[0053] (3) 0.2 g of TiO2 was dispersed in 20 mL of PBS buffer solution and ultrasonically stirred at room temperature for 30 min to obtain a pre-electrolyte. The pre-electrolyte was centrifuged and filtered to obtain a Ti solution, which served as the electrolyte for the prepared sensor. An MCH / Apt / Au / TiO2 / ITO electrode was placed in the electrolyte to obtain an aptamer photoelectrochemical sensor for detecting toxins in food.

[0054] Comparative Example 1

[0055] The difference from Example 1 is that TiO2 was not added to the 20 mL PBS buffer solution. The experimental results show that even under ultrasonic stirring treatment, the photocurrent of MCH / Apt / Au / TiO2 / ITO still shows a downward trend over time.

[0056] Performance Characterization

[0057] Five aptamer photoelectrochemical sensors were prepared using Example 1. The MCH / Apt / Au / TiO2 / ITO in the five aptamer photoelectrochemical sensors was heated to a concentration of 1 μg mL -1 The aflatoxin B1 was incubated separately, and then the signal was detected. The concentration of the analyte OTC was 1 μg mL -1 .like Figure 3 As shown in the figure, the sensors prepared with 5 electrodes have similar signal responses, indicating that the prepared sensors have good reproducibility.

[0058] The photocurrent test was performed using the aptamer photoelectrochemical sensor for detecting toxins in food prepared in Example 1 and the aptamer photoelectrochemical sensor for detecting toxins in food prepared in Comparative Example 1. Figure 4 As shown, it was found that the photocurrent of the MCH / Apt / Au / TiO2 / ITO working electrode decreased rapidly in the electrolyte of Comparative Example 1, but the photocurrent remained basically unchanged under long-term testing in the electrolyte of Comparative Example 1, indicating that the MCH / Apt / Au / TiO2 / ITO working electrode has good stability in the electrolyte containing saturated Ti.

Claims

1. A method for preparing an aptamer photoelectrochemical sensor for detecting toxins in food, characterized in that: include: Step 1: drop-coating a TiO2 dispersion on the surface of an ITO conductive glass electrode and drying the TiO2 / ITO electrode, and then adding the TiO2 / ITO electrode to a chloroauric acid solution for electrodeposition to obtain an Au / TiO2 / ITO electrode; Step 2: Modify the food toxin aptamer on the surface of the Au / TiO2 / ITO electrode and incubate it. After drying with nitrogen, obtain the apt / Au / TiO2 / ITO electrode. Wherein, apt is the food toxin aptamer. 6-mercaptohexanol is used as a blocking agent to incubate it on the surface of the apt / Au / TiO2 / ITO electrode. After drying, obtain the MCH / apt / Au / TiO2 / ITO electrode. Step 3: Using the MCH / apt / Au / TiO2 / ITO electrode as the working electrode and the TiO2 saturated solution as the electrolyte, an aptamer photoelectrochemical sensor for detecting toxins in food was obtained; The preparation method of the electrolyte comprises: dispersing TiO2 in a PBS buffer solution, ultrasonicating for 20-30 minutes, stirring at room temperature to obtain a pre-electrolyte, and centrifugally filtering the pre-electrolyte to obtain an electrolyte, wherein the mass / volume ratio of the TiO2 to the PBS buffer solution is 1:10-20 g / mL.

2. The method for preparing the aptamer photoelectrochemical sensor for detecting toxins in food according to claim 1, characterized in that: The method for preparing the TiO2 dispersion comprises: adding TiO2 into ultrapure water to form a TiO2 solution, and ultrasonically dispersing the TiO2 solution to obtain the TiO2 dispersion.

3. The method for preparing the aptamer photoelectrochemical sensor for detecting toxins in food according to claim 1, characterized in that: The concentration of the TiO2 dispersion is 0.2-2 mg / mL.

4. The method for preparing the aptamer photoelectrochemical sensor for detecting toxins in food according to claim 1, characterized in that: In step 1, the parameters of the electrodeposition are: scanning range: -0.3 to 0 V; scanning rate: 40 to 60 mV / s; and number of scanning cycles: 2 to 5 cycles.

5. The method for preparing the aptamer photoelectrochemical sensor for detecting toxins in food according to claim 1, characterized in that: In step 2, the food toxin aptamer is an aflatoxin B1 aptamer, and the concentration of the aflatoxin B1 aptamer is 2-5 μM.

6. The method for preparing the aptamer photoelectrochemical sensor for detecting toxins in food according to claim 1, characterized in that: PBS buffer solution is 0.1-0.5M.

7. An aptamer photoelectrochemical sensor prepared according to the method for preparing an aptamer photoelectrochemical sensor for detecting toxins in food according to any one of claims 1 to 6.

8. An application of the aptamer photoelectrochemical sensor prepared by the method for preparing the aptamer photoelectrochemical sensor for detecting toxins in food according to claim 7 in toxin analysis and detection, comprising: Step 1: applying aflatoxin B1 solution to the surface of the MCH / apt / Au / TiO2 / ITO electrode for incubation; Step 2: Immerse the MCH / apt / Au / TiO2 / ITO electrode obtained in step 1 in an electrolyte of a TiO2 saturated solution and perform a current-time scan to obtain a photocurrent signal, and detect the concentration of aflatoxin B1 based on the photocurrent signal.

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