Bio-aptamer sensor with double photoelectrodes and application of bio-aptamer sensor in ochratoxin A detection

Abstract

The invention discloses a dual-photoelectrode biological aptamer sensor and application thereof in ochratoxin A. A portable electrochemical workstation and a color signal converter are used as output devices, ZnO / ZnIn2S4 / Bi2S3 and Cu2O / Cu2S are used as a photoanode material and a photocathode material respectively, and PFC is constructed through an enzymatic reaction induced by prepared platinum nano-enzyme; prussian blue (PB) is prepared in an adjacent region of a photoelectric cathode material by utilizing an electrodeposition technology to construct the electrochromic biological aptamer sensor. According to the photoelectric / electrochromic dual-photoelectrode biological aptamer sensor, concept verification is provided for integration of a target object, and then portable detection of ochratoxin A is achieved through a photoelectrochemical / electrochromic dual-mode biological aptamer sensing platform.

Description

Technical Field

[0001] The present invention relates to the technical field of photoelectrochemical sensors, and specifically to a portable dual-photoelectrode aptasensor and its application in the detection of ochratoxin A. Background Art

[0002] With the continuous development of contemporary society, people's attention to food safety is also rising continuously. Among the many links in food processing and storage and transportation, it is possible to suffer from varying degrees of pollution. Therefore, it is necessary to strictly control the pollution risks of pathogenic microorganisms and mycotoxins to food during the processing and storage and transportation processes. Among them, mycotoxins refer to a class of secondary metabolites produced by molds in contaminated grains, which usually have teratogenic, mutagenic, and carcinogenic properties, and enter the human and animal bodies directly or indirectly through various channels, causing serious and irreversible health damage. Among the many mycotoxins, ochratoxin A (OTA) has cytotoxicity and genotoxicity even at low concentrations. When humans consume food contaminated with OTA for a long time, the toxicity of OTA poses a great threat to the health and safety of the kidneys.

[0003] Among modern detection technologies, high performance liquid chromatography-mass spectrometry (HPLC-MS) is usually used for quantitative analysis of biotoxins. However, this technology requires the deployment of relevant technical personnel, complex sample pretreatment, relatively high costs, and has many limitations in actual operation. Therefore, there is an urgent need to establish a portable, sensitive, fast, and low-cost method for detecting low-abundance mycotoxins.

[0004] Among the related patents for detecting OTA toxin that have been disclosed, CN118604098A is a preparation method of an electrochemical aptasensor for detecting ochratoxin A in traditional Chinese medicine. By using digoxin labeling to replace the traditional biotin labeling, the non-specific recognition of streptavidin-modified magnetic beads is avoided, the detection performance of the sensor is improved, and the sensitive detection of OTA in the complex matrix of traditional Chinese medicine is realized. However, false positive or false negative signals may occur in single-mode detection.

[0005] CN118914320A is a method for detecting ochratoxin A by a dual-mode bipolar electrode electrochemiluminescence sensor. By adopting a dual-aptamer sandwich immune strategy and introducing ZnCoN-C to regulate the emission potential of bipolar electrode electrochemiluminescence, the sensitive detection and imaging analysis of OTA are realized, but this device fails to achieve portable detection.

[0006] CN115711926B Preparation method of a molecularly imprinted electrochemical sensor for detecting ochratoxin A. By generating a polymer film on the surface of the Mxene-CNTs nanocomposite to obtain a molecular imprinting template, the enrichment and sensitive detection of OTA are realized. This method has good selectivity and anti-interference ability. However, this method fails to achieve visual and portable detection. Summary of the Invention

[0007] In view of the deficiencies of the prior art, the present invention provides a portable dual-photoelectrode photocatalytic fuel (PFC) and an electrochromic dual-mode to construct a biosensor for sensitive detection of ochratoxin A (OTA).

[0008] A preparation method of a dual-photoelectrode biosensor of the present invention includes the following steps:

[0009] (1) Prepare the photoanode material ZnO / ZnIn2S4 / Bi2S3, including the following steps:

[0010] (1.1) Take ZnO and place it in ethylene glycol, and dissolve it by ultrasonic treatment;

[0011] (1.2) Subsequently, add ZnCl2, InCl3·4H2O, Bi(NO3)3·5H2O and TAA. Each time a substance is added, stir and dissolve it first, and then add another substance and continue to stir and dissolve it. Finally, pour the mixture into a polytetrafluoroethylene reaction kettle for reaction;

[0012] (1.3) Centrifuge to collect the black precipitate obtained in the reaction kettle, wash it several times with water and ethanol respectively, and dry it overnight to obtain the photoanode material ZnO / ZnIn2S4 / Bi2S3;

[0013] (2) Prepare the photocathode material Cu2O / Cu2S, including the following steps:

[0014] (2.1) Take Cu2O and dissolve it in water, and then add Na2S and stir;

[0015] (2.2) Centrifuge to collect the obtained black precipitate, wash it several times with water and ethanol respectively, and dry it overnight to obtain the photocathode material Cu2O / Cu2S;

[0016] (3) Prepare Pt nanozyme and OTA-Apt-Pt probe, including the following steps:

[0017] (3.1) Take H2PtCl6 and place it in water, add trisodium citrate, stir, and then slowly dropwise add NaBH4, and stir at room temperature to obtain Pt nanozyme;

[0018] (3.2) Mix the Pt nanozyme suspension with the aptamer OTA-Apt solution, and incubate after mixing to obtain the OTA-Apt-Pt probe;

[0019] (4) Prepare the modified photoanode and photocathode, including the following steps:

[0020] (4.1) Immerse the indium tin oxide glass in ultrapure water and anhydrous ethanol successively, and perform ultrasonic pretreatment, then dry for subsequent use;

[0021] (4.2) Drop the suspension ZnO / ZnIn2S4 / Bi2S3 and the Cu2O / Cu2S dispersion on the prepared ITO, then dry. The obtained modified electrodes ZnO / ZnIn2S4 / Bi2S3 / ITO and Cu2O / Cu2S / ITO are used as the photoanode and photocathode respectively;

[0022] (5) Construct a dual-photoelectrode PFC aptasensor, including the following steps:

[0023] (5.1) Cast the ZnO / ZnIn2S4 / Bi2S3 suspension on the cleaned ITO electrode, and dry after casting;

[0024] (5.2) Then drop chitosan, and dry after dropping;

[0025] (5.3) Take glutaraldehyde and cast it on the electrode in step (5.2), and store it in the dark;

[0026] (5.4) Then, drop the aminated cDNA solution and incubate overnight at low temperature;

[0027] (5.5) Take the BSA solution and coat it on the electrode, and let it stand for a period of time to occupy the non-specific active sites;

[0028] (5.6) Take the OTA-apt-Pt suspension and cast it on the modified electrode, and fix it at a certain temperature for a period of time after casting;

[0029] (5.7) Incubate the sensor constructed in step (5.6) with a series of concentrations of OTA at a certain temperature for a period of time;

[0030] (5.8) Put the sensor into the mixed solution of H2O2 and 4-CN, and incubate for a period of time. After each step of modification, wash the electrode surface with PBS buffer solution;

[0031] (5.9) Immerse the prepared optoelectronic anode aptamer ZnO / ZnIn2S4 / Bi2S3 / ITO and the optoelectronic cathode Cu2O / Cu2S / ITO into a single-chamber quartz electrolytic cell filled with PBS electrolyte. Use a xenon lamp to vertically irradiate the dual optoelectronic electrodes. The irradiation generates transient photocurrent, and use a portable electrochemical workstation to record the change of the transient photocurrent;

[0032] (6) Construct an electrochromic visualization sensor, including the following steps:

[0033] (6.1) Take HCl and place it in a beaker of deionized water, stir vigorously, and then add FeCl3 and stir to dissolve;

[0034] (6.2) Take KCl and dissolve it in another beaker filled with deionized water, and then add potassium ferricyanide and stir to dissolve;

[0035] (6.3) Mix the solutions in the beakers, add deionized water and stir to obtain a PB solution;

[0036] (6.4) Under a three-electrode system, use linear cyclic voltammetry to electrodeposit PB on the ITO electrode to obtain a PB / ITO electrode;

[0037] (6.5) Cast the Cu2O / Cu2S suspension near the PB / ITO electrode. While recording the change of the transient photocurrent in step (5), PB receives electrons and is converted into PW. Use a mobile phone software color signal converter to read the RGB value and record the color change.

[0038] Further, in the preparation method step (1.1), the dosage ratio of ZnO to ethylene glycol is 0.1 - 1 g:10 - 50 mL;

[0039] In step (1.2), the addition amount of ZnCl2 is 0.001 - 0.01 g;

[0040] The addition amount of InCl3·4H2O is 0.1 - 1 g;

[0041] The addition amount of Bi(NO3)3·5H2O is 0.1 - 1 g;

[0042] The addition amount of TAA is 0.1 - 1 g;

[0043] After adding substances, the stirring time is 2 - 30 min;

[0044] Finally, pour the mixture into a 50 mL polytetrafluoroethylene reaction kettle for reaction. The reaction temperature is 150 - 180 °C, and the reaction time is 10 - 15 h.

[0045] Further, in the preparation method step (2.1), the dosages of Cu2O and water are 0.01 - 0.1 g: 10 - 50 mL;

[0046] The addition amount of Na2S is 10 - 20 mL, the concentration is 0.001 - 0.01 M, and after adding Na2S, stir for 20 - 30 min.

[0047] Further, in the preparation method step (3.1), the dosage ratio of H2PtCl6 to water is 1 - 10 mL: 30 - 40 mL, and the concentration of H2PtCl6 is 16 mM;

[0048] The addition amount of trisodium citrate is 1 - 10 mL, the concentration is 40 mM, and after adding, stir for 20 - 30 min;

[0049] The addition amount of NaBH4 is 100 - 1000 μL, the concentration is 50 mM, and after adding, stir for 30 - 60 min;

[0050] In step (3.2), the dosages of the Pt nanozyme suspension and the aptamer solution are 1 - 10 mL: 1 - 10 mL;

[0051] The concentration of the aptamer solution is 1.0 μM;

[0052] After mixing the Pt nanozyme suspension and the aptamer solution, incubate at 4 °C for 10 - 12 h.

[0053] Further, in the preparation method step (4.2), the ZnO / ZnIn2S4 / Bi2S3 suspension and the Cu2O / Cu2S dispersion are prepared by dissolving ZnO / ZnIn2S4 / Bi2S3 and Cu2O / Cu2S respectively in a mixed solution of 5 wt% Nafion and absolute ethanol;

[0054] The volume ratio of Nafion to absolute ethanol is 1:9;

[0055] The dosage of the ZnO / ZnIn2S4 / Bi2S3 suspension is 10 - 100 μL, and the concentration is 1 - 5 mg / mL;

[0056] The dosage of the Cu2O / Cu2S dispersion is 10 - 100 μL, and the concentration is 1 - 5 mg / mL;

[0057] The geometric area of the exposed ITO is 0.16π cm 2 .

[0058] Further, in the preparation method step (5.1), the dosage of the ZnO / ZnIn2S4 / Bi2S3 suspension is 10 - 50 μL, and the concentration is 1 - 5 mg / mL;

[0059] In step (5.2), chitosan is first dissolved in a 1% acetic acid solution. After dissolution, it is added dropwise, with the addition amount being 10 - 100 μL and the concentration being 0.1 wt%. After addition, it is dried at 40 - 50 °C for 1 h;

[0060] In step (5.3), the amount of glutaraldehyde used is 10 - 100 μL and the concentration is 5 wt%;

[0061] In step (5.4), the addition amount of the cDNA solution is 10 - 100 μL and the concentration is 1 μM. After addition, it is incubated overnight at 4 °C;

[0062] In step (5.5), the amount of the BSA solution used is 10 - 100 μL;

[0063] In step (5.6), the amount of the OTA-apt-Pt suspension used is 10 - 100 μL and the concentration is 1 μM. After the OTA-apt-Pt suspension is cast on the modified electrode, it is fixed at 37 °C for 1 h;

[0064] In step (5.7), the concentration of OTA is 1×10 -4 ~1×10 2 ng mL -1 , and the sensor is incubated with OTA at 37 °C for 1 h;

[0065] In step (5.8), the concentration of H2O2 is 0.1 - 1 mM, the concentration of 4-CN is 1 - 10 mM, and the sensor is incubated in the solution for 30 min,

[0066] the concentration of the PBS buffer solution is 0.01 M, P and the pH is 7.40;

[0067] In step (5.9), the amount of the PBS electrolyte used is 20 mL and the concentration is 10 mM;

[0068] The power of the xenon lamp is 200 - 800 W.

[0069] Furthermore, in step (6.1) of the preparation method, the usage ratio of HCl to deionized water is 10 - 100 μL:10 - 50 mL, the concentration of HCl is 35%, and the addition amount of FeCl3 is 0.01 - 0.1 g;

[0070] In step (6.2), the usage ratio of KCl to deionized water is 0.1 - 1 g:10 - 50 mL, and the addition amount of potassium ferricyanide is 0.01 - 0.1 g;

[0071] In step (6.3), the addition amount of deionized water is 10 - 50 mL and the stirring time is 30 min;

[0072] In step (6.4), the electrodeposition parameters are set between 0.4 - 0.8 V, the scanning rate is 0.1 V / S, and the cycle is 150 times.

[0073] In the present invention, a portable electrochemical workstation and a color signal converter are used as output devices. ZnO / ZnIn2S4 / Bi2S3 and Cu2O / Cu2S are used as the photoanode and photocathode materials respectively. A PFC is constructed by inducing an enzymatic reaction with the prepared platinum nanozyme. A Prussian blue (PB) is prepared in the adjacent area of the photocathode material by electrodeposition technology to construct an electrochromic aptasensor.

[0074] The present invention also provides the application of the dual-photoelectrode aptasensor prepared by the above preparation method in detecting ochratoxin A (OTA).

[0075] The principle of detecting ochratoxin A with the dual-photoelectrode aptasensor of the present invention is that, in the photoelectrochemical mode, under the irradiation of a xenon lamp, the two photoanodes are simultaneously excited, and the photo-generated electrons are transferred from the photoanode to the photocathode through an external circuit. The platinum nanozyme has catalytic peroxidase-like activity. In the presence of hydrogen peroxide, the platinum nanozyme can catalyze the conversion of 4-chloro-1-naphthol (4-CN) into an insoluble precipitate, benzo-4-chloro-hexadienone (4-CD), which inhibits the output of the photocurrent signal.

[0076] When the OTA aptamer modified with platinum nanozyme and its complementary strand are modified on the photoanode, since the binding force between OTA toxin and the aptamer is better than that between the aptamer and the complementary strand, after the aptamer specifically recognizes OTA, OTA and the aptamer modified with platinum nanozyme fall off from the electrode surface, and the amount of platinum nanozyme with induced enzymatic reaction on the electrode surface decreases. The amount of 4-CD generated decreases accordingly, the steric hindrance on the electrode surface caused by catalysis decreases, and the electron transfer rate increases, thus forming a "signal-on" detection of OTA.

[0077] In the electrochromic mode, since the photocurrent signal intensity in the photoelectrochemical mode increases with the increase of OTA concentration, more electrons are received by PB on the photocathode, and PB undergoes a redox reaction, causing PB to transform into Prussian white (PW), resulting in a gradual increase in the R + B value. With a portable electrochemical workstation and a color signal converter of a smartphone as the signal output device, accurate detection of OTA by the photoelectrochemical / electrochromic dual-photoelectrode aptasensor is realized.

[0078] The photoelectric / electrochromic dual-photoelectrode aptasensor of the present invention provides a proof of concept for the integration of the target substance, and further enables the sensor to realize the portable detection of ochratoxin A. Description of the Drawings

[0079] Figure 1Standard curve of the sensor prepared in the embodiment for detecting the content of OTA in the standard solution in the optoelectronic mode;

[0080] Figure 1 The inset in is the logarithmic linear relationship curve between the photocurrent signal intensity and the OTA concentration;

[0081] In the figure, the OTA concentrations are 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100 ng mL -1 ;

[0082] Figure 2 Standard curve of the sensor prepared in the embodiment for detecting the content of OTA in the standard solution in the electrochromic mode;

[0083] Figure 2 The inset in is the logarithmic linear relationship curve between the R + B value and the OTA concentration;

[0084] In the figure, the OTA concentrations are 0.01, 0.05, 0.001, 0.1, 0.5, 1, 5, 10, 50, 100 ng mL -1 . Detailed implementation manners

[0085] The content of the present invention will be further described below in conjunction with the embodiments and the drawings, but it is not a limitation of the present invention.

[0086] Embodiment

[0087] A preparation method of a dual-photoelectrode aptasensor includes the following steps:

[0088] Step (1) Prepare the optoelectronic anode material ZnO / ZnIn2S4 / Bi2S3, including the following steps:

[0089] (1.1) Take 0.0061 g of ZnO and place it in 30 mL of ethylene glycol, and dissolve it by ultrasonic treatment;

[0090] (1.2) Subsequently, add 0.14 g of ZnCl2, stir for 2 min to dissolve, add 0.59 g of InCl3·4H2O, stir for 15 min to dissolve, add 0.4851 g of Bi(NO3)3·5H2O, stir for 2 min to dissolve, add 0.45 g of TAA and stir for 30 min to dissolve. Finally, pour the mixture into a 50 mL polytetrafluoroethylene reaction kettle and react at 160 °C for 12 h;

[0091] (1.3) Centrifuge to collect the black precipitate obtained in the reaction kettle, wash it 3 times with water and ethanol respectively, and dry it overnight at 60 °C to obtain the optoelectronic anode material ZnO / ZnIn2S4 / Bi2S3.

[0092] Step (2) Prepare the photocathode material Cu2O / Cu2S, including the following steps:

[0093] (2.1) Take 0.04 g of Cu2O and dissolve it in 30 mL of water. Then add 20 mL of 0.006 M Na2S and stir for 30 min;

[0094] (2.2) Centrifuge to collect the obtained black precipitate, wash it 3 times with water and ethanol respectively, and dry it overnight at 60 °C to obtain the photocathode material Cu2O / Cu2S.

[0095] Step (3) Prepare Pt nanozyme and OTA-Apt-Pt probe, including the following steps:

[0096] (3.1) Take 1 mL of 16 mM H2PtCl6 and place it in 38 mL of water. Add 1 mL of 40 mM trisodium citrate, stir for 30 min, and then slowly add 600 μL of 50 mM NaBH4 and stir at room temperature for 1 h to obtain Pt nanozyme;

[0097] (3.2) Take 2 mL of the Pt nanozyme suspension and mix it with 2 mL of 1.0 μM aptamer OTA-Apt solution, and incubate at 4 °C for 12 h to obtain OTA-Apt-Pt probe.

[0098] Step (4) Prepare the modified photoanode and photocathode, including the following steps:

[0099] (4.1) Immerse indium tin oxide (ITO) glass in ultrapure water and absolute ethanol successively and perform ultrasonic pretreatment, and dry it for subsequent use;

[0100] (4.2) Disperse 40 μL of the suspension (dissolved in a V:V = 1:9 Nafion (5 wt%) : absolute ethanol solution) ZnO / ZnIn2S4 / Bi2S3 and Cu2O / Cu2S on the prepared ITO (the exposed geometric area is 0.16π cm -1 ) and dry at 40 °C. The obtained modified electrodes ZnO / ZnIn2S4 / Bi2S3 / ITO and Cu2O / Cu2S / ITO are used as photoanode and photocathode respectively. 2 ) respectively.

[0101] Step (5) Construct a dual-photoelectrode PFC aptasensor, including the following steps:

[0102] (5.1) Cast 10 - 50 μL of the ZnO / ZnIn2S4 / Bi2S3 suspension (4 mg mL -1 ) on the cleaned ITO electrode and dry at 40 °C;

[0103] (5.2) Chitosan was first dissolved in a 1% acetic acid solution. After dissolution, 20 μL of chitosan with a concentration of 0.1 wt% was added dropwise. After the addition, it was dried at 40 °C for 1 h;

[0104] (5.3) 20 μL of glutaraldehyde with a concentration of 5 wt% was cast on the electrode in step (5.2) and stored in the dark for 1 h;

[0105] (5.4) 20 μL of an amine - modified cDNA solution with a concentration of 1 μM was added dropwise. After the addition, it was incubated overnight at 4 °C;

[0106] (5.5) 10 μL of BSA solution was coated on the electrode and allowed to stand for 1 h to occupy non - specific active sites;

[0107] (5.6) 20 μL of an OTA - apt - Pt suspension with a concentration of 1 μM was cast on the modified electrode and fixed at 37 °C for 1 h;

[0108] (5.7) The sensor constructed in step (5.6) was incubated with a series of concentrations of OTA (1×10 -4 -1×10 2 ng mL -1 ) at 37 °C for one hour;

[0109] (5.8) The sensor was placed in a mixed solution containing 0.15 mM of H2O2 and 1 mM of 4 - CN and incubated for 30 min. After each modification step, the electrode surface was washed with PBS (0.01 M, pH 7.40) buffer solution;

[0110] (5.9) The prepared photoanode aptamer ZnO / ZnIn2S4 / Bi2S3 / ITO and photocathode Cu2O / Cu2S / ITO were immersed in a single - chamber quartz electrolytic cell containing 20 mL of 10 mM PBS electrolyte. A 500 W xenon lamp was used to vertically irradiate the double - photo - electrode, and the transient photocurrent was generated for 20 s. The change of the transient photocurrent was recorded using a portable electrochemical workstation.

[0111] Step (6) to construct an electrochromic visualization sensor includes the following steps:

[0112] (6.1) 10 μL of 35% HCl was placed in a beaker containing 40 mL of deionized water and stirred vigorously. Subsequently, 0.02 g of FeCl3 was added and dissolved;

[0113] (6.2) 0.15 g of KCl was placed in another beaker containing 15 mL of deionized water and dissolved. Subsequently, 0.04 g of potassium ferricyanide was added and stirred until dissolved;

[0114] (6.3) Mix the solutions in the beaker, add 20 mL of deionized water and stir for 30 min to obtain Prussian blue (PB) solution;

[0115] (6.4) Under a three - electrode system, electro - deposit PB on the ITO electrode using linear cyclic voltammetry. The electro - deposition parameters are set between 0.4 - 0.8 V, the scanning rate is 0.1 V / s, and cycle 150 times to obtain the PB / ITO electrode;

[0116] (6.5) Cast the Cu2O / Cu2S suspension near the PB / ITO electrode. While recording the instantaneous photocurrent change in step (5), PB receives electrons and is converted into Prussian white (PW). Use a mobile phone software color signal converter to read the RGB value and record the color change.

[0117] The example analyzes and determines different concentrations of OTA toxin through the immunosensing platform of the prepared optoelectronic and electrochromic sensors.

[0118] Refer to Figure 1 and Figure 1 As shown in the illustration, the detection range of OTA toxin in the optoelectronic mode is 0.0001 - 100 ng mL -1 , and its photocurrent signal intensity increases with the increase of OTA concentration. This is because the binding force between OTA toxin and the aptamer is better than that between the aptamer and the complementary strand. When OTA toxin is added dropwise to the sensor for incubation, the aptamer specifically recognizes OTA, and OTA and the aptamer modified with platinum nanozyme fall off from the electrode surface. The amount of platinum nanozyme with induced enzymatic reaction on the electrode surface decreases, the generation amount of 4 - CD decreases accordingly, the catalytic - induced steric hindrance on the electrode surface decreases, and the electron transfer rate increases, thus forming a "signal - on" detection of OTA. And within this linear range, the photocurrent signal intensity has a linear relationship with the logarithm of OTA concentration (the illustration is the logarithmic linear relationship curve of photocurrent signal intensity and OTA concentration). The linear equation is I=-64.047×lgC |OTA| - 594.18, (R 2 = 0.9981), and the lowest detection limit (LOD) is 0.091 pg mL -1 (where LOD = 3SD / K, SD is the standard deviation of the PFC current signals measured from 10 blank samples, and K is the slope of the linear equation).

[0119] Refer to Figure 2 and Figure 2 As shown in the illustration, the detection range of OTA toxin in the electrochromic mode is 0.01 - 100 ngmL -1, its R + B value increases with the increase of OTA concentration. This is because under the optoelectronic mode, the intensity of the photocurrent signal increases with the increase of OTA concentration. PB on the optoelectrode receives more electrons, and PB undergoes a redox reaction, with the color changing from dark blue to white gradually, resulting in a gradual increase in the R + B value. In this linear range, the R + B has a linear relationship with the logarithm of the OTA concentration (the inset shows the curve of the linear relationship between the R + B value and the logarithm of the OTA concentration). The linear equation is CI = 38.130×lgC |OTA| + 225.29, (R 2 = 0.9974), and the lowest detection limit (LOD) is 1.147 pg mL -1 .

[0120] To further evaluate the practical application potential of this optoelectrochemical / electrochromic dual-mode biosensing platform, the optoelectronic / electrochromic dual-mode biosensing platform was selected to detect OTA in moldy corn samples.

[0121] OTA toxin standard solutions with different concentrations were added to moldy corn samples, and the extracts of moldy corn were detected using this sensor. The results are shown in Table 1 below.

[0122] Table 1 Results of constructing an optoelectronic / electrochromic dual-mode immunosensing platform for analyzing OTA toxin in moldy corn samples:

[0123]

[0124] As shown in Table 1, the spiked recovery results of the sensor under the optoelectronic mode are between 94.56% and 109.18%, and the maximum RSD does not exceed 7.4%. Under the electrochromic mode, the spiked recovery results are between 93.27% and 102.76%, and the maximum RSD does not exceed 7.0%. Comparing the measurement results of this sensing platform with those of HPLC, it can be seen that the detection results of the two methods are basically the same, further verifying the reliability and accuracy of this sensor, and proving that this sensor can be used for the detection of OTA toxin in moldy corn samples.

Claims

1. A method for preparing a dual-photoelectrode bioaptamer sensor, characterized in that: The steps include: (1) Preparing a photoanode material ZnO / ZnIn2S4 / Bi2S3, comprising the following steps: (1.1) Place ZnO in ethylene glycol and dissolve by ultrasonication; (1.2) Then, ZnCl2, InCl3·4H2O, Bi(NO3)3·5H2O and TAA were added. Each time a substance was added, it was stirred and dissolved before another substance was added and stirred and dissolved. Finally, the mixture was poured into a polytetrafluoroethylene reactor for reaction. (1.3) The black precipitate obtained in the reactor was collected by centrifugation, washed several times with water and ethanol, and dried overnight to obtain the photoanode material ZnO / ZnIn2S4 / Bi2S3; (2) Preparing the photocathode material Cu2O / Cu2S, comprising the following steps: (2.1) Dissolve Cu2O in water, then add Na2S and stir; (2.2) Collect the resulting black precipitate by centrifugation, wash it several times with water and ethanol, and dry it overnight to obtain the photocathode material Cu2O / Cu2S; (3) Preparing Pt nanozymes and OTA-Apt-Pt probes, comprising the following steps: (3.1) Place H2PtCl6 in water, add trisodium citrate, stir, then slowly add NaBH4 dropwise, stir at room temperature to obtain Pt nanozyme; (3.2) mixing the Pt nanozyme suspension with the aptamer OTA-Apt solution, and incubating the mixture to obtain the OTA-Apt-Pt probe; (4) Preparing a modified photoanode and photocathode, comprising the following steps: (4.1) Immersing the indium tin oxide glass in ultrapure water and anhydrous ethanol in sequence for ultrasonic pretreatment, and drying for subsequent use; (4.2) dropping the suspension ZnO / ZnIn2S4 / Bi2S3 and Cu2O / Cu2S dispersion on the prepared ITO, and then drying, and using the obtained modified electrodes ZnO / ZnIn2S4 / Bi2S3 / ITO and Cu2O / Cu2S / ITO as photoanode and photocathode, respectively; (5) Constructing a dual-photoelectrode PFC aptasensor, comprising the following steps: (5.1) Casting the ZnO / ZnIn2S4 / Bi2S3 suspension on the cleaned ITO electrode and drying it after casting; (5.2) Then chitosan is added dropwise and dried after the addition; (5.3) Cast glutaraldehyde on the electrode in step (5.2) and store in a dark place; (5.4) Then, add the aminated cDNA solution dropwise and incubate overnight at low temperature; (5.5) Apply BSA solution on the electrode and let it stand for a while to occupy non-specific active sites; (5.6) Casting the OTA-apt-Pt suspension on the modified electrode and fixing it at a certain temperature for a period of time after casting; (5.7) incubating the sensor constructed in step (5.6) with a series of concentrations of OTA at a certain temperature for a period of time; (5.8) Place the sensor in a mixed solution of H2O2 and 4-CN and incubate for a period of time. After each modification step, wash the electrode surface with PBS buffer; (5.9) The prepared photoanode aptamer ZnO / ZnIn2S4 / Bi2S3 / ITO and the photocathode Cu2O / Cu2S / ITO were immersed in a single-chamber quartz electrolytic cell filled with PBS electrolyte, and a xenon lamp was used to vertically irradiate the dual photoelectrodes to generate instantaneous photocurrent, and the changes in the instantaneous photocurrent were recorded using a portable electrochemical workstation; (6) Constructing an electrochromic visual sensor, comprising the following steps: (6.1) Place HCl in a beaker of deionized water and stir vigorously. Then add FeCl3 and stir to dissolve. (6.2) Dissolve KCl in another beaker filled with deionized water, then add potassium ferrocyanide and stir to dissolve; (6.3) Mix the solutions in the beaker, add deionized water and stir to obtain PB solution; (6.4) In a three-electrode system, PB is electrodeposited on the ITO electrode using linear cyclic voltammetry to obtain a PB / ITO electrode; (6.5) Cast a Cu2O / Cu2S suspension near the PB / ITO electrode. While recording the instantaneous photocurrent change in step (5), the PB receives electrons and converts them into PW. The RGB value is read using a mobile phone software color signal converter to record the color change.

2. The preparation method according to claim 1, characterized in that: In step (1.1), the ratio of ZnO to ethylene glycol is 0.1-1 g:10-50 mL; In step (1.2), the amount of ZnCl2 added is 0.001 to 0.01 g; The amount of InCl3·4H2O added is 0.1 to 1 g; The amount of Bi(NO3)3·5H2O added is 0.1 to 1 g; The amount of TAA added is 0.1 to 1 g; After adding the substance, the stirring time is 2-30min; The mixture was finally poured into a 50 mL polytetrafluoroethylene reactor for reaction at a temperature of 150-180° C. for 10-15 h.

3. The preparation method according to claim 1, characterized in that In step (2.1), the ratio of Cu2O to water is 0.01-0.1 g:10-50 mL; The amount of Na2S added is 10-20mL, the concentration is 0.001-0.01M, and stirring is performed for 20-30 minutes after adding Na2S.

4. The preparation method according to claim 1, characterized in that: In step (3.1), the ratio of H2PtCl6 to water is 1-10mL:30-40mL, and the concentration of H2PtCl6 is 16mM; The amount of trisodium citrate added is 1-10 mL, the concentration is 40 mM, and it is stirred for 20-30 minutes after addition; The amount of NaBH4 added is 100-1000 μL, the concentration is 50 mM, and it is stirred for 30-60 minutes after addition; In step (3.2), the amount of Pt nanozyme suspension and aptamer solution is 1-10mL:1-10mL; the concentration of aptamer OTA-Apt solution is 1.0μM; The Pt nanozyme suspension was mixed with the aptamer solution and incubated at 4 °C for 10-12 h.

5. The preparation method according to claim 1, characterized in that: In step (4.2), the ZnO / ZnIn2S4 / Bi2S3 suspension and the Cu2O / Cu2S dispersion are prepared by dissolving ZnO / ZnIn2S4 / Bi2S3 and Cu2O / Cu2S in a mixed solution of 5 wt% Nafion and anhydrous ethanol, respectively; The volume ratio of Nafion and anhydrous ethanol is 1:9; The amount of ZnO / ZnIn2S4 / Bi2S3 suspension is 10-100 μL, and the concentration is 1-5 mg / mL; The amount of Cu2O / Cu2S dispersion is 10-100 μL, and the concentration is 1-5 mg / mL; The geometric area of ​​ITO exposure is 0.16πcm 2 .

6. The preparation method according to claim 1, characterized in that: In step (5.1), the amount of ZnO / ZnIn2S4 / Bi2S3 suspension is 10-50 μL, and the concentration is 1-5 mg / mL; In step (5.2), chitosan is first dissolved with 1% acetic acid solution, and then added dropwise, the amount of which is 10 to 100 μL, the concentration is 0.1 wt%, and then dried at 40-50° C. for 1 h; In step (5.3), the amount of glutaraldehyde used is 10-100 μL, and the concentration is 5 wt%; In step (5.4), the amount of cDNA solution added is 10 to 100 μL, the concentration is 1 μM, and incubation is carried out overnight at 4°C; in step (5.5), the amount of BSA solution used is 10 to 100 μL; In step (5.6), the amount of OTA-apt-Pt suspension is 10-100 μL, the concentration is 1 μM, and the OTA-apt-Pt suspension is cast on the modified electrode and fixed at 37°C for 1 h; In step (5.7), the concentration of OTA is 1×10 -4 ~1×10 2 ng mL -1 , the sensor and OTA were incubated at 37 °C for 1 h; In step (5.8), the concentration of H2O2 is 0.1-1 mM, the concentration of 4-CN is 1-10 mM, the sensor is incubated in the solution for 30 min, the concentration of PBS buffer is 0.01 M, and the pH is 7.40; In step (5.9), the amount of PBS electrolyte used is 20 mL, and the concentration is 10 mM; The power of xenon lamp is 200~800W.

7. The preparation method according to claim 1, characterized in that: In step (6.1), the ratio of HCl to deionized water is 10-100 μL:10-50 mL, the concentration of HCl is 35%, and the amount of FeCl3 added is 0.01-0.1 g; In step (6.2), the ratio of KCl to deionized water is 0.1-1 g:10-50 mL, and the amount of potassium ferrocyanide added is 0.01-0.1 g; In step (6.3), the amount of deionized water added is 10-50 mL, and the stirring time is 30 min; In step (6.4), the electrodeposition parameters were set between 0.4 and 0.8 V, the scan rate was 0.1 V / S, and the cycle was repeated 150 times.

8. Use of the dual-photoelectrode bioaptamer sensor prepared according to the preparation method according to any one of claims 1 to 7 in detecting ochratoxin A.

9. The use according to claim 8, characterized in that: The detection range of ochratoxin A in photoelectric mode is 0.0001~100ng mL -1 The photocurrent signal intensity increases with the increase of OTA concentration, and the logarithmic linear equation of photocurrent signal intensity and OTA concentration is ΔI = -64.047 × lgC |OTA| -594.18, R 2 =0.9981, the minimum detection limit LOD is 0.091pg mL -1 ; The detection range of ochratoxin A in electrochromic mode is 0.01~100ng mL -1 The R+B value increases with the increase of OTA concentration, and the logarithmic linear equation of R+B value and OTA concentration is ΔI=38.130×lgC |OTA| +225.29, R 2 =0.9974, the minimum detection limit LOD is 1.147pg mL -1 .

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