Polarity turning type photoelectrode constructed by polyindole-5-formaldehyde and preparation method and application of polarity turning type photoelectrode
By using polyindole-5-formaldehyde and WO3/Pt composite materials in photoelectrochemical sensors to construct polar flip-type photoelectrodes, the shortcomings of traditional photoelectrochemical sensors in distinguishing signals are solved, and the accuracy and anti-interference are achieved, while simplifying the equipment and operation.
Patent Information
- Application Number
- CN202411812846.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-10
- Publication Date
- 2025-03-14
Smart Images
Figure CN119619248A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of photoelectrochemical aptamer sensors, and in particular to a polarity reversal photoelectrode constructed with the participation of polyindole-5-carboxaldehyde, and a preparation method and application thereof. Background Art
[0002] Ochratoxin A (OTA) is an important component of mycotoxins, which are secondary metabolites of fungi. Its teratogenic and carcinogenic effects are far more serious than the hazards of pesticide residues or food additives in food. This toxin is most likely to be hidden in agricultural products such as corn, oats, grains, nuts, coffee, beer, and grape juice. Because of its outstanding chemical and thermal stability, it is not affected by hydrolysis and heat treatment procedures during food processing. If related contaminated foods are consumed, their toxicity will continue to accumulate in the food chain, which is very likely to cause stubborn diseases such as cancer. In order to protect human health from the toxicity of OTA, it is necessary to develop a sensitive and reliable analytical method to monitor the level of OTA toxins and evaluate the safety of food.
[0003] In recent years, among the many analytical techniques used to quantify OTA, the most authoritative ones are high performance liquid chromatography (HPLC) and gas chromatography (GC), especially the chromatography technology combined with advanced diode array or mass spectrometry detectors, which significantly improves accuracy and sensitivity and can meet the needs of the industry. However, chromatography / color quality detection requires large, expensive and complex instruments, and the cleaning and maintenance of chromatographic columns require experienced professionals. Sample pretreatment is very delicate to prevent column contamination, which is not friendly to new researchers or units with scarce resources. Therefore, it is very important to develop more convenient, economical and sensitive OTA analysis methods for food testing.
[0004] Photoelectrochemistry (PEC) has the advantages of simple operation, time saving, economy, and high sensitivity, and has been widely studied in the field of sensors. However, almost all reported PEC sensor studies use the "signal on" or "signal off" phenomenon to demonstrate the sensor's responsiveness. In fact, whether the "signal on" or "signal off" phenomenon is used, the test data can only be reflected by a single photoelectric signal. Therefore, PEC sensors with this detection mode cannot distinguish between background noise, interference signals of real samples and detection matrices, and the response signal sometimes has false positive or false negative phenomena.
[0005] The photocurrent polarity reversal model that has emerged in recent years has been used to construct PEC biosensors, which can eliminate the threat of false positives or false negatives faced by the three-electrode test system and effectively improve the selectivity of PEC sensors. At present, the key component of the polarity reversal PEC biosensors reported is the "polarity switcher", which is mainly concentrated in organic dyes (such as heme, methylene blue) and inorganic semiconductor materials (CdTe quantum dots, Cu2O / CuO). No reports have been found on the research of conductive polymers / polymer materials acting as "polarity switches". Although there are also literature reports on the participation of polypyrrole (PPy) in the construction of molecular imprinting (MIP)-PEC sensors, it has not been found that it has a polarity switching function.
[0006] The present invention focuses on indole-5-carboxaldehyde (5FIn), because polyindole-5-carboxaldehyde (P5FIn) is also a conductive polymer with good biocompatibility and can be used to fix biomolecules. The document "Simple'signal-on'photoelectrochemical aptasensor for ultrasensitive detecting AFB1 based one electrochemically reduced graphene oxide / poly(5-formylindole) / Aunanocomposites" (Bin Zhang, Biosensors and Bioelectronics, 134 (2019) 42-48) discloses a PEC sensor constructed with the participation of P5FIn, but its photocurrent polarity is not reversed.
[0007] The present invention utilizes P5FIn and WO3 / Pt to jointly construct a PEC aptamer (Apt) sensor with photocurrent polarity reversal for application in OTA detection, and there is no related report yet. Summary of the invention
[0008] The purpose of the present invention is to provide a polarity reversal photoelectrode constructed with the participation of polybenzole-5-carboxaldehyde, a preparation method and a use thereof in view of the above-mentioned problems. The present invention is a polarity reversal aptamer sensor, which can eliminate false negative or false positive signals caused by interfering substances and improve the accuracy of PEC sensors.
[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows:
[0010] The invention provides a polarity reversal photoelectrode constructed with the participation of polyindole-5-carboxaldehyde. The base electrode of the polarity reversal photoelectrode is an ITO electrode, which is obtained by sequentially modifying the surface of the ITO electrode with a WO3 / Pt composite material, indole-5-carboxaldehyde, an ochratoxin A aptamer and bovine serum albumin; the sequence of the ochratoxin A aptamer is 5′-NH2-(CH2)6-GAT CGGGTG TGG GTG GCG TAAAGG GAG CAT CGGACA-3′.
[0011] In the present invention, preferably, the WO3 / Pt composite material is WO 2.72 Disperse in deionized water, dissolve H2PtCl6·6H2O in deionized water, and drop H2PtCl6·6H2O solution into WO 2.72 The dispersion is reacted, and is obtained through solid-liquid separation, washing, and drying.
[0012] In the present invention, preferably, the WO 2.72 The mass ratio of H2PtCl6·6H2O is 1:0.028~0.040.
[0013] The present invention also provides a method for preparing the polarity reversal photoelectrode, comprising the following steps:
[0014] (1) Prepare the raw material WO 2.72 ;
[0015] (2) Will WO 2.72 Disperse in deionized water, dissolve H2PtCl6·6H2O in deionized water, and drop H2PtCl6·6H2O solution into WO 2.72 The dispersion is reacted, and the WO3 / Pt composite material is obtained through solid-liquid separation, washing and drying;
[0016] (3) dispersing the WO3 / Pt composite material in deionized water to obtain a WO3 / Pt dispersion, applying the WO3 / Pt dispersion to a cleaned ITO electrode, and drying at room temperature to obtain an ITO|WO3 / Pt electrode;
[0017] (4) Weigh indole-5-carboxaldehyde (5FIn) and tetrabutylammonium tetrafluoroborate and dissolve them in acetonitrile to obtain an electropolymerization solution; place the ITO|WO3 / Pt electrode prepared in the previous step in the electropolymerization solution, rinse the electrode with acetonitrile after the polymerization is completed, and dry it at room temperature to obtain an ITO|WO3 / Pt|P5FIn electrode;
[0018] (5) preparing an OTA aptamer Apt solution, transferring the Apt solution and applying it dropwise on the surface of the ITO|WO3 / Pt|P5FIn electrode, and obtaining the ITO|WO3 / Pt|5FIn|Apt electrode after incubation;
[0019] (6) Bovine serum albumin was transferred to the surface of the ITO|WO3 / Pt|5FIn|Apt electrode, and then the electrode was rinsed with TE buffer solution to obtain an ITO|WO3 / Pt|5FIn|Apt|BSA electrode.
[0020] In the above preparation process, preferably, in step (3), the mass concentration of the WO3 / Pt dispersion is 0.25-4 mg·mL -1 The drop volume is 5-20 μL, and the drop area is kept at 1 cm 2 .
[0021] In the above preparation process, preferably, in step (4), the concentration of indole-5-carboxaldehyde in the electropolymerization solution is 5 to 12 mg·mL -1 , the concentration of tetrabutylammonium tetrafluoroborate is 22~38g·mL -1 .
[0022] In the above preparation process, preferably, in step (5), the concentration of the Apt solution is 1-10 μM, the drop coating amount is 5-20 μL, the incubation temperature is 4° C., and the incubation time is 6-12 h.
[0023] In the above preparation process, preferably, in step (6), the mass concentration of the bovine serum albumin is 0.5-5%, and the dosage is 2-8 μL.
[0024] The present invention also provides an application of a polarity reversal photoelectrode constructed with the participation of polybenzazole-5-carboxaldehyde, which refers to using the photoelectrode for the detection of ochratoxin A.
[0025] Specifically, the specific steps of using the photoelectrode to detect ochratoxin A are:
[0026] (1) preparing multiple ochratoxin A standard solutions with concentration gradients;
[0027] (2) A quantitative amount of ochratoxin A standard solution was transferred to the surface of the ITO|WO3 / Pt|5FIn|Apt|BSA electrode for incubation. After a certain period of incubation, the electrode was rinsed with TE buffer solution to obtain multiple ITO|WO3 / Pt|5FIn|Apt|BSA|OTA electrodes. A three-electrode system was used, with ITO|WO3 / Pt|5FIn|Apt|BSA|OTA as the working electrode, Pt electrode as the auxiliary electrode, and Ag / AgCl electrode as the reference electrode. In a TE buffer solution with a pH of 6.5 to 8.5, the I–t technique was used at a bias voltage of 0.0 V, with an LED light as the excitation light source, and the light was switched on and off every 50 seconds to test the photocurrent intensity, and obtain a linear relationship between the photocurrent intensity and the logarithm of the concentration of the ochratoxin A standard solution.
[0028] (3) Under the same conditions as in step (2), a quantitative amount of the sample solution of ochratoxin A to be tested is transferred to the surface of the ITO|WO3 / Pt|5FIn|Apt|BSA electrode for incubation, and after elution, a three-electrode system is established. In the same TE buffer as in step (2), the photocurrent intensity of the sample solution to be tested is detected under the same conditions as in step (2), and the concentration of ochratoxin A in the sample solution to be tested is obtained according to the measured photocurrent intensity using the linear relationship obtained in step (2).
[0029] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:
[0030] 1. In the present invention, polyindole-5-carboxaldehyde (P5FIn) is a conductive polymer with good biocompatibility. It can not only fix the photoelectric active material WO3 / Pt, but also the aldehyde group (-CHO) on it connects the imino group (-NH2) at the top of the aptamer to produce a C=N double bond, further fix the aptamer on the electrode surface, and finally use the electrode to identify the OTA target molecule. P5FIn and WO3 / Pt form a three-component all-solid-state Z-type heterojunction, which changes the transmission path of the charge carriers and the polarity of the generated photocurrent is reversed, which is completely opposite to the photocurrent generated when P5FIn is not involved in the construction of the photoelectrode. It is inferred that P5FIn is not just a simple fixation of the electrode, but also participates in the charge transfer. Due to the reversal of the photocurrent polarity, the present invention can easily distinguish through the current polarity signal under the premise of ensuring the stability of the sensor, breaking through the limitation of the background signal, eliminating the false negative or false positive signals caused by interfering substances, and further improving the anti-interference of the sensor.
[0031] 2. The detection conditions of the ochratoxin A aptamer sensor constructed by ITO|WO3 / Pt|5FIn|Apt|BSA photoelectrode of the present invention are mild (~465nm, 0.0V bias), the activity of the aptamer is maintained, and the detection limit of ochratoxin A is 0.0229pg·mL -1 (S / N=3), linear range: 0.0004~400ng·mL -1 and 400~12μg·mL -1 . After storage for 20 days, the photoelectric electrode activity remained at about 91.8%, and the relative standard deviation of multiple parallel electrodes was about 1.18%, indicating good stability and reproducibility. When tested in combination with interfering substances, it showed good selectivity and anti-interference. The recovery rate of corn OTA applied to the actual sample was 96.9%-101%, and the RSD was 0.95%-3.77%, indicating that the PEC aptamer sensor has strong applicability and high accuracy. The ITO|WO3 / Pt|5FIn|Apt|BSA photoelectrode constructed by the present invention is expected to be used for monitoring the OTA toxin levels in other agricultural products or foods. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 WO prepared in Example 1 2.72 and SEM images of WO3 / Pt samples;
[0033] Figure 2 This is the EDS image of WO3 / Pt prepared in Example 1;
[0034] Figure 3 WO prepared in Example 1 2.72 and X-ray diffraction analysis of WO3 / Pt samples;
[0035] Figure 4 WO prepared in Example 1 2.72 and Fourier transform infrared spectra of WO3 / Pt samples;
[0036] Figure 5 Electrochemical impedance spectroscopy (EIS) characterization diagram of different modified electrodes;
[0037] Figure 6 The photocurrent response diagram of different modified electrodes to ochratoxin A;
[0038] Figure 7 The photocurrent response of ITO|WO3 / Pt|P5FIn|Apt|BSA photoelectrode to different concentrations of OTA;
[0039] Figure 8 is the linear calibration curve corresponding to OTA concentration and photocurrent logarithm;
[0040] Fig. 9 The selectivity and anti-interference properties of ITO|WO3 / Pt|P5FIn|Apt|BSA photoelectrode. DETAILED DESCRIPTION
[0041] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0042] The instruments, reagents, and materials involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, and materials already in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods involved in the following embodiments, unless otherwise specified, are all conventional experimental methods and detection methods already in the prior art. Ochratoxin A aptamers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The preparation method of TE buffer solution is as follows: 0.0931 g of sodium ethylenediaminetetraacetate (EDTA) and 0.3028 g of trishydroxymethylaminomethane (Tris) were weighed separately, and the mixture was dissolved in deionized water and fixed to volume in a 250 mL volumetric flask.
[0043] 1. Preparation Example
[0044] Example 1
[0045] The polarity reversal photoelectrode constructed with the participation of polyindole-5-carboxaldehyde has an ITO electrode as its base electrode, which is obtained by sequentially modifying the surface of the ITO electrode with a WO3 / Pt composite material, indole-5-carboxaldehyde, an OTA aptamer, and bovine serum albumin; the OTA aptamer is 5′-NH2-(CH2)6-GAT CGG GTG TGG GTG GCG TAAAGG GAG CAT CGG ACA-3′. The preparation method of the polarity reversal photoelectrode comprises the following steps:
[0046] (1) Raw material WO 2.72 Preparation: 1.2 g WCl6 was dissolved in 50 mL anhydrous ethanol and alcoholyzed to obtain a yellow solution; the yellow solution was then transferred to a 100 mL autoclave, heated at 160 °C for 24 h, cooled naturally to room temperature, centrifuged, and washed with deionized water; finally, dried in a vacuum oven at 80 °C for 12 h to obtain WO 2.72 Blue powder;
[0047] (2) Preparation of WO3 / Pt composite material: 0.5 g WO3 / Pt composite material was stirred. 2.72 Dissolve in 100 mL of deionized water and stir to obtain WO 2.72 Suspension, 0.5mL of concentration 0.01g mL -1 The H2PtCl6·6H2O solution was dropped into WO 2.72 In the suspension, the color of the reactant changed from blue to milky yellow. After 4 h, 0.9 mL of 0.01 g mL -1 The H2PtCl6·6H2O solution was dropped into the suspension and stirred for 12 hours. The precipitate was washed alternately with ethanol and deionized water, and the product was collected by centrifugation. The product was placed in a 160°C forced air drying oven for 24 hours to obtain WO3 / Pt light yellow powder.
[0048] (3) Preparation of ITO|WO3 / Pt electrode
[0049] Weigh 1 mg of WO3 / Pt powder and disperse it in 1 mL of deionized water. Ultrasonic dispersion was performed for 30 min to obtain WO3 / Pt dispersion. 15 μL of WO3 / Pt dispersion was drop-coated on the cleaned ITO electrode, and the drop-coating area was kept at 1 cm. 2 , slowly dried at room temperature to obtain ITO|WO3 / Pt electrode;
[0050] (4) Preparation of ITO|WO3 / Pt|P5FIn electrode
[0051] Weigh 0.8 g of indole-5-carboxaldehyde (5FIn) and 3 g of tetrabutylammonium tetrafluoroborate and dissolve them in acetonitrile. Dose to a 100 mL volumetric flask to obtain a 5FIn concentration of 8 g mL -1 The ITO|WO3 / Pt electrode prepared in the previous step is placed in the electropolymerization solution, and after the polymerization is completed, the electrode is rinsed with acetonitrile and dried at room temperature to obtain the ITO|WO3 / Pt|P5FIn electrode;
[0052] (5) Preparation of ITO|WO3 / Pt|5FIn|Apt electrode
[0053] Prepare the ochratoxin A aptamer Apt solution, transfer 12 μL and 5 μM Apt solution to the surface of the ITO|WO3 / Pt|P5FIn electrode for incubation at 4°C for 8 h. The imino group at the top of Apt and the aldehyde group of P5FIn on the electrode surface undergo valence bond bonding to fix Apt on the electrode surface, thereby preparing the ITO|WO3 / Pt|5FIn|Apt electrode.
[0054] (6) Preparation of ITO|WO3 / Pt|5FIn|Apt|BSA Electrode
[0055] 5 μL of 2% bovine serum albumin was transferred to the surface of the ITO|WO3 / Pt|5FIn|Apt electrode for 30 min, and then the electrode was rinsed with TE buffer solution to obtain an ITO|WO3 / Pt|5FIn|Apt|BSA electrode.
[0056] Example 2
[0057] The polarity reversal photoelectrode constructed with the participation of polyindole-5-carboxaldehyde has an ITO electrode as its base electrode, which is obtained by sequentially modifying the surface of the ITO electrode with a WO3 / Pt composite material, indole-5-carboxaldehyde, an OTA aptamer, and bovine serum albumin; the OTA aptamer is 5′-NH2-(CH2)6-GAT CGG GTG TGG GTG GCG TAAAGG GAG CAT CGG ACA-3′. The preparation method of the polarity reversal photoelectrode comprises the following steps:
[0058] (1) Raw material WO 2.72 Preparation: 1.0 g WCl6 was dissolved in 40 mL of anhydrous ethanol and alcoholyzed to obtain a yellow solution; the yellow solution was then transferred to a 100 mL autoclave, heated at 130 °C for 30 h, cooled naturally to room temperature, centrifuged, and washed with deionized water; finally, dried in a vacuum oven at 80 °C for 12 h to obtain WO 2.72 Blue powder;
[0059] (2) Preparation of WO3 / Pt composite material: 0.3 g WO3 / Pt composite material was stirred. 2.72 Dissolve in 100 mL of deionized water and stir to obtain WO 2.72 Suspension, 0.4 mL of concentration 0.01 g mL -1 The H2PtCl6·6H2O solution was dropped into WO 2.72 In the suspension, the color of the reactant changed from blue to milky yellow. After 2 h, 0.8 mL of 0.01 g mL -1 The H2PtCl6·6H2O solution was dropped into the suspension and stirred for 8 h. The precipitate was washed alternately with ethanol and deionized water, the product was collected by centrifugation and dried to obtain WO3 / Pt light yellow powder.
[0060] (3) Preparation of ITO|WO3 / Pt electrode
[0061] Weigh 0.5 mg of WO3 / Pt powder and disperse it in 2 mL of deionized water. Ultrasonic dispersion was performed for 30 min to obtain WO3 / Pt dispersion. 20 μL of WO3 / Pt dispersion was drop-coated on the cleaned ITO electrode, and the drop-coating area was kept at 1 cm. 2 , slowly dried at room temperature to obtain ITO|WO3 / Pt electrode;
[0062] (4) Preparation of ITO|WO3 / Pt|P5FIn electrode
[0063] Weigh 0.5 g of indole-5-carboxaldehyde (5FIn) and 2.2 g of tetrabutylammonium tetrafluoroborate and dissolve them in acetonitrile. Dose the mixture to a 100 mL volumetric flask to obtain a 5FIn concentration of 5 g mL -1The ITO|WO3 / Pt electrode prepared in the previous step is placed in the electropolymerization solution, and after the polymerization is completed, the electrode is rinsed with acetonitrile and dried at room temperature to obtain the ITO|WO3 / Pt|P5FIn electrode;
[0064] (5) Preparation of ITO|WO3 / Pt|5FIn|Apt electrode
[0065] Prepare the ochratoxin A aptamer Apt solution, transfer 20 μL of 1 μM Apt solution and drop-coat it on the surface of the ITO|WO3 / Pt|P5FIn electrode for incubation at 4°C for 6 h. The imino group at the top of Apt and the aldehyde group of P5FIn on the electrode surface undergo valence bond bonding to fix Apt on the electrode surface, thus preparing the ITO|WO3 / Pt|5FIn|Apt electrode.
[0066] (6) Preparation of ITO|WO3 / Pt|5FIn|Apt|BSA Electrode
[0067] 8 μL of 0.5% bovine serum albumin was transferred to the surface of the ITO|WO3 / Pt|5FIn|Apt electrode for 30 min, and then the electrode was rinsed with TE buffer solution to obtain an ITO|WO3 / Pt|5FIn|Apt|BSA electrode.
[0068] Example 3
[0069] The polarity reversal photoelectrode constructed with the participation of polyindole-5-carboxaldehyde has an ITO electrode as its base electrode, which is obtained by sequentially modifying the surface of the ITO electrode with a WO3 / Pt composite material, indole-5-carboxaldehyde, an OTA aptamer, and bovine serum albumin; the OTA aptamer is 5′-NH2-(CH2)6-GAT CGG GTG TGG GTG GCG TAAAGG GAG CAT CGG ACA-3′. The preparation method of the polarity reversal photoelectrode comprises the following steps:
[0070] (1) Raw material WO 2.72 Preparation: 2.0 g WCl6 was dissolved in 70 mL anhydrous ethanol, and alcoholysis was performed to obtain a yellow solution; the yellow solution was then transferred to a 100 mL autoclave, heated at 180 °C for 20 h, cooled naturally to room temperature, centrifuged, and washed with deionized water; finally, dried in a vacuum oven at 80 °C for 12 h to obtain a blue powder;
[0071] (2) Preparation of WO3 / Pt composite material: 1.0 g WO3 / Pt composite material was stirred. 2.72 Dissolve in 100 mL of deionized water and stir to obtain WO 2.72 Suspension, 1mL concentration of 0.01g mL -1The H2PtCl6·6H2O solution was dropped into WO 2.72 In the suspension, the color of the reactant changed from blue to milky yellow. After 6 h, 2.5 mL of 0.01 g mL -1 The H2PtCl6·6H2O solution was dropped into the suspension and stirred for 20 h. The precipitate was washed alternately with ethanol and deionized water, the product was collected by centrifugation and dried to obtain WO3 / Pt light yellow powder.
[0072] (3) Preparation of ITO|WO3 / Pt electrode
[0073] Weigh 2 mg of WO3 / Pt powder and disperse it in 0.5 mL of deionized water. Ultrasonic dispersion was performed to obtain WO3 / Pt dispersion. 5 μL of WO3 / Pt dispersion was drop-coated on the cleaned ITO electrode, and the drop-coating area was kept at 1 cm. 2 , slowly dried at room temperature to obtain ITO|WO3 / Pt electrode;
[0074] (4) Preparation of ITO|WO3 / Pt|P5FIn electrode
[0075] Weigh 1.2 g of indole-5-carboxaldehyde (5FIn) and 3.8 g of tetrabutylammonium tetrafluoroborate and dissolve them in acetonitrile. Dose the mixture to a 100 mL volumetric flask to obtain a 5FIn concentration of 12 g mL -1 The ITO|WO3 / Pt electrode prepared in the previous step is placed in the electropolymerization solution, and after the polymerization is completed, the electrode is rinsed with acetonitrile and dried at room temperature to obtain the ITO|WO3 / Pt|P5FIn electrode;
[0076] (5) Preparation of ITO|WO3 / Pt|5FIn|Apt electrode
[0077] Prepare the ochratoxin A aptamer Apt solution, transfer 5 μL and 10 μM Apt solution to the surface of the ITO|WO3 / Pt|P5FIn electrode for incubation at 4°C for 12 h. The imino group at the top of Apt and the aldehyde group of P5FIn on the electrode surface undergo valence bond bonding to fix Apt on the electrode surface, thereby preparing the ITO|WO3 / Pt|5FIn|Apt electrode.
[0078] (6) Preparation of ITO|WO3 / Pt|5FIn|Apt|BSA Electrode
[0079] 2 μL of 5% bovine serum albumin was transferred to the surface of the ITO|WO3 / Pt|5FIn|Apt electrode for 30 min, and then the electrode was rinsed with TE buffer solution to obtain an ITO|WO3 / Pt|5FIn|Apt|BSA electrode.
[0080] Comparative Example 1
[0081] Raw material WO 2.72 Preparation: 1.2 g WCl6 was dissolved in 50 mL anhydrous ethanol and alcoholyzed to obtain a yellow solution; the yellow solution was then transferred to a 100 mL autoclave, heated at 160 °C for 24 h, cooled naturally to room temperature, centrifuged, and washed with deionized water; finally, dried in a vacuum oven at 80 °C for 12 h to obtain WO 2.72 Blue powder;
[0082] Weigh 1 mg WO 2.72 Disperse in 1 mL of deionized water, ultrasonically disperse for 30 min, and apply 15 μL of the dispersion to the cleaned ITO electrode, keeping the coating area at 1 cm 2 , slowly dried at room temperature to obtain ITO|WO 2.72 electrode.
[0083] Comparative Example 2
[0084] Weigh 0.8 g of indole-5-carboxaldehyde (5FIn) and 3 g of tetrabutylammonium tetrafluoroborate and dissolve them in acetonitrile. Dose to a 100 mL volumetric flask to obtain a 5FIn concentration of 8 g mL -1 The electropolymerization solution was prepared; the cleaned ITO electrode was placed in the electropolymerization solution, and after completion, the electrode was rinsed with acetonitrile and dried at room temperature to obtain an ITO|P5FIn electrode.
[0085] 2. Characterization of Materials
[0086] 1. Morphology and component characterization
[0087] WO was analyzed by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). 2.72 And the microstructure and composition of the composite WO3 / Pt. Figure 1 WO prepared in Example 1 2.72 The SEM images of WO3 / Pt show that WO 2.72 The surface of the urchin is similar to that of a sea urchin and has spines of about 5 to 20 nm. This result is consistent with the research conclusion of the prior art. 2.72 When it meets H2PtCl6·6H2O, a spontaneous redox reaction occurs. During the reaction, high-speed stirring causes the sea urchin spines to become significantly passivated. The surface of the sea urchin is relatively rough, and the Pt element may have been loaded on the precursor.
[0088] In order to explore the chemical composition of WO3 / Pt composite materials, EDS characterization was introduced. Figure 1 The SEM image of WO3 / Pt in the Figure 2 The EDS diagram of WO3 / Pt shows the presence of W, O and Pt elements.
[0089] 2. X-ray diffraction
[0090] In Example 1, WO 2.72 The X-ray diffraction analysis results of the sample and WO3 / Pt sample are shown in Figure 3 As shown, WO 2.72 The typical peaks of the X-ray diffraction (XRD) of the sample are located at 23.516°, 23.836°, 24.502°, 33.744° and 34.195°, which correspond to the crystal planes (010), (103), (-111), (113) and (-512), respectively, which are consistent with the standard card (JCPDS No.05-0392). For the WO3 / Pt composite material, its characteristic peak position has slightly shifted, and the intensity has also slightly increased. The characteristic peaks are located at 23.143°, 23.643°, 24.366°, 26.831° and 28.870°, which are consistent with the WO3 standard card (JCPDS No.20-1323) (002), (020), (200), (120) and (112) crystal planes, which indicates that the spontaneous redox reaction changes the low oxygen compound WO 2.72 In addition, the metal Pt of the WO3 / Pt composite is located at 39.7°, and its corresponding crystal plane (111) is marked by ◆. The peak is broad and the intensity is relatively weak, indicating that it is difficult to find large Pt clusters or Pt nanoparticles in the WO3 / Pt composite. It is very likely that the metal Pt is dispersed in the precursor.
[0091] 3. Infrared spectroscopy analysis
[0092] In Example 1, WO 2.72 The Fourier transform infrared spectra (FTIR) of WO3 / Pt samples are shown in Figure 4 , WOW at 615cm -1 There is stretching vibration at 772cm -1 There is bending vibration at 1592cm -1 and 3451cm -1 The peaks at 1360 cm-1 and 1360 cm-2 can be attributed to the bending vibration of -OH and the stretching vibration of -OH, respectively. -1 The peak at is attributed to the bending vibration of CO.
[0093] 3. Performance Test
[0094] 1. Electrochemical impedance spectroscopy (EIS) characterization of different modified electrodes
[0095] Electrochemical impedance spectroscopy (EIS) was used to characterize whether the modified material was assembled on the electrode as required and the charge transfer capacity after assembly. -1 [Fe(CN)6] 3- / [Fe(CN)6] 4- +0.1mol·L-1 The electrochemical impedance of different modified electrodes was tested in KCl solution without light. The applied voltage was the open circuit voltage, the frequency range was 0.01-105 Hz, and the amplitude was 5 mV. The test objects included bare ITO electrodes, ITO|WO prepared in Comparative Example 1, 2.72 electrode, an ITO|WO3 / Pt electrode prepared by the same preparation method as Example 1, an ITO|P5FIn electrode prepared by the method of Comparative Example 2, an ITO|WO3 / Pt|P5FIn electrode prepared by the same method as Example 1, an ITO|WO3 / Pt|P5FIn|Apt electrode prepared by the same method as Example 1, an ITO|WO3 / Pt|P5FIn|Apt|BSA electrode prepared by the same method as Example 1, and an ITO|WO3 / Pt|P5FIn|Apt|BSA|OTA combined with OTA target molecules (12 μL OTA target molecule solution was transferred for incubation for 6 hours, and then the electrode was rinsed with TE buffer solution).
[0096] The test results are presented in the form of a Nyquist curve, which consists of a straight line in the low-frequency region (representing the diffusion-controlled process) and a semicircle in the high-frequency region (representing the electron transfer process). The diameter of the semicircle represents the charge transfer resistance (R ct ), you can use the impedance meter (AUT88103) to build an equivalent circuit diagram for fitting. Figure 5 As shown, the fitted R ct The value is 11.0Ω (curve a), which is close to the true value provided by Hengxinyang Glass Co., Ltd., proving that the test link and the simulated equivalent circuit are reliable. 2.72 After that, the electrode R ct is 22.0Ω (curve b); after doping with Pt, the conductivity of the composite WO3 / Pt increases significantly, R ct The conductivity of pure P5FIn polymer film is weak, and its R ct Close to 70.1Ω (curve d), it was polymerized on the WO3 / Pt surface and found that the R of the electrode ITO|WO3 / Pt|P5FIn ct The R value of the unpolymerized P5FIn electrode is reduced to 32.8Ω (curve e), which is greater than the R value of the unpolymerized P5FIn electrode. ct (12.9Ω), indicating that P5FIn has been constructed on the electrode surface. The R of the ITO|WO3 / Pt|P5FIn|Apt electrode was obtained by valence bond fixation of Apt. ct The R of the ITO|WO3 / Pt|P5FIn|Apt|BSA electrode stabilized by non-conductive bovine serum albumin (BSA) is 46.4Ω (curve f), which is attributed to the non-conductive single-stranded oligonucleotide and the steric hindrance. ctThe electrode can recognize the OTA target molecule by specificity and affinity, and its R ct Increased to 79.1Ω (curve h).
[0097] 2. Photocurrent response of different modified electrodes
[0098] In order to study the photocurrent response of ochratoxin A (OTA) on different modified electrodes, each modified electrode was placed in TE buffer (pH 5.5-8.5) and the I-t technique was used at 0.0V bias and LED light (λ=465nm) as the excitation light source. The light was turned on and off every 50s to investigate the photoelectric response performance of the photoelectrode. Each modified electrode was a bare ITO electrode, an ITO|WO prepared in Comparative Example 1, and a 2.72 electrode, an ITO|WO3 / Pt electrode prepared by the same preparation method as in Example 1, an ITO|P5FIn electrode prepared by the method of Comparative Example 2, an ITO|WO3 / Pt|P5FIn electrode prepared by the same method as in Example 1, an ITO|WO3 / Pt|P5FIn|Apt electrode prepared by the same method as in Example 1, an ITO|WO3 / Pt|P5FIn|Apt|BSA electrode prepared by the same method as in Example 1, and ITO|WO3 / Pt|P5FIn|Apt|BSA|OTA combined with OTA target molecules.
[0099] Test results see Figure 6 From the comparison of different electrode currents in the figure, it can be seen that although the R ct The value is the minimum, but the photocurrent generated is not the maximum (curve a, I = -12.7nA). 2.72When modified, the photocurrent increased significantly (curve b, I = -94nA), while the photocurrent of the WO3 / Pt modified electrode increased significantly (curve c, I = -232.4nA), reflecting that the precious metal Pt can effectively promote electron transfer, but the Pt content must be controlled. If it is too high, obvious water electrolysis will occur, which is not conducive to sensor research. The photocurrent of the pure P5FIn polymer film electrode (curve d, I = -28nA) is higher than that of bare ITO, which may be due to the separation of photogenerated electrons and holes in the photoexcited polymer film. However, when it is polymerized on the WO3 / Pt surface, it is found that the photocurrent of the electrode ITO|WO3 / Pt|P5FIn reverses its polarity (curve e, I = 4227nA), and the photocurrent intensity is 150 times that of curve d and 18 times that of curve c, indicating that a heterojunction may be formed between the P5FIn polymer film and the substrate material, constructing a new electron transfer path, and playing the dual functions of "switch" and current signal amplification. When the imino group (-NH2) at the top of Apt forms a valence bond with the aldehyde group (-CHO) of P5FIn on the electrode surface, the photocurrent of the resulting ITO|WO3 / Pt|P5FIn|Apt electrode begins to decrease (curve f, I=1816nA), which may be attributed to the insulation effect of single-stranded DNA protein and the steric hindrance effect. The photocurrent of the BSA-stabilized ITO|WO3 / Pt|P5FIn|Apt|BSA electrode dropped to 1816nA (curve g). After the electrode specifically and affinity-directedly recognized the OTA target molecule, its current dropped to 891nA (curve h). If the concentration of the OTA target molecule is changed and the electrode is continued to be incubated, it is found that the photocurrent of curve h changes accordingly, indicating that the electrode can be used to prepare a PEC sensor for OTA.
[0100] 4. Application Examples
[0101] The specific steps of the present invention for detecting ochratoxin A using the above-mentioned photoelectrode are as follows:
[0102] (1) preparing multiple ochratoxin A standard solutions with concentration gradients;
[0103] (2) A quantitative amount of ochratoxin A standard solution was transferred to the surface of an ITO|WO3 / Pt|5FIn|Apt|BSA electrode for incubation. After a certain period of incubation, the electrode was rinsed with a TE buffer solution to obtain a plurality of ITO|WO3 / Pt|5FIn|Apt|BSA|OTA electrodes. A three-electrode system was used, with ITO|WO3 / Pt|5FIn|Apt|BSA|OTA as the working electrode, a Pt electrode as the auxiliary electrode, and an Ag / AgCl electrode as the reference electrode. In a TE buffer solution, the I–t technique was used at a bias voltage of 0.0 V, with an LED light as the excitation light source, and the light was switched on and off every 50 seconds to test the photocurrent intensity, and obtain a linear relationship between the photocurrent intensity and the logarithm of the concentration of the ochratoxin A standard solution.
[0104] (3) Under the same conditions as step (2), a certain amount of ochratoxin A sample solution is transferred to the surface of the ITO|WO3 / Pt|5FIn|Apt|BSA electrode for incubation. After elution, the same three-electrode system is established. In TE buffer, the photocurrent intensity of the sample solution is detected under the same conditions as step (2). The concentration of ochratoxin A in the sample solution is obtained according to the measured photocurrent intensity using the linear relationship obtained in step (2).
[0105] When the method of the present invention is used in a specific application, the amount of ochratoxin A standard solution can be selected to be 5 to 20 μL. When the pH of the TE electrolyte solution changes from 6.5 to 8.5, the best photoelectric response effect is observed in a solution with a pH of 7.5. Therefore, the best choice is a TE buffer with a pH of 7.5. In order to study the effect of the aptamer OTA incubation time on the sensor, different aptamer incubation times are given to the working electrode. The photocurrent intensity decreases continuously in the range of 0.3 to 12 hours, but the decreasing trend after incubation for 6 hours is close to being flat. Therefore, the main incubation time is optimally set to 6 hours. It should be noted that the ochratoxin A sample solution, TE buffer, and incubation conditions in step (3) should be consistent with those in step (2) to maintain test consistency and accuracy. The following is a preferred application example.
[0106] Example 4
[0107] (1) Prepare multiple ochratoxin A standard solutions with a concentration gradient, with a specific concentration range of 0.0001 ng·mL -1 ~100 μg mL -1 ;
[0108] (2) 12 μL of ochratoxin A standard solution was transferred to the surface of the ITO|WO3 / Pt|5FIn|Apt|BSA electrode for incubation for 6 h, and then the electrode was rinsed with TE buffer solution to obtain multiple ITO|WO3 / Pt|5FIn|Apt|BSA|OTA electrodes; a three-electrode system was used, with ITO|WO3 / Pt|5FIn|Apt|BSA|OTA as the working electrode, Pt electrode as the auxiliary electrode, and Ag / AgCl electrode as the reference electrode. In a TE buffer solution with a pH of 7.5, the I–t technique was used at a bias of 0.0 V, with an LED lamp as the excitation light source (λ=465 nm), and the light was switched on and off every 50 s to test the photocurrent intensity, and the linear relationship between the photocurrent intensity and the logarithm of the concentration of the ochratoxin A standard solution was obtained; see Figure 7 It can be seen that the photocurrent intensity of the ITO|WO3 / Pt|5FIn|Apt|BSA|OTA electrode decreases with the increase of OTA concentration, see Figure 8OTA concentration is between 0.0004 and 400 ng·mL -1 and 400~12μg·mL -1 There are two linear relationships in the range, and the corresponding linear regression equations are I = -86logC + 905 (R2 = 0.9978) and I = -388logC + 9051766 (R 2 =0.9967), the detection limit of the sensor is 0.0229pg·mL -1 (S / N=3).
[0109] (3) Under the same conditions as in step (2), 12 μL of the ochratoxin A sample solution was transferred to the surface of the ITO|WO3 / Pt|5FIn|Apt|BSA electrode for incubation. After elution, the same three-electrode system was established. In a TE buffer solution of pH 7.5, the photocurrent intensity of the sample solution was detected under the same conditions as in step (2). The concentration of ochratoxin A in the sample solution was obtained according to the measured photocurrent intensity using the linear relationship obtained in step (2).
[0110] After consulting the literature and comparing with other reports, the polarity switching PEC sensor used in the present invention has the advantages of higher sensitivity and lower detection limit in detecting OTA.
[0111] 1. Reproducibility and stability
[0112] Stability and reproducibility over time are key factors in determining whether a sensor can measure effectively. According to the method in step (2) of Example 4, an ITO|WO3 / Pt|P5FIn|Apt|BSA|OTA aptamer sensor was constructed to observe its short-term stability. The light was turned on and off once every 50 seconds, and repeated for 25 cycles to test its photocurrent intensity. The current change value ΔI with or without light in the 25th cycle was 25th The current change value ΔI with or without light in the first cycle 1st In comparison, it was found that the photocurrent in the 25th cycle could stabilize to 96.0% of the initial photocurrent, indicating that the PEC aptamer sensor has a relatively ideal short-term stability. The newly modified ITO|WO3 / Pt|5FIn|Apt|BSA|OTA electrode was stored in a 4°C refrigerator for 20 days. During this period, the photocurrent response value was measured every 5 days. When comparing, the change value of the switching light ΔI in the last cycle was taken. It was found that the photocurrent continued to decrease and dropped to 91.8% of the initial value on the 20th day, indicating that the PEC aptamer sensor has a relatively good long-term stability. Using five freshly modified ITO|WO3 / Pt|5FIn|Apt|BSA|OTA electrodes, the reproducibility of the sensor was investigated under the same conditions as step (2) of Example 4. The relative standard deviation (RSD) was 1.18%, indicating that the proposed PEC aptamer sensor has excellent reproducibility.
[0113] 2. Selectivity and anti-interference
[0114] In order to study the anti-interference ability of aptamer sensors in practical applications, zearalenone (ZEN), aflatoxin B1 (AFB1), and ochratoxin AB (OTB), which are close to OTA in structure and may coexist in real samples, were selected as interfering molecules. Two concentration gradients of 0.4 and 4000 ng·mL were set respectively. -1 , the concentration of OTA and the concentration of the interfering substance were 1:1, and the mixture was mixed with 3 kinds of interfering substances and OTA at equal concentrations, and the electrode photocurrent was tested after incubation according to the method of step (3) of Example 4. Fig. 9 The results show that the photocurrent changes of the ITO|WO3 / Pt|5FIn|Apt|BSA|OTA electrode of the present invention to interfering substances account for 2.9% and 4.8% of the initial current (ZEN), 2.8% and 5.3% aflatoxin B1 (AFB1), 2.6% and 5.0% ochratoxin AB (OTB); while the photocurrent changes before and after the recognition of the single target molecule OTA account for 29.4% and 72.1% of the original, and the photocurrent changes after OTA mixed with interfering substances account for 25% and 69.6% of the original; the above data show that the coexistence of interfering substances and target molecules has a relatively weak effect on the determination of OTA, indicating that the PEC aptamer sensor has good selectivity for OTA.
[0115] 3. Actual sample testing
[0116] Bulk corn was purchased from the market, crushed with a grinder, and sieved with a test sieve with a pore size of 1 mm. Accurately weigh 25.0 g corn powder and disperse it in 100.0 mL of methanol / water extract (80:20, v / v), seal the tube mouth, place it on an oscillator, and shake it at high speed for 30 minutes; secondly, the extract was divided into batches and centrifuged at high speed (10,000 rpm·min -1 ), and the supernatant was filtered through a 0.45 μm filter membrane; finally, it was diluted with TE buffer (10 mL:50 mL, v / v) to obtain corn extract for OTA detection.
[0117] Add 0.1, 10.0 and 50 ng·mL -1The OTA standard solution was subjected to PEC and HPLC recovery tests respectively to evaluate the feasibility and reliability of the OTA aptamer sensor proposed in this experiment. As shown in Table 1, the HPLC test showed that the recovery of OTA in the spiked corn sample was 93.8% to 96.8%, with an RSD of 0.53% to 1.32%, while the PEC test showed that the recovery of OTA in the spiked corn sample was 96.9% to 101%, with an RSD of 0.95% to 3.77%. The results showed that the sensor had good accuracy, indicating that the constructed aptamer sensor was accurate and feasible and could be applied to actual sample analysis.
[0118] Table 1 Detection results of OTA in corn samples
[0119]
[0120]
[0121] The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.
Claims
1. A polarity-reversing photoelectrode constructed with the participation of polybenzole-5-carboxaldehyde, characterized in that: The base electrode of the polarity reversal photoelectrode is an ITO electrode, which is obtained by sequentially modifying the surface of the ITO electrode with a WO3 / Pt composite material, indole-5-carboxaldehyde, an ochratoxin A aptamer and bovine serum albumin; the sequence of the ochratoxin A aptamer is 5′-NH2-(CH2)6-GAT CGGGTG TGG GTG GCG TAAAGG GAG CAT CGGACA-3′.
2. The polarity reversal photoelectrode according to claim 1, characterized in that: The WO3 / Pt composite material is WO 2.72 Disperse in deionized water, dissolve H2PtCl6·6H2O in deionized water, and drop H2PtCl6·6H2O solution into WO 2.72 The dispersion is reacted, and is obtained through solid-liquid separation, washing, and drying.
3. The polarity reversal photoelectrode according to claim 2, characterized in that: The WO 2.72 The mass ratio of H2PtCl6·6H2O is 1:0.028~0.
040.
4. The method for preparing a polarity reversal photoelectrode according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Prepare the raw material WO 2.72 ; (2) Will WO 2.72 Dissolve H2PtCl6·6H2O in deionized water and drop the H2PtCl6·6H2O solution into WO 2.72 The dispersion is reacted, and the WO3 / Pt composite material is obtained through solid-liquid separation, washing and drying; (3) dispersing the WO3 / Pt composite material in deionized water to obtain a WO3 / Pt dispersion, applying the WO3 / Pt dispersion to a cleaned ITO electrode, and drying at room temperature to obtain an ITO|WO3 / Pt electrode; (4) Weigh indole-5-carboxaldehyde (5FIn) and tetrabutylammonium tetrafluoroborate and dissolve them in acetonitrile to obtain an electropolymerization solution; place the ITO|WO3 / Pt electrode prepared in the previous step in the electropolymerization solution, rinse the electrode with acetonitrile after the polymerization is completed, and dry it at room temperature to obtain an ITO|WO3 / Pt|P5FIn electrode; (5) preparing an ochratoxin A aptamer Apt solution, transferring the Apt solution and applying it dropwise on the surface of the ITO|WO3 / Pt|P5FIn electrode, and obtaining the ITO|WO3 / Pt|5FIn|Apt electrode after incubation; (6) Bovine serum albumin was transferred to the surface of the ITO|WO3 / Pt|5FIn|Apt electrode, and then the electrode was rinsed with TE buffer solution to obtain an ITO|WO3 / Pt|5FIn|Apt|BSA electrode.
5. The preparation method according to claim 4, characterized in that: In step (3), the mass concentration of the WO3 / Pt dispersion is 0.25-4 mg·mL -1 The drop volume is 5-20 μL, and the drop area is kept at 1 cm 2 .
6. The preparation method according to claim 4, characterized in that: In step (4), the concentration of indole-5-carboxaldehyde in the electropolymerization solution is 5 to 12 mg·mL -1 , the concentration of tetrabutylammonium tetrafluoroborate is 22~38g·mL -1 .
7. The preparation method according to claim 4, characterized in that: In step (5), the concentration of the Apt solution is 1-10 μM, the drop coating amount is 5-20 μL, the incubation temperature is 4° C., and the incubation time is 6-12 h.
8. The preparation method according to claim 4, characterized in that: In step (6), the mass concentration of the bovine serum albumin is 0.5-5%, and the dosage is 2-8 μL.
9. Application of polarity reversal photoelectrode constructed with the participation of polybenzazole-5-carboxaldehyde, characterized in that: The photoelectrode is used for the detection of ochratoxin A.
10. The use according to claim 9, characterized in that: The specific steps of using the photoelectrode to detect ochratoxin A are as follows: (1) preparing multiple ochratoxin A standard solutions with concentration gradients; (2) A quantitative amount of ochratoxin A standard solution was transferred to the surface of the ITO|WO3 / Pt|5FIn|Apt|BSA electrode for incubation. After a certain period of incubation, the electrode was rinsed with TE buffer solution to obtain multiple ITO|WO3 / Pt|5FIn|Apt|BSA|OTA electrodes. A three-electrode system was used, with ITO|WO3 / Pt|5FIn|Apt|BSA|OTA as the working electrode, Pt electrode as the auxiliary electrode, and Ag / AgCl electrode as the reference electrode. In a TE buffer solution with a pH of 6.5 to 8.5, the I–t technique was used at a bias voltage of 0.0 V, with an LED light as the excitation light source, and the light was switched on and off every 50 seconds to test the photocurrent intensity, and obtain a linear relationship between the photocurrent intensity and the logarithm of the concentration of the ochratoxin A standard solution. (3) Under the same conditions as in step (2), a quantitative amount of the sample solution of ochratoxin A to be tested is transferred to the surface of the ITO|WO3 / Pt|5FIn|Apt|BSA electrode for incubation, and after elution, a three-electrode system is established. In the same TE buffer as in step (2), the photocurrent intensity of the sample solution to be tested is detected under the same conditions as in step (2), and the concentration of ochratoxin A in the sample solution to be tested is obtained according to the measured photocurrent intensity using the linear relationship obtained in step (2).