Visual photoelectrochemical sensing device based on electrochromic supercapacitor and use thereof

Abstract

The application belongs to the technical field of biosensors, and relates to a visual photoelectrochemical sensing device based on an electrochromic super capacitor and an application thereof. A working electrode of a PEC sensor is a type-II MgTi2O5 / CdSe heterojunction as a photosensitive base, and the photoelectric conversion efficiency is improved. Meanwhile, an electrochromic super capacitor (ESC) composed of tungsten oxide and polyaniline is used as a signal display that can be repeatedly cycled; a designed circuit board is used to connect the PEC sensor and the ESC, the photocurrent generated by the PEC sensor is converted into voltage, and the ESC produces color changes with the changes of the photocurrent; a portable sensing device is constructed based on the PEC sensor and the ESC, and is successfully applied to precise and rapid analysis of Cry1Ab protein in genetically modified crops, and plays a demonstration role in other miniaturized visual PEC sensing systems in the fields of environmental monitoring and food safety evaluation, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, specifically relating to a visual photoelectrochemical sensing device based on an electrochromic supercapacitor and its use in detecting Cry1Ab protein. Background Technology

[0002] The presence of insecticidal crystal proteins (Cry) endows genetically modified (GM) crops with highly specific insecticidal activity. Among them, Cry1Ab protein is the most widely distributed insecticidal crystal protein in GM crops. Studies have shown that Cry1Ab protein can be detected in soil from GM corn planted for four consecutive years, and even in corn residues several months after harvest. Furthermore, during the growth of GM crops, Cry1Ab protein continuously enters the soil from plant residues, root exudates, and pollen. The long-term presence of Cry1Ab protein in the soil affects soil biodiversity and soil fertility, which in turn further impacts crop growth. Because the impact of GM agricultural products on human health is still unclear, many countries have set threshold values ​​for GM foods, requiring labeling of foods exceeding these limits. Therefore, it is necessary to develop accurate, rapid, and portable biosensors to detect Cry1Ab protein in GM crops for application in fields such as agricultural ecological protection and agricultural product quality monitoring.

[0003] Visualized photoelectrochemical (PEC) biosensors based on the principle of electrochromism have become a research hotspot due to their advantages such as high sensitivity, fast response, simple operation, and low detection cost. Their working mechanism involves stimulating electrochromic materials with current or potential, causing different redox states and thus generating a visual signal readout. Traditional visualized PEC biosensors based on the principle of electrochromism rely on a single electrochromic material. Since the color of the electrochromic material is irreversible, its use as a visual signal readout is limited to single-use. Research has found that electrochromic supercapacitors (ESCs) composed of electrochromic materials have the advantage of reversible color, allowing for repeated use as signal displays. However, a key unresolved issue is how to combine the highly reusable ESCs with PEC sensors to establish a visualized PEC sensing platform. Summary of the Invention

[0004] This invention introduces a designed circuit board between a photoelectrochemical (PEC) sensor and a reusable photoelectrochemical sensor (ESC) to construct an ESC-based visualized PEC sensing device for detecting the Cry1Ab protein. In the PEC sensor, MgTi2O5 / CdSe, as an excellent photosensitive material, generates a type-II heterojunction through its cross-matched bandgap structure, improving photoelectric conversion efficiency and amplifying the photocurrent. In the designed circuit board, the reference electrode (RE), counter electrode (CE), and solution resistance form a negative feedback circuit to stabilize the electrode potential; working electrode one (WE-B, without Cry1Ab protein) and working electrode two (WE-C, with Cry1Ab protein) are connected to a current / voltage conversion circuit that converts the photocurrent of WE into a voltage.

[0005] The voltages output from WE-B and WE-C are applied to both ends of the ESC, causing different color changes and thus revealing the concentration of Cry1Ab protein. For portable detection, an LED is used as the excitation light source, a portable electrochemical workstation and colorimeter are used as PEC and visualization signal acquisition devices, and a smartphone is used as the signal reader. Finally, all detection devices are integrated into a single portable sensing device. This sensing device serves as a model for other miniaturized visualization PEC sensing systems in the fields of environmental monitoring and food safety assessment.

[0006] To achieve the above objectives, the present invention includes the following technical solutions:

[0007] I. PEC Sensor:

[0008] S1. The preparation method of the working electrode is as follows:

[0009] (1) Preparation of MgTi2O5:

[0010] First, under stirring, Mg(CH3COO)2·4H2O was dissolved in ethylene glycol, and then tetrabutyl titanate was added. After stirring at room temperature, a mixed solution was obtained. The mixed solution was centrifuged to collect the precipitate, which was washed with ethanol by centrifugation, and then dried and calcined to obtain MgTi2O5 solid powder. Then it was dispersed in ultrapure water to obtain MgTi2O5 solution.

[0011] In step (1), the ratio of Mg(CH3COO)2·4H2O, ethylene glycol and tetrabutyl titanate is 0.1341g:60mL:0.2121mL; the stirring time at room temperature is 2h; the drying temperature is 80℃ and the time is 4h; the calcination temperature is 600℃ and the time is 2h, with a rate of 2℃ / min.

[0012] (2) Preparation of CdSe QDs:

[0013] (a) First, under nitrogen protection, selenium powder (Se) and sodium borohydride (NaBH4) are dissolved in ultrapure water (H2O) to obtain sodium selenide (NaHSe) precursor;

[0014] In step (2) (a), the ratio of selenium powder, sodium borohydride and ultrapure water is 7.9 mg: 7.6 mg: 5 mL.

[0015] (b) Then, under nitrogen protection, chromium chloride (CdCl2·2.5H2O) was dissolved in ultrapure water, 3-mercaptopropionic acid (MPA) was added, and the pH was adjusted to obtain a solution, which was denoted as solution A. After purging nitrogen into solution A for a period of time, the NaHSe precursor prepared in step (a) was added, and after reacting in a microwave synthesizer, the solution was washed with anhydrous ethanol by centrifugation. The precipitate after washing was dried and redispersed in ultrapure water to obtain a CdSe QDs solution.

[0016] In step (2) (b), the ratio of chromium chloride, ultrapure water and 3-mercaptopropionic acid is 45.7 mg: 50 mL: 44 μL; the pH adjustment is achieved by adjusting the solution pH to 11 using 1 M sodium hydroxide (NaOH).

[0017] The nitrogen gas purging time is 30 minutes;

[0018] The reaction in the microwave synthesizer was carried out at a temperature of 100°C for 4 hours.

[0019] The volume ratio of 3-mercaptopropionic acid (MPA) to ultrapure water in the sodium selenide (NaHSe) precursor in solution A is 11:1250.

[0020] (3) Modify the MgTi2O5 solution prepared in step (1) onto the surface of the ITO electrode and dry it in an incubator to obtain ITO / MgTi2O5;

[0021] Preferably, in step (3), the concentration of the MgTi2O5 solution is 0.4 mg / mL and the volume is 20 μL.

[0022] (4) Modify the ITO / MgTi2O5 prepared in step (3) with the CdSe QDs solution prepared in step (2) and dry it in an incubator to obtain ITO / MgTi2O5 / CdSe;

[0023] Preferably, in step (4), the concentration of the CdSe QDs solution is 0.1 mg / mL and the volume is 20 μL.

[0024] S2. A PEC sensor is constructed using ITO / MgTi2O5 / CdSe as the working electrode, Ag / AgCl as the reference electrode, and a platinum wire electrode as the counter electrode.

[0025] II. The fabrication method of electrochromic supercapacitor (ESC) is as follows:

[0026] (1) Electrodeposition of tungsten oxide (WO3) on a fluorine-doped tin oxide (FTO) electrode;

[0027] WO3 / FTO was prepared by depositing an FTO electrode in an electrodeposition solution at a certain potential.

[0028] Preferably, in step (1), the electrodeposition solution is composed of Na2WO4 and H2O2, with a final concentration of Na2WO4 of 12.5 mM and a mass concentration of H2O2 of 0.1%; wherein the pH of the electrodeposition solution is adjusted to 1.3 using HClO4.

[0029] The set potential is -0.7V (vs. Ag / AgCl), and the deposition time is 10 min.

[0030] (2) Electrodeposit polyaniline (PANI) on the FTO electrode;

[0031] A constant current is applied to the FTO electrode in an electrolyte solution, and after deposition for a period of time, PANI / FTO is prepared.

[0032] Preferably, in step (2), the electrolyte solution is composed of Na2SO4, H2SO4 and aniline, with the final concentration of Na2SO4 being 0.5M, the final concentration of H2SO4 being 0.5M, and the final concentration of aniline being 0.05M.

[0033] The constant current is 0.1 mA / cm. 2 (vs.Ag / AgCl), deposition time was 30 min.

[0034] (3) Mix polyvinyl alcohol (PVA) and H2SO4 solution under certain temperature conditions to obtain H2SO4 / PVA gel electrolyte. After cooling to room temperature, sandwich the H2SO4 / PVA gel electrolyte between PANI / FTO and WO3 / FTO. After drying, ESC is obtained.

[0035] Preferably, in step (3), the temperature is 90°C, the ratio of polyvinyl alcohol to H2SO4 solution is 1.5g:10mL, and the concentration of H2SO4 solution is 1M.

[0036] The drying temperature is 40℃, and the time is 12-24 hours;

[0037] III. A Visualized Photoelectrochemical Sensing Device Based on Electrochromic Supercapacitors

[0038] It consists of the PEC sensor prepared in step one, the light source module, the circuit board, the first lithium battery, the control power switch, the ESC prepared in step two, the colorimeter, the portable electrochemical workstation, the second lithium battery, and the smartphone.

[0039] The circuit board is electrically connected to the PEC sensor, lithium battery 1, and ESC.

[0040] The lithium battery is electrically connected to the light source module and the control power switch;

[0041] The portable electrochemical workstation is electrically connected to the PEC sensor, the lithium battery, and the smartphone.

[0042] The colorimeter is electrically connected to a smartphone.

[0043] Preferably, the circuit board includes a voltage reference source (denoted as U4), an operational amplifier, and a current / voltage conversion circuit, wherein there are two operational amplifiers, denoted as U0 and U2 respectively; there are two current / voltage conversion circuits, denoted as U1 and U3 respectively; the voltage reference source is located at the top of the circuit board and provides a 2.5V reference voltage;

[0044] The PEC sensor and the light source module are each provided in two units, which are respectively referred to as PEC sensor one, PEC sensor two, light source module one and light source module two 2';

[0045] Independent connection points are provided at the bottom of the circuit board, and are named WE_B, RE_B, CE_B, U_BLANK, U_Cry1Ab, WE_C, RE_C and CE_C from one end of the circuit board to the other.

[0046] A connection point P0 is also provided on the upper side of the circuit board, and P0 is electrically connected to a lithium battery.

[0047] RE_C and CE_C are electrically connected to the negative input and output terminals of operational amplifier U0, respectively; RE_B and CE_B are electrically connected to the negative input and output terminals of operational amplifier U2, respectively; WE_C and U_Cry1Ab are electrically connected to the negative input and output terminals of current / voltage conversion circuit U1; WE_B and U_BLANK are electrically connected to the negative input and output terminals of current / voltage conversion circuit U3.

[0048] Meanwhile, WE_C, RE_C, and CE_C are electrically connected to the working electrode, reference electrode, and counter electrode of PEC sensor one, respectively; WE_B, RE_B, and CE_B are electrically connected to the working electrode, reference electrode, and counter electrode of PEC sensor two, respectively; U_Cry1Ab and U_BLANK are electrically connected to both ends of ESC;

[0049] The lithium battery is electrically connected to the first light source module, the second light source module 2', the circuit board, and the control power switch.

[0050] The light source module includes an LED lamp and a constant current / constant voltage drive circuit. The function of the constant current / constant voltage drive circuit is to limit the current of the LED lamp.

[0051] The application of a visual photoelectrochemical sensing device based on an electrochromic supercapacitor for detecting Cry1Ab protein follows these steps:

[0052] I. Photoelectrochemical detection:

[0053] (1) Cry1Ab aptamer was modified on the surface of the working electrode (ITO / MgTi2O5 / CdSe) of the PEC sensor and incubated at a certain temperature for a period of time. After incubation, it was washed with ultrapure water. The working electrode was labeled as ITO / MgTi2O5 / CdSe / aptamer after washing.

[0054] Preferably, in step (1), the concentration of Cry1Ab aptamer is 2 μM and the amount is 20 μL; the incubation temperature is 4℃ and the incubation time is 12h.

[0055] (2) First, prepare Cry1Ab protein standard solutions of different concentrations, then modify ITO / MgTi2O5 / CdSe / aptamer, incubate at a certain temperature for a period of time, and then clean the electrode with ultrapure water. The product is labeled as ITO / MgTi2O5 / CdSe / aptamer / Cry1Ab.

[0056] Preferably, in step (2), the concentration of the Cry1Ab protein standard solution is 0.3 to 3000 ng / mL, the incubation temperature is 37°C, and the incubation time is 80 min.

[0057] (3) Using the ITO / MgTi2O5 / CdSe / aptamer / Cry1Ab prepared in step (2) as the working electrode; the light source module (2) is used as the excitation light source for the working electrode. The photoelectric signal generated by the working electrode is detected by a portable electrochemical workstation, and the obtained PEC signal is transmitted to a smartphone terminal and displayed on the smartphone. A standard curve is established with the concentration of Cry1Ab protein standard solution as the abscissa and the generated PEC signal as the ordinate.

[0058] (4) First, obtain the test solution, and then follow the operation of step (2), except that the Cry1Ab protein standard solution is replaced with the test solution. Continue to follow the operation of step (3), obtain the PEC signal and input it into the corresponding standard curve to realize the detection of Cry1Ab protein in the unknown sample.

[0059] II. Visual Inspection:

[0060] (1) Cry1Ab aptamer was modified on the working electrode surfaces of PEC sensor 1 and PEC sensor 2 respectively, and incubated at a certain temperature for a period of time. After incubation, it was washed with ultrapure water. The working electrodes were labeled as ITO / MgTi2O5 / CdSe / aptamer-1 and ITO / MgTi2O5 / CdSe / aptamer-1′ after cleaning.

[0061] Preferably, in step (1), the concentration of Cry1Ab aptamer is 2 μM, and the amount of modification is 20 μL; the incubation temperature is 4 °C, and the incubation time is 12 h.

[0062] (2) First, Cry1Ab protein standard solutions of different concentrations were prepared. Different concentrations of Cry1Ab protein standard solutions were modified on the surface of ITO / MgTi2O5 / CdSe / aptamer-1. After incubation and washing, the final product was labeled as ITO / MgTi2O5 / CdSe / aptamer-1 / Cry1Ab-N; where N is the Cry1Ab protein standard solution with a concentration of 0 to 1000 ng / mL. Only Cry1Ab protein standard solution with a concentration of 0 was modified on the surface of ITO / MgTi2O5 / CdSe / aptamer-1′. After incubation and washing, the final product was labeled as ITO / MgTi2O5 / CdSe / aptamer-1′ / Cry1Ab-0.

[0063] Then, ITO / MgTi2O5 / CdSe / aptamer-1 / Cry1Ab-N was used as the first working electrode, and ITO / MgTi2O5 / CdSe / aptamer-1′ / Cry1Ab-0 was used as the second working electrode. Light source module 1 was used as the excitation source for the first working electrode, and light source module 2′ was used as the excitation source for the second working electrode. The photocurrent generated by the first and second working electrodes was transmitted to both ends of the ESC through the circuit board. The color and RGB values ​​of the ESC were collected by a colorimeter and transmitted to a smartphone to display a visual signal. A standard curve was established with the concentration of the Cry1Ab protein standard solution as the x-axis and the visual signal as the y-axis.

[0064] Preferably, in step (2), the incubation conditions are 37°C for 80 minutes.

[0065] (3) Actual sample testing:

[0066] First, obtain the test solution, and then follow the operation in step (2), except that the Cry1Ab protein standard solution of different concentrations is replaced with the test solution and modified on the surface of ITO / MgTi2O5 / CdSe / aptamer-1. Obtain the visual signal, substitute it into the corresponding standard curve, and the detection of Cry1Ab protein in unknown samples can be realized.

[0067] The beneficial effects of this invention are:

[0068] (1) This invention designs and prepares a type-II MgTi2O5 / CdSe heterojunction by utilizing the energy level band gap difference of semiconductors. As an excellent photosensitive material, it effectively improves the photoelectric conversion efficiency and amplifies the photocurrent.

[0069] (2) The ESC-based visual PEC sensing device proposed in this invention successfully connects the PEC sensor to the ESC for the first time by introducing a designed circuit board, and uses the reusable ESC as a signal display screen for visual PEC sensing detection, which effectively improves the detection efficiency.

[0070] (3) This invention proposes to use solid-state ESC as a signal display screen and develop a solid-state module color display method to effectively eliminate the problem of interference of the visual signal by the solution environment.

[0071] (4) This invention combines the sensitive detection of PEC with the rapid detection of visualization and introduces miniaturized detection instruments, so as to realize the accurate and rapid analysis of Cry1Ab protein while making the detection device portable.

[0072] (5) The visualization PEC sensing device constructed in this invention is used for Cry1Ab protein detection. It has high sensitivity, good selectivity, good stability, wide linear range (PEC: 0.3~3000ng / mL; visualization: 1~1000ng / mL), and low detection limit (PEC: 0.11ng / mL; visualization: 0.14ng / mL). Attached Figure Description

[0073] Figure 1 A schematic diagram illustrating the use of the PEC sensing device for Cry1Ab protein detection.

[0074] Figure 2 To monitor the (A) color switching response and (B) cycle stability of the ESC using the chronoamperometry method.

[0075] Figure 3This is a diagram of a visual photoelectrochemical sensing device based on an electrochromic supercapacitor; where 1—PEC sensor one, 1′—PEC sensor two, 2—light source module one, 2′—light source module two, 3—circuit board, 4—lithium battery one, 5—control power switch, 6—electrochromic supercapacitor, 7—colorimeter, 8—portable electrochemical workstation, 9—lithium battery two, 10—smartphone.

[0076] Figure 4 This is a schematic diagram of the circuit board.

[0077] Figure 5 This is the schematic diagram of the circuit board.

[0078] Figure 6 This section provides a block diagram and explanation of the working principle of the entire system.

[0079] Figure 7 (A) is a physical image of the light source module; (B) is the cover of the electrolytic cell with the three electrodes fixed; (C) is a physical image of the three-electrode system and the electrolytic cell.

[0080] Figure 8 (A) shows the PEC response of the sensor to different concentrations of Cry1Ab protein (0.3–3000 ng / mL); (B) shows the linear regression curve for detecting Cry1Ab protein (the logarithm of photocurrent I to Cry1Ab protein concentration); (C) shows the visual response of the sensor to different concentrations of Cry1Ab protein (1–1000 ng / mL); and (D) shows the corresponding calibration curve for detecting Cry1Ab protein (ΔC to the logarithm of Cry1Ab protein concentration). Detailed implementation method:

[0081] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments are based on the technical solution of the present invention and provide detailed implementation steps and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0082] The aptamers used in this invention were purchased from Sangon Biotech (Shanghai) Co., Ltd.; the sequence of the Cry1Ab aptamer is: 5'-SH-CCG CGG TTC CTC GGC CCC CTA TCC ACG CCG AGT CCC GAA TAC TCC CCAGAT GTA GTA GCC CTC AGC ATAG-3'

[0083] This application does not involve sequence listing applications. The above sequences are for illustrative purposes only and are all conventional reagent sequences, belonging to primer sequences.

[0084] Example 1:

[0085] I. A PEC sensor consists of a working electrode, a reference electrode, and a counter electrode.

[0086] The specific steps for preparing the working electrode are as follows:

[0087] (1) Preparation of MgTi2O5:

[0088] First, 0.1341 g of Mg(CH3COO)2·4H2O was dissolved in 60 mL of ethylene glycol under stirring, and then 0.2121 mL of tetrabutyl titanate was added. After stirring at room temperature for 2 h, a mixed solution was obtained. The mixed solution was centrifuged to collect the precipitate, washed with ethanol, and dried at 80 °C for 4 h. Finally, the sample was calcined at 600 °C at a heating rate of 2 °C / min for 2 h to obtain MgTi2O5 solid powder, which was then dispersed in ultrapure water to obtain MgTi2O5 solution.

[0089] (2) Preparation of CdSe QDs:

[0090] First, under nitrogen protection, 7.9 mg of selenium powder (Se) and 7.6 mg of sodium borohydride (NaBH4) were dissolved in 5 mL of ultrapure water (H2O) to obtain sodium selenide (NaHSe) precursor;

[0091] Then, under nitrogen protection, 45.7 mg of chromium chloride (CdCl2·2.5H2O) was dissolved in 50 mL of H2O, and 44 μL of 3-mercaptopropionic acid (MPA) was added. The pH of the solution was adjusted to 11 using 1 M sodium hydroxide (NaOH), and this solution was denoted as solution A. After purging nitrogen into solution A for 30 min, the prepared NaHSe precursor was quickly added, and the reaction was carried out at 100 °C for 4 h in a microwave synthesizer. Finally, the product was centrifuged and washed with anhydrous ethanol, and the precipitate was dried and redispersed in ultrapure water to obtain a CdSeQDs solution.

[0092] (3) The MgTi2O5 (20 μL, 0.4 mg / mL) solution prepared in step (1) was modified onto the surface of the ITO electrode and dried in an incubator. At this time, the product was labeled as ITO / MgTi2O5.

[0093] (4) The CdSe QDs (20 μL, 0.1 mg / mL) solution prepared in step (2) was used to modify the surface of the ITO / MgTi2O5 electrode prepared in step (3); and the electrode was dried in an incubator. The dried product was labeled as ITO / MgTi2O5 / CdSe.

[0094] A PEC sensor is constructed using ITO / MgTi2O5 / CdSe as the working electrode, Ag / AgCl as the reference electrode, and a platinum wire electrode as the counter electrode.

[0095] II. The preparation method of ESC is as follows:

[0096] (1) Electrodeposition of tungsten oxide (WO3) on a fluorine-doped tin oxide (FTO) electrode;

[0097] The FTO electrode was deposited in an electrodeposition solution (composed of Na2WO4 and H2O2, with a final Na2WO4 concentration of 12.5 mM and a H2O2 mass concentration of 0.1%), wherein the pH of the electrodeposition solution was adjusted to 1.3 by HClO4; WO3 / FTO was prepared after deposition at a potential of -0.7 V (vs. Ag / AgCl) for 10 min.

[0098] (2) Electrodeposit polyaniline (PANI) on the FTO electrode.

[0099] The FTO electrode is placed in an electrolyte solution (composed of Na2SO4, H2SO4, and aniline, with a final concentration of Na2SO4 of 0.5 M, a final concentration of H2SO4 of 0.5 M, and a final concentration of aniline of 0.05 M) and an applied current of 0.1 mA / cm. 2 PANI / FTO was prepared by deposition at a constant current (vs. Ag / AgCl) for 30 min.

[0100] (3) Dissolve 1.5g of polyvinyl alcohol (PVA) in 10mL of 1M H2SO4 solution at 90℃ to prepare H2SO4 / PVA gel electrolyte. After cooling to room temperature, sandwich the gel electrolyte between PANI / FTO and WO3 / FTO. Finally, dry at 40℃ for 12h to evaporate excess water. After drying, ESC is obtained.

[0101] The color switching response and cycle stability of the ESC were monitored using the chronoamperometry method. For example... Figure 2 As shown in Figure (A), the coloring time for ESC is 1.1 seconds, and the bleaching time is 1.1 seconds. Figure 2 As shown in Figure (B), the ESC was subjected to 200 cycles of testing between 0 and 0.9V using the chronoamperometry method. The experimental results show that the ESC has the advantages of fast response time and reusability.

[0102] III. Figure 3 As shown, a visual photoelectrochemical sensing device based on an electrochromic supercapacitor is constructed. The device consists of a PEC sensor prepared in step one, a light source module, a circuit board 3, a lithium battery 4, a control power switch 5, an ESC prepared in step two, a colorimeter 7, a portable electrochemical workstation 8, a lithium battery 9, and a smartphone 10. There are two PEC sensors and two light source modules, referred to as PEC sensor 1, PEC sensor 2', light source module 2, and light source module 2'.

[0103] like Figure 4-6 As shown, the circuit board 3 includes a voltage reference source (denoted as U4), an operational amplifier, and a current / voltage conversion circuit. There are two operational amplifiers, denoted as U0 and U2 respectively; there are two current / voltage conversion circuits, denoted as U1 and U3 respectively; the voltage reference source U4 is located at the top of the circuit board and provides a 2.5V reference voltage.

[0104] Independent connection points are provided at the bottom of circuit board 3, and are named WE_B, RE_B, CE_B, U_BLANK, U_Cry1Ab, WE_C, RE_C and CE_C from left to right.

[0105] A connection point P0 is provided on the upper right side of the circuit board 3, and P0 is electrically connected to the lithium battery 4.

[0106] RE_C and CE_C are electrically connected to the negative input and output terminals of operational amplifier U0, respectively; RE_B and CE_B are electrically connected to the negative input and output terminals of operational amplifier U2, respectively; WE_C and U_Cry1Ab are electrically connected to the negative input and output terminals of current / voltage conversion circuit U1; WE_B and U_BLANK are electrically connected to the negative input and output terminals of current / voltage conversion circuit U3.

[0107] Meanwhile, WE_C, RE_C, and CE_C are electrically connected to the working electrode, reference electrode, and counter electrode of the PEC sensor 1, respectively; WE_B, RE_B, and CE_B are electrically connected to the working electrode, reference electrode, and counter electrode of the PEC sensor 2', respectively; and U_Cry1Ab and U_BLANK are electrically connected to both ends of ESC6.

[0108] The circuit board 3 is electrically connected to the PEC sensor, lithium battery 4, and ESC6.

[0109] The lithium battery 4 is electrically connected to the light source module 2, the light source module 2', and the control power switch 5.

[0110] The portable electrochemical workstation 8 is electrically connected to the PEC sensor, lithium battery 2 9, and smartphone 10;

[0111] The colorimeter 7 is electrically connected to the smartphone 10.

[0112] IV. Figure 7 As shown in (A), the light source module 2 includes an LED lamp and a constant current / constant voltage driving circuit. The function of the constant current / constant voltage driving circuit is to limit the current of the LED lamp.

[0113] V. The application of the above-mentioned visual photoelectrochemical sensing device based on electrochromic supercapacitor for detecting Cry1Ab protein is as follows: Figure 1 As shown, the specific steps are as follows:

[0114] S1, photoelectrochemical detection:

[0115] (1) 20 μL and 2 μM of Cry1Ab aptamer were modified on the surface of the working electrode ITO / MgTi2O5 / CdSe of PEC sensor-1 and incubated at 4℃ for 12 h. After incubation, the electrode was washed with ultrapure water and labeled as ITO / MgTi2O5 / CdSe / aptamer.

[0116] (2) First, prepare Cry1Ab protein standard solutions (concentrations of 0.3, 1, 3, 10, 30, 100, 300, 1000, and 3000 ng / mL, respectively), and then modify them on the surface of the working electrode ITO / MgTi2O5 / CdSe / aptamer. Incubate at 37°C for 80 min, and then clean the electrode with ultrapure water. The product is labeled as ITO / MgTi2O5 / CdSe / aptamer / Cry1Ab.

[0117] (3) PEC detection: The ITO / MgTi2O5 / CdSe / aptamer / Cry1Ab prepared in step (2) is used as the working electrode; the light source module 2 is used as the excitation light source of the working electrode. The photoelectric signal generated by the working electrode is detected by the portable electrochemical workstation 8, and the obtained PEC signal is transmitted to the smartphone 10 terminal and displayed on the smartphone 10. A standard curve is established with the concentration of Cry1Ab protein standard solution as the abscissa and the generated PEC signal as the ordinate.

[0118] (4) First, obtain the test solution, and then follow the operation of step (2), except that the Cry1Ab protein standard solution is replaced with the test solution. Continue to follow the operation of step (3), obtain the PEC signal and input it into the corresponding standard curve to realize the detection of Cry1Ab protein in the unknown sample.

[0119] S2, Visual Inspection:

[0120] (1) Cry1Ab aptamer was modified on the working electrode surfaces of PEC sensor 1 and PEC sensor 2 1′, respectively. The amount of Cry1Ab aptamer used was 20 μL and the concentration was 2 μM. The electrodes were incubated at 4 °C for 12 h. After incubation, they were washed with ultrapure water. The working electrodes were labeled as ITO / MgTi2O5 / CdSe / aptamer-1 and ITO / MgTi2O5 / CdSe / aptamer-1′.

[0121] (2) First, prepare Cry1Ab protein standard solution. Modify the surface of ITO / MgTi2O5 / CdSe / aptamer-1 with Cry1Ab protein standard solution (concentrations of 0, 1, 3, 10, 30, 100, 300, 1000, and 1000 ng / mL, respectively). After incubation (37℃ for 80 min) and washing, the final product is recorded as ITO / MgTi2O5 / CdSe / aptamer-1 / Cry1Ab-N; (where N is the concentration of Cry1Ab protein standard solution).

[0122] The surface of ITO / MgTi2O5 / CdSe / aptamer-1′ was modified with Cry1Ab protein standard solution at a concentration of 0. After incubation (37℃ for 80 min) and washing, the final product was labeled as ITO / MgTi2O5 / CdSe / aptamer-1′ / Cry1Ab-0.

[0123] Then, ITO / MgTi2O5 / CdSe / aptamer-1 / Cry1Ab-N was used as the first working electrode, and ITO / MgTi2O5 / CdSe / aptamer-1′ / Cry1Ab-0 was used as the second working electrode. Light source module 1 2 served as the excitation source for the first working electrode, and light source module 2′ served as the excitation source for the second working electrode. The photocurrent generated by the first and second working electrodes was transmitted to both ends of ESC6 through circuit board 3. The color and RGB values ​​of ESC6 were collected by colorimeter 7, and the collected data was transmitted to smartphone 10, where the visualized signal was displayed. Finally, a standard curve was established with the concentration of Cry1Ab protein standard solution as the x-axis and the visualized signal as the y-axis.

[0124] (3) Actual sample testing:

[0125] First, obtain the test solution, and then follow the operation in step (2), except that the Cry1Ab protein standard solution of different concentrations is replaced with the test solution and modified on the surface of ITO / MgTi2O5 / CdSe / aptamer-1; after detection, obtain the visualized signal, and substitute it into the corresponding standard curve to realize the detection of Cry1Ab protein in unknown samples.

[0126] Example 2:

[0127] Performance analysis of a visual photoelectrochemical sensing device based on an electrochromic supercapacitor for detecting Cry1Ab protein:

[0128] For photoelectrochemical detection, working electrodes, reference electrodes, and counter electrodes modified with different concentrations of Cry1Ab protein standard solutions (concentrations of 0.3, 1, 3, 10, 30, 100, 300, 1000, and 3000 ng / mL, respectively) were fixed to the electrolytic cell lid. Figure 7 In sections B and C), the electrolyte was 0.1M pH 7.4 PBS (containing 0.1M AA) buffer solution, the excitation source was a 365nm LED, and the applied voltage was 0V. A standard curve was constructed by plotting the logarithm of the Cry1Ab protein standard solution concentration on the x-axis and the obtained photocurrent signal on the y-axis. From... Figure 8 As shown in Figure A, the photocurrent gradually decreases with increasing Cry1Ab protein concentration. Figure 8 As shown in Figure B, the linear regression equation for photoelectrochemical detection is I = -1.810lg C. Cry1Ab +9.271(R 2 =0.9996).

[0129] For visual detection, ITO / MgTi2O5 / CdSe / aptamer-1 / Cry1Ab-N (N represents the corresponding concentration values, with concentrations of 0, 1, 3, 10, 30, 100, 300, 1000, and 1000 ng / mL) was used as the first working electrode, and ITO / MgTi2O5 / CdSe / aptamer-1′ / Cry1Ab-0 was used as the second working electrode. The electrolyte was 0.1M pH 7.4 PBS (containing 0.1 MAA) buffer solution, the excitation light source was a 365nm LED, and the applied voltage was 0V.

[0130]

[0131] Wherein ΔR, ΔG, and ΔB are the R, G, and B values ​​generated by working electrode one corresponding to different concentrations of Cry1Ab protein standard solution (N = 1, 3, 10, 30, 100, 300, 1000, 1000 ng / mL), minus the R, G, and B values ​​generated by working electrode one with a Cry1Ab protein standard solution of N concentration of 0 ng / mL.

[0132] from Figure 8 As can be seen from the data, ΔC gradually increases with increasing Cry1Ab protein concentration; for example... Figure 8 As shown in Figure D, the linear regression equation for the visual detection is ΔC = 21.53lg C. Cry1Ab +6.378(R 2 =0.9996).

[0133] Example 3:

[0134] The constructed visualization PEC sensing device based on electrochromic supercapacitor was used to detect and analyze Cry1Ab protein in actual samples. Different concentrations of Cry1Ab protein standard solution were added to non-GMO maize (1-4) and maize field soil (5-8) for PEC and visualization detection.

[0135] The non-GMO corn was divided into four groups, numbered 1-4; the soil from the cornfield was also divided into four groups, numbered 5-8; and the GMO corn MON810 was divided into two groups, numbered 9-10.

[0136] Sample description: The non-GMO corn came from cornfields in Dongying City, Shandong Province; the soil also came from cornfields in Dongying City, Shandong Province.

[0137] The genetically modified corn MON810 comes from Shanghai Zhenzhun Biotechnology Co., Ltd.

[0138] As shown in Table 1, the RSD is less than 5.62%, indicating that the developed biosensor has good reproducibility. Its recovery rate ranges from 90.9% to 104%, which is basically consistent with the detection results of enzyme-linked immunosorbent assay (ELISA). Furthermore, the developed biosensor was applied to the quantitative determination of transgenic maize MON810 samples (9-10). For PEC / visualization assays, the Cry1Ab protein content in transgenic maize was 2.75 / 2.58 ng / mL and 12.85 / 12.83 ng / mL, which is consistent with the ELISA results (2.47 and 12.79 ng / mL), indicating that the developed biosensor has good reliability in actual sample analysis.

[0139] Table 1. Determination of Cry1Ab protein in non-GMO maize, soil, and GMO maize MON810 (n=3)

[0140]

[0141] Note: "-" indicates not detected.

[0142] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A visual photoelectrochemical sensing device based on an electrochromic supercapacitor, characterized in that, The device consists of a PEC sensor, a light source module, a circuit board (3), a lithium battery (4), a control power switch (5), an ESC (6), a colorimeter (7), a portable electrochemical workstation (8), a lithium battery (9), and a smartphone (10). The circuit board (3) is electrically connected to the PEC sensor, lithium battery (4), and ESC (6). The lithium battery (4) is electrically connected to the light source module and the control power switch (5); The portable electrochemical workstation (8) is electrically connected to the PEC sensor, lithium battery II (9), and smartphone (10); The colorimeter (7) is electrically connected to the smartphone (10); The PEC sensor includes a working electrode, a reference electrode, and a counter electrode. The reference electrode is an Ag / AgCl electrode, and the counter electrode is a platinum wire electrode. The working electrode is ITO / MgTi2O5 / CdSe, and its preparation method is as follows: (1) First, under stirring, Mg(CH3COO)2·4H2O was dissolved in ethylene glycol, and then tetrabutyl titanate was added. After stirring at room temperature, a mixed solution was obtained. The mixed solution was centrifuged to collect the precipitate, washed with ethanol by centrifugation, and then dried and calcined to obtain MgTi2O5 solid powder. Then, it was dispersed in ultrapure water to obtain MgTi2O5 solution. The ratio of the amounts of Mg(CH3COO)2·4H2O, ethylene glycol and tetrabutyl titanate was 0.1341 g: 60 mL: 0.2121 mL. The stirring time at room temperature was 2 h. The drying temperature was 80 ℃ and the time was 4 h. The calcination temperature was 600 ℃ and the time was 2 h. The calcination rate was 2 ℃ / min. (2) Preparation of CdSe QDs: (a) First, under nitrogen protection, selenium powder and sodium borohydride are dissolved in ultrapure water to obtain sodium selenide precursor; (b) Then, under nitrogen protection, chromium chloride was dissolved in ultrapure water, 3-mercaptopropionic acid was added, and the pH was adjusted to obtain a solution, which was denoted as solution A. After passing nitrogen gas through solution A for a period of time, sodium selenide precursor prepared in step (a) was added. After reacting in a microwave synthesizer, the solution was washed with anhydrous ethanol by centrifugation. The precipitate was dried and redispersed in ultrapure water to obtain CdSe QDs solution. (3) The MgTi2O5 solution prepared in step (1) is modified onto the surface of the ITO electrode and dried in an incubator to obtain ITO / MgTi2O5; (4) Modify the ITO / MgTi2O5 prepared in step (3) with the CdSe QDs solution prepared in step (2) and dry it in an incubator to obtain ITO / MgTi2O5 / CdSe; The method for preparing the ESC in the device includes the following steps: S1. WO3 / FTO is prepared by depositing an FTO electrode in an electrodeposition solution at a certain potential. S2. Apply a constant current to the FTO electrode in the electrolyte solution and deposit it for a period of time to prepare PANI / FTO; S3. Mix polyvinyl alcohol and H2SO4 solution under certain temperature conditions to obtain H2SO4 / PVA gel electrolyte. After cooling to room temperature, sandwich the H2SO4 / PVA gel electrolyte between PANI / FTO and WO3 / FTO, and obtain ESC after drying.

2. The visual photoelectrochemical sensing device based on an electrochromic supercapacitor according to claim 1, characterized in that, The circuit board (3) includes a voltage reference source U4, an operational amplifier and a current / voltage conversion circuit, wherein there are two operational amplifiers, denoted as U0 and U2 respectively; there are two current / voltage conversion circuits, denoted as U1 and U3 respectively; The PEC sensor and the light source module are each provided in two units, which are respectively referred to as PEC sensor one (1), PEC sensor two (1′), light source module one (2) and light source module two (2′). Independent connection points are provided at the bottom of the circuit board (3), and are named WE_B, RE_B, CE_B, U_BLANK, U_Cry1Ab, WE_C, RE_C and CE_C from one end of the circuit board (3) to the other end. A connection point P0 is also provided on the upper side of the circuit board (3), and P0 is electrically connected to the lithium battery (4). RE_C and CE_C are electrically connected to the negative input and output terminals of operational amplifier U0, respectively; RE_B and CE_B are electrically connected to the negative input and output terminals of operational amplifier U2, respectively; WE_C and U_Cry1Ab are electrically connected to the negative input and output terminals of current / voltage conversion circuit U1; WE_B and U_BLANK are electrically connected to the negative input and output terminals of current / voltage conversion circuit U3. Meanwhile, WE_C, RE_C, and CE_C are electrically connected to the working electrode, reference electrode, and counter electrode of PEC sensor one (1), respectively; WE_B, RE_B, and CE_B are electrically connected to the working electrode, reference electrode, and counter electrode of PEC sensor two (1′), respectively; U_Cry1Ab and U_BLANK are electrically connected to both ends of ESC (6); The lithium battery (4) is electrically connected to the light source module (2), the light source module (2'), the circuit board (3), and the control power switch (5).

3. The visual photoelectrochemical sensing device based on an electrochromic supercapacitor according to claim 1, characterized in that, In step (2) (a), the ratio of selenium powder, sodium borohydride, and ultrapure water is 7.9 mg: 7.6 mg: 5 mL; in (b), the ratio of chromium chloride, ultrapure water, and 3-mercaptopropionic acid is 45.7 mg: 50 mL: 44 µL; the pH adjustment is achieved by using 1 M sodium hydroxide (NaOH) to adjust the solution pH to 11. The nitrogen gas purging time is 30 min; the reaction temperature in the microwave synthesizer is 100 ℃ and the time is 4 h; The volume ratio of 3-mercaptopropionic acid to ultrapure water in the sodium selenide precursor in solution A is 11:1250.

4. The visual photoelectrochemical sensing device based on an electrochromic supercapacitor according to claim 1, characterized in that, In step (3), the concentration of the MgTi2O5 solution is 0.4 mg / mL and the volume is 20 μL; in step (4), the concentration of the CdSe QDs solution is 0.1 mg / mL and the volume is 20 μL.

5. The visual photoelectrochemical sensing device based on an electrochromic supercapacitor according to claim 1, characterized in that, In step S1, the electrodeposition solution is composed of Na2WO4 and H2O2, with a final concentration of Na2WO4 of 12.5 mM and a mass concentration of H2O2 of 0.1%; the pH of the electrodeposition solution is adjusted to 1.3 using HClO4; the fixed potential is −0.7 V, and the deposition time is 10 min. In step S2, the electrolyte solution consists of Na₂SO₄, H₂SO₄, and aniline, with a final concentration of 0.5 M for Na₂SO₄, 0.5 M for H₂SO₄, and 0.05 M for aniline; the constant current is 0.1 mA / cm². 2 The deposition time was 30 minutes. In step S3, the temperature is 90 °C, the ratio of polyvinyl alcohol to H2SO4 solution is 1.5 g: 10 mL, and the concentration of H2SO4 solution is 1 M; the drying temperature is 40 °C, and the time is 12~24 h.

6. The use of the visual photoelectrochemical sensing device based on an electrochromic supercapacitor according to any one of claims 1-5 for detecting Cry1Ab protein, characterized in that, The steps are as follows: I. Photoelectrochemical detection: (1) The Cry1Ab aptamer was modified on the surface of the working electrode of the PEC sensor (1) and incubated at a certain temperature for a period of time. After incubation, it was washed with ultrapure water. After washing, the working electrode was labeled as ITO / MgTi2O5 / CdSe / aptamer. (2) First, prepare Cry1Ab protein standard solutions of different concentrations, then modify them on ITO / MgTi2O5 / CdSe / aptamer, incubate them at a certain temperature for a period of time, and then clean the electrodes with ultrapure water. The product is labeled as ITO / MgTi2O5 / CdSe / aptamer / Cry1Ab. (3) The ITO / MgTi2O5 / CdSe / aptamer / Cry1Ab prepared in step (2) is used as the working electrode; the light source module (2) is used as the excitation light source of the working electrode, so that the photoelectric signal generated by the working electrode is detected by the portable electrochemical workstation (8), and the obtained PEC signal is transmitted to the smartphone (10) terminal and displayed on the smartphone (10). A standard curve was established with the concentration of Cry1Ab protein standard solution as the x-axis and the generated PEC signal as the y-axis. (4) First, obtain the test solution, and then follow the operation of step (2), except that the Cry1Ab protein standard solution is replaced with the test solution. Continue to follow the operation of step (3), obtain the PEC signal and input it into the corresponding standard curve to realize the detection of Cry1Ab protein in the unknown sample. II. Visual Inspection: (1) Cry1Ab aptamers were modified on the working electrode surfaces of PEC sensor one (1) and PEC sensor two (1′) respectively, and incubated at a certain temperature for a period of time. After incubation, they were washed with ultrapure water. After washing, the working electrodes were labeled as ITO / MgTi2O5 / CdSe / aptamer-1 and ITO / MgTi2O5 / CdSe / aptamer-1′. (2) First, Cry1Ab protein standard solutions of different concentrations were prepared. Different concentrations of Cry1Ab protein standard solutions were modified on the surface of ITO / MgTi2O5 / CdSe / aptamer-1. After incubation and washing, the final product was labeled as ITO / MgTi2O5 / CdSe / aptamer-1 / Cry1Ab-N; where N is the Cry1Ab protein standard solution with a concentration of 0~1000 ng / mL. Only Cry1Ab protein standard solution with a concentration of 0 was modified on the surface of ITO / MgTi2O5 / CdSe / aptamer-1′. After incubation and washing, the final product was labeled as ITO / MgTi2O5 / CdSe / aptamer-1′ / Cry1Ab-0. Then, ITO / MgTi2O5 / CdSe / aptamer-1 / Cry1Ab-N is used as the first working electrode, and ITO / MgTi2O5 / CdSe / aptamer-1′ / Cry1Ab-0 is used as the second working electrode. The first light source module (2) is used as the excitation light source for the first working electrode, and the second light source module (2′) is used as the excitation light source for the second working electrode. The photocurrent generated by the first working electrode and the second working electrode is transmitted to both ends of the ESC (6) through the circuit board (3). The color and RGB value of the ESC (6) are collected by the colorimeter (7) and transmitted to the smartphone (10) to display the visual signal. A standard curve was constructed using the concentration of Cry1Ab protein standard solution as the x-axis and the visualized signal as the y-axis. (3) Actual sample testing: First, obtain the test solution, and then follow the operation in step (2), except that the Cry1Ab protein standard solution of different concentrations is replaced with the test solution and modified on the surface of ITO / MgTi2O5 / CdSe / aptamer-1. Obtain the visual signal, substitute it into the corresponding standard curve, and the detection of Cry1Ab protein in the unknown sample can be realized.

7. The use according to claim 6, characterized in that, In step (1) of photoelectrochemical detection, the concentration of Cry1Ab aptamer is 2 μM and the volume is 20 μL; the incubation temperature is 4 ℃ and the incubation time is 12 h; in step (2), the concentration of Cry1Ab protein standard solution is 0.3~3000 ng / mL, the incubation temperature is 37 ℃ and the incubation time is 80 min.

8. The use according to claim 6, characterized in that, In step (1) of the visualization detection, the concentration of Cry1Ab aptamer was 2 μM, and the amount of modification was 20 μL; the incubation temperature was 4 ℃, and the incubation time was 12 h.

9. The use according to claim 6, characterized in that, In step (2) of the visualization detection, the incubation conditions are: 37 ℃, 80 min.

Images