Method for specifically detecting environmental pollutant microcystin by using organic photoelectrochemical transistor

Abstract

A novel organic photoelectrochemical transistor aptamer (OPECT) biosensor is developed, photoelectrochemical analysis and an organic electrochemical transistor are combined, AgI-TiO2 (AgI-TNs) is used as a photoactive material, target specific DNA chain reaction hybridization is used as a signal amplifier, and the sensor is used for detecting microcystin-LR in the environment. The developed sensor is high in detection sensitivity, the linear range is 0.1 fg mL <-1 > to 10 pg mL <-1 >, and the detection limit is as low as 0.079 fg mL <-1 >. Development of a biological sensitive OPECT platform brings hope for MC-LR detection, and meanwhile the biological sensitive OPECT platform can be used for detecting other marine pollutants.

Description

[0001] In this invention, we developed a novel organic photoelectrochemical transistor aptamer (OPECT) biosensor, which combines photoelectrochemical analysis and organic electrochemical transistor, using AgI-TiO2 (AgI-TNs) as the photoactive material and target-specific DNA chain reaction hybridization as the signal amplifier for the detection of microcystin-LR. In addition, since the amplitude of the signal drop directly depends on the amount of the target, the device I D The changes can be used to measure quantitatively. Background Art

[0002] Since its invention in 1947, the transistor has been regarded as the beginning of the microelectronics revolution. Organic electrochemical transistors (OECTs) that can achieve mixed ion and electron transport have attracted widespread attention due to their bio-system-friendly characteristics such as low operating voltage and good biocompatibility. The development of OPECT is based on the interaction between light and the photoactive gate. The change in the gate interface potential caused by the excitation light is used to adjust the doping state of the channel semiconductor, thereby bringing about different responses of the channel current, and the photoelectrochemical process is used to control the behavior of the OECT. Therefore, OPECT inherits the advantages of PEC and OECT, such as easy manufacturing, low operating voltage, strong light controllability, etc., and has great application potential. Summary of the invention

[0003] The present invention designs a novel organic photoelectrochemical transistor aptamer (OPECT) biosensor based on OPECT which combines the advantages of PEC and OECT. The device can realize the high-sensitivity detection of trace amounts of marine algae toxin (MC-LR).

[0004] To achieve the above purpose, the design idea of ​​the present invention is:

[0005] The essence of OPECT converting the light signal received by the gate into a large channel current is the change in the conductivity of the channel polymer induced by light. Specifically, the channel semiconductor is p-type and the carriers are holes. Under normal conditions, the holes on PEDOT are composed of PSS. - Doping compensation, at this time the semiconductor is in an oxidized state, and the channel carrier density is large. Under light, the forward photovoltage generated by the n-type semiconductor drives the cations in the solution into the semiconductor, making the PEDOT in the oxidized state + Transformed into reduced PEDOT 0 , which leads to I D When illuminated, the gate photosensitive semiconductor electron transition is accompanied by charge transfer, and the generated photovoltage continues to be distributed at the two interfaces. Assume that the G / E interface partial pressure change caused by light irradiation is V ph oto, at a fixed V G Under this condition, the channel / electrolyte interface partial pressure increases to V CIn the OPECT-based biosensor, as long as the biochemical reaction related to MC-LR can induce V photo Changes occur, changes V e G ff , producing a readable I D The change can realize the detection of the MC-LR to be tested.

[0006] The advantages of the present invention are:

[0007] The device has a low detection limit for detecting environmental pollutants and its application, and can achieve sensitive detection of trace amounts of MC-LR. It has broad application prospects in biosensing and environmental monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 It is a schematic diagram of the present invention.

[0009] Figure 2 Schematic diagram of the fabrication of OECT devices.

[0010] Figure 3 The morphology of TNs and AgI-TNs of the present invention is characterized. (a) SEM image of TNs. (b) TEM images of TNs and (c) AgI-TNs. (d) Mapping diagram of AgI-TNs.

[0011] Figure 4 This is a mapping diagram of the SEM images of Ti, O, Ag and I in the AgI-TNs of the present invention.

[0012] Figure 5 The structural characterization images of TNs and AgI-TNs of the present invention. (a) X-ray photoelectron spectroscopy (XPS) spectra of TNs and AgI-TNs. (b) UV-visible DRS of TNs and AgI-TNs. (c, d) Tauc plots and (e, f) Mott-Schottky plots of TNs and AgI-TNs.

[0013] Figure 6 The feasibility test diagram of PEC of the present invention. (a) Schematic diagram of the ET mechanism between TiO2 and AgI. (b) Photocurrent stability of the PEC electrode sensing platform under multi-cycle light irradiation. (c) PEC response of TNs, AgI-TNs, BSA / Apt / TNs-AgI and MC-LR / BSA / Apt / TNs-AgI. (d) Nyquist plot of TNs, AgI-TNs, BSA / Apt / TNs-AgI and MC-LR / BSA / Apt / TNs-AgI.

[0014] Figure 7The experimental conditions of the present invention are optimized. (a) Optimization of aptamer concentration (b) Optimization of target incubation time.

[0015] Figure 8 Figure 1 is a feasibility test diagram of the organic phototransistor in the present invention. (a) I of the PEDOT / PSS device measured in PBS solution D -V G curve, up to 750 minutes. (b) Different V G Output characteristic curve under different V G Next I DS (d) TNs as device I G and I D Transient response of AgI-TNs device I. G and I D (f) The corresponding current gain.

[0016] Fig. 9 The image of the phototransistor sensing analysis of the present invention. (a) Transfer curves at each stage under dark conditions. (b) Photocurrent response of the gate at different modification stages. (c) Transfer curves of Apt / TNs-AgI and MC-LR / Apt / TNs-AgI gate electrodes under irradiation and without irradiation. Ion transfer diagram of MC-LR / Apt / TNs-AgI gated device (d) in the dark and (e) under illumination, and (f) ion transfer diagram of Apt / TNs-AgI gated device under illumination. (g) Potential distribution of enhanced OPECT system.

[0017] Fig.10 Linear and selective analysis images for the present invention. (a) I D Response to different concentrations of MC-LR. (b) The corresponding calibration curve (error bars, n=3). (c) The corresponding selectivity test histogram.

[0018] Fig.11 This is the channel current measured every five days by the present invention.

[0019] Fig.12 Six electrodes were used to test the repeatability of the method.

[0020] Fig.13 This is the actual sample recovery rate determination of the present invention. DETAILED DESCRIPTION

[0021] 1. Preparation of Materials

[0022] 1. Synthesis of TiO2 nanorod arrays using FTO

[0023] TiO2 nanotubes (TNs) were grown in situ on FTO (TNs-FTO) by hydrothermal method. Equal amounts (15 mL) of hydrochloric acid (35 wt.%) and deionized water were mixed and stirred in a water bath heated at 50 °C for 30 min. Then 0.32 mL of tetrabutyl titanate was quickly added and heating and stirring were continued for about 60 min. After stirring evenly, the homogenous solution was transferred to a Teflon-coated stainless steel titanium dioxide autoclave with a 0.7 cm × 5 cm FTO sheet, with the conductive surface of the FTO sheet facing down to avoid excessive precipitation on the surface. The hydrothermal reaction was carried out in an oven at 150 °C for 4 h, and then the precursor grown on FTO was washed with secondary water and ethanol in turn. Finally, the TNs-FTO electrode was sintered at 450 °C for 2 h in a muffle furnace at a heating rate of 2 °C per minute.

[0024] 2. AgI decorated TNs-FTO electrode

[0025] The resulting TNs-FTO electrodes were decorated by depositing AgI nanoparticles using a simple ultrasonic dipping strategy. Typically, the TNs-FTO electrodes were first immersed in a AgNO3 (5 mM) solution for 15 min and then transferred to a KI (5 mM) solution for 15 min to promote the AgI nanoparticles. + and I - In situ coprecipitation on the TNs surface. After each ultrasonication, the electrode surface was rinsed with deionized water to remove the unadsorbed ions. This process can be repeated to obtain a certain amount of AgI on the TNs-FTO electrode. Finally, the composite electrode was rinsed with deionized water to remove the residue and dried at room temperature. The preparation method of TNs is basically the same as that of AgI-TNs, except that no ultrasonic AgI was added to TNs. + and I - .

[0026] 2. Sensor Construction

[0027] Before making the PEC sensor substrate, the area of ​​all prepared AgI-TNs and TNs photoelectrodes was controlled to 1.0 cm using insulating glue. 2 Chitosan solution (1%, w / v) was coated on AgI-TNs and TNs with a surface area of ​​1.0 cm 2 , and then dried at 40°C. Then 5% glutaraldehyde solution (50μL) was injected. Leave at room temperature for 30 minutes. Then 40μL of 2.5μM aptamer was placed on the AgI-TNs functionalized with glutaraldehyde. Store at 4°C for 12 hours. To remove unbound aptamers, it should be rinsed thoroughly. Then, to prevent nonspecific adsorption, treated 1wt% BSA was added. Finally, 1 cm2 of the PEC sensing platform was washed and stored at 4°C.

[0028] 3. Feasibility of PEC signal detection

[0029] The time-based photocurrent response of the AgI-TNs optoelectronic material when the excitation light was repeatedly turned on and off was continuously recorded at 0.1 mAA. Figure 6 b shows some typical results. Under the action of light, the photocurrent rises immediately and stabilizes within a few seconds. The photocurrent remains stable for 15 cycles. This clearly shows that the photoelectric sensing material of the present invention has high stability and good repeatability in PEC sensors. In order to evaluate the photoelectrochemical reaction and examine the effect of different fabrication steps on the performance of PEC sensors, the It curves of the same electrode under different fabrication steps were recorded in 0.1MAA solution ( Figure 6 c). Under visible light irradiation, pure TNs showed a weak current signal. However, after the AgI nanoparticles were modified onto Ns, the current signal of AgI-TNs was greatly enhanced. After the addition of MC-LR amino acid, the signal dropped sharply to a weak signal. After the addition of MC-LR, the signal was slightly enhanced. This proves the feasibility of the optoelectronic material of the present invention.

[0030] 4. Feasibility Characterization of Organic Phototransistors

[0031] like Figure 8 As shown in a, a typical Ag / AgCl reference electrode is used to demonstrate V G Adjustment I D When V G When the voltage rises from 0V to 0.6V, I D The increase in the conductivity of the polymer channel was obvious, indicating that the fabricated device has good transport properties. In addition, the long-term test of the continuous curve from 0 to 750 minutes shows that the polymer channel has good stability. Figure 8 b shows the range from 0V to 0.6V with different V G The corresponding output characteristics under the value of I D With V G Changes in response. Figure 8 c, at V G Fast and reproducible signals were recorded during some of the pulsation phases, indicating that V G The induced reversibility and good functional stability of the polymer channel. Figure 8 As shown in d, the I D The response is 375μA, I G The signal is 0.5μA. Figure 8 e shows that the I D is about 1040μA, and the corresponding I G About 0.883μA. Figure 5f, a high current gain of three orders of magnitude is obtained, which clearly demonstrates the ability of the AgI-TNs device to amplify the input current. The above results show that as I G The increase in I D will also increase, which indicates that within a certain range, I D Related to I G There is a positive correlation between them. Fig. 9 a shows the corresponding changes in the transfer curves under dark conditions. In the dark state, the transfer curve after Apt modification is slightly biased towards a higher gate voltage compared to AgI-TNs, because its charge changes the electrode surface potential, which in turn affects the gate voltage; while the transfer curve after MC-LR treatment is between these two curves, because of the reduction in the DNA content on the electrode surface. Fig. 9 b shows the corresponding transient I when the light is turned off and on D Response. It can be seen that the original I D It is about 0.95mA, and it quickly decreases to about 0.45mA after Apt modification. D The level increases to about 0.58 mA. Fig. 9 As shown in Figure c, under illumination, the transfer characteristics of Apt / TNs-AgI and MC-LR / Apt / TNs-AgI both shift significantly toward higher gate voltages, but the change of Apt / TNs-AgI is smaller than that of MC-LR / Apt / TNs-AgI, demonstrating the feasibility of the sensing of the present invention in organic phototransistors.

[0032] 5. Optimization of experimental conditions

[0033] The experimental conditions of the present invention are optimized as follows Figure 7 As shown. In order to improve the signal strength of the sensor, the aptamer concentration and incubation time of MC-LR were optimized. When the aptamer concentration was 2.5 μM and the target incubation time was 20 minutes, the experiment reached the optimal conditions. Under the optimal experimental conditions, linear analysis was performed.

[0034] VI. Linearity Measurement Analysis and Selectivity Evaluation

[0035] In the determination of MC-LR by an organic phototransistor, the linear range of MC-LR concentration is as in the present invention Fig.10 As shown in A. The increase in MC-LR concentration leads to I D increase. Figure 7 b shows the relationship between MC-LR concentration and I D Concentration of 10 pg mL -1 To 0.1 fg mL -1The linear relationship between them. The regression equation of the calibration curve is (I-I0) / I0=0.1471+0.0801log C(R 2 =0.996). In the selectivity assessment, Fig.10 As shown in Figure c, by adding the interferents shown in the figure respectively, only the target MC-LR or its mixture containing MC-LR can cause significant changes in signal transduction, which shows that the sensing platform of the present invention has good selectivity.

[0036] VII. Real sample determination

[0037] In order to evaluate the ability of the sensor to detect real samples, the samples were measured using the spike recovery method. Fig.13 As shown, the recovery rate is between 97.45% and 101.39%, and the RSD is less than 5%. The results show that the sensing platform constructed by the present invention has a good ability to detect real samples, and it is expected to be applied in environmental monitoring in the near future.

Claims

1. A method for detecting microcystin using an organic photoelectrochemical transistor is characterized by: The device integrates PEC and OECT to achieve high-sensitivity detection and visual imaging of microcystin (MC-LR). While the device detects the target, it breaks the traditional signal amplification strategy. After adding MC-LR, the target binds to the aptamer chain, and MC-LR is oxidized on the electrode surface to generate current, resulting in an effective gate voltage (V G ) changes, which leads to the enhanced doping of PEDOT:PSS, resulting in I D The response changes.

2. The method for detecting MC-LR using an organic photoelectrochemical transistor according to claim 1, characterized in that: A gate potential (V G ) can trigger electrochemical doping of the channel (C) material through ion migration, thereby modulating the electronic conductivity of the channel, which can be achieved by applying a channel voltage (V D ) when detecting the drain (D) current (I D ) for monitoring. Therefore, the OPECT biosensor uses the change in the potential drop of one of the two interfaces (i.e., C / E and G / E) caused by the target, resulting in I D The change is proportional to the target concentration. Therefore, the device can achieve sensitive detection of MC-LR.

3. The method for detecting MC-LR using an organic photoelectrochemical transistor according to claims 1-2, characterized in that: The optimal driving photovoltage of the gate electrode is 3.0V.

4. The method for detecting MC-LR using an organic photoelectrochemical transistor according to claims 1 to 2, characterized in that: The optimal concentration of the aptamer was 2.5 μM.

5. The method for detecting MC-LR using an organic photoelectrochemical transistor according to claims 1 to 2, characterized in that: The optimal incubation time for the target was 20 min.

6. A method for detecting MC-LR using an organic photoelectrochemical transistor according to claims 1 to 2, characterized in that: The sensitivity, flexibility, controllability and selectivity of the sensor are significantly improved.

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