Construction method and application of organic phototransistor biosensor based on BiVO4-ZnIn2S4

Abstract

A high-sensitivity CTCs detection sensing system is developed for realizing detection and analysis of human breast cancer cells (MCF-7). The BiVO4-ZnIn2S4 photoelectric material is prepared by adopting a simple reaction method. Gold nanoparticles are directly integrated on the surface of an electrode, and an OPECT photoelectric sensing platform is constructed. DNANTH which is functionalized by sulfydryl and provided with an aptamer at the top is fixed on the surface of a photoelectric material to capture cells, and compared with traditional single-stranded DNA, the DNANTH has higher stability and directional regulation and control. As the MUC1 protein is over-expressed on the cell membrane, the MCF-7 cell can be specifically captured. After MCF-7 cells are introduced, a current signal is obviously reduced. The organic photoelectrochemical transistor biosensor has relatively high selectivity and sensitivity, and realizes sensitive detection of MCF-7. The sensing system provides a new thought for improving the detection rate of cancer patients, and has wide application prospects in biosensing and clinical detection.

Description

Technical Field

[0001] In the present invention, a novel organic photoelectrochemical transistor (OPECT) device is designed based on photoelectrochemistry (PEC) and organic electrochemical transistor (OECT), and an OPECT biosensor platform is successfully constructed to realize the analysis and detection of human breast cancer cells (MCF-7). This technology has better amplification ability and high sensitivity, and achieves the effect of reducing background and bias to a large extent. DNA NTH with an aptamer on the top that has been functionalized with thiol groups is fixed on the surface of the photoelectric material to capture cells. Compared with traditional single-stranded DNA, DNA NTH has higher stability and directional regulation. The constructed OPECT photoelectric biosensor has high selectivity and sensitivity, realizes sensitive detection of MCF-7, and provides a new idea for improving the survival rate of cancer patients. Background Art

[0002] With the continuous exploration of PEC and OECT, a new concept of sensitive biosensor has been proposed. The concept is an organic photoelectrochemical transistor (OPECT) that combines photoelectrochemistry (PEC) and organic electrochemical transistor (OECT). It combines the advantages of separation of excitation source and signal detection system in PEC, and the inherent signal amplification function of OECT. Compared with traditional PEC and OECT biosensors, it greatly improves the sensitivity of the sensor, reduces the background signal, and does not require an external gate voltage. Organic photoelectrochemical transistor sensing has a wide range of applications and has been successfully applied in many fields, including disease diagnosis, bioanalysis, supercapacitors, etc., leaving unlimited potential. The device is expected to inspire the exploration of future material biocompatibility and photobiomaterial interactions, and further promote the application and research of advanced organic electronics. Summary of the invention

[0003] In the present invention, a novel organic photoelectrochemical transistor (OPECT) device was designed based on photoelectrochemistry (PEC) and organic electrochemical transistor (OECT), and an OPECT biosensor platform was successfully constructed to realize the analysis and detection of human breast cancer cells (MCF-7).

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

[0005] BiVO4-ZnIn2S4 optoelectronic material was prepared by a simple reaction method. Gold nanoparticles were directly integrated on the electrode surface to construct the OPECT optoelectronic sensing platform. DNA NTH with an aptamer on the top that had been functionalized with thiol groups was fixed on the surface of the optoelectronic material to capture cells. Compared with traditional single-stranded DNA, DNA NTH has higher stability and directional regulation. Since MUC1 protein is overexpressed on the cell membrane, MCF-7 cells can be specifically captured. After the introduction of MCF-7 cells, the current signal was significantly reduced. This sensor greatly improves the sensitivity of the sensor, reduces the background signal, and does not require an external gate voltage. It has broad application prospects in biosensing, clinical testing, and environmental monitoring.

[0006] The advantages of the present invention are:

[0007] DNANTH with aptamers on the top that have been functionalized with thiol groups is fixed on the surface of optoelectronic materials to capture cells. Compared with traditional single-stranded DNA, DNANTH has higher stability and directional regulation. Compared with traditional PEC and OECT biosensors, it greatly improves the sensitivity of the sensor, reduces the background signal, and does not require an external gate voltage. The device has broad application prospects in biosensing, clinical testing, and environmental monitoring, and is expected to inspire the exploration of future material biocompatibility and photobiomaterial interactions, and further promote the application and research of advanced organic electronics. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0009] Figure 2 Characterization diagram of the materials of the present invention. (A) Synthesis of BiVO4-ZnIn2S4. (B) SEM image of BiVO4-ZnIn2S4. (C, D) TEM image of BiVO4-ZnIn2S4. (E) Elemental mapping of BiVO4-ZnIn2S4. (F) XRD spectrum of BZIS. (G) XPS spectrum of BZIS. (H) UV-visible diffuse reflectance spectra of BiVO4, ZnIn2S4 and BZIS. (I) (Ahν) of BiVO4 and ZnIn2S4 n Relationship diagram with photon energy (hν). (J) Mott-Schottky diagram of BiVO4 and ZnIn2S4.

[0010] Figure 3The PEC experimental diagrams of the present invention. (A) PEC response of BiVO4 (grey curve), ZnIn2S4 (red curve) and BZIS (blue curve). (B) PEC stability of BZIS through repeated on / off illumination cycles. (C) Charge transfer mechanism of BZIS heterojunction under light irradiation. (D) Agarose gel electrophoresis of DNANTH structure. (E) PEC response of BZIS (grey curve), BZIS / AuNPs (red curve), BZIS / Au NPs / DNA NTH (blue curve) and BZIS / AuNPs / DNANTH / MCF-7 (green curve). (F) 5 mM [Fe(CN)6] 3- / 4- EIS of the gradual modification process. (G) Transfer characteristic curve measured at 1440 min using Ag / AgCl gate. (H) I DS (I) OPECT operation mechanism based on light-induced ion modulation channel.

[0011] Figure 4 The OPECT feasibility of the photoelectric material BiVO4-ZnIn2S4 of the present invention. (A) OPECT of BiVO4 / ITO gate electrode DS and I GS Signal changes over time. (B) I of ZnIn2S4 / ITO gate electrode OPECT DS and I GS Signal changes over time. (C) I of BZIS / ITO gate electrode OPECT DS and I GS Signal variation over time. Experiments were performed in an electrolyte consisting of 0.1 M TEOA. (D) Derived current gain.

[0012] (E) The corresponding modulation mechanism.

[0013] Figure 5 The OPECT experiment diagram of the present invention. (A) Transfer curve of BZIS gate OPECT device before and after MCF-7 reaction. (B) Potential distribution of OPECT system. (C) I of OPECT biosensor changing with time DS Signal. (D)I DS Response to different MCF-7 cell concentrations. (E) Corresponding derived calibration curve. ΔI / I0 as a function of MCF-7 concentration (ΔI / I0=0.15973Log C-0.22083,

[0014] R 2 =0.99546). Error bars are obtained from three replicates. (F) Selectivity test of three cell disruptors and their mixtures.

[0015] Figure 6 The XPS peaks of the elements in the present invention are fitted. High-resolution XPS spectra of (A) Bi 4f and S2p, (B) In 3d, (C) V 2p and (D) Zn 2p.

[0016] Figure 7 This is a schematic diagram of the manufacturing principle of the OECT device of the present invention.

[0017] Figure 8 The experimental conditions of the present invention are optimized. (A) Effects of incubation time and (B) aptamer concentration on the photocurrent response.

[0018] Fig. 9 Figure 1 shows the repeatability and reproducibility of the present invention. (A) Repeatability of OPECT signal. (B) Reproducibility of OPECT sensor. DETAILED DESCRIPTION

[0019] 1. Synthesis of BiVO4-ZnIn2S4 Photoelectric Material

[0020] 1. Preparation of BiVO4

[0021] 1.5mmol Bi(NO3)3·5H2O was dissolved in 30mL ethylene glycol, and 1.5mmol KI was dissolved in 30mL deionized water. The Bi(NO3)3·5H2O solution was added dropwise to the KI solution and stirred evenly. BiOI on the electrode was prepared by electrodeposition using the conventional three-electrode method. ITO was placed in the prepared solution as the working electrode, and the deposition process was carried out at an electrostatic potential of -0.3V for 10s and at an electrostatic potential of -0.1V for 300s to obtain an orange product.

[0022] Using BiOI as a precursor, 10 μL of VO(acac)2 dissolved in dimethyl sulfoxide was dropped onto the BiOI surface and dried at room temperature. After that, the dried electrode was annealed at 450°C for 2 h (1°C / min). Finally, 0.1 mol / L NaOH was used to clean the electrode surface to remove V2O5 on the electrode surface.

[0023] 2. Preparation of BiVO4-ZnIn2S4

[0024] 0.055mmol ZnCl2, 0.11mmol InCl3 and 0.22mmol thioacetamide were dissolved in 20mL ethylene glycol and transferred to a reactor. The modified electrode was then immersed in the reactor solution and reacted at 90°C for 2h. After being taken out, it was washed with anhydrous ethanol and dried under vacuum.

[0025] 2. PEC signal measurement of photoelectric material BiVO4-ZnIn2S4

[0026] The PEC signal of the photoelectric material was detected using a traditional three-electrode system. The photoelectric material BiVO4-ZnIn2S4 was used as the working electrode, the Pt wire was used as the counter electrode, and the Ag / AgCl electrode was used as the reference electrode to implement the detection on the electrochemical workstation. The three electrodes were immersed in a pool of 0.1M TEOA solution, and the It curve was used for real-time detection after the light source was turned on.

[0027] 3. Biosensor Construction and Detection

[0028] 1. ITO electrode pretreatment

[0029] The ITO electrode was ultrasonically cleaned with acetone, ethanol and ultrapure water for 30 min respectively and dried for later use.

[0030] 2. Construction of OPECT biosensor platform

[0031] The prepared BiVO4-ZnIn2S4 electrode was immersed in a 5% MPTMS ethanol solution overnight. After being taken out and washed with ethanol, it was placed in an oven at 80°C for 30 minutes to remove unbound MPTMS molecules and prevent AuNPs from aggregating in the next step. After that, the electrode was immersed in AuNPs for 10 hours at room temperature and rinsed with deionized water. Then, DNANTH solution (10μL, 1μΜ) was added dropwise to the AuNPs / BZIS / ITO surface, incubated at 4°C for 12 hours, and the electrode surface was washed with deionized water. After that, 10μL (1mM) of MCH solution was added, incubated for 30 minutes, nonspecific binding sites were blocked, and rinsed with deionized water. Then, 30μL MCF-7 cells (1×10 4 cell mL -1 ) was dropped on the electrode surface and incubated at 37°C for 40 min to complete the recognition process. Unbound cells were removed by washing with deionized water.

[0032] 3. OPECT signal detection

[0033] The modified electrode was used as the gate of OPECT and immersed in a pool of 0.1 MTEOA solution together with the source and drain of the OPECT device. After turning on the light source, the electrochemical workstation was connected and the It curve was used for real-time monitoring. When the baseline of the It curve was stable, the light was irradiated on the electrode to collect the OPECT signal. A standard curve was established based on the relationship between different concentrations of MCF-7 cells and the corresponding OPECT signal intensity.

[0034] The morphology, size, peak position, and elements contained in the optoelectronic material BiVO4-ZnIn2S4 were characterized by SEM, TEM, XRD, XPS, infrared, ultraviolet, and Mott-Schottky to prove that the material was successfully prepared.

[0035] 5. Feasibility Characterization of Biosensing Platform

[0036] Amplification I based on OPECT system DS Signal and I DS with I GS A biosensor capable of sensitively detecting MCF-7 cells was constructed. Figure 5 C records the time-varying I of the OPECT system after the preparation of the optoelectronic material BiVO4-ZnIn2S4, DNANTH modification and the gradual addition of MCF-7. DS As shown in the figure, the original BZIS heterojunction can generate 1.031mA of I D-step After DNANTH modification, I D-step After adding MCF-7 cells, I D-step It further significantly decreased to around 0.395mA.

[0037] 6. Optimization of experimental conditions

[0038] The experimental conditions of the present invention are optimized as follows Figure 8 In order to improve the sensitivity of the immunosensor, the reaction time after the instillation of MCF-7 cells and the reaction time of the DNANTH tetrahedron aptamer were optimized. Under the optimal experimental conditions, linear analysis was performed.

[0039] 7. Linear and Visual Imaging Analysis

[0040] The linear range of the concentration of MCF-7 in the present invention is as follows Figure 5 D. Since I D-step The response of I D-step To achieve the detection of MCF-7, thus achieving high-sensitivity biological detection of MCF-7 as a model target. The increase in MCF-7 cell concentration leads to I D-step The present invention Figure 5 E for I D-step The target cell concentration is between 50 and 5 × 10 5 cell mL -1 The figure shows a good linear relationship with the target concentration. The regression equation of the calibration curve is ΔI / I0=0.15973LogC-0.22083(R 2=0.99546), the detection limit is 43 cell mL -1 .

[0041] 8. Immunosensor Performance Evaluation

[0042] Stability is an important indicator for evaluating the performance of biosensors. As shown in 3B of the present invention, the PEC signal of the photoelectric material BiVO4-ZnIn2S4 was continuously detected for 10 minutes. The results show that it has good stability. Secondly, the selectivity of the biosensor of the present invention was studied. Figure 5 As shown in F. Three different cells, HepG2, Hela, and A549, were selected as interferences to study their selectivity. The ΔI / I0 induced by MCF-7 cells was significantly higher than that of other interference cells. In addition, the reaction induced by MCF-7 mixed with other interference substances was almost the same as that induced by MCF-7 cells. The results showed that this method has good selectivity.

[0043] In addition, the repeatability and reproducibility of the present invention were studied. Fig. 9 As shown, first, the same experiment was performed on different electrodes, and the experimental results were detected for signal to obtain better signal data repeatability, and the RSD of the corresponding experiment was 0.98%. Secondly, the reproducibility was the experiment using the electrode, and the signal was detected at the same time interval, and the RSD of the corresponding experiment was 1.94%. This shows that the biosensing platform has excellent reproducibility and reproducibility.

Claims

1. A method for constructing an organic photoelectrochemical transistor biosensor based on BiVO4-ZnIn2S4, characterized in that: The device uses the prepared BiVO4-ZnIn2S4 as the photoelectrically active material to create an ultra-sensitive OPECT sensing platform, and uses it for the detection of human breast cancer cells (MCF-7). The sensitivity of detection is greatly improved by utilizing the property of channel current amplifying gate current. When BiVO4-ZnIn2S4 is photoexcited, efficient electron transfer occurs. Since DNANTH with an aptamer on the top has a large steric effect, the OPECT response intensity is reduced. Afterwards, the further increase in the number of MCF-7 cells captured by the aptamer further reduces the OPECT response intensity. Thus, the relationship between the MCF-7 concentration and the OPECT response value is established to achieve rapid and sensitive detection of MCF-7 in actual blood samples.

2. A method for constructing an organic photoelectrochemical transistor biosensor based on BiVO4-ZnIn2S4, characterized in that: The steps include: Step 1: Preparation of BiVO4-ZnIn2S4 composite material by simple hydrothermal method: First, Bi(NO3)3·5H2O and KI were dissolved in ethylene glycol and deionized water, respectively. After that, the two solutions were mixed and stirred evenly to obtain a BiOI solution. BiOI on the electrode was prepared by electrodeposition. Using BiOI as a precursor, a VO(acac)2 solution dissolved in dimethyl sulfoxide was added dropwise. After drying, it was placed in a muffle furnace for annealing. After taking it out, it was washed with NaOH. Finally, ZnCl2, InCl3 and thioacetamide were dissolved in ethylene glycol, and then transferred to a polytetrafluoroethylene reactor for solvothermal reaction. After taking it out, it was washed with anhydrous ethanol and dried under vacuum. Step 2: Construction of the grid in the OPECT sensor: Immerse the BiVO4-ZnIn2S4 electrode prepared in step 1 in the MPTMS ethanol solution overnight. After taking it out and washing it with ethanol, place it in an oven to react to remove unbound MPTMS molecules and prevent Au NPs from aggregating in the next step. After that, immerse the electrode in AuNPs at room temperature for reaction and rinse with deionized water. Then, drop the DNANTH solution on the surface of Au NPs / BZIS / ITO, refrigerate it in a refrigerator, and rinse it with deionized water. After that, add MCH solution for incubation to block nonspecific binding sites and rinse it with deionized water. Then, drop MCF-7 cells on the electrode surface and incubate it at room temperature to complete the recognition process. Wash with deionized water to remove unbound cells. Step 3: Construction of OPECT sensor: The electrode modified in step 2 was used as the gate electrode of OPECT. MCF-7 / DNA NTH / Au NPs / BiVO4-ZnIn2S4 was immersed in an electrolytic cell containing TEOA PBS solution together with the OPECT device, and an organic photoelectrochemical transistor biosensor based on BiVO4-ZnIn2S4 was successfully constructed.

3. The construction method according to claim 2, characterized in that: In step 1, the Bi(NO3)3·5H2O and KI are both 1.5 mmol, and the ethylene glycol and deionized water are both 30 mL; the electrodeposition process is carried out at a constant potential of -0.3 V for 10 s and at a constant potential of -0.1 V for 300 s; the reaction process in the muffle furnace is annealing at 450°C for 2 h (1°C / min); the concentration of NaOH is 0.1 mol / L; the electrode is reacted at 90°C in the reactor for 2 h.

4. The construction method according to claim 2, characterized in that: In step 2, the volume ratio of MPTMS to ethanol is 1:95; after washing with ethanol, the electrode is placed in an oven at 80°C for 30 minutes; the electrode is immersed in AuNPs for 10 hours; the added DNANTH solution has a volume of 10 μL and a concentration of 1 μΜ, and is reacted at 4°C for 12 hours; the concentration of MCH is 1 mM, and the reaction time is 30 minutes; the added MCF-7 cells have a volume of 30 μL and a concentration of 1×10 4 cell mL -1 The reaction time at room temperature is 40 min.

5. The construction method according to claim 2, characterized in that: In step 3, the concentration of TEOA was 0.1 M.

6. Use of the organic photoelectrochemical transistor biosensor based on BiVO4-ZnIn2S4 prepared by the construction method according to any one of claims 2 to 5 for detecting MCF-7.

7. A method for detecting MCF-7 using the organic photoelectrochemical transistor biosensor according to claims 1 to 2, characterized in that: The sensitivity and selectivity of the sensor are significantly improved.

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