An organic photoelectrochemical transistor sensor for detecting ovarian cancer marker HE-4 and a preparation method and a detection method thereof

Abstract

The application discloses an organic photoelectrochemical transistor sensor for detecting an ovarian cancer marker HE-4 and a preparation method and a detection method thereof, wherein the organic photoelectrochemical transistor sensor comprises an electrolytic cell loaded with an electrolyte solution, a substrate inserted into the electrolyte solution at one end, and a gate electrode; the substrate is provided with a source electrode and a drain electrode inserted into the electrolyte solution at one end and spacedly distributed; the source electrode and the drain electrode are coated with an organic semiconductor thin film on surfaces; the gate electrode is provided with a photoelectric active layer at one end inserted into the electrolyte solution; and the material of the photoelectric active layer is a covalent organic framework material; and the organic photoelectrochemical transistor sensor further comprises a well plate loaded with a sample solution to be detected. The ovarian cancer marker HE-4 detection method based on the organic photoelectrochemical transistor sensor has high sensitivity, is simple to operate, and has high detection efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of organic photoelectrochemical transistors, in particular to an organic photoelectrochemical transistor sensor for detecting ovarian cancer marker HE-4 and a preparation method and a detection method thereof. BACKGROUND

[0002] Ovarian cancer (OC) is one of the common malignant tumors of the female reproductive system, with the third highest incidence and the highest mortality rate. The overall 5-year relative survival rate is generally between 30-40%. The reason for the high mortality rate of ovarian cancer is that the tumor develops in a hidden manner, the symptoms appear slowly, and there is a lack of effective early detection means. Most women with OC are diagnosed in the late stage of the disease, therefore, early detection, early diagnosis and early treatment of ovarian cancer are very important, which can significantly improve the survival rate of ovarian cancer patients.

[0003] The tumor markers of ovarian cancer mainly include CA125 (carbohydrate antigen 125), CA-19-9 (carbohydrate antigen 199), AFP (alpha-fetoprotein), HE-4 (human epididymal protein 4), CEA (carcinoembryonic antigen) and the like. If a single marker is used, HE-4 has the best specificity and sensitivity; if CA125 is used in combination with HE-4 to diagnose ovarian cancer, the sensitivity can reach 91.67% and the specificity can reach 85%. At present, the detection of HE-4 mostly uses chemiluminescence, electrochemical method, giant magnetoresistance effect method and the like, but these methods either extremely depend on professional detection instruments and personnel and are expensive, or the detection limit and sensitivity of HE-4 are limited. Therefore, designing a simple operation, daily detection, affordable and high sensitivity HE-4 detection method is still a difficult problem to be solved.

[0004] The prior art still needs to be improved and developed. SUMMARY

[0005] In view of the above deficiencies of the prior art, the purpose of the present application is to provide a design idea, a preparation method and a detection method of an organic photoelectrochemical transistor sensor for detecting ovarian cancer marker HE-4, aiming to solve the problems of complicated operation and low detection sensitivity of the existing methods for detecting ovarian cancer marker HE-4.

[0006] The technical scheme of the present application is as follows:

[0007] The application discloses an organic photoelectrochemical transistor sensor for detecting an ovarian cancer marker HE-4, wherein the sensor comprises an electrolytic cell containing an electrolyte solution, a substrate inserted into the electrolyte solution and a gate electrode, the substrate is provided with source electrodes and drain electrodes inserted into the electrolyte solution and arranged at intervals, surfaces of the source electrodes and the drain electrodes are coated with an organic semiconductor film, the gate electrode is provided with a photoelectric active layer at one end inserted into the electrolyte solution, and the photoelectric active layer is made of a covalent organic framework material; and the sensor further comprises a well plate containing a sample solution to be detected, the well plate is modified with a double-antibody sandwich structure, the double-antibody sandwich structure comprises a first antibody combined on the well plate, HE-4 specifically combined with the first antibody, a second antibody specifically combined with the HE-4, a gold nanoparticle and an ALP enzyme combined on the gold nanoparticle, and the sample solution to be detected is an ascorbic acid solution generated after L-ascorbic acid-2-phosphoric acid trisodium salt reacts with the ALP enzyme in the sandwich structure.

[0008] The organic photoelectrochemical transistor sensor for detecting the ovarian cancer marker HE-4, wherein the material of the organic semiconductor film layer is at least one of poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid, a polypyrrole, a polythiophene, a polyaniline, a polycarbazole and a copolymer thereof.

[0009] The organic photoelectrochemical transistor sensor for detecting the ovarian cancer marker HE-4, wherein the covalent organic framework (COF) material is one or more of a polymer of trialdehyde-based phloroglucinol and 2,5-dimethyl-1,4-phenylenediamine, a polymer of 2,6-dialdehyde-1,5-dihydroxynaphthalene and tris(4-aminophenyl)amine, a polymer of 2,4,6-tris(4-aminophenyl)-1,3,5-triazine and 2,5-dihydroxyterephthalic acid and an aminotetraphenylporphyrin.

[0010] The organic photoelectrochemical transistor sensor for detecting the ovarian cancer marker HE-4, wherein the material of the source electrodes and the drain electrodes is selected from one or more of Ti, Pt, Au, Ti3C2 and deposited carbon, and the material of the gate electrode is one of ITO glass and FTO glass.

[0011] A preparation method of an organic photoelectrochemical transistor sensor for detecting an ovarian cancer marker HE-4, wherein the method comprises the following steps:

[0012] The application provides a hole plate provided with a sample solution to be detected, wherein the hole plate is provided with a double-antibody sandwich structure, the double-antibody sandwich structure comprises a first antibody combined on the hole plate, HE-4 specifically combined with the first antibody, and a second antibody complex specifically combined with the HE-4, the second antibody complex comprises a gold nanoparticle and a second antibody and an ALP enzyme combined on the gold nanoparticle, and the sample solution to be detected is an ascorbic acid solution generated after L-ascorbic acid-2-phosphoric acid trisodium salt reacts with the ALP enzyme in the sandwich structure.

[0013] A source electrode and a drain electrode are prepared on a substrate, and an organic semiconductor thin film is prepared on the surface of the source electrode and the drain electrode;

[0014] A gate electrode is provided, and a photoelectric active layer is prepared on the gate electrode, wherein the material of the photoelectric active layer is a covalent organic framework material;

[0015] The substrate and the gate electrode provided with the photoelectric active layer are inserted into an electrolytic cell provided with an electrolyte solution, so as to obtain the organic photoelectrochemical transistor sensor for detecting the ovarian cancer marker HE-4.

[0016] The preparation method of the organic photoelectrochemical transistor sensor for detecting the ovarian cancer marker HE-4, wherein the preparation of the hole plate provided with the sample solution to be detected comprises the following steps:

[0017] The first antibody solution is added into the hole plate and incubated overnight, and the first antibody is firmly combined with the hole plate through physical adsorption between hydrophobic groups;

[0018] After the incubation of the first antibody is completed, the PBS solution containing Tween 20 is used for cleaning three times to remove the uncombined first antibody, then the BSA solution is added to block the uncombined sites in the hole plate, and after the blocking is completed, the washing solution is used for cleaning;

[0019] After the cleaning, the sample to be detected containing unknown concentration HE-4 is added, and the incubation is performed to specifically combine the HE-4 with the first antibody;

[0020] After the cleaning, the second antibody complex is added for incubation to specifically combine the second antibody in the second antibody complex with the HE-4, and after the washing solution is used for cleaning, the double-antibody sandwich structure is formed in the hole plate;

[0021] The PBS solution containing AAP is added into the hole plate, and the ascorbic acid solution is generated after the incubation reaction, so as to obtain the hole plate provided with the sample solution to be detected.

[0022] The preparation method of the organic photoelectrochemical transistor sensor for detecting the ovarian cancer marker HE-4, wherein the preparation of the second antibody complex comprises the following steps:

[0023] The pH of the gold nanoparticles is adjusted to 8 using a potassium carbonate solution to obtain a gold nanoparticle solution;

[0024] The second antibody and ALP enzyme are sequentially added to the gold nanoparticle solution, and after incubation overnight, centrifugation is performed to obtain wine-red oil droplets, followed by the addition of PBS buffer and uniform mixing, and then centrifugation is performed to collect the unbound proteins, and finally the second antibody complex is obtained by dispersing in a PBS solution containing bovine serum albumin.

[0025] A detection method of an organic photoelectrochemical transistor sensor for detecting an ovarian cancer marker HE-4, comprising the steps of:

[0026] After preparing HE-4 with different known concentrations into a double-antibody sandwich structure in a well plate, a sufficient amount of PBS solution containing AAP is added, and after incubation and reaction, ascorbic acid solutions with different concentrations are obtained;

[0027] The ascorbic acid solutions with different concentrations are added to an electrolyte solution, the substrate and the gate electrode are simultaneously placed in the electrolyte solution, the channel current signal is read under light shielding conditions, and the channel current step signal is obtained by comparing the channel current under the excitation of light at a wavelength of 420 nm, and an HE-4 concentration-channel current step signal curve is drawn;

[0028] After preparing a sample to be detected containing HE-4 with an unknown concentration into a double-antibody sandwich structure in a well plate, a sufficient amount of PBS solution containing AAP is added, and after incubation and reaction, ascorbic acid solutions with unknown concentrations are obtained;

[0029] The ascorbic acid solutions with unknown concentrations are added to an electrolyte solution, the substrate and the gate electrode are simultaneously placed in the electrolyte solution, the channel current signal is read under light shielding conditions, and the current channel current step signal is obtained by comparing the channel current under the excitation of light at a wavelength of 420 nm;

[0030] According to the HE-4 concentration-channel current step signal curve and the current channel current step signal, the concentration of HE-4 in the sample to be detected is calculated.

[0031] Beneficial effects: the application provides an organic photoelectrochemical transistor sensor for detecting ovarian cancer marker HE-4, after generating a COF film on the gate in situ, different amounts of HE-4 are captured to different amounts of secondary antibodies and ALP enzymes thereon through a "double-antibody sandwich structure", and different amounts of strong electron donors AA are catalytically generated and added to the electrolyte; due to the different amounts of strong electron donors in the electrolyte, the degree of PEC cathode photocurrent to anode current conversion of the COF material is different, which further affects the interface potential distribution of the device, so that the channel current changes, and the detection of different concentrations of HE-4 is realized by measuring the change amount of the channel current. The ovarian cancer marker HE-4 detection method based on the organic photoelectrochemical transistor sensor is simple to operate, does not need to use a chemiluminescence detector spectrometer, and the current signal data is intuitive; the method has high sensitivity and high detection efficiency, and has good application prospect in the field of biological sensing. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 It is a structural schematic diagram of an organic photoelectrochemical transistor sensor for detecting ovarian cancer marker HE-4.

[0033] Figure 2 It is a schematic diagram of generating AA in a "sandwich" structure of primary antibody-antigen-secondary antibody on a 96-well plate.

[0034] Figure 3 It is a schematic diagram of loading secondary antibodies and ALP enzymes on gold nanoparticles.

[0035] Figure 4 It is a flow chart of the preparation method of the organic photoelectrochemical transistor sensor for detecting ovarian cancer marker HE-4.

[0036] Figure 5 It is a schematic diagram of synthesizing a COF film on the gate electrode in situ

[0037] Figure 6 It is a transfer curve diagram of OPECT under "off-on" light irradiation in a 0.1M AA solution.

[0038] Figure 7 It is a detection result of different concentrations of HE-4.

[0039] Figure 8 It is a PEC step current detection result diagram of TpPa-(CH3)2 electrode under different concentrations of AA. DETAILED DESCRIPTION

[0040] The application provides an organic photoelectrochemical transistor sensor for detecting an ovarian cancer marker HE-4 and a preparation method and a detection method thereof.

[0041] Please refer to Figures 1-3 The application provides an organic photoelectrochemical transistor sensor for detecting an ovarian cancer marker HE-4, as shown in the figure, which comprises an electrolytic cell 2 containing an electrolyte solution 1, a substrate 3 inserted into the electrolyte solution 1 at one end, and a gate electrode 4, the substrate 3 is provided with a source electrode 5 and a drain electrode 6 inserted into the electrolyte solution 1 at one end and spaced apart, the surface of the source electrode 5 and the drain electrode 6 is coated with an organic semiconductor film 7, the gate electrode 4 inserted into the electrolyte solution 1 at one end is provided with a photoelectric active layer 8, and the material of the photoelectric active layer 8 is a covalent organic framework material; further comprising a well plate containing a sample solution to be detected, the well plate is modified with a double-antibody sandwich structure 10, the double-antibody sandwich structure comprises a first antibody 11 combined on the well plate, HE-4 (human epididymal protein 4) 12 specifically combined with the first antibody, a second antibody complex 13 specifically combined with the HE-4, the second antibody complex 13 comprises gold nanoparticles 131 and a second antibody 132 and an ALP enzyme 133 combined on the gold nanoparticles 131, and the sample solution to be detected is an ascorbic acid solution generated after L-ascorbic acid-2-phosphoric acid trisodium salt 134 reacts with the ALP enzyme 133 in the sandwich structure.

[0042] Specifically, the organic photoelectrochemical transistor (OPECT) is a new detection method in the field of biosensing, which uses the working electrode of photoelectrochemistry (PEC) as the gate of the organic electrochemical transistor (OECT), the photoactive material on the gate is excited by light to produce electron transfer and generate an electrical signal, based on the change of the effective gate voltage and channel current of the device caused by the biological recognition reaction, high-sensitivity detection of biomolecules (such as enzymes, antigens-antibodies, DNA, cells, etc.) can be realized. Therefore, OPECT has the advantages of PEC and OECT, which has extremely low or even zero background signal and signal amplification function. OPECT can be divided into cathodic current system and anodic current system according to the difference in the electron transfer path of the gate. However, the common OPECT detection method is basically limited to the anodic current system, the reason is that the PEC current change of the photoactive material of the cathodic current system before and after light irradiation is small (mostly in the order of 10 -9 A), resulting in small channel current change and weak regulation ability of the device.

[0043] Covalent organic framework (COF) materials are a new type of crystalline porous material assembled by organic molecules through covalent bonds, which has the advantages of low density, large surface area, adjustable pore size and structure, and convenient customization. Among them, two-dimensional COF materials usually have a wide optical absorption spectrum and strong visible light capture ability due to the characteristics of large conjugated system within the layer and strong π-π interaction between adjacent layers. In addition, the organic nature of COF also provides an opportunity to improve the separation of photo-generated carriers by designing and constructing molecular polarity, so the photoelectric active material constructed by two-dimensional COF has great potential. The PEC sensing system with two-dimensional COF as the photoactive material can achieve a change in photocurrent of 10 -6 A order of magnitude, combined with the signal amplification function of the OECT device, can further improve the sensitivity and detection limit of the sensing system. In addition, the COF material is very sensitive to the amount of strong electron donor (such as ascorbic acid AA) contained in the electrolyte, and the increase in the amount of strong electron donor will reduce the PEC cathodic photocurrent generated by the COF material.

[0044] The application applies covalent organic framework (COF) materials to organic photoelectrochemical transistors (OPECT), generates a COF film in situ on a conductive glass as a gate electrode of an OPECT device, and the COF material generates an electron transfer under the illumination of 420 nm wavelength light to generate a PEC cathodic photocurrent. The ALP enzyme is modified on the secondary antibody, and the content of the HE-4 to be measured is converted into the amount of ALP enzyme by the double-antibody sandwich structure of the primary antibody-antigen-secondary antibody, and then the content of ascorbic acid (AA) generated in the 96-well plate is changed. Finally, the liquid in the 96-well plate is transferred to the electrolyte, and the PEC cathodic photocurrent generated by the COF material is reduced by the characteristic that the increase in the amount of strong electron donor. By the strategy of catalyzing the generation of AA by the ALP enzyme and the characteristics of the OECT that small gate voltage changes are converted into large channel current changes, the purpose of signal amplification is achieved, and high-sensitivity detection of HE-4 is achieved. The detection of the photocurrent signal in the OPECT biosensor is mainly through the 4200 semiconductor analyzer, and a three-electrode test system composed of a gate, a source and a drain is established to detect the size of the channel current. The application develops a new type of HE-4 detection biosensor, which has the advantages of simple operation, low price, high sensitivity, strong specificity, miniaturization, etc., and has a wide range of applications, and can be applied to the immunodetection of different antigens.

[0045] In some embodiments, the covalent organic framework material is one or more of, but not limited to, a polymer of triformylphloroglucinol and 2,5-dimethyl-1,4-phenylenediamine (TpPa-(CH3)2), a polymer of 2,6-diformyl-1,5-dihydroxynaphthalene and tris(4-aminophenyl)amine (D-TA), a polymer of 2,4,6-tris(4-aminophenyl)-1,3,5-triazine and 2,5-dihydroxyterephthalic acid (TTA-DHTA), and amino tetraphenyl porphyrin (TAPP). In this embodiment, the covalent organic framework material is a new type of organic porous framework material connected by covalent bonds between light atoms (such as hydrogen, boron, carbon, nitrogen, etc.). As preferred, the covalent organic framework material is TpPa-(CH3)2, which is derived from TpPa-1 and inherits its narrow band gap and photocatalytic reduction ability. The TpPa-(CH3)2 is a classic covalent organic framework with many phenyl rings and planar structures, and its fluffy porous structure can be used as a carrier for doping other particles and can be used for catalytic applications. Moreover, it not only has oxygen vacancies, which can enhance visible light capture and reduce the recombination of photo-generated carriers at the inter-band level, but also has a π-electron conjugated structure, which has a wide visible light absorption and excellent photocatalytic performance.

[0046] In some embodiments, the material of the organic semiconductor thin film layer is at least one of, but not limited to, poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid, polypyrrones, polythiophenes, polyanilines, polycarbazoles, and copolymers thereof.

[0047] In some embodiments, the material of the source electrode and the drain electrode is selected from one or more of Ti, Pt, Au, Ti3C2, and deposited carbon, and the gate electrode material is one of ITO glass and FTO glass, but not limited thereto.

[0048] In some embodiments, a preparation method of an organic photoelectrochemical transistor sensor for detecting an ovarian cancer marker HE-4 is also provided, as shown in Figure 4 The method comprises the following steps:

[0049] S10, providing a well plate loaded with a sample solution to be detected, the well plate being modified with a double-antibody sandwich structure, the double-antibody sandwich structure comprising a first antibody bound to the well plate, HE-4 specifically bound to the first antibody, and a second antibody complex specifically bound to the HE-4, the second antibody complex comprising a gold nanoparticle and a second antibody and an ALP enzyme bound to the gold nanoparticle, the sample solution to be detected being an ascorbic acid solution generated after the L-ascorbic acid-2-phosphoric acid trisodium salt reacts with the ALP enzyme in the sandwich structure;

[0050] S20, preparing a source electrode and a drain electrode on a substrate, and preparing an organic semiconductor thin film on the surface of the source electrode and the drain electrode, for standby;

[0051] S30, providing a gate electrode, and preparing a photoelectric active layer on the gate electrode, wherein the material of the photoelectric active layer is a covalent organic framework material;

[0052] S40, inserting the substrate and the gate electrode provided with the photoelectric active layer into an electrolytic cell containing an electrolyte solution, to obtain the organic photoelectrochemical transistor sensor for detecting the ovarian cancer marker HE-4.

[0053] The preparation method of the organic photoelectrochemical transistor sensor of the present application is further explained and described below through specific embodiments:

[0054] Preparation of the OECT device: first, filter PEDOT for more than 7 times, then add DMSO (PEDOT:DMSO = 20:1), and magnetically stir and mix for 2 h to prepare a PEDOT:PSS spin coating solution; cut the soda-lime glass, and then clean it by ultrasonic washing with detergent, acetone, ethanol, and water, and blow dry with a nitrogen gun; then, paste a mask on the clean glass sheet, and then paste the glass sheet on the chamber of a thermal evaporator, and add an appropriate amount of cadmium and gold, and start thermal evaporation according to the preset program; after thermal evaporation, wash and dry the obtained electrode with ethanol and water, and then perform Plasma plasma cleaning treatment; place the cleaned electrode on a spin coater, and drop 80 μL of the PEDOT:PSS spin coating solution uniformly, and start spinning after trying to cover the entire gate electrode and ensuring that there is no air bubble; after spinning, wipe off the excess PEDOT:PSS layer, and heat treat the device at 180℃ for 1 h to obtain the prepared OECT device.

[0055] In-situ synthesis of COF thin film on ITO electrode: as shown in Figure 5 shown, ultrasonically clean the ITO electrode (1 cm x 3.5 cm) in a mixed solution of acetone, ethanol, and NaOH (1 M) in ethanol / water (v / v, 1:1) and ultrapure water for 15 min, and blow dry with a nitrogen gun. Then, dissolve Tp (0.7 mg) and Pa-(CH3)2 (0.7 mg) in a mixed solution of mesitylene, ethanol, and acetic acid (v / v / v 5:5:1, 550 μL); mix Tp and Pa-(CH3)2 in a volume ratio of 1:1. Take 60 μL of the mixed solution and drop it on the surface of the ITO glass (1 cm x 3.5 cm), and seal and store at room temperature for 10 min. Next, immerse the ITO glass in dichloromethane for 5 minutes to remove unreacted residues, and dry at room temperature to obtain a uniform reddish-brown thin film on the ITO surface. Then, heat treat at 100℃ for 1 h under a nitrogen atmosphere to obtain an ITO glass gate electrode with a COF thin film.

[0056] Preparation of gold nanoparticles: Gold nanoparticles (Au NPs) were prepared by the common method of sodium borohydride (NaBH4) reduction of chloroauric acid (HAuCl4). The specific experimental steps are as follows: 20 mL of 2.5 x 10 -4 The M chloroauric acid (HAuCl4) solution was placed in an ice water bath and continuously stirred; 0.1 M sodium borohydride (NaBH4) was prepared using ice water, and 0.6 mL of the NaBH4 solution was taken and added to the HAuCl4 solution; the solution quickly turned orange, representing the formation of Au NPs, and the solution was continuously stirred in the ice water bath for 10 minutes and then at room temperature for 3 h; after the end of stirring, the gold nanoparticle sol was stored in a 4°C refrigerator;

[0057] Labeling of the secondary antibody with ALP enzyme: As shown in Figure 2 , first the pH of the Au NPs was adjusted to 8 using a 0.2 M potassium carbonate (K2CO3) solution; 1 mL of the Au NPs with a pH of 8 was taken into a centrifuge tube, 100 μL of 0.05 mg / mL secondary antibody (Ab2) and 200 μL of 0.5 mg / mL alkaline phosphatase (ALP) were sequentially added, and after 4°C incubation overnight, centrifugation was performed to obtain wine-red oil droplets; 1 mL of 10 mM PBS buffer was then added and mixed uniformly, and centrifugation was continued to collect, and unbound proteins were removed; finally, the mixture was dispersed in 1 mL of 10 mM PBS solution containing 1 wt% BSA to obtain the bioconjugate Ab2-Au NPs-ALP.

[0058] Construction of a "double antibody sandwich structure" of primary antibody-antigen-secondary antibody: A double antibody sandwich immunological structure for the detection of the substance HE-4 was established in a 96-well plate, as shown in Figure 3 , first 50 μL of 0.2 mg / mL primary antibody Ab1 solution was added to the 96-well plate and incubated overnight; the primary antibody Ab1 was firmly combined with the 96-well plate through physical adsorption between hydrophobic groups. After the end of the primary antibody incubation, 10 mM PBS solution containing 0.05% Tween 20 (washing solution) was used to wash three times to remove unbound Ab1, and then 50 μL of 1 wt% BSA solution was added and incubated at 37°C for 1 h to block unbound sites in the 96-well plate and prevent non-specific adsorption; after the completion of blocking, the washing solution was used for washing; 50 μL of HE-4 with different concentrations was added, and specific binding with Ab1 was achieved by incubation at 37°C for 0.5 h; after washing, 50 μL of Ab2-Au NPs-ALP bioconjugate was added, and a double antibody sandwich immunological structure was formed by incubation at 37°C for 1 h and washing with the washing solution. In the 96-well plate containing Ab1 / HE-4 / Ab2-Au NPs-ALP, 300 μL of 100 mM PBS solution containing 10 mM AAP was added, and incubation was performed at 37°C for 0.5 h.

[0059] The grid of the OPECT is made of ITO glass for generating COF film in situ, the source electrode and the drain electrode are made of gold electrode prepared by thermal evaporation on a glass substrate, and PEDOT:PSS is coated on specific areas of the gold electrode, so as to obtain the device structure as shown in Figure 1 Thus, the HE-4 sensor based on the organic photoelectrochemical transistor is constructed.

[0060] In some embodiments, a detection method of the organic photoelectrochemical transistor sensor for detecting the ovarian cancer marker HE-4 is also provided, which comprises the following steps: after preparing HE-4 with different known concentrations into a double-antibody sandwich structure in a well plate, adding sufficient PBS solution containing AAP, and obtaining ascorbic acid solutions with different concentrations after incubation, adding the ascorbic acid solutions with different concentrations into an electrolyte solution, placing the OECT device and the gate electrode into the electrolyte solution at the same time, reading the channel current signal under the light-proof condition, comparing the channel current under the light excitation condition at a wavelength of 420 nm to obtain the channel current step signal, and drawing the HE-4 concentration-channel current step signal curve; after preparing a sample to be detected containing HE-4 with an unknown concentration into a double-antibody sandwich structure in a well plate, adding sufficient PBS solution containing AAP, and obtaining ascorbic acid solution with an unknown concentration after incubation, adding the ascorbic acid solution with the unknown concentration into an electrolyte solution, placing the OECT device and the gate electrode into the electrolyte solution at the same time, reading the channel current signal under the light-proof condition, comparing the channel current under the light excitation condition at a wavelength of 420 nm to obtain the current channel current step signal, and calculating the concentration of HE-4 in the sample to be detected according to the HE-4 concentration-channel current step signal curve and the current channel current step signal.

[0061] In the embodiment, the principle of the organic photoelectrochemical transistor sensor for detecting the ovarian cancer marker HE-4 is shown as follows: when the light of 420 nm wavelength is directly irradiated on the COF film of the gate electrode, the electrons on the valence band of the COF film are excited to jump to the conduction band, the electrons on the valence band move to the solution, and the electrons on the ITO glass move to the valence band of the COF film and combine with the holes thereon, thereby forming a stable PEC cathode photocurrent. Meanwhile, the movement of the charges changes the partial pressure of the gate-electrolyte interface and the electrolyte-channel interface in the device. For the PEC cathode photocurrent, the movement of the electrons to the solution causes the charge accumulation of the gate-electrolyte interface of the OPECT device to be more, the partial pressure to be larger, and the partial pressure of the electrolyte-channel interface, which has the greatest influence on the actual channel current regulation due to the unchanged gate voltage, to be smaller, and the effective gate voltage to be smaller. Since the device with PEDOT:PSS as the semiconductor is a depletion-type device, the smaller effective gate voltage causes the channel current to increase. Therefore, the overall effect is that the light causes the channel current of the device to increase. In the 96-well plate, the "double-antibody sandwich structure" of the primary antibody-antigen-secondary antibody can specifically convert the content of HE-4 into the content of ALP enzyme in the solution. Under the same reaction conditions and the concentration of AAP, the greater the amount of ALP enzyme, the greater the amount of AA generated. The sandwich solution is added to the electrolyte, AA is a strong electron donor, which can inhibit the transfer of the electrons in the COF material to the solution and guide the transfer of the electrons to the electrode, so that the PEC cathode photocurrent of the COF film is smaller. Therefore, the amount of the channel current of the device caused by the light is also smaller, and even under a certain concentration, the channel current can be reduced, as shown in Figure 6 . Thus, the conversion of the content of HE-4 to the content of AA in the electrolyte is realized, and the change of the visible channel current of the device is caused.

[0062] The present application tests the channel current step signals corresponding to different concentrations of HE-4 in advance, and draws the HE-4 concentration-channel current step signal curve, as shown in Figure 7 . Finally, the HE-4 concentration of the sample to be tested is obtained by comparing the channel current step signal of the sample to be tested. Figure 7 It can also be seen that the detection range of the detection method provided in the embodiment is 1×10 -12 g / mL to 1×10 -6 g / mL, and the minimum detection limit is 1×10 -12 g / mL. In the embodiment, the channel voltage V DS added during the test is 0.1 V, and the gate voltage V GS is 0 V.

[0063] In order to verify that the PEC cathode current signal of the COF electrode gradually becomes smaller with the increase of the AA concentration in the solution, the PEC electric signal of the COF electrode is detected in the present embodiment: the PEC electric signal detection of the COF electrode is carried out in a quartz electrolytic cell, a platinum electrode is used as a counter electrode, an Ag / AgCl electrode is used as a reference electrode, the COF electrode is used as a working electrode, and the three electrodes are simultaneously put into 100 mM PBS solution to construct a three-electrode system; the current signal is read by a CHI600E electrochemical workstation under light-proof conditions, and the cathode current step signal is obtained by comparing the current under the excitation of light at a wavelength of 420 nm. Figure 8 In order to verify that the PEC cathode current signal of the COF electrode gradually becomes smaller with the increase of the AA concentration in the solution, the PEC electric signal of the COF electrode is detected in the present embodiment: the PEC electric signal detection of the COF electrode is carried out in a quartz electrolytic cell, a platinum electrode is used as a counter electrode, an Ag / AgCl electrode is used as a reference electrode, the COF electrode is used as a working electrode, and the three electrodes are simultaneously put into 100 mM PBS solution to construct a three-electrode system; the current signal is read by a CHI600E electrochemical workstation under light-proof conditions, and the cathode current step signal is obtained by comparing the current under the excitation of light at a wavelength of 420 nm. Figure 8 It can be seen that the PEC cathode current signal of the COF electrode gradually becomes smaller with the increase of the AA concentration in the solution, and even turns into an anode current signal when the AA concentration in the solution reaches 10 -2 M.

[0064] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. An organic photoelectrochemical transistor sensor for the detection of the ovarian cancer marker HE-4, characterized in that, The device includes an electrolytic cell containing an electrolyte solution, a substrate with one end inserted into the electrolyte solution, and a gate electrode. A source electrode and a drain electrode, each with one end inserted into the electrolyte solution and spaced apart, are disposed on the substrate. The surfaces of the source and drain electrodes are coated with an organic semiconductor thin film. A photoactive layer, made of a covalent organic framework material, is disposed on the end of the gate electrode inserted into the electrolyte solution. The device also includes a well plate containing a sample solution to be tested. The well plate is modified with a dual-antibody sandwich structure. The dual-antibody sandwich structure includes a first antibody bound to the well plate, HE-4 specifically bound to the first antibody, and a secondary antibody complex specifically bound to the HE-4. The secondary antibody complex includes gold nanoparticles and a second antibody and an ALP enzyme bound to the gold nanoparticles. The sample solution to be tested is an ascorbic acid solution generated by the reaction of L-ascorbic acid-2-phosphate trisodium salt with the ALP enzyme in the sandwich structure. The covalent organic framework material is a polymer of trialdehyde phloroglucinol and 2,5-dimethyl-1,4-phenylenediamine.

2. The organic photoelectrochemical transistor sensor for detecting the ovarian cancer marker HE-4 according to claim 1, characterized in that, The material of the organic semiconductor thin film layer is at least one of poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid, polypyrrole, polythiophene, polyaniline, polycarbazole and their copolymers.

3. The organic photoelectrochemical transistor sensor for detecting the ovarian cancer marker HE-4 according to claim 1, characterized in that, The source electrode and drain electrode are made of one or more of Ti, Pt, Au, Ti3C2 and deposited carbon, and the gate electrode is made of one of ITO glass and FTO glass.

4. A method for preparing an organic photoelectrochemical transistor sensor for detecting the ovarian cancer marker HE-4 as described in any one of claims 1-3, characterized in that, Including the following steps: A well plate containing a sample solution to be tested is provided. The well plate is modified with a double antibody sandwich structure. The double antibody sandwich structure includes a first antibody bound to the well plate, HE-4 specifically bound to the first antibody, and a secondary antibody complex specifically bound to the HE-4. The secondary antibody complex includes gold nanoparticles and a second antibody and ALP enzyme bound to the gold nanoparticles. The sample solution to be tested is an ascorbic acid solution generated by reacting L-ascorbic acid-2-phosphate trisodium salt with the ALP enzyme in the sandwich structure. A source electrode and a drain electrode are fabricated on a substrate, and an organic semiconductor thin film is fabricated on the surface of the source electrode and the drain electrode for later use; A gate electrode is provided, and a photoactive layer is fabricated on the gate electrode, wherein the photoactive layer is made of a covalent organic framework material; The substrate and the gate electrode with the photoactive layer are inserted into an electrolytic cell containing an electrolyte solution to obtain the organic photoelectrochemical transistor sensor for the detection of the ovarian cancer marker HE-4.

5. The method for preparing the organic photoelectrochemical transistor sensor for detecting the ovarian cancer marker HE-4 according to claim 4, characterized in that, The steps for preparing a well plate containing the sample solution to be tested are as follows: The first antibody solution was added to the well plate and incubated overnight. The first antibody was firmly bound to the well plate through physical adsorption between hydrophobic groups. After the primary antibody incubation was completed, the plate was washed three times with PBS solution containing Tween 20 to remove unbound primary antibody. Then, BSA solution was added to block the unbound sites in the plate. After blocking, the plate was washed with washing buffer. After washing, a sample to be tested containing an unknown concentration of HE-4 is added, and incubation is carried out to allow the HE-4 to specifically bind to the first antibody; After washing, the secondary antibody complex was added and incubated to allow the second antibody in the secondary antibody complex to specifically bind to HE-4. After washing with washing solution, a double antibody sandwich structure was formed in the well plate. Add a PBS solution containing AAP to the well plate, and after incubation, an ascorbic acid solution is generated, thus obtaining a well plate containing the sample solution to be tested.

6. The method for preparing the organic photoelectrochemical transistor sensor for detecting the ovarian cancer marker HE-4 according to claim 5, characterized in that, The preparation of the secondary antibody complex includes the following steps: The pH of the gold nanoparticles was adjusted to 8 using potassium carbonate solution to obtain a gold nanoparticle solution. The second antibody and ALP enzyme were added sequentially to the gold nanoparticle solution, incubated overnight, and then centrifuged with PBS buffer to collect the secondary antibody complex.

7. A detection method using an organic photoelectrochemical transistor sensor for detecting the ovarian cancer marker HE-4 as described in any one of claims 1-3, characterized in that, Including the following steps: After preparing a double-antibiotic sandwich structure with known concentrations of HE-4 in a well plate, a sufficient amount of PBS solution containing AAP was added, and after incubation, ascorbic acid solutions of different concentrations were obtained. The ascorbic acid solutions of different concentrations were added to the electrolyte solution. The OECT device and the gate electrode were placed in the electrolyte solution at the same time. The channel current signal was read under light-shielding conditions and compared with the channel current under 420nm wavelength light excitation conditions to obtain the channel current step signal. The HE-4 concentration-channel current step signal curve was plotted. After preparing the test sample containing an unknown concentration of HE-4 into a double-antibody sandwich structure in a well plate, a sufficient amount of PBS solution containing AAP was added, and after incubation, an ascorbic acid solution of an unknown concentration was obtained. The unknown concentration of ascorbic acid solution was added to the electrolyte solution. The OECT device and the gate electrode were placed in the electrolyte solution at the same time. The channel current signal was read under light-shielding conditions and compared with the channel current under 420nm wavelength light excitation conditions to obtain the current channel current step signal. The HE-4 concentration in the sample to be tested is calculated based on the HE-4 concentration-channel current step signal curve and the current channel current step signal. The detection method described is not for the purpose of treating or diagnosing diseases.

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