Microfluidic photoelectrochemical immunosensor based on ZnIn2S4-In2S3 heterojunction photosensitive material and application thereof

Abstract

The invention discloses a micro-fluidic photoelectrochemical immunosensor based on a ZnIn2S4-In2S3 heterojunction photosensitive material and application of the micro-fluidic photoelectrochemical immunosensor based on the ZnIn2S4-In2S3 heterojunction photosensitive material, synthesized In2S3 is used as a precursor, and the ZnIn2S4-In2S3 heterojunction photosensitive material is synthesized in one step. A ZnIn2S4-In2S3 heterojunction photosensitive material is sprayed on the tin oxyfluoride electrode subjected to photoetching of the micro-fluidic channel, and an ascorbic acid solution is used as a solution for enhancing a light absorption signal; the preparation method comprises the following steps: preparing a placenta growth factor monoclonal antibody, activating amino and carboxyl of the sensor through chitosan (CTS) and glutaraldehyde (GA), and fixing the placenta growth factor monoclonal antibody on a heterojunction photosensitive material through a covalent bond to obtain the microfluidic photoelectrochemical immunosensor. And the kit has good clinical sample detection capability and has important clinical application value for human precision medical research.

Description

Technical Field

[0001] The invention relates to the field of detection, in particular to a microfluidic photoelectrochemical immunosensor based on ZnIn2S4-In2S3 heterojunction photosensitive material, and also to the application of the sensor. Background Art

[0002] Pre-eclampsia (PE) is one of the conditions of pregnancy, and it is also an urgent and serious complication of pregnancy-induced hypertension in pregnant women. It is a systemic multi-system disease unique to pregnancy. Placental growth factor (PLGF) and the anti-angiogenic factor-soluble FMS-like tyrosine kinase-1 (sFlt-1) have high sensitivity and specificity for the diagnosis of preeclampsia. However, studies have shown that PLGF is the best predictive factor, and there are relatively few studies on PLGF. The clinical evaluation method of PLGF level is: PLGF ≥ 100pg mL -1 Indicates normal placental function; PLGF <100pg mL -1 This indicates placental insufficiency and requires treatment to support placental perfusion. PLGF < 12 pg mL -1 This indicates severe placental insufficiency and strongly predicts premature birth within 14 days.

[0003] As the number of older and high-risk pregnant women increases, the incidence of high-risk pregnancies such as gestational hypertension, fetal growth restriction, miscarriage and trisomy 21 will gradually increase. At present, there is no effective treatment for preeclampsia in clinical practice. The only treatment method is to terminate the pregnancy at the right time, which is extremely harmful to the health of pregnant women. Therefore, it is very important to accurately diagnose the PLGF content in the blood of pregnant women with preeclampsia; if preeclampsia can be identified as early as possible, early intervention and treatment can greatly reduce the serious obstetric complications and perinatal maternal and fetal deaths caused by preeclampsia.

[0004] The content of PLGF in the blood is at the picogram level, so it is of great significance to accurately quantify low concentrations of PLGF, and a highly sensitive method is needed for detection. Currently, the methods reported for detecting PLGF include enzyme-linked immunosorbent assay, fluorescent immunochromatography, time resolved fluoroimmunoassay (TRFIA) and electrochemiluminescence.

[0005] Large and small-scale clinical studies mainly use ELISA to detect serum free PLGF levels. Enzyme-catalyzed chemical reactions are highly efficient and have good specificity. ELISA has the disadvantages of instability of enzyme markers and a narrow quantitative range, and it takes a long time and cannot achieve rapid detection.

[0006] Fluorescence immunochromatography is easy to operate and fast, but the precision is not high, and the intra-batch and inter-batch differences are large. The preparation of fluorescent microsphere immunochromatography test strips for quantitative detection of PLGF usually only has a sensitivity of nanograms, which has become an important limiting factor for its widespread use without sacrificing its own advantages. At present, the electrochemiluminescence method is a closed system, and large-scale detection equipment is expensive and the reagent cost is high. Time-resolved fluoroimmunoassay (TRFIA) is an ultra-trace chemiluminescence method that combines time-resolved technology with fluorescent immunoassay technology, but the instrument requirements and costs are relatively high, and it is easily contaminated by lanthanide ions in the environment, reagents and containers, which increases the background.

[0007] Among the current biosensor detection technologies, optical detection and electrochemical detection are the most widely used, while photoelectrochemical detection is relatively less used. Among biosensors, photoelectrochemical (PEC) biosensor is a new analytical method based on the PEC process and specific biorecognition process. On ordinary photoelectrochemical sensor electrodes, it is difficult for light stimulation alone to convert the absorbed light energy into driving force on the bare electrode, so it is necessary to coat the conductive glass with a photocatalyst to amplify the light energy. Among various visible light responsive photocatalysts, ZnIn2S4 ternary metal sulfide has outstanding advantages such as simple synthesis, excellent stability, and suitable band structure. However, low solar energy utilization, rapid recombination of photogenerated charges, and poor redox ability are still obvious disadvantages that seriously hinder the improvement of the photocatalytic efficiency of ZnIn2S4 photocatalysts. The above shortcomings can be solved simultaneously by constructing a heterojunction between ZnIn2S4 and other semiconductors. It is known that the lattice matching of In2S3 and ZnIn2S4 has excellent visible light response; thus, a larger specific surface area and a stronger ohmic contact with the conductive substrate are obtained, which provides more exposed active sites and accelerates the reaction kinetics. In addition, the photoexcited holes in In2S3 / ZnIn2S4 can oxidize water to produce hydrogen peroxide (H2O2), significantly improving the separation efficiency of electron-hole pairs. It can then serve as a strong photocatalyst to further amplify biological signals.

[0008] Microfluidic chips are a type of science and technology that is mainly characterized by the manipulation of fluids in micrometer-scale space. At present, the mainstream form of microfluidic chips refers to the integration or basic integration of sample basic operation units involved in fields such as chemistry and biology on a chip of a few square centimeters or even smaller, with a network formed by microchannels and controllable fluids running through the entire system. Its advantages are high sensitivity and small size.

[0009] Therefore, there is an urgent need for an efficient and low-cost biosensor that can assist in the early screening of eclampsia-related diseases in pregnant women. Summary of the invention

[0010] In view of this, one of the objects of the present invention is to provide a microfluidic photoelectrochemical immunosensor based on ZnIn2S4-In2S3 heterojunction photosensitive material; the second object of the present invention is to provide the application of a microfluidic photoelectrochemical immunosensor based on ZnIn2S4-In2S3 heterojunction photosensitive material.

[0011] In order to achieve the above object, the present invention provides the following technical solutions:

[0012] 1. A microfluidic photoelectrochemical immunosensor based on ZnIn2S4-In2S3 heterojunction photosensitive material, wherein the microfluidic photoelectrochemical immunosensor uses synthesized In2S3 as a precursor, and further synthesizes the ZnIn2S4-In2S3 heterojunction photosensitive material in a further step, sprays the ZnIn2S4-In2S3 heterojunction photosensitive material on a tin fluoride oxide electrode that has been photoetched into a microfluidic channel, and uses an ascorbic acid solution as a solution for enhancing the light absorption signal; then, chitosan (CTS) and glutaraldehyde (GA) are used to activate the amino and carboxyl groups of the sensor, and finally, the placenta growth factor monoclonal antibody is fixed to the heterojunction photosensitive material by a covalent bond to obtain a microfluidic photoelectrochemical immunosensor.

[0013] Preferably, the ZnIn2S4-In2S3 heterojunction photosensitive material is prepared by using the synthesized IS as a precursor, adding a ZnIn2S4 layer at a material ratio of 1:1 and 1:1, respectively, and reacting at 180°C for 4 hours to obtain a heterostructure In2S3 / ZnIn2S4. 3和 ZnIn2S4 was prepared by a two-step hydrothermal method.

[0014] Preferably, the method for preparing the tin fluoride oxide electrode of the photolithographic microfluidic channel is as follows:

[0015] (1) Clean with acetone, alcohol, and deionized water in sequence, and blow dry under nitrogen;

[0016] (2) Carving out a pre-designed microfluidic channel pattern on a silicon wafer;

[0017] (3) After the substrate is cooled to room temperature, the photoresist is evenly spin-coated on the wafer, and the wafer is left to stand for a period of time to allow bubbles to escape before use; after the spin coating is completed, the wafer is dried and cooled to room temperature;

[0018] (4) The designed mask pattern and photoresist pattern are lithographed in a 1:1 ratio. The mask is made of film and the light source is a mercury lamp ultraviolet light source. The exposure gap is aligned to 50 μm, and then the alignment is adjusted by the base knob. After the alignment is completed, press the lift plate. After exposure for 15 seconds, remove the silicon wafer and mask respectively.

[0019] (5) Dry in an oven at 65°C for 5 min;

[0020] (6) removing the unexposed area at a temperature range of 20-24°C, using an immersion development method and an alkaline aqueous solution as a developer for the UV photoresist, leaving behind a circuit pattern;

[0021] (7) Hard film: Drying in a 65°C oven for 5 minutes can improve the stability of the photoresist.

[0022] Preferably, the ZnIn2S4-In2S3 heterojunction photosensitive material spraying method of the present invention is as follows: after the fluorine tin oxide electrode is continuously cleaned with ultrasound in acetone, ethanol and deionized water, the ZnIn2S4-In2S3 heterojunction photosensitive material is sprayed onto the fluorine tin oxide electrode to obtain a microfluidic fluorine tin oxide electrode.

[0023] Preferably, the activation is to push 8 μL of 10 mM / 2 mM CTS and GA onto the electrode area to activate the surface groups, react for 2 hours; then add 10 μL of 1 μg·mL -1 PLGF monoclonal antibody was fixed on the above electrode at 4°C overnight. The next day, the electrode was washed with 0.1M PBS, 8μL 1wt% BSA was pumped into the electrode and blocked at nonspecific binding sites at 37°C for 2 hours, and unbound biomolecules were removed with 0.1M PBS.

[0024] 2. Application of the microfluidic photoelectrochemical immunosensor in detecting PLGF concentration.

[0025] The beneficial effects of the present invention are as follows: the present invention provides a microfluidic photoelectrochemical immunosensor based on ZnIn2S4-In2S3 heterojunction photosensitive material, by combining microfluidic integration technology with a photoelectrochemical platform, using fluorine tin oxide (FTO) as an electrode substrate, two compounds In2S3 and ZnIn2S4 as photosensitive materials, a new hydrothermal synthesis method is used to synthesize the heterojunction of the two and spray it on a new photoanode with a single-channel microfluidic channel, and by integrating the photoelectrode into a microfluidic chip, automatic sampling and detection are successfully achieved, the photoanode is modified with In2S3 / ZnIn2S4 heterojunction nanomolecules as photosensitive materials, and the PLGF catalyzing the analyte is fixed on the biocathode, and only the enzymatic biocathode is incubated in the sample to catalyze the target reaction, while the photoanode does not contact the sample, thereby avoiding the interference of electron donors in the actual sample and enhancing the stability and anti-interference ability of the sensor; in addition, studies have shown that the PEC platform has achieved significant sensing performance in sensitivity, selectivity, stability and a wide detection range. At present, there are no reports on the use of this technology for the detection of placental growth factor antigen. A new type of high-sensitivity and high-stability PLGF photoelectrochemical biosensor detection technology based on In2S3 / ZnIn2S4 heterojunction nanomolecular photosensitive materials and PEC platform was prepared, which has the advantages of good stability, fast response speed, high specificity, small size, less consumables, and economy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0027] Figure 1 SEM characterization of the photocatalyst (ac: SEM of ZnIn2S4; df are SEM images of In2S3; gi are SEM images of heterojunction IS / ZIS).

[0028] Figure 2 EDS layered images of IS / ZIS (a is the electron distribution diagram of S, Zn, and In; bd are the EDS of the three elements S, Zn, and In, respectively; e is the total distribution spectrum of each element; and f is the mass ratio and atomic ratio of each element).

[0029] Figure 3 XRD patterns of IS, ZIS and IS / ZIS.

[0030] Figure 4 XPS measurement spectra (a: XPS measurement spectra of IS, ZIS, and IS-ZIS; bd are Zn 2p (b), In3d (c), and S2p (d) are high-resolution XPS spectra, respectively).

[0031] Figure 5Diagram for the establishment of photoelectrochemical microfluidic sensor.

[0032] Figure 6 Heterojunction mechanism study (a is the UV diagram of IS, ZIS and IS / ZIS, b is the lauc curve diagram of IS and ZIS, c is the model Schottky diagram of IS and ZIS).

[0033] Figure 7 The mechanism of e- / h+ transfer in PEC immunosensors.

[0034] Figure 8 These are the sensor optimization results (a is the ratio of heterojunction materials; b is the optimization diagram of spraying heterojunction content; c is the optimization diagram of ascorbic acid concentration; d is the pH optimization diagram of the test solvent used).

[0035] Fig. 9 Changes in the electrode interface properties during the preparation of the immunosensor (a is the impedance curve of IS / ZIS heterojunction; b is the impedance curve of IS heterojunction; c is the impedance curve of ZIS heterojunction; d is the impedance curve of IS / ZIS-Ab; e is the impedance curve of IS / ZIS-Ab-BSA; f is the impedance curve of IS / ZIS-Ab-BSA-Ag).

[0036] Fig.10 Performance test of the immunosensor (a is the It diagram of the photoelectrode modification process under 0.5 M ascorbic acid and light conditions (af are the photocurrents when the sensors are modified with IS / ZIS, ZIS, IS / ZIS-Ab, IS / ZIS-Ab-BSA, IS / ZIS-Ab-BSA-Ag, and IS, respectively), b is the original It diagram of the photoelectrode modification process of IS under 0.5 M ascorbic acid and light conditions).

[0037] Fig.11 The detection performance test of the immunosensor (a is the photocurrent of the immunosensor when measuring different concentrations of PLGF, b is the photocurrent I and PLGF concentration (0.1 pg mL -1 -1 μg mL -1 ) logarithmic linear fitting curve; c is the IT diagram of the sensor testing different samples; d is the stability performance test diagram of the immunosensor in different time periods; e is the 400S cycle IT diagram of repeated testing of the same sample; f is the IT diagram of 5 electrodes measuring the same concentration sample for 3 times).

[0038] Fig.12 IT plots of clinical samples tested for the immunosensor. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0040] Example 1. Material Synthesis

[0041] Dissolve 0.05M InCl3·4H2O and 0.1M CH4N2S in 30mL of deionized water in turn, and completely dissolve to obtain a clear solution, which is transferred to a polytetrafluoroethylene-sealed autoclave; the autoclave is sealed and reacted in a drying oven at 180°C for 4 hours, and then naturally cooled to room temperature (RT). Take out the sample, wash it with water and anhydrous ethanol 3 times, and dry it at 60°C overnight. The obtained sample is named In2S3 (denoted as IS). ZnIn2S4 is synthesized in the same way. A thin layer of ZnIn2S4 (denoted as ZIS) is synthesized by the same method as above with the addition of 0.025M Zn(NO3)2·6H2O zinc source. On this basis, the synthesized IS was used as a precursor and added into the synthesis process of ZIS in the molar ratios (1:2, 1:1, 2:1), respectively. The reaction was carried out at 180 °C for 4 h to obtain the heterostructure In2S3 / ZnIn2S4 (IS / ZIS), which were recorded as 1-ZnIn2S4 / In2S3, 2-ZnIn2S4 / In2S3, and 3-ZnIn2S4 / In2S3, respectively.

[0042] In order to further understand the individual photocatalysts In2S3, ZnIn2S4 and In2S 3 / The heterogeneous structure of ZnIn2S4 was characterized by SEM. Figure 1 shown. Figure 1 The middle ac is the SEM of ZnIn2S4, showing a three-dimensional flower-like microsphere structure assembled in a regular and orderly manner. The surface of the flower-like microsphere is composed of countless thin and dense flakes. Figure 1 Figures df are SEM images of In2S3, which exist in the form of irregular solid nanoparticles with a smooth surface and graphene-like morphology. Figure 1 Figure gi is the SEM image of the heterojunction IS / ZIS. It can be seen from the figure that there are obviously two characteristic IS and ZIS structures in the heterojunction. When IS is used as a precursor and reacts again under the same conditions, it changes from an irregular solid to a regular tetrahedron and exists in the flower ball ZIS, and the two are in close contact.

[0043] In addition, to determine the element distribution in the IS / ZIS heterojunction, the EDS mapping of the sample ( Figure 2 ) shows the elemental distribution of S, Zn, and In atoms. Elemental mapping image Figure 2The ad proves that the elements S, Zn, and In are in In2S 3 / Uniform distribution in ZnIn2S4 heterojunction. Figure 2 The results of the EF show that Zn, In and S elements are uniformly present in the 0-4 region, with atomic proportions of 10.8%, 34.92% and 54.28%, respectively, confirming the low content of ZnIn2S4 in IS / ZIS. The results further confirm that In2S 3 / Successful preparation of ZnIn2S4.

[0044] The powder XRD spectra of IS, ZIS synthesized by one step hydrothermal method and IS / ZIS composite samples synthesized by two steps are shown in Figure 2. Figure 3 As shown. X-ray diffraction analysis shows that the characteristic diffraction peaks of IS at 27.7°, 33.5°, 43.8° and 47.9° correspond to the (311), (400), (511) and (440) crystal planes of β-In2S3; each diffraction peak completely corresponds to the standard card (PDF#03-065-0459), and has good crystallinity. The characteristic diffraction peaks of ZIS at 21.5°, 27.6° and 41.1° match the (006), (102) and (110) crystal planes of the ZnIn2S4 standard card (PDF#-03-065-2023). For the IS / ZIS sample, the characteristic diffraction peaks of IS and ZIS appear simultaneously, indicating that the IS / ZIS composite material was successfully prepared.

[0045] X-ray photoelectron spectroscopy (XPS) is also used to indicate the elemental composition and chemical valence states. Figure 4 The full scan XPS spectra of In2S3, ZnIn2S4, and In2S3 / ZnIn2S4 show that the characteristic peaks are Zn 2p, In 3d, and S2p orbitals. In the high-resolution XPS spectrum of Zn 2p of the synthesized heterojunction, two characteristic peaks at 1022.08 eV and 1045.24 eV are attributed to Zn 2p, respectively. 3 / 2 and Zn2p 1 / 2 The binding energy of Zn 2+ In the high-resolution XPS spectrum of In 3d, two characteristic peaks at 452.56eV and 445.03eV are respectively attributed to In 3d 3 / 2 and In 3d 5 / 2 The binding energy of In 3+ In the high-resolution XPS spectrum of S2p, the two characteristic peaks at 161.79 eV and 163.06 eV are respectively attributed to S2p 3 / 2 and S2p 1 / 2 The binding energy of S2 -The results show that the In2S3 / ZnIn2S4 heterojunction composite material was successfully prepared.

[0046] Heterojunction mechanism research: Light absorption performance is a key factor in testing the photocatalytic performance of semiconductors, which can be characterized by ultraviolet-visible absorption spectroscopy (UV), such as Figure 6 As shown in a, the visible light range is generally 390nm-760nm, and the heterojunction IS / ZIS and the single materials ZIS and IS can absorb most of the visible light. According to the Kubelka-Munk function, the lauc curve can be obtained, and the band gap energy (Eg) of ZIS and IS can be calculated. The band gap energy can be calculated by formula (1):

[0047] (αhν) n =A(hν-Eg) (1)

[0048] Where α is the absorption coefficient, h and ν are Planck constant and light frequency respectively, k is a constant, and the band gap energy can be represented by Eg. For direct band gap semiconductors, n is 2. The final calculation results show that the Eg values ​​of IS and ZIS are 2.05 and 2.44 eV respectively. Figure 6 As shown in b and c, it shows that the heterojunction has a lower band gap energy and higher proton utilization after surface sulfurization.

[0049] To further explore the catalytic mechanism of the IS / ZIS heterojunction, a Mott-schottky plots test was performed on the heterojunction using a platinum sheet as the counter electrode, Ag / AgCl as the reference electrode, and 0.2M sodium sulfate solution as the electrolyte solution. Figure 6 In c, since the tangent slope is positive, it can be inferred that IS and ZIS are n-type semiconductors. According to equations (2) and (3), the flat band potentials of IS and ZIS relative to Ag / AgCl electrode (vs.Ag / AgCl) are -1.25 and -1.06 eV, respectively, and the flat band potentials relative to normal hydrogen electrode (NHE) are -1.05 and -0.86 eV, respectively.

[0050] E(RHE)=E(Ag / AgCl)+0.0591pH+0.2 (2)

[0051] E(RHE)=E(NHE)+0.0591pH (3)

[0052] From equation (4), we can see that the valence band potentials of IS and ZIS are 0.8 eV and 1.38 eV, respectively.

[0053] E g =E VB -E CB (4)

[0054] Example 2. Preparation of microfluidic channels

[0055] The production of PDMS microfluidic channels includes microchannel patterning, microchannel etching, PDMS casting, PDMS demolding, etc., and a 1 cm × 1 cm silicon wafer is selected.

[0056] (1) Cleaning: Use acetone, alcohol, and deionized water to clean in sequence, and blow dry under nitrogen.

[0057] (2) A pre-designed microfluidic channel pattern is engraved on a silicon wafer. The photoresist used here is for patterning PDMS microfluidics. The thickness is 150 μm, and negative photoresist SU82075 is selected.

[0058] 1) Use the spin coating method. After the substrate cools to room temperature, slowly drip SU82005 negative photoresist on the wafer to prevent bubbles from forming. Increase the rotation speed in a step-by-step manner, set the rotation speed and time, and spin-coat the photoresist evenly on the wafer. Let it stand for a while to allow bubbles to escape before use. After spin coating, put the wafer into a 95°C drying oven for 15 minutes and cool it to room temperature.

[0059] 2) Photolithography: The designed mask pattern and photoresist pattern are photolithographically processed in a 1:1 ratio. The mask is made of film. The photolithography machine used is a URE-2000 / 30AL UV photolithography machine, and the light source is a mercury lamp UV light source. The exposure gap is aligned to 50μm, and then the alignment is adjusted using the base knob. After the alignment is completed, press the lift button. After exposure for 15s, remove the silicon wafer and mask respectively.

[0060] 3) Post-baking: Dry in a 65°C oven for 5 minutes. Since the photoresist film has not yet closed, post-baking can make the film tighter, minimize the standing wave effect as much as possible, improve the corrosion resistance of the photoresist, and avoid pattern deformation in the subsequent etching process.

[0061] 4) Development: Remove the unexposed area at a temperature range of 20-24°C. Use an immersion development method and an alkaline aqueous solution as the developer of the UV photoresist to leave the circuit pattern.

[0062] 5) Hard film: Drying in a 65℃ oven for 5 minutes can improve the stability of the photoresist.

[0063] Example 3: Establishment of microfluidic photoelectrochemical immunosensor

[0064] Before modification, the fluorine tin oxide (FTO) electrode (working area: 1cm×1cm) was continuously cleaned with ultrasound in acetone, ethanol and deionized water for 5 minutes. The heterojunction photosensitive material obtained by the hydrothermal synthesis method in Example 1 was sprayed onto the FTO after photolithography, and the microfluidic channel was bonded to the electrode to form an IS / ZIS / FTO electrode. An In2S3 / ZnIn2S4 / FTO photoanode was installed in the upper chamber of the microfluidic channel, and the biocathode was placed in the bottom chamber to specifically identify the analyte (PLGF); during the detection process, after the catalytic operation on the bioanode, PBS was pumped into the chip from inlet No. 1 to clean the biocathode, and then AA solution was pumped in as an electrolyte to increase light absorption. When the upper and bottom chambers were filled with electrolytes in turn, the response between the biocathode and the photoanode was recorded using a semiconductor test analyzer 4200. The structure diagram is shown in the figure. Figure 5 shown.

[0065] Next, 8 μL of CTS / GA (10 mM / 2 mM) was pushed onto the IS / ZIS / FTO electrode area using a peristaltic pump to activate the surface groups and reacted at room temperature for 2 h. Then, 10 μL of PLGF monoclonal antibody (1 μg mL -1 ) was fixed on the above electrode at 4°C overnight. The next day, after washing the electrode with 0.1M PBS, 8μL 1wt% BSA was pumped into the electrode and blocked at nonspecific binding sites at 37°C for 2 hours, and unbound biomolecules were removed with 0.1M PBS. Finally, the resulting sensor was stored in a 4°C refrigerator until testing. The results are shown in Figure 7 As shown. The results show that under visible light illumination, photoelectrons transfer from In2S3 to ZnIn2S4 through CB, and holes migrate from ZnIn2S4 to In2S3 through VB. Obviously, the thin nanosheets shorten the charge diffusion path of photoholes in water. In addition, ZnIn2S4 has a high specific surface area, which not only enhances visible light absorption but also provides more exposed active sites for photocatalytic hydrogen production. The results show that the IS / ZIS sample forms a nn-type II heterojunction, which is beneficial to carrier separation.

[0066] Sensor optimization: Among the three synthesized heterojunctions, e.g. Figure 8 As shown in a, the photocurrent response is arranged from large to small as 2-ZnIn2S4 / In2S3, 3-ZnIn2S4 / In2S3, and 1-ZnIn2S4 / In2S3, so 2-ZnIn2S4 / In2S3 with a synthesis ratio of 1:1 was selected as the optimal photocatalyst. In addition, in order to obtain the best PLGF analysis performance, the amount of sprayed photocatalyst, the concentration of ascorbic acid, and the pH value of PBS were optimized. Figure 8As shown in (b), when the concentration of 2-ZnIn2S4 / In2S3 is 15 μg, the photocurrent response reaches the maximum value, which is due to the provision of sufficient 2-ZnIn2S4 / In2S3 to bind to PLGF. Ascorbic acid, as an effective electron donor, can capture photogenerated holes and is an important factor affecting the photocurrent response. Figure 8 Figure c shows that as the concentration of ascorbic acid increases, the detection rate of 1 μg mL -1 The photocurrent reached a maximum of 40 μA·cm when PLGF antigen was applied -2 The pH value of the solution affects the properties of the antigen and antibody, and is therefore also the main factor affecting the photocurrent of the PEC immunosensor. Figure 8 In middle d, as the pH value increases from 6.5 to 8.5, the photocurrent increases first and then decreases, and the optimal pH value is 7.4. This is because excess acid or excess base will destroy the structure of the protein.

[0067] EIS can characterize the changes in the electrode interface properties during the preparation of the immunosensor. In the EIS Nyquist plot, the linear part is related to the diffusion process, and the semicircular part is the electron transfer resistance (Ret), which reflects the process of limiting electron transfer. The resistance is used to monitor each step of electrode surface modification. Fig. 9 The EIS curve in the graph shows a semicircle, indicating that the sensor is essentially a parallel circuit. The semicircle diameter of the IS / ZIS modified FTO electrode is the smallest and the resistance is the smallest (curve a); the resistance of IS (curve b) and the resistance of ZIS (curve c) decrease successively. After the electrode surface is gradually modified by PLGF antibody Ab (curve d), 1% BSA (curve e), and PLGF antigen Ag (curve f), the semicircle diameter increases successively, indicating that there is resistance in the modified layer, which may be caused by steric hindrance, which prevents a large number of electrons from transferring from the active material to the electrode surface. The EIS test proves the successful layered modification of the immunosensor.

[0068] Example 4: Biological Response of Sensor

[0069] The prepared sensor was pumped into PLGF antigen solutions of different concentrations and incubated at 37°C for 0.5 h. The photocurrent response of the microfluidic photoelectrochemical immunosensor to different concentrations of PLGF antigen in 0.5 M AA solution was studied using IT diagrams.

[0070] (1) Immunosensor detection performance test

[0071] To explore the performance of PEC immunosensor, the results are as follows Fig.10 The results show that the change of photocurrent is related to the modification of PEC immunosensor. Fig.10In Figures ab and b, after the PEC immunosensor was modified with PLGF monoclonal antibody, 1% BSA, and PLGF antigen, the measured photocurrent decreased in turn, indicating that the sensor can well fix biological samples such as PLGF monoclonal antibody, which is beneficial for subsequent quantitative testing.

[0072] The change in photocurrent is also related to the concentration of PLGF, so the concentration of PLGF can be detected by the change in photocurrent response. Under the optimal conditions, PLGF was quantitatively analyzed. Fig.11 As shown in (a), the photocurrent decreased with the increase of PLGF concentration, which was because a large amount of Ab was captured and participated in the PEC process.

[0073] a. Sensitivity test

[0074] PLGF antigen was diluted into different gradients (1 μg mL -1 、100ng·mL -1 , 10ng·mL -1 , 1ng·mL -1 , 100pg·mL -1 , 10pg·mL -1 , 1pg·mL -1 , 0.1pg·mL -1 ) were pumped into the prepared PEC microfluidic sensor and incubated in a 37°C constant temperature incubator for 30 minutes. After washing with PBS, the photocurrent was tested with a semiconductor tester with a bias voltage between -0.5 and 0.5 V, repeated 3 times, and the data was recorded. The results are shown in Fig.11 As shown in b. The results show that at 0.1pg·mL -1 -1μg·mL -1 Within the range, a good linear relationship was obtained between the change of photocurrent intensity (I) and the logarithm of PLGF concentration. The fitting curve is I = 283.456-39.956lgc (R 2 =0.997), and the detection limit of PLGF was 0.1 pg·mL -1 The detection limit was 0.0879 pg·mL -1 (S / N=3). The constructed PEC immunosensor for PLGF detection has better analytical performance than other detection methods.

[0075] b. Specificity test

[0076] The concentration was 1 μg mL -1Different antigens (PLGF, sFlt-1, NES, VEGF-165, AMH, LP-PLA2) and the mixture were pumped into the prepared PEC microfluidic sensor and incubated in a 37°C constant temperature incubator for 30 minutes. After washing with PBS, the photocurrent was tested with a semiconductor tester with a bias voltage between -0.5 and 0.5 V, repeated 3 times, and the data was recorded. The results are shown in Fig.11 As shown in c. The results show that the photocurrent is reduced only for the target PLGF solution or mixture, indicating that the PEC biosensor has good selectivity and anti-interference for the detection of PLGF.

[0077] C. Stability test

[0078] The prepared PEC microfluidic sensor was tested for photocurrent using a semiconductor tester at different time periods (0 days, 1 day, 2 days, 4 days, 8 days, and 16 days) with a bias voltage between -0.5 and 0.5 V, repeated 3 times, and the data were recorded. Fig.11 As shown in Figure d, the results show that the photocurrent of the immunosensor stored at 4°C was measured at different times. The photocurrent remained relatively stable after 16 days, indicating that the immunosensor has good stability. -1 The PLGF antigen was pumped into the prepared PEC microfluidic sensor and incubated in a 37°C constant temperature incubator for 30 minutes. After washing with PBS, the photocurrent was tested with a semiconductor tester. The light and dark were alternated 20 times, the cycle was 400 seconds, the bias voltage was between -0.5-0.5V, repeated 3 times, and the data was recorded. The results are shown in Fig.11 As shown in e. The results show that the test 1μg·mL -1 It curve of PLGF with adjustable light on or off for 20 irradiation cycles of 400 s. PLGF (1 μg·mL -1 ) has no obvious fluctuation in the photocurrent response, indicating that the PEC immunosensor has good stability. In addition, its storage stability was also tested.

[0079] D. Repeatability test

[0080] Five prepared PEC microfluidic sensors were randomly selected and the concentration was 1 μg mL -1 The PLGF antigen was pumped into the prepared PEC microfluidic sensor and incubated in a 37°C constant temperature incubator for 30 minutes. After washing with PBS, the photocurrent was tested with a semiconductor tester with a bias voltage between -0.5 and 0.5 V, repeated 3 times, and the data was recorded. Fig.11 As shown in figure f, the current values ​​I of the five parallel electrodes are relatively close, proving that the immunosensor has reproducibility.

[0081] Example 5: Clinical testing of immunosensors

[0082] In order to evaluate the performance of PEC immunosensors in practical applications, clinical samples were tested. Because the PlGF level is evaluated clinically as follows: PlGF ≥ 100 pg mL -1 Indicates normal placental function; PlGF < 100 pg·mL -1 This indicates placental insufficiency and requires appropriate treatment. If this photoelectrochemical immunosensor is used to evaluate clinical positive and negative results, the theoretical critical value should be 200 μA·cm -2 , and if it is lower than this value, it is negative. In the clinical sample test (the hospital has used chemiluminescence to determine its concentration and positive and negative) 7 samples were tested, of which 4 were positive clinical samples (concentrations were 9.5pg·mL -1 , 27.88pg·mL -1 , 57.5 pg·mL -1 , 75.9pg·mL -1 ), 3 negative clinical samples (concentrations were 463.5 pg mL -1 , 485.1pg·mL -1 , 800.7pg·mL -1 ) were pumped into the prepared PEC microfluidic sensor and incubated in a 37°C constant temperature incubator for 30 minutes. After washing with PBS, the photocurrent was tested with a semiconductor tester with a bias voltage between -0.5 and 0.5 V, repeated 3 times, and the data was recorded. The results are shown in Fig.12 The results showed that all four positive clinical samples were successfully detected, and Table 1 shows the concentration comparison between the PEC test value and the actual value. The relative error and relative standard deviation (RSD) of the measurement data were within 10%, indicating that the test was relatively accurate and that the immunosensor had good clinical testability.

[0083] Table 1. Comparison of clinical sample test data

[0084] Group name <![CDATA[C(pg mL -1 )]]> Test concentration Relative standard deviation Relative error S1 9.5 9.28 2.25％ 0.33％ S2 27.88 27.99 0.30％ 7.79％ S3 57.5 57.59 0.50％ 5.53％ S4 75.9 75.64 0.57％ 9.66％ G1 463.5 463.36 0.03％ 4.95％ G2 485.1 484.72 2.24％ 8.33％ G3 800.7 800.19 2.72％ 6.95％

[0085] In summary, this paper prepared a microfluidic photoelectrochemical immunosensor for PLGF antigen detection that combines a photoelectrochemical platform that converts light energy into electrical energy with a microfluidic channel. Compared with traditional PEC sensors, the advantages of this work can be simply summarized as follows: (1) The heterojunction structure of ZnIn2S4-In2S3 synthesized in one step using In2S3 as a precursor improves the photoelectric conversion efficiency, provides a signal amplification effect, and significantly reduces the detection limit. (2) The newly designed single-channel microfluidic channel provides space for capturing antigens and antibodies, reduces the injection volume, and reduces consumables. (3) Among the existing PLGF antigen detection methods, the microfluidic photoelectrochemical immunosensor in this work has a wider response range of 0.1 pg·mL -1 -1ug·mL -1 The detection limit (LOD) was 0.0879 pg·mL -1 (S / N=3). The results showed that the prepared PLGF microfluidic photoelectrochemical immunosensor has strong selectivity, good stability and low consumables.

[0086] In addition, the PLGF microfluidic photoelectrochemical immunosensor can not only be used as an effective tool for laboratory monitoring of eclampsia and its complications, but can also detect other disease-related biomarkers by changing the enzyme on the biocathode. By replacing the enzyme on the biocathode, the biosensor chip can play a role in monitoring a wide range of disease markers and has good application prospects in photoelectrochemistry and biosensing.

[0087] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A microfluidic photoelectrochemical immunosensor based on ZnIn2S4-In2S3 heterojunction photosensitive material, characterized by: The microfluidic photoelectrochemical immunosensor uses synthesized In2S3 as a precursor, and further synthesizes a ZnIn2S4-In2S3 heterojunction photosensitive material in a step, and sprays the ZnIn2S4-In2S3 heterojunction photosensitive material on a tin fluoride oxide electrode that has been photoetched into a microfluidic channel, and uses an ascorbic acid solution as a solution to enhance the light absorption signal; then, chitosan (CTS) / glutaraldehyde (GA) is used to activate the amino and carboxyl groups of the sensor, and finally, the placenta growth factor monoclonal antibody is fixed to the heterojunction photosensitive material through a covalent bond to obtain a microfluidic photoelectrochemical immunosensor.

2. The microfluidic photoelectrochemical immunosensor based on ZnIn2S4-In2S3 heterojunction photosensitive material according to claim 1, characterized in that: The ZnIn2S4-In2S3 heterojunction photosensitive material is prepared by using the synthesized IS as a precursor, adding a ZnIn2S4 layer at a material ratio of 1 to 2:1 to 2, and reacting at 180° C. for 4 hours to obtain a heterostructure In2S3 / ZnIn2S4. 3和 ZnIn2S4 was prepared by hydrothermal method.

3. The microfluidic photoelectrochemical immunosensor based on ZnIn2S4-In2S3 heterojunction photosensitive material according to claim 1, characterized in that: The preparation method of the tin fluoride oxide electrode of the photolithographic microfluidic channel is as follows: the preparation of the PDMS microfluidic channel includes microchannel patterning, microchannel etching, PDMS casting, PDMS demoulding, etc.

4. The microfluidic photoelectrochemical immunosensor based on ZnIn2S4-In2S3 heterojunction photosensitive material according to claim 1, characterized in that: The ZnIn2S4-In2S3 heterojunction photosensitive material spraying method is as follows: after the fluorine tin oxide electrode is continuously cleaned with ultrasound in acetone, ethanol and deionized water, the ZnIn2S4-In2S3 heterojunction photosensitive material is sprayed onto the fluorine tin oxide electrode to obtain a microfluidic fluorine tin oxide electrode.

5. The microfluidic photoelectrochemical immunosensor based on ZnIn2S4-In2S3 heterojunction photosensitive material according to claim 1, characterized in that: The activation step is to push 8 μL of 10 mM / 2 mM chitosan (CTS) and glutaraldehyde (GA) onto the electrode area to activate the surface groups and react for 2 hours; then 10 μL of 1 μg mL -1 PLGF monoclonal antibody was fixed on the above electrode at 4°C overnight. The next day, the electrode was washed with 0.1M PBS, 8μL 1wt% BSA was pumped into the electrode and blocked at nonspecific binding sites at 37°C for 2 hours, and unbound biomolecules were removed with 0.1M PBS.

6. Use of the microfluidic photoelectrochemical immunosensor according to any one of claims 1 to 5 in detecting PLGF concentration.

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