CN114577883A - Construction method of multichannel-chip type self-powered sensor for high-throughput detection of porcine diarrheal coronavirus - Google Patents
Construction method of multichannel-chip type self-powered sensor for high-throughput detection of porcine diarrheal coronavirus Download PDFInfo
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Abstract
The invention belongs to the technical field of electrochemical biosensing, and discloses a construction method of a multichannel-chip type self-powered sensor for detecting porcine diarrhea coronavirus at high flux. First, ZnIn is prepared2S4Modifying the photo-anode region and Cu2O-CuO modifies the integrated chip electrode in the photocathode area; then, a multichannel-chip self-powered sensor capable of detecting the three porcine diarrhea coronavirus is constructed. The invention adopts laser etching technology, integrates the photo-anode and the photo-cathode on the same chip, and separates the immune recognition elements of various targets according to regions based on a space resolution strategy, so that each target can recognize and reactAnd signal output do not interfere with each other, so that high-throughput specific detection of three viruses, namely PEDV, PDCoV and TGEV, is realized. The chip integrated electrode promotes the miniaturization and integration development of the sensing equipment, and has the advantages of high-flux detection capability, short detection time, small sample consumption and low test cost.
Description
Technical Field
The invention belongs to the technical field of electrochemical biosensing, and relates to a multi-channel-chip type self-powered sensor based on a spatial resolution strategy and a construction method thereof.
Technical Field
The incidence rate of the porcine viral diarrhea is high, the distribution is wide and the harm is large. Coronavirus is the main pathogen causing porcine viral diarrhea, and includes Porcine Epidemic Diarrhea Virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine delta coronavirus (PDCoV), etc. The sick pigs infected by the viruses all have the main clinical symptoms of vomiting, diarrhea, dehydration and intestinal bleeding, and the difficulty of etiological diagnosis is increased. Meanwhile, the sick pigs are often accompanied by recessive infection or multi-pathogen mixed infection, so that the illness state is more serious, the infectivity is stronger, and the fatality rate is higher. The traditional method for detecting the porcine diarrheal coronavirus comprises the following steps: pathogen separation and electron microscope observation, serum neutralization experiment, immunofluorescence technology, loop-mediated isothermal amplification technology and the like. However, these detection methods are limited in practical application due to the problems of complicated operation, long detection time, low selectivity, high cost, difficulty in realizing high-throughput accurate detection, and the like. Therefore, the development of an analysis method which is simple, rapid and low in cost and can realize high-throughput detection of various viruses for diagnosing the porcine viral diarrhea is urgently needed, and technical support is provided for vast farmers.
The electrochemical analysis method has the advantages of simple operation, rapid detection, high sensitivity, low cost and the like. A self-powered sensor based on a photo-assisted fuel cell is used as a novel electrochemical sensing technology, depends on a double-electrode system, and supplies power to a self-sensing process by simultaneously converting light energy and chemical energy into electric energy. The device is simple, the energy utilization rate is high, and the development prospect is wide. However, due to the inherent mechanism of the photo-assisted fuel cell, it is difficult to distinguish the power densities generated by different photoactive materials or identification elements in one detection process, so that the existing self-powered sensor still lacks the capability of multi-target substance quantitative analysis and is difficult to realize high-throughput detection.
The multichannel sensing chip based on the spatial resolution strategy can separate the identification elements of various targets according to regions, and mutual interference does not exist between identification reaction and signal output of the targets, so that the multichannel sensing chip is an effective method for realizing high-flux accurate detection. And the photo-anode and the photo-cathode are integrated on the same electrode chip, so that the miniaturization and portability development of the sensing equipment is remarkably promoted. However, to date, research on multichannel-chip self-powered sensors based on spatial resolution strategies has not been carried out. Therefore, the development of a multichannel-chip self-powered sensor with high sensitivity, good selectivity and quick response for diagnosing the porcine viral diarrhea is very important.
Disclosure of Invention
The invention aims to provide a multi-channel-chip self-powered sensor based on a spatial resolution strategy.
The invention also aims to provide a preparation method of the sensor.
The technical problem to be solved by the invention is to provide a method for detecting Porcine Epidemic Diarrhea Virus (PEDV), porcine transmissible gastroenteritis virus (TGEV) and porcine delta coronavirus (PDCoV) in high throughput.
The purpose of the invention is realized by the following technical scheme:
the multichannel-chip type self-powered sensor based on the spatial resolution strategy is characterized in that an electrode chip is etched to form a photoanode region and a photocathode region by adopting a laser etching technology, and a photoactive material indium sulfide (ZnIn) is modified in the photoanode region2S4) Cathode region modified cuprous oxide-copper oxide composite nano microsphere (Cu)2O-CuO) as an integrated dual-photo electrode. Modification of antibodies (Ab) to PEDV, TGEV and PDCoV in three separate anodic regions based on a spatial resolution strategyPEDV，AbPDCoV，AbTGEV) The specific biological recognition reaction between the antibody and the target is carried out by an immunological method. And then based on the steric hindrance effect generated by the immune complex on the surface of the electrode, the high-flux specific detection of the three porcine diarrhea coronavirus is realized.
The preparation method of the multi-channel chip type self-powered sensor comprises the following steps:
step 1, preparing a photoanode material of zinc indium sulfide (ZnIn)2S4)：
Adding zinc acetate dihydrate (Zn (CH)3COO)2·2H2O) and indium trichloride tetrahydrate (InCl)3·4H2O) is dissolved in ethylene glycol ((CH)2OH)2) Uniformly stirring to obtain a solution A; then slowly adding Thioacetamide (TAA) into the solution A, uniformly stirring to obtain a solution B, transferring the solution B into a stainless steel high-pressure reaction kettle for solvothermal reaction, washing with deionized water and ethanol after the reaction is finished, and drying to obtain a solid product ZnIn2S4。
Mixing copper nitrate trihydrate (Cu (NO)3)2·3H2O) dissolving in deionized water, and uniformly stirring to obtain a solution C; then, ammonium hydroxide (25 wt%) was added dropwise to the solution C to obtain a solution D, and the solution D was transferred to a stainless steel autoclave for hydrothermal reaction. After the reaction is finished, washing the reaction product by deionized water and ethanol, and drying the reaction product to obtain a solid product Cu2O-CuO。
Step 3, preparing an integrated chip electrode:
ZnIn obtained in the step 1 and the step 22S4And Cu2Dispersing O-CuO in N, N-Dimethylformamide (DMF) to respectively obtain ZnIn2S4Dispersion liquid, Cu2O-CuO dispersion. ITO conductive glass with the thickness of 40mm multiplied by 25mm multiplied by 2mm is designed into an electrode chip with four parallel channels through laser etching, wherein three conductive channels are used as a photo-anode area, and one conductive channel is used as a photo-cathode area. ZnIn is mixed with a solvent2S4、Cu2And respectively dripping O-CuO dispersion liquid on a photo-anode region and a photo-cathode region with fixed areas, and drying under an infrared lamp to obtain the integrated chip electrode.
Step 4, constructing a multi-channel-chip type self-powered sensor:
first, in ZnIn2S4Dripping Chitosan (CHIT) solution in an ITO photo-anode area, and drying under an infrared lamp. Then, the Glutaraldehyde (GA) solution is dropped on the surface of the electrode and is placed at room temperature for reaction, after the reaction is finished, PBS (pH 7.4,0.01M) is used for leaching, and the redundant GA on the surface of the electrode is removed. Antibody solution Ab of three viruses was prepared using PBS (pH 7.4,0.01M) as solventPEDV、AbPDCoV、AbTGEVDropping three antibodies into respective biological recognition subareas (area A, area B and area C) in the photo-anode area, leaching with PBS (pH 7.4,0.01M) after reacting for a period of time to remove excessive unadsorbed antibodies, then dropping Bovine Serum Albumin (BSA) solution to block nonspecific active sites, and finally obtaining the photo-anode (AbAbAb) modified by the three antibodiesPEDV/ZnIn2S4/ITO，Ab PDCoV/ZnIn2S4/ITO，AbTGEV/ZnIn2S4ITO), and photocathode Cu2O-CuO/ITO constitutes the multi-channel-chip self-powered sensor.
In step 1, Zn (CH)3COO)2·2H2O、InCl3·4H2O、(CH2OH)2The dosage ratio of the thioacetamide to the thioacetamide is 0.0732-0.2196 g: 0.1953-0.5859 g: 10mL of: 0.1-0.3 g;
the temperature of the solvothermal reaction is 140-180 ℃, and the reaction time is 14-18 h.
In the step 2, the dosage ratio of the copper nitrate trihydrate, the deionized water and the ammonium hydroxide is 0.5825-1.7475 g: 40mL of: 1-3 mL; wherein the mass percentage concentration of the ammonium hydroxide is 25 wt%;
the temperature of the hydrothermal reaction is 90 ℃, and the reaction time is 3 h.
In the step 3, the step of the method is that,
ZnIn2S4dispersion liquid, Cu2The concentration of the O-CuO dispersion liquid is 2 mg/mL; ZnIn2S4、Cu2The dropwise adding amount of the O-CuO dispersion liquid is 20-40 mu L; the fixed area of the chip type ITO conductive glass is 0.09 pi cm2。
In the step 4, the process of the method,
the mass percentage concentration of the CHIT is 0.1 percent, and the dropping amount is 10 mu L;
the volume percentage concentration of the GA is 2.5 percent, and the dropping amount is 20 mu L; the reaction time of CHIT and GA is 1-2 h;
AbPEDV、AbPDCoV、AbTGEVthe antibody concentration is 1-5 mug/mL, 1-5 mug/mL and 1-5 mug/mL respectively, the dropping amount is 20-40 muL, and the reaction time is 10-14 h; the mass percentage concentration of BSA is 3%.
The multi-channel-chip self-powered sensor prepared by the invention is used for high-flux detection of PEDV, PDCoV and TGEV. When high flux detection is carried out, the integrated chip electrode is put into a single-chamber electrolytic cell, and the high flux detection is controlled by an electrode clamp switch, which comprises the following steps:
(1) different concentrations of PEDV (PEDV concentration 10)2～105TCID50/mL) solution drop to AbPEDV/ZnIn2S4On the ITO photo-anode area A; different concentrations of PDCoV (PDCoV concentration is 10)3～106.5 TCID50/mL) solution drop to AbPDCoV/ZnIn2S4On the ITO photo-anode area B; TGEV was added at various concentrations (TGEV concentration 10)2～106TCID50/mL) solution drop to AbTGEV/ZnIn2S4On the ITO photo-anode region C; and the dripping amount of the virus is 10-30 mu L, incubating for a period of time at room temperature,
(2) putting the multi-channel chip type self-powered sensor processed in the step (1) into a single-chamber electrolytic cell containing PBS (0.1M, pH 7.4), vertically irradiating the multi-channel chip by a xenon lamp light source, connecting a photoanode area A and a photocathode by using an electrochemical workstation, and directly collecting an electric signal; making a standard curve (the PBS dosage is 20-30 mL) of the logarithmic value of the maximum output power density and the PEDV concentration;
(3) and (3) collecting the maximum output power density of the PEDV solution with unknown concentration by adopting the method in the step (2), and substituting the maximum output power density into the standard curve to obtain the concentration of the PEDV solution.
(4) Putting the integrated chip electrode processed in the step (1) into a single-chamber electrolytic cell containing a PBS (phosphate buffer solution) (pH 7.4,0.1M), vertically irradiating the multi-channel chip by a xenon lamp light source, connecting a photoanode area B and a photocathode by using an electrochemical workstation, and directly collecting an electric signal; making a standard curve (the PBS dosage is 20-30 mL) on the logarithmic value of the maximum output power density and the PDCoV concentration;
(5) and (5) collecting the maximum output power density of the PDCoV solution with unknown concentration by adopting the method in the step (4), and substituting the maximum output power density into a standard curve to obtain the concentration of the PDCoV solution.
(6) Putting the integrated chip electrode processed in the step (1) into a single-chamber electrolytic cell containing PBS (0.1M, pH 7.4), vertically irradiating the multi-channel chip by a xenon lamp light source, connecting a photoanode area C and a photocathode by an electrochemical workstation, and directly collecting an electric signal; making a standard curve (the PBS dosage is 20-30 mL) of the logarithmic value of the maximum output power density and the TGEV concentration;
(7) and (4) collecting the maximum output power density of the TGEV solution with unknown concentration by adopting the method in the step (6), and substituting the maximum output power density into the standard curve to obtain the concentration of the TGEV solution.
The invention has the beneficial effects that:
ZnIn prepared by the invention2S4Modifying the photo-anode region of the integrated chip, Cu2The O-CuO modified integrated chip photocathode area successfully constructs a multi-channel-chip self-powered sensor to realize high-flux detection of PEDV, PDCoV and TGEV, and the characteristics and advantages are expressed as follows:
(1) the invention prepares ZnIn2S4As photoanode active material, Cu2O-CuO is used as a photocathode active material to construct a multi-channel integrated chip, the Fermi level matching between the photocathode and the photocathode is good, and the detection performance of the sensor is excellent;
(2) the integrated chip prepared by the invention integrates the photo-anode and the photo-cathode on the same electrode chip, replaces the traditional independently connected photo-anode and photo-cathode, effectively reduces the size of equipment and promotes the miniaturization and integration of the sensor;
(3) the multichannel-chip type self-powered sensing device provided by the invention realizes sensitive detection of PEDV, PDCoV and TGEV, and the concentration of PEDV is 102～105TCID50The PDCoV concentration is 10 in the concentration interval of/mL3～106.5TCID50Within the concentration interval of/mL, the TGEV concentration is 102～106TCID50The concentration range of/mL; logarithm of the concentration of the three(lg(c/TCID50mL-1) Maximum output power density (P) with self-powered sensing platformmax) The values showed good linearity, and the detection limits were 33.33TCID respectively50/mL、333.3TCID50mL and 33.33TCID50/mL；
(4) The multi-channel-chip self-powered sensor constructed by the invention does not need an external power supply, and compared with the traditional single-target detection sensor, the multi-channel-chip self-powered sensor can realize high-flux detection of three targets, thereby greatly saving the test time and cost.
Drawings
FIG. 1 is a schematic diagram of a multi-channel-chip self-powered sensor;
FIG. 2 shows ZnIn2S4And Cu2Scanning electron micrographs of O-CuO;
FIG. 3 shows ZnIn in step (3) of example 12S4Voltage-current curve (A) (V-I) diagram of chip electrode composed of photoanode material and different photocathode materials, and (B) power-current curve (P-I) diagram, wherein (a) Pt and (B) Cu2O-CuO；
FIG. 4(A) is a graph of PEDV concentration versus output power from a self-powered sensing platform (linear plot is shown); (B) the PDCoV concentration is plotted as a function of the output power of the self-powered sensing platform (the linear relation is shown in an inset); (C) the TGEV concentration is plotted (linearly) against the output power of the self-powered sensing platform.
Detailed Description
The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.
Fig. 1 is a construction diagram of a constructed multichannel-chip type self-powered sensing device.
Example 1:
(1)ZnIn2S4the preparation of (1):
0.0732g Zn (CH) were measured3COO)2·2H2O with 0.1953g InCl3·4H2O dissolved in 10mL (CH)2OH)2Stirring uniformly to obtain a solution A; weighing 0.2g TAA, adding into the solution A, and stirringUniformly mixing to obtain a solution B, transferring the solution B into a stainless steel high-pressure reaction kettle for solvothermal reaction, reacting for 16 hours at 160 ℃, washing with deionized water and ethanol after the reaction is finished, and drying to obtain a solid product ZnIn2S4。
(2)Cu2Preparation of O-CuO:
first, 1.165g (Cu (NO)3)2·3H2O) is dissolved in 40mL of deionized water and is uniformly stirred to obtain a solution C; then, 2mL of ammonium hydroxide (25 wt%) was added dropwise to solution C to obtain solution D, which was transferred to a stainless steel autoclave for hydrothermal reaction. After the reaction is finished, washing the reaction product by deionized water and ethanol, and drying the reaction product to obtain a solid product Cu2O-CuO。
FIG. 2 shows ZnIn in example 12S4And Cu2Scanning electron microscope picture of O-CuO, can see the ZnIn prepared2S4Is a silver ear-like nanometer flower; for Cu2O-CuO is a nano microsphere with the diameter of about 500nm and is composed of sheets.
(3) Preparing an integrated modified chip electrode:
ITO is pretreated before preparing the photo-anode and the photo-cathode. Placing ITO conductive glass of 40mm × 25mm × 2mm in 1M sodium hydroxide solution, boiling for 30min, then ultrasonically cleaning with acetone, distilled water and ethanol in sequence, and blow-drying with nitrogen for later use. Packaging the laser etched ITO electrode by using polyimide adhesive tape (gold finger) to ensure that the exposed geometric area of each conductive channel of the electrode chip is 0.09 pi cm2. Weighing 2mg ZnIn2S4And Cu2O-CuO is respectively dispersed in 1mL of DMF to obtain ZnIn2S4、Cu2Transferring 20 μ L of ZnIn from O-CuO dispersion2S4、 Cu2And respectively and uniformly dripping O-CuO dispersion liquid on a photo-anode region and a photo-cathode region with fixed areas, and drying under an infrared lamp to obtain the integrated chip electrode.
In an electrolytic cell of Phosphate Buffered Saline (PBS) contained in the integrated chip electrode, a xenon lamp light source vertically irradiates the multi-channel chip electrode, and an electric signal is collected. Wherein the concentration of PBS is 0.1M, and the pH value is 7.4;
FIG. 3 shows a composition of ZnIn2S4Voltage-current curve (A) (V-I) diagram of chip electrode composed of photoanode material and different photocathode materials, and (B) power-current curve (P-I) diagram, wherein (a) Pt and (B) Cu2O-CuO. As can be seen from FIG. 3, ZnIn is formed by the photo-anode2S4ITO and photocathode Cu2The self-powered platform formed by O-CuO/ITO has the best electrical output performance;
step 4, constructing a multi-channel-chip type self-powered sensor:
firstly, in ZnIn2S410 mu L of 0.1% CHIT solution is dripped into the ITO photo-anode area and dried under an infrared lamp. Subsequently, 20. mu.L of 2.5% GA solution was dropped on the electrode surface and allowed to react at room temperature for 1 hour, and after completion of the reaction, the electrode surface was rinsed 2 times with PBS (pH 7.4,0.01M) to remove the excess GA on the electrode surface. Antibody solutions (Ab) of three viruses were prepared using PBS (pH 7.4,0.01M) as solventPEDV、AbPDCoV、AbTGEVRespectively having antibody concentrations of 3. mu.g/mL, 2. mu.g/mL and 3. mu.g/mL), adding dropwise the three antibodies into respective biological recognition partitions (region A, region B and region C) in the photo-anode region, reacting for a period of time, rinsing with PBS (pH 7.4,0.01M) to remove excessive unadsorbed antibodies, adding dropwise Bovine Serum Albumin (BSA) solution to block non-specific active sites, and obtaining photo-anode (Ab) modified by the three antibodiesPEDV/ZnIn2S4/ITO，AbPDCoV/ZnIn2S4/ITO，Ab TGEV/ZnIn2S4ITO), and photocathode Cu2O-CuO/ITO constitutes the multi-channel-chip self-powered sensor.
The multichannel-chip self-powered sensor detects three viruses PEDV, PDCoV and TGEV at high flux:
thereafter, 20. mu.L of 10 concentration was added2，102.5，103，103.5，104，104.5And 105TCID50PEDV/mL was dispensed on the photo-anode area A (Ab)PEDV/ZnIn2S4ITO); 20 μ L of the extract was added to a concentration of 103，103.5，104， 104.5，105，106And 106.5TCID50/mL of PDCoV was applied by dropping on the photo-anode region B (Ab)PDCoV/ZnIn2S4ITO); 20 μ L of 102，103，103.5，104，104.5，105And 106TCID50TGEV/mL was applied by drop coating to the photo-anode region C (Ab)TGEV/ZnIn2S4ITO); and incubated at room temperature for a period of time. Finally, putting the incubated integrated chip electrode into an electrolytic cell containing 20mL of PBS (pH 7.4,0.1M), and performing electrochemical analysis under the condition that a xenon lamp light source (with the intensity of 25-100%) vertically irradiates a multichannel-photoelectric chip through a two-electrode system of an electrochemical workstation;
the detection results are shown in FIG. 4:
FIG. 4(A) is a graph of PEDV concentration versus output power density of a self-powered sensing platform (linear plot shown in inset) at 102～105TCID50Lg (c/TCID) of maximum power density value and PEDV concentration in concentration interval of/mL50mL-1) The linear relation between the two can be well shown, and the detection limit can reach 33.33TCID50Per mL; FIG. 4(B) is a graph of PDCoV concentration versus output power density of a self-powered sensing platform (linear plot shown in inset) at 103～106.5TCID50Lg (c/TCID) of maximum power density value and PDCoV concentration in concentration interval of/mL50mL-1) Has good linear relationship, and the detection limit can reach 333.3TCID50Per mL; FIG. 4C is a graph of TGEV concentration versus output power density of a self-powered sensing platform (linear plot shown in inset) at 102～106TCID50Lg (c/TCID) of maximum power density value and TGEV concentration in concentration interval of/mL50 mL-1) The linear relation between the two can be well shown, and the detection limit can reach 33.33TCID50/mL。
Example 2:
(1)ZnIn2S4the preparation of (1):
0.1464g Zn (CH) were measured3COO)2·2H2O with 0.1953g InCl3·4H2O dissolved in 10mL (CH)2OH)2Stirring uniformly to obtain a solution A; measuring 0.2g of TAA, adding the TAA into the solution A, uniformly stirring to obtain a solution B, transferring the solution B into a stainless steel high-pressure reaction kettle for solvothermal reaction, reacting for 16 hours at 140 ℃, washing with deionized water and ethanol after the reaction is finished, and drying to obtain a solid product ZnIn2S4。
(2)Cu2Preparation of O-CuO:
first, 1.7475g (Cu (NO)3)2·3H2O) is dissolved in 40mL of deionized water and is uniformly stirred to obtain a solution C; then, 2mL of ammonium hydroxide (25 wt%) was added dropwise to solution C to obtain solution D, which was transferred to a stainless steel autoclave for hydrothermal reaction. After the reaction is finished, washing the reaction product by deionized water and ethanol, and drying the reaction product to obtain a solid product Cu2O-CuO。
Steps (3) and (4) were the same as Steps (3) and (4) of example 1.
Example 3:
(1)ZnIn2S4the preparation of (1):
0.0732g Zn (CH) were measured3COO)2·2H2O with 0.3906g InCl3·4H2O dissolved in 10mL (CH)2OH)2Stirring uniformly to obtain a solution A; measuring 0.2g of TAA, adding the TAA into the solution A, uniformly stirring to obtain a solution B, transferring the solution B into a stainless steel high-pressure reaction kettle for solvothermal reaction, reacting at 180 ℃ for 16 hours, washing with deionized water and ethanol after the reaction is finished, and drying to obtain a solid product ZnIn2S4。
(2)Cu2Preparation of O-CuO:
first, 0.5825g (Cu (NO)3)2·3H2O) is dissolved in 40mL of deionized water and is uniformly stirred to obtain a solution C; then, 2mL of ammonium hydroxide (25 wt%) was added dropwise to solution C to obtain solution D, which was transferred to a stainless steel autoclave for hydrothermal reaction. After the reaction is finished, washing the reaction product by deionized water and ethanol, and drying the reaction productObtaining a solid product Cu2O-CuO。
Steps (3) and (4) were the same as Steps (3) and (4) of example 1.
Claims (10)
1. A construction method of a multichannel-chip type self-powered sensor for detecting porcine diarrheal coronavirus at high flux is characterized by comprising the following steps:
step 1, preparing a photoanode material ZnIn2S4：
Dissolving zinc acetate dihydrate and indium trichloride tetrahydrate in ethylene glycol to obtain a solution A;
slowly adding thioacetamide into the solution A to obtain a solution B;
transferring the solution B into a stainless steel high-pressure reaction kettle for solvothermal reaction, washing with deionized water and ethanol after the reaction is finished, and drying to obtain a solid product ZnIn2S4；
Step 2, preparing a photocathode material cuprous oxide-copper oxide composite nano microsphere Cu2O-CuO：
First, copper nitrate trihydrate Cu (NO)3)2·3H2Dissolving O in deionized water, and uniformly stirring to obtain a solution C;
then, dropwise adding ammonium hydroxide into the solution C to obtain a solution D;
and finally, transferring the solution D into a stainless steel high-pressure reaction kettle for hydrothermal reaction, washing with deionized water and ethanol after the reaction is finished, and drying to obtain a solid product Cu2O-CuO；
Step 3, preparing an integrated chip electrode:
ZnIn obtained in the step 1 and the step 22S4And Cu2O-CuO is dispersed in N, N-dimethylformamide DMF to respectively obtain ZnIn2S4Dispersion liquid, Cu2The O-CuO dispersion liquid is prepared by laser etching ITO conductive glass to obtain four parallel-channel electrode chips, wherein three conductive channels are used as photo-anode regions, one conductive channel is used as a photo-cathode region, and ZnIn is used as a cathode region2S4、Cu2The O-CuO dispersion is respectively coated on the surface of the substratePlacing the photo-anode region and the photo-cathode region with fixed areas under an infrared lamp for drying to obtain an integrated chip electrode;
step 4, constructing a multi-channel-chip type self-powered sensor:
firstly, in ZnIn2S4Dripping chitosan CHIT solution in an ITO photo-anode area, and drying under an infrared lamp;
dripping a glutaraldehyde GA solution on the surface of the electrode, placing the electrode at room temperature for reaction, leaching the electrode with a PBS solution after the reaction is finished, and removing redundant GA on the surface of the electrode;
preparing antibody solutions of three viruses by using PBS (phosphate buffer solution) as a solvent, respectively dripping the three antibodies into respective biological recognition subareas, namely a region A, a region B and a region C, reacting for a period of time, leaching by using the PBS solution to remove excessive unadsorbed antibodies, respectively dripping Bovine Serum Albumin (BSA) solution to seal non-specific active sites, and finally obtaining the photoanode Ab modified by the three antibodiesPEDV/ZnIn2S4/ITO，Ab PDCoV/ZnIn2S4/ITO，Ab TGEV/ZnIn2S4ITO, and Cu2The O-CuO/ITO photocathode area forms a multi-channel-chip type self-powered sensor.
2. The method of construction according to claim 1, wherein in step 1: zn (CH)3COO)2·2H2O、InCl3·4H2O、(CH2OH)2The dosage ratio of the thioacetamide to the thioacetamide is 0.0732-0.2196 g: 0.1953-0.5859 g: 10mL of: 0.1-0.3 g; the temperature of the solvothermal reaction is 140-180 ℃, and the reaction time is 14-18 h.
3. The method of construction according to claim 1, wherein in step 2: the using amount ratio of the copper nitrate trihydrate, the deionized water and the ammonium hydroxide is 0.5825-1.7475 g: 40mL of: 1-3 mL; wherein the mass percentage concentration of the ammonium hydroxide is 25 wt%; the temperature of the hydrothermal reaction is 90 ℃, and the reaction time is 3 h.
4. The method of claim 1, wherein in step 3:
ZnIn2S4dispersion liquid, Cu2The concentration of the O-CuO dispersion liquid is 2 mg/mL; ZnIn2S4、Cu2The dropwise adding amount of the O-CuO dispersion liquid is 20-40 mu L, the size of the ITO conductive glass is 40mm multiplied by 25mm multiplied by 2mm, and the fixed area of the chip type ITO conductive glass is 0.09 pi cm2。
5. The method of construction according to claim 1, wherein in step 4:
the mass percentage concentration of the CHIT is 0.1 percent, and the dropping amount is 10 mu L;
the volume percentage concentration of the GA is 2.5 percent, and the dropping amount is 20 mu L; the reaction time of CHIT and GA is 1-2 h.
6. The method of claim 1, wherein, in step 4,
Ab PEDV、Ab PDCoV、Ab TGEVthe antibody concentration is 1-5 mug/mL, 1-5 mug/mL and 1-5 mug/mL respectively, the dropping amount is 20-40 muL, and the reaction time is 10-14 h; the mass percentage concentration of BSA is 3%;
the PBS solution had a pH of 7.4 and a concentration of 0.01M.
7. The application of the multichannel-chip self-powered sensor for detecting porcine diarrheal coronavirus at high flux, which is constructed by the construction method of any one of claims 1-6, in detecting PEDV, PDCoV and TGEV.
8. The use according to claim 7, characterized by the specific steps of:
(1) different concentrations of PEDV solution were dropped to the AbPEDV/ZnIn2S4On the ITO photo-anode area A; dropping different concentrations of PDCoV solution to AbPDCoV/ZnIn2S4On the ITO photo-anode area B; dropping TGEV solutions of different concentrations to AbTGEV/ZnIn2S4On the ITO photo-anode region C; incubating at room temperature for a period of time;
(2) putting the multi-channel chip type self-powered sensor processed in the step (1) into a single-chamber electrolytic cell containing a PBS solution, vertically irradiating the multi-channel chip by a xenon lamp light source, connecting a photoanode area A and a photocathode by using an electrochemical workstation, and directly collecting an electric signal; making a standard curve of the logarithm value of the maximum output power density and the PEDV concentration;
(3) collecting the maximum output power density of the PEDV solution with unknown concentration by adopting the method in the step (2), and substituting the maximum output power density into a standard curve to obtain the concentration of the PEDV solution;
(4) putting the integrated chip electrode processed in the step (1) into a single-chamber electrolytic cell containing a PBS solution, vertically irradiating a multi-channel chip by a xenon lamp light source, connecting a photo-anode region B and a photo-cathode by using an electrochemical workstation, and directly collecting an electric signal; making a standard curve of the logarithm value of the maximum output power density and the PDCoV concentration;
(5) and (4) collecting the maximum output power density of the PDCoV solution with unknown concentration by adopting the method in the step (4), and substituting the maximum output power density into a standard curve to obtain the concentration of the PDCoV solution.
(6) For TGEV detection, different concentrations of TGEV solutions were dropped into AbTGEV/ZnIn2S4The ITO photo-anode area C is incubated for a period of time at room temperature;
(7) putting the integrated chip electrode processed in the step (1) into a single-chamber electrolytic cell containing PBS (phosphate buffer solution), vertically irradiating the multi-channel chip by a xenon lamp light source, connecting a photo-anode region C and a photo-cathode by using an electrochemical workstation, and directly collecting an electric signal; making a standard curve of the logarithmic value of the maximum output power density and the TGEV concentration;
(8) and (4) collecting the maximum output power density of the TGEV solution with unknown concentration by adopting the method in the step (7), and substituting the maximum output power density into the standard curve to obtain the concentration of the TGEV solution.
9. The use according to claim 8,
in the step (1), the concentration of PEDV is 102～105TCID50Per mL, the dropping amount is 10-30 mu L;
in the step (4), the PDCoV concentration is 103～106.5TCID50Per mL, the dropping amount is 10-30 mu L;
in the step (7), the TGEV concentration is 102～106TCID50The amount of the dripping agent is 10-30 mu L/mL.
10. The use of claim 8, wherein in steps (2), (4) and (7), the PBS solution has a pH of 7.4 and a concentration of 0.1M.
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