CN115656304A - Immune biosensor for detecting PEDV and preparation method and application thereof - Google Patents

Abstract

The invention relates to an immunobiosensor for detecting PEDV, a preparation method and application thereof, and relates to the field of rapid pathogen detection. The immune biosensor comprises an electrode substrate, a graphene oxide layer, a chitosan layer and a porcine epidemic diarrhea virus monoclonal antibody layer; the graphene oxide layer, the chitosan layer and the porcine epidemic diarrhea virus monoclonal antibody layer are sequentially laminated and cover the electrode substrate. The immunosensor has excellent sensitivity, accuracy and specificity for detecting PEDV.

Description

Immune biosensor for detecting PEDV and preparation method and application thereof

Technical Field

The invention relates to the field of rapid pathogen detection, in particular to an immunobiosensor for detecting PEDV and a preparation method and application thereof.

Background

Porcine Epidemic Diarrheic Virus (PEDV) is the major pathogen of PED, a enveloped RNA Virus belonging to the family coronaviridae, the genus alphacoronavirus. PEDV can infect pigs of many ages, but mainly causes the symptoms of diarrhea, vomit, anorexia, dehydration, weight loss and the like of newborn piglets, and the fatality rate of piglets below 7 days can even reach 100%. Since PEDV was first isolated in 1978 by Pensaert, which is equal to Belgium, and then spread to many countries and regions, causing serious economic losses, the current main prevention and control means for PEDV is vaccination, and due to the lack of corresponding therapeutic drugs, timely detection of PEDV is a prerequisite and basis for prevention and control of the disease. Establishing a rapid, accurate, easy-to-operate, low-cost detection method will help to diagnose and control the prevalence of the disease early in the production practice.

Currently, methods for detecting PEDV mainly include virus isolation and identification, quantitative Real-time PCR (qPCR), enzyme Linked Immunosorbent Assay (ELISA), and the like.

The virus separation and identification is the most accurate and reliable virus detection method, is the 'gold standard' of virus detection, and is also the earliest virus detection method. The method comprises the steps of collecting pathological materials, culturing and separating viruses by using cells or chick embryos, and determining the viruses by performing virulence determination, physicochemical property determination and the like on the viruses. The method has high detection accuracy, but has long detection period, complex operation, high cost and higher requirement on the quality of operators, and is not suitable for large-scale application and popularization in actual production.

The Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR, qPCR) has the characteristics of high sensitivity, short detection time and the like, and the basic principle is that in the in-vitro nucleic acid amplification process, a fluorescent substance capable of being inserted into a nucleic acid sequence is added, the quantity of the fluorescent substance is continuously increased along with continuous amplification, and the quantity of viruses is detected through the strength of a fluorescent signal. The method is one of the most widely used methods at present, compared with Polymerase Chain Reaction (PCR), the method further improves the sensitivity, is efficient and saves time, but detection equipment and reagents are expensive, the method is easy to pollute false positive results, and the requirement on the quality of operators is high.

Enzyme-linked Immunosorbent Assay (ELISA) has the characteristics of simple operation and low requirement on test conditions, and is widely applied to detection of a large number of samples. The basic principle is to fix antigen or antibody on solid phase carrier in advance by utilizing the specific combination of antigen and antibody, and to judge the detection result by observing precipitate with naked eyes or using an enzyme-labeling instrument after the combination of antigen and antibody. Compared with molecular biological diagnosis methods such as qPCR and the like, ELISA has the advantages of lower cost, lower requirements on test conditions and simple operation. But the detection sensitivity is poor, the incubation time is long and the like.

Disclosure of Invention

In view of the above technical problems, the present invention provides an immunobiosensor for detecting PEDV, which has excellent sensitivity, accuracy and specificity for the detection of PEDV.

In order to achieve the above objects, the present invention provides an immunobiosensor for detecting PEDV, comprising an electrode substrate, a graphene oxide layer, a chitosan layer, a porcine epidemic diarrhea virus monoclonal antibody layer; the graphene oxide layer, the chitosan layer and the porcine epidemic diarrhea virus monoclonal antibody layer are sequentially laminated and cover the electrode substrate.

The immune biosensor has the characteristics of low preparation cost, simplicity in operation and the like, can be used only after simple training, and is more suitable for application in actual production. Therefore, the inventors of the present invention have made use of the specific binding characteristics of antigen and antibody to detect PEDV, fix a modified material on a working electrode to improve the detection performance of a sensor, fix an antibody on the working electrode, and detect the PEDV by the change in current detected by the sensor after the binding of antigen and antibody.

Furthermore, the present inventors adopted graphene oxide/chitosan as a modification material of the electrode. Graphene (GR) has attracted much attention in the fields of electrochemical catalysis, electrochemical analysis, and the like because of its excellent physicochemical properties such as large specific surface area, high thermal conductivity, good mechanical properties, and high charge mobility. But the GR has irreversible agglomeration, thereby limiting the application of the GR in various electrochemical fields. Graphene Oxide (GO) is an important derivative of GR, and can also be regarded as a typical functionalized GR, the structure of which is very similar to that of GR, and GO is simple and easy to prepare, has good hydrophilic property and can make up for the defects of GR.

Chitosan (CS) has the characteristics of abundant sources, low price, nontoxicity, good biocompatibility, biodegradability, antibacterial performance and the like. A large number of amino groups contained in the molecular chain of the electrode undergo sol-gel transition through the change of pH, so that the electrode is self-assembled into hydrogel on the surface of a cathode, when a certain voltage or current is applied, electrochemical reaction can occur on the surface of the electrode, and OH is generated at the cathode due to the electrochemical reaction of water ⁻ Or consume H ⁺ The method causes a local high pH gradient on the surface of the cathode, and CS molecules near the cathode in the solution are subjected to sol-gel transformation due to the local high pH generated on the surface of the cathode to form CS gel on the surface of the cathode.

In one embodiment, the electrode substrate is a glassy carbon electrode.

The electrochemical electrodes commonly used in the field mainly include disk electrodes (glassy carbon electrodes, gold electrodes, platinum electrodes), indium tin oxide electrodes (ITO electrodes), screen printing electrodes (screen printing carbon electrodes, screen printing gold electrodes, screen printing nickel electrodes, etc.), interdigitated array microelectrodes, and the like. The Glassy Carbon Electrode (GCE) selected by the inventor has the advantages of good conductivity, high chemical stability, small thermal expansion coefficient, hard texture and good air tightness, can be made into Electrode shapes such as cylinders, discs and the like, and can be used as a substrate to be made into mercury film Glassy Carbon electrodes, chemically modified electrodes and the like. According to the invention, the GCE is used as the working electrode of the sensor, so that the sensitivity of the sensor can be better improved, and the GCE has stable properties and is convenient to store, and compared with other types of working electrodes, the GCE is relatively low in price and wide in application.

In one embodiment, the immunobiosensor further comprises: and the bovine serum albumin sealing layer covers the surface of the monoclonal antibody layer of the porcine epidemic diarrhea virus.

The redundant non-specific active sites on the electrode are closed by using bovine serum albumin solution, so that the immune biosensor has better specificity.

The invention also provides a preparation method of the immunobiosensor for detecting PEDV, which comprises the following steps:

preparing a graphene oxide layer: depositing graphene oxide on the surface of an electrode substrate by an electrochemical reduction method to form a graphene oxide layer;

preparing a chitosan layer: depositing chitosan on the surface of the graphene oxide layer by an electrochemical deposition method to form a chitosan layer;

preparing a monoclonal antibody layer of porcine epidemic diarrhea virus: and dripping the porcine epidemic diarrhea virus monoclonal antibody on the surface of the chitosan layer, and incubating to form a porcine epidemic diarrhea virus monoclonal antibody layer, thereby obtaining the immune biosensor for detecting PEDV.

In the prior art, a method for fixing Graphene Oxide (GO) to an electrode generally adopts a dripping coating method, namely, prepared GO or functional GO, such as Reduced Graphene Oxide (rGO), is dripped on the surface of the electrode, and the electrode is modified after the electrode is dried. Moreover, various oxygen-containing groups rich in a Graphene Oxide (GO) structure can reduce the electron transport capability of a GO modified interface, so that the application of the GO modified interface in an electrochemical sensor is limited.

In the research process, the inventor finds that the number of oxygen-containing groups on the surface of GO can be reduced and controlled by adopting a proper reduction method so as to recover a more perfect plane conjugated structure, thereby improving the conductivity of GO and adjusting the band gap, and further achieving the purpose of regulating and controlling the electrocatalytic performance of the material. Therefore, the graphene oxide is deposited on the surface of the electrode substrate by adopting an electrochemical reduction method, and the method has the advantages of simple and rapid preparation process, green and environment-friendly process, high-efficiency and controllable reduction degree, no introduction of byproducts and impurities and the like. The Electrochemical deposition method has good reproducibility, and a product obtained by deposition on the surface of the electrode is Electrochemical Reduced Graphene Oxide (ERGO), so that the conductivity of the electrode can be stably improved. And the number of layers deposited on the surface of the electrode can be accurately controlled by controlling the deposition time, so that the influence of the modification material on the conductivity of the electrode is controlled. In addition, the deposition method used in the invention is short in time, can finish modification of the electrode in about five minutes, and can convert GO into ERGO and deposit the ERGO onto the electrode without additional operation.

Meanwhile, the Chitosan (CS) has pH stimulation responsiveness, can be deposited through electrochemical reaction, adopts an electrochemical deposition technology to ensure that the deposition process has time and space selectivity and controllability, can realize the deposition of the CS in a specific area and the codeposition with other substances, and can prepare CS hydrogel with different thicknesses and layers through electrodeposition. CS is low in price, non-toxic and good in biocompatibility, can generate a good synergistic effect with ERGO, keeps good conductivity of ERGO on one hand, keeps good biocompatibility of CS on the other hand, and is more beneficial to fixation of an antibody on CS. Similar to electrochemical deposition of GO, electrochemical deposition of CS can form a uniform and controllable thickness film compared to a dispensing method, preventing an excessively thick CS film from affecting conductivity of the sensor, and preventing an excessively thin CS film from adversely affecting adhesion of antibodies.

The two main modified materials of the invention both adopt electrochemical deposition, the final detection capability of the sensor can be simultaneously and accurately controlled, and the ERGO and the CS can play a synergistic role to the maximum extent by determining the optimal deposition thickness ratio of the ERGO and the CS on the electrodes, thereby influencing the final detection capability of the sensor. On the basis of determining the deposition conditions of two key modified materials, the minimum detection limit of the sensor is directly influenced by controlling the fixed amount of the antibody and the optimal proportion of the antibody cross-linking agent.

In one embodiment, the concentration of the porcine epidemic diarrhea virus monoclonal antibody is 200 ng/. Mu.L.

The immobilized antibody is used as a core element for recognizing the antigen of the biosensor, and the amount of the immobilized antibody loaded on the surface of the electrode directly influences the detection capability of the sensor. The sensitivity of the sensor can be improved due to the high antibody loading capacity, but the effective utilization rate of the antibody can be influenced after the antibody on the surface of the electrode is supersaturated, and the preparation cost of the sensor is greatly increased. The amount of antibody which can be loaded on the electrode is related to various factors such as electrode surface modification materials, selected antibody fixing methods, buffer systems during antibody fixing, temperature and the like. After a large number of experiments, the inventor finds that the concentration of 200 ng/. Mu.L has a better detection limit under the condition that EDC/NHS is taken as a cross-linking agent, the antibody is dissolved in PBS and incubated on ice.

In one embodiment, the step of preparing the porcine epidemic diarrhea virus monoclonal antibody layer further comprises: before the monoclonal antibody of the epidemic diarrhea virus is dripped, the electrode substrate is soaked in a mixed solution of N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride for 40-60min.

The chemical coupling by using the mixed solution has the following advantages: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide [1- (3-dimethylamino propyl) -3-ethyl carboxyl diimide, EDC ] belongs to carbodiimide chemical coupling agents, and EDC can activate carboxyl to enable the carboxyl to react with protein, peptide, antibody and small molecules with amino to form stable covalent amido bond. However, the O-acylurea, which is an intermediate formed in the EDC-mediated coupling reaction, is unstable and easily hydrolyzed, and thus it is necessary to react in the presence of N-hydroxysuccinimide (NHS) to form a stable amino-reactive intermediate sulfo-NHS ester, which is then reacted with a primary amine to form a stable amide bond. The chemical coupling reaction does not need to be carried out under the anhydrous condition, the reagent does not need drying treatment, the reaction time is shorter, the efficiency is higher, the operation is easy, complex equipment or instruments are not needed, the activation of the electrode can be completed only by soaking the modified electrode in the mixed solution for a period of time so as to fix the antibody, the cost is low, the effect is exact, and the reproducibility is better.

In one embodiment, the preparation method further comprises: after the step of preparing the porcine epidemic diarrhea virus monoclonal antibody layer, incubating the electrode substrate modified with the porcine epidemic diarrhea virus monoclonal antibody layer with bovine serum albumin to form a bovine serum albumin sealing layer.

In one embodiment, the electrochemical reduction process comprises the steps of: soaking the electrode base material in the graphene oxide dispersion liquid, wherein the pH value is 5.8-6.4, and scanning for 25-35 circles within a potential interval of-1.5V-0.3V at a scanning rate of 80-120mV/S by adopting a cyclic voltammetry method.

Although the electrochemical deposition method has many advantages compared with the conventional drop coating method, the deposition conditions are difficult to determine, and a plurality of deposition condition variables greatly influence the deposition effect in the deposition process. The inventor finds that graphene oxide can be electrochemically reduced into reduced graphene oxide within a pH range of 2.0-10.0 and deposited on an electrode, but the optimum deposition pH value of the graphene oxide may be different in different deposition environments, and finds that a graphene oxide solution gradually aggregates with the increase of the pH value in a test process, so that the deposition effect of the graphene oxide on the electrode is influenced; on the other hand, in a lower pH value environment, hydrogen ions can compete with GO for electrons, so that the deposition efficiency is influenced, and due to the occurrence of a large number of bubbles, the formation of a microstructure on the surface of an electrode can be influenced, and the two jointly act to influence the deposition of GO. Through a large number of experiments, the inventor finds that weak acidity, namely pH 6.4, is a more suitable deposition condition, and when the pH is more than 6.4, the deposition effect begins to decrease, and after the pH exceeds 6.8 or the deposition effect is remarkably reduced due to the agglomeration of GO.

Meanwhile, the inventor also finds that the reduction degree of GO is higher along with the prolonging of the reduction time until most of the oxygen-containing functional groups in the structure are removed, and the conductivity is improved along with the reduction. However, in the reduction process, the number of layers of GO converted into ERGO deposited on the electrode is gradually increased, and the conductivity is reduced, so that the control of the deposition time plays a key role in controlling the modification of the electrode material. After a lot of experiments, the inventor finds that after scanning for 30 cycles, the current of the electrode is obviously improved compared with the electrode scanning for 20 cycles, but the electrode scanning for 40 cycles is not obviously improved compared with the electrode scanning for 30 cycles, and the number of 30 cycles is the deposition time with better effect.

In one embodiment, the electrochemical deposition method comprises the following steps: and soaking the electrode substrate modified with the graphene oxide in a chitosan solution, and depositing for 250-350s by adopting a chronoamperometry at a potential of (-3V) - (-2V).

In one embodiment, in the mixed solution of N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, the molar ratio of N-hydroxysuccinimide to 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 1; the electrode base material is a glassy carbon electrode.

The invention also provides a PEDV detection method, which comprises the following steps: preparing a biological sample into a supernatant, dropwise adding the supernatant into the immune biosensor for detecting PEDV, incubating, detecting a response current value by adopting a differential pulse voltammetry method, and calculating to obtain the antigen concentration of the PEDV in the biological sample.

Compared with the prior art, the invention has the following beneficial effects:

the immune biosensor for detecting PEDV adopts graphene oxide/chitosan as a modification material of an electrode, fixes two modification materials on the surface of the electrode in an electrochemical deposition mode, dropwise coats a porcine epidemic diarrhea virus monoclonal antibody on the surface of the electrode, and seals redundant non-specific active sites on the electrode by using bovine serum albumin solution, so that the finally prepared biosensor has excellent sensitivity, accuracy and specificity. The preparation method adopts a mode of fixing the modified material by electrochemical deposition, is simple and efficient, has low cost, has the characteristics of good repeatability, short modification time and good modification effect compared with other common fixing modes, greatly improves the performance of the sensor, and is consistent with a qPCR result in the detection of clinical pathological materials. The method for detecting the PEDV by adopting the immune biosensor is suitable for clinical detection of the porcine epidemic diarrhea virus in a non-laboratory, and can be used for qualitatively and quantitatively detecting the porcine epidemic diarrhea virus antigen in a biological sample according to the response current value measured in the differential pulse voltammetry.

Drawings

FIG. 1 is a schematic diagram showing the operation of the immunobiosensor in example 1;

FIG. 2 is a CV plot for GO electrochemical reduction deposition in example 1;

FIG. 3 is a CV diagram of each modification stage of the immunobiosensor in example 1;

FIG. 4 is a graph of DPV after incubation with different concentrations of PEDV in example 2;

FIG. 5 is a graph of peak current versus standard antigen concentration after incubation with different concentrations of PEDV in example 2;

FIG. 6 is a graph showing the peak current at different scanning times in example 2;

FIG. 7 is a graph of the peak current of the immunobiosensor after the different antigens were measured in example 2;

FIG. 8 is a CV diagram for different pH values in the experimental examples;

FIG. 9 is a graph of DPV curves for different concentrations of antibody in the experimental examples.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Defining:

the DPV of the invention: referred to as differential pulse voltammetry.

Reagents, materials and equipment used in the embodiment are all commercially available sources unless otherwise specified; unless otherwise specified, all the experimental methods are routine in the art.

Example 1

An immunobiosensor for detecting PEDV was prepared, and the working principle of the immunobiosensor is shown in fig. 1.

1. Al having a particle size of 0.3 μm is used ₂ O ₃ Polishing powder the GCE surface to a mirror surface by using chamois, ultrasonically cleaning by using absolute ethyl alcohol, then rinsing by using ultrapure water, and drying at room temperature. The three-electrode working system adopts a calomel electrode as a reference electrode, a platinum wire electrode as a counter electrode and GCE as a working electrode. Using a catalyst containing 0.1 mol/L ^-1 KCl、5.0 mmol/L ^-1 K ₄ [Fe (CN) ₆ ]、5.0 mmol/L ^-1 K ₃ [Fe (CN) ₆ ]The PBS buffer (PH 7.4) is used as a measurement buffer, and a Cyclic Voltammetry (CV) is adopted to scan in a potential range of-0.6-1.2V at a scanning rate of 100 mV/S, and a CV curve is recorded.

2. Preparing Graphene Oxide (GO) dispersion liquid with the concentration of 1mg/mL by using PBS buffer solution, adjusting the PH to 6.4, taking 10 mL of GO dispersion liquid, soaking an electrode, scanning 30 circles by adopting CV at the scanning speed of 100 mV/S in a potential interval of-1.5V-0.3V for electrochemical reduction (figure 2), obtaining reduced graphene oxide (rGO) and depositing the reduced graphene oxide (rGO) on GCE. As the sweep progresses, the response current gradually rises, indicating that rGO has been deposited onto the GCE. After the scanning, the extra GO dispersion liquid on the surface of the electrode is washed by ultrapure water, the electrode working system, the measurement buffer solution and the CV parameters which are the same as those in the step 1 in the embodiment are selected for measurement, and the CV curve is recorded.

3. A Chitosan (CS) solution having a concentration of 2mg/mL was prepared using a 1% acetic acid solution, and the pH thereof was adjusted to 6.0. Soaking the electrode in 10 mL of CS solution, performing electrochemical deposition by a Chronoamperometry (CA) at a potential of-2.5V for 300s, washing excessive CS solution on the surface of the electrode by using ultrapure water after the deposition is finished, and drying at room temperature. The same electrode working system, measurement buffer and CV parameters as those in step 1 in this example were selected for measurement, and the CV curve was recorded.

4. A mixed solution of 100 mM N-hydroxysuccinimide (NHS) and 400 mM 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC. HCl) was prepared as a crosslinking agent using ultrapure water. The electrodes were immersed in the solution and activated for 50 min for immobilization of the antibody. Then 10. Mu.L of 200. Mu.g/mL PEDV N protein monoclonal antibody was added dropwise to the electrode surface and incubated at 37 ℃ for 50 min. After the incubation, the electrode working system, the measurement buffer solution and the CV parameters which are the same as those in the step 1 in the embodiment are selected for measurement, and the CV curve is recorded.

5. A30 mg/mL Bovine Serum Albumin (BSA) solution was prepared using a PBS solution of pH7.4, and the electrode was immersed in 2 mL BSA solution and incubated at 37 ℃ for 50 min. After the incubation is completed, the electrode is washed by using 0.05% Tween-20 Phosphate Buffer (PBST) to wash off the excess BSA solution on the surface of the electrode, then the electrode working system, the measurement buffer and the CV parameters which are the same as those in step 1 in this embodiment are selected to perform measurement, the CV curve is recorded, and the result is compared with the CV curves obtained in the modification stages from steps 1 to 4, wherein the result is shown in fig. 3, in which Ag in rGO-CS-Ab-BSA-Ag is a CV curve after the immune biosensor (rGO-CS-Ab-BSA) prepared in example 1 is used to detect pathogenic PEDV. After rGO deposition, the response current increased compared to unmodified GCE, indicating that rGO had been deposited on the electrode and could improve the conductivity of the GCE. As the electrode is further modified, there is a gradual decrease in response current, indicating that the modifying material has been immobilized onto the GCE and affected the electrical conductivity of the GCE. A CV curve of the immune biosensor (rGO-CS-Ab-BSA) after detecting the pathogenic PEDV shows obvious current reduction, and the immune biosensor (rGO-CS-Ab-BSA) in the embodiment can be used for detecting the PEDV.

Example 2

The immunobiosensor of example 1 was used in a method for PEDV detection.

1. Sensitivity test: using several immunobiosensors (rGO-CS-Ab-BSA) prepared in example 1, 20. Mu.L PEDV antigen concentration 10 was added drop-wise to the immunobiosensor electrodes ^6.059 、10 ^5.059 、10 ^4.059 、10 ^3.059 、10 ^2.059 、10 ^1.059 copies/. Mu.L of PBS buffer and the same volume of PBS buffer was used as a blank and incubated at 37 ℃ for 50 min. After the incubation was completed, the electrodes were washed with PBST buffer to remove unbound antigens from their surfaces, and then the response current values of the electrodes at different PEDV antigen concentrations after incubation were determined using DPV using the same electrode working system, measurement buffer as in example 1 (fig. 4). At an antigen concentration of 10 ^2.059 A significant drop in response current at copies/mL occurred, so the lowest detection Limit (LOD) of the sensor was 10 ^1.059 copies/mL. The peak current (I,. Mu.A) was plotted against antigen concentration (C, copies/. Mu.L) (FIG. 5), with a peak current of 10 ^1.059 copies/μL ~ 10 ^6.059 The relationship between copies/mu L is linear, the standard equation is I = -31.16 logC + 277.08, and the correlation coefficient (R) ² ) Is 0.9935.

2. And (3) repeatability experiment: the response current of the sensor after binding to PEDV antigen was continuously measured using DPV using the same electrode working system, measurement buffer as in example 1, and the peak currents at 1, 3, 5, 7, 9, 11 th times were recorded and compared (fig. 6). The Relative Standard Deviation (RSD) was calculated to be 0.384%.

3. Specific experiments: PRRSV antigen, CSFV antigen, JEV antigen, and PRV antigen were selected as control groups, the same electrode working system and measurement buffer as in example 1 were used, and the response current value of the sensor after binding of different antigens was measured by DPV, and the peak current thereof was recorded (fig. 7). The results show that there was a significant decrease in response current after incubation of PEDV antigen with the electrode, whereas the decrease in current was not significant in the control.

Example 3

The immunobiosensor of example 1 was applied to the clinical detection of PEDV.

1. Respectively selecting intestinal tissues of different sick pigs, fully grinding, diluting and dissolving by using sterile PBS buffer solution (PH 7.4), repeatedly freezing and thawing for 3 times, centrifuging for 10 min at the rotating speed of 5000 r/min, taking supernate, subpackaging in sterile centrifuge tubes, and storing in a refrigerator at 4 ℃.

2. The antigen concentration of each clinical sample was determined using real-time fluorescent quantitative PCR (qPCR).

3. Several immunobiosensors of example 1 were used, followed by dropping 20. Mu.L of clinical sample supernatant onto the electrodes of the immunobiosensors, respectively, and incubation at 37 ℃ for 50 min. After completion of incubation, the electrodes were washed with PBST buffer, and the response current values of the electrodes after incubation for each clinical sample were determined using DPV using the same electrode working system, measurement buffer as in example 1. The corresponding antigen concentration was calculated from its peak current by a standard equation and compared to the qPCR results, the results are shown in the table below. The result shows that the measurement result of the immune biosensor and the measurement result of qPCR have better consistency.

TABLE 1 comparison of the results of the immunobiosensors with the qPCR

Sample numbering	Tissue source	qPCR assay (copies/. Mu.L)	Immunobiotonsor assay	Consistency
					1	Jejunum	8.45×107	+	Is that
2	Jejunum	7.41×106	+	Is that
					3	Jejunum	9.70×106	+	Is that
4	Ileum	2.16×107	+	Is that
					5	Ileum	4.56×107	+	Is that
6	Ileum	9.78×106	+	Is that

Comparative example 1

An immunobiosensor for detecting PEDV.

The preparation method of the immunobiosensor is substantially the same as that of example 1, except that in step 2 of example 1, a Graphene Oxide (GO) dispersion solution with a concentration of 1mg/mL is prepared using a PBS buffer solution, and then the pH is adjusted to 6.8.

Comparative example 2

An immunobiosensor for detecting PEDV.

The preparation method of the immunobiosensor is substantially the same as that of example 1, except that in step 2 of example 1, a Graphene Oxide (GO) dispersion solution with a concentration of 1mg/mL is prepared using a PBS buffer solution, and then the pH is adjusted to 7.0.

Comparative example 3

An immunobiosensor for detecting PEDV.

The preparation method of the immunobiosensor is substantially the same as that of example 1, except that in step 2 of example 1, a Graphene Oxide (GO) dispersion solution with a concentration of 1mg/mL is prepared using a PBS buffer solution, and then the pH is adjusted to 7.2.

Comparative example 4

An immunobiosensor for detecting PEDV.

The preparation method of the immune biosensor is basically the same as that of example 1, except that in the 4 th step of example 1, the electrode is immersed in the solution to be activated for 50 min for fixing the antibody, 10 μ L of PBS solution is adopted to replace PEDV N protein monoclonal antibody, the PEDV N protein monoclonal antibody is dripped on the surface of the electrode, and the electrode is placed in 37 ℃ to be incubated for 50 min.

Comparative example 5

An immunobiosensor for the detection of PEDV.

The immunobiosensor was prepared in substantially the same manner as in example 1, except that in the 4 th step of example 1, the electrode was immersed in the solution to activate for 50 min for antibody immobilization, and then 10. Mu.L of 50. Mu.g/mL PEDV N protein monoclonal antibody was dropped onto the surface of the electrode, and incubated at 37 ℃ for 50 min.

Comparative example 6

An immunobiosensor for the detection of PEDV.

The immunobiosensor was prepared in substantially the same manner as in example 1, except that in step 4 of example 1, the electrode was immersed in the solution to activate for 50 min for immobilization of the antibody, and then 10. Mu.L of 100. Mu.g/mL PEDV N protein monoclonal antibody was dropped onto the surface of the electrode, and incubated at 37 ℃ for 50 min.

Comparative example 7

An immunobiosensor for detecting PEDV.

The immunobiosensor was prepared in substantially the same manner as in example 1, except that in step 4 of example 1, the electrode was immersed in the solution to activate for 50 min for immobilization of the antibody, and then 10. Mu.L of 150. Mu.g/mL PEDV N protein monoclonal antibody was dropped onto the surface of the electrode, and incubated at 37 ℃ for 50 min.

Comparative example 8

An immunobiosensor for detecting PEDV.

The preparation method of the immunobiosensor is substantially the same as that of example 1, except that in the 4 th step of example 1, the electrode is immersed in the solution for activation for 50 min for antibody immobilization, then 10 μ L of 250 μ g/mL PEDV N protein monoclonal antibody is added dropwise to the surface of the electrode, and the electrode is incubated at 37 ℃ for 50 min.

Examples of the experiments

1. The immunobiosensors prepared in example 1 and comparative examples 1 to 3 were measured using the same electrode working system, measurement buffer, and CV parameters as those in step 1 of this example, and their CV curves were recorded and compared, with the results shown in fig. 8.

2. The immunosensors prepared in example 1 and comparative examples 4 to 8 were measured using the same electrode working system, measurement buffer, and CV parameters as in step 1 of this example, and the response current values were measured using DPV and compared, and the results are shown in fig. 9.

All possible combinations of the technical features of the above embodiments may not be described for the sake of brevity, but should be considered as within the scope of the present disclosure as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (10)

1. An immunobiosensor for detecting PEDV, comprising an electrode substrate, a graphene oxide layer, a chitosan layer, a porcine epidemic diarrhea virus monoclonal antibody layer; the graphene oxide layer, the chitosan layer and the porcine epidemic diarrhea virus monoclonal antibody layer are sequentially laminated and cover the electrode substrate.

2. The immunobiosensor of claim 1, wherein the electrode substrate is a glassy carbon electrode.

3. The immunobiosensor for detecting PEDV according to any one of claims 1-2, wherein the immunobiosensor further comprises: and the bovine serum albumin sealing layer is covered on the surface of the porcine epidemic diarrhea virus monoclonal antibody layer.

4. A method of making an immunobiosensor for detecting PEDV, comprising the steps of:

preparing a graphene oxide layer: depositing graphene oxide on the surface of an electrode substrate by an electrochemical reduction method to form a graphene oxide layer;

preparing a chitosan layer: depositing chitosan on the surface of the graphene oxide layer by an electrochemical deposition method to form a chitosan layer;

preparing a monoclonal antibody layer of the porcine epidemic diarrhea virus: and dripping the porcine epidemic diarrhea virus monoclonal antibody on the surface of the chitosan layer, and incubating to form a porcine epidemic diarrhea virus monoclonal antibody layer, thereby obtaining the immune biosensor for detecting PEDV.

5. The method for preparing a monoclonal antibody layer against porcine epidemic diarrhea virus according to claim 4, wherein the step of preparing the monoclonal antibody layer against porcine epidemic diarrhea virus further comprises: before the monoclonal antibody of the epidemic diarrhea virus is dripped, the electrode substrate is soaked in a mixed solution of N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride for 40-60min.

6. The method of manufacturing according to claim 4, further comprising: after the step of preparing the porcine epidemic diarrhea virus monoclonal antibody layer, the electrode substrate which modifies the porcine epidemic diarrhea virus monoclonal antibody layer is incubated with bovine serum albumin to form a bovine serum albumin sealing layer.

7. The production method according to any one of claims 4 to 6, characterized in that the electrochemical reduction method comprises the steps of: soaking the electrode base material in the graphene oxide dispersion liquid, wherein the pH value is 5.8-6.4, and scanning for 25-35 circles within a potential interval of-1.5V-0.3V at a scanning rate of 80-120mV/S by adopting a cyclic voltammetry method.

8. The method of claim 7, wherein the electrochemical deposition method comprises the steps of: and soaking the electrode substrate modified with the graphene oxide in a chitosan solution, and depositing for 250-350s by adopting a timing current method at a potential of (-3V) - (-2V).

9. The method according to claim 5, wherein in the mixed solution of N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, the molar ratio of N-hydroxysuccinimide to 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 1; the electrode substrate is a glassy carbon electrode.

10. Use of an immunobiosensor according to any of claims 1-3 for the detection of PEDV, comprising the steps of: preparing a biological sample into a supernatant, dropwise adding the supernatant into the immune biosensor for detecting PEDV according to any one of claims 1-3, incubating, detecting the response current value by adopting differential pulse voltammetry, and calculating to obtain the antigen concentration of the PEDV in the biological sample.

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