Electrochemical immunosensor for Clostridium perfringens alpha toxin and its preparation and application

Abstract

The present invention provides a method for preparing an electrochemical immunosensor for alpha toxin of Clostridium perfringens, an electrochemical immunosensor for alpha toxin of Clostridium perfringens prepared by the method, and a method for applying the electrochemical immunosensor for alpha toxin of Clostridium perfringens to detect alpha toxin of Clostridium perfringens. The preparation method comprises: using chloroauric acid solution to deposit gold nanoparticles on the surface of a substrate electrode by a multi-potential step electrodeposition method to modify the surface of the substrate electrode to obtain a gold modified electrode; using an activation solution containing N-hydroxysuccinimide to activate the gold modified electrode to obtain an activated electrode; coating the working electrode surface of the activated electrode with an antibody for alpha toxin of Clostridium perfringens to obtain a coated electrode; and sealing the coated electrode to obtain a detection electrode of the electrochemical immunosensor. The present invention can realize rapid, specific and sensitive detection of alpha toxin of Clostridium perfringens.

Description

Technical Field

[0001] The present invention relates to the field of biological toxin detection and electrochemical immunotechnology, and in particular to a method for preparing a Clostridium perfringens alpha toxin electrochemical immunosensor, a Clostridium perfringens alpha toxin electrochemical immunosensor prepared by the method, and a method for using the Clostridium perfringens alpha toxin electrochemical immunosensor for Clostridium perfringens alpha toxin detection. Background Art

[0002] Clostridium perfringens is the most common clostridium in clinical gas gangrene pathogens. It can decompose sugars in muscles and connective tissues, produce a large amount of gas, cause severe tissue emphysema, and then affect blood supply, causing large-area tissue necrosis. In addition, this bacterium can form a capsule in the body, so it is named Clostridium perfringens. Clostridium perfringens can infect human and animal wounds and cause gas gangrene, with a mortality rate of up to 30%. The bacterium can produce four major lethal exotoxins: α, β, ε and ι. According to the different types of exotoxins produced, Clostridium perfringens can be divided into 5 toxin types: A, B, C, D and E.

[0003] Clostridium perfringens α-toxin (CPA) is one of the most important toxins of Clostridium perfringens and the most important virulence factor of type A Clostridium perfringens. The essence of α-toxin is an enzyme, which has the characteristics of cytotoxicity, hemolytic activity, lethality, skin necrosis, platelet aggregation and increased vascular permeability, so its detection and analysis technology is crucial. However, the current traditional detection methods for CPA, such as high-performance liquid chromatography (HPLC) and gas chromatography (GC), are not satisfactory in terms of sensitivity and accuracy. Therefore, it is urgent to develop a sensitive and accurate CPA detection and analysis technology. Summary of the invention

[0004] In view of the above problems, the present invention proposes a method for preparing an electrochemical immunosensor for Clostridium perfringens alpha toxin that overcomes the above problems or at least partially solves the above problems, an electrochemical immunosensor for Clostridium perfringens alpha toxin prepared by the method, and a method for using the Clostridium perfringens alpha toxin electrochemical immunosensor for detecting Clostridium perfringens alpha toxin.

[0005] One object of the present invention is to achieve rapid, specific and sensitive detection of Clostridium perfringens alpha toxin.

[0006] Another object of the present invention is to simplify the operation of the step of modifying the surface of the substrate electrode with gold nanoparticles in the preparation process of the electrochemical immunosensor for Clostridium perfringens alpha toxin and to shorten the time of the step.

[0007] Another object of the present invention is to immobilize more target antibodies on the electrode surface.

[0008] According to one aspect of the present invention, the present invention provides a method for preparing an electrochemical immunosensor for Clostridium perfringens alpha toxin, comprising:

[0009] Step S102: using chloroauric acid solution to deposit gold nanoparticles on the surface of the substrate electrode by a multi-potential step electrodeposition method to modify the surface of the substrate electrode to obtain a gold-modified electrode;

[0010] Step S104: activating the gold modified electrode using an activation solution containing N-hydroxysuccinimide to obtain an activated electrode;

[0011] Step S106: coating the surface of the working electrode of the activated electrode with Clostridium perfringens alpha toxin antibody to obtain a coated electrode; and

[0012] Step S108: performing sealing treatment on the coated electrode to obtain a detection electrode of the electrochemical immunosensor.

[0013] Optionally, step S102 specifically includes:

[0014] Dissolving gold (III) chloride hydrate having a gold content of ≥48% by mass in pure water to prepare a chloroauric acid solution having a molar concentration of 0.5-2.5%;

[0015] The base electrode is placed in a chloroauric acid solution, and multi-potential step electrodeposition is performed for 10-50 seconds at an electrodeposition voltage of 1-10 V to obtain a gold-modified electrode.

[0016] Optionally, the substrate electrode is a screen-printed carbon electrode based on PET;

[0017] The molar concentration of chloroauric acid solution is 1%;

[0018] The electrodeposition voltage was 4 V;

[0019] The multi-potential step electrodeposition was carried out for 20 s.

[0020] Optionally, step S104 specifically includes:

[0021] Dissolve morpholineethanesulfonic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in pure water at a molar ratio of (3-5):(3-5):1 to prepare an activation solution;

[0022] The gold modified electrode is placed in an activation solution, and the electrode is activated at 23-31° C. for 40-80 min to obtain an activated electrode;

[0023] The molar content of morpholineethanesulfonic acid in the activation solution is 0.05-0.15 mmol / mL, the molar content of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 0.05-0.15 mmol / mL, and the molar content of N-hydroxysuccinimide is 0.01-0.05 mmol / mL;

[0024] The temperature for electrode activation was 29°C, and the time for electrode activation was 70 min.

[0025] Optionally, step S106 specifically includes:

[0026] Coating the activated electrode with 0.5-3 μL of 0.1-15 mg / mL Clostridium perfringens alpha toxin antibody solution;

[0027] The activated electrode coated with the antibody was placed in a humidified box at 2-6°C overnight to obtain a coated electrode.

[0028] Optionally, the concentration of the Clostridium perfringens alpha toxin antibody solution is 5 mg / mL, and the dosage is 1 μL.

[0029] Optionally, step S108 specifically includes:

[0030] The coated electrode was blocked with a 0.5-5% bovine serum albumin solution at room temperature for 30-120 min.

[0031] According to another aspect of the present invention, the present invention further provides an electrochemical immunosensor for Clostridium perfringens alpha toxin, wherein the electrochemical immunosensor is prepared by any of the aforementioned preparation methods.

[0032] According to another aspect of the present invention, the present invention also provides a method for applying the aforementioned Clostridium perfringens alpha toxin electrochemical immunosensor to detect Clostridium perfringens alpha toxin, comprising:

[0033] The assembled electrochemical immunosensor is placed in a sample solution to be tested to carry out an immune reaction at room temperature;

[0034] placing the detected electrode after the reaction in a potassium ferrocyanide solution to perform a cyclic voltammetry test to obtain an electrochemical response signal of the sample; and

[0035] The content of Clostridium perfringens alpha toxin in the sample solution to be tested is determined according to the electrochemical response signal of the sample and the standard working curve of the electrochemical immunosensor obtained in advance.

[0036] Optionally, the duration of the immune response is 20-50 min;

[0037] The potassium ferrocyanide solution is a solution prepared with a 10×PBS solution and contains 0.01-0.02 mmol / mL potassium ferrocyanide and 0.01-0.02 mmol / mL potassium ferrocyanide.

[0038] Optionally, the electrochemical response signal is a current signal, and the standard working curve is obtained by:

[0039] A plurality of Clostridium perfringens alpha toxin standard solutions with different concentrations ranging from 0.001 ng / mL to 1000 ng / mL were prepared with 1×PBS solution;

[0040] Place the detection electrodes in each standard solution and incubate at room temperature for 30-60 minutes;

[0041] The detection electrode before and after incubation was tested using cyclic voltammetry in potassium ferrocyanide solution;

[0042] Collect the difference Δi between the oxidation peak current of the detection electrode before and after incubation corresponding to each standard solution p The logarithm of the concentration of α-toxin of Clostridium perfringens in each standard solution is used as the horizontal axis, Δi p / i p,0 As the ordinate, the standard working curve of the electrochemical immunosensor is drawn, where i p,0 is the oxidation peak current of the detection electrode before incubation.

[0043] In the preparation method of the electrochemical immunosensor for alpha toxin of Clostridium perfringens provided by the present invention, the detection electrode of the electrochemical immunosensor for alpha toxin of Clostridium perfringens is prepared by the steps of electrode modification by multi-potential step (STEP) electrodeposition method, electrode activation based on N-hydroxysuccinimide, antibody coating and sealing treatment, so as to complete the construction of the electrochemical immunosensor for alpha toxin of Clostridium perfringens. The prepared electrochemical immunosensor for alpha toxin of Clostridium perfringens combines the high specific reaction of antigen-antibody with the high sensitivity of electrochemical signal, and is applied to the detection of alpha toxin of Clostridium perfringens, so as to achieve the purpose of rapid, specific and sensitive detection of alpha toxin of Clostridium perfringens.

[0044] Furthermore, the preparation method of the electrochemical immunosensor for Clostridium perfringens alpha toxin of the present invention simplifies the operation of the step of modifying the surface of the base electrode with gold nanoparticles in the preparation process of the electrochemical immunosensor for Clostridium perfringens alpha toxin and shortens the time of this step by optimizing the electrodeposition voltage and time of the multi-potential step electrodeposition method, thereby improving production efficiency.

[0045] Furthermore, the preparation method of the electrochemical immunosensor for Clostridium perfringens alpha toxin of the present invention activates the carboxyl groups on the electrode surface through a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS) activation system, and then fixes the target antibody by coupling the activated carboxyl groups with the amino groups on the target antibody, thereby fixing more target antibodies on the electrode surface, and preventing the target antibodies on the electrode surface from falling off due to subsequent cleaning or blocking, thereby improving the detection effect of the prepared Clostridium perfringens alpha toxin electrochemical immunosensor.

[0046] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented according to the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are listed below.

[0047] Based on the following detailed description of specific embodiments of the present invention in conjunction with the accompanying drawings, those skilled in the art will become more aware of the above and other objects, advantages and features of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0049] Figure 1 1 is a schematic flow chart of a method for preparing an electrochemical immunosensor for Clostridium perfringens alpha toxin according to an embodiment of the present invention;

[0050] Figure 2 is a schematic diagram of the assembly and use process of the Clostridium perfringens alpha toxin electrochemical immunosensor according to an embodiment of the present invention;

[0051] Figure 3 is a curve diagram of optimization results of electrodeposition voltage of a multi-potential step electrodeposition method according to an embodiment of the present invention, wherein the electrodeposition time is fixed at 10 s;

[0052] Figure 4 is a curve diagram of optimization results of electrodeposition time of a multi-potential step electrodeposition method according to an embodiment of the present invention, wherein the electrodeposition voltage is fixed at 4V;

[0053] Figure 5 is a curve diagram of optimization results of activation temperature in an electrode activation step according to an embodiment of the present invention, wherein the activation time is fixed at 60 min;

[0054] Figure 6 is a curve diagram of optimization results of activation time in an electrode activation step according to an embodiment of the present invention, wherein the activation temperature is fixed at 29° C.;

[0055] Figure 7 is a curve diagram of optimization results of antibody coating concentration in the antibody coating step according to one embodiment of the present invention;

[0056] Figure 8 1 is a flow chart of a method for applying a Clostridium perfringens alpha toxin electrochemical immunosensor to detect Clostridium perfringens alpha toxin according to an embodiment of the present invention;

[0057] Fig. 9 is a curve chart of the time optimization result of the immune response according to one embodiment of the present invention;

[0058] Fig.10 : is a schematic diagram of a standard working curve of an electrochemical immunosensor for Clostridium perfringens alpha toxin according to an embodiment of the present invention, wherein the standard working curve is a change in oxidation peak current (Δi p / i p,0 ) percentage and the logarithmic value of CPA concentration. DETAILED DESCRIPTION

[0059] The exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0060] Clostridium perfringens alpha toxin (CPA) is one of the most important toxins of Clostridium perfringens and the main virulence factor of Clostridium perfringens type A. It is crucial to develop sensitive and accurate CPA detection and analysis technology.

[0061] Immunosensor is a biosensor that uses the affinity of antigens and antibodies to combine a series of chemical and physical changes produced during specific immune reactions with appropriate signal conversion technology to detect antigens and antibodies to achieve biomolecular recognition. As an important branch of immunoassay technology, immunosensors show good application prospects due to their specificity and sensitivity, and are widely used in clinical, bioelectrochemical and environmental analysis. Electrochemical immunosensors are biosensors that use electrochemical technology (such as current, voltage, resistance, etc.) and immunological technology (such as antibodies, antigens, immune reactions) to detect targets. Compared with traditional detection methods, such as high performance liquid chromatography (HPLC) and gas chromatography (GC), electrochemical immunosensors have the advantages of simple operation, rapid detection, high sensitivity, low cost and real-time monitoring, so they are increasingly used in biological toxin detection.

[0062] In view of the shortcomings of the existing CPA detection methods and in combination with the characteristics of electrochemical immunosensors, the present invention proposes an electrochemical immunosensor technology for detecting Clostridium perfringens alpha toxin.

[0063] In particular, the embodiments of the present invention provide a method for preparing an electrochemical immunosensor for Clostridium perfringens alpha toxin.

[0064] Figure 1 FIG. 1 is a flow chart of a method for preparing an electrochemical immunosensor for Clostridium perfringens alpha toxin according to an embodiment of the present invention. Figure 1 As shown, the method for preparing the Clostridium perfringens alpha toxin electrochemical immunosensor may at least include the following steps S102 to S108:

[0065] Step S102: using chloroauric acid solution to deposit gold nanoparticles on the surface of the substrate electrode by a multi-potential step electrodeposition method to modify the surface of the substrate electrode to obtain a gold-modified electrode;

[0066] Step S104: activating the gold modified electrode using an activation solution containing N-hydroxysuccinimide to obtain an activated electrode;

[0067] Step S106: coating the surface of the working electrode of the activated electrode with Clostridium perfringens alpha toxin antibody to obtain a coated electrode; and

[0068] Step S108: performing sealing treatment on the coated electrode to obtain a detection electrode of the electrochemical immunosensor, thereby completing the construction of the Clostridium perfringens alpha toxin electrochemical immunosensor.

[0069] In the preparation method of the electrochemical immunosensor for alpha toxin of Clostridium perfringens provided in the embodiment of the present invention, the detection electrode of the electrochemical immunosensor for alpha toxin of Clostridium perfringens is prepared by the steps of electrode modification by multi-potential step electrodeposition method (referred to as STEP electrodeposition), electrode activation based on N-hydroxysuccinimide, antibody coating and sealing treatment, so as to complete the construction of the electrochemical immunosensor for alpha toxin of Clostridium perfringens. The prepared electrochemical immunosensor for alpha toxin of Clostridium perfringens combines the high specific reaction of antigen-antibody with the high sensitivity of electrochemical signal, and is applied to the detection of alpha toxin of Clostridium perfringens, so as to achieve the purpose of rapid, specific and sensitive detection of alpha toxin of Clostridium perfringens.

[0070] Figure 2 Schematic diagram of the assembly and use process of the electrochemical immunosensor for Clostridium perfringens alpha toxin according to an embodiment of the present invention. Figure 1 and Figure 2 Each step of the method for preparing the Clostridium perfringens alpha toxin electrochemical immunosensor of the present invention is described in detail.

[0071] In some embodiments, step S102 (electrode modification step) may specifically include:

[0072] Dissolving gold (III) chloride hydrate having a gold content of ≥48% by mass in pure water to prepare a chloroauric acid solution having a molar concentration of 0.5-2.5%;

[0073] The substrate electrode is placed in the chloroauric acid solution, and multi-potential step electrodeposition is performed at an electrodeposition voltage of 1-10V for 10-50s to deposit gold nanoparticles (CuNPs for short) on the surface of the substrate electrode to modify the electrode surface, thereby obtaining a gold-modified electrode.

[0074] Optionally, the gold modified electrode can be washed with ultrapure water and then air-dried for subsequent electrode activation.

[0075] In some specific embodiments, the molar concentration of the chloroauric acid solution is 1%.

[0076] In some specific embodiments, Figure 2 As shown, the substrate electrode is a screen-printed carbon electrode (SPCE) based on PET (Polyethylene Terephthalate). Thus, the corresponding gold-modified electrode can be expressed as a SPCE / Au electrode.

[0077] In the embodiment of the present invention, the STEP electrodeposition voltage and time in the electrode modification step are also optimized.

[0078] Figure 3 : is a graph showing the optimization result of the electrodeposition voltage of the multi-potential step electrodeposition method according to an embodiment of the present invention, wherein the electrodeposition voltage (in V) is used as the abscissa, and the difference Δi between the oxidation peak current before and after the electrode incubation is detected. p (unit: A) is the vertical axis, and the electrodeposition time is fixed at 10 s.

[0079] Reference Figure 3 As shown, preferably, the STEP electrodeposition voltage is a value in the range of 2-9 V. More preferably, the STEP electrodeposition voltage is a value in the range of 3-8 V. Still more preferably, the STEP electrodeposition voltage is a value in the range of 4-7 V. Most preferably, the STEP electrodeposition voltage is 4 V.

[0080] Figure 4 : is a graph showing the optimization result of the electrodeposition time of the multi-potential step electrodeposition method according to an embodiment of the present invention, wherein the electrodeposition time (in seconds) is used as the abscissa, and the difference Δi between the oxidation peak current before and after the electrode incubation is detected. p (unit: A) is the vertical axis, and the electrodeposition voltage is fixed at 4V.

[0081] Reference Figure 4 As shown, preferably, the STEP electrodeposition time is a value in the range of 10-40 s. More preferably, the STEP electrodeposition time is a value in the range of 10-20 s. Most preferably, the STEP electrodeposition time is 20 s.

[0082] In the embodiment of the present invention, by optimizing the electrodeposition voltage and time of the multi-potential step electrodeposition method, the operation of the step of modifying the surface of the substrate electrode with gold nanoparticles in the preparation process of the electrochemical immunosensor for Clostridium perfringens alpha toxin is simplified and the time of this step is shortened, thereby improving the production efficiency.

[0083] In some embodiments, step S104 (electrode activation step) may specifically include:

[0084] Morpholineethanesulfonic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in a molar ratio of (3-5):(3-5):1 are dissolved in pure water to prepare an activation solution (expressed as EDC / NHS solution); a gold modified electrode is placed in the activation solution and the electrode is activated at 23-31°C for 40-80 minutes to activate the carboxyl groups on the electrode surface, thereby obtaining an activated electrode. In the case where the base electrode is a SPCE electrode, the activated electrode is expressed as a SPCE / Au / NHS electrode.

[0085] Optionally, the activated electrode is washed with ultrapure water and then air-dried to prepare for subsequent antibody coating.

[0086] In some specific embodiments, the molar content of morpholineethanesulfonic acid in the activation solution is 0.05-0.15 mmol / mL, the molar content of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 0.05-0.15 mmol / mL, and the molar content of N-hydroxysuccinimide is 0.01-0.05 mmol / mL.

[0087] In the embodiment of the present invention, the activation temperature and activation time in the electrode activation step are also optimized.

[0088] Figure 5 : is a curve diagram of the optimization result of the activation temperature in the electrode activation step according to one embodiment of the present invention, wherein the activation temperature (in ° C) is used as the horizontal axis, and the difference Δi between the oxidation peak current before and after the electrode incubation is detected. p (unit: A) is the vertical axis, and the activation time is fixed at 60 min.

[0089] Reference Figure 5 As shown, preferably, the activation temperature is in the range of 25-29°C. More preferably, the activation temperature is in the range of 27-29°C. Most preferably, the activation temperature is 29°C.

[0090] Figure 6 is a curve diagram of the optimization result of the activation time in the electrode activation step according to one embodiment of the present invention, wherein the activation time (in min) is used as the horizontal axis, and the difference Δi between the oxidation peak current before and after the electrode incubation is detected. p (unit is A) is the vertical axis, and the activation temperature is fixed at 29°C.

[0091] Reference Figure 6 As shown, preferably, the activation time is in the range of 50-70 min. More preferably, the activation time is in the range of 60-70 min. Most preferably, the activation time is 70 min.

[0092] In some embodiments, step S106 (antibody coating step) may specifically include:

[0093] The activated electrode is coated with 0.5-3 μL of 0.1-15 mg / mL Clostridium perfringens alpha toxin antibody (abbreviated as anti-CPA) solution; the antibody-coated activated electrode is placed in a wet box at 2-6°C overnight to obtain a coated electrode. In the case where the substrate electrode is a SPCE electrode, the coated electrode is represented as a SPCE / Au / NHS / anti-CPA electrode.

[0094] Optionally, the coated electrode is washed with ultrapure water and then air-dried to prepare for subsequent sealing treatment.

[0095] In some specific embodiments, the amount of anti-CPA solution used is 1 μL. The activated electrode coated with the antibody is placed in a wet box at 4° C. overnight.

[0096] In the embodiment of the present invention, the concentration of the anti-CPA solution in the antibody coating step is also optimized.

[0097] Figure 7 : is a curve diagram of the optimization result of the antibody coating concentration (i.e., the concentration of the anti-CPA solution) in the antibody coating step according to one embodiment of the present invention, wherein the antibody coating concentration (in mg / mL) is used as the abscissa, and the difference Δi between the oxidation peak current before and after the electrode incubation is detected. p (unit is A) is the vertical coordinate.

[0098] Reference Figure 7 As shown, preferably, the concentration of the anti-CPA solution is a value within the range of 1-10 mg / mL, such as 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL. Most preferably, the concentration of the anti-CPA solution is 5 mg / mL.

[0099] In an embodiment of the present invention, the carboxyl groups on the electrode surface are activated by a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS) activation system, and then the target antibody is fixed by coupling the activated carboxyl groups with the amino groups on the target antibody, thereby fixing more target antibodies on the electrode surface, and the target antibodies on the electrode surface will not fall off due to subsequent cleaning or blocking, thereby improving the detection effect of the prepared Clostridium perfringens alpha toxin electrochemical immunosensor.

[0100] In some embodiments, step S108 (sealing treatment step) may specifically include: sealing the coated electrode with a 0.5-5% bovine serum albumin (BSA) solution at room temperature (e.g., 25° C.) for 30-120 minutes. In the case where the substrate electrode is a SPCE electrode, the electrode after the sealing treatment is represented as a SPCE / Au / NHS / anti-CPA / BSA electrode.

[0101] Optionally, the sealed electrode is washed with ultrapure water and naturally air-dried for later use.

[0102] In some specific embodiments, the concentration of the BSA solution used for the blocking treatment is 1%, and the blocking treatment time is 1 hour.

[0103] Based on the same technical concept, an embodiment of the present invention also provides an electrochemical immunosensor for alpha toxin of Clostridium perfringens, which is prepared by the preparation method of the electrochemical immunosensor for alpha toxin of Clostridium perfringens of any of the above embodiments or combinations of embodiments. The electrochemical immunosensor for alpha toxin of Clostridium perfringens combines the high specific reaction of antigen-antibody with the high sensitivity of electrochemical signal, and is applied to the detection of alpha toxin of Clostridium perfringens, so as to achieve the purpose of rapid, specific and sensitive detection of alpha toxin of Clostridium perfringens.

[0104] Based on the same technical concept, an embodiment of the present invention further provides a method for applying the aforementioned Clostridium perfringens alpha toxin electrochemical immunosensor to the detection of Clostridium perfringens alpha toxin.

[0105] Figure 8 FIG. 1 is a flow chart of a method for detecting alpha toxin of Clostridium perfringens using an electrochemical immunosensor for detecting alpha toxin of Clostridium perfringens according to an embodiment of the present invention. Figure 8 As shown, the method for applying the Clostridium perfringens alpha toxin electrochemical immunosensor to detect Clostridium perfringens alpha toxin includes the following steps S802 to S806.

[0106] Step S802: placing the assembled electrochemical immunosensor in a sample solution to be tested to perform an immune reaction at room temperature.

[0107] like Figure 2 As shown in step (e), through immune reaction, the Clostridium perfringens alpha toxin in the sample solution is combined with the Clostridium perfringens alpha toxin antibody fixed on the detection electrode. After the immune reaction is completed, the detection electrode is washed with ultrapure water and naturally air-dried.

[0108] Step S804: placing the detected electrode after the reaction in a potassium ferrocyanide solution to perform a cyclic voltammetry test to obtain an electrochemical response signal of the sample.

[0109] In this step, an electrochemical workstation is used to collect signals through cyclic voltammetry testing.

[0110] Step S806: Determine the content of Clostridium perfringens alpha toxin in the sample solution to be tested according to the electrochemical response signal of the sample and the pre-obtained standard working curve of the electrochemical immunosensor.

[0111] The present invention uses an electrochemical immunosensor for the quantitative detection of Clostridium perfringens alpha toxin by cyclic voltammetry (CV). This method has the following advantages: (1) the test time is short, which is more conducive to the purpose of rapid on-site detection; (2) it is more widely used and the detection method is simple; (3) the specific binding of antigen and antibody is utilized to improve the accuracy of detection.

[0112] In the embodiment of the present invention, the immune response time in step S802 is optimized. Fig. 9 is a graph of the time optimization result of the immune response according to an embodiment of the present invention, wherein the immune response time (in min) is used as the horizontal axis, and the difference Δi between the oxidation peak current before and after the electrode incubation is detected. p (unit is A) is the vertical coordinate.

[0113] Reference Fig. 9 As shown, in some preferred embodiments, the immune response time is 20-50 min. More preferably, the immune response time is 30-40 min. Most preferably, the immune response time is 30 min.

[0114] In some embodiments, the potassium ferrocyanide solution is a solution prepared with a 10×PBS solution and containing 0.01-0.02 mmol / mL potassium ferrocyanide and 0.01-0.02 mmol / mL potassium ferrocyanide.

[0115] In some specific embodiments, the electrochemical response signal collected in the cyclic voltammetry test is a current signal. When the Clostridium perfringens alpha toxin antibody on the detection electrode specifically binds to the antigen in the sample, a corresponding change in the current signal will be generated, thereby achieving quantitative detection of Clostridium perfringens alpha toxin.

[0116] In some specific embodiments, the standard working curve of the Clostridium perfringens alpha toxin electrochemical immunosensor can be obtained by drawing in the following manner:

[0117] A plurality of Clostridium perfringens alpha toxin standard solutions with different concentrations ranging from 0.001 ng / mL to 1000 ng / mL were prepared with 1×PBS solution;

[0118] The detection electrodes of the Clostridium perfringens alpha toxin electrochemical immunosensor were placed in each standard solution and incubated at room temperature for 20-50 minutes;

[0119] The detection electrode before and after incubation was tested using cyclic voltammetry in potassium ferrocyanide solution;

[0120] Collect the difference Δi between the oxidation peak current of the detection electrode before and after incubation corresponding to each standard solution p The logarithm of the concentration of α-toxin of Clostridium perfringens in each standard solution is used as the horizontal axis, Δi p / i p,0 As the ordinate, the standard working curve of the electrochemical immunosensor is drawn, where i p,0 is the oxidation peak current of the detection electrode before incubation.

[0121] Those skilled in the art will appreciate that the detection electrode before incubation has not yet bound to the Clostridium perfringens alpha toxin (ie, the target). p,0 It can also be called the oxidation peak current before adding the target. In actual operation, for each standard solution, the detection electrode before incubation can be tested by cyclic voltammetry in a potassium ferrocyanide solution, and then the detection electrode is placed in a standard solution for incubation, and then the detection electrode after incubation is tested by cyclic voltammetry in a potassium ferrocyanide solution, so as to collect the difference Δi between the oxidation peak current of the detection electrode before and after incubation corresponding to each standard solution. p .

[0122] In addition, those skilled in the art should recognize that in order to ensure the accuracy and consistency of the test results, the conditions for incubation of the detection electrode (i.e., immune reaction) and cyclic voltammetry testing of the sample solution to be tested should be the same as those for incubation and cyclic voltammetry testing of the standard solution.

[0123] In a specific embodiment, standard solutions with concentrations of 1000 ng / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.1 ng / mL, 0.01 ng / mL, and 0.001 ng / mL were prepared, and these standard solutions were used for testing and a standard working curve was drawn. Fig.10 In order to intuitively show the response of the electrochemical immunosensor to CPA concentration, Fig.10 The percentage change of the oxidation peak current Δi is plotted in p / i p,0 (100%) versus the logarithmic value of CPA concentration.

[0124] like Fig.10 As shown, the percentage change of oxidation peak current Δi p / i p,0 The curve of the logarithmic value of CPA concentration (expressed as C) showed a good linear relationship in the concentration range of 0.001-10 ng / mL, and the linear relationship was Δi p / i p,0 =4.4489+1.3092log 10 C, linear correlation coefficient (R 2 ) is 0.9982. This indicates that the detection linear range of the electrochemical immunosensor is 0.001-10 ng / ml. The minimum response value is calculated by adding the average measurement value of the blank solution to the standard deviation of 3 times the blank. After substituting it into the equation, the detection limit (LOD) of the electrochemical immunosensor is 0.417 pg / mL.

[0125] The following is an explanation of the embodiments of the present invention by specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0126] Example 1

[0127] In Example 1, a method for preparing an electrochemical immunosensor for detecting alpha toxin of Clostridium perfringens is provided, comprising the following steps:

[0128] In the first step, the disposable screen-printed carbon electrode (SPCE) was washed with ultrapure water and air-dried naturally. The electrode was then placed in a 1% chloroauric acid solution to modify the electrode surface by the STEP electrodeposition method, wherein the STEP electrodeposition parameters were set as follows: electrodeposition voltage 4V, electrodeposition time 20s, to obtain the SPCE / Au electrode. The SPCE / Au electrode was washed with ultrapure water and air-dried naturally.

[0129] In the second step, the SPCE / Au electrode is placed in the prepared activation solution to activate the carboxyl groups on the electrode surface. The activation temperature is 29°C and the activation time is 70 minutes to obtain the SPCE / Au / NHS electrode. The SPCE / Au / NHS electrode is washed with ultrapure water and air-dried naturally. The specific preparation method of the activation solution is: take 10.6625g of morpholineethanesulfonic acid, 9.59g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1.44g of N-hydroxysuccinimide (NHS), dissolve them with ultrapure water, and dilute to 500mL to obtain the required activation solution.

[0130] The third step is to coat the working electrode surface of the SPCE / Au / NHS electrode with Clostridium perfringens alpha toxin antibody, the antibody coating concentration is 5 mg / mL, and the coating amount is 1 μL. The electrode coated with the antibody is placed in a wet box at 4°C overnight to obtain the SPCE / Au / NHS / anti-CPA electrode. The SPCE / Au / NHS / anti-CPA electrode is washed with ultrapure water and air-dried naturally.

[0131] Step 4: The SPCE / Au / NHS / anti-CPA electrode was sealed with 1% BSA at room temperature (25°C) for 1 h to obtain the SPCE / Au / NHS / anti-CPA / BSA electrode. The SPCE / Au / NHS / anti-CPA / BSA electrode was washed with ultrapure water, air-dried, and stored at 4°C for later use.

[0132] Example 2

[0133] In Example 2, a method for drawing a standard working curve of an electrochemical immunosensor for detecting alpha toxin of Clostridium perfringens is provided, comprising the following steps:

[0134] First, CPA toxin standard was diluted with 1×PBS, and CPA toxin standard solutions with concentrations of 1000ng / mL, 100ng / mL, 10ng / mL, 1ng / mL, 0.1ng / mL, 0.01ng / mL, and 0.001ng / ml were added to different test tubes, and the assembled SPCE / Au / NHS / anti-CPA / BSA electrodes were placed therein (two parallel groups were set for each condition), and incubated at 25°C (room temperature) for 30 minutes. After the reaction was completed, the electrodes were taken out and CV tests were performed in the prepared potassium ferrocyanide solution to analyze the electrochemical response caused by the sensor binding to different concentrations of CPA. The specific preparation method of potassium cyanide solution is: take 1.6465g potassium ferrocyanide and 2.1120g potassium ferrocyanide powder, dissolve them with 10×PBS solution, and dilute to 500mL to obtain the required potassium ferrocyanide solution.

[0135] Then, the difference in the oxidation peak current (Δi p ), with the logarithm of CPA concentration as the horizontal axis, Δi p / i p,0 As the ordinate, draw the response curve of target concentration-electrical signal change, where i p,0 is the oxidation peak current before adding the target. The response curve is shown in Fig.10 shown.

[0136] Through analysis, it can be seen that after optimizing the relevant parameters, the electrochemical immunosensor of Clostridium perfringens alpha toxin of the present invention has an obvious response to the CPA concentration in the range of 0.001-10 ng / mL, and the curve of the percentage change of the oxidation peak current versus the logarithmic value of the CPA concentration shows a good linear relationship in the concentration range of 0.001-10 ng / mL, and the linear relationship is Δi p / i p,0 =4.4489+1.3092log 10 C, linear correlation coefficient (R 2 ) is 0.9982. The minimum response value is calculated by adding the average measurement value of the blank solution to the standard deviation of 3 times the blank. After substituting it into the equation, the detection limit (LOD) of the electrochemical immunosensor is 0.417 pg / mL.

[0137] In the description provided herein, a large number of specific details are described. However, it is understood that embodiments of the present invention can be practiced without these specific details. In some instances, well-known methods, structures and techniques are not shown in detail so as not to obscure the understanding of this description.

[0138] At this point, those skilled in the art should recognize that, although multiple exemplary embodiments of the present invention have been shown and described in detail herein, many other variations or modifications that conform to the principles of the present invention can still be directly determined or derived based on the content disclosed in the present invention without departing from the spirit and scope of the present invention. Therefore, the scope of the present invention should be understood and recognized as covering all these other variations or modifications.

Claims

1. A method for preparing an electrochemical immunosensor for Clostridium perfringens alpha toxin, comprising: Step S102: using chloroauric acid solution to deposit gold nanoparticles on the surface of the substrate electrode by a multi-potential step electrodeposition method to modify the surface of the substrate electrode to obtain a gold-modified electrode; Step S104: activating the gold modified electrode using an activation solution containing N-hydroxysuccinimide to obtain an activated electrode; Step S106: coating the surface of the working electrode of the activated electrode with Clostridium perfringens alpha toxin antibody to obtain a coated electrode; as well as Step S108: performing sealing treatment on the coated electrode to obtain the detection electrode of the electrochemical immunosensor; Wherein, the step S102 specifically includes: Dissolving gold (III) chloride hydrate having a gold content of ≥48% by mass in pure water to prepare a chloroauric acid solution having a molar concentration of 0.5-2.5%; The substrate electrode is placed in the chloroauric acid solution, and multi-potential step electrodeposition is performed for 10-20 seconds at a constant electrodeposition voltage in the range of 4-5 V to obtain the gold modified electrode.

2. The preparation method according to claim 1, characterized in that: The substrate electrode is a screen-printed carbon electrode based on PET; The molar concentration of the chloroauric acid solution is 1%; The electrodeposition voltage is 4V; The multi-potential step electrodeposition was carried out for 20 s.

3. The preparation method according to claim 1, characterized in that: The step S104 specifically includes: Dissolving morpholineethanesulfonic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a molar ratio of (3-5):(3-5):1 in pure water to prepare the activation solution; placing the gold modified electrode in the activation solution, and performing electrode activation at 23-31° C. for 40-80 min to obtain the activated electrode; Wherein, the molar content of morpholineethanesulfonic acid in the activation solution is 0.05-0.15 mmol / mL, the molar content of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 0.05-0.15 mmol / mL, and the molar content of N-hydroxysuccinimide is 0.01-0.05 mmol / mL; The electrode activation temperature is 29° C., and the electrode activation time is 70 min.

4. The preparation method according to claim 1, characterized in that: The step S106 specifically includes: Coating the activated electrode with 0.5-3 µL of 0.1-15 mg / mL Clostridium perfringens alpha toxin antibody solution; The activated electrode coated with the antibody was placed in a humidified box at 2-6° C. overnight to obtain the coated electrode.

5. The preparation method according to claim 4, characterized in that: The concentration of the Clostridium perfringens alpha toxin antibody solution is 5 mg / mL, and the dosage is 1 µL.

6. The preparation method according to claim 1, characterized in that: The step S108 specifically includes: The coated electrode is blocked with a bovine serum albumin solution having a concentration of 0.5-5% at room temperature for 30-120 minutes.

7. An electrochemical immunosensor for Clostridium perfringens alpha toxin, characterized in that: The electrochemical immunosensor is prepared by the preparation method according to any one of claims 1-6.

8. A method for detecting alpha toxin of Clostridium perfringens for non-diagnostic and therapeutic purposes, comprising: placing the assembled electrochemical immunosensor in a sample solution to be tested to perform an immune reaction at room temperature, wherein the electrochemical immunosensor is the Clostridium perfringens alpha toxin electrochemical immunosensor according to claim 7; The detection electrode after the reaction is placed in a potassium ferrocyanide solution to perform a cyclic voltammetry test to obtain an electrochemical response signal of the sample; as well as The content of Clostridium perfringens alpha toxin in the sample solution to be tested is determined according to the electrochemical response signal of the sample and the pre-obtained standard working curve of the electrochemical immunosensor.

9. The method for detecting alpha toxin of Clostridium perfringens for non-diagnostic and therapeutic purposes according to claim 8, characterized in that: The immune response time is 20-50 minutes; The potassium ferrocyanide solution is a solution prepared with 10×PBS solution and contains 0.01-0.02 mmol / mL potassium ferrocyanide and 0.01-0.02 mmol / mL potassium ferrocyanide.

10. The method for detecting alpha toxin of Clostridium perfringens for non-diagnostic and therapeutic purposes according to claim 8 or 9, characterized in that: The electrochemical response signal is a current signal, and the standard working curve is obtained in the following manner: A plurality of Clostridium perfringens alpha toxin standard solutions with different concentrations ranging from 0.001 ng / mL to 1000 ng / mL were prepared with 1×PBS solution; The detection electrodes are placed in the standard solutions and incubated at room temperature for 30-60 minutes; Testing the detection electrode before and after incubation using cyclic voltammetry in the potassium ferrocyanide solution; Collect the difference Δi between the oxidation peak current of the detection electrode before and after incubation corresponding to each standard solution p The logarithm of the concentration of α-toxin of Clostridium perfringens in each standard solution is taken as the abscissa, Δi p / i p,0 As the ordinate, the standard working curve of the electrochemical immunosensor is obtained by drawing, wherein i p,0 is the oxidation peak current of the detection electrode before incubation.

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