Electrochemical immunosensing module for instantly detecting escherichia coli

Abstract

The invention discloses an electrochemical immunosensing module for instantly detecting escherichia coli. The electrochemical immunosensing module comprises a substrate and an immunosensor array arranged on the substrate; the immunosensor array comprises three working electrodes, a reference electrode and a carbon counter electrode which are all screen-printed electrodes; the working electrode is obtained by sequentially modifying carbon nanosphere aerogel with three-dimensional hierarchical pores, an escherichia coli antibody labeled by horse radish peroxidase and bovine serum albumin on a carbon electrode. The electrochemical immunosensing module can be used for three-channel detection, and the carbon nanosphere aerogel with three-dimensional hierarchical pores is used as one of core components of a working electrode, has a unique three-dimensional hierarchical structure and a high specific surface area, is used as an electrode substrate material of the working electrode, and has excellent performance in the aspect of detecting E.coli O157: H7. The electrochemical immunosensing module disclosed by the invention can be used for realizing portable analysis and detection of E.coli O157: H7 in food.

Description

Technical Field

[0001] The present invention relates to an electrochemical immunosensing module for the rapid detection of Escherichia coli, belonging to the technical field of electrochemical biosensing. Background Art

[0002] With the development of rapid detection technology, portable electronic products have brought valuable achievements to personalized diagnosis, environmental monitoring, food safety, and traffic management, greatly improving the quality of people's daily lives and becoming an indispensable tool. Traditional laboratory testing requires a long turnaround time, bulky equipment, and trained staff in a centralized laboratory. In contrast, portable sensing technology has advanced biological sample processing technology, rapid analysis technology, and convenient information extraction technology, showing great potential in achieving home / on-site diagnosis. Pathogens can be transmitted through media such as air, water, or food, thus posing a huge threat to human survival throughout history. Usually, pathogens cause approximately 50,000 deaths globally every day. Among all pathogens, Escherichia coli (E. coli) O157:H7 is considered a common foodborne pathogen and has received sufficient attention because its bacterial colonies can cause various diseases, including respiratory diseases, diarrhea, urinary tract infections, pneumonia, and other fatal complications. Therefore, at the point of care, especially in resource-limited areas, there is an urgent need for miniaturized, affordable, and easy-to-use instruments to detect E. coli O157:H7.

[0003] Electrochemical sensors have been widely used in multiple scientific disciplines such as physics, biology, and environmental testing for various reasons, including high sensitivity, fast detection speed, low cost, simple operation, and the potential for miniaturization on a portable platform. By introducing the concept of portability into electrochemical sensors for pathogen detection, portable electrochemical sensors can achieve the portable analysis of E. coli O157:H7 in water and food, thereby realizing food safety and environmental protection in the most convenient way. Summary of the Invention

[0004] The purpose of the present invention is to provide an electrochemical immunosensing module capable of achieving the portable analysis and detection of Escherichia coli (E. coli) O157:H7 in food.

[0005] The electrochemical immunosensing module provided by the present invention includes a substrate and an immunosensor array disposed on the substrate;

[0006] The immunosensor array includes three working electrodes, a reference electrode, and a carbon counter electrode, all of which are screen-printed electrodes;

[0007] The working electrode is obtained by successively modifying a carbon electrode with a carbon nanosphere aerogel with three-dimensional hierarchical pores, an antibody of Escherichia coli labeled with horseradish peroxidase, and bovine serum albumin;

[0008] The preparation method of the carbon nanosphere aerogel with three-dimensional hierarchical pores is as follows:

[0009] S1. After the guava is peeled and pitted, the residue is made; the residue and water are placed in a high-pressure reactor for reaction to obtain a biomass-derived hydrogel;

[0010] S2. The biomass-derived hydrogel is placed in a tubular furnace after vacuum freeze-drying and reacted in a nitrogen atmosphere to obtain it;

[0011] Preferably, the biomass-derived hydrogel is frozen and then subjected to the vacuum freeze-drying;

[0012] In the method for preparing the carbon nanosphere aerogel with three-dimensional hierarchical pores, in step S1, the residue and double-distilled water are placed in a stainless-steel high-pressure reactor with a polytetrafluoroethylene lining for reaction;

[0013] The temperature of the reaction is 160-200 °C, preferably 180 °C, and the time is 8-12 h, preferably 10 h.

[0014] Before step S2, the method further includes a step of immersing the biomass-derived hydrogel in hot water (~60 °C) to remove impurities;

[0015] In step S2, the conditions of the vacuum freezing are -48 to -42 °C, preferably -44 °C, and the time is 44-52 h, preferably 48 h;

[0016] In step S2, the temperature of the reaction is 800-1000 °C, preferably 900 °C, and the time is 1-3 h, preferably 1 h;

[0017] The rates during the heating and cooling processes are both preferably 5 °C min -1 .

[0018] The three-dimensional hierarchical porous carbon nanosphere aerogel (3D-HPCNAs) adopted in the present invention presents a three-dimensional interconnected nanosphere structure, has a large surface area, and also has a hierarchical microporous-mesoporous structure. This nanostructure makes it an ideal carrier for antibody loading, and may achieve high-quality and compact antibody loading. At the same time, it also provides continuous and unobstructed channels for the diffusion and transmission of molecules, thereby facilitating the enhancement of electron transfer. Therefore, the 3D-HPCNAs with a 3D interconnected nanosphere structure provided by the present invention is a very attractive electrode material and can be used to construct a sensing module.

[0019] In the electrochemical immunosensing module provided by the present invention, the steps for modifying the three-dimensional hierarchically porous carbon nanosphere aerogel solution, the horseradish peroxidase-labeled Escherichia coli antibody, and the bovine serum albumin are as follows:

[0020] Drop the solution of the three-dimensional hierarchically porous carbon nanosphere aerogel onto the carbon electrode; after drying, drop the solution of the horseradish peroxidase-labeled Escherichia coli antibody; after incubation, continue to drop the solution of the bovine serum albumin; preferably, rinse the carbon electrode and then drop the solution of the bovine serum albumin.

[0021] Among them, the solution of the three-dimensional hierarchically porous carbon nanosphere aerogel is prepared from a chitosan solution with a concentration of 0.5 - 2.5 mg / mL, preferably 2 mg / mL, and the chitosan solution is prepared from an acetic acid solution;

[0022] The solution of the horseradish peroxidase-labeled Escherichia coli antibody is prepared from PBS;

[0023] The temperature of the incubation is 0 - 5 °C, preferably 4 °C, and the time is 10 - 20 h, preferably 16 h;

[0024] The solution of the bovine serum albumin is prepared from PBS.

[0025] In the electrochemical immunosensing module of the present invention, the substrate is preferably a polyethylene terephthalate film, and an insulating layer is covered thereon;

[0026] The reference electrode is preferably an Ag / AgCl electrode.

[0027] The electrochemical immunosensing module of the present invention can be used to detect Escherichia coli, preferably Escherichia coli O157:H7.

[0028] The present invention also provides a method for detecting Escherichia coli, including the following steps:

[0029] Place the electrochemical immunosensing module in the solution of the sample to be tested, incubate it, and then place it in an acetic acid buffer solution containing thionine and hydrogen peroxide for detection; the content of Escherichia coli in the sample to be tested can be obtained by using chronoamperometry;

[0030] Among them, in the acetic acid buffer solution, the concentration of thionine can be 0.5 - 3 mM, preferably 1 mM, and the concentration of hydrogen peroxide can be 0.1 - 0.8 mM, preferably 0.6 mM.

[0031] The applied potential of chronoamperometry is preferably -0.14 V (vs. Ag / AgCl);

[0032] The sample to be tested can be diluted with PBS.

[0033] The electrochemical immunosensing module of the present invention can perform three-channel detection. Among them, 3D-HPCNAs, as one of the core components of the working electrode, have a unique three-dimensional hierarchical structure and a high specific surface area, and are used as the electrode substrate material of the working electrode, showing excellent performance in detecting E. coli O157:H7.

[0034] E. coli O157:H7 is a very dangerous foodborne bacterium that can cause hemorrhagic colitis, hemolytic uremic syndrome, and other similar life-threatening problems, indicating that the rapid detection of the pathogenic bacterium E. coli O157:H7 is of great significance for ensuring food safety and protecting the health of the general population. The electrochemical immunosensing module of the present invention can detect E. coli O157:H7 in samples and uses the chronoamperometry method for detection. After the antigen-antibody interaction, the cathodic current density decreases with the increase in the concentration of E. coli O157:H7. The current density shows an excellent linear relationship with the E. coli O157:H7 level, and the linear range is 10 2 ~10 9 CFU mL -1 , and the detection limit can be as low as 30 CFU mL -1 (S / N = 3). In addition, the sensing module of the present invention has high selectivity for some other common bacteria that may exist in actual samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the flow chart for the preparation of the electrochemical immunosensing module of the present invention.

[0036] Figure 2 is the schematic diagram for the preparation of the PET film in the electrochemical immunosensing module of the present invention.

[0037] Figure 3 is the schematic diagram for the preparation of the insulating layer in the electrochemical immunosensing module of the present invention.

[0038] Figure 4 is the schematic diagram of different layers and their corresponding functions in the electrochemical immunosensing module of the present invention.

[0039] Figure 5 is the SEM image (left figure, scale bar: 650 nm) and TEM image (right figure, scale bar: 300 nm) of the 3D-HPCNAs used in the present invention.

[0040] Figure 6 is the diameter distribution diagram of 3D-HPCNA obtained from the SEM image and TEM image (statistically, the average diameter is ~148 nm).

[0041] Figure 7The N2 adsorption - desorption isotherm curve of 3D - HPCNAs used in the present invention, and the inset is the pore size distribution diagram of 3D - HPCNAs.

[0042] Figure 8 The Raman spectrum of 3D - HPCNAs used in the present invention.

[0043] Figure 9 The XPS spectrum of 3D - HPCNAs used in the present invention.

[0044] Figure 10 The high - resolution XPS spectrum of O1s of 3D - HPCNAs used in the present invention.

[0045] Figure 11 The chronoamperometry method was used to detect E. coli O157:H7 by the sensing module of the present invention, and the inset is the corresponding calibration curve.

[0046] Figure 12 The selectivity of the sensing module of the present invention for E. coli O157:H7 (the concentration of each bacterium: 1.0×10 6 CFU mL -1 ).

[0047] Figure 13 The results of detecting E. coli O157:H7 in actual samples by the sensing module of the present invention. Among them, Figure A is the detection of E. coli O157:H7 in tap water (5.0×10 6 CFU mL -1 ) added with E. coli O157:H7 by the sensing module and a commercial enzyme - linked immunosorbent assay kit, and Figure B is the detection of E. coli O157:H7 in watermelon juice (5.0×10 6 CFU mL -1 ) added with E. coli O157:H7 by the sensing module and a commercial enzyme - linked immunosorbent assay kit. Detailed implementation mode

[0048] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0049] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0050] Example 1. Preparation of three - dimensional hierarchical - pore carbon nanosphere aerogel (3D - HPCNAs)

[0051] First, peel and pit the guava and cut it into small pieces. Then, put the small pieces of guava into a juicer and blend them, and filter the blended guava through a filter to obtain guava filter residue. Subsequently, put the guava filter residue and double-distilled water into a stainless-steel autoclave with a polytetrafluoroethylene lining, and heat the autoclave at 180 °C for 10 h to obtain a biomass-derived hydrogel. Then, first wash the obtained biomass-derived hydrogel three times with a mixed solution of double-distilled water and ethanol, and then immerse it in hot water (~60 °C) for 48 hours to remove soluble impurities. Then store the sample at -80 °C for 5 hours and then vacuum freeze-dry it at -44 °C for 48 hours. Put the above product into a high-temperature tube furnace and hold it at 900 °C for 1 hour under a N2 atmosphere (the heating and cooling rates are both 5 °C min -1 ) to obtain 3D-HPCNAs.

[0052] Example 2. Preparation of an electrochemical immunosensing module

[0053] The preparation flow chart is as Figure 1 shown.

[0054] 1. Preparation of the PET membrane

[0055] After cutting a polyethylene terephthalate (PET) layer (1.75 cm × 1.10 cm × 200.00 μm) using a digital cutting machine, a PET membrane is obtained, as Figure 2 shown.

[0056] 2. Preparation of the screen-printed electrode (SPE)

[0057] Use a commercial manual screen printer and a custom template to prepare the SPE by screen printing technology.

[0058] First, print the silver paste on the PET membrane. After curing it in an oven at 93 °C for 15 minutes, a PET membrane with a silver layer is obtained. Next, print the carbon paste on the PET membrane with the silver layer. After curing it in an oven at 120 °C for 10 minutes, a PET membrane with a carbon layer and a silver layer is obtained. After pasting the insulating layer, as Figure 3 shown, a PET membrane with an insulating layer, a carbon layer, and a silver layer is obtained. Next, drop 0.6 μL of 0.1 M FeCl3 solution onto part of the silver layer and keep it for 5 h, and then wash the remaining FeCl3 with water. Finally, dry it under infrared light to obtain the SPE.

[0059] 3. Preparation of the immunosensor array

[0060] First, 2 mg of 3D-HPCNAs powder was dispersed in 1 mL of chitosan solution (0.2 wt%, with the solvent being 1 wt% acetic acid solution), sonicated for 2 h, and stirred for 4 h to obtain a uniform 3D-HPCNAs solution. Then, 0.8 μL of the 3D-HPCNAs solution was dropped onto each circular carbon electrode of the SPE. After drying under ambient conditions, a solution of horseradish peroxidase-labeled E. coli O157:H7 antibody (HRP-anti-E. coli O157:H7) (0.5 μg mL -1 , with the solvent being 0.01 M pH 7.4 PBS) was drop-coated onto the three electrodes (i.e., 3D-HPCNAs / SPE). The obtained electrodes were incubated overnight at 4 °C to immobilize HRP-anti-E. coli O157:H7, and then rinsed with 0.01 M pH 7.4 phosphate buffer solution (PBS) to remove the unimmobilized HRP-anti-E. coli O157:H7. After air-drying under ambient conditions, three HRP-anti-E. coli O157:H7 / 3D-HPCNAs / SPEs were obtained. Then, 1.0 μL of bovine serum albumin (BSA) solution (5 mg mL -1 , with the solvent being 0.01 M pH 7.4 PBS) was dropped onto each of the three HRP-anti-E. coli O157:H7 / 3D-HPCNAs / SPEs to eliminate the non-specific binding effect and block the remaining active groups, and then they were air-dried under ambient conditions. After careful rinsing with double-distilled water, an immunosensor array within the sensing module was obtained (i.e., three BSA / HRP-anti-E. coli O157:H7 / 3D-HPCNAs / SPEs as working electrodes), as Figure 1 shown.

[0061] The electrochemical immunosensing module prepared in this example can be used to detect and analyze E. coli O157:H7, and the sample to be tested was diluted with 0.1 M pH 7.4 PBS (1:9, v / v). After incubating the electrochemical immunosensing module in the diluted actual sample solution at 37 °C for 30 minutes, the sensing module was carefully washed with 0.1 M pH 7.4 PBS. The detection of E. coli O157:H7 can be carried out in 0.1 M pH 6.0 acetate buffer solution containing thionine (such as 1.0 mM) and H2O2 (0.6 mM).

[0062] Example 3. Design and Characterization of Electrochemical Immunosensing Module

[0063] The electrochemical immunosensing module prepared in Example 2 of the present invention includes two parts: an immunosensor array and a PET membrane. Among them, the immunosensor array is used for the detection of E. coli O157:H7; the PET membrane is used for the fixation of the sensing module. The immunosensor array includes three working electrodes, an Ag / AgCl reference electrode, and a carbon counter electrode. This immunosensor array can perform three-channel detection. Among them, 3D-HPCNAs, as one of the core components of the working electrode, has a unique three-dimensional hierarchical structure and a high specific surface area, and is used as the electrode substrate material of the working electrode, and has excellent performance in the detection of E. coli O157:H7, such as Figure 4 shown.

[0064] To prepare the immunosensor array, 3D-HPCNAs, horseradish peroxidase-labeled anti-E. coli O157:H7 antibody (HRP-anti-E. coli O157:H7), and bovine serum albumin (BSA) are sequentially modified on each circular screen-printed electrode (SPE) in the sensing module. The multi-channel array improves the accuracy by averaging the detection signals and can be modified into different pathogen sensors to achieve the simultaneous detection of multiple pathogens. E. coli O157:H7 in the culture medium binds to HRP-anti-E. coli O157:H7 and forms an immune complex, which can block the electron transfer between the active center of horseradish peroxidase and the mediator thionine. Thionine is used as a mediator, and the cathodic current generated by the reaction of horseradish peroxidase reducing hydrogen peroxide (H2O2) is negatively correlated with the concentration of E. coli O157:H7 in the actual sample.

[0065] Due to the large specific surface area, unique pore structure, and fast mass transfer performance of the hierarchically porous three-dimensional carbonaceous nanomaterials, they are very suitable as electrode materials for electronic devices. In order to accurately identify the presence of E. coli O157:H7, the present invention uses 3D-HPCNAs with a unique nanostructure and excellent electrochemical performance and uses it as the electrode material of the sensing module. 3D-HPCNAs presents a three-dimensional interconnected nanosphere structure, such as Figure 5 and Figure 6 shown, with a large specific surface area, and the specific surface area can reach 650.69 m 2 g -1 . 3D-HPCNAs also has a hierarchical microporous-mesoporous structure (mainly concentrated at 0.55 nm, 1.50 nm, and 4.0 nm), such as Figure 7 shown. This nanostructure makes it an ideal carrier for antibody loading and may achieve high-quality and compact antibody loading.

[0066] Meanwhile, it also provides a continuous and unobstructed channel for the diffusion and transmission of molecules, thus facilitating the enhancement of electron transfer. The Raman spectrum shows that 3D-HPCNAs have a relatively high I D / I G value (2.14), as Figure 8 shown, indicating that there are a large number of defect sites in 3D-HPCNAs. These defect sites can serve as channels for electron transfer, potentially accelerating electron transfer and thus improving the performance of the sensor.

[0067] In the X-ray photoelectron spectroscopy (XPS) spectrum of 3D-HPCNAs, two characteristic peaks at 284.67 eV and 533.63 eV appear, corresponding to the electron binding energies of the C1s and O1s orbitals of 3D-HPCNAs, respectively, as Figure 9 shown. This means that there are C and O elements in 3D-HPCNAs, and the C content is 80.95 at.%, indicating that 3D-HPCNAs are mainly composed of carbon atoms and are a carbon-based material. The presence of the O 1s peak indicates that there may be oxygen-containing functional groups on the surface of 3D-HPCNAs, as Figure 10 shown. These functional groups can enhance the hydrophilicity of 3D-HPCNAs and reduce the charge transfer resistance at the electrode interface, thereby improving the performance of the entire device.

[0068] The chronoamperometry method was used to verify the detection performance of the electrochemical immunosensing module of the present invention for E. coli O157:H7. The results are as Figure 11 shown. It can be seen that after the antigen-antibody interaction, the cathodic current density decreases with the increase in the concentration of E. coli O157:H7. The current density shows an excellent linear relationship with the concentration of E. coli O157:H7, and the linear range is 10 2 ~10 9 CFU mL -1 , and the detection limit can be as low as 30 CFU mL -1 (S / N = 3).

[0069] As Figure 12 shown, the detection results of other bacteria using the electrochemical immunosensing module of the present invention are presented. It can be seen that the electrochemical immunosensing module of the present invention has high selectivity for some other common bacteria that may exist in actual samples.

[0070] Example 4. Verification of the Detection of E. coli O157:H7 in Actual Samples by the Electrochemical Immunosensing Module

[0071] To verify the applicability and accuracy of the electrochemical immunosensing module of the present invention in the detection of E. coli O157:H7, the electrochemical immunosensing module of the present invention and a commercial enzyme-linked immunosorbent assay kit (purchased from Wuxi Ditenmin Biotechnology Co., Ltd.) were used to detect tap water added with E. coli O157:H7 and watermelon juice added with E. coli O157:H7.

[0072] The tap water or watermelon juice was diluted with 0.1 M PBS at pH 7.4 in a ratio of 1:9 (V 实际样品 :V PBS ). After incubating the sensing module in the diluted actual sample solution at 37 °C for 30 minutes, the sensing module was carefully washed with 0.1 M PBS at pH 7.4. Detection was carried out in a 0.1 M acetic acid buffer solution at pH 6.0 containing 1.0 mM thionine and 0.6 mM H2O2. The applied potential for chronoamperometry was -0.14 V (vs. Ag / AgCl).

[0073] As Figure 13 can be seen, compared with the results detected by the commercial enzyme-linked immunosorbent assay kit, the relative deviations of the detection results of E. coli O157:H7 in the tap water sample added with E. coli O157:H7 and the watermelon juice sample added with E. coli O157:H7 by the sensing module of the present invention were 8.51% and -5.08% respectively, which proves that the sensing module of the present invention has high applicability and accuracy in detecting E. coli O157:H7 in actual samples.

Claims

1. An electrochemical immunosensing module, comprising a substrate and an immunosensor array disposed on the substrate; The immunosensor array includes three working electrodes, a reference electrode, and a carbon counter electrode, all of which are screen-printed electrodes; The working electrode is obtained by sequentially modifying a carbon electrode with carbon nanosphere aerogel with three-dimensional hierarchical pores, horseradish peroxidase-labeled Escherichia coli antibody, and bovine serum albumin; The preparation method of the carbon nanosphere aerogel with three-dimensional hierarchical pores is as follows: S1. After the guava is peeled and pitted, it is made into filter residue; the filter residue and water are placed in a high-pressure reaction kettle for reaction to obtain a biomass-derived hydrogel; S2. The biomass-derived hydrogel is placed in a tube furnace after vacuum freeze-drying and reacted in a nitrogen atmosphere to obtain it.

2. The electrochemical immunosensing module according to claim 1, wherein: In step S1, the temperature of the reaction is 160-200 °C, and the time is 8-12 h.

3. The electrochemical immunosensing module according to claim 1 or 2, characterized in that: Before step S2, the method further includes a step of immersing the biomass-derived hydrogel in hot water to remove impurities; In step S2, the conditions of vacuum freezing are -48 to -42 °C, and the time is 44-52 h.

4. The electrochemical immunosensing module according to any one of claims 1-3, characterized in that: In step S2, the temperature of the reaction is 800-1000 °C, and the time is 1-3 h.

5. The electrochemical immunosensing module according to any one of claims 1-4, characterized in that: The steps of modifying the carbon nanosphere aerogel with three-dimensional hierarchical pores, the horseradish peroxidase-labeled Escherichia coli antibody, and the bovine serum albumin are as follows: The solution of the carbon nanosphere aerogel with three-dimensional hierarchical pores is dropped onto the carbon electrode; after drying, the solution of the horseradish peroxidase-labeled Escherichia coli antibody is dropped; after incubation, the solution of bovine serum albumin is continuously dropped.

6. The electrochemical immunosensing module according to claim 5, wherein: The solution of the carbon nanosphere aerogel with three-dimensional hierarchical pores is prepared from a chitosan solution, with a concentration of 0.5-2.5 mg / mL, and the chitosan solution is prepared from an acetic acid solution; The solution of the horseradish peroxidase-labeled Escherichia coli antibody is prepared from PBS; The temperature of the incubation is 0-5 °C, and the time is 10-20 h; The solution of bovine serum albumin is prepared from PBS.

7. The electrochemical immunosensing module according to any one of claims 1-6, characterized in that: The substrate is a polyethylene terephthalate film; The reference electrode is an Ag / AgCl electrode.

8. The application of the electrochemical immunosensing module according to any one of claims 1-7 in detecting Escherichia coli.

9. A method for detecting Escherichia coli, comprising the following steps: The electrochemical immunosensing module is placed in a solution of a sample to be tested, incubated, and then placed in an acetic acid buffer solution containing thionine and hydrogen peroxide for detection; the content of Escherichia coli in the sample to be tested is obtained by chronoamperometry.

10. The application according to claim 8 or the method according to claim 9, characterized in that: The Escherichia coli is Escherichia coli O157:H7.

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