Electrochemical immunosensing module based on laser-induced graphene and application of electrochemical immunosensing module in instant detection of escherichia coli
The electrochemical immunosensing module prepared by laser-induced graphene solves the time and equipment cost problems of the existing method for detecting E. coli O157:H7, and realizes portable high-sensitive detection, with high selectivity and rapid response capabilities.
Patent Information
- Application Number
- CN202410227951.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-02-29
- Publication Date
- 2025-09-02
AI Technical Summary
The existing methods for detecting E. coli O157:H7 take several days, labor or expensive equipment, and are not suitable for on-site testing, with a risk of false alarms and lack of fast, simple and low-cost detection methods.
The electrochemical immunosensing module was prepared using laser-induced graphene (LIG), including laser-printed graphene electrodes, chitosan-modified and horseradish peroxidase-labeled E. coli antibodies, and was detected in combination with the timing current method.
A portable high sensitivity detection of E. coli O157:H7 is achieved, with a linear range of 2.5×103~108CFU mL-1, and the detection limit is as low as 100CFU mL-1, with high selectivity and rapid response capabilities.
Smart Images
Figure CN120577375A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to an electrochemical immunosensor module based on laser-induced graphene and application thereof in real-time detection of Escherichia coli, belonging to the technical field of electrochemical biosensor. Background Art
[0002] Escherichia coli O157:H7 (E. coli O157:H7) is a pathogenic Shiga toxin-producing Escherichia coli that can contaminate food and water resources and cause severe gastrointestinal complications and even death, such as hemolytic uremic syndrome. Current methods for detecting E. coli O157:H7 mainly include traditional plate culture, polymerase chain reaction, enzyme-linked immunosorbent assay, and surface plasmon resonance. Although these methods have demonstrated satisfactory sensitivity, specificity, and throughput, several challenges remain. For example, these methods require several days and extensive labor, rely on expensive and power-hungry laboratory equipment, or are relatively prone to false positives, making them unsuitable for field testing. Therefore, the development of a rapid, simple, sensitive, and low-cost method for on-site monitoring of E. coli O157:H7 is imperative, especially in resource-poor settings.
[0003] Electrochemical sensors have been widely used in various scientific disciplines, including physics, biology, and environmental testing, due to their high sensitivity, rapid detection speed, low cost, ease of operation, and potential for miniaturization on portable platforms. Electrode materials are considered a crucial component in achieving high-sensitivity in electrochemical sensors. Laser-induced graphene (LIG) is a 3D porous graphene formed on a polyimide (PI) film under ambient conditions using a commercial CO2 laser cutter. This one-step preparation of LIG exhibits high surface area, high thermal stability, and excellent conductivity. Furthermore, the entire process can be performed in ambient air without the need for any solvents, significantly simplifying the process and reducing costs. Therefore, LIG-based electrochemical immunosensor modules, characterized by their simple and low-cost material preparation, hold broad application prospects for the point-of-care detection of E. coli O157:H7 and provide a novel approach for the development of highly sensitive immunoassay platforms. Summary of the Invention
[0004] The purpose of the present invention is to provide an electrochemical immunosensor module based on laser-induced graphene, which can realize portable analysis and detection of Escherichia coli (E. coli) O157:H7 in food.
[0005] The electrochemical immunosensor module provided by the present invention comprises a substrate and an immunosensor array arranged on the substrate;
[0006] The immunosensor array includes a working electrode, a reference electrode and a counter electrode;
[0007] The working electrode is obtained by sequentially modifying chitosan, horseradish peroxidase-labeled Escherichia coli antibody and bovine serum albumin on a laser-printed graphene electrode;
[0008] The counter electrode is a laser-printed graphene electrode.
[0009] Wherein, the laser-printed graphene electrode is obtained by laser etching a PI film;
[0010] The conditions of the laser etching are as follows:
[0011] At room temperature and air environment, a CO2 laser system is used with a laser power of 9 to 15 W, preferably 12 W, and a laser speed of 500 to 700 mm s -1 , preferably 600 mm s -1 .
[0012] Preferably, the steps of modifying the chitosan, the horseradish peroxidase-labeled Escherichia coli antibody and the bovine serum albumin are as follows:
[0013] The chitosan solution is dripped onto the laser-printed graphene electrode; after drying, the horseradish peroxidase-labeled Escherichia coli antibody solution is dripped; and after incubation, the bovine serum albumin solution is continuously dripped.
[0014] Wherein, the chitosan solution is prepared from acetic acid solution with a mass concentration of 0.1-0.3%;
[0015] The horseradish peroxidase-labeled Escherichia coli antibody solution is prepared from PBS;
[0016] The incubation temperature is 0-5°C and the time is 10-20h;
[0017] The bovine serum albumin solution is prepared from PBS.
[0018] In the present invention, the substrate is a PI film;
[0019] The reference electrode is an Ag / AgCl electrode.
[0020] The electrochemical immunosensor module of the present invention can be used to detect Escherichia coli, preferably Escherichia coli O157:H7.
[0021] The laser-printed graphene used in the present invention has a large specific surface area, a unique pore structure and a fast mass transfer performance, and is very suitable as an electrode material for electronic devices.
[0022] The present invention also provides a method for detecting Escherichia coli, comprising the following steps:
[0023] The electrochemical immunosensor module is placed in a solution of a sample to be tested, incubated, and then placed in an acetate buffer solution containing thionine and hydrogen peroxide for detection; the content of Escherichia coli in the sample to be tested is obtained using a chronoamperometry method;
[0024] Wherein, in the acetate buffer solution, the concentration of thionine may be 0.3-1.1 mM, preferably 0.7 mM, and the concentration of hydrogen peroxide may be 0.1-0.8 mM, preferably 0.4 mM.
[0025] The applied potential for chronoamperometry is preferably -0.29 V (vs. Ag / AgCl);
[0026] The test sample can be diluted with PBS.
[0027] The electrochemical immunosensor module of the present invention can perform three-channel detection. Among them, laser-printed graphene (LEG) is one of the core components of the working electrode. It has a unique three-dimensional porous structure and a high specific surface area. It is used as the electrode substrate material of the working electrode and has excellent performance in detecting Escherichia coli O157:H7.
[0028] E. coli O157:H7 is a highly dangerous foodborne bacterium that can cause hemorrhagic colitis, hemolytic uremic syndrome, and other similar life-threatening conditions. This suggests that immediate detection of the pathogenic E. coli O157:H7 is crucial for ensuring food safety and protecting the health of the general population. The electrochemical immunosensor module of the present invention is capable of detecting E. coli O157:H7 in samples using a chronoamperometric method. Following antigen-antibody interaction, the cathode current density decreases with increasing E. coli O157:H7 concentration, and the current density exhibits an excellent linear relationship with E. coli O157:H7 levels, with a linear range of 2.5 × 10 3 ~10 8 CFU mL -1 The detection limit can be as low as 100 CFU mL -1 (S / N=3) In addition, the sensor module of the present invention has high selectivity for some other common bacteria that may exist in actual samples. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 This is a flow chart of the preparation of the electrochemical immunosensor module of the present invention.
[0030] Figure 2 Schematic diagram of the preparation of the PI layer in the electrochemical immunosensor module of the present invention.
[0031] Figure 3 Schematic diagram of the preparation of the insulating layer in the electrochemical immunosensor module of the present invention.
[0032] Figure 4 Schematic diagram of different layers and corresponding functions in the electrochemical immunosensor module of the present invention.
[0033] Figure 5 SEM images (Figure A is the SEM image of the top of LEG, Figure B is the SEM image of the cross-section of LEG) and TEM images (Figure C is the TEM image of LEG, Figure D is the high-resolution TEM image of LEG) of the laser-printed graphene (LEG) used in the present invention.
[0034] Figure 6 Figure 2 shows the N2 adsorption-desorption isotherm (Figure A) and pore size distribution (Figure B) of the laser-printed graphene (LEG) used in the present invention.
[0035] Figure 7 The XPS spectrum (Figure A) and O1s high-resolution XPS spectrum (Figure B) of the laser-printed graphene (LEG) used in the present invention.
[0036] Figure 8 This is the Raman spectrum of the laser-printed graphene (LEG) used in the present invention.
[0037] Figure 9 The sensor module of the present invention uses chronoamperometry to detect E. coli O157:H7 (Figure A) and the corresponding calibration curve (Figure B).
[0038] Figure 10 The selectivity of the sensor module of the present invention to E. coli O157:H7 (concentration of each bacterium: 1.0×10 6 CFU mL -1 ). DETAILED DESCRIPTION
[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0040] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0041] Example 1. Preparation of electrochemical immunosensor module
[0042] Preparation flow chart Figure 1 shown.
[0043] 1. Preparation of polyimide (PI) layer
[0044] The PI layer was obtained by cutting the PI film (2.40 cm × 1.30 cm × 200.00 μm) using a digital cutting machine. Figure 2 shown.
[0045] 2. Preparation of laser-engraved graphene (LEG) sensors
[0046] The PI film was ultrasonically cleaned in ethanol and ultrapure water to remove surface impurities and then dried. A CO2 laser system was used at room temperature and air environment with a laser power of 12W and a laser speed of 600 mm s -1 The PI film was laser etched under the conditions of 100 nm, and the laser irradiated the surface of the PI film (orange) to convert it into LEG (black). Next, silver ink was printed on the PI layer by screen printing using a commercial manual screen printer and a custom template. After curing in a 93°C oven for 15 minutes, a PI layer with a silver layer pattern and LEG was obtained. After the insulating layer was pasted on ( Figure 3 ), resulting in a PI layer with an insulating layer, a silver layer, and LEG. 0.5 μL of a 0.1 M FeCl₃ solution was then dripped onto part of the silver layer and left for 5 hours. The remaining FeCl₃ was then rinsed with water. Finally, the solution was dried under infrared light to obtain the LEG sensor.
[0047] 3. Preparation of immunosensor
[0048] First, 0.6 μL chitosan solution (0.2 wt %, solvent: 1% acetic acid solution) was dropped onto the LEG sensor. After drying under ambient conditions, 0.3 μL HRP-anti-E. coli O157:H7 solution (0.5 μg mL -1 The resulting electrode was incubated overnight at 4°C to immobilize HRP-anti-E.coli O157:H7, and then rinsed with 0.01M pH 7.4 PBS to remove unimmobilized HRP-anti-E.coli O157:H7. After drying under ambient conditions, HRP-anti-E.coli O157:H7 / CS / / LEG was obtained. Then, 0.5 μL of BSA solution (5 mg mL -1 , the solvent was 0.01M pH7.4 PBS) to eliminate nonspecific binding effects and block the remaining active groups, and then it was left to dry under ambient conditions. After careful rinsing with double distilled water, the immunosensor in the sensing module (i.e., BSA / HRP-anti-E. coli O157:H7 / CS / LEG as the working electrode) was obtained, as shown in Figure 1 shown.
[0049] The electrochemical immunosensor module prepared in this example can be used to detect and analyze E. coli O157:H7. After incubating the electrochemical immunosensor module in a diluted real-world sample solution at 37°C for 30 minutes, the module was carefully rinsed with 0.1M pH 7.4 PBS. E. coli O157:H7 can be detected in a 0.1M pH 6.0 acetate buffer solution containing thionine (e.g., 0.7mM) and H2O2 (0.4mM).
[0050] Example 2: Design and characterization of electrochemical immunosensor module
[0051] The sensor module prepared in Example 1 of the present invention comprises two parts: an immunosensor and a polyimide (PI) layer. The immunosensor is used for the detection of E. coli O157:H7; the PI layer is used for the fixation of the immunosensor, such as Figure 4 The immunosensor consists of a working electrode, an Ag / AgCl reference electrode, and a counter electrode. Laser-printed graphene (LEG), one of the core components of the working electrode, has a unique three-dimensional porous structure and a high specific surface area. It serves as the electrode substrate for the working electrode and demonstrates excellent performance in detecting E. coli O157:H7.
[0052] To fabricate the immunosensor, a circular LEG electrode within the sensor module was modified sequentially with chitosan (CS), horseradish peroxidase-labeled Escherichia coli O157:H7 antibody (HRP-anti-E. coli O157:H7), and bovine serum albumin (BSA). E. coli O157:H7 in the culture medium binds to HRP-anti-E. coli O157:H7, forming an immunocomplex. This immunocomplex blocks electron transfer between the active site of horseradish peroxidase and the mediator thionine. The cathodic current generated by the enzyme's reduction of hydrogen peroxide (H2O2) mediated by thionine is negatively correlated with the concentration of E. coli O157:H7 in the actual sample.
[0053] Laser-printed graphene (LEG) is very suitable as an electrode material for electronic devices due to its large specific surface area, unique pore structure and fast mass transfer performance. In order to accurately identify the presence of E. coli O157:H7, the present invention uses PI film as a carbon source and uses laser to prepare porous graphene (LEG) on the surface of PI film, with a laser power of 12W, and uses it to construct a sensing module. LEG has a 3D porous structure ( Figure 5 After laser irradiation, a LEG layer with a thickness of about 128.24 μm was clearly formed on the surface of the PI film ( Figure 5This unique structure is likely due to the extremely high local temperatures (>2500°C) generated by laser irradiation on the PI film surface. This triggers the depolymerization of the PI, which then rearranges the carbon atoms on the PI surface. The remaining atoms also recombine and are released as gas. Furthermore, because the entire laser etching process takes place in air, the oxygen and water molecules in the air burn off a certain amount of carbon as the local temperature rises, resulting in the porous structure of the prepared LEG.
[0054] The microstructure of LEG was characterized by TEM. As shown in the figure, LEG has a layered structure ( Figure 5 Figure C in the middle). High-resolution TEM (HRTEM) images show that LEG has clear lattice fringes with an average lattice spacing of 0.34 nm, corresponding to the (002) crystal plane of graphite material ( Figure 5 Figure D in the middle). LEG has a high specific surface area (322.91m 2 g -1 ) and hierarchical microporous-mesoporous structures (mainly concentrated at 0.82nm, 2.19nm and 3.83nm, Figure 6 This nanostructure makes it an ideal carrier for antibody loading, potentially enabling high-quality and compact antibody loading. It also provides continuous and unobstructed pathways for molecular diffusion and transport, enabling the sensor to rapidly respond and detect changes in the environment.
[0055] In order to further study the elemental composition of LEG, the present invention uses XPS to analyze the elements on the surface of LEG. The XPS spectrum shows that LEG has three characteristic peaks: C1s (285.2eV), N 1s (402.2eV) and O1s (533.2eV). Figure 7 In addition, the XPS spectrum shows that the C content of LEG is 91.26 at.%, which indicates that LEG has achieved a high degree of carbonization and is a carbon-based material. The O1s high-resolution spectrum of LEG shows that LEG contains oxygen-containing functional groups ( Figure 7 This is very beneficial to enhancing hydrophilicity and reducing charge transfer resistance at the electrode interface. In addition, Raman spectra show that LEG has a relatively high I D / I G Value (0.89, Figure 8 ), which indicates that LEG has a high density of defect sites, which may accelerate electron transfer and thus improve sensing performance. In summary, LEG with 3D porous structure is an attractive electrode material for constructing sensing modules.
[0056] The chronoamperometry method was used to verify the analytical performance of the portable integrated sensing platform of the present invention for the detection of E. coli O157:H7. Figure 9As shown in Figure A, it can be seen that after antigen-antibody interaction, the cathode current density decreases with the increase of E. coli O157:H7 concentration. Under optimal conditions, the current density shows an excellent linear relationship with the E. coli O157:H7 level, and the linear range is 2.5×10 3 ~10 8 CFU mL -1 The detection limit can be as low as 100 CFU mL -1 (S / N=3)( Figure 9 (Middle Figure B).
[0057] like Figure 10 As shown in FIG, the detection results of other bacteria using the electrochemical immunosensor module of the present invention are shown. It can be seen that the electrochemical immunosensor module of the present invention has a high selectivity for some other common bacteria that may exist in actual samples.
Claims
1. An electrochemical immunosensor module comprising a substrate and an immunosensor array disposed on the substrate; The immunosensor array includes a working electrode, a reference electrode and a counter electrode; The working electrode is obtained by sequentially modifying chitosan, horseradish peroxidase-labeled Escherichia coli antibody and bovine serum albumin on a laser-printed graphene electrode; The counter electrode is a laser-printed graphene electrode.
2. The electrochemical immunosensor module according to claim 1, characterized in that: The laser-printed graphene electrode is obtained by laser etching a PI film.
3. The electrochemical immunosensor module according to claim 2, characterized in that: The conditions of the laser etching are as follows: At room temperature and air environment, a CO2 laser system is used with a laser power of 9 to 15 W and a laser speed of 500 to 700 mm s -1 .
4. The electrochemical immunosensor module according to any one of claims 1 to 3, characterized in that: The steps of modifying the chitosan, the horseradish peroxidase-labeled Escherichia coli antibody and the bovine serum albumin are as follows: The chitosan solution is dripped onto the laser-printed graphene electrode; after drying, the horseradish peroxidase-labeled Escherichia coli antibody solution is dripped; and after incubation, the bovine serum albumin solution is continuously dripped.
5. The electrochemical immunosensor module according to claim 4, characterized in that: The chitosan solution is prepared from acetic acid solution with a mass concentration of 0.1-0.3%; The horseradish peroxidase-labeled Escherichia coli antibody solution is prepared from PBS; The incubation temperature is 0-5°C and the time is 10-20h; The bovine serum albumin solution is prepared from PBS.
6. The electrochemical immunosensor module according to any one of claims 1 to 5, characterized in that: The substrate is a PI film; The reference electrode is an Ag / AgCl electrode.
7. Use of the electrochemical immunosensor module according to any one of claims 1 to 6 in detecting Escherichia coli.
8. A method for detecting Escherichia coli, comprising the following steps: The electrochemical immunosensor module is placed in a solution of a sample to be tested, and after incubation, is placed in an acetate buffer solution containing thionine and hydrogen peroxide for detection; the content of Escherichia coli in the sample to be tested is obtained using a chronoamperometry method.
9. The use according to claim 7 or the method according to claim 8, characterized in that: The Escherichia coli is Escherichia coli O157:H7.