Label-free immunosensor for helicobacter pylori and preparation method and application thereof

Abstract

This application relates to a label-free immunosensor for Helicobacter pylori, its preparation method, and its application. The method involves directly incubating the Helicobacter pylori multivalent epitope antigen FvpE on a modified electrode, eliminating the need for hybridization labeling during the preparation of the label-free immunosensor. This simplifies the preparation process, making it simple and easy to operate. Furthermore, the prepared label-free immunosensor can directly detect Helicobacter pylori in samples, greatly simplifying the detection process and reducing detection time. Simultaneously, the prepared label-free immunosensor can detect polyclonal antibodies anti-Hp and different antibody genotypes such as CagA, UreA, UreB, and VacA, and can simultaneously detect multiple cytotoxins or virulence factors. This improves the universality of the label-free immunosensor, thereby reducing workload, shortening detection time, and lowering detection costs.

Description

Technical Field

[0001] This application relates to the field of Helicobacter pylori detection technology, and in particular to a label-free immunosensor for Helicobacter pylori, its preparation method, and its application. Background Technology

[0002] Helicobacter pylori (H. pylori) is a spiral-shaped or S-shaped, microaerophilic Gram-negative bacillus. H. pylori was first isolated and cultured in 1982 and classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IRAC) under the World Health Organization (WHO) in 1994. Humans, as the only natural host of H. pylori, are universally susceptible, but the severity and onset of symptoms vary significantly, demonstrating marked differences in pathogenicity. The type of disease in H. pylori-infected individuals is closely related to the type of bacterial strain infecting them. H. pylori strains can produce various cytotoxins and virulence factors, such as cytotoxin-associated gene A (CagA), vacuole toxin gene A (VacA), urease (Ure), and neutrophil activating protein (NAP). Among them, type I virulent H. pylori strains (which produce CagA and VacA virulence factors) are significantly associated with the occurrence of peptic ulcers and gastric cancer, while type II attenuated H. pylori strains (which do not produce CagA and VacA virulence factors) generally only cause mild gastritis. Therefore, rapid and sensitive diagnosis of H. pylori infection is of great significance for preventing the occurrence of related diseases and for timely treatment.

[0003] Methods for detecting H. pylori infection can be broadly categorized into non-invasive and invasive methods. Non-invasive methods include the urea breath test, fecal antigen detection, serological antibody detection, and urine antibody detection; invasive methods include endoscopy, histological examination, and rapid urease test (RUT). The development of electrochemical methods has provided new theories and approaches for clinical testing. Electrochemical sensors have attracted widespread attention due to their high sensitivity, low cost, low detection limits, and ease of operation. Furthermore, DNA detection has advantages in electrochemical and piezoelectric fields compared to optical detection. DNA electrochemical hybridization can construct simple and sensitive detection platforms. Therefore, in recent years, DNA electrochemical sensors for detecting Helicobacter pylori have seen some development.

[0004] Currently, various electrochemical analysis methods for detecting Helicobacter pylori have been published. For example, Chinese invention patent No. 202010597081.3 provides a Helicobacter pylori nucleic acid sensor and detection method and application, which detects Helicobacter pylori through a constructed DNA electrochemical sensor. Other existing technologies also disclose papers on the detection of Helicobacter pylori using constructed DNA electrochemical sensors. It is evident that the technology for detecting Helicobacter pylori using DNA electrochemical sensors is relatively mature. However, as revealed by the published technical solutions, the process of constructing DNA electrochemical sensors is currently relatively complex and cumbersome. It requires hybridization labeling, a complex preparation process, and sample pretreatment labeling before detection. It cannot directly detect urine or blood samples. Monitoring the immunoassay reaction through changes in the amount of the detected label makes the detection process complex and time-consuming. Furthermore, since each constructed DNA electrochemical sensor can only target a specific DNA sequence, each constructed DNA electrochemical sensor can only target one type of cytotoxin or virulence factor, resulting in poor universality of the DNA electrochemical sensor. When detecting Helicobacter pylori in samples, it is necessary to construct DNA electrochemical sensors for each cytotoxin or virulence factor in order to accurately detect which specific cytotoxin or virulence factor is present in the sample. This results in a large workload, long detection time, and high detection cost. Summary of the Invention

[0005] Therefore, it is necessary to address the fact that the construction of DNA electrochemical sensors in existing technologies is relatively complex and cumbersome, requiring hybridization labeling, complicated preparation processes, and lengthy detection times. Furthermore, each constructed DNA electrochemical sensor can only target one cytotoxin or virulence factor, resulting in poor universality. When detecting Helicobacter pylori in samples, it is necessary to construct DNA electrochemical sensors targeting each cytotoxin or virulence factor, leading to a large workload, long detection time, and high detection costs. This paper provides a label-free immunosensor for Helicobacter pylori, its preparation method, and its application. The Helicobacter pylori multivalent epitope antigen FvpE is directly incubated on a modified electrode, eliminating the need for hybridization labeling during the preparation of the label-free immunosensor, simplifying the preparation process, making the method simple and easy to operate. Moreover, the prepared label-free immunosensor can directly detect Helicobacter pylori in samples, greatly simplifying the detection process and reducing detection time. Meanwhile, the prepared label-free immunosensor for Helicobacter pylori can detect polyclonal antibodies anti-Hp and different antibody genotypes such as CagA, UreA, UreB and VacA. It can simultaneously detect multiple cytotoxins or virulence factors, thereby improving the universality of the label-free immunosensor for Helicobacter pylori, which can reduce the workload, shorten the detection time and reduce the detection cost.

[0006] A method for preparing a label-free immunosensor for Helicobacter pylori includes the following steps:

[0007] S10. Take carbon nanofibers into nano-gold sol, stir evenly, and then dry to obtain nano-gold modified carbon nanofibers.

[0008] S20. Take the gold-modified carbon nanofibers, carbon nanopowder, titanium carbide and ionic liquid and place them in a mortar, add a binder, grind evenly to obtain a paste;

[0009] S30. The paste is filled into a container and heated to obtain an electrode column. The surface of the electrode column is polished smooth to obtain a modified electrode.

[0010] S40. Helicobacter pylori multivalent epitope antigen FVpE was dropped onto the surface of the modified electrode, incubated, rinsed with ultrapure water, dried, and bovine serum albumin was added for further incubation. After incubation, a label-free immunosensor for Helicobacter pylori was obtained.

[0011] Preferably, in the above preparation method, in step S10, 80 to 120 parts of the carbon nanofibers and 160 to 240 parts of the gold nanosol are taken and stirred evenly.

[0012] Preferably, in the above preparation method, in step S20, 23 to 27 parts of the gold-modified carbon nanofibers, 18 to 22 parts of the carbon nanoparticles, 4 to 6 parts of the titanium carbide, and 23 to 27 parts of the ionic liquid are taken.

[0013] Preferably, in the above preparation method, in step S20, the binder is paraffin oil, and 18 to 22 parts of the paraffin oil are added dropwise; the ionic liquid is 1-octylpyridine hexafluorophosphate.

[0014] Preferably, in the above preparation method, in step S30, the paste is filled into a polytetrafluoroethylene tube.

[0015] Preferably, in the above preparation method, in step S40, 4 to 5 parts of the Helicobacter pylori multivalent epitope antigen FVpE are dropped onto the surface of the modified electrode, incubated for 35 to 45 minutes, rinsed with ultrapure water, dried, and then 4 to 5 parts of bovine serum albumin are dropped onto the electrode and incubated for 35 to 45 minutes. After incubation, the Helicobacter pylori electrochemical immunosensor is obtained, and the concentration of the Helicobacter pylori multivalent epitope antigen FvpE immobilized on the surface of the modified electrode is 1 mg / mL to 1.5 mg / mL.

[0016] Preferably, in the above preparation method, before step S40, the following step is further included:

[0017] An artificial gene NCV was synthesized, wherein the artificial gene NCV contains conserved antigenic epitope enrichment segments from NAP, CagA302-437 and VacA1-46 / 332-494, and the artificial gene NCV has Nco I and Hind III restriction sites at both ends.

[0018] The artificial gene NCV was inserted into plasmid pET-28a to construct the recombinant plasmid pET-NCV.

[0019] The UE gene was amplified from pET-CUE using PCR technology, and the UE gene was inserted into the recombinant plasmid pET-NCV to construct the recombinant expression vector pET-FVpE.

[0020] The recombinant expression vector pET-FVpE was transformed into Escherichia coli, and expression was induced by IPTG at a preset temperature. The Helicobacter pylori multivalent epitope antigen FvpE was obtained by protein purification techniques such as Ni-NTA affinity chromatography.

[0021] A label-free immunosensor for Helicobacter pylori is prepared using a method for preparing a label-free immunosensor for Helicobacter pylori as described in any one of the above methods.

[0022] An application of the aforementioned label-free immunosensor for Helicobacter pylori in the field of Helicobacter pylori detection is characterized by comprising the following steps:

[0023] Construct a label-free immunosensor for Helicobacter pylori as described above;

[0024] An electrolyte system was constructed, which consisted of the sample solution to be tested, a K3[Fe(CN)6] / K4[Fe(CN)6] mixed probe solution, and a PBS buffer solution.

[0025] The label-free immunosensor for Helicobacter pylori was incubated in an electrolyte system, and the concentration of Helicobacter pylori was detected after incubation.

[0026] Preferably, in one of the above applications, the electrolyte system consists of the sample solution to be tested, a 5mM K3[Fe(CN)6] / K4[Fe(CN)6] solution containing 0.1M KCl, and a 0.01M PBS buffer solution, and the pH of the electrolyte system is 6 to 8. The label-free immunosensor for Helicobacter pylori is incubated in the electrolyte system for 40 to 50 minutes. After incubation, the concentration of Helicobacter pylori is detected by differential pulse voltammetry, with a scanning range of -0.2V to 0.5V.

[0027] The technical solution adopted in this application can achieve the following beneficial effects:

[0028] The label-free immunosensor for Helicobacter pylori disclosed in this application, its preparation method, and its application involve directly incubating the Helicobacter pylori multivalent epitope antigen FvpE on a modified electrode. This eliminates the need for hybridization labeling during the preparation of the label-free immunosensor, simplifying the preparation process. The method is simple and easy to operate. Furthermore, the prepared label-free immunosensor can directly detect Helicobacter pylori in samples by directly measuring the physical and chemical changes during antigen-antibody complex formation, greatly simplifying the detection process and reducing detection time. Simultaneously, the prepared label-free immunosensor can detect polyclonal antibodies anti-Hp and different antibody genotypes such as CagA, UreA, UreB, and VacA. The detection limits for anti-Hp, CagA, UreA, and UreB are all 0.001 ng / mL. -1 The detection limit for VacA is 0.01 ng / mL. -1As can be seen, the label-free immunosensor for Helicobacter pylori can simultaneously detect multiple cytotoxins or virulence factors, thereby improving its universality, reducing workload, shortening detection time, and lowering detection costs. After storing the prepared label-free immunosensor at 4℃ for two months, antibody detection results showed that the current difference before and after the immune reaction decreased by only 11.51% compared to two months prior, indicating good long-term stability and reproducibility of the constructed label-free immunosensor. The relative standard deviation of six measurements was only 2.37%. When the prepared label-free immunosensor was applied to the detection of Helicobacter pylori, the high specificity of antigen-antibody binding reduced non-specific interference, resulting in high sensitivity and selectivity. The spiked recovery rate ranged from 68.5% to 100.5%, and the constructed label-free immunosensor can be used for the detection of Helicobacter pylori in actual samples. Attached Figure Description

[0029] Figure 1 This is a schematic diagram illustrating the preparation process and response mechanism of the label-free immunosensor for Helicobacter pylori disclosed in the embodiments of this application;

[0030] Figure 2 This is a schematic diagram of the SEM surface morphology characterization of the label-free immunosensor for Helicobacter pylori disclosed in the embodiments of this application;

[0031] Figure 3 This is a schematic diagram of the DPV electrochemical characterization of the label-free immunosensor for Helicobacter pylori disclosed in the embodiments of this application;

[0032] Figure 4 This is a schematic diagram of the EIS electrochemical characterization of the label-free immunosensor for Helicobacter pylori disclosed in the embodiments of this application;

[0033] Figure 5 This is a schematic diagram illustrating the FVpE fixation capabilities of different modified electrodes disclosed in the embodiments of this application;

[0034] Figure 6 This is a schematic diagram illustrating the binding ability of the label-free immunosensor for Helicobacter pylori against various antibody types of Helicobacter pylori disclosed in the embodiments of this application.

[0035] Figure 7 This is a schematic diagram illustrating the effect of different pH values ​​on the response of the label-free immunosensor for Helicobacter pylori disclosed in the embodiments of this application;

[0036] Figure 8This is a schematic diagram illustrating the effect of different fixed antigen concentrations on the response of the label-free immunosensor for Helicobacter pylori, as disclosed in the embodiments of this application.

[0037] Figure 9 This is a schematic diagram illustrating the effect of different incubation times on the response of the label-free immunosensor for Helicobacter pylori, as disclosed in the embodiments of this application.

[0038] Figure 10 This is a schematic diagram illustrating the electrochemical analysis of different concentrations of different antibodies against Helicobacter pylori using differential pulse voltammetry under optimal conditions, based on the label-free immunosensor for Helicobacter pylori disclosed in this application.

[0039] Figure 11 This is a schematic diagram of the detection results of the label-free immunosensor for Helicobacter pylori disclosed in the embodiments of this application during the specificity study process. Detailed Implementation

[0040] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of this application.

[0041] It should be noted that when an element is referred to as being "set on" another element, it can be directly on the other element or there may be an intervening element. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or there may be an intervening element. The terms "vertical," "horizontal," "left," "right," "top," "bottom," "end," "top," and similar expressions used herein are for illustrative purposes only and do not represent the only possible implementation.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0043] This application discloses a method for preparing a label-free immunosensor for Helicobacter pylori, comprising the following steps:

[0044] S10. Take carbon nanofibers into nano-gold sol, stir evenly, and then dry to obtain nano-gold modified carbon nanofibers.

[0045] Nanofibers and gold nanoparticle sol were separately placed in a stirring container and stirred until homogeneous. After stirring, the mixture was allowed to air dry naturally to obtain gold-modified carbon nanofibers. These were then removed from the stirring container for later use. Specifically, 80 to 120 parts of carbon nanofibers and 160 to 240 parts of gold nanoparticle sol could be taken and stirred until homogeneous. It should be noted that each part of carbon nanofiber is measured in grams (g), and each part of gold nanoparticle sol is measured in milliliters (mL). For example, 0.8 to 1.2 g of carbon nanofibers and 1.6 to 2.4 mL of gold nanoparticle sol could be taken, for instance. 1 g of carbon nanofibers and 2 mL of gold nanoparticle sol could be stirred until homogeneous and then allowed to air dry naturally to obtain gold-modified carbon nanofibers.

[0046] S20. Take nano-gold modified carbon nanofibers, nano-carbon powder, titanium carbide and ionic liquid and place them in a mortar, add binder dropwise, grind evenly to obtain a paste;

[0047] Nano-gold modified carbon nanofibers, nano-carbon powder, titanium carbide, and ionic liquid were weighed separately and placed in an agate mortar. An appropriate amount of binder was added to the agate mortar, and the mixture was ground evenly to obtain a paste. The ionic liquid can be 1-octylpyridine hexafluorophosphate. Preferably, 23 to 27 parts of gold-modified carbon nanofibers, 18 to 22 parts of carbon nanoparticles, 4 to 6 parts of titanium carbide, and 23 to 27 parts of ionic liquid can be taken respectively. It should be noted that each part of gold-modified carbon nanofibers, carbon nanoparticles, titanium carbide, and ionic liquid is in g. Specifically, 0.023 g to 0.027 g of gold-modified carbon nanofibers, 0.018 g to 0.022 g of carbon nanoparticles, 0.004 g to 0.006 g of titanium carbide, and 0.023 g to 0.027 g of ionic liquid can be weighed. For example, 0.025 g of gold-modified carbon nanofibers, 0.02 g of carbon nanoparticles, 0.005 g of titanium carbide, and 0.025 g of ionic liquid can be weighed and placed in an agate mortar. An appropriate amount of binder can be added to the agate mortar, and the mixture can be mixed and ground evenly to obtain a paste. Furthermore, the binder can be paraffin oil. Under the above raw material dosage standards, the amount of binder, i.e., paraffin oil, added can be 18 to 22 parts. It should be noted that each part of paraffin oil is in μL. Specifically, the amount of paraffin oil added can be 18 μL to 22 μL, so that the above raw materials can be fully dispersed while facilitating the formation of carbon nanofiber paste.

[0048] S30. Fill a container with a paste, heat it to obtain an electrode column, and polish the surface of the electrode column to obtain a modified electrode;

[0049] The prepared paste is filled into a polytetrafluoroethylene (PTFE) tube or glass tube, heated, and an electrode column is obtained. This column is then pressed onto polishing paper and polished smooth to obtain a modified electrode, also known as a titanium carbide / gold nanoparticle sol / ionic liquid composite modified carbon nanofiber paste electrode (Ti3C2Tx / AuNPs@CNFILPE). The PTFE tube can be 2 mm thick.

[0050] S40. Helicobacter pylori multivalent epitope antigen FVpE was dropped onto the surface of the modified electrode, incubated, rinsed with ultrapure water, dried, and bovine serum albumin was added for further incubation. After incubation, a label-free immunosensor for Helicobacter pylori was obtained.

[0051] Preferably, 4 to 5 parts of Helicobacter pylori multivalent epitope antigen FvpE are dropped onto the surface of the modified electrode, incubated for 35 to 45 minutes, rinsed with ultrapure water, and air-dried to immobilize the Helicobacter pylori multivalent epitope antigen FvpE on the modified electrode surface. Then, 4 to 5 parts of bovine serum albumin are added and incubated for 35 to 45 minutes to obtain a label-free Helicobacter pylori immunosensor. It should be noted that the units for each part of Helicobacter pylori multivalent epitope antigen FvpE and bovine serum albumin are μL.

[0052] As the concentration of antigen immobilized on the modified electrode surface increases, the response of the label-free Helicobacter pylori immunosensor is enhanced. Therefore, the concentration of Helicobacter pylori multivalent epitope antigen FvpE immobilized on the modified electrode surface can be from 1 mg / mL to 1.5 mg / mL.

[0053] The Helicobacter pylori multivalent epitope antigen FvpE mentioned above was artificially prepared before the preparation of the Helicobacter pylori label-free immunosensor. Specifically, an artificial gene NCV was first synthesized. The artificial gene NCV contains conserved antigenic epitope enrichment segments from NAP, CagA302-437 and VacA1-46 / 332-494, and the artificial gene NCV has NcoI and Hind III restriction sites at both ends.

[0054] The artificial gene NCV was inserted into plasmid pET-28a to construct the recombinant plasmid pET-NCV;

[0055] The UE gene was amplified from pET-CUE using PCR technology, and the UE gene was inserted into the recombinant plasmid pET-NCV to construct the recombinant expression vector pET-FVpE.

[0056] The recombinant expression vector pET-FVpE was transformed into Escherichia coli, and expression was induced by IPTG at a preset temperature (e.g., 37°C). The multivalent epitope antigen FvpE of Helicobacter pylori was obtained by protein purification techniques such as Ni-NTA affinity chromatography.

[0057] The purity and physicochemical properties of Helicobacter pylori multivalent epitope antigen FvpE can be detected by high performance liquid chromatography (HPLC) Model-L2000, acid-base titration, and other techniques.

[0058] This application also discloses a label-free immunosensor for Helicobacter pylori, which is prepared using the method described above for preparing a label-free immunosensor for Helicobacter pylori.

[0059] This application also discloses the application of the label-free immunosensor for Helicobacter pylori as described above in the field of Helicobacter pylori detection, specifically for the detection of Helicobacter pylori. The application process includes the following steps:

[0060] Construct a label-free immunosensor for Helicobacter pylori as described above;

[0061] A label-free immunosensor for Helicobacter pylori was prepared using the method described above. A three-electrode system was constructed with a saturated calomel electrode (SCE) as the reference electrode, a platinum wire as the auxiliary electrode, and the prepared label-free immunosensor for Helicobacter pylori as the working electrode.

[0062] An electrolyte system was constructed, consisting of the sample solution to be tested, a K3[Fe(CN)6] / K4[Fe(CN)6] mixed probe solution, and a PBS buffer solution.

[0063] Preferably, the electrolyte system consists of the sample solution to be tested, a 5mM K3[Fe(CN)6] / K4[Fe(CN)6] solution containing 0.1M KCl, and a 0.01M PBS buffer solution, and the pH of the electrolyte system is 6 to 8.

[0064] A label-free immunosensor for Helicobacter pylori was incubated in an electrolyte system, and the concentration of Helicobacter pylori was detected after incubation.

[0065] Specifically, the label-free immunosensor for Helicobacter pylori was incubated in an electrolyte system for 40 to 50 minutes. After incubation, the concentration of Helicobacter pylori was detected using differential pulse voltammetry, with a scanning range of -0.2V to 0.5V. Differential pulse voltammetry (DPV) was recorded within the -0.2V to 0.5V scanning range to study the antigen-antibody immune response. Based on the relationship between the response current of the label-free immunosensor and the concentration of Helicobacter pylori, the concentration of Helicobacter pylori was obtained directly or indirectly.

[0066] The label-free immunosensor for Helicobacter pylori disclosed in this application, its preparation method, and its application involve directly incubating the Helicobacter pylori multivalent epitope antigen FvpE on a modified electrode. This eliminates the need for hybridization labeling during the preparation of the label-free immunosensor, simplifying the preparation process. The method is simple and easy to operate. Furthermore, the prepared label-free immunosensor can directly detect Helicobacter pylori in samples by directly measuring the physical and chemical changes during antigen-antibody complex formation, greatly simplifying the detection process and reducing detection time. Simultaneously, the prepared label-free immunosensor can detect polyclonal antibodies anti-Hp and different antibody genotypes such as CagA, UreA, UreB, and VacA. The detection limits for anti-Hp, CagA, UreA, and UreB are all 0.001 ng / mL. -1 The detection limit for VacA is 0.01 ng / mL. -1 As can be seen, the label-free immunosensor for Helicobacter pylori can simultaneously detect multiple cytotoxins or virulence factors, thereby improving its universality, reducing workload, shortening detection time, and lowering detection costs. After storing the prepared label-free immunosensor at 4℃ for two months, antibody detection results showed that the current difference before and after the immune reaction decreased by only 11.51% compared to two months prior, indicating good long-term stability and reproducibility of the constructed label-free immunosensor. The relative standard deviation of six measurements was only 2.37%. When the prepared label-free immunosensor was applied to the detection of Helicobacter pylori, the high specificity of antigen-antibody binding reduced non-specific interference, resulting in high sensitivity and selectivity. The spiked recovery rate ranged from 68.5% to 100.5%, and the constructed label-free immunosensor can be used for the detection of Helicobacter pylori in actual samples.

[0067] The detection principle of the label-free immunosensor for Helicobacter pylori is as follows: antigens or antibodies are immobilized on electrodes. When they bind to the specific antigens or antibodies to be tested in the solution, they cause a change in the charge density at the interface between the electrode surface membrane and the solution, resulting in a change in membrane potential. The degree of change is proportional to the concentration of the antigens or antibodies to be tested in the solution. Thus, the change in membrane potential is used to analyze the concentration of the antigens or antibodies to be tested in the solution.

[0068] The following detailed experimental process further illustrates the technical solution and technical effects of this application.

[0069] The sources, specifications, and types of experimental instruments used in the following experiments are as follows:

[0070] Bovine serum albumin (BSA) (Shanghai Aladdin Biotechnology Co., Ltd.), 1-octylpyridine hexafluorophosphate (Lanzhou Institute of Chemical Physics), carbon nanofibers (Shanghai Aladdin Biotechnology Co., Ltd.), nano-carbon powder (Nanjing Xianfeng Nanomaterials Technology Co., Ltd.), titanium carbide (Nanjing Xianfeng Nanomaterials Technology Co., Ltd.), nano-gold sol (Nanjing Xianfeng Nanomaterials Technology Co., Ltd.), CagA Ab (2.8 mg / mL) -1 , Fitzgerald Industries International, Inc., USA), VacA Ab (2mg mL -1 )), UreA Ab (4mg mL) -1 ), UreB Ab (1mg mL) -1 (LifeSpan BioSciences, Inc.), Rabbit anti-H. pylori polyclonal antibody (12 mg / mL) -1 (Shanghai Linc-BioScience Co., Ltd.). Differential pulse voltammetry (DPV) was performed on a CHI660E electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd., China), and electrochemical impedance spectroscopy (EIS) was performed on a PARSTAT 4000 electrochemical workstation (Princeton, USA). A three-electrode system was used with a saturated calomel electrode (SCE) as the reference electrode, a platinum wire as the auxiliary electrode, and the prepared label-free immunosensor for Helicobacter pylori as the working electrode. The system was operated by a Hitachi S-3400N scanning electron microscope (Tokyo, Japan).

[0071] I. Obtaining high-purity H. pylori multivalent epitope antigen FVpE

[0072] First, an artificial gene named NCV was synthesized. NCV contains conserved epitope enrichment regions from NAP, CagA302-437, and VacA1-46 / 332-494, and has NcoI and HindIII restriction sites at both ends. Then, NCV was inserted into plasmid pET-28a to construct the recombinant plasmid pET-NCV. Next, the UE gene was amplified from pET-CUE using PCR and inserted into the recombinant plasmid pET-NCV to construct the recombinant expression vector pET-FVpE. The recombinant expression vector pET-FVpE was transformed into *E. coli*, and expression was induced by IPTG at 37°C. High-purity *H. pylori* multivalent epitope antigen FvpE (hereinafter referred to as FvpE) was obtained using protein purification techniques such as Ni-NTA affinity chromatography.

[0073] II. Preparation of Helicobacter pylori electrochemical immunosensor

[0074] First, 1g of carbon nanofibers and 2mL of nano-gold sol were stirred evenly and then air-dried to obtain nano-gold modified carbon nanofibers. Preparation of the modified electrode: 0.025g of nano-gold modified carbon nanofibers, 0.02g of nano-carbon powder, 0.005g of titanium carbide, and 0.025g of ionic liquid were placed in an agate mortar, and 20μL of paraffin oil was added. The mixture was ground evenly. Then, a certain amount of the paste was inserted into a 2mm polytetrafluoroethylene tube, heated for 2 to 3 minutes, pressed firmly on polishing paper, and polished smooth. The resulting electrode is called a titanium carbide / nano-gold sol / ionic liquid composite modified carbon nanofiber paste electrode (Ti3C2Tx / AuNPs@CNFILPE). Preparation of the Helicobacter pylori electrochemical immunosensor: 5 μL of FVpE was added to the surface of the modified electrode, incubated for 40 min, rinsed with ultrapure water, and dried. Then, 5 μL of BSA was added and incubated for another 40 min to obtain the Helicobacter pylori electrochemical immunosensor (hereinafter referred to as the immunosensor). The preparation process and response mechanism of this immunosensor are as follows: Figure 1 As shown. For comparative studies, carbon nanofiber paste electrodes (CNFPE) and titanium carbide-modified carbon nanofiber paste electrodes (Ti3C2T) were also prepared. X / CNFPE), gold nanofiber modified carbon nanofiber paste electrode (AuNPs@CNFPE) and carbon nanofiber ionic liquid paste electrode (CNFILPE).

[0075] III. Analytical Methods

[0076] In a 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution containing 0.1 M KCl, electrochemical impedance spectroscopy (EIS) was used at a scan frequency of 0.1 Hz–10 Hz. 5 Electrochemical impedance spectroscopy (Niquist plots) were recorded in the Hz range to study the electrochemical performance of the modified electrode. The immunosensor was incubated in a Helicobacter pylori antibody solution for 45 min to allow for a full immune response. Then, differential pulse voltammetry (DPV) was recorded in 0.01 M phosphate buffered saline (PBS) containing 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] within a scan range of -0.2 V to 0.5 V to study the antigen-antibody immune response.

[0077] IV. Surface Morphology Characterization

[0078] Scanning electron microscopy (SEM) was used to characterize the surface morphology of the nanocomposite materials and the sensor, and to study the microstructure of the modified electrode surface. Figure 2Image A is a scanning electron microscope (SEM) image of Ti3C2Tx / AuNPs@CNFILPE. The surface shows a uniform, particulate distribution of the titanium carbide / gold nanoparticle / ionic liquid composite material, which effectively increases the antigen loading. Figure 2 As shown in Figure B, after adding antigen FVpE, a thin film was observed covering the electrode surface on FVpE / Ti3C2Tx / AuNPs@CNFILPE, indicating that the antigen had been successfully immobilized onto the modified electrode. After blocking non-specific binding sites with BSA (…),… Figure 2 C), BSA / FVpE / Ti3C2T x The film thickness on the / AuNPs@CNFILPE surface is significantly increased. After the immunosensor is immersed in the CagA antibody solution, an immune reaction occurs, generating an immune complex; the surface morphology after this is as follows. Figure 2 As shown in D (CagA Ab / BSA / FVpE / Ti3C2T) x / AuNPs@CNFILPE), this structure provides a good biocompatible platform and a large number of binding sites for the immobilization of immune components.

[0079] V. Electrochemical Characterization

[0080] The modified electrode was characterized using differential pulse voltammetry (DPV) in a 5 mM potassium ferricyanide and potassium ferrocyanide mixed probe solution containing 0.1 M KCl. The results are as follows: Figure 3 As shown, the peak current on the carbon nanofiber paste electrode (CNFPE, curve h) is relatively small. The peak current increases significantly after the addition of modifiers titanium carbide (curve g), gold nanoparticles (curve f), and ionic liquid (curve b), respectively. The peak current reaches its maximum on the titanium carbide / gold nanoparticle-modified carbon nanofiber ionic liquid paste electrode (curve a), indicating that this modified electrode can provide a biocompatible platform for signal amplification. When antigen FVpE is added to the modified electrode (curve c), the peak current decreases significantly due to the electrochemical inertness of the protein. Next, after blocking the non-specific adsorption sites on the modified electrode surface with BSA (curve d), the peak current decreases further. Finally, due to the immune reaction between CagA Ab and antigen FVpE (curve e), the current value continues to decrease, indicating that this immunosensor can selectively detect CagA Ab.

[0081] The electrochemical performance of the electrochemical immunosensor was further characterized using electrochemical impedance spectroscopy (EIS). The size of the semicircle diameter in the impedance spectrum reflects the electron transfer impedance on the modified electrode; a larger semicircle diameter indicates a greater impedance. Figure 4 As shown, the modified electrode Ti3C2T XThe approximate straight line of / AuNPs@CNFILPE (curve a) indicates a low charge transfer impedance of the modified electrode, suggesting that the composite material of titanium carbide, gold nanoparticles, and ionic liquids possesses good conductivity and can promote the electron transfer process of the modified electrode. When the non-electroactive FVpE is immobilized on the modified electrode (curve b), the resistance increases, indicating successful immobilization of FVpE. After blocking with BSA (curve c), the impedance further increases, indicating successful construction of the Helicobacter pylori electrochemical immunosensor. Incubation in a probe solution containing CagA Ab (curve d) results in a significant increase in impedance due to the highly sensitive and specific binding between FVpE and CagA Ab, forming an immune complex that hinders charge transfer. This result is consistent with the DPV study.

[0082] VI. The ability of the modified electrode to immobilize FVpE

[0083] To verify that the composite material can provide a good biocompatible platform for FVpE, we investigated the immobilization ability of FVpE by different modified electrodes. Figure 5 It can be seen that FVpE is dropped into carbon nanofiber paste electrode (CNFPE). Figure 5 The peak current before and after (A) did not decrease, indicating that CNFPE has a poor adsorption and fixation capacity for FVpE. However, the peak current before and after (A) was not lower. Figure 5 B), Ti3C2Tx@CNFPE( Figure 5 C), CNFILPE( Figure 5 D), Ti3C2Tx / AuNPs@CNFILPE( Figure 5 The peak current decreased to varying degrees before and after FVpE fixation on CNFILPE, indicating that CNFILPE and Ti3C2Tx / AuNPs@CNFILPE have good FVpE fixation capabilities. Given that the immunosensor fabrication process involves several peak current reductions, the modified electrode Ti3C2Tx / AuNPs@CNFILPE, which exhibits the highest peak current before FVpE fixation, was selected as the substrate electrode.

[0084] VII. The binding ability of immune sensors to antibodies

[0085] like Figure 6 As shown, the prepared immunosensors were respectively reacted with Helicobacter pylori polyclonal antibodies (anti-H. pylori, Figure 6 A), Cytotoxin-associated gene A antibody (CagA Ab, Figure 6 B), Urease A antibody (UreA Ab, Figure 6 C), Urease B antibody (UreB Ab, Figure 6 D), Vacuole toxin-associated gene A antibody (VacA Ab, Figure 6 E) After binding, the current values ​​of the antibodies decreased significantly, indicating that the immunosensor has the ability to bind to all antibody types of Helicobacter pylori.

[0086] VIII. Optimization of Experimental Conditions

[0087] (1) Effect of pH

[0088] We used DPV to study the effect of different pH values ​​on the response of the immunosensor. Figure 7 As shown, the current difference before and after the immune response increased continuously as the solution pH changed from 5 to 7.4. However, the current difference decreased as the pH continued to increase, indicating that the immunosensor had the best response at pH 7.4. This phenomenon may be due to the fact that both excessively low and high pH values ​​can affect the activity of protein molecules. Therefore, this experiment selected PBS with pH 7.4 containing 5 mM mixed probes as the optimal electrolyte solution.

[0089] (2) Antigen concentration

[0090] Figure 8 The effect of FVpE antigen concentration immobilized on the electrode surface on the responsiveness of the immunosensor was investigated. Results showed that within the range of 0.1–1.0 mg / mL... -1 As the range of antigen concentration changes, the response of the immunosensor increases with increasing antigen concentration; ultimately, 1 mg / mL was selected. -1 The concentration of the fixed antigen.

[0091] (3) Incubation time

[0092] The degree of completion of specific immune recognition is related to the time of the immune response. We immersed the constructed immunosensor in a probe solution containing Helicobacter pylori antibodies and discussed the effect of different incubation times (5 min-60 min) on the response current. The results are as follows: Figure 9 As shown, the response current of the immunosensor decreases with increasing incubation time, but after incubation time exceeds 45 minutes, the current value no longer shows a significant decrease, indicating that the immune response is complete at 45 minutes. Therefore, 45 minutes was selected as the optimal incubation time for the immune response.

[0093] IX. Electrochemical Detection of Several Different Antibodies

[0094] The prepared immunosensor was electrochemically analyzed under optimal conditions using differential pulse voltammetry to differentiate Helicobacter pylori antibodies at different concentrations, and the current values ​​(IL) at the corresponding concentrations were recorded. x The current values ​​(I0) of the blank sample and the current difference (ΔI) are given by the blank sample peak current and the peak current at the response concentration, i.e., ΔI = I0 - I x .like Figure 10 As shown, with the increase of antibody concentration, the immune complex on the sensor also increases, and the difference in peak current before and after the immune response gradually increases. Figure 10 As shown in A, the polyclonal antibody anti-Hp is effective in doses ranging from 0.1 to 5 ng / mL. -1 Within the specified range, the peak current difference (ΔI) showed a good linear relationship with the antibody concentration, with the linear equation being: ΔI = 5.4468C (ng / mL). -1 )+17.558(R 2 =0.9848). Similarly, from Figure 10 B, 10C, and 10E indicate that the linear range for CagA, UreA, and VacA is 0.1 to 5 ng / mL. -1 The linear equations are as follows: ΔI (CagA) = 4.4905C (ng / mL) -1 )+8.1628(R 2 =0.9742); ΔI (UreA) =3.8242C (ng / mL) -1 )+11.018(R 2 =0.9714); ΔI (VacA) =2.4508C (ng / mL) -1 )+9.4367(R 2 =0.8744). For example... Figure 10 As shown in D, the linear range of UreB is 0.1 to 4.5 ng / mL. -1 The linear equation is: ΔI (UreB) = 8.6666C (ng / mL) -1 )+21.018(R 2 =0.9268). Among them, the detection limits for anti-Hp, CagA, UreA, and UreB were all 0.001 ng / mL. -1 The detection limit for VacA is 0.01 ng / mL. -1 The results show that the constructed Helicobacter pylori electrochemical immunosensor can analyze polyclonal antibodies against Hp and different antibody genotypes such as CagA, UreA, UreB, and VacA.

[0095] 10. Specificity Studies

[0096] To investigate the selectivity of the immune sensor, we detected 0.2 ng mL⁻¹ of anti-Hp, CagA Ab, UreA Ab, UreB Ab, VacA Ab, and 20-fold concentrations of dopamine transporter monoclonal antibody (DAT), growth-associated protein antibody (GAP-43), G protein-coupled receptor 75 antibody (GRP75), and tyrosine-DNA phosphodiesterase antibody (TDP) in PBS solution containing 5 mM probe. The results are as follows: Figure 11 As shown, the peak current difference (ΔI) of each antibody against Helicobacter pylori before and after the immune response is relatively large, but the ΔI value of high concentrations of interfering substances does not change significantly, indicating that this immunosensor can be applied to the high-sensitivity and high-selectivity detection of Helicobacter pylori.

[0097] XI. Reproducibility and Stability Studies

[0098] The same Helicobacter pylori electrochemical immunosensor was incubated in a solution containing 0.2 ng mL⁻¹ CagA antibody, followed by detection in PBS containing 5 mM mixed probes. The relative standard deviation of six measurements was 2.37%, indicating good reproducibility of the immunosensor. Furthermore, after storing the prepared electrochemical immunosensor at 4°C for two months, antibody detection showed that the current difference before and after the immune reaction decreased by only 11.51% compared to two months prior. This means that after two months, the response value of the immunosensor was 88.49% of the initial value, indicating good long-term stability of the constructed electrochemical immunosensor.

[0099] 12. Actual Sample Testing

[0100] To verify its practical application value, the constructed electrochemical immunosensor was used to analyze Helicobacter pylori in human serum. Human serum was diluted 100-fold with 0.01M PBS containing mixed probes and stored at 4°C for later use. The constructed immunosensor was incubated in the test solution for 45 min, and detection was performed using DPV. Then, 2.00 ng / mL of the solution was added. -1 The anti-Hp assay was performed again, and the results are shown in Table 1. A concentration of 0.36 ng / mL was detected in sample 1. -1 Anti-Hp antibodies were not detected in samples 2 and 3, and the recoveries ranged from 68.5% to 100.5%. These results indicate that the constructed immunosensor can be used for the detection of Helicobacter pylori in real samples.

[0101] Table 1. Helicobacter pylori in samples determined by label-free immunosensor (n=3) a )

[0102]

[0103] a. Measure the average of three measurements.

[0104] This application document constructs a Ti3C2T-based... x / AuNPs@CNFILPE label-free immunosensor for Helicobacter pylori. Ti3C2T x The / AuNPs / IL composite material provides a good biocompatibility platform for FVpE immobilization, and its good loading capacity provides more binding sites for the immune response of various Helicobacter pylori antibodies. The prepared label-free immunosensor for Helicobacter pylori can be used for the detection of various antibodies such as anti-Hp, CagA, UreA, UreB, and VacA, exhibiting high sensitivity and excellent selectivity. This label-free immunosensor can be used for serum sample analysis, indicating its potential clinical application value.

[0105] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0106] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A method for preparing a Helicobacter pylori label-free immunosensor, characterized by, Includes the following steps: S10. Take carbon nanofibers into nano-gold sol, stir evenly, and then dry to obtain nano-gold modified carbon nanofibers. S20. Take the gold-modified carbon nanofibers, carbon nanopowder, titanium carbide and ionic liquid and place them in a mortar, add a binder, grind evenly to obtain a paste; S30. The paste is filled into a container and heated to obtain an electrode column. The surface of the electrode column is polished smooth to obtain a modified electrode. S40. Helicobacter pylori multivalent epitope antigen FVpE was dropped onto the surface of the modified electrode, incubated, rinsed with ultrapure water, dried, and bovine serum albumin was added for further incubation. After incubation, a label-free immunosensor for Helicobacter pylori was obtained. Before S40, the following steps are also included: An artificial gene NCV was synthesized, wherein the artificial gene NCV contains conserved antigenic epitope enrichment segments from NAP, CagA302-437 and VacA1-46 / 332-494, and the artificial gene NCV has Nco I and Hind III restriction sites at both ends. The artificial gene NCV was inserted into plasmid pET-28a to construct the recombinant plasmid pET-NCV. The UE gene was amplified from pET-CUE using PCR technology, and the UE gene was inserted into the recombinant plasmid pET-NCV to construct the recombinant expression vector pET-FVpE. The recombinant expression vector pET-FVpE was transformed into Escherichia coli, and expression was induced by IPTG at a preset temperature. The Helicobacter pylori multivalent epitope antigen FvpE was obtained by Ni-NTA affinity chromatography protein purification technology.

2. The production method according to claim 1, characterized by, In step S10, 80 to 120 parts of the carbon nanofibers and 160 to 240 parts of the nano-gold sol are taken and stirred evenly.

3. The preparation method according to claim 1, characterized in that, In step S20, 23 to 27 parts of the gold-modified carbon nanofibers, 18 to 22 parts of the carbon nanoparticles, 4 to 6 parts of the titanium carbide, and 23 to 27 parts of the ionic liquid are taken.

4. The method of claim 1, wherein, In S20, the binder is paraffin oil, and 18 to 22 parts of the paraffin oil are added dropwise; the ionic liquid is 1-octylpyridine hexafluorophosphate.

5. The preparation method according to claim 1, characterized in that, In step S30, the paste is filled into a polytetrafluoroethylene tube.

6. The preparation method according to claim 1, characterized in that, In step S40, 4 to 5 parts of the Helicobacter pylori multivalent epitope antigen FVpE are dropped onto the surface of the modified electrode, incubated for 35 to 45 minutes, rinsed with ultrapure water, and dried. Then, 4 to 5 parts of the bovine serum albumin are dropped onto the electrode and incubated for 35 to 45 minutes. After incubation, the label-free immunosensor of Helicobacter pylori is obtained, and the concentration of the Helicobacter pylori multivalent epitope antigen FvpE immobilized on the surface of the modified electrode is 1 mg / mL to 1.5 mg / mL.

7. A label-free immunosensor for Helicobacter pylori, characterized in that, It was prepared using the method for preparing a label-free immunosensor for Helicobacter pylori as described in any one of claims 1 to 6.

8. The application of a label-free immunosensor for Helicobacter pylori as described in claim 7 in the field of Helicobacter pylori detection, characterized in that, Includes the following steps: Construct the label-free immunosensor for Helicobacter pylori as described in claim 7; An electrolyte system was constructed, which consisted of the sample solution to be tested, a K3[Fe(CN)6] / K4[Fe(CN)6] mixed probe solution, and a PBS buffer solution. The label-free immunosensor for Helicobacter pylori was incubated in an electrolyte system, and the concentration of Helicobacter pylori was detected after incubation.

9. An application according to claim 8, characterized in that, The electrolyte system consists of the sample solution to be tested, a 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution containing 0.1 M KCl, and a 0.01 M PBS buffer solution. The pH of the electrolyte system is 6 to 8. The label-free immunosensor for Helicobacter pylori is incubated in the electrolyte system for 40 to 50 minutes. After incubation, the concentration of Helicobacter pylori is detected by differential pulse voltammetry, with a scanning range of -0.2 V to 0.5 V.

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