Kit for infectious igg antibody hypersensitivity detection and application

Abstract

The application specifically relates to a kit for infectious IgG antibody hypersensitive detection and application. The kit comprises a first oligonucleotide chain, a second oligonucleotide chain, a third oligonucleotide chain, a fourth oligonucleotide chain, a first hairpin probe, a second hairpin probe, H. pylori antigen functionalized magnetic beads, enzyme fluorescent substrate, a secondary antibody-flipper complex, a buffer solution, a droplet generation chip and a droplet detection chip, the first oligonucleotide chain, the second oligonucleotide chain, the third oligonucleotide chain and the fourth oligonucleotide chain can be self-assembled to form a rectangular DNA frame, the flipper is modified with azide at one end, one end of the first hairpin probe and the second hairpin probe is modified with biotin-triglycol, the H. pylori antigen functionalized magnetic beads are formed by incubation of H. pylori antigens and magnetic beads, and the secondary antibody-flipper complex is formed by connection of a secondary antibody and a flipper. The system is used for detection of H. pylori antibodies, and has high sensitivity and strong specificity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a kit and its application for the ultrasensitive detection of infectious IgG antibodies. Background Technology

[0002] Helicobacter pylori (Hp) is one of the most common human bacterial pathogens, with a global infection rate exceeding 50%. Hp infection can lead to chronic peptic gastritis, peptic ulcers, and in severe cases, gastric cancer. Early detection and diagnosis are key to improving the prognosis of Hp-infected individuals. Serological testing for infectious antibodies is one of the most important non-invasive methods for detecting Hp infection, as infectious antibodies appear early in the infection and persist in the body, unaffected by medication, making it particularly suitable for timely diagnosis and continuous monitoring of infection.

[0003] Enzyme-linked immunosorbent assay (ELISA) is the mainstream method for clinical detection of serum infectious antibodies, with a detection limit of approximately 10. -12 mol / L. However, in the early stages of many diseases, the concentration of infectious antibodies in the blood can be as low as 10 mol / L. -16 At concentrations of mol / L, traditional ELISA can no longer meet the needs of early detection.

[0004] Droplet digital ELISA (ddELISA) is an ultrasensitive single-molecule immunoassay technique based on droplet microfluidics. It uses pillary-level "water-in-oil" microdroplets to disperse a large-volume reaction into small, independent reaction units, achieving ultrasensitive quantification of protein targets based on Poisson distribution counting, with a detection limit down to the aM level.

[0005] However, existing ddELISA has the following technical problems: (1) low signal-to-noise ratio (SNR) of microdroplet fluorescence; (2) high cost of methods to improve the SNR of ddELISA (such as using a more precise microfluidic system or PCR amplification); and (3) no ddELISA system that can be used for ultrasensitive detection of serological infectious antibodies.

[0006] Hybridization chain reaction (HCR) is an isothermal self-assembly of nucleic acids without the involvement of enzymes, and the final product is long double-stranded DNA (dsDNA), which has a strong signal amplification capability. However, the hairpin probes of traditional HCR exist in solution in monomeric form, resulting in low local concentrations and limiting reaction efficiency. Summary of the Invention

[0007] In view of this, the present invention provides a kit for the ultrasensitive detection of infectious IgG antibodies and its application, in order to solve the above-mentioned technical problems.

[0008] This application provides a kit for detecting Helicobacter pylori antibodies, comprising a first oligonucleotide chain, a second oligonucleotide chain, a third oligonucleotide chain, a fourth oligonucleotide chain, a first hairpin probe, a second hairpin probe, Helicobacter pylori antigen-functionalized magnetic beads, an enzyme fluorescent substrate, a secondary antibody-trigger complex, a buffer solution, a droplet generation chip, and a droplet detection chip. The first, second, third, and fourth oligonucleotide chains can self-assemble to form a rectangular DNA framework. One end of the trigger is modified with an azide. One end of both the first and second hairpin probes is modified with biotin-triethylene glycol. The Helicobacter pylori antigen-functionalized magnetic beads are formed by co-incubating Helicobacter pylori antigen and magnetic beads. The secondary antibody-trigger complex is formed by linking a secondary antibody and a trigger.

[0009] Optionally, the nucleotide sequence of the first oligonucleotide chain is shown in SEQ ID NO.1, the nucleotide sequence of the second oligonucleotide chain is shown in SEQ ID NO.2, the nucleotide sequence of the third oligonucleotide chain is shown in SEQ ID NO.3, and the nucleotide sequence of the fourth oligonucleotide chain is shown in SEQ ID NO.4.

[0010] Optionally, the nucleotide sequence of the first hairpin probe is shown in SEQ ID NO.8, and the nucleotide sequence of the second hairpin probe is shown in SEQ ID NO.9.

[0011] Optionally, the oligonucleotide sequence of the trigger is shown in SEQ ID NO.7.

[0012] Optionally, both the first hairpin probe and the second hairpin probe are fixed to the rectangular DNA framework.

[0013] Optionally, the molar ratio of the first oligonucleotide chain, the second oligonucleotide chain, the third oligonucleotide chain and the fourth oligonucleotide chain is 0.8-1.1:0.8-1.1:0.8-1.1:0.8-1.1.

[0014] Optionally, the molar ratio of the first hairpin probe to the rectangular DNA frame is 0.8-1.1:0.8-1.1.

[0015] Optionally, the molar ratio of the second hairpin probe to the rectangular DNA frame is 0.8-1.1:0.8-1.1.

[0016] Optionally, the molar ratio of the trigger to the secondary antibody is 2-4:1.

[0017] Optionally, the Helicobacter pylori antigen-functionalized magnetic beads are used to capture Helicobacter pylori antibodies.

[0018] Optionally, the Helicobacter pylori antigen-functionalized magnetic beads are formed by covalently coupling the Helicobacter pylori antigen to the surface of the magnetic beads.

[0019] Optionally, the enzyme fluorescent substrate is used to generate a fluorescent signal through an enzymatic reaction.

[0020] Optionally, the droplet generation chip is used to generate microdroplets and then perform an enzymatic reaction, and the droplet detection chip is used to quantitatively detect the microdroplets after the enzymatic reaction.

[0021] Optionally, the trigger in the secondary antibody-trigger complex is used to initiate a hybridization chain reaction.

[0022] Optionally, the buffer solution is a TNaK buffer solution.

[0023] This application also provides a method for detecting Helicobacter pylori antibodies, wherein the method utilizes the kit described above to detect biological samples.

[0024] The beneficial effects of this invention are: (1) Ultra-high sensitivity: The detection limit (LOD) of Helicobacter pylori antibodies using the system proposed in this application is as low as 0.02 pg / mL (approximately 133 aM), which is about 10% higher than that of traditional ELISA. 4 It is approximately 10 times faster than the traditional ddELISA; the linear range spans four orders of magnitude (R0). 2 >0.99); (2) High specificity: The system of this application has extremely low cross-reactivity with other common infectious antibodies such as HBs-Ab, HBe-Ab, HBc-Ab, HCV-Ab, CS-Ab, TP-Ab, and HIV-Ab; the detection accuracy of clinical samples reaches 100%; (3) High signal-to-noise ratio: RF-HCR increases the local concentration of hairpin probe by about 20,551 times through the DRF framework, and the reaction efficiency is about twice that of traditional HCR, which significantly improves the fluorescence SNR of microdroplets; (4) Easy to operate: The entire process is at a constant temperature, requiring no complicated temperature control instruments, making it suitable for primary healthcare institutions and point-of-care testing (POCT) scenarios; (5) Good economic efficiency: RF-HCR signal amplification does not require enzyme participation, which greatly reduces the detection cost compared with existing ddELISA methods to improve SNR (precision microfluidic equipment or PCR amplification); (6) High versatility: By replacing Helicobacter pylori antigen with other pathogen antigens, ultrasensitive detection of a variety of infectious antibodies and other target proteins can be achieved. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the detection principle of RF-HCR-ddELISA. RF-HCR-ddELISA represents a rectangular frame-mediated hybridization chain reaction-droplet digital enzyme-linked immunosorbent assay. Sequantial Hybridization represents sequential hybridization. Assembly represents assembly. DRF represents a DNA rectangular frame. H1 / H2 represents hairpin probes. RF-HCR represents a rectangular frame-hybridization chain reaction. Trigger represents a trigger oligonucleotide chain. Step represents a step. DNA Rectangular Frame-Supported Hybrid Chain Reaction represents a DNA rectangular frame-supported hybridization chain reaction. sRF-HCR represents a tandem rectangular frame-hybridization chain reaction. Partitioning by microfluidics represents microfluidic dispersion using droplets. FDG represents the enzyme fluorescent substrate. Oil represents droplet-generated oil. Water-in-oil microdroplet represents a water-in-oil microdroplet. Incubation represents incubation. Recognition represents recognition. Microdroplet Signal Recognition represents microdroplet signal recognition. Negative and Empty Microdroplets represent negative and empty droplets; Positivemicrodroplets represent positive droplets; Analysis represents analysis; Positive represents positive; Negative represents negative; Threshole line represents the threshold line; Event number represents the number of events; Fluorescence amplitude represents the fluorescence amplitude; Antigen-coated magnetic beads represent antigen-coated magnetic beads; Target antibody represents the target antibody; Trigger-modified secondary antibody represents a secondary antibody modified with a trigger oligonucleotide chain; Streptavidin-β-galactosidase represents a galactosidase modified with streptavidin. Figure 2 The results of agarose gel electrophoresis of RF-HCR and HCR are shown. Figure 3 The images show the transmission electron microscopy observations of DRF, RF-HCR, and sRF-HCR. Figure 4 The diagram shows a comparison of particle size and potential for DRF, RF-HCR, and sRF-HCR. Figure 5 The plot shows the concentrated region results of traditional HCR and RF-HCR calculated based on collision theory, with fold indicating the factor. Figure 6 The image shows the fluorescence characterization results of RF-HCR and conventional HCR. FL intensity represents fluorescence intensity, Trigger represents the trigger oligonucleotide chain, H1 / H2 represents the hairpin probe, and DRF represents the DNA rectangular frame. Figure 7 The graph shows the optimization results of RF-HCR reaction temperature and time. SNR represents the signal-to-noise ratio, Temperature represents the temperature, and Time represents the time. Figure 8 The graph shows the results of RF-HCR response to the trigger. FL intensity represents fluorescence intensity, Wavelength represents wavelength, and Trigger concentration represents the concentration of the trigger oligonucleotide chain. Figure 9 This is a peak diagram of the excitation and emission wavelengths of fluorescein, where FL intensity represents fluorescence intensity and Wavelength represents wavelength. Figure 10 The graph shows the results of the responsiveness verification of FDG to β-Gal. FL intensity represents fluorescence intensity, Wavelength represents wavelength, and Enzyme concentration represents enzyme concentration. Figure 11 The image shows the fluorescence characterization results of RF-HCR-ELISA. RF-HCR-ELISA represents a rectangular frame-mediated hybridization chain reaction-enzyme-linked immunosorbent assay. Target antibody represents the target antibody, Trigger-modified secondary antibody represents the trigger oligonucleotide chain modified secondary antibody, Streptavidin-β-galactosidase represents streptavidin-modified galactosidase, Helicobacter pylori antigen represents Helicobacter pylori antigen, Wavelength represents wavelength, FL intensity represents fluorescence intensity, Secondary antibody represents the secondary antibody, Trigger represents the trigger oligonucleotide chain, H1 / H2 represents the hairpin probe, and DRF represents the DNA rectangular frame. Figure 12The graph shows the optimized working concentrations of the coating antigen and secondary antibody-Trigger for RF-HCR-ELISA. SNR represents the signal-to-noise ratio, and Concentration of coated antigen represents the antigen coating concentration. Figure 13 The diagram shows the optimization results of the optimal connection ratio between the secondary antibody and the trigger. SNR represents the signal-to-noise ratio, and Secondary antibody: Trigger represents the oligonucleotide chain of the secondary antibody: trigger. Figure 14 The figure shows the optimization results of RF-HCR in the RF-HCR-ELISA system. SNR represents the signal-to-noise ratio, RF-HCR reaction time represents the RF-HCR reaction time, RF-HCR reaction temperature represents the RF-HCR reaction temperature, and RF-HCR concentration represents the RF-HCR concentration. Figure 15 The figure shows the optimization results of β-Gal and fluorescent substrate FDG in the RF-HCR-ELISA system. β-Gal reaction time represents the enzyme reaction time, β-Gal reaction temperature represents the enzyme reaction temperature, and FDG concentration represents the concentration of substrate FDG. Figure 16 The standard curve of concentration versus fluorescence intensity constructed for RF-HCR-ELISA to detect serially diluted Hp-IgG under optimal conditions is shown in the figure. FL intensity represents fluorescence intensity, and Target antibody represents target antibody. Figure 17 This is a graph showing the specificity verification results of RF-HCR-ELISA. FL intensity represents fluorescence intensity, Hp-IgG represents Helicobacter pylori IgG antibody, HBs-Ab represents hepatitis B surface antibody, HBe-Ab represents hepatitis B e antibody, HBc-Ab represents hepatitis B core antibody, HCV-Ab represents hepatitis C antibody, CS-Ab represents liver fluke antibody, TP-Ab represents Treponema pallidum antibody, and HIV-Ab represents human immunodeficiency virus antibody. Figure 18 The graph shows the accuracy and precision validation results of RF-HCR-ELISA. Recovery represents the recovery rate, Coefficient of variation represents the coefficient of variation, and Spike concentration represents the doping concentration. Figure 19The image shows the fluorescence response peaks and corresponding standard curves of the magnetic bead-based RF-HCR-ELISA. Antigen-coated magnetic beads represent antigen-coated magnetic beads, Target antibody represents the target antibody, Trigger-modified secondary antibody represents the trigger oligonucleotide chain modified secondary antibody, streptavidin-β-galactosidase represents streptavidin-modified galactosidase, FL intensity represents fluorescence intensity, Wavelength represents wavelength, and Target antibody represents the target antibody. Figure 20 The images show the surface morphology of the magnetic beads before and after the RF-HCR-ELISA reaction, as observed by scanning electron microscopy. Figure 21 The image shows the microdroplet reaction results of RF-HCR-ddELISA observed by fluorescence optical microscopy. Bright indicates the bright field, and Fluorescence indicates the fluorescence field. Figure 22 The image shows the microdroplet fluorescence characterization results of RF-HCR-ddELISA. Magnetic beads represent magnetic beads, secondary antibody represents secondary antibody, trigger represents trigger oligonucleotide chain, H1 / H2 represents hairpin probe, and DRF represents DNA rectangular frame. Figure 23 The microdroplet fluorescence SNR is the signal-to-noise ratio of the microdroplet in RF-HCR-ddELISA. Figure 24 The figure shows the optimization results of the reaction time of β-Gal in the RF-HCR-ddELISA system. MicrodropletSNR represents the signal-to-noise ratio of the microdroplets, and Time represents the time. Figure 25 The figure shows the optimization results of the reaction temperature of β-Gal in the RF-HCR-ddELISA system. MicrodropletSNR represents the signal-to-noise ratio of microdroplets, and Temperature represents the temperature. Figure 26 The graph shows the optimized reaction concentration of FDG in the RF-HCR-ddELISA system. Microdroplet SNR represents the signal-to-noise ratio of the microdroplets, and FDG concentration represents the FDG concentration. Figure 27The graph shows the sensitivity verification results of the RF-HCR-ddELISA system. Average enzyme per bead represents the average enzyme per bead, and Target antibody represents the target antibody. Figure 28 The image shows the specificity verification results of the RF-HCR-ddELISA system. NC represents the negative control, HBs-Ab represents hepatitis B surface antibody, HCV-Ab represents hepatitis C antibody, CS-Ab represents liver fluke antibody, TP-Ab represents Treponema pallidum antibody, Hp-IgG represents Helicobacter pylori IgG antibody, and Concentration represents the concentration. Figure 29 This is a performance evaluation graph for clinical sample detection of RF-HCR-ddELISA. Positive indicates positive, Weakly positive indicates weakly positive, Negative indicates negative, Sample concentration indicates sample concentration, C13Positive indicates C13 positive, C13 Negative indicates C13 negative, Sensitivity indicates sensitivity, and 1-Specificity indicates 1-specificity. Detailed Implementation

[0026] The present invention will be further illustrated by specific examples below. However, it should be noted that the specific material ratios, process conditions and results described in the embodiments of the present invention are only for illustrating the present invention and cannot be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

[0027] To address the shortcomings of existing detection technologies in detecting infectious antibodies, such as insufficient sensitivity, limited quantification capabilities, and inability to detect early low-abundance infections, this application develops a novel droplet digital ELISA (ddELISA) ultrasensitive detection system based on rectangular frame-guided hybridization chain reaction (RF-HCR) for ultrasensitive, digital quantitative detection of Helicobacter pylori IgG antibodies (Hp-IgG).

[0028] This application designed and synthesized four oligonucleotide chains (S1, S2, S3, S4) capable of self-assembling to form a DNA rectangular frame (DRF). HCR hairpin probes (H1, H2) were immobilized on the DRF, increasing the local concentration of the hairpin probes (approximately 20,551 times higher than conventional HCR), thus constructing an RF-HCR signal amplification system. When a trigger is present in the system, the HCR reaction can be initiated, forming a long-chain DNA polymer carrying a large amount of biotin labeling (Series-connected RF-HCR, sRF-HCR), achieving cascaded signal amplification. In short, RF-HCR uses the DRF as a scaffold, anchoring hairpin probes H1 and H2 to the four ends of the DRF, increasing the local probe concentration by approximately 20,000 times and significantly improving reaction efficiency. The first and second hairpin probes can self-complementarily pair to form hairpin structures.

[0029] Specifically, such as Figure 1 As shown, this application utilizes antigen-functionalized magnetic beads to capture target antibodies, which are then bound to them using secondary antibodies modified with triggers. After adding pre-synthesized RF-HCRs to the reaction system, they are triggered by the triggers on the positive magnetic beads to generate a hybridization chain reaction, resulting in a large number of RF-HCRs being linked to the positive magnetic beads. Since each RF-HCR is linked to two biotin molecules, the positive magnetic beads carry a large amount of biotin. After incubation with streptavidin-modified galactosidase in the reaction system, the binding of streptavidin to biotin causes a large concentration of galactosidase on the positive magnetic beads. Subsequently, these magnetic beads, along with the fluorescent substrate FDG, are dispersed into microdroplets using a droplet microfluidic system. After a brief incubation, the microdroplets containing the positive magnetic beads produce a significant fluorescent signal. Finally, these microdroplets are identified and counted using a droplet reader to calculate the concentration of the target antibody. Therefore, the method of this application enables the digital detection of target antibodies.

[0030] In summary, this application innovatively combines RF-HCR with ddELISA to develop a novel method for the ultrasensitive immunoassay of infectious antibodies—Rectangular frame-guided hybridization chain reaction-droplet digitalenzyme-linked immunosorbent assay (RF-HCR-ddELISA).

[0031] The present invention will be described in detail below through specific examples and embodiments. It should also be understood that the following embodiments are only for specific illustration of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0032] Sample Source: Ten serum samples that were positive for Hp-IgG using clinical indirect ELISA qualitative detection, six serum samples that were weakly positive for Hp-IgG, and eight serum samples that were negative for Hp-IgG were selected from December 2025 to February 2026 at the First Affiliated Hospital of Guangxi Medical University. All samples were stored at -80℃. Inclusion Criteria: ① Samples with definitive Hp-IgG qualitative detection results using our hospital's clinical testing methods; ② Positive or weakly positive samples with clear C13 breath test results. Exclusion Criteria: ① Samples with abnormal conditions such as lipemia or hemolysis; ② Samples lacking basic information such as gender and age.

[0033] (I) Construction and Optimization of RF-HCR Signal Amplification System 1.1 Primer Design and Synthesis This application designs four oligonucleotide chains (S1, S2, S3, S4) that can self-assemble to form a DRF, and the nucleotide sequences are shown in Table 1. Two HCR hairpin probes (H1, H2) that can be fixed on a rectangular DNA framework were designed, and their nucleotide sequences are shown in Table 1. HCR hairpin probes (H1, H2) were fixed on the DRF to increase the local concentration of the hairpin probes (by about 20,551 times), and finally the RF-HCR signal amplification system was obtained. When a trigger is present in the system, the HCR reaction can be initiated to form an sRF-HCR carrying a large amount of biotin labeling, thereby achieving cascaded signal amplification. All sequences in this application were synthesized by a biotechnology company.

[0034] Table 1 Sequence List 1.2 Preparation of RF-HCR All nucleic acid sequences were dissolved in TE buffer and stored at 4°C.

[0035] (1) Preparation of TNaK buffer: It is composed of Tris-HCl (pH 7.4), NaCl, KCl and ultrapure water. The final concentrations of each component are: Tris-HCl 20 mmol / L, NaCl 125 mmol / L and KCl 20 mmol / L. TNaK buffer is used as the working solution for the reaction.

[0036] (2) Preparation of H1 and H2: H1 and H2 need to undergo denaturation-annealing reactions before use. That is, use a regular PCR instrument to heat H1 solution and H2 solution to 95℃ respectively, and treat them at 95℃ for 5 min. Then, slowly cool them to room temperature over 60 min to allow H1 and H2 to fully complement each other and form hairpin structures. After the reaction is completed, store at 4℃ for later use.

[0037] (3) DRF preparation: Mix S1, S2, S3, and S4 in a molar ratio of 1:1:1:1 and dilute with TNaK buffer. Specifically, mix the S1 solution prepared with TNaK buffer, the S2 solution prepared with TNaK buffer, the S3 solution prepared with TNaK buffer, and the S4 solution prepared with TNaK buffer to obtain a mixture with a final concentration of 1 μM for each of S1, S2, S3, and S4. Heat the mixture at 95°C for 5 min using a standard PCR instrument, and then slowly cool it to room temperature over 60 min to allow the four synthesized sequences to fully complement each other and form the DRF. Store the synthesized DRF at 4°C for later use.

[0038] (4) Preparation of RF-HCR: Mix the prepared H1 and H2 and dilute with TNaK buffer. Specifically, mix the H1 solution and H2 solution obtained after treatment (2) to obtain an H1 / H2 mixture with a final concentration of 1 μM for both H1 and H2. Then, mix the H1 / H2 mixture with DRF at a molar ratio of 1:1, i.e., the molar ratio of H1 to S1 in step (3) is 1:1. Incubate at room temperature (25℃) for 60 min to allow H1 and H2 to pair complementaryly with DRF through their tails, ultimately forming RF-HCR. After synthesis, RF-HCR is stored at 4℃ for later use.

[0039] It should be noted that in this application, H1 and H2 in Sections 1.6-1.8 refer to H1-FAM and H2, respectively. H1 and H2 mentioned in other sections refer to H1-Biotin and H2-Biotin, respectively.

[0040] 1.3 Lipoglycosylation gel electrophoresis to verify RF-HCR and HCR (1) Six RF-HCR systems were prepared for validation. The reaction components of each system were diluted with TNaK buffer to a final concentration of 1 μM, as follows: ①S1 (final concentration 1μM): Dilute S1 with TNaK buffer to a final concentration of 1μM; ②S1+S2 (1:1, both with a final concentration of 1μM): The solutions of S1 and S2 prepared with TNaK buffer, both with a final concentration of 1μM, were heated at 95℃ for 5 min and then slowly cooled to room temperature over 60 min. ③S1+S2+S3 (1:1:1, with a final concentration of 1 μM for each of the three): The solutions of S1, S2 and S3 prepared with TNaK buffer, each with a final concentration of 1 μM, were heated at 95 °C for 5 min and then slowly cooled to room temperature over 60 min. ④DRF: Prepared according to step 1.2, with the final concentrations of S1, S2, S3, and S4 all being 1 μM; ⑤RF-HCR: Prepared according to step 1.2, with the final concentrations of S1, S2, S3, S4, H1, and H2 all being 1 μM; ⑥ The product of the reaction of Trigger (0.5 μM) and RF-HCR at room temperature for 40 min - sRF-HCR: Add a trigger to the RF-HCR (1 μM) obtained in step 1.2 to make the final concentration of the trigger 0.5 μM, and then react at room temperature for 40 min to obtain sRF-HCR; (2) Five HCR groups were prepared for verification. The reaction components of each group were diluted to 1 μM with TNaK buffer, as follows: ①Trigger (final concentration 1μM): Add Trigger to TNaK buffer to make the final concentration of Trigger 1μM; ②H1 (final concentration 1μM): Add H1 to TNaK buffer to make the final concentration of H1 1μM; ③H2 (final concentration 1μM): Add H1 to TNaK buffer to make the final concentration of H1 1μM; ④ H1 / H2 mixture (final concentration of H1 and H2 is 1 μM): Prepared according to step 1.2, with final concentration of H1 and H2 of 1 μM; ⑤ The product obtained by reacting the Trigger (0.5 μM) with the H1 / H2 mixture at room temperature for 40 min is as follows: the Trigger is added to the H1 / H2 mixture (1 μM) obtained in step 1.2 to make the final concentration of the Trigger 0.5 μM, and then the mixture is reacted at room temperature for 40 min to obtain the reaction product.

[0041] (3) Perform gel electrophoresis on the 6 systems prepared in (1) and the 5 systems prepared in (2) respectively. The specific steps are as follows: 1) Dilute 10×TBE buffer to prepare 0.5×TBE working solution. Weigh 2g of agarose powder and pour it into a clean Erlenmeyer flask. Add 100mL of 0.5×TBE buffer and shake gently to mix. 2) Place the conical flask in the microwave oven and heat for 30 seconds until it boils. Remove it and shake well. Repeat this process 4 times until the agarose powder is completely dissolved and the flask is clear and transparent. 3) After the conical flask has cooled slightly, add 5µL of 10000×GelstainRed nucleic acid dye and mix thoroughly. Then pour the mixture into a mold suitable for the electrophoresis tank, insert a comb with an appropriate number of wells, and leave it at room temperature for 20 minutes until the agarose gel is completely solidified. 4) After the agarose gel has completely solidified, remove the comb, place the gel block in the electrophoresis tank, and add 0.5×TBE buffer until the buffer covers the gel surface; 5) Mix the Loading Buffer (6×) with each group of RF-HCR and HCR at a ratio of 1:6 by pipetting until homogeneous. Slowly add 10 μL of this mixture to the sample wells on the gel. Add 10 µL of 50 bp DNA ladder marker to the blank well next to the RF-HCR preparation group, and add 10 µL of 20 bp DNA ladder marker to the blank well next to the HCR preparation group as indicator bands. 6) Connect the electrophoresis tank to the power supply of the electrophoresis apparatus and perform electrophoresis at a constant voltage of 140V for 45 minutes; 7) After electrophoresis, the gel block is placed in a gel imaging system, and the electrophoresis results are visualized using a gel imaging system. The results are as follows: Figure 2 As shown.

[0042] Depend on Figure 2 It can be seen that DRF is assembled stepwise with the four strands, and the molecular weight of the bands increases sequentially; the molecular weight of the RF-HCR band is significantly larger than that of DRF. These results indicate that H1 and H2 are successfully anchored to the DNA rectangular framework; after triggering, ultra-high molecular weight polymers are generated, and the bands are retained in the sample well, resulting in a chain amplification efficiency that is significantly higher than that of traditional HCR.

[0043] 1.4 Transmission electron microscopy observation of RF-HCR and its reaction products (1) Configure 3 groups to be observed: ① DRF: Prepared according to step 1.2, with the final concentration of S1, S2, S3 and S4 all being 10 μM; ② RF-HCR: Prepared according to step 1.2, with the final concentrations of S1, S2, S3, S4, H1, and H2 all being 10 μM; ③ sRF-HCR: Add a trigger (5 μM) to the RF-HCR (10 μM) obtained in step 1.2 to achieve a final trigger concentration of 5 μM. Then react at room temperature for 40 min to obtain sRF-HCR. (2) Observation of samples under transmission electron microscopy: Dilute each group of samples 10 times, then take 5µL of the sample dilution and drop it onto the copper grid for electron microscopy. Let it stand at room temperature for 10 minutes. Use filter paper to absorb the liquid from the side of the copper grid, then invert the copper grid onto a drop of 20µL of 1% phosphotungstic acid staining solution and let it stand at room temperature for 5 minutes. Use filter paper to absorb the phosphotungstic acid staining solution from the side of the copper grid, and let it stand at room temperature for 10 minutes until the copper grid is dry. At this time, the copper grid has been adsorbed with the stained sample. Use tweezers to pick up the copper grid loaded with the sample and place it into the sample mounting slot of the transmission electron microscope. Install the gasket and fix it. Then align the sample tube with the sample inlet of the transmission electron microscope and gently push it in with your thumb at the bayonet. After hearing a sound, the red light will turn on. Turn on the switch below. After seeing the green light turn on, turn the sample tube to the right. The sample will be automatically sucked in. Then turn it back to the left by about 5 degrees to suck it into the bottom. Then you can observe the sample morphology under the H-7650 transmission electron microscope. The results are as follows. Figure 3 As shown, the sample position is located by rotating the knob and the magnification is adjusted to obtain the sample morphology image. The result is as follows. Figure 3 As shown, the average particle size was measured using a ZetaView nanoparticle tracking analyzer, and Zeta potential analysis was performed. The results are as follows. Figure 4 As shown; Depend on Figure 3 As can be seen from the transmission electron microscopy observation results, the DRF is a regular rectangular frame with a size of less than 20 nm; the RF-HCR is a composite structure of frame and hairpin probe with a size of about 20-30 nm; after triggering, a long chain-like sRF-HCR is formed with a length of more than 100 nm.

[0044] Depend on Figure 4 The average particle size of DRF was 203.36 nm; the average particle size of RF-HCR was 251.01 nm; and the average particle size of sRF-HCR was 367.63 nm. Zeta potential analysis showed that the negative potential of RF-HCR was significantly enhanced, approximately 2.7 times that of DRF and RF-HCR.

[0045] 1.5 Efficiency Calculation of RF-HCR Reaction The reaction efficiency of RF-HCR was confirmed based on collision theory: In the formula, N is Avogadro's constant, c is the concentration of the reaction, and V represents the relative volume of the sphere. The calculation results are as follows: Figure 5As shown, the local concentration of H1 / H2 in RF-HCR reached 2055.17 μM, which is 20551.7 times higher than that of conventional HCR.

[0046] 1.6 Fluorescence characterization of RF-HCR (1) Six RF-HCR reaction systems were designed using H1-FAM modified with fluorescent groups (FAM) (as shown in Table 2) for characterization experiments. The concentration of the reaction component in each group was 0.2 μM, and the reaction time was 60 min, as detailed below: ① H1-FAM only: Dilute H1-FAM with TNaK buffer to a final concentration of 0.2 μM; ②H1 / H2 mixture: Except that H1 is replaced with H1-FAM decorated with fluorescent group (FAM), prepare H1 / H2 mixture according to step 1.2. The final concentration of H1 and H2 is 0.2μM. ③RF-HCR: Except that H1 is replaced with H1-FAM decorated with fluorescent group (FAM), RF-HCR is prepared according to step 1.2. The final concentrations of S1, S2, S3, S4, H1 and H2 are all 0.2 μM. ④ Trigger reaction with H1-FAM: Add Trigger (0.1 μM, the final concentration here, the same below) to H1-FAM (0.2 μM), and then react at room temperature for 60 min; ⑤ Trigger reaction with H1 / H2 mixture: Except that H1 is replaced with H1-FAM decorated with fluorescent group (FAM), prepare H1 / H2 mixture according to step 1.2, add Trigger (0.1μM) to the prepared H1 / H2 mixture, and then react at room temperature for 60min; ⑥ Trigger and RF-HCR reaction: Except that H1 was replaced with H1-FAM decorated with a fluorescent group (FAM), an H1 / H2 mixture was prepared according to step 1.2. A trigger (0.1 μM) was added to the prepared H1 / H2 mixture, and then the reaction was carried out at room temperature for 60 min. Each group had 3 replicates. The settings for each group are shown in Table 2. + represents the component contained in the group, and - represents the component not added in the group. (2) The fluorescence changes of each group were detected using a quantitative real-time polymerase chain reaction (qPCR) instrument. The average of three replicates for each group was used as the test result. "+" indicates the component contained in the group, and "-" indicates the component not added to the group. The results are as follows: Figure 6 As shown.

[0047] Table 2 Characterization of the reaction components in RF-HCR like Figure 6 As shown, in the absence of a trigger, the fluorescence values ​​of H1, HCR, and RF-HCR in groups 1, 2, and 3 were all low, indicating good stability of the reaction system. After reaction with a trigger, H1, HCR, and RF-HCR all showed significant fluorescence growth, with RF-HCR in group 6 producing the highest fluorescence intensity, approximately twice that of HCR in group 5, followed by H1 in group 4. These results suggest that the reaction efficiency of RF-HCR is significantly superior to that of conventional HCR.

[0048] 1.7 Optimization of RF-HCR Reaction Conditions: Key parameters of the RF-HCR reaction system were optimized, as follows: (1) Optimization of reaction temperature: Trigger was added to RF-HCR (0.2 μM) to make the final concentration of Trigger 0.1 μM. Several groups were set up, and the reaction temperatures of each group were 4℃, 8℃, 25℃, 37℃, 42℃, 50℃, 55℃ and 60℃, respectively. Each reaction temperature was set up in triplicate. The fluorescence change of each group was detected by qPCR. The average value of the three replicates of each group was used as the test result. The reaction time was selected as 60 min to ensure complete reaction. The fluorescence SNR was used as the selection criterion. The temperature corresponding to the maximum SNR was taken as the optimal reaction temperature of RF-HCR. The results are as follows. Figure 7 As shown.

[0049] (2) Optimization of reaction time: A trigger was added to RF-HCR (0.2 μM) to bring the final concentration of the trigger to 0.1 μM. The reaction was then carried out at 25 °C. Several groups were set up with reaction times of 5 min, 15 min, 30 min, 45 min, 60 min, 75 min, 90 min, and 120 min, respectively. Each reaction time was repeated in triplicate. The fluorescence changes in each group were detected using qPCR, and the average of the three replicates was used as the test result. The fluorescence SNR was used as the selection criterion, and the time corresponding to the maximum SNR was taken as the optimal reaction time for RF-HCR. The results are as follows: Figure 7 As shown.

[0050] Depend on Figure 7 It can be seen that the optimal reaction temperature for RF-HCR is 25℃ and the optimal reaction time is 30 min.

[0051] 1.8 Verification of RF-HCR Response to Triggers A series of black ELISA plates were prepared, and each well was filled with 0.5 μM RF-HCR (prepared according to step 1.2, with final concentrations of S1, S2, S3, S4, H1, and H2 all at 0.5 μM). Triggers were then added to achieve final concentrations of 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, and 0 nM, respectively. The plates were reacted under optimal conditions, with three replicates for each trigger concentration. Detection was performed using a Synergy H1 multi-mode microplate reader, with a fixed excitation wavelength of 480 nm. The fluorescence emission wavelength range was 510 nm–640 nm, and fluorescence signals were detected every 5 nm.

[0052] The results are as follows Figure 8 As shown, the trigger concentration and peak fluorescence exhibit a good linear relationship, with a correlation coefficient of R. 2 =0.91. This result suggests that RF-HCR has a good response to trigger concentration, and the RF-HCR response is related to the amount of trigger.

[0053] (II) Establishment and validation of a rectangular frame-guided hybridization chain reaction-enzyme-linked immunosorbent assay (RF-HCR-ELISA) detection system 2.1 Validation of β-Gal with the fluorescent substrate FDG (1) Finding the optimal excitation and emission wavelengths of fluorescein, the product of the reaction between β-Gal and the fluorescent substrate FDG: Black ELISA plates were used, and commercially available fluorescent substrate FDG was added to each well to a final concentration of 10 μM. Then, β-galactosidase (β-Gal) was added to a final concentration of 100 ng / mL, and the plates were incubated at 37°C for 60 min. Detection was performed using a Synergy H1 multi-mode microplate reader. For excitation wavelength measurement, the emission wavelength was fixed at 570 nm, starting at 250 nm and stopping at 540 nm, with fluorescence signals detected every 2 nm. For emission wavelength measurement, the excitation wavelength was fixed at 418 nm, starting at 465 nm and stopping at 680 nm, with fluorescence signals detected every 2 nm. The average of three replicates for each group was used as the test result. The fluorescence values ​​obtained at the excitation and emission wavelengths were plotted as a line graph. The wavelength corresponding to the peak fluorescence is the optimal excitation and emission wavelength of the fluorophore. The results are shown below. Figure 9 As shown.

[0054] Depend on Figure 9 It is known that β-Gal catalyzes the formation of fluorescein from FDG, with the optimal excitation wavelength being 486 nm and the emission wavelength being 518 nm.

[0055] (2) Verification of the responsiveness of the fluorescent substrate FDG to β-Gal: A series of black ELISA plates were used. FDG was added to each well to a final concentration of 10 μM, followed by the addition of β-Gal to final concentrations of 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, and 0 ng / mL. The plates were incubated at 37°C for 60 min. Fluorescence detection was performed using a Synergy H1 multi-mode microplate reader. The excitation wavelength was fixed at 486 nm, and the fluorescence emission wavelength range was 500 nm–680 nm. Fluorescence signals were detected every 5 nm. Three replicates were set for each β-Gal concentration.

[0056] Depend on Figure 10 It is known that FDG has a concentration-dependent response to β-Gal, and the fluorescence intensity can reflect the amount of β-Gal in the system, which can be applied to the RF-HCR-ELISA system.

[0057] 2.2 Preparation of secondary antibody-Trigger (1) Pretreatment Zeba desalination centrifuge column: 1) Take one Zeba desalting centrifuge column, break off the end of the column and loosen the cap, and put the column into a 15mL centrifuge tube; 2) Make a mark on the top of the slant of the centrifuge column beforehand, with this side facing outwards, and centrifuge at 1000*g for 2 minutes. Then discard the storage liquid that comes out from the first centrifugation in the centrifuge tube. 3) Add 1 mL of PBS buffer to the column, let stand for 2 min to allow the solution to be absorbed by the high-efficiency resin, centrifuge at 1000*g for 2 min with the labeled side facing out, and discard the liquid inside the centrifuge tube; repeat this step 3 times. (2) Secondary antibody buffer replacement: 1) Transfer the centrifuge column to a new 15mL centrifuge tube, slowly add the secondary antibody (Goat anti-Human IgG Fc Highly Cross-Adsorbed Secondary Antibody, 1mL) to the high-efficiency resin in the center of the column, and let it stand for 2 minutes to allow the solution to be absorbed; 2) Centrifuge at 1000*g for 2 min with the labeled side facing out to collect the sample into a centrifuge tube. At this time, the buffer solution of the two antigens has been replaced with PBS, and the centrifuge column can be discarded.

[0058] (3) Copper-free click chemistry method for linking secondary antibody and trigger 1) Dissolve 50 mg of commercially available DBCO-PEG4-NHS ester (i.e., diphenylcyclooctyn-tetraethylene glycol-active ester) in 1.54 mL of freshly opened dimethyl sulfoxide (DMSO) to obtain a 50 mM solution as a stock solution. 2) Dilute the DBCO-PEG4-NHS ester stock solution with PBS to prepare the working solution. The dilution factor is 10 times. Prepare and use immediately. Mix the secondary antibody with the DBCO-PEG4-NHS ester working solution. The molar ratio of the secondary antibody to the DBCO-PEG4-NHS ester working solution is 1:20. After mixing, gently mix the two solutions. 3) Using a rotary mixer, the reaction was carried out at room temperature for 60 min. The carboxyl group on the secondary antibody reacted with the NHS of DBCO-PEG4-NHSester to form an amide bond, thus connecting the secondary antibody to DBCO-PEG4-NHS ester and synthesizing the secondary antibody-PEG4-DBCO. 4) After the reaction is complete, use the pretreated Zeba desalting centrifuge column to replace the buffer solution and remove the unbound DBCO-PEG4-NHS ester. The operation procedure is the same as (1) and (2). 5) The secondary antibody-PEG4-DBCO was mixed with a trigger linked to azide(N3) (the trigger linked to azide(N3) was synthesized by a biotechnology company) at a molar concentration of 1:1, and then the mixture was incubated overnight at 4°C in the dark. DBCO undergoes a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction with the azide group, achieving rapid coupling without copper catalysis, thus connecting the secondary antibody and the trigger to synthesize the secondary antibody-Trigger.

[0059] 2.3 Construction and fluorescence characterization of RF-HCR-ELISA (1) Coating antigen: CagA antigen and VacA antigen were mixed at a mass ratio of 1:1, and the antigen mixture was diluted with PBS buffer to obtain an antigen dilution solution with a concentration of 10 μg / mL (i.e., the final concentration of CagA antigen was 10 μg / mL, and the final concentration of VacA antigen was 10 μg / mL). 50 μL of antigen dilution solution was added to each well of a black ELISA plate. The plate was incubated overnight at 4°C. After washing, 300 μL of protein-free blocking buffer (commercially available) was added to each well, and the plate was incubated at 37°C for 60 min. After washing, the plate was stored at 4°C.

[0060] (2) Construct a complete detection system for RF-HCR-ELISA: 1) Using the black ELISA plate coated with antigen from the previous step (10 μg / mL), add commercially available Hp-IgG to each well to make the final concentration of Hp-IgG 50 μL. Set up a negative control well and add 50 μL of PBS buffer to the negative control well. After sealing the plate with sealing film, incubate at 37°C for 30 min. Set up 3 replicates for each well. 2) Pat the liquid out of the holes, fill each hole with detergent and soak for 30 seconds, then discard and pat dry again; repeat this step 5 times. 3) Add 50 μL of secondary antibody-Trigger (diluted 10 times) to each reaction well, seal the plate with sealing film, and incubate at 37°C for 30 min; 4) The operation is the same as 2); 5) Add 50 μL of RF-HCR (0.5 μM) to each reaction well, seal the plate with a sealing film, and incubate at room temperature (25 °C) for 30 min; 6) The operation is the same as 2); 7) Add 50 μL of β-Gal to each reaction well, seal the plate with a sealing film, and incubate at 37°C for 30 min; 8) The operation is the same as 2); 9) Add 50 μL of fluorescent substrate FDG (as above) to each reaction well to make a final concentration of 10 μM. Seal the plate with sealing film and incubate at 37 °C for 30 min. 10) Use the Synergy H1 multi-functional microplate reader for fluorescence detection, with an excitation wavelength of 486nm and an emission wavelength of 518nm.

[0061] (3) Fluorescence characterization of RF-HCR-ELISA Six groups of reaction component characterization experiments were designed and RF-HCR-ELISA detection was performed. The nominal composition of each reaction system is shown in Table 3. The detection concentration of Hp-IgG was 25 pg / mL, as detailed below: ① No secondary antibody: The difference from the other groups is that no secondary antibody was added; ②No Trigger: The difference from the group is that no trigger is added; ③ No H1: The difference from the group is that H1 is not included; ④ No H2: The difference from the group is that H2 was not added; ⑤ No DRF: The difference from the groups is that DRF was not included; ⑥ Complete RF-HCR-ELISA: i.e. the system of step (2).

[0062] Each group had three replicates, and the average of the three replicates was used as the test result for each group. The settings for each group are shown in Table 3. "+" indicates the component present in the group, and "-" indicates the component not added to the group. The test results are as follows: Figure 11 As shown.

[0063] Depend on Figure 11 It is known that RF-HCR is compatible with the ELISA system, and RF-HCR-ELISA has been successfully constructed. In the ELISA environment, RF-HCR has better reaction efficiency than traditional HCR.

[0064] Table 3 Characterization of reaction components in RF-HCR-ELISA Note: Secondary antibody refers to a secondary antibody.

[0065] 2.4 Optimization of RF-HCR-ELISA reaction conditions (1) Optimization of the optimal working concentrations of antigen and detection antibody: Six antigen coating concentrations were set: 1 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 40 μg / mL. Six secondary antibody-trigger dilutions were also set: 1:10, 1:100, 1:1000, 1:2000, 1:4000, and 1:8000. For each antigen-coated well, a complete RF-HCR-ELISA detection procedure was performed using the six dilutions of the secondary antibody-trigger, with other conditions remaining unchanged (i.e., group ⑥ in the complete RF-HCR-ELISA detection system, hereinafter the same). The detection concentration was 25 pg / mL of Hp-IgG, with three replicates per group. Using fluorescence SNR as the selection criterion, the SNR was plotted as a dotted-line graph. The antigen coating concentration and dilution corresponding to the highest SNR point in the dotted-line graph were the optimal working concentrations for both. The results are as follows: Figure 12 As shown.

[0066] Depend on Figure 12 It is known that the optimal antigen coating concentration for RF-HCR-ELISA is 5 μg / mL, and the optimal secondary antibody-Trigger dilution is 1:100.

[0067] (2) Optimization of the secondary antibody-trigger conjugation ratio: Conjugation ratios of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, and 1:7 were prepared according to the molar concentrations of the secondary antibody and trigger. Complete RF-HCR-ELISA experiments were performed using these various secondary antibody-trigger ratios, with other conditions remaining constant. Hp-IgG at a concentration of 25 pg / mL was detected, with three replicates per group. The fluorescence SNR was used as the selection criterion; the ratio corresponding to the highest SNR value was considered the optimal secondary antibody-trigger conjugation ratio. The results are as follows: Figure 13 As shown.

[0068] Depend on Figure 13 It can be seen that the optimal ligation ratio of secondary antibody to trigger in the RF-HCR-ELISA system is 1:3.

[0069] (3) Optimization of reaction conditions for RF-HCR in the RF-HCR-ELISA system: 1) Optimization of RF-HCR reaction time: Under otherwise unchanged conditions, a complete RF-HCR-ELISA experiment was performed to detect Hp-IgG at a concentration of 25 pg / mL. RF-HCR reaction times were set to 1 min, 5 min, 15 min, 30 min, 45 min, 60 min, 75 min, and 90 min, with three replicates for each group. The fluorescence spot rate (SNR) was used as the selection criterion; the time corresponding to the highest SNR value was considered the optimal reaction time for RF-HCR in the RF-HCR-ELISA system. The results are as follows: Figure 14 As shown.

[0070] 2) Optimization of RF-HCR reaction temperature: With other conditions unchanged, a complete RF-HCR-ELISA experiment was performed to detect Hp-IgG at a concentration of 25 pg / mL. The RF-HCR reaction temperatures were set at 4℃, 8℃, 25℃, 37℃, 42℃, 50℃, 55℃, and 60℃, with three replicates for each group. The fluorescence spot rate (SNR) was used as the selection criterion; the temperature corresponding to the highest SNR value was considered the optimal reaction temperature for RF-HCR in the RF-HCR-ELISA system. The results are as follows: Figure 14 As shown.

[0071] 3) Optimization of RF-HCR reaction concentration: With other conditions unchanged, a complete RF-HCR-ELISA experiment was performed to detect Hp-IgG at a concentration of 25 pg / mL. The RF-HCR reaction concentrations (i.e., the final concentrations of S1, S2, S3, S4, H1, and H2 during RF-HCR preparation) were set to 0.025 μM, 0.05 μM, 0.1 μM, 0.25 μM, 0.5 μM, 1 μM, 2 μM, and 4 μM, with three replicates for each group. The fluorescence SNR was used as the selection criterion; the concentration corresponding to the highest SNR value was considered the optimal reaction concentration of RF-HCR in the RF-HCR-ELISA system. The results are as follows: Figure 14 As shown.

[0072] like Figure 14 As shown, the optimal reaction time for RF-HCR in the RF-HCR-ELISA system is 5 min, the optimal reaction temperature is 25℃, and the optimal reaction concentration is 0.25 μM.

[0073] (4) Optimization of reaction conditions for β-Gal and its fluorescent substrate FDG in the RF-HCR-ELISA system: 1) Optimization of β-Gal reaction time: Under otherwise unchanged conditions, a complete RF-HCR-ELISA experiment was performed to detect Hp-IgG at a concentration of 25 pg / mL. The reaction times for β-Gal were set to 1 min, 5 min, 15 min, 30 min, 45 min, 60 min, 75 min, and 90 min, with three replicates for each group. The fluorescence spot rate (SNR) was used as the selection criterion; the time corresponding to the highest SNR value was considered the optimal reaction time for β-Gal in the RF-HCR-ELISA system. The results are as follows: Figure 15 As shown.

[0074] 2) Optimization of β-Gal reaction temperature: With other conditions unchanged, a complete RF-HCR-ELISA experiment was performed to detect Hp-IgG at a concentration of 25 pg / mL. The reaction temperatures for β-Gal were set at 4℃, 8℃, 25℃, 37℃, 42℃, 50℃, 55℃, and 60℃, with three replicates for each group. The fluorescence spot rate (SNR) was used as the selection criterion; the temperature corresponding to the highest SNR value was considered the optimal reaction temperature for β-Gal in the RF-HCR-ELISA system. The results are as follows: Figure 15 As shown.

[0075] 3) FDG reaction concentration optimization: With other conditions unchanged, a complete RF-HCR-ELISA experiment was performed to detect Hp-IgG at a concentration of 25 pg / mL. FDG reaction concentrations were set at 0.1 μM, 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, and 30 μM, with three replicates for each group. The fluorescence spot rate (SNR) was used as the selection criterion; the concentration corresponding to the highest SNR value was considered the optimal reaction concentration of FDG in the RF-HCR-ELISA system. The results are as follows: Figure 15 As shown.

[0076] like Figure 15 As shown, the optimal reaction time for β-Gal in the RF-HCR-ELISA system is 30 min, the optimal reaction temperature is 37 °C, and the optimal reaction concentration for FDG is 15 μM.

[0077] 2.5 System Performance Verification: (1) Sensitivity verification With other conditions remaining unchanged, the complete RF-HCR-ELISA system was used to detect serially diluted Hp-IgG target antibodies under the optimal conditions determined in section 2.4: 1000 pg / mL, 200 pg / mL, 100 pg / mL, 50 pg / mL, 25 pg / mL, 12.5 pg / mL, 6.25 pg / mL, 3.13 pg / mL, 1.56 pg / mL, 0.78 pg / mL, 0.39 pg / mL, 0.20 pg / mL, 0.10 pg / mL, 0.05 pg / mL, and 0 pg / mL, with three replicates for each concentration. A standard curve was plotted with Hp-IgG concentration as the X-axis and the detected fluorescence intensity as the Y-axis.

[0078] According to formula A LOD Sensitivity is calculated as M + 3 × SD. In the formula, A... LOD Here, A represents the fluorescence intensity value corresponding to the limit of detection (LOD), M represents the average fluorescence intensity of the negative control, and SD represents the standard deviation of the negative control. LOD The LOD can be obtained by substituting the input into the standard curve, and the result is as follows: Figure 16 As shown.

[0079] like Figure 16 As shown, the calculated LOD was 0.30 pg / mL, and the linear range was 0.78 pg / mL-100 pg / mL (R²>0.98). RF-HCR-ELISA demonstrated excellent detection performance and high sensitivity.

[0080] (2) Specificity verification The specificity of the system in this application was verified by quantitative analysis of fluorescence intensity. The specific steps are as follows: With other conditions remaining unchanged, the complete RF-HCR-ELISA system prepared according to the optimal conditions determined in section 2.4 was used to perform quantitative analysis of fluorescence intensity in the following 8 groups, which are as follows: ①Hp-IgG; ②Hepatitis B surface antibody (HBs-Ab); ③Hepatitis B e antibody (HBe-Ab); ④ Hepatitis B core antibody (HBc-Ab); ⑤ Hepatitis C antibody (HCV-Ab); ⑥ Liver fluke antibody (CS-Ab); ⑦ Syphilis antibody (TP-Ab); ⑧ Human immunodeficiency virus antibody (HIV-Ab); The concentration of the corresponding antibody in each group was 100 pg / mL (the PBS negative control was replaced with an equal volume of PBS buffer). Three replicates were set for each group, and the average of the three replicates was used as the test result. The specificity of the method was evaluated by comparing the detection fluorescence intensity of each group. Results are as follows: Figure 17 As shown.

[0081] Depend on Figure 17 It is evident that the RF-HCR-ELISA system of this application exhibits no cross-reactivity or extremely weak cross-reactivity with other infectious antibodies such as HBs-Ab, HBe-Ab, HBc-Ab, HCV-Ab, CS-Ab, TP-Ab, and HIV-Ab. This result demonstrates the high specificity of the RF-HCR-ELISA detection.

[0082] (3) Accuracy and precision verification The accuracy and precision of the system in this application were verified by quantitative analysis of fluorescence intensity. The specific steps are as follows: Spiked sera were prepared by adding known concentrations of Hp-IgG to negative sera: 100 pg / mL, 50 pg / mL, 25 pg / mL, 12.5 pg / mL, 6.25 pg / mL, 3.13 pg / mL, 1.56 pg / mL, and 0.78 pg / mL. These spiked sera were then tested using an optimized RF-HCR-ELISA system (i.e., a complete RF-HCR-ELISA system prepared according to the optimal conditions determined in section 2.4, with all other conditions unchanged). Each group was tested in triplicate.

[0083] The accuracy and precision of this method were evaluated by calculating the recovery rate and coefficient of variation. The results are as follows: Figure 18 As shown.

[0084] like Figure 18 As shown, the recovery rate of the system proposed in this application can reach 79.37%-118.69% and the coefficient of variation is 1.36%-8.21%, both of which meet the requirements for clinical testing.

[0085] (III) Establishment and validation of the RF-HCR-ddELISA detection system 3.1 Theoretical Feasibility of RF-HCR-ddELISA The RF-HCR-ddELISA achieves digital detection primarily by utilizing two independent Poisson distribution probability theories, specifically the following two parts: (1) Capture of a single Hp-IgG target antibody based on Poisson distribution probability In the RF-HCR-ddELISA detection system, using magnetic beads to disperse and capture target antibodies in the sample is one of the key steps in achieving single-molecule target antibody detection. During the capture of dispersed target antibodies using magnetic beads, according to Poisson distribution probability theory, the probability of capturing x target antibodies on each magnetic bead is: In the formula, λIb represents the average number of target antibodies captured on each magnetic bead, i.e., the total number of target antibodies N in the sample. I Divide by the number of magnetic beads N b , λ Ib=N I / N b Calculations showed that only when λIb < 0.3 can it be ensured that at most one antibody is captured on 96.31% of the magnetic beads.

[0086] (2) Dispersion of a single magnetic bead based on the probability theory of Poisson distribution During the dispersion of magnetic beads and the fluorescent substrate FDG into microdroplets using a droplet microfluidic chip, it is necessary to ensure that each microdroplet contains only 0 or 1 magnetic beads. Therefore, according to the Poisson probability theory, the probability that each microdroplet contains y magnetic beads is: In the formula, λbd represents the average number of magnetic beads in each microdroplet, i.e., the total number of magnetic beads N. b Divide by the number of microdroplets, λbd = N b / N d Calculations show that only when λbd < 0.1 can it be ensured that 99.53% of the microdroplets contain at most one magnetic bead. In the RF-HCR-ddELISA detection system, this application introduces approximately 10... 4 One Hp antigen-functionalized magnetic bead, generating approximately 10 5 With a microdroplet size of λbd < 0.1, the Poisson distribution probability ensures that 99.53% of the microdroplets contain ≤ 1 magnetic bead. Under these conditions, the RF-HCR-ddELISA detection system can achieve digital detection of Hp-IgG.

[0087] 3.2 Preparation of Hp antigen-functionalized magnetic beads (1) Invert the magnetic beads in the reagent bottle to suspend and mix them, and pipette 100 μL of magnetic beads into a 1.5 mL EP tube; (2) Add 300 μL of MES buffer with a concentration of 25 mM and pH of 5.0, mix thoroughly, and then place on a rotary mixer for 10 min to rotate and incubate. (3) Insert the EP tube into the magnetic frame and let it stand for 1 minute. After the magnetic beads are attracted to the wall, remove the liquid. (4) Add 400 μL of 25 mM MES buffer solution with pH 5.0 to the EP tube, mix thoroughly, and then place it on a rotary mixer for 10 min. Repeat once. (5) Insert the EP tube into the magnetic frame and let it stand for 1 minute. After the magnetic beads are attracted to the wall, remove the liquid. (6) Add 60 μL of the prepared Hp antigen dilution solution with a concentration of 1 mg / mL to the above EP tube, mix thoroughly with the magnetic beads, and slowly rotate and incubate at room temperature for 30 min; (7) Immediately dissolve EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) in cold 100mM MES buffer solution with pH 5.0 to obtain an EDC solution with a concentration of 100mg / mL. Prepare and use immediately. (8) Add 30 μL of EDC solution to the above EP tube and mix thoroughly with the magnetic bead suspension; (9) Add 10 μL of 25 mM MES buffer with pH 5.0 to the EP tube and mix thoroughly. Then incubate slowly at 4°C for 2 h by rotating. (10) Insert the EP tube into the magnetic frame and let it stand for 1 minute. After the magnetic beads are attracted to the wall, remove the liquid. (11) Add 400 μL of Tris-HCl with a concentration of 50 mM and pH of 7.4, mix thoroughly, and then place on a rotary mixer for 15 min to rotate and incubate. (12) Insert the EP tube into the magnetic frame and let it stand for 1 minute. After the magnetic beads are attracted to the wall, remove the liquid. (13) Add 400 μL of 50 mM Tris-HCl with pH 7.4 containing 0.1% Tween-20 and vortex thoroughly; (14) Insert the EP tube into the magnetic frame and let it stand for 2 minutes. After the magnetic beads are attracted to the wall, remove the liquid. Repeat steps (13) and (14) 4 times. (15) Add 100 μL of PBS to resuspend the magnetic beads. At this point, the concentration of the magnetic beads is approximately 2 × 10⁻⁶. 6 beads / μL.

[0088] 3.3 Constructing a magnetic bead-based RF-HCR-ELISA detection system (1) Take a series of sealed black ELISA plates and set up several groups as follows: Group ①: Add 50 μL of Hp-IgG to the well to make the final concentration of Hp-IgG 100 pg / mL; Group ②: Add 50 μL of Hp-IgG to the well to make the final concentration of Hp-IgG 50 pg / mL; Group ③: Add 50 μL of Hp-IgG to the well to make the final concentration of Hp-IgG 25 pg / mL; Group ④: Add 50 μL of Hp-IgG to the wells to make the final concentration of Hp-IgG 12.5 pg / mL; Group ⑤: Add 50 μL of Hp-IgG to the wells to make the final concentration of Hp-IgG 6.25 pg / mL; Group 6: Add 50 μL of Hp-IgG to the wells to make the final concentration of Hp-IgG 3.13 pg / mL; Also set up a negative control well, and add 50 μL of PBS buffer to the negative control well; Add 10 to all holes 4 Functionalized magnetic beads were used. After sealing with a sealing film, the plates were incubated at 37°C for 30 min, with three replicates for each reaction well. (2) Carefully peel off the sealing film and place the well plate on the magnetic plate for 30 seconds. Then, keeping the well plate in close contact with the magnetic plate, tilt the plate to remove excess liquid. Remove the well plate, add 100 μL of washing solution to each well, let it stand for 30 seconds, then place the well plate back on the magnetic plate and keep it in close contact, let it stand for another 30 seconds, then tilt the plate to remove the washing solution and discard it. Repeat this process 5 times; (3) Add 50 μL of secondary antibody-Trigger (1:100 dilution, as above) to each reaction well, seal the plate with sealing film, and incubate at 37°C for 30 min; (4) The operation is the same as (2); (5) Add 50 μL of RF-HCR to each reaction well (prepared according to the above procedure, with the final concentrations of S1, S2, S3, S4, H1, and H2 all being 0.25 μM), seal the plate with a sealing film, and incubate at room temperature (25 °C) for 5 min. (6) The operation is the same as (2); (7) Add 50 μL of β-Gal to each reaction well, seal the plate with a sealing film, and incubate at 37°C for 30 min; (8) The operation is the same as (2); (9) Add 50 μL of fluorescent substrate FDG to each reaction well (the concentration of fluorescent substrate FDG was diluted to 15 μM using PBS buffer), seal the plate with sealing film, and incubate at 37°C for 30 min. (10) Fluorescence detection was performed on all wells using a Synergy H1 multi-functional microplate reader. The excitation wavelength was fixed at 486 nm, and the fluorescence emission wavelength range was 500 nm-680 nm. Fluorescence signals were detected every 5 nm. The average of three replicates for each group was used as the test result. A fluorescence response curve was plotted with the target antibody concentration on the x-axis and the fluorescence intensity on the y-axis. The results are as follows: Figure 19 As shown.

[0089] Depend on Figure 19 The fluorescence response curve showed R² = 0.99. This result indicates that magnetic bead-based RF-HCR-ELISA is feasible, and Hp antigen-functionalized magnetic beads have been successfully prepared and can form an immune complex -sRF-HCR-β-Gal structure on them.

[0090] 3.4 Observation of surface changes of magnetic beads using scanning electron microscopy (1) Prepare two groups to be observed: ① Magnetic beads before the reaction; ②Magnetic beads after complete RF-HCR-ELISA detection under the aforementioned optimal conditions.

[0091] (2) Scanning electron microscopy observation of the surface of each group of magnetic beads: The magnetic bead solution was thoroughly mixed and diluted with distilled water. Then, a small copper sheet was used as a carrier and wrapped with a layer of tin foil. Areas were marked on the tin foil with a pen. Approximately 10 μL of diluted magnetic bead suspension was added to the marked areas for each group. The copper sheet was placed in a vacuum chamber and vacuumed with a vacuum pump to quickly dry the liquid. After drying, the copper sheet was placed in an ion sputtering instrument for approximately 1 minute of surface gold sputtering. The treated copper sheet was carefully installed on the sample stage of the Apreo 2S scanning electron microscope, and then the sample stage was pushed into the pre-vacuum chamber and locked. The vacuum button was clicked on the dedicated software, and the process was waited for approximately 5 minutes until the software showed that the vacuuming progress had reached 100%. The complete copper sheet and the marked areas could be directly seen in the software. The desired observation position was selected with the mouse, and the electron microscope voltage was adjusted to 10.00 kV, and the distance from the sample to the probe was adjusted to 10 mm. Use the magnification knob to gradually increase the magnification, the focus knob to adjust the sharpness, and the astigmatism reduction knob to make the image sharper and clearer. Adjust the brightness and contrast to make the details on the magnetic bead surface bright and visible. After adjustment, click to take a picture in the software to obtain an image of the details on the magnetic bead surface, as shown in the image. Figure 20 As shown.

[0092] like Figure 20 As shown, the surface of the magnetic beads before the reaction has many obvious gaps and voids. Figure 20 A), and the surface of the magnetic beads after the reaction is smoother overall ( Figure 20B). Combined with the results of the previous experiment, the analysis shows that these viscous components are mainly immune complex-sRF-HCR-β-Gal structures. This result further suggests that Hp-functionalized magnetic beads are effective and can be used to construct subsequent RF-HCR-ddELISA detection systems.

[0093] 3.5 Verify the feasibility of RF-HCR-ddELISA using fluorescence optical microscopy Experimental group: Hp-IgG was detected at a concentration of 10 pg / mL; Control group: PBS was used for detection. The detection steps for (1)-(8) are the same as in 3.3. (9) Resuspend the magnetic beads in 30 μL of PBS buffer for subsequent dispersion into microdroplets; (10) Add droplet-generating oil to the oil phase wells of the droplet generation chip, and add fluorescent substrate FDG (10 μM) and magnetic bead resuspension to the sample wells, respectively. Mount the chip into the droplet generator, which pressurizes the oil phase wells and sample wells, causing the magnetic beads and FDG to disperse into microdroplets with a diameter of approximately 100 μm through microfluidic channels. This study generated approximately 10... 5 Each microdroplet; (11) After the microdroplets are generated, incubate them at 37°C for 30 min; The incubated microdroplets were placed on a glass slide and arranged in a monolayer. They were observed under low magnification using a BX51 fluorescence optical microscope. Bright-field observation was used to examine the microdroplets and the magnetic beads within them, while fluorescence observation (blue excitation light) was used to observe the fluorescence reaction of the microdroplets. Images of the droplets were acquired using Olympus software. The results are shown below. Figure 21 As shown.

[0094] like Figure 21 As shown, the number of magnetic beads in the microdroplets in the experimental group was ≤1 under bright field conditions, which is consistent with the theoretical calculation. After switching to a fluorescent field, the microdroplets containing magnetic beads emitted obvious green fluorescence, while the microdroplets without magnetic beads showed no fluorescence signal. Figure 21 B represents the negative control group. It was observed that no fluorescence signal was generated even when the microdroplets contained magnetic beads under a fluorescent field. This result indicates that an effective immune complex – sRF-HCR-β-Gal structure – was formed on the magnetic beads in the experimental group, and the β-Gal in this structure can effectively hydrolyze FDG in the microdroplets to produce fluorescein. Therefore, the RF-HCR-ddELISA system is feasible for digital detection of Hp-IgG target antibodies.

[0095] 3.6 Characterizing RF-HCR-ddELISA using a droplet reading instrument Design 7 sets of experiments: ① Group without magnetic beads: Compared with group ⑦, it lacks magnetic beads; ② Group without secondary antibody: lacked secondary antibody compared to group ⑦; ③ No Trigger Group: Lacking a Trigger compared to Group ⑦; ④ Group without H1: Group ⑦ lacks H1; ⑤ Group without H2: Group ⑦ lacks H2; ⑥ No DRF group: lacked DRF compared to group ⑦; ⑦ Complete RF-HCR-ddELISA.

[0096] Each group was tested three times. The settings for each group are shown in Table 4, where + indicates the component contained in the group, and - indicates the component not added to the group. Table 4. Characterization of reaction components in RF-HCR-ddELISA The detection steps (1)-(11) are the same as in 3.5. (12) The microdroplets and the droplet reading chip are loaded onto the droplet reader. The droplet reader automatically loads the microdroplets into the droplet reading chip and arranges them into a single layer. Then, the microdroplets are identified and counted. The results are as follows: Figure 22 and 23 As shown.

[0097] like Figure 22 As shown, the fluorescence signal of positive microdroplets in the RF-HCR-ddELISA system with complete characteristics was stronger than that in other groups.

[0098] like Figure 23 As shown, the trend of microdroplet fluorescence SNR in each group is consistent with the droplet analysis diagram. The first 4 groups have no SNR, the SNR of groups 5 / 6 / 7 gradually increases, and the 7th group has the highest SNR. This suggests that the droplet reader can accurately identify and distinguish the microdroplet signal of RF-HCR-ddELISA. Further optimization of RF-HCR-ddELISA can be carried out in the future.

[0099] 3.7 Optimization of RF-HCR-ddELISA reaction conditions (1) Optimization of the reaction time of β-Gal in the RF-HCR-ddELISA system: Under the same conditions, a complete RF-HCR-ddELISA experiment was performed to detect Hp-IgG at a concentration of 10 pg / mL. The reaction time of β-Gal was set to 1 min, 5 min, 15 min, 30 min, 45 min, 60 min, 75 min and 90 min, with 3 replicates for each group. The fluorescence signal of microdroplets was read using a droplet reader. The fluorescence SNR of microdroplets was used as the selection criterion. The time corresponding to the highest value of microdroplet SNR was the optimal reaction time of β-Gal in the RF-HCR-ddELISA system. The results are as follows: Figure 24As shown.

[0100] (2) Optimization of the reaction temperature of β-Gal in the RF-HCR-ddELISA system: Under the same conditions, a complete RF-HCR-ddELISA experiment was performed to detect Hp-IgG at a concentration of 10 pg / mL. The reaction temperatures of β-Gal were set to 4℃, 8℃, 25℃, 37℃, 42℃, 50℃, 55℃ and 60℃, with 3 replicates for each group. The fluorescence signal of microdroplets was read using a droplet reader. The microdroplet fluorescence SNR was used as the selection criterion. The temperature corresponding to the highest microdroplet SNR value was the optimal reaction temperature of β-Gal in the RF-HCR-ddELISA system. The results are as follows: Figure 25 As shown.

[0101] (3) Optimization of the reaction concentration of fluorescent substrate FDG in the RF-HCR-ddELISA system: Under the same conditions, a complete RF-HCR-ddELISA experiment was performed to detect Hp-IgG at a concentration of 10 pg / mL. The reaction concentrations of FDG were set to 0.1 μM, 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM and 30 μM, with 3 replicates for each group. The fluorescence signal of the microdroplets was read using a droplet reader. The fluorescence SNR of the microdroplets was used as the selection criterion. The concentration corresponding to the highest value of the microdroplet SNR was the optimal reaction concentration of FDG in the RF-HCR-ddELISA system. The results are as follows: Figure 26 As shown.

[0102] Depend on Figure 24-26 It can be seen that the optimal reaction time for β-Gal in the RF-HCR-ddELISA system is 60 min and the optimal reaction temperature is 37℃; the optimal concentration of FDG in the RF-HCR-ddELISA system is 5 μM.

[0103] 3.8 Evaluation of the detection performance of RF-HCR-ddELISA (1) Sensitivity verification The optimized RF-HCR-ddELISA system was used to detect serially diluted Hp-IgG target antibodies: 100 pg / mL, 50 pg / mL, 10 pg / mL, 1 pg / mL, 0.1 pg / mL, 0.01 pg / mL, 0.001 pg / mL, and 0 pg / mL, with three replicates for each concentration. In the RF-HCR-ddELISA, the target concentration is related to the average enzymes per bead (AEB), calculated as the negative logarithm of the ratio of negative droplets to total droplets: AEB = -ln(Negative droplets / Total droplets). When the total number of droplets is fixed, a higher number of positive droplets and a lower number of negative droplets results in a larger AEB value. Therefore, the Poisson distribution probability theory and the application of AEB allow the target concentration to be obtained by simply counting the number of positive droplets and the total number of droplets. A fitted curve was constructed with Hp-IgG concentration as the X-axis and AEB as the Y-axis. According to formula A... LOD Sensitivity is calculated as M + 3 × SD. In the formula, A... LOD Let A be the AEB corresponding to LOD, M be the mean AEB corresponding to the negative control, and SD be the standard deviation of the negative control. LOD Substituting the values ​​into the obtained curve, the LOD of RF-HCR-ddELISA can be calculated, and the results are as follows: Figure 27 As shown.

[0104] Depend on Figure 27 The detection limit (LOD) of RF-HCR-ddELISA was found to be 0.02 pg / mL (≈133 aM), with a linear range spanning four orders of magnitude and R² > 0.99. These results indicate that RF-HCR-ddELISA possesses excellent detection performance and extremely high sensitivity, enabling single-molecule immunoassay of target antibodies.

[0105] (2) Specificity verification Eight assays were performed using the optimized RF-HCR-ELISA system, as follows: ① PBS negative control (NC); ②HBs-Ab; ③HCV-Ab; ④CS-Ab; ⑤TP-Ab; ⑥Hp-IgG; ⑦HBs-Ab+HCV-Ab+CS-Ab+TP-Ab; ⑧HBs-Ab+HCV-Ab+CS-Ab+TP-Ab+Hp-IgG; Each antibody concentration was 20 pg / mL, and each group was configured with three replicates. The specificity of the method was evaluated by comparing the detection values ​​of each group. The results are as follows: Figure 28 As shown.

[0106] Depend on Figure 28 It can be seen that the RF-HCR-ddELISA system has good specificity and the ability to specifically detect Hp-IgG target antibodies from serum matrices containing a large number of interfering antibodies.

[0107] 2.3.5 Clinical Sample Testing Performance Test (1) Centrifuge 2000 g for 5 min of the 24 collected clinical serum samples (including 10 positive, 6 weakly positive and 8 negative) to precipitate the remaining cells and cell debris, and store the supernatant at -80℃ for later use.

[0108] (2) Dilute the serum samples 5-fold using the General Assay Diluent. Add 50 μL of diluted serum to the optimized RF-HCR-ddELISA detection system for detection, and calculate and analyze the Hp-IgG concentration in each sample.

[0109] (3) The RF-HCR-ddELISA detection results were compared and analyzed with the clinical plate ELISA qualitative detection results to evaluate the clinical sample detection capability of this method. The results are as follows: Figure 29 As shown.

[0110] like Figure 29 As shown, the difference in detection concentration between the positive and negative sample groups was significant. P <0.001), the difference in detection concentration between the positive sample group and the weakly positive sample group was statistically significant ( P <0.05, the difference in detection concentration between the weakly positive sample group and the negative sample group was significant ( P <0.001). ROC analysis showed that, compared with clinical serum antibody qualitative detection results, RF-HCR-ddELISA could accurately distinguish between positive, weakly positive, and negative samples, with 100% accuracy, 100% sensitivity, and 100% specificity, with cutoff values ​​of 45.63 pg / mL, 65.19 pg / mL, and 15.38 pg / mL, respectively. These results indicate that RF-HCR-ddELISA is suitable for clinical sample detection, accurately distinguishing between positive, weakly positive, and negative samples, and is expected to provide strong support for the diagnosis and monitoring of Helicobacter pylori infection in clinical practice.

[0111] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A kit for detecting Helicobacter pylori antibodies, characterized in that, The device includes a first oligonucleotide chain, a second oligonucleotide chain, a third oligonucleotide chain, a fourth oligonucleotide chain, a first hairpin probe, a second hairpin probe, Helicobacter pylori antigen-functionalized magnetic beads, an enzyme fluorescent substrate, a secondary antibody-trigger complex, a buffer solution, a droplet generation chip, and a droplet detection chip. The first, second, third, and fourth oligonucleotide chains can self-assemble to form a rectangular DNA framework. One end of the trigger is modified with an azide. One end of both the first and second hairpin probes is modified with biotin-triethylene glycol. The Helicobacter pylori antigen-functionalized magnetic beads are formed by co-incubation of Helicobacter pylori antigen and magnetic beads. The secondary antibody-trigger complex is formed by connecting a secondary antibody and a trigger.

2. The kit for detecting Helicobacter pylori antibodies as described in claim 1, characterized in that, The nucleotide sequence of the first oligonucleotide chain is shown in SEQ ID NO.1, the nucleotide sequence of the second oligonucleotide chain is shown in SEQ ID NO.2, the nucleotide sequence of the third oligonucleotide chain is shown in SEQ ID NO.3, and the nucleotide sequence of the fourth oligonucleotide chain is shown in SEQ ID NO.

4.

3. The kit for detecting Helicobacter pylori antibodies as described in claim 1, characterized in that, The nucleotide sequence of the first hairpin probe is shown in SEQ ID NO.8, and the nucleotide sequence of the second hairpin probe is shown in SEQ ID NO.

9.

4. The kit for detecting Helicobacter pylori antibodies as described in claim 1, characterized in that, The oligonucleotide sequence of the trigger is shown in SEQ ID NO.

7.

5. The kit for detecting Helicobacter pylori antibodies as described in claim 1, characterized in that, Both the first hairpin probe and the second hairpin probe are fixed to the rectangular DNA framework.

6. The kit for detecting Helicobacter pylori antibodies as described in claim 1, characterized in that, The molar ratio of the first oligonucleotide chain, the second oligonucleotide chain, the third oligonucleotide chain, and the fourth oligonucleotide chain is 0.8-1.1:0.8-1.1:0.8-1.1:0.8-1.1; And / or, the molar ratio of the first hairpin probe to the rectangular DNA frame is 0.8-1.1:0.8-1.

1.

7. The kit for detecting Helicobacter pylori antibodies as described in claim 1, characterized in that, The Helicobacter pylori antigen-functionalized magnetic beads are used to capture Helicobacter pylori antibodies; And / or, the Helicobacter pylori antigen-functionalized magnetic beads are formed by covalently coupling the Helicobacter pylori antigen to the surface of the magnetic beads.

8. The kit for detecting Helicobacter pylori antibodies as described in claim 1, characterized in that, The molar ratio of the second hairpin probe to the rectangular DNA framework is 0.8-1.1:0.8-1.1; And / or, the molar ratio of the trigger to the secondary antibody is 2-4:

1.

9. The kit for detecting Helicobacter pylori antibodies as described in claim 1, characterized in that, The droplet generation chip is used to generate microdroplets and then perform an enzymatic reaction, and the droplet detection chip is used to quantitatively detect the microdroplets after the enzymatic reaction. And / or, the enzyme fluorescent substrate is used to generate a fluorescent signal via an enzymatic reaction; And / or, the trigger in the secondary antibody-trigger complex is used to initiate a hybridization chain reaction; And / or, the buffer solution is a TNaK buffer solution.

10. A method for detecting Helicobacter pylori antibodies, characterized in that, The method utilizes the kit described in any one of claims 1-9 to detect biological samples.

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