Magnetic immune micro-fluidic helicobacter pylori typing detection chip and method

Abstract

The invention discloses a magnetic immune micro-fluidic helicobacter pylori typing detection chip and method, and relates to the technical field of biomedical detection, the magnetic immune micro-fluidic helicobacter pylori typing detection chip comprises: a magnetic probe: core-shell structure Fe3O4 (at) SiO2 nanoparticles, the core diameter is 70-90 nm, the shell thickness is 15-25 nm, and the surface is covalently bound with a helicobacter pylori typing antibody combination; the antibody combination comprises a CagA antibody and a VacA antibody in a molar ratio of (1: 1)-(1: 3); the magnetic immune micro-fluidic helicobacter pylori typing detection chip is efficient in detection, the whole process can be completed within 45 minutes, and the detection speed is far faster than that of a traditional method. A core-shell structure Fe3O4 (at) SiO2 magnetic probe is adopted, a zigzag pre-enrichment channel with an inclination angle of 30-0 degree and PluronicF127 modification treatment are combined, the enrichment efficiency is high, the detection limit reaches 10 < 2 > CFU / mL, and the sensitivity is excellent.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to a magnetic immunoassay microfluidic chip and method for detecting Helicobacter pylori typing. Background Technology

[0002] Helicobacter pylori (Hp), a Gram-negative microaerophilic bacillus, primarily colonizes the surface of gastric mucosal epithelial cells and is a key pathogenic factor in chronic gastritis, peptic ulcers, and even gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer. Based on virulence phenotypes, Hp can be divided into type I (highly virulent strains) and type II (lowly virulent strains): Type I strains, carrying the cytotoxin-associated gene A (CagA) and the vacuole toxin gene (VacA), stably express CagA and VacA proteins, exhibiting stronger tissue invasiveness and immunogenicity; Type II strains lack these virulence genes, resulting in significantly reduced pathogenicity. Therefore, accurate Hp genotyping is of irreplaceable value for clinical treatment strategy development, disease prognosis assessment, and early warning of gastric cancer risk.

[0003] Existing Hp genotyping technologies have significant limitations: bacterial isolation and culture, as the gold standard, requires a strictly anaerobic environment and specific culture media, with an operation cycle of 3-7 days, and is affected by the quality of sample collection, with a detection rate often below 60%; polymerase chain reaction (PCR) can achieve high-sensitivity detection through gene amplification, but is prone to false positives due to cross-contamination, and requires high levels of laboratory hardware and operator skills; while immunoblotting can achieve protein-level genotyping through antibody recognition, it suffers from problems such as high reagent consumption, cumbersome operation steps, and a detection cycle exceeding 24 hours, making it difficult to meet the clinical demand for real-time testing.

[0004] Therefore, we proposed a magnetic immunomicrofluidic Helicobacter pylori typing detection chip and method to solve the problems mentioned above.

[0005] The information disclosed above in this background section is only for enhancing the understanding of the background section of this invention, and therefore may include prior art that is not known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a magnetic immunomicrofluidic Helicobacter pylori typing detection chip and method to solve the limitations of current Hp typing detection technologies on the market as mentioned in the background.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A magnetic immunoassay microfluidic Helicobacter pylori typing detection chip includes:

[0009] Magnetic probe: core-shell structured Fe3O4@SiO2 nanoparticles with a core diameter of 70-90 nm and a shell thickness of 15-25 nm, covalently bound to a Helicobacter pylori typing antibody combination on the surface; the antibody combination includes CagA antibody and VacA antibody in a molar ratio of 1:1-1:3.

[0010] Microfluidic substrate: It includes a three-inlet design with a serum inlet, a lysis buffer inlet, and a buffer inlet, which are sequentially connected to a sample mixing area, a serrated pre-enrichment channel, and a cross-shaped genotyping reaction cell; the serrated pre-enrichment channel has an inclination angle of 30°-60°, and the inner wall of the channel is modified with 0.5-2% Pluronic F127.

[0011] Detection module: A giant magnetoresistive sensor array embedded at the bottom of the typing reaction cell, with a sensor spacing ≤ 1.5 times the diameter of the antibody binding site, and the resolution of the giant magnetoresistive sensor array ≥ 0.1 mT.

[0012] Preferably, the magnetic probe has a particle size range of 85-115 nm.

[0013] Preferably, the sample mixing zone is designed to allow the gastric fluid sample to be fully mixed and incubated with the magnetic probe for 5-15 minutes.

[0014] A detection method for a magnetic immunoassay microfluidic Helicobacter pylori typing detection chip includes the following steps:

[0015] Step 1: Incubate the gastric fluid sample and the magnetic probe in the sample mixing area for 5-15 minutes;

[0016] Step 2: Apply a gradient magnetic field of 0.5-1.5T to enrich the magnetic probe bound to the target bacteria in the serrated pre-enrichment channel;

[0017] Step 3: After removing the magnetic field, use a pulsed fluid flow rate of 0.05-0.2 mL / min to allow the complex to flow into the typing reaction cell and undergo a sandwich reaction with the immobilized detection antibody;

[0018] Step 4: Detect the magnetic signal intensity using a giant magnetoresistive sensor. When CagA is positive and the VacA positive signal is ≥3 times the baseline value, it is determined to be type I bacteria. When CagA is negative and VacA is negative, it is determined to be type II bacteria.

[0019] Preferably, the immobilized detection antibody is a specific antibody against Helicobacter pylori CagA and VacA antigens, which is immobilized in different regions of the cross-type reaction pool to achieve a multi-target immune response.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] The magnetic immunomicrofluidic Helicobacter pylori typing detection chip of this invention exhibits high detection efficiency, completing the entire process within 45 minutes, significantly faster than traditional methods. It employs a core-shell structured Fe3O4@SiO2 magnetic probe, combined with a serrated pre-enrichment channel with a 30°-60° tilt angle and Pluronic F127 modification treatment, resulting in high enrichment efficiency and a detection limit of 10. 2 CFU / mL, with excellent sensitivity.

[0022] This invention achieves accurate typing by using a sandwich reaction of CagA and VacA antibodies combined with a giant magnetoresistive sensor array with a resolution ≥0.1mT. Type I is determined by double positivity of CagA and VacA, and Type II is determined by double negative, with reliable results.

[0023] This invention features a three-inlet design combined with pulse-driven operation, making it easy to operate, highly automated, and its integrated and miniaturized design saves reagents, making it easy to promote at the grassroots level and providing a rapid and reliable basis for clinical diagnosis.

[0024] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the invention will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the microfluidic substrate in this invention.

[0026] In the diagram: 101, serum inlet; 102, lysis buffer inlet; 103, buffer solution inlet; 200, sample mixing area; 300, serrated pre-enrichment channel; 400, cross-shaped typing reaction chamber. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1: Fabrication of a magnetic immunomicrofluidic Helicobacter pylori typing detection chip

[0029] 1. The specific steps for preparing magnetic probes are as follows:

[0030] First, Fe3O4 nanoparticles were prepared by coprecipitation: 0.1 mol / L FeCl2 solution and 0.2 mol / L FeCl3 solution were mixed at a volume ratio of 1:2, and 5 mol / L NaOH solution was slowly added dropwise under nitrogen protection to adjust the pH to 10-11. The reaction was carried out at 60℃ for 30 min to generate Fe3O4 nanoparticles. After washing and centrifugation, Fe3O4 cores with a core diameter of 80±10 nm were obtained.

[0031] Then, the SiO2 shell was encapsulated using the sol-gel method: the above Fe3O4 nanoparticles were dispersed in a mixed solution of ethanol and water with a volume ratio of 4:1, ammonia was added to adjust the pH to 9-10, and then tetraethyl orthosilicate was slowly added dropwise. The mixture was stirred at 30°C for 2 hours. The amount of tetraethyl orthosilicate was controlled to make the thickness of the generated SiO2 shell 20±5nm, thus obtaining core-shell structured Fe3O4@SiO2 nanoparticles.

[0032] Finally, the surface-modified typing antibody combination was prepared: core-shell Fe3O4@SiO2 nanoparticles were washed with 0.1 mol / L LMES and pH 5.5 buffer, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added. The mixture was activated at room temperature for 30 min, and then CagA antibody and VacA antibody were added in a molar ratio of 1:2. The mixture was incubated at 37 °C for 2 h. After centrifugation and washing, the magnetic probe of the surface covalently bound typing antibody combination was obtained.

[0033] 2. Integration of the detection module

[0034] A giant magnetoresistive sensor array with a resolution ≥0.1mT and a sensor spacing 1.2 times the diameter of the antibody binding site is embedded in the bottom of the cross-shaped typing reaction cell 400 and connected to an external detection circuit via wires to complete the integration of the detection module.

[0035] The PDMS microfluidic substrate prepared above was bonded to a substrate with an integrated detection module to obtain a magnetic immunomicrofluidic Helicobacter pylori typing detection chip.

[0036] Example 2: Helicobacter pylori typing detection using the chip from Example 1.

[0037] First, sample processing is performed by collecting gastric fluid samples, centrifuging them, and obtaining the supernatant for later use.

[0038] From the appendix Figure 1 As shown, the specific testing steps are as follows:

[0039] Step 1: Inject the gastric fluid sample and the magnetic probe prepared in Example 1 into the sample mixing area 200 through the serum inlet 101 and the corresponding channel, respectively, and incubate them in the sample mixing area 200 for 10 min.

[0040] Step 2: Apply a 1.0T gradient magnetic field to the outside of the serrated pre-enrichment channel 300 to enrich the magnetic probe bound to the target bacteria in the serrated pre-enrichment channel 300 for 5 minutes.

[0041] Step 3: After removing the magnetic field, inject buffer solution through buffer inlet 103. Using a pulsed fluid drive at 0.1 mL / min, allow the complex to enter the cross-shaped typing reaction chamber 400, where it undergoes a sandwich reaction with the immobilized detection antibody. The reaction time is 15 min. The immobilized detection antibody is a specific antibody against Helicobacter pylori CagA and VacA antigens, immobilized in different regions of the cross-shaped typing reaction chamber.

[0042] Step 4: Detect the magnetic signal intensity using a giant magnetoresistive sensor array. When CagA positivity is detected and the VacA positivity signal is ≥ 3 times the baseline value, it is determined to be type I bacteria; when CagA negative and VacA negative are detected, it is determined to be type II bacteria.

[0043] In summary, the integrated design of microfluidic chips combines sample mixing, enrichment, reaction, and detection into one process. Combined with a pulsed fluid drive, this significantly shortens the detection time, which can be controlled within 45 minutes, much faster than traditional bacterial culture and PCR methods, thus meeting the needs of rapid clinical testing.

[0044] This invention utilizes core-shell Fe3O4@SiO2 nanoparticles as a magnetic probe. Their superparamagnetic properties ensure efficient enrichment under a gradient magnetic field. Combined with a serrated pre-enrichment channel (30°-60° tilt angle) and Pluronic F127 modification, the capture efficiency of target bacteria is significantly improved, achieving a detection limit of 10. 2 CFU / mL; at the same time, through the specific combination of CagA antibody and VacA antibody with a molar ratio of 1:1-1:3 and the sandwich reaction design, it can identify only the characteristic antigens of Helicobacter pylori, effectively avoiding cross-reaction and improving detection specificity.

[0045] Based on the biological characteristics of Helicobacter pylori type I containing CagA and VacA and type II without CagA and VacA, this invention designs a standard to identify type I bacteria as CagA-positive and VacA-positive with a signal ≥ 3 times the baseline value, and type II bacteria as CagA-negative and VacA-negative. Combined with the precise detection of magnetic signals by a giant magnetoresistive sensor array with a resolution ≥ 0.1 mT, this invention achieves accurate differentiation between the two types, providing a reliable basis for disease diagnosis and treatment planning.

[0046] The chip in this invention adopts a three-inlet design, which, combined with the automated fluid control of the microfluidic system, reduces manual operation steps and human error; at the same time, no complicated instruments and equipment are required during the detection process, making it easy to promote and apply in primary medical institutions.

[0047] This invention integrates a magnetic probe, a pre-enrichment channel, a typing reaction cell, and a giant magnetoresistive sensor array onto a single chip, which not only saves on sample and reagent usage and reduces detection costs, but also makes rapid on-site detection of Helicobacter pylori possible.

[0048] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0049] The embodiments of the present invention are given for the purposes of illustration and description. Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Any changes, modifications, substitutions and variations made by those skilled in the art to the above embodiments within the scope of the present invention should be included within the protection scope of the present invention.

Claims

1. A magnetic immunoassay microfluidic Helicobacter pylori typing detection chip, characterized in that, include: Magnetic probe: core-shell structured Fe3O4@SiO2 nanoparticles with a core diameter of 70-90 nm and a shell thickness of 15-25 nm, covalently bound to a Helicobacter pylori typing antibody combination on the surface; the antibody combination includes CagA antibody and VacA antibody in a molar ratio of 1:1-1:

3. Microfluidic substrate: It includes a three-inlet design with a serum inlet (101), a lysis buffer inlet (102), and a buffer inlet (103), which are sequentially connected to a sample mixing area (200), a serrated pre-enrichment channel (300), and a cross-shaped genotyping reaction cell (400); the serrated pre-enrichment channel has an inclination angle of 30°-60°, and the inner wall of the channel is modified with 0.5-2% Pluronic F127; Detection module: a giant magnetoresistive sensor array embedded at the bottom of the typing reaction cell (400), with a sensor spacing ≤ 1.5 times the diameter of the antibody binding site, and the resolution of the giant magnetoresistive sensor array ≥ 0.1 mT.

2. The magnetic immunoassay microfluidic Helicobacter pylori typing detection chip according to claim 1, characterized in that: The magnetic probe has a particle size range of 85-115 nm.

3. The magnetic immunoassay microfluidic Helicobacter pylori typing detection chip according to claim 1, characterized in that: The sample mixing zone (200) is used to thoroughly mix and incubate the gastric fluid sample with the magnetic probe for 5-15 minutes.

4. A detection method using the magnetic immunomicrofluidic Helicobacter pylori typing detection chip according to claim 1, characterized in that, Includes the following steps: Step 1: Incubate the gastric fluid sample and the magnetic probe in the sample mixing area for 5-15 minutes; Step 2: Apply a gradient magnetic field of 0.5-1.5T to enrich the magnetic probe bound to the target bacteria in the serrated pre-enrichment channel; Step 3: After removing the magnetic field, use a pulsed fluid flow rate of 0.05-0.2 mL / min to allow the complex to flow into the typing reaction cell and undergo a sandwich reaction with the immobilized detection antibody; Step 4: Detect the magnetic signal intensity using a giant magnetoresistive sensor. When CagA is positive and the VacA positive signal is ≥ 3 times the baseline value, it is determined to be type I bacteria. When CagA is negative and VacA is negative, it is determined to be type II bacteria.

5. The detection method of the magnetic immunomicrofluidic Helicobacter pylori typing detection chip according to claim 4, characterized in that: The immobilized detection antibodies are specific antibodies against Helicobacter pylori CagA and VacA antigens, which are immobilized in different regions of the cross-type reaction pool to achieve a multi-target immune response.

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