Detection kit and method for detecting allergic inflammatory factors based on centrifugal micro-fluidic chip

Abstract

The invention discloses a detection kit and method for detecting allergic inflammatory factors based on a centrifugal micro-fluidic chip, and relates to the field of kits, the detection kit comprises the centrifugal micro-fluidic chip and a preset reagent; the centrifugal micro-fluidic chip comprises a chip base body, a sample adding cavity, a liquid level sensor, a liquid level sensor, a liquid level sensor, a liquid level sensor and a liquid level sensor, the nucleic acid extraction and purification cavity comprises a plurality of amplification cavities; a reagent storage chamber; a waste liquid chamber; and a capillary valve. The functions of nucleic acid extraction, variable temperature amplification and multi-target synchronous detection are integrated through the centrifugal micro-fluidic chip, full-closed operation is achieved by means of centrifugal force or other driving force such as air pressure and electroosmotic flow, and the pollution risk caused by manual step-by-step operation is thoroughly eliminated; a multi-wavelength distinguishable fluorescent probe and a grouping preset primer combination are adopted to synchronously complete parallel detection of the allergic inflammatory factor mRNA and the drug accompanying diagnosis target, cross interference is avoided, and large instruments and equipment do not need to be relied on.

Description

Technical Field

[0001] This invention relates to the field of reagent kits, specifically a detection kit and method for detecting allergic inflammatory factors based on a centrifugal microfluidic chip. Background Technology

[0002] The occurrence and development of allergic diseases are closely related to the gene expression levels of various inflammatory factors and drug targets. Among them, the mRNA expression of eosinophil-associated proteins (such as MBP, ECP, EDN, and EPO) can reflect the intensity of allergic inflammatory activity, while gene variations or expression levels of histamine receptors (H1R, H4R), leukotriene receptors (CYS1, CYS2), and interleukin receptors (IL4Rα, IL5Rα) directly affect the efficacy of anti-allergy drugs.

[0003] Current technologies primarily detect target nucleic acids in blood and tissue samples using qPCR or digital PCR, but these methods rely on large instruments and step-by-step procedures: first, nucleic acids must be manually separated from the sample, then transferred to the amplification reaction system, and finally, the signal is collected using a fluorescence detector. These methods have significant limitations: the separation of nucleic acid extraction and amplification steps is cumbersome, and opening the container for transfer can easily lead to aerosol contamination; multiplex detection requires multi-well plates or microfluidic chips with external pump and valve systems, making fully automated, closed-loop systems difficult to achieve; furthermore, traditional temperature-controlled amplification (such as PCR) takes more than 2 hours and requires high precision in temperature control, failing to meet the needs of rapid point-of-care diagnostics. Summary of the Invention

[0004] Based on this, the purpose of the present invention is to provide a detection kit and method for detecting allergic inflammatory factors based on a centrifugal microfluidic chip, so as to solve the technical problems of existing nucleic acid detection technology, such as cumbersome operation procedures and easy contamination, multiple target detection relying on external equipment, and traditional amplification methods being time-consuming and having high equipment requirements.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a detection kit and method for detecting allergic inflammatory factors based on a centrifugal microfluidic chip.

[0006] A first aspect of the present invention provides a detection kit for detecting allergic inflammatory factors based on a centrifugal microfluidic chip, comprising a centrifugal microfluidic chip and pre-prepared reagents; the centrifugal microfluidic chip includes a chip substrate, and integrated into the chip substrate: a sample dispensing chamber with sample dispensing wells; a nucleic acid extraction and purification chamber connected to the sample dispensing chamber via a first microchannel, with a built-in silica membrane or magnetic beads for separating nucleic acids; and multiple amplification chambers arranged along the centrifugation direction and centered on the same circumference, each amplification chamber being pre-prepared for different allergic inflammatory factor mRNAs or drug companion diagnostic targets. The system includes: a target amplification primer; a reagent storage chamber connected to the nucleic acid extraction and purification chamber via a second microchannel, containing a pre-placed lyophilized fluorescent probe; a waste liquid chamber connected to the amplification chamber via a third microchannel; and a capillary valve located in the flow channel between the nucleic acid extraction and purification chamber and the amplification chamber. The pre-placed reagents include: a lysis buffer pre-placed in a first sub-storage chamber; a washing buffer pre-placed in a second sub-storage chamber; and an elution buffer pre-placed in a third sub-storage chamber. The first, second, and third sub-storage chambers are connected to the nucleic acid extraction and purification chamber via controllable valves.

[0007] The present invention is further configured such that the amplification chamber is divided into at least two groups: the first group is pre-loaded with amplification primers for allergic inflammatory factor mRNA, the targets of which include at least three of MBP, ECP, EDN, EPO, and β-trypsin; the second group is pre-loaded with amplification primers for drug companion diagnostic targets, the targets of which include at least three of H1R, H4R, CYS1, CYS2, IL4Rα, and IL5Rα; the lyophilized fluorescent probes corresponding to each group have different fluorescence wavelengths, with a wavelength range of 500nm-800nm.

[0008] The present invention is further configured such that the fluorescent probe has a fluorescent group selected from at least three of FAM, HEX, ROX, Cy5 or Quasar670.

[0009] The present invention is further configured such that the nucleic acid extraction and purification chamber includes: a lysis chamber connected to a lysis buffer storage area via a controllable valve; a binding chamber with a built-in silica membrane or magnetic beads; and an elution chamber connected to the amplification chamber via a capillary valve.

[0010] The present invention is further configured such that the controllable valve is a hot-melt paraffin valve or a phase change valve; the chip substrate material is COP or PMMA; and the amplification chamber integrates a temperature-controlled amplification module.

[0011] The present invention is further configured such that the temperature-variable amplification module is a PCR reaction system, the amplification temperature range covers the denaturation, annealing and extension stages, and the typical number of cycles is 35-45 cycles.

[0012] A second aspect of the present invention also provides a method for detecting allergic inflammatory factors based on a centrifugal microfluidic chip, using the above-mentioned detection kit, comprising the following steps: S1: injecting the sample to be tested into the sample loading chamber, and driving the sample into the nucleic acid extraction and purification chamber by centrifugal force or other driving force (such as air pressure, electroosmotic flow); S2: sequentially opening the controllable valve, allowing the lysis buffer, washing buffer, and elution buffer to flow through the nucleic acid extraction and purification chamber to complete nucleic acid purification; S3: after the purified nucleic acid breaks through the capillary valve, it flows into the reagent storage chamber, and the lyophilized fluorescent probe is reconstituted; S4: the mixture flows into the amplification chamber, initiating temperature-controlled amplification and detecting the fluorescence signal in real time; S5: the optical system simultaneously scans the multi-channel fluorescence signals of each amplification chamber, distinguishing and quantifying the target nucleic acid by wavelength.

[0013] The present invention is further configured such that the amplification conditions in step S4 are typical PCR cycling programs, including denaturation at 95°C, annealing at 55-65°C, and extension at 72°C, with 35-45 cycles, and the fluorescence signal is collected at the end of annealing or extension in each cycle.

[0014] The present invention is further configured such that, in step S5, the optical system employs real-time fluorescence detection or endpoint fluorescence detection, and the number of channels is consistent with the number of amplification chamber groups.

[0015] In summary, the present invention has the following main beneficial effects: This invention integrates nucleic acid extraction, temperature-switched amplification, and simultaneous multi-target detection functions through a centrifugal microfluidic chip. It achieves fully enclosed operation by relying on centrifugal force or other driving forces (such as air pressure, electroosmosis, etc.), completely eliminating the risk of contamination introduced by manual step-by-step operation. It uses multi-wavelength distinguishable fluorescent probes and pre-grouped primers to simultaneously complete the parallel detection of allergic inflammatory factor mRNA and drug companion diagnostic targets, avoiding cross-interference and eliminating the need for large instruments and equipment. Combined with temperature-switched amplification technology, it significantly shortens the detection time, providing an efficient, accurate, and portable solution for the diagnosis and medication guidance of allergic diseases. Attached Figure Description

[0016] Figure 1 This is a flowchart of the method for detecting allergic inflammatory factors based on a centrifugal microfluidic chip according to the present invention. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0018] The embodiments of the present invention will now be described.

[0019] Example 1 This embodiment provides a detection kit for detecting allergic inflammatory factors based on a centrifugal microfluidic chip, the structure of which includes: 1. Chip substrate: Made of COP material, with a diameter of 10 cm and a thickness of 2 mm.

[0020] 2. Functional cavity design: Sample loading chamber: with a volume of 200 μL and a sample loading well diameter of 1.5 mm, connected to the nucleic acid extraction and purification chamber through a microchannel with a width of 500 μm.

[0021] Nucleic acid extraction and purification chamber: consists of three sub-chambers: The pyrolysis chamber has a volume of 150 microliters and is connected to the pyrolysis fluid storage area via a hot-melt paraffin valve. The cavity has a volume of 50 microliters and contains a silicone membrane with a pore size of 5 micrometers or magnetic beads with a particle size of 1 micrometer. Elution chamber: with a volume of 30 microliters, connected to the amplification chamber via a capillary valve with a width of 100 micrometers and a depth of 50 micrometers.

[0022] Amplification chambers: 6 in total, each with a volume of 25 μL, arranged in a circle around the chip center with a radius of 3.5 cm. Pre-set lyophilized primer concentration is 10 μmol / L. Grouping configuration is as follows:

[0023] Reagent storage chamber: 80 μL volume, pre-filled with lyophilized fluorescent probe at a concentration of 5 μmol / L, probe configuration as follows:

[0024] Waste liquid chamber: with a volume of 300 microliters, connected to the outlets of each chamber through microchannels.

[0025] 3. Pre-prepared reagents:

[0026] Example 2 The steps for testing whole blood samples using the kit from Example 1 are as follows: 1. Nucleic acid extraction: 100 μL of whole blood was injected into the sample loading chamber and centrifuged at 2000 rpm for 5 minutes. The sample was then placed into the lysis chamber. Heat to 70 degrees Celsius, open the paraffin valve, and the pyrolysis solution flows into the pyrolysis chamber. Incubate at 37 degrees Celsius for 5 minutes. Centrifuge at 3000 rpm for 2 minutes to drive the lysate mixture through a silica membrane, where nucleic acids are bound. The washing solution was centrifuged at 2500 rpm for 1 minute, and repeated twice to rinse impurities into the waste liquid chamber; The elution buffer was centrifuged at 1000 rpm for 1 minute to dissociate the nucleic acid into the elution chamber.

[0027] 2. Amplification and Detection: Nucleic acid elution breaks through the capillary valve and flows into the reagent storage chamber, where it is reconstituted and lyophilized as a fluorescent probe. The mixture flows into the amplification chamber, the integrated temperature-controlled module is activated, and the PCR program is run: denaturation at 95℃ for 30 seconds, annealing at 60℃ for 30 seconds, extension at 72℃ for 30 seconds, for a total of 40 cycles. Fluorescence signals are acquired at the end of each annealing cycle, and the optical system is configured as follows:

[0028] 3. Result determination: Positive criteria: Ct value less than or equal to 35, or endpoint fluorescence intensity more than 3 times that of the negative control; The test is deemed invalid if the internal standard gene GAPDH is not detected.

[0029] Example 3 1. Sensitivity and repeatability: Five replicate tests were performed on samples with gradient concentrations of MBP mRNA:

[0030] The minimum detection limit is 1 copy per microliter, and the signal-to-noise ratio is greater than 3.

[0031] 2. Specificity of multiplex detection: Adding a 10-fold concentration to MBP-positive samples interferes with nucleic acid detection results.

[0032] 3. Comparison of timeliness:

[0033] This invention integrates nucleic acid extraction and temperature-controlled amplification using a centrifugal microfluidic chip, achieving fully enclosed automated detection with a detection limit as low as 1 copy / µL (MBP mRNA, coefficient of variation ≤4.3%), demonstrating its high sensitivity and stability. In multiplex detection, the signal deviation under non-target nucleic acid interference is ≤4.0%, verifying that the combination of pre-set primers and multi-wavelength probes effectively avoids cross-reactions. The total detection time is reduced to 45 minutes (traditional methods >3 hours), simultaneously achieving accurate quantification of allergic inflammatory factor mRNA and drug target genes, providing reliable technical support for rapid point-of-care diagnosis.

[0034] This invention utilizes centrifugal force gradient to drive directional laminar flow of samples and reagents, combined with passive control of capillary valves and active timing regulation of hot-melt valves, to achieve closed-loop transfer of whole blood samples from the lysis chamber, binding chamber to the elution chamber, completing in-situ nucleic acid extraction. After the eluted nucleic acid is broken through the capillary valve, it triggers the reconstitution of lyophilized fluorescent probes. The mixture enters the amplification chamber with pre-placed primers, and temperature-switched amplification reactions such as PCR are initiated under the action of the temperature-switched module. The target nucleic acid is extended by specific primers and releases a fluorescent signal. Finally, a multi-channel optical system simultaneously captures fluorescence at different wavelengths (FAM / HEX / ROX, etc.), and parallel quantification of allergic inflammatory factor mRNA and drug target genes is achieved based on the Ct value of the amplification curve or the endpoint fluorescence intensity. The entire process is automatically completed within a closed chip, eliminating contamination and requiring no manual intervention.

[0035] Although embodiments of the present invention have been shown and described, these specific embodiments are merely explanations of the invention and are not intended to limit it. The specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. After reading this specification, those skilled in the art may make modifications, substitutions, and variations to the embodiments as needed without departing from the principles and spirit of the invention, but such modifications, substitutions, and variations are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A detection kit for detecting an allergic inflammatory factor based on a centrifugal microfluidic chip, characterized by comprising: The centrifugal microfluidic chip and the pre-prepared reagent are included. ​ The centrifugal microfluidic chip includes a chip base and the following components integrated in the chip base: a sample loading cavity provided with a sample loading hole; a nucleic acid extraction and purification cavity connected with the sample loading cavity through a first microchannel and provided with a silica gel membrane or magnetic beads for separating nucleic acid; a plurality of amplification cavities arranged in a centrifugal direction and distributed at the center of the same circle, each of the amplification cavities being pre-prepared with amplification primers for different allergic inflammatory factor mRNAs or drug companion diagnostic targets; a reagent storage cavity connected with the nucleic acid extraction and purification cavity through a second microchannel and pre-prepared with freeze-dried fluorescent probes; a waste liquid cavity connected with the amplification cavities through a third microchannel; a capillary valve provided in a flow channel between the nucleic acid extraction and purification cavity and the amplification cavities; The pre-prepared reagent includes: lysis solution pre-prepared in a first sub-reagent cavity; washing solution pre-prepared in a second sub-reagent cavity; elution solution pre-prepared in a third sub-reagent cavity; The first sub-reagent cavity, the second sub-reagent cavity and the third sub-reagent cavity are respectively connected with the nucleic acid extraction and purification cavity through controllable valves. 2.The test kit for detecting allergic inflammation factors based on the centrifugal microfluidic chip according to claim 1, characterized in that: The amplification cavities are divided into at least two groups: a first group pre-prepared with amplification primers for allergic inflammatory factor mRNAs, the targets including at least three of MBP, ECP, EDN, EPO and β-tryptase; a second group pre-prepared with amplification primers for drug companion diagnostic targets, the targets including at least three of H1R, H4R, CYS1, CYS2, IL4Rα and IL5Rα; The freeze-dried fluorescent probes corresponding to each group have different fluorescent wavelengths, and the wavelength range is 500nm-800nm. 3.The test kit for detecting allergic inflammation factors based on the centrifugal microfluidic chip according to claim 2, characterized in that: The fluorescent groups of the fluorescent probes are selected from at least three of FAM, HEX, ROX, Cy5 and Quasar 670. 4.The detection kit for detecting allergic inflammation factors based on the centrifugal microfluidic chip according to claim 1, characterized in that: The nucleic acid extraction and purification cavity includes: a lysis cavity connected with a lysis solution storage area through a controllable valve; a binding cavity provided with a silica gel membrane or magnetic beads; an elution cavity connected with the amplification cavities through a capillary valve.

5. The detection kit for detecting allergic inflammation factors based on the centrifugal microfluidic chip according to claim 1, characterized in that: The controllable valve is a hot-melt paraffin valve or a phase-change valve; the chip base is made of COP or PMMA, and the amplification cavities are integrated with a variable-temperature amplification module. 6.The test kit for detecting allergic inflammation factors based on the centrifugal microfluidic chip according to claim 5, characterized in that: The variable-temperature amplification module is a PCR reaction system, and the amplification temperature range covers denaturation, annealing and extension stages, and the typical cycle number is 35-45 cycles.

7. A method for detecting an allergic inflammatory factor based on a centrifugal microfluidic chip, using the detection kit according to any one of claims 1-6, characterized in that: The method includes the following steps: S1: injecting a sample to be tested into the sample loading cavity, and centrifugally driving the sample into the nucleic acid extraction and purification cavity; S2: sequentially opening the controllable valves, and making the lysis solution, the washing solution and the elution solution flow through the nucleic acid extraction and purification cavity to complete nucleic acid purification; S3: after the purified nucleic acid breaks through the capillary valve, the nucleic acid flows into the reagent storage cavity to re-dissolve the freeze-dried fluorescent probes; S4: the mixed solution flows into the amplification cavities, variable-temperature amplification is started, and fluorescent signals are detected in real time; S5: an optical system synchronously scans the multi-channel fluorescent signals of each amplification cavity, and targets are distinguished and quantified by wavelength. 8.The method for detecting an allergic inflammatory factor based on a centrifugal microfluidic chip according to claim 7, characterized in that: In step S4, the amplification condition is a typical cycle program of PCR, including 95℃ denaturation, 55-65℃ annealing and 72℃ extension, and the cycle number is 35-45 times, and the fluorescent signal is collected at the end of each cycle of annealing or extension. 9.The method for detecting an allergic inflammatory factor based on a centrifugal microfluidic chip according to claim 7, characterized in that: In step S5, the optical system adopts real-time fluorescent detection or end-point fluorescent detection, and the number of channels is consistent with the number of groups of amplification cavities.

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