Microfluidic centrifugal field-based multiphase microsphere separation and analysis system and application thereof

Abstract

The application discloses a kind of based on microfluidic centrifugal field multiphase microsphere separation analysis system and application thereof.The application provides a kind of based on microfluidic centrifugal field multiphase microsphere separation analysis system, the based on microfluidic centrifugal field multiphase microsphere separation analysis system includes: microfluidic chip, multiphase microsphere separation system and microfluidic centrifugal field platform;Wherein the multiphase microsphere separation system includes first microsphere and second microsphere.The based on microfluidic centrifugal field multiphase microsphere separation analysis system provided in the application, non-target detection object (for example impurity) in sample is removed and target detection object (for example nucleic acid) enrichment etc.are completed by multiphase microsphere cooperation effect step by step.And, the application analysis speed is fast, detection precision is high, can be realized in 45 minutes nucleic acid of sample high sensitivity detection, solves the problem, such as low detection sensitivity of traditional method, multiple test operation is complicated, detection time is long, accuracy is poor, etc., provides new technical scheme for POCT.

Description

Technical Field

[0001] This invention belongs to the field of analytical testing technology, specifically relating to a multiphase microsphere separation and analysis system based on a microfluidic centrifugal field and its application. Background Technology

[0002] Traditional nucleic acid extraction and detection rely on large-scale equipment and cumbersome manual operations, making it difficult to meet the needs of on-site testing at ports, warehouses, or fields. The emergence of microfluidic technology offers a new solution for nucleic acid detection. Microfluidic chips can achieve rapid sample processing and reactions by precisely controlling fluid flow. Combined with centrifugation-driven separation technology, it can effectively improve the efficiency of nucleic acid extraction and purification, shorten detection time, and enhance detection sensitivity and accuracy. However, existing microfluidic systems still have bottlenecks in sample pretreatment, fluid manipulation, and separation efficiency, especially in achieving efficient and specific nucleic acid extraction, enrichment, and separation in complex biological samples, which remains a challenging task. Therefore, developing an integrated and automated microfluidic nucleic acid analysis system can effectively solve many problems existing in traditional detection methods and is one of the important directions for the current development of nucleic acid detection technology.

[0003] Microsphere separation technology, as an emerging bioseparation method, can effectively enrich and separate target biomarkers. The surface of microspheres can be functionalized to specifically bind target molecules, thereby improving detection sensitivity and selectivity. Combining microsphere separation technology with LAMP and microfluidics enables rapid enrichment, separation, and detection of nucleic acids in biological samples, achieving automated and integrated nucleic acid extraction and detection. Summary of the Invention

[0004] The problem the invention aims to solve

[0005] The purpose of this invention is to provide a microfluidic centrifugal microsphere separation and analysis system that is easy to operate, fast in analysis, and has high detection accuracy. It can integrate functions such as sample lysis, impurity removal, nucleic acid enrichment, isothermal amplification, and real-time fluorescence monitoring within a single chip.

[0006] Solution for solving the problem

[0007] [1]. A multiphase microsphere separation and analysis system based on a microfluidic centrifugal field, wherein the multiphase microsphere separation and analysis system based on a microfluidic centrifugal field includes: a microfluidic chip, a multiphase microsphere separation system, and a microfluidic centrifugal field platform; wherein:

[0008] The multiphase microsphere separation system includes a first microsphere and a second microsphere, wherein,

[0009] The first microsphere is used to adsorb non-target analytes present in the sample to be tested.

[0010] The second microsphere is used to adsorb the target analyte present in the sample to be tested;

[0011] The microfluidic chip includes a sample loading region, a microsphere separation region, and a reaction detection region, wherein...

[0012] The sample loading area is provided with a sample application port for loading the sample to be tested, the first microsphere, and / or the second microsphere. The sample loading area is connected to the microsphere separation area. The second microsphere, which adsorbs the target analyte in the sample loading area, enters the microsphere separation area by centrifugation, while the first microsphere, which adsorbs non-target analytes, remains in the sample loading area.

[0013] The microsphere separation zone is connected to the reaction detection zone via a microchannel, and the second microsphere that adsorbs the target analyte enters the reaction detection zone by centrifugation.

[0014] [2]. According to the multiphase microsphere separation and analysis system based on microfluidic centrifugation field described in [1], wherein the microfluidic centrifugation field platform includes a centrifugation drive module, a temperature control module, and a real-time fluorescence monitoring module, wherein,

[0015] The centrifugal drive module is used to provide controllable centrifugal force to achieve the directional migration and separation of different microspheres within the microfluidic chip.

[0016] The real-time fluorescence monitoring module is used to monitor fluorescence in real time.

[0017] The temperature control module is used to control the temperature of each region of the microfluidic chip.

[0018] [3]. According to the multiphase microsphere separation and analysis system based on microfluidic centrifugation field described in [1] or [2], wherein the multiphase microsphere separation system further includes a lysis buffer, which is used to destroy the cell or virus structure in the sample to be tested, thereby inactivating the protein and releasing the nucleic acid;

[0019] Optionally, the first microspheres adsorb non-target analytes present in the sample to be tested treated with the lysis buffer, the non-target analytes including impurities other than nucleic acids.

[0020] The second microsphere adsorbs target analytes present in the sample to be tested treated with the lysis buffer, the target analytes including nucleic acids;

[0021] Preferably, the lysis buffer includes urea, guanidine hydrochloride, Tris solution, EDTA solution, CaCl2 solution, SDS solution, and Tween 20.

[0022] More preferably, the volume ratio of the sample to be tested to the lysis solution is (2~8):1, more preferably (3~8):1, and even more preferably (3~5):1.

[0023] [4]. According to any one of [1]-[3], the multiphase microsphere separation and analysis system based on microfluidic centrifugation field, wherein the particle size of the first microsphere is larger than that of the second microsphere, such that during centrifugation, the first microsphere cannot enter the microsphere separation zone, while the second microsphere enters the microsphere separation zone;

[0024] Optionally, the first microsphere comprises resin particles with chelating resin groups modified on their surface, and the second microsphere comprises silica microspheres with hydroxyl groups modified on their surface.

[0025] Optionally, the particle size of the first microsphere is 150~300 μm;

[0026] Optionally, the particle size of the second microsphere is 200 nm to 10 μm, preferably 600 nm to 5 μm, more preferably 800 nm to 3 μm, and even more preferably 800 nm to 1.2 μm;

[0027] Optionally, the volume ratio of the sample to be tested to the suspension of the first microsphere is (2~10):1, preferably (4~10):1, and more preferably (6~10):1;

[0028] Optionally, the volume ratio of the sample to be tested to the suspension of the second microsphere is (1~8):1, preferably (3~8):1, and more preferably (3~5):1.

[0029] Optionally, the mass fraction of the first microspheres in the suspension of the first microspheres is 2% to 10%, preferably 3% to 8%;

[0030] Optionally, the density of the second microsphere in the suspension of the second microsphere is 10-50 mg / mL, preferably 15-40 mg / mL.

[0031] [5]. A multiphase microsphere separation and analysis system based on a microfluidic centrifugal field as described in any one of [1]-[4], wherein,

[0032] The sample loading area is a chamber with a volume of 65~75 μL; and / or

[0033] The microsphere separation zone is equipped with pores that communicate with the atmosphere, ensuring equal pressure inside and outside the microchannel, allowing the microspheres and liquid to move within the microchannel; and / or

[0034] The reaction detection region includes at least one detection well, each detection well being a chamber with a volume of 3-8 μL. Preferably, primers are immobilized in the reaction detection region; and / or,

[0035] The microchannel has the following dimensions: diameter of 0.1~0.2mm and length of 0.8~1.2cm.

[0036] [6]. A nucleic acid detection method, wherein the nucleic acid in the sample to be tested is detected using a multiphase microsphere separation and analysis system based on a microfluidic centrifugal field as described in any one of [1]-[5];

[0037] Preferably, the nucleic acid detection method includes:

[0038] The microfluidic chip pretreatment step includes adding primers and nucleic acid amplification solution to the reaction detection region of the microfluidic chip;

[0039] The steps for preparing the sample to be tested include mixing the sample to be tested with a lysis buffer, a first microsphere, and a second microsphere to form a mixture, and adding the mixture through a sample loading port into the pretreated microfluidic chip in the microfluidic chip pretreatment step; and

[0040] The detection steps include the following steps: in the preparation of the sample to be tested, the microfluidic chip after sample addition is loaded onto the microfluidic centrifuge platform, parameters are set, and the lysis, centrifugation and reaction processes are carried out to obtain the detection results.

[0041] [7]. According to the nucleic acid detection method described in [6], wherein,

[0042] The microfluidic chip preprocessing steps include:

[0043] (a) Primers corresponding to the target gene are added to the reaction detection area of ​​the microfluidic chip and dried to fix the primers in the reaction detection area; and

[0044] (b) Add nucleic acid amplification solution through the sample loading wells in the sample loading area and centrifuge to allow the nucleic acid amplification solution to enter the detection wells in the reaction detection area for later use;

[0045] Preferably, in step (a), the volume of primer added is 2~4 μL;

[0046] Preferably, in step (a), the temperature used for primer drying is 50~70 °C;

[0047] Preferably, primers corresponding to the target gene are added to the reaction detection area of ​​the microfluidic chip, dried to fix the primers in the reaction detection area, and then sealed with a bottom film. Optionally, the bottom film is a transparent PVDC film.

[0048] Preferably, in step (b), the volume of nucleic acid amplification solution added to each well in the sample loading area is 25~40 μL;

[0049] Preferably, in step (b), the centrifugation parameters for centrifuging the nucleic acid amplification solution into the detection well of the reaction detection area are: centrifugation at 56~126 g for 10~30 s, followed by centrifugation at 894~1132 g for 10~30 s.

[0050] [8]. According to the nucleic acid detection method described in [6] or [7], wherein, in the step of preparing the sample to be tested, the volume of the mixture loaded into the microfluidic chip is 55~75 μL.

[0051] [9]. According to any one of [6]-[8], in the detection step, the lysis temperature is 60~65 °C and the lysis time is 3~5 min; and / or

[0052] In the detection steps described above, the centrifugation includes: centrifuging at 0.6~5 g for 10 s, followed by centrifugation at 224~503 g for 30 s twice.

[0053]

[10] . A nucleic acid detection method according to any one of [6]-[9], wherein the nucleic acid detection method qualitatively or quantitatively detects the target gene in the sample to be tested.

[0054]

[11] . Application of the multiphase microsphere separation and analysis system based on microfluidic centrifugation field according to any one of [1]-[5] in detecting target genes in the sample to be tested.

[0055]

[12] . A kit comprising a multiphase microsphere separation and analysis system based on a microfluidic centrifugal field as described in any one of [1]-[5].

[0056] The effects of the invention

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] In some implementation schemes, the multiphase microsphere separation and analysis system and nucleic acid detection method based on microfluidic centrifugation field provided by the present invention can achieve the removal of non-target analytes (e.g., impurities) and enrichment of target analytes (e.g., nucleic acids) in a sample in one step through the synergistic effect of multiphase microspheres.

[0059] In some implementation schemes, the multiphase microsphere separation and analysis system and nucleic acid detection method based on microfluidic centrifugal field provided by the present invention uses centrifugal field drive as the microfluidic driving method, which does not require external pumps or valves.

[0060] In some implementation schemes, the multiphase microsphere separation and analysis system and nucleic acid detection method based on microfluidic centrifugation field provided by the present invention uses a disposable chip design, which is convenient to operate and low in cost. A single chip can simultaneously detect 8 samples and obtain results within 45 minutes, meeting the requirements of POCT.

[0061] In some implementation schemes, the multiphase microsphere separation and analysis system and nucleic acid detection method based on microfluidic centrifugation field provided by the present invention can be used for gene detection of animal, plant and microbial samples, and is also suitable for rapid detection of nucleic acids of various pathogens such as SARS-CoV-2, influenza virus and HPV. It has a wide range of applications and meets the needs of multiple scenarios. Attached Figure Description

[0062] Figure 1 This is a schematic diagram of a centrifugal microfluidic chip.

[0063] Figure 2 This is a schematic diagram illustrating the integrated detection process of the present invention.

[0064] Figure 3 These are the COMSOL simulation results of the motion of the multiphase microsphere separation system of this invention in a microfluidic centrifugal field; Figure 3 In the figure, 'a' represents the simulation of microsphere trajectory. Figure 3 b in the text refers to the simulation of fluid motion.

[0065] Figure 4 This is the result of laser confocal fluorescence microscopy characterization of the liquid in the reaction detection well after centrifugation of the sample using the multiphase microsphere separation system of this invention; Figure 4 'a' in the image represents the bright field image. Figure 4 b: Fluorescence image, Figure 4 c: Overlay of bright-field and fluorescence images.

[0066] Figure 5 This demonstrates the performance of this invention in the detection of the 87701 sequence in genetically modified soybeans; Figure 5 In Figure 'a': Amplification curves of plasmids from genetically modified soybean 87701 at different concentrations. Figure 5 b: Standard curve showing the relationship between plasmid concentration (87701) and nucleic acid amplification Tt value. Figure 5 c in the figure represents the amplification curve of the genetically modified soybean sample.

[0067] Figure 6 This is the performance of the present invention in the detection of Escherichia coli uidA sequence in clinical samples; Figure 6 a: Agarose gel electrophoresis results of Escherichia coli nucleic acid separation and extraction from different clinical matrices using a multiphase microsphere separation system; Figure 6 b: Amplification curves of the uidA sequence of Escherichia coli in different clinical matrices. Detailed Implementation

[0068] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0069] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0070] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0071] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0072] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0073] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B. [Detailed Description of the Invention]

[0075] <Multiphase Microsphere Separation and Analysis System Based on Microfluidic Centrifugation Field>

[0076] In some aspects of the present invention, a multiphase microsphere separation and analysis system based on a microfluidic centrifugation field is provided, comprising: a microfluidic chip, a multiphase microsphere separation system, and a microfluidic centrifugation field platform; wherein:

[0077] The multiphase microsphere separation system includes a first microsphere and a second microsphere, wherein the first microsphere is used to adsorb non-target analytes present in the sample to be tested, and the second microsphere is used to adsorb target analytes present in the sample to be tested.

[0078] The microfluidic chip includes a sample loading area, a microsphere separation area, and a reaction detection area. The sample loading area is provided with a sample loading port for loading the sample to be tested, a first microsphere, and / or a second microsphere. The sample loading area (e.g., through multiple microvalves) is connected to the microsphere separation area, so that the second microsphere adsorbing the target analyte in the sample loading area enters the microsphere separation area by centrifugation, while the first microsphere adsorbing a non-target analyte remains in the sample loading area. The microsphere separation area is connected to the reaction detection area through a microchannel, so that the second microsphere adsorbing the target analyte enters the reaction detection area by centrifugation.

[0079] In the multiphase microsphere separation and analysis system based on microfluidic centrifugation field provided by the present invention, the removal of non-target analytes (e.g., impurities) and the enrichment of target analytes (e.g., nucleic acids) in the sample are completed in one step through the synergistic effect of multiphase microspheres.

[0080] (Microfluidic centrifuge platform)

[0081] In some implementations, the microfluidic centrifuge platform includes a centrifugation drive module, a temperature control module, and a real-time fluorescence monitoring module. The centrifugation drive module provides controllable centrifugal force to achieve the directional migration and separation of different microspheres within the centrifugal microfluidic chip. The real-time fluorescence monitoring module monitors fluorescence in real time. The temperature control module controls the temperature of each region of the microfluidic chip to ensure the smooth progress of the reaction.

[0082] In this invention, there are no special limitations on the microfluidic centrifugation platform and its centrifugation drive module, temperature control module, and real-time fluorescence monitoring module, as long as they are suitable for centrifugation and fluorescence monitoring of centrifugal microfluidic chips. The microfluidic centrifugation platform, its centrifugation drive module, temperature control module, and real-time fluorescence monitoring module provided by this invention can be commercially available, for example, purchased from Shanghai Suchuang Diagnostic Products Co., Ltd., product model: MA3000.

[0083] (pyrolysis solution)

[0084] In some embodiments, the multiphase microsphere separation system further includes a lysis buffer used to disrupt the cellular or viral structures in the sample to be tested, thereby inactivating proteins and releasing nucleic acids.

[0085] In some implementations, when the sample to be tested is lysed using a lysis buffer, the first microspheres adsorb non-target analytes present in the sample treated with the lysis buffer. Exemplarily, these non-target analytes include impurities other than nucleic acids, such as proteins that may interfere with subsequent detection (amplification).

[0086] In some implementations, when the sample to be tested is lysed using a lysis buffer, the second microsphere adsorbs the target analyte present in the sample treated with the lysis buffer. For example, the target analyte includes nucleic acids.

[0087] In some preferred embodiments, the lysis buffer includes urea, guanidine hydrochloride, Tris solution, EDTA solution, CaCl2 solution, SDS solution, and Tween 20.

[0088] In some preferred embodiments, when the sample to be tested is lysed using a lysis buffer, the volume ratio of the sample to the lysis buffer is (2~8):1, preferably (3~8):1, more preferably (3~5):1, for example 3:1, 4:1 or 5:1. This invention creatively discovers that, at the above ratios, the stability and repeatability of subsequent detection can be improved.

[0089] (First microsphere and second microsphere)

[0090] In some implementations, the particle size of the first microsphere is larger than that of the second microsphere, such that during centrifugation, the first microsphere cannot enter the microsphere separation zone through the microvalve, while the second microsphere enters the microsphere separation zone through the microsphere.

[0091] In some optional embodiments, the particle size of the first microsphere is 150~300 μm.

[0092] In some optional embodiments, the particle size of the second microsphere is 200 nm to 10 μm, preferably 600 nm to 5 μm, more preferably 800 nm to 3 μm, and even more preferably 800 nm to 1.2 μm, for example 800 nm, 1 μm, and 1.2 μm. The present invention creatively discovers that using the first and / or second microspheres with the above-mentioned particle sizes is more advantageous for removing non-target analytes (e.g., impurities) from samples and enriching target analytes (e.g., nucleic acids), thereby improving the sensitivity and specificity of subsequent detections, as well as enhancing the stability and repeatability of subsequent detections.

[0093] In some optional embodiments, the first microsphere comprises resin particles with a surface modified with chelating resin groups, which can effectively adsorb impurities such as proteins present in the lysed sample that may interfere with subsequent amplification.

[0094] In some optional embodiments, the second microsphere comprises silica microspheres with hydroxyl-modified surfaces, which can effectively adsorb nucleic acids released from sample lysis.

[0095] In this invention, there are no special restrictions on the source of the first and second microspheres; for example, they can be obtained through commercial purchase.

[0096] In some optional embodiments, the volume ratio of the sample to be tested to the suspension of the first microsphere is (2~10):1, preferably (4~10):1, more preferably (6~10):1, for example 7:1, 8:1 or 9:1.

[0097] In some optional embodiments, the volume ratio of the sample to be tested to the suspension of the second microsphere is (1~8):1, preferably (3~8):1, more preferably (3~5):1, for example 3:1, 4:1 or 5:1.

[0098] The present invention creatively discovers that using the above-mentioned ratio of sample to microspheres (or further combining the above-mentioned first microspheres and / or second microspheres of the above-mentioned particle size) is more conducive to removing non-target analytes (e.g., impurities) from the sample and enriching target analytes (e.g., nucleic acids), thereby improving the sensitivity and specificity of subsequent detection, as well as improving the stability and repeatability of subsequent detection.

[0099] In some optional embodiments, the mass fraction of the first microspheres in the suspension is 2% to 10%, preferably 3% to 8%. For example, 3%, 4%, 5%, 6%, 7%, and 8%.

[0100] In some optional embodiments, the density of the second microspheres in the suspension is 10-50 mg / mL, preferably 15-40 mg / mL, more preferably 15-30 mg / mL, and even more preferably 20-30 mg / mL, such as 20 mg / mL, 25 mg / mL, and 30 mg / mL.

[0101] At the aforementioned mass fraction or density, combined with the aforementioned ratio of the sample to be tested to the microspheres and / or the aforementioned particle size of the first and / or second microspheres, it can further facilitate the removal of non-target analytes (e.g., impurities) from the sample and the enrichment of target analytes (e.g., nucleic acids).

[0102] (Microfluidic chip)

[0103] In this invention, there are no particular limitations as long as the microfluidic chip can be used in conjunction with the multiphase microsphere separation system to achieve the technical effects of this invention.

[0104] In this invention, the microfluidic chip can be any microscale physical structure system capable of separating the first microsphere and the second microsphere, or any microscale physical structure capable of separating the first microsphere and the second microsphere under centrifugal field control. It is not limited to the centrifugal microfluidic chip described in the embodiments.

[0105] In some specific implementations, the microfluidic chip is a centrifugal microfluidic chip. For example, the centrifugal microfluidic chip is as follows: Figure 1 As shown, it can be obtained commercially, for example, from Shanghai Suchuang Diagnostic Products Co., Ltd., product model: SC-1001.

[0106] In some implementations, the sample loading area is a chamber with a volume of 65-75 μL.

[0107] like Figure 1 As shown, in some preferred embodiments, the sample loading area has a fan-shaped chamber.

[0108] In some implementations, the microsphere separation zone is provided with pores that communicate with the atmosphere, so that the pressure inside and outside the microchannel is equal, allowing the microspheres and liquid to move within the microchannel.

[0109] In some embodiments, the reaction detection zone includes at least one detection well, each detection well being a chamber with a volume of 3 to 8 μL, preferably 3 to 6 μL, for example 3 μL or 5 μL.

[0110] In some preferred embodiments, each detection hole is a circular chamber.

[0111] In some specific implementations, the microsphere separation zone is connected to multiple detection holes of the reaction detection zone through multiple microchannels, so that the second microsphere adsorbing the target analyte enters each detection hole of the reaction detection zone by centrifugation.

[0112] In some implementations, the reaction detection region is pre-fixed with primers.

[0113] In some implementations, the microchannel has the following dimensions: a diameter of 0.1~0.2 mm and a length of 0.8~1.2 cm.

[0114] <Nucleic Acid Detection Methods>

[0115] In some aspects of the present invention, a nucleic acid detection method is provided, which uses the multiphase microsphere separation and analysis system based on microfluidic centrifugation field described above to detect nucleic acids in the sample to be tested.

[0116] In some preferred embodiments, the nucleic acid detection method includes:

[0117] The microfluidic chip pretreatment step includes adding primers and nucleic acid amplification solution to the reaction detection region of the microfluidic chip;

[0118] The steps for preparing the sample to be tested include mixing the sample to be tested with a lysis buffer, a first microsphere, and a second microsphere to form a mixture, and adding the mixture through a sample loading port into the pretreated microfluidic chip in the microfluidic chip pretreatment step; and

[0119] The detection steps include loading the microfluidic chip after sample addition in the sample preparation step onto the microfluidic centrifuge platform, setting parameters, and performing lysis, centrifugation, and reaction processes to obtain the detection results.

[0120] In some specific implementations, the microfluidic chip preprocessing steps include:

[0121] (a) Primers corresponding to the target gene are added to the reaction detection area of ​​the microfluidic chip and dried to fix the primers in the reaction detection area; and

[0122] (b) Add nucleic acid amplification solution through the sample loading well in the sample loading area and centrifuge to allow the nucleic acid amplification solution to enter the detection wells in the reaction detection area for later use.

[0123] In some preferred embodiments, in step (a), the volume of primer added is 2–4 μL. Those skilled in the art can select appropriate primer concentrations according to different nucleic acid reaction types, as long as the volume is within the above range.

[0124] In some preferred embodiments, in step (a), the temperature used for primer drying is 50-70 °C.

[0125] In some preferred embodiments, primers corresponding to the target gene are dropped into the reaction detection area of ​​the microfluidic chip, dried to fix the primers in the reaction detection area, and then sealed with a bottom film. Optionally, the bottom film is a transparent PVDC film.

[0126] In some preferred embodiments, in step (b), the volume of nucleic acid amplification solution added to each well of the sample loading area is 25-40 μL.

[0127] In some preferred embodiments, in step (b), the centrifugation parameters for centrifuging the nucleic acid amplification solution into the detection well of the reaction detection area are: centrifugation at 56~126 g for 10~30 s, followed by centrifugation at 894~1132 g for 10~30 s.

[0128] In some specific implementations, during the preparation of the sample to be tested, the volume of the mixed liquid loaded into the microfluidic chip is 55~75 μL.

[0129] In some specific implementations, during the detection step, the pyrolysis temperature is 60-65 °C and the pyrolysis time is 3-5 min.

[0130] In some specific implementations, the lysis occurs within the sample loading region of the microfluidic chip. It is understood that while the sample is lysed in the sample loading region, the first and second microspheres adsorb impurities and nucleic acids from the sample, respectively.

[0131] In some specific implementations, the centrifugation in the detection step includes: centrifuging at 0.6~5 g for 10 s, followed by centrifugation at 224~503 g for 30 s twice.

[0132] In some specific implementations, the centrifugation causes the second microspheres adsorbing the target analyte to enter the reaction detection zone, thereby carrying out the reaction.

[0133] In this invention, there are no special limitations on the type of reaction, as long as it can detect nucleic acids. Examples include PCR, RPA, CRISPR, NASBA, TMA, and LAMP. These methods are known to those skilled in the art. Those skilled in the art can select appropriate reaction conditions based on different nucleic acid reaction types.

[0134] In some exemplary embodiments, for LAMP, the nucleic acid amplification solution may include Bst enzyme, dATP, dTTP, dGTP, dCTP, BSA-A, MgSO4, SYTO9, taurine, and ddH2O.

[0135] In some specific implementations, the reaction is a LAMP amplification reaction. Preferably, the LAMP amplification reaction conditions are: temperature: 60~65 ℃; amplification time: 30~60 min.

[0136] In some alternative implementations, the nucleic acid detection method qualitatively or quantitatively detects the target gene in the sample to be tested.

[0137] In some specific implementation schemes, the nucleic acid detection method quantitatively detects target genes in the sample to be tested.

[0138] Establishing a standard curve determines the basis for quantitative analysis comparison, and also determines the detection limit and quantitation limit of the detection system or method.

[0139] For quantitative detection of target genes in a sample, the nucleic acid detection method further includes the step of establishing a standard curve. Specifically, a series of standard substance working solutions of different concentrations are prepared using standard substances (e.g., plasmid standards). Following the steps of the nucleic acid detection method described above, a linear relationship between the calibration peak area and the standard substance concentration is established, and the content of the target gene in the sample is then calculated.

[0140] In some specific implementation schemes, the steps for establishing the standard curve include:

[0141] Step 1: Detect the gradient concentration plasmid standards corresponding to the target gene according to the aforementioned nucleic acid detection method;

[0142] Step 2: Establish a standard curve by relating the logarithm of the nucleic acid concentration of the plasmid standard to the Tt value of nucleic acid amplification;

[0143] Step 3: Analyze the sample to be tested according to the aforementioned nucleic acid testing method;

[0144] Step 4: Substitute the Tt value of the sample to be tested obtained in Step 3 into the standard curve obtained in Step 2 to calculate the concentration of the target gene in the sample to be tested.

[0145] <Application>

[0146] In some aspects of the present invention, the application of the multiphase microsphere separation and analysis system based on microfluidic centrifugation field described above in the detection of target genes in a sample to be tested is provided.

[0147] <Reagent Kit>

[0148] In some aspects of the present invention, a kit is provided that includes the multiphase microsphere separation and analysis system based on a microfluidic centrifugation field as described above.

[0149] In the above aspects of the present invention, the target gene may be DNA or RNA. In some embodiments, the target gene is single-stranded. In some embodiments, the target gene is double-stranded. In some embodiments, the target gene is genomic DNA. In some embodiments, the target gene is genomic RNA.

[0150] In the above aspects of the invention, the target gene can be from any source. In some embodiments, the target gene is the genomic nucleic acid (e.g., DNA or RNA) of a microorganism, such as a pathogenic microorganism, including but not limited to viruses, bacteria, fungi, protozoa, and algae. In some embodiments, the target gene is the genomic DNA of bacteria, such as pathogenic bacteria. In some embodiments, the target gene is the genomic DNA of a DNA virus. In some embodiments, the target gene is the genomic RNA of an RNA virus. Exemplary detectable viruses include, but are not limited to, coronaviruses (e.g., SARS, MERS, and 2019-nCoV, i.e., SARS-CoV2), papillomaviruses (e.g., human papillomavirus (HPV), polyomavirus); alpha influenza viruses (e.g., influenza A virus H1N1 subtype); hepatitis viruses (e.g., hepatitis B virus (HBV)); herpesviruses (e.g., herpes simplex virus (HSV)), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV)); adenoviruses (e.g., atadenovirus, avian adenovirus, fish adenovirus, mammalian adenovirus, simian adenovirus); poxviruses (e.g., smallpox, vaccinia virus). Viruses include: vaccinia virus, shrimppox virus, sheeppox virus, pseudovaccinia virus, bovine papular stomatitis virus; tannapox virus, aba monkey tumor virus; molluscum contagiosum virus (MCV); parvoviruses (e.g., adeno-associated virus (AAV), parvovirus B19, human bocavirus, bufavirus, human parv4 Gi); arboviruses (e.g., hepatitis C virus (HCV), Zika virus, dengue virus); lentiviruses (e.g., human immunodeficiency virus (HIV)); Geminiviridae; Dwarfviridae; Phycodnaviridae; etc. In some implementations, the target gene is derived from a parasite.

[0151] In the foregoing aspects of this invention, the terms "sample," "sample," or "sample to be tested" are used herein to mean any sample containing nucleic acids. Samples can be derived from any source; for example, a sample can be a synthetic combination of purified nucleic acids, or a sample can be cell lysate. Samples can be obtained from a patient (e.g., for diagnostic purposes). Suitable samples include, but are not limited to, saliva, blood, serum, plasma, urine, extracts, and biopsy tissue samples. In some embodiments, samples obtained from a patient are selected from the group consisting of whole blood, plasma, serum, or combinations thereof. In some embodiments, the sample is an archived blood sample, a fresh blood sample, or a frozen blood sample. In some embodiments, the sample of this invention is a tissue sample. The samples of this invention are derived from or include: amniotic fluid, blood, plasma, serum, lymph, cerebrospinal fluid, ocular fluid, urine, saliva, feces, mucus, sweat, blood, skin, hair, hair follicles, saliva, oral mucus, vaginal mucus, sweat, tears, epithelial tissue, urine, semen, seminal fluid, seminal plasma, prostatic fluid, Cowper's fluid, excrement, living tissue, ascites, cerebrospinal fluid, and / or lymph. In some embodiments, the sample is a solid sample. In some embodiments, the sample is a liquid sample.

[0152] In the above aspects of the invention, the sample is derived from diseased or suspected diseased cells, fluids, tissues, or organs. In some embodiments, the sample is derived from normal (non-diseased) cells, fluids, tissues, or organs. In some embodiments, the sample is derived from an individual suspected of having a microbial infection, such as a viral infection. For example, the sample may be derived from an individual who may or may not be infected—and the sample may be any biological sample collected from the individual (e.g., blood, saliva, live tissue, plasma, serum, bronchoalveolar lavage, sputum, fecal sample, cerebrospinal fluid, fine needle aspirate, swab samples (e.g., oral swabs, cervical swabs, nasal swabs), interstitial fluid, synovial fluid, nasal discharge, tears, erythrocyte sedimentation rate (ESR) buffy coat, mucosal samples, epithelial cell samples (e.g., epithelial cell scraping), etc.). In some cases, the sample is a cell-free liquid sample. In some cases, the sample is a liquid sample that may contain cells. Pathogens include viruses, fungi, bacteria, protozoa, and microbial parasites.

[0153] Example

[0154] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0155] Example 1: Centrifugal Microfluidic Chip

[0156] The centrifugal microfluidic chip used in the following examples was purchased from Shanghai Suchuang Diagnostic Products Co., Ltd., model: SC-1001, and its structure is as follows. Figure 1 As shown.

[0157] Briefly, the centrifugal microfluidic chip is a circular chip injection-molded from polycarbonate, comprising a sample loading area, a microsphere separation area, and a reaction detection area. The sample loading area has sample loading ports for loading the sample to be tested. The sample loading area is connected to the microsphere separation area via several capillary microvalves, allowing the sample to enter the microsphere separation area under low-speed centrifugation. The entire area is a fan-shaped chamber with a volume of approximately 65-75 μL. The microsphere separation area has vents that communicate with the atmosphere, ensuring equal pressure inside and outside the microchannel and guaranteeing smooth movement of the microspheres and liquid within the microchannel. Each detection port in the reaction detection area is a circular chamber with a volume of approximately 5 μL, connected to the microsphere separation area via a microchannel (100 (length) × 100 (width) μm). The diameter of this centrifugal microfluidic chip is 80 mm.

[0158] The microfluidic centrifugation platform includes a centrifugation drive module and a real-time fluorescence monitoring module, purchased from Shanghai Suchuang Diagnostic Products Co., Ltd., product model: MA3000.

[0159] Example 2: Construction of a Multiphase Microsphere Separation and Analysis System and Detection Method Based on Microfluidic Centrifugation Field

[0160] This embodiment constructs a multiphase microsphere separation and analysis system based on a microfluidic centrifugal field, such as... Figure 2 As shown, it includes the following steps:

[0161] (1) Prepare LAMP primers according to a certain ratio and add trehalose (final concentration of about 0.1%) to maintain the stability of the primer structure. Take a centrifugal microfluidic chip from Example 1, and add 2 μL of LAMP primers (prepared by dd H2O, wherein the final concentration of primers F3 and B3 is 0.66 μM, the final concentration of primers FIP and BIP is 5.33 μM, and the final concentration of LB and LF is 2.66 μM) to each detection well in the reaction detection area of ​​the chip. Place it in a 60 ℃ oven and dry for 10 min to fix the primers in each detection well in the reaction detection area of ​​the chip. Then seal the bottom of the chip with a PVDC film to construct a microchannel for later use.

[0162] (2) Add 30 μL of nucleic acid amplification solution (including Bst DNA polymerase, SYTO 9 nucleic acid fluorescent dye, dNTP, etc., see Table 1 below) to each sample loading area through the sample loading well of the chip sample loading area. After sealing the sample loading well with an aluminum mold, load the chip onto the microfluidic centrifuge platform (Shanghai Suchuang Diagnostic Products Co., Ltd., MA3000) and centrifuge at low speed (1500 rpm × 10 s) and high speed (4500 rpm × 10 s) to fill each detection well of the reaction detection area with nucleic acid amplification solution and rehydrate the primers.

[0163] Table 1: Composition of Nucleic Acid Amplification Buffer

[0164]

[0165] (3) Mix 40 μL of sample with 10 μL of lysis buffer (including urea, guanidine hydrochloride, Tris solution, EDTA solution, CaCl2 solution, SDS solution, Tween20, the specific formula is shown in Table 2 below), 10 μL of 25 mg / mL hydroxylated silica microspheres with a particle size of 1 μm and 5 μL of 5% Chelex-100 resin particles (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., item number C7901, 50~100 mesh). Take 60 μL of the resulting sample mixture and add it to the chip through the sample loading well in the sample loading area. Seal the sample loading well with an aluminum film to seal the chip and prevent aerosol contamination and liquid overflow.

[0166] Table 2: Composition of lysis buffer

[0167]

[0168] After completely dissolving the above raw materials, adjust the pH to 6.40±0.05 with HCl, bring the volume to 1000mL with sterile DEPC water, filter the solution through a 0.22 μm filter for sterilization, and store it at room temperature to obtain the lysate.

[0169] (4) Load the chip with the sample added in step (3) onto the microfluidic centrifuge platform and set the parameters as follows: lysis temperature: 65 ℃; lysis time: 5 min; low speed centrifugation: 100 rpm × 10 s, high speed centrifugation: 2500 rpm; LAMP amplification reaction temperature: 65 ℃; amplification time: 40 min. Start the instrument and perform integrated operation of sample lysis, centrifugation separation, and real-time fluorescence curve monitoring. Obtain the detection results within 45 min.

[0170] Example 3: Characterization and Optimization of Multiphase Microsphere Separation and Analysis System Based on Microfluidic Centrifugation Field

[0171] The performance verification of the multiphase microsphere separation and analysis system of this invention was carried out through multiple characterization and optimization tests, including the following steps:

[0172] (1) The system was simulated using COMSOL Multiphysics 6.3 software to track the motion trajectory and process of fluid and microspheres at a centrifugation speed of 2500 rpm.

[0173] like Figure 3 The COMSOL simulation results show that as the rotation process occurs, the microsphere moves outward from the position near the center of rotation, and the change in angular velocity also affects the particle motion. Observing the particle distribution, the darker the color, the more particles there are at that position. As the particles move, the cumulative distribution also changes, with the number of particles gradually increasing in the end circular hole region.

[0174] (2) According to the detection method of the present invention, the nucleic acid of Escherichia coli is isolated, extracted and characterized.

[0175] Take a centrifugal microfluidic chip and seal the bottom with a PVDC membrane for later use. Add 30 μL of nucleic acid amplification solution to each sample well (this experiment does not involve amplification, but only characterizes the separation and extraction effect). After sealing the sample well with an aluminum mold, load the chip onto the microfluidic centrifuge platform and centrifuge at low speed (1500 rpm × 10 s) and high speed (4500 rpm × 10 s) to fill the reaction detection wells with nucleic acid amplification solution.

[0176] 40 μL of E. coli sample was mixed with 10 μL of lysis buffer, 10 μL of 25 mg / mL hydroxylated silica microspheres with a particle size of 1 μm, and 5 μL of 5% Chelex-100 resin particles. 60 μL of the resulting sample mixture was added to the chip through the sample well and the well was sealed with an aluminum film to seal the chip.

[0177] Load the chip onto the microfluidic centrifuge platform and set the parameters: lysis temperature: 65 ℃; lysis time: 5 min; low-speed centrifugation: 0.6 g (100 rpm) × 10 s, high-speed centrifugation: 349 g (2500 rpm) × 30 s × 2; LAMP amplification reaction temperature: 65 ℃; amplification time: 0 min. Start the instrument, perform sample lysis and centrifugation, then remove the chip and aspirate the liquid from the outer reaction detection wells. Add 2 μL of wwGreen 1000x nucleic acid dye, mix, and prepare the sample. Image using a laser confocal fluorescence microscope. Figure 4 As shown, the laser confocal fluorescence image demonstrates that the microsphere separation system of this invention successfully separated and extracted nucleic acids from the sample.

[0178] (3) Optimize the parameters of the multiphase microsphere system. Use serially diluted plasmids as samples for nucleic acid amplification, and select the optimal parameters based on the stability and repeatability of nucleic acid amplification.

[0179] Specifically, the fluorescence threshold is set to 300, and the time when the fluorescence value of the amplification curve reaches 500 is the Tt value (time threshold). The Tt value of the highest concentration sample and the detection limit are used as the evaluation criteria for the system optimization test results, and cost is also taken into consideration.

[0180] The experiment used different concentrations (10) 6 10 5 10 4 10 3 10 2 10 1 10 0 The uidA nucleic acid plasmid standard (copies / μL) was tested.

[0181] (3.1) Hydroxylated silica microspheres with different particle sizes (200 nm / 600 nm / 800 nm / 1 μm / 10 μm) were tested, and the optimal particle size was about 1 μm.

[0182] The specific experimental results are shown in Table 3 below:

[0183] Table 3:

[0184]

[0185] (3.2) Different amounts of hydroxylated silica microspheres (5 μL / 10 μL / 15 μL / 20 μL / 25 μL) were used for testing. Considering cost, the optimal amount was about 10 μL.

[0186] The specific experimental results are shown in Table 4 below:

[0187] Table 4:

[0188]

[0189] (3.3) Different amounts of lysis buffer (5 μL / 10 μL / 15 μL / 20 μL) were used for testing, and the optimal amount was about 10 μL.

[0190] The specific experimental results are shown in Table 5 below:

[0191] Table 5:

[0192]

[0193] (3.4) Different amounts of 50-100 mesh Chelex-100 (5 μL / 10 μL / 15 μL / 20 μL) were used for testing, and the optimal amount was about 5 μL.

[0194] The specific experimental results are shown in Table 6 below:

[0195] Table 6:

[0196]

[0197] (3.5) Different high-speed centrifugal forces (56 g / 123 g / 224 g / 349 g / 438 g / 572 g / 894 g) were used to centrifuge the silica microspheres to the outer reaction detection well. The optimal centrifugal force was about 349 g.

[0198] The specific experimental results are shown in Table 7 below:

[0199] Table 7:

[0200]

[0201] Application Example 1: Detection of the 87701 Sequence in Transgenic Soybean Using a Multiphase Microsphere Separation and Analysis System Based on Microfluidic Centrifugation Field

[0202] The detection of the 87701 transgenic sequence in soybeans includes the following steps:

[0203] (1) Dilute plasmid 87701 (synthesized by Shanghai Sangon Biotech Co., Ltd.) serially to 10 with dd H2O. 6 ~10 1 As nucleic acid standards, copies / μL were used, and the nucleic acid standards were tested according to the detection method in Example 2, such as... Figure 5 In step 'a', the amplification curve of the nucleic acid standard is obtained; for example... Figure 5 As shown in b, a standard curve is established to establish the relationship between the logarithm of the concentration of standard nucleic acid and the Tt value of nucleic acid amplification;

[0204] (2) Weigh 5 mg of soybean powder into a 1.5 ml centrifuge tube, add 200 μL of dd H2O, shake for 10 s, centrifuge briefly, incubate at 99℃ for 10 min, then centrifuge at 12000 rpm for 1 min. Take the supernatant and dilute it 10 times with dd H2O as the soybean sample. Detect the soybean sample according to the detection method in Example 2 to obtain the nucleic acid amplification curve and Tt value of the soybean sample.

[0205] like Figure 5 As shown in c, the soybean sample was successfully amplified, and an amplification curve appeared within 20 minutes, indicating that the soybean sample was a transgenic soybean containing the 87701 sequence. Further substituting the Tt value of the soybean sample into the standard curve yielded the approximate content of the 87701 sequence in the soybean sample.

[0206] Table 8: Transgenic Sequence of 87701

[0207]

[0208] Table 9: Primers used in this application example

[0209]

[0210] Application Example 2: Detection of E. coli uidA sequence in clinical samples using a multiphase microsphere separation and analysis system.

[0211] To verify the ability of the multiphase microsphere separation and analysis system to detect nucleic acids in various clinical samples, the uidA sequence of Escherichia coli in different clinical matrices was detected and characterized.

[0212] (1) Take 1.2 g of agarose powder, dissolve it in 60 ml of 1× TAE buffer to prepare 2% agarose, shake and microwave it three times, heating for 50s, 30s and 20s respectively, shaking well between heating. Cool to about 60℃, add 8 μL of SYBR Green I nucleic acid dye and mix well. Pour into a mold and cool for 30 min. After solidification, place it in an electrophoresis tank and pour in 1× TAE buffer to submerge the gel.

[0213] Take 40 μL of different clinical matrix samples containing E. coli (serum / vaginal swab / urine / pharyngeal swab / fecal supernatant) as the test samples. Perform lysis and centrifugation on the samples according to the system and detection method in Example 2. Then, puncture the wells of the chip to remove the liquid from the outer reaction detection wells. Mix 10 μL of the mixture with 2 μL of loading buffer, and load 10 μL of the mixture onto the prepared agarose gel for agarose gel electrophoresis. This is used to characterize the separation and extraction effect of the multiphase microsphere separation and analysis system of this invention on E. coli nucleic acid in clinical matrices. Figure 6As shown in a, the multiphase microsphere separation and analysis system of the present invention can extract a certain amount of nucleic acid into the reaction wells of the outer ring of the chip in different clinical matrices. Compared with the traditional magnetic bead method, this method omits multiple washing steps and shortens the sample processing time to within 6 minutes.

[0214] (2) Take 40 μL of different clinical matrix samples containing Escherichia coli (serum / vaginal swab / urine / pharyngeal swab / fecal supernatant) as the test samples, and perform nucleic acid amplification on the samples according to the detection method in Example 2, such as... Figure 6 As shown in b, the system and detection method of the present invention have high sensitivity and specificity for the uidA sequence of Escherichia coli in different clinical sample matrices, and can be used as a powerful method for identifying Escherichia coli in clinical samples.

[0215] Table 10: uidA sequence

[0216]

[0217] Table 11: Primers used in this application example

[0218]

[0219] In summary, this invention develops an integrated nucleic acid detection system, which mainly includes a microfluidic nucleic acid analyzer, a microfluidic chip, and related reagents (sample lysis buffer, nucleic acid amplification buffer, microspheres, etc.). It enables a one-step detection mode of "sample in - result out," with the entire detection process automated by the instrument. A single instrument can process eight clinical samples simultaneously within one hour, and its detection performance meets the needs of point-of-care testing. This invention offers fast analysis speed and high detection accuracy, achieving highly sensitive detection of nucleic acids in samples within 45 minutes. It solves the problems of low detection sensitivity, cumbersome multiple testing operations, long detection time, and poor accuracy associated with traditional methods, providing a new technological solution for point-of-care testing (POCT).

[0220] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0221] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A multiphase microsphere separation and analysis system based on a microfluidic centrifugal field, characterized in that, The multiphase microsphere separation and analysis system based on a microfluidic centrifugation field includes: a microfluidic chip, a multiphase microsphere separation system, and a microfluidic centrifugation field platform; wherein: The multiphase microsphere separation system includes a first microsphere and a second microsphere, wherein, The first microsphere is used to adsorb non-target analytes present in the sample to be tested. The second microsphere is used to adsorb the target analyte present in the sample to be tested; The microfluidic chip includes a sample loading region, a microsphere separation region, and a reaction detection region, wherein... The sample loading area is provided with a sample application port for loading the sample to be tested, the first microsphere, and / or the second microsphere. The sample loading area is connected to the microsphere separation area. The second microsphere, which adsorbs the target analyte in the sample loading area, enters the microsphere separation area by centrifugation, while the first microsphere, which adsorbs non-target analytes, remains in the sample loading area. The microsphere separation zone is connected to the reaction detection zone via a microchannel, and the second microsphere that adsorbs the target analyte enters the reaction detection zone by centrifugation.

2. The multiphase microsphere separation and analysis system based on a microfluidic centrifugal field according to claim 1, characterized in that, The microfluidic centrifuge platform includes a centrifugation drive module, a temperature control module, and a real-time fluorescence monitoring module, wherein... The centrifugal drive module is used to provide controllable centrifugal force to achieve the directional migration and separation of different microspheres within the microfluidic chip. The real-time fluorescence monitoring module is used to monitor fluorescence in real time. The temperature control module is used to control the temperature of each region of the microfluidic chip.

3. The multiphase microsphere separation and analysis system based on a microfluidic centrifugal field according to claim 1 or 2, characterized in that, The multiphase microsphere separation system also includes a lysis buffer, which is used to destroy the cell or virus structure in the sample to be tested, thereby inactivating proteins and releasing nucleic acids. Optionally, the first microspheres adsorb non-target analytes present in the sample to be tested treated with the lysis buffer, the non-target analytes including impurities other than nucleic acids. The second microsphere adsorbs target analytes present in the sample to be tested treated with the lysis buffer, the target analytes including nucleic acids; Preferably, the lysis buffer includes urea, guanidine hydrochloride, Tris solution, EDTA solution, CaCl2 solution, SDS solution, and Tween 20. More preferably, the volume ratio of the sample to be tested to the lysis solution is (2~8):1, more preferably (3~8):1, and even more preferably (3~5):

1.

4. The multiphase microsphere separation and analysis system based on a microfluidic centrifugal field according to any one of claims 1-3, characterized in that, The particle size of the first microsphere is larger than that of the second microsphere, so that during centrifugation, the first microsphere cannot enter the microsphere separation zone, while the second microsphere does. Optionally, the first microsphere comprises resin particles with chelating resin groups modified on their surface, and the second microsphere comprises silica microspheres with hydroxyl groups modified on their surface. Optionally, the particle size of the first microsphere is 150~300 μm; Optionally, the particle size of the second microsphere is 200 nm to 10 μm, preferably 600 nm to 5 μm, more preferably 800 nm to 3 μm, and even more preferably 800 nm to 1.2 μm; Optionally, the volume ratio of the sample to be tested to the suspension of the first microsphere is (2~10):1, preferably (4~10):1, and more preferably (6~10):1; Optionally, the volume ratio of the sample to be tested to the suspension of the second microsphere is (1~8):1, preferably (3~8):1, more preferably (3~5):1; Optionally, the mass fraction of the first microspheres in the suspension of the first microspheres is 2% to 10%, preferably 3% to 8%; Optionally, the density of the second microsphere in the suspension of the second microsphere is 10-50 mg / mL, preferably 15-40 mg / mL.

5. The multiphase microsphere separation and analysis system based on a microfluidic centrifugal field according to any one of claims 1-4, characterized in that, The sample loading area is a chamber with a volume of 65~75 μL; and / or The microsphere separation zone is equipped with pores that communicate with the atmosphere, ensuring equal pressure inside and outside the microchannel, allowing the microspheres and liquid to move within the microchannel; and / or The reaction detection region includes at least one detection well, each detection well being a chamber with a volume of 3-8 μL. Preferably, primers are immobilized in the reaction detection region; and / or, The microchannel has the following dimensions: diameter of 0.1~0.2mm and length of 0.8~1.2cm.

6. A nucleic acid detection method, wherein the multiphase microsphere separation and analysis system based on microfluidic centrifugation field according to any one of claims 1-5 is used to detect nucleic acids in the sample to be tested; Preferably, the nucleic acid detection method includes: The microfluidic chip pretreatment step includes adding primers and nucleic acid amplification solution to the reaction detection region of the microfluidic chip; The steps for preparing the sample to be tested include mixing the sample to be tested with lysis buffer, first microspheres, and second microspheres to form a mixture, and adding the mixture through the sample addition port into the microfluidic chip that has been pretreated in the microfluidic chip pretreatment step. as well as The detection steps include the following steps: in the preparation of the sample to be tested, the microfluidic chip after sample addition is loaded onto the microfluidic centrifuge platform, parameters are set, and the lysis, centrifugation and reaction processes are carried out to obtain the detection results.

7. The nucleic acid detection method according to claim 6, characterized in that, The microfluidic chip preprocessing steps include: (a) Primers corresponding to the target gene are added to the reaction detection area of ​​the microfluidic chip and dried to fix the primers in the reaction detection area; and (b) Add nucleic acid amplification solution through the sample loading wells in the sample loading area and centrifuge to allow the nucleic acid amplification solution to enter the detection wells in the reaction detection area for later use; Preferably, in step (a), the volume of primer added is 2~4 μL; Preferably, in step (a), the temperature used for primer drying is 50~70 °C; Preferably, primers corresponding to the target gene are added to the reaction detection area of ​​the microfluidic chip, dried to fix the primers in the reaction detection area, and then sealed with a bottom film. Optionally, the bottom film is a transparent PVDC film. Preferably, in step (b), the volume of nucleic acid amplification solution added to each well in the sample loading area is 25~40 μL; Preferably, in step (b), the centrifugation parameters for centrifuging the nucleic acid amplification solution into the detection well of the reaction detection area are: centrifugation at 56~126 g for 10~30 s, followed by centrifugation at 894~1132 g for 10~30 s.

8. The nucleic acid detection method according to claim 6 or 7, characterized in that, In the step of preparing the sample to be tested, the volume of the mixed liquid loaded into the microfluidic chip is 55~75 μL.

9. The nucleic acid detection method according to any one of claims 6-8, characterized in that, In the detection step described above, the pyrolysis temperature is 60~65 ℃, and the pyrolysis time is 3~5 min; and / or In the detection steps described above, the centrifugation includes: centrifuging at 0.6~5 g for 10 s, followed by centrifugation at 224~503 g for 30 s twice.

10. The nucleic acid detection method according to any one of claims 6-9, characterized in that, The nucleic acid detection method qualitatively or quantitatively detects the target gene in the sample to be tested.

11. The application of the multiphase microsphere separation and analysis system based on microfluidic centrifugation field according to any one of claims 1-5 in detecting target genes in a sample to be tested.

12. A kit comprising the multiphase microsphere separation and analysis system based on a microfluidic centrifugation field according to any one of claims 1-5.

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