Magnetically-driven micro-fluidic chip for nucleic acid detection and application of magnetically-driven micro-fluidic chip

Abstract

The invention discloses a magnetic drive micro-fluidic chip for nucleic acid detection and an analysis method. The chip comprises a cover plate layer, an oil phase cavity layer, a water phase cavity layer and a bottom plate layer which are sequentially bonded from top to bottom. The cover plate layer is provided with an oil phase cavity liquid inlet hole, an oil phase cavity liquid outlet hole and a water phase cavity liquid inlet hole. The oil-phase cavity is positioned above each water-phase cavity and is used for separating each reaction system; the water-phase cavity layer is provided with a magnetic bead storage cavity, a nucleic acid amplification cavity, a combination cavity and a signal output cavity which are arranged in sequence. The functionalized magnetic beads sequentially penetrate through the water-phase cavities through magnetic force driving, so that specific amplification products and signal report molecules are sequentially captured, finally, a chromogenic reaction is carried out in the signal output cavity, and reading of a detection result is completed. By using the micro-fluidic chip and the analysis method disclosed by the invention, high-sensitivity and high-specificity detection aiming at a nucleic acid target is expected to be realized, and the micro-fluidic chip does not need to be assisted by a large instrument and is low in cost, so that the micro-fluidic chip and the analysis method have a relatively good practical application prospect.

Description

Technical Field

[0001] The present invention relates to the fields of biomedical detection and microfluidic chips, and more specifically to a magnetically driven microfluidic chip and an analysis method for nucleic acid detection. Background Art

[0002] Nucleic acid testing uses the theories and techniques of molecular biology and biochemistry to directly or indirectly detect the existence or integrity of nucleic acids, and analyze the structure and function of nucleic acids from the structure, replication, transcription or translation level of nucleic acids. It is currently the gold standard for screening and prevention of infectious diseases. Nucleic acid testing technology represented by qPCR requires each step to be completed in a partitioned laboratory, and requires professional operators and expensive analytical instruments, so its application scenarios are greatly limited. ( Sci. China Chem. , 2021, 64, 171-203.) CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), that is, regularly clustered short palindromic repeat sequences, together with its associated Cas (CRISPR Associated Protein) protein form the CRISPR-Cas system, which is an acquired immune mechanism for bacteria and archaea to resist phage infection. Among them, Cas12a is a type 2 V-type Cas protein. It is self-inhibited when not activated. When it binds to a specific crRNA (CRISPR RNA), its conformation will change to form a Cas12a-crRNA binary complex. This complex can specifically recognize the target DNA and activate its endonuclease activity. When Cas12a sequentially cleaves the DNA double strands cis After cleavage, the active site remains exposed, showing a striking trans Based on the above principle, scientists such as Doudna designed fluorescent probes as tran s cutting substrate, thus designing a new high-sensitivity nucleic acid detection platform for human papillomavirus. In addition, a variety of nucleic acid detection platforms based on CRISPR system have emerged, and such nucleic acid detection technology is playing an increasingly important role. Mol. Cell , 2019, 73, 589-600. Cell ,2016, 165, 949-962. Science , 2017, 356, 438-442. Science, 2018, 360, 436-439.). However, the existing CRISPR nucleic acid detection technology has similar limitations as qPCR, such as relying on high-quality nucleic acid molecular probes and highly sensitive fluorescence detection instruments, and requiring professionals to perform complex operations. These defects limit the detection sites of the above methods to central laboratories. How to develop a more portable and inexpensive new nucleic acid detection method while maintaining the advantages of the existing CRISPR system such as high sensitivity and high specificity, and achieve a wide spectrum of application scenarios, will be an urgent problem to be solved in this field ( Biotechnol. Bioeng. , 2021, 118, 1587-1596. Anal. Chem. , 2021,93(50): 16965-16973.).

[0003] Point-of-care testing (POCT) refers to rapid testing and diagnosis performed at the patient's side. Nucleic acid testing based on POCT technology combines the high sensitivity and high specificity of molecular diagnosis with the advantages of POCT, which is small, portable and fast, and is more conducive to application scenarios such as primary medical institutions, airports, and communities ( Surgery , 2016, 34(2): 91-93.). Microfluidic technology can integrate the basic operation units of the analysis process, such as sample preparation, reaction, separation, and detection, into a chip of a few centimeters in size. It has the advantages of small size, low sample consumption, fast analysis speed, and simple operation. Therefore, it has broad application prospects in the field of POCT ( Biosens. Bioelectron. , 2017, 96: 332-338.). Therefore, it is of great significance to develop a new platform for instant detection of nucleic acid targets by combining CRISPR Cas system and microfluidic chip technology. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the existing CRISPR nucleic acid detection technology, combine the CRISPR nucleic acid recognition system and the magnetically driven microfluidic chip, and provide a new integrated nucleic acid detection platform with high sensitivity, high specificity, and no need for large-scale instrument assistance, which is portable, rapid, and low-cost.

[0005] Specifically, the purpose of the present invention is achieved through the following technical solutions: A magnetically driven microfluidic chip for nucleic acid detection, the chip consisting of a cover layer, an oil phase cavity layer, a water phase cavity layer and a bottom layer bonded in sequence from top to bottom, the cover layer being provided with an oil phase cavity liquid inlet, an oil phase cavity liquid outlet and a water phase cavity liquid inlet; the oil phase cavity layer being provided with one or more oil phase cavities, the water phase cavity layer being provided with a plurality of water phase cavities; the oil phase cavity being located above each of the water phase cavities to separate the reaction systems in each water phase cavity; the water phase cavity being one or more groups of magnetic bead storage cavities, nucleic acid amplification cavities, binding cavities and signal output cavities arranged in sequence; the oil phase cavity liquid inlet being connected to the oil phase cavity, the magnetic bead storage cavities, nucleic acid amplification cavities, binding cavities and signal output cavities being respectively connected to the water phase liquid inlet on the cover layer; The magnetic bead storage chamber contains functionalized magnetic beads; the functionalized magnetic beads are magnetic microspheres coated with functional molecules; the functional molecules are a complex of programmable nuclease and its guiding nucleic acid.

[0006] When the magnetically driven microfluidic chip is used for nucleic acid detection, it includes the following reaction steps: Step 1: Add the sample to be tested into the nucleic acid amplification chamber, amplify the target nucleic acid fragment by nucleic acid amplification technology, and obtain a target nucleic acid fragment amplification product with biotin labeling; Step 2: Through magnetic drive, the functionalized magnetic beads in the magnetic bead storage chamber are dragged through the oil phase into the nucleic acid amplification chamber, and the target nucleic acid fragment amplification product is captured through the specific recognition ability of the functional molecules to the target nucleic acid fragment amplification product; Step 3: Through magnetic drive, the functionalized magnetic beads are dragged through the oil phase into the binding cavity, and the signal reporter molecules are captured by binding with biotin; Step 4: Through magnetic drive, the functionalized magnetic beads are dragged through the oil phase to the signal output cavity. The signal reporter molecules react with the color reaction substrate, and the detection results are output by reading the color signal.

[0007] In a preferred embodiment of the present invention, the magnetically driven microfluidic chip further comprises a primary cleaning chamber located between the nucleic acid amplification chamber and the binding chamber. When the magnetically driven microfluidic chip is used for nucleic acid detection, the functionalized magnetic beads are dragged from the nucleic acid amplification chamber through the oil phase to the primary cleaning chamber for cleaning by magnetic drive, and then dragged to the binding chamber through the oil phase again.

[0008] In a preferred embodiment of the present invention, the magnetically driven microfluidic chip further comprises a secondary cleaning chamber located between the binding chamber and the signal output chamber. When the magnetically driven microfluidic chip is used for nucleic acid detection, the functionalized magnetic beads are dragged from the binding chamber through the oil phase to the secondary cleaning chamber for cleaning by magnetic drive, and then dragged again through the oil phase to the signal output chamber.

[0009] In a preferred embodiment of the present invention, the number of the primary cleaning chambers is 0-48.

[0010] In a preferred embodiment of the present invention, the number of the secondary cleaning chambers is 0-48.

[0011] In a preferred embodiment of the present invention, the number of the water phase cavities is 4-100.

[0012] In a preferred embodiment of the present invention, the cover layer, the oil phase cavity layer, the water phase cavity layer and the bottom layer all contain an alignment hole for easy bonding.

[0013] In a preferred embodiment of the present invention, the magnetic drive includes the following modes: by controlling the movement of the magnet in the vertical direction, the functionalized magnetic beads can shuttle back and forth between the oil phase and the water phase; by controlling the movement of the magnet in the horizontal direction, the functionalized magnetic beads can move in the connected oil phase cavity; by controlling the rotation of the magnet in the horizontal direction, the functionalized magnetic beads and the reaction solution in the water phase cavity are mixed, thereby achieving different reactions in different chambers.

[0014] In a preferred embodiment of the present invention, the nucleic acid amplification technology is selected from PCR, LAMP, RPA, RAA, SDA, NASAB, RCA, RT-PCR, RT-LAMP, RT-RPA, RT-RAA, RT-SDA, RT-NASAB, and RT-RCA.

[0015] In the nucleic acid amplification chamber, the primers used in the nucleic acid amplification technology are labeled with biotin, so the amplified product contains the biotin label.

[0016] In a preferred embodiment of the present invention, the target nucleic acid fragment is selected from DNA or RNA.

[0017] In a preferred embodiment of the present invention, the number of biotin labels on the target nucleic acid fragment amplification product is ≥1.

[0018] In a preferred embodiment of the present invention, the functional molecule is selected from dCas12a-crRNA complex, Cas9-sgRNA complex, Cas12-crRNA complex, Cas13a-crRNA complex, Argonaute proteins-gDNA complex, Argonaute proteins-gRNA complex.

[0019] The dCas12a-crRNA complex refers to a complex formed by the combination of dCas12a nuclease and crRNA by means of a spatial structure, and other complexes are named in a similar way.

[0020] The specific recognition relationship between the programmable nuclease and its guide nucleic acid complex and the target nucleic acid sequence is well known to those skilled in the art.

[0021] In a preferred embodiment of the present invention, the functional molecule is a dCas12a-crRNA complex, the target nucleic acid fragment is an HIV-1 viral genome; the crRNA is HIV-1-crRNA, and the nucleotide sequence of the HIV-1-crRNA is shown in SEQ ID No.1; the nucleic acid amplification technology is RT-RAA, wherein the primers used are HIV-1-FP and HIV-1-RP, respectively, the nucleotide sequence of the HIV-1-FP is shown in SEQ ID No.2, and the nucleotide sequence of the HIV-1-RP is shown in SEQ ID No.3; HIV-1-crRNA, i.e., SEQ ID No. 1 in the sequence list: 5'-UAAUUUCUACUAAGUGUAGAUUUGCCACACAAUCAUCACCU-3' HIV-1-FP, i.e., SEQ ID No. 2 in the sequence list: 5'-Biotin-TTTTTCGGGTTTATTACAGGGACAGCAGAAA-3' HIV-1-RP, i.e., SEQ ID No. 3 in the sequence list: 5'-ATGTTCTAATCCTCATCCTGTTTTCTTGCC-3'.

[0022] In a preferred embodiment of the present invention, the functional molecule is a dCas12a-crRNA complex, the target nucleic acid fragment is a SARS-CoV-2 viral genome; the crRNA is CoV-crRNA, and the nucleotide sequence of the CoV-crRNA is shown in SEQ ID No.4; the nucleic acid amplification technology is RT-RAA, wherein the primers used are CoV-FP and CoV-RP, respectively, the nucleotide sequence of the CoV-FP is shown in SEQ ID No.5, and the nucleotide sequence of the CoV-RP is shown in SEQ ID No.6; CoV-crRNA, i.e., sequence list SEQ ID No.4: 5'-UAAUUUCUACUAAGUGUAGAUCCCCCAGCGCUUCAGCGUUC-3' CoV-FP, i.e., SEQ ID No.5 in the sequence list: 5'-Biotin-AACTAATCAGACAAGGAACTGATTAAAAC-3' CoV-RP, i.e., sequence ID No.6: 5'-CTTATTCAGCAAATGACTTGATCTTTGAA-3' In a preferred embodiment of the present invention, the signal reporter molecule is a horseradish peroxidase-streptavidin complex, and the color development reaction substrate is selected from tetramethylbenzidine, diaminobenzidine, o-phenylenediamine, 5-aminosalicylic acid or 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt.

[0023] In a preferred embodiment of the present invention, the signal reporter molecule is an alkaline phosphatase-streptavidin complex, and the color development reaction substrate is selected from 4-nitrophenyl phosphate or 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt.

[0024] The horseradish peroxidase-streptavidin complex refers to a complex formed by co-expression or coupling of horseradish peroxidase and streptavidin.

[0025] The alkaline phosphatase-streptavidin complex refers to a complex formed by alkaline phosphatase and streptavidin by co-expression or coupling.

[0026] In a preferred embodiment of the present invention, the method of reading the color signal is selected from naked eye reading, absorbance instrument reading or image recognition software reading.

[0027] In a preferred embodiment of the present invention, the material of the chip can be selected from glass, polydimethylsiloxane, polymethyl methacrylate, polycarbonate, polyvinyl chloride, polystyrene, polytetrafluoroethylene or cycloolefin copolymers.

[0028] In a preferred embodiment of the present invention, the chip has a material thickness of 0.1-3.0 mm, and no obvious deformation occurs under the action of external force.

[0029] The present invention also provides a use of the magnetically driven microfluidic chip in nucleic acid detection.

[0030] The present invention also provides a nucleic acid detection method, which uses any of the above-mentioned magnetically driven microfluidic chips to perform nucleic acid detection.

[0031] Compared with the prior art, the present invention has the following beneficial effects: the magnetically driven microfluidic chip for nucleic acid detection provided by the present invention has high sensitivity, high specificity, and high accuracy, and does not require the assistance of large instruments. It is a portable, rapid, low-cost, and widely applicable new integrated nucleic acid detection platform. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : The chip design diagram of the present invention. AD is the layered structure diagram of the chip, and E is the structural diagram of the chip.

[0033] Figure 2 : Magnetic drive mode diagram.

[0034] Figure 3 : Schematic diagram of the working principle of magnetically driven microfluidic chip.

[0035] Figure 4 :Characterization of the feasibility of detection using dsDNA as a simulated target nucleic acid fragment. A is the color change produced by the chip of the present invention when detecting dsDNA of different concentrations. B is the absorbance change (652 nm) produced by the chip of the present invention when detecting dsDNA of different concentrations.

[0036] Figure 5 :Characterized by the detection sensitivity of HIV-1 genomic target nucleic acid fragments. A is the image of the signal output cavity 17 obtained by the chip of the present invention combined with the thermal lysis sample treatment method to detect HIV-1 of different concentrations. B is the image of the signal output cavity 17 obtained by the chip of the present invention combined with the kit sample treatment method to detect HIV-1 of different concentrations. C is the brightness difference between the image of the signal output cavity 17 obtained by the chip of the present invention combined with two different pretreatment methods to detect HIV-1 of different concentrations and the negative control.

[0037] Figure 6 :Characterized by the universality of detecting HIV-1 genomic target nucleic acid fragments. A is the sequence alignment of common HIV-1 variants (numbered 2-17) and standard strains (numbered 1), as well as the mismatch with crRNA and RT-RAA primers. B is the signal output cavity 17 image obtained by the chip of the present invention for detecting 5000 CPs / mL HIV-1 variants or standard strains. C is the brightness difference between the signal output cavity 17 image obtained by the chip of the present invention for detecting 5000 CPs / mL HIV-1 variants or standard strains and the negative control.

[0038] Figure 7 :Characterized by the detection specificity of HIV-1 genome target nucleic acid fragments. The concentration of 50000 CPs / mL C. albicans , A. fumigatus , C. neoformans , P. aeruginosa, S. aureus and HIV-2 as negative controls, with a concentration 10 times that of the HIV-1 positive control.

[0039] Figure 8 : Characterized by the detection accuracy of HIV-1 genomic target nucleic acid fragments. A is the response between the color change in the signal output cavity 17 and each HIV-1 sample (including 28 positive samples and 28 negative samples). B is the receiver operating characteristic curve of the chip of the present invention for HIV-1 detection. If each parallel test is regarded as an independent measurement, the area under the curve (AUC) is 0.9993. C is the response between the brightness difference of the signal output cavity 17 image and the HIV-1 sample. Blank+3*SD is set as the diagnostic cutoff. D is an analysis of the sensitivity, specificity and overall accuracy of the chip of the present invention for HIV-1 detection compared with qRT-PCR. Fig. 9 :Characterization of the feasibility of SARS-CoV-2 genome target nucleic acid fragments. This figure shows the brightness difference between the signal output cavity 17 image and the negative control obtained by the chip of the present invention for detecting different concentrations of SARS-CoV-2 genome target nucleic acid fragments. Implementation

[0040] The technical solution of the present invention is further described below through specific implementation modes in combination with the accompanying drawings, but these embodiments are only used as exemplary descriptions of the present invention and are not intended to limit the scope of the present invention.

[0041] 1. Chip Structure like Figure 1 Shown is a magnetically driven microfluidic chip for nucleic acid detection of the present invention, which is composed of a cover layer A, an oil phase cavity layer B, an aqueous phase cavity layer C and a bottom layer D. The cover layer A comprises an oil phase liquid inlet hole 1, an oil phase liquid outlet hole 2, and aqueous phase liquid inlet holes 3, 4, 5, 6, 7, 8, 9; the oil phase cavity layer B comprises a connected arc-shaped oil phase cavity 10; the aqueous phase cavity layer C comprises seven independent aqueous phase cavities, which are a magnetic bead storage cavity 11, a nucleic acid amplification cavity 12, a primary cleaning cavity 13, a binding cavity 14, a secondary cleaning cavity 15, a secondary cleaning cavity 16, and a signal output cavity 17; each of the above layers comprises a rectangular alignment hole 18 for easy bonding. The top view of the prepared chip is shown as follows. Figure 1 As shown in E.

[0042] 2. Chip Preparation The magnetically driven microfluidic chip is composed of four layers of polymethyl methacrylate, e.g. Figure 1 As shown in AD. After the chip was cleaned with deionized water, soaked in 2% fat-free milk solution and dried, it was assembled layer by layer using the adhesive force of optical double-sided tape. Finally, the top view of the prepared chip is shown in Figure 1As shown in E, store it in a desiccator and take it out when needed.

[0043] 3. Nucleotide sequences of crRNA and primers The target nucleic acid fragment of this embodiment is the HIV-1 viral genome, and the functional molecule coated by the functionalized magnetic beads is a dCas12a-crRNA complex, wherein the crRNA is HIV-1-crRNA, and its nucleotide sequence is shown in SEQ ID No. 1. The nucleic acid amplification technology used is RT-RAA, wherein the primers used are HIV-1-FP and HIV-1-RP, respectively, and their nucleotide sequences are shown in SEQ ID No. 2 and SEQ ID No. 3, respectively.

[0044] HIV-1-crRNA, i.e., SEQ ID No. 1 in the sequence list: 5'-UAAUUUCUACUAAGUGUAGAUUUGCCACACAAUCAUCACCU-3' HIV-1-FP, i.e., SEQ ID No. 2 in the sequence list: 5'-Biotin-TTTTTCGGGTTTATTACAGGGACAGCAGAAA-3' HIV-1-RP, i.e., SEQ ID No. 3 in the sequence list: 5'-ATGTTCTAATCCTCATCCTGTTTTCTTGCC-3' 4. Preparation of dCas12a-crRNA functionalized magnetic beads Take 1 mL of 25% Ni-NTA magnetic microsphere (MBs) suspension (purchased from GenScript Biotech Co., Ltd., Catalog No.: L00295), remove the storage solution by magnetic separation, and wash several times with 1×PBS buffer. The above magnetic microspheres were mixed with 1 mL 400 nM His-tagged dCas12a and incubated with rotation at room temperature for 20 minutes to form dCas12a-MBs. After washing several times with 1×PBS buffer, dCas12a-MBs were incubated with 1 mL 2% skim milk at 4°C overnight. Finally, dCas12a-MBs were washed again with 1×PBS buffer several times and resuspended in 1 mL storage buffer (10 mM Tris-HCl, 50 mM NaCl, 10mM MgCl2, 0.05% Tween20, pH7.9) to obtain dCas12a-MBs stock solution. Take 100 μL of dCas12a-MBs stock solution, mix it thoroughly with 2 μL of 2 μM crRNA, wash the magnetic beads several times with 1× PBS buffer, and resuspend them in 100 μL of storage buffer to prepare dCas12a-crRNA functionalized magnetic beads.

[0045] 5. Liquid inflow process of water phase and oil phase 100 μL dCas12a-crRNA functionalized magnetic beads, 80 μL RT-RAA reaction solution (including 67 μL RT-RAA buffer, 8 μL 5 μM primer mixture and 5 μL amplification initiator), 100 μL 250 nM horseradish peroxidase-streptavidin complex solution and 100 μL soluble diaminobenzidine substrate solution were added to the magnetic bead storage chamber 11, nucleic acid amplification chamber 12, binding chamber 14 and signal output chamber 17 through the inlet holes 3, 4, 6 and 9 of the aqueous phase chamber. Washing buffer (1×PBS buffer, containing 0.75% Tween 20) was added to the primary washing chamber-1 13, washing chamber-2 15 and washing chamber-3 16 through the inlet holes 5, 7 and 8 of the aqueous phase chamber. Light mineral oil is added into the oil phase cavity 10 through the oil phase cavity liquid inlet hole 1 until the entire arc-shaped cavity is filled and overflows from the oil phase cavity liquid outlet hole 2.

[0046] 6. Basic process of nucleic acid detection using magnetically driven microfluidic chips The sample to be tested is added into the nucleic acid amplification chamber 12 through the liquid inlet hole 4 of the aqueous phase chamber, the chip is incubated at 37° C. for 20 minutes, and the nucleic acid amplification technology RT-RAA is used to achieve isothermal nucleic acid amplification. Figure 2The movement mode of the functionalized magnetic beads under magnetic drive when the chip is placed horizontally is shown. After continuous magnetic drive using a magnet located above the magnetic bead storage chamber 11 and below the nucleic acid amplification chamber 12, the functionalized magnetic beads are migrated to the nucleic acid amplification chamber 12. Subsequently, the solution is mixed by rotating the magnet located below the nucleic acid amplification chamber 12, so that the dCas12a guided by crRNA can capture the target amplification product. After 10 minutes of incubation, the magnetic beads are transferred using the same method and enter the binding chamber 14 after washing for 2 minutes in the primary cleaning chamber 13. After 10 minutes of mixing in the binding chamber 14, the horseradish peroxidase-streptavidin complex is completely bound to the biotin group marked by the amplification product. The subsequent magnetic beads are transferred to the secondary cleaning chamber 15 and the secondary cleaning chamber 16 in turn, and washed for a total of 4 minutes. Finally, the functional magnetic beads are transferred to the signal output chamber 17, and the oxidation of diaminobenzidine is catalyzed by horseradish peroxidase to form a blue product, and the reaction lasts for 20 minutes. The color reaction signal is read with the naked eye, spectrophotometer or image recognition software to output the test results.

[0047] 7. Basic principles of nucleic acid detection using the magnetically driven microfluidic chip provided by the present invention Detection principle Figure 3 As shown. Among them, I is in the nucleic acid amplification chamber, the HIV-1 nucleic acid fragment in the sample to be tested is reverse transcribed and exponentially amplified by the RT-RAA nucleic acid amplification technology to obtain a target nucleic acid fragment amplification product with a biotin label; II is in the nucleic acid amplification chamber, the functional molecule uses the specific recognition ability of the target nucleic acid fragment amplification product to capture the target nucleic acid fragment amplification product; III is in the binding chamber, the biotin on the target nucleic acid fragment amplification product is combined with the signal reporter molecule horseradish peroxidase-streptavidin complex to capture the signal reporter molecule; IV is in the signal output chamber, the horseradish peroxidase reacts with diaminobenzidine to produce a color reaction, and the detection result is output by reading the color signal.

[0048] Biotin-modified dsDNA was used as a simulated target, and an equal volume of RT-RAA reaction solution was replaced by a washing buffer in the nucleic acid amplification chamber 12 to investigate the feasibility of the magnetic drive chip for nucleic acid detection. Figure 4 As shown in the figure, with the increase of dsDNA concentration, the chip signal output cavity 17 produces obvious color change (blue) and has a characteristic absorption peak at 652 nm. Without the help of any nucleic acid amplification technology, the color change caused by dsDNA as low as 0.98 nM can be directly distinguished by the naked eye. With the help of a spectrophotometer, the sensitivity is increased to 0.49 nM, and the detected absorbance is positively correlated with the dsDNA concentration within a certain range.

[0049] like Figure 5 As shown in A and 5B, for HIV-1 samples, by combining thermal lysis and kit extraction, the magnetically driven microfluidic chip described in Implementation 1 can identify HIV-1 viral genomes of at least 1000 CPs / mL and 250 CPs / mL, respectively. According to the "National Technical Specifications for AIDS Testing (2020 Edition)", the cutoff for preliminary diagnosis of HIV-1 is 5000 CPs / mL, indicating that the detection sensitivity of these two methods can meet clinical testing requirements. Figure 5 As shown in FIG. 3 , by analyzing the brightness of the signal output cavity 17 through image recognition software, qualitative / semi-quantitative output of the detection results can also be achieved.

[0050] In the above detection process, dCas9-sgRNA complex, dCas13a-crRNA complex, Argonaute proteins-gDNA complex, Argonaute proteins-gRNA complex, and other types of dCas12-crRNA complexes (the guide nucleic acid and amplification primer sequences are conventionally designed according to the target nucleic acid sequence) can be used to replace the dCas12a-crRNA complex used in Example 1, and similar detection effects can be achieved.

[0051] HIV-1 is an RNA virus with many variants, the most common of which include subtype B and epidemic recombinant strains 01_AE, 07_BC, and 08_BC. Therefore, in this example, the genomes of HIV-1 viruses of different subtypes (5000 CPs / mL) were used as target nucleic acid fragments (e.g. Figure 6 A), investigate the universality of the magnetically driven microfluidic chip described in Example 1 for nucleic acid detection. Figure 6 As shown in B and 6C, the combined effect of RT-RAA primers, the chip can effectively identify variants (numbers 2, 4-15) with base substitutions in the primer fragment, PAM site or upstream region of the target fragment (1-5 bases), and the color change produced in the signal output cavity 17 is not significantly different from the standard strain (number 1). When the target fragment in the key region mutates, it may cause changes in the binding ability of dCas12a, and the color changes caused by these variants (numbers 3, 16-17) are not as obvious as the standard strain (number 1). Since the color changes caused by the above virus strains at 5000 CPs / mL can be directly observed by the naked eye, it reflects that the magnetically driven microfluidic chip has good versatility in HIV-1 detection and does not affect its potential application in clinical screening.

[0052] HIV-1 infection can significantly weaken the human immune system, often leading to opportunistic infections (OIs). To evaluate the specificity of the magnetically driven microfluidic chip, common OI pathogens were tested, including Candida albicans ( Candida albicans )、Aspergillus fumigatus( Aspergillus fumigatus )、Cryptococcus neoformans( Cryptococcus neoformans ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Staphylococcus aureus ( Staphylococcus aureus ) and HIV-2. Figure 7 As shown in the figure, even though the concentration of the above OI pathogens was 10 times that of the positive control HIV-1 (5000 CPs / mL), no obvious color change was observed in these negative control groups. The corresponding image brightness difference was also not significantly different from that of the blank group, indicating that the detection system has good specificity.

[0053] In order to investigate the accuracy of nucleic acid detection by the magnetically driven microfluidic chip, this embodiment used the chip and qRT-PCR to detect 28 HIV-1 positive samples (10 6 -250 CPs / mL) and 28 negative samples (0 CPs / mL) were tested. Figure 8 A shows the detection results obtained from the signal output cavity 17 of each chip. Figure 8 B constructed the receiver operating characteristic curve (ROC curve). These results show that as a new detection system, the magnetically driven microfluidic chip of the present invention has good diagnostic accuracy, and the area under the curve (AUC) is 0.9993. In addition, Figure 8 As shown in Figures C and 8D, compared with qRT-PCR technology, the sensitivity, specificity, and overall accuracy of HIV-1 detection using image recognition technology were 96.4%, 100%, and 98.2%, respectively.

[0054] To characterize the versatility of the patent of the present invention, the SARS-CoV-2 genome was detected in this embodiment. In this embodiment, the functional molecule coated with the functionalized magnetic beads is a dCas12a-crRNA complex, wherein the crRNA is CoV-crRNA, and its nucleotide sequence is shown in SEQ ID No.4. The nucleic acid amplification technology used is RT-RAA, wherein the primers used are CoV-FP and CoV-RP, respectively, and their nucleotide sequences are shown in SEQ ID No.5 and SEQ ID No.6, respectively.

[0055] CoV-crRNA, i.e., sequence list SEQ ID No.4: 5'-UAAUUUCUACUAAGUGUAGAUCCCCCAGCGCUUCAGCGUUC-3' CoV-FP, i.e., SEQ ID No.5 in the sequence list: 5'-Biotin-AACTAATCAGACAAGGAACTGATTAAAAC-3' CoV-RP, i.e., sequence ID No.6: 5'-CTTATTCAGCAAATGACTTGATCTTTGAA-3' like Fig. 9 As shown, by analyzing the brightness of the signal output cavity 17 through image recognition software, qualitative / semi-quantitative output of the detection results can be achieved. The results show that this method can at least identify SARS-CoV-2 genome fragments as low as 10 CPs / mL.

Claims

1. A magnetically driven microfluidic chip for nucleic acid detection, the chip is composed of a cover layer, an oil phase cavity layer, a water phase cavity layer and a bottom layer bonded in sequence from top to bottom, the cover layer is provided with an oil phase cavity liquid inlet, an oil phase cavity liquid outlet and a water phase cavity liquid inlet; the oil phase cavity layer is provided with one or more oil phase cavities, and the water phase cavity layer is provided with a plurality of water phase cavities; the oil phase cavity is located above each of the water phase cavities to separate the reaction systems in each water phase cavity; the water phase cavity is one or more groups of magnetic bead storage cavities, nucleic acid amplification cavities, binding cavities and signal output cavities arranged in sequence; the oil phase cavity liquid inlet is connected to the oil phase cavity, and the magnetic bead storage cavity, nucleic acid amplification cavity, binding cavity and signal output cavity are respectively connected to the water phase liquid inlet on the cover layer; preferably, the number of the water phase cavities is 4 to 100; The magnetic bead storage chamber contains functionalized magnetic beads; the functionalized magnetic beads are magnetic microspheres coated with functional molecules; the functional molecules are programmable nucleases and complexes of their guide nucleic acids; When the magnetically driven microfluidic chip is used for nucleic acid detection, it includes the following reaction steps: Step 1: Add the sample to be tested into the nucleic acid amplification chamber, amplify the target nucleic acid fragment by nucleic acid amplification technology, and obtain a target nucleic acid fragment amplification product with biotin labeling; Step 2: Through magnetic drive, the functionalized magnetic beads in the magnetic bead storage chamber are dragged through the oil phase into the nucleic acid amplification chamber, and the target nucleic acid fragment amplification product is captured through the specific recognition ability of the functional molecules to the target nucleic acid fragment amplification product; Step 3: Through magnetic drive, the functionalized magnetic beads are dragged through the oil phase into the binding cavity, and the signal reporter molecules are captured by binding with biotin; Step 4: Through magnetic drive, the functionalized magnetic beads are dragged through the oil phase to the signal output cavity. The signal reporter molecules react with the color reaction substrate, and the detection results are output by reading the color signal.

2. The magnetically driven microfluidic chip for nucleic acid detection according to claim 1, characterized in that: The magnetically driven microfluidic chip also includes a primary cleaning chamber located between the nucleic acid amplification chamber and the binding chamber. When the magnetically driven microfluidic chip is used for nucleic acid detection, the functionalized magnetic beads are dragged from the nucleic acid amplification chamber through the oil phase to the primary cleaning chamber for cleaning by magnetic drive, and then dragged to the binding chamber through the oil phase again; preferably, the number of the primary cleaning chambers is 1-X.

3. The magnetically driven microfluidic chip for nucleic acid detection according to claim 1, characterized in that: The magnetically driven microfluidic chip also includes a secondary cleaning chamber located between the binding chamber and the signal output chamber. When the magnetically driven microfluidic chip is used for nucleic acid detection, the functionalized magnetic beads are dragged from the binding chamber through the oil phase to the secondary cleaning chamber for cleaning through magnetic drive, and then dragged to the signal output chamber through the oil phase again; preferably, the number of the secondary cleaning chambers is 1-Y.

4. The magnetically driven microfluidic chip for nucleic acid detection according to claims 1-3, characterized in that: The cover plate layer, the oil phase cavity layer, the water phase cavity layer and the bottom plate layer all contain an alignment hole for easy bonding.

5. The magnetically driven microfluidic chip for nucleic acid detection according to claims 1-3, characterized in that: The magnetic drive includes the following modes: by controlling the movement of the magnet in the vertical direction, the functionalized magnetic beads can shuttle back and forth between the oil phase and the water phase; by controlling the movement of the magnet in the horizontal direction, the functionalized magnetic beads can move in the connected oil phase cavity; by controlling the rotation of the magnet in the horizontal direction, the functionalized magnetic beads and the reaction solution in the water phase cavity are mixed, thereby achieving different reactions in different chambers.

6. The magnetically driven microfluidic chip for nucleic acid detection according to claim 1, characterized in that: The nucleic acid amplification technology is selected from PCR, LAMP, RPA, RAA, SDA, NASAB, RCA, RT-PCR, RT-LAMP, RT-RPA, RT-RAA, RT-SDA, RT-NASAB, and RT-RCA.

7. The magnetically driven microfluidic chip for nucleic acid detection according to claim 1, characterized in that: The target nucleic acid fragment is selected from DNA or RNA; preferably, the number of biotin labels on the amplified product of the target nucleic acid fragment is ≥1.

8. The magnetically driven microfluidic chip for nucleic acid detection according to claim 1, characterized in that: The functional molecule is selected from a dCas12a-crRNA complex, a Cas9-sgRNA complex, a Cas12-crRNA complex, a Cas13a-crRNA complex, an Argonaute proteins-gDNA complex or an Argonaute proteins-gRNA complex.

9. The magnetically driven microfluidic chip for nucleic acid detection according to claim 1, characterized in that: The functional molecule is a dCas12a-crRNA complex, and the target nucleic acid fragment is an HIV-1 viral genome; preferably, the crRNA is HIV-1-crRNA, and the nucleotide sequence of the HIV-1-crRNA is shown in SEQ ID No.1; the nucleic acid amplification technology is RT-RAA, wherein the primers used are HIV-1-FP and HIV-1-RP, respectively, the nucleotide sequence of the HIV-1-FP is shown in SEQ ID No.2, and the nucleotide sequence of the HIV-1-RP is shown in SEQ ID No.

3.

10. The magnetically driven microfluidic chip for nucleic acid detection according to claim 1, characterized in that: The functional molecule is a dCas12a-crRNA complex, and the target nucleic acid fragment is a SARS-CoV-2 viral genome; preferably, the crRNA is CoV-crRNA, and the nucleotide sequence of the CoV-crRNA is shown in SEQ ID No.4; the nucleic acid amplification technology is RT-RAA, wherein the primers used are CoV-FP and CoV-RP, respectively, the nucleotide sequence of the CoV-FP is shown in SEQ ID No.5, and the nucleotide sequence of the CoV-RP is shown in SEQ ID No.

6.

11. The magnetically driven microfluidic chip for nucleic acid detection according to claim 1, characterized in that: The signal reporter molecule is a horseradish peroxidase-streptavidin complex, and the color reaction substrate is selected from tetramethylbenzidine, diaminobenzidine, o-phenylenediamine, 5-aminosalicylic acid or 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt; or the signal reporter molecule is an alkaline phosphatase-streptavidin complex, and the color reaction substrate is selected from 4-nitrophenyl phosphate or 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt.

12. The magnetically driven microfluidic chip for nucleic acid detection according to claim 1, characterized in that: The method of reading the color signal is selected from naked eye reading, absorbance instrument reading or image recognition software reading.

13. The magnetically driven microfluidic chip for nucleic acid detection according to claim 1, characterized in that: The material of the chip can be selected from glass, polydimethylsiloxane, polymethyl methacrylate, polycarbonate, polyvinyl chloride, polystyrene, polytetrafluoroethylene or cycloolefin copolymer; Preferably, the chip has a material thickness of 0.1-3.0 mm, and no obvious deformation occurs under the action of external force.

14. Use of the magnetically driven microfluidic chip according to any one of claims 1 to 13 in nucleic acid detection.

15. A method for nucleic acid detection, characterized in that: Use the magnetically driven microfluidic chip as described in any one of claims 1-13 to perform nucleic acid detection.

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