Multi-channel micro-fluidic chip for detecting multiple drug-resistant mycobacterium tuberculosis as well as use method and application of multi-channel micro-fluidic chip

Abstract

The invention relates to the technical field of micro-fluidic electrochemical detection, in particular to a multi-channel micro-fluidic chip for detecting various drug-resistant mycobacterium tuberculosis as well as a use method and application of the multi-channel micro-fluidic chip. The multi-channel micro-fluidic chip comprises a splitting area and a sample mixing area, the splitting area comprises a sample injection hole and a DNA splitting chamber which are communicated in sequence, the sample mixing area comprises a sample mixing chamber, and the DNA splitting chamber and the sample mixing chamber are communicated through a first micro-channel; the electrode array area comprises a plurality of reaction detection chambers and detection electrodes, the detection electrodes are in one-to-one correspondence with the reaction detection chambers and are in contact with the inner spaces of the reaction detection chambers, the electrodes are modified with signal amplification probes, Cas12a-crRNA compounds are preloaded on the surfaces of the electrodes, and the reaction detection chambers are communicated with the sample mixing chamber through a second micro-channel. Multiple channels are designed, synchronous detection of multiple drug-resistant genes is achieved, integration of clinical sample treatment, DNA acquisition, DNA thermal denaturation treatment and DNA detection is achieved, operation is easy and smooth, and clinical diagnosis and medication guidance of tuberculosis drug resistance can be well served.

Description

Technical Field

[0001] This invention relates to the field of microfluidic electrochemical detection technology, specifically to a multichannel microfluidic chip for detecting various drug-resistant Mycobacterium tuberculosis, and its usage and application. Background Technology

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis. It is an ancient disease that remains a significant threat to human health and a major public health problem, ranking among the top 10 causes of death worldwide. Genetic mutations in Mycobacterium tuberculosis can easily reduce or even eliminate the bactericidal efficiency of antibiotics, and the existence of drug-resistant strains further increases the difficulty of treatment. Based on the type of drug resistance, TB is classified into single-drug resistant TB, multidrug-resistant TB, multidrug-resistant TB, and extensively multidrug-resistant TB. The "gold standard" for assessing drug-resistant TB is the phenotypic drug susceptibility test, which takes about a month, delaying timely treatment and increasing mortality and the risk of infection transmission. Molecular diagnostic technologies rapidly assess TB drug resistance by detecting gene mutations associated with drug resistance, such as GeneXpert MTB / RIF testing, whole-genome sequencing, and targeted next-generation sequencing. However, current research mainly focuses on rifampicin-only resistance and has limitations in detecting multidrug resistance.

[0003] The CRISPR / Cas system, an adaptive immune system derived from bacteria and archaea, has been developed as a gene-editing tool. Its high efficiency, precision, and ease of operation have rapidly made it a popular research area in life sciences, bioengineering, and the medical-engineering intersection. The core of the CRISPR / Cas system is the Cas nuclease, with Cas12a requiring only one crRNA to guide it to the target DNA site, simplifying the construction and delivery of gene-editing tools. This enzyme can recognize and cleave both double-stranded and single-stranded DNA, expanding the application scope of gene editing. CRISPR / Cas12a has become a promising nucleic acid detection platform, demonstrating application potential in medical diagnostics and environmental monitoring.

[0004] Microfluidic chips are miniature systems capable of precisely processing small volumes of fluids. They can perform complex unit operations such as separation, transfer, extraction, adsorption, and washing at the micrometer and millimeter scale, enabling the separation and detection of complex molecules such as proteins and nucleic acids. They offer advantages such as high throughput, rapid response, and portability. Multi-channel microfluidic designs can achieve parallel operation, high-throughput processing, and the integration of multiple sensors for real-time diagnostics. The combination of microfluidic chips with the CRISPR / Cas12a system will have a profound impact on gene expression analysis, disease diagnosis, and drug efficacy evaluation. However, how to combine microfluidic chips with the CRISPR / Cas system to develop highly sensitive biosensors for detecting biomolecules and pathogens has not yet been reported in research. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a multi-channel microfluidic chip for detecting various drug-resistant Mycobacterium tuberculosis, as well as its usage method and application.

[0006] The multi-channel microfluidic chip of the present invention for detecting drug resistance of Mycobacterium tuberculosis assesses the drug resistance of Mycobacterium tuberculosis by calculating changes in electrochemical signals, and simultaneously detects multiple drug resistances through multi-channel design, achieving rapid and high-throughput combined detection of mycobacterial drug resistance.

[0007] To achieve the above and other related objectives, a first aspect provides a multi-channel microfluidic chip for amplification-free detection, comprising a microfluidic chip functional layer, wherein the functional layer includes a lysis region, a sample mixing region, and an electrode array region;

[0008] The lysis zone includes a sample inlet and a DNA lysis chamber connected in sequence, and the sample mixing zone includes a sample mixing chamber. The DNA lysis chamber and the sample mixing chamber are connected through a first microchannel.

[0009] The electrode array region includes several reaction detection chambers and detection electrodes. The detection electrodes correspond one-to-one with the reaction detection chambers and are in contact with the internal space of the reaction detection chambers. The detection electrodes are modified with signal amplification probes, and the surface of the detection electrodes is preloaded with Cas12a-crRNA complexes. The reaction detection chambers and the sample mixing chambers are connected through a second microfluidic channel.

[0010] The second aspect of this invention provides the use of the above-mentioned multi-channel microfluidic chip detection platform in the preparation of various drug-resistant Mycobacterium tuberculosis detection products.

[0011] A third aspect of the present invention provides a method for using the above-mentioned multi-channel microfluidic chip, comprising the following steps:

[0012] 1) After DNA is extracted from the sample in the lysis chamber, it is lysed into single-stranded DNA and then enters the CRISPR reaction chamber;

[0013] 2) The target recognition probe is added to the CRISPR reaction chamber and mixed with single-stranded DNA to form a CRISPR-Cas12a reaction solution. The solution is then incubated on the electrode surface through the detection chamber. The electrode is modified with a signal amplification probe.

[0014] 3) The electrodes were scanned using an electrochemical workstation, and the differential pulse voltammetry (DPV) method was used to detect drug resistance in Mycobacterium tuberculosis.

[0015] In summary, this invention discloses a multichannel microfluidic chip for detecting various drug-resistant Mycobacterium tuberculosis, its usage method, and applications, and achieves the following beneficial effects:

[0016] 1) This invention is designed with multiple chambers to achieve integrated processing from clinical sample processing, DNA acquisition, DNA thermal denaturation treatment to DNA detection. The operation is simple and smooth, and avoids the possibility of contamination.

[0017] 2) This invention is designed with multiple channels to achieve simultaneous detection of multiple drug resistance genes, which is comprehensive and rapid, and can well serve the clinical diagnosis and medication guidance of tuberculosis drug resistance.

[0018] 3) By using a multi-channel microfluidic chip, different crRNA sequences can be placed in the reaction chamber to achieve highly sensitive detection of multiple targets simultaneously. This invention successfully achieved ultrasensitive detection of multiple targets, including katG, embB, and rpoB, with detection limits reaching 3 × 10⁻⁶. -14 2.5×10 -15 2.5×10 -17 Moles per liter. Attached Figure Description

[0019] Figure 1 The diagram shows the structure and physical image of the microfluidic chip; where 11-sample inlet, 12-DNA lysis chamber, 13-sample mixing chamber, 14-reaction detection chamber, and 15-liquid outlet.

[0020] Figure 2 A comparative diagram of the katG, embB, and rpoB gene sequences of drug-resistant Mycobacterium tuberculosis;

[0021] Figure 3 The response results of single, double, and triple crRNA systems were used to verify the trans-cleavage experiment.

[0022] Figure 4 Schematic diagram of a multichannel microfluidic chip for drug-resistant Mycobacterium tuberculosis;

[0023] Figure 5 The current response curve of the multichannel microfluidic chip to the wild-type Mycobacterium tuberculosis gene;

[0024] Figure 6 The current response curves of the multichannel microfluidic chip to the katG, embB, and rpoB genes of drug-resistant Mycobacterium tuberculosis are shown.

[0025] Figure 7 Standard curves for the detection signals of the katG and embB genes in drug-resistant Mycobacterium tuberculosis using a multi-channel microfluidic chip;

[0026] Figure 8 The image shows the detection signal results of the rpoB gene in drug-resistant Mycobacterium tuberculosis using a multi-channel microfluidic chip.

[0027] Figure 9 This image shows the results of specific detection of the katG, embB, and rpoB genes in drug-resistant Mycobacterium tuberculosis using a multichannel microfluidic chip.

[0028] Figure 1 The labels in the attached figures are as follows:

[0029] 11. Sample inlet; 12. DNA lysis chamber; 13. Sample mixing chamber; 14. Reaction detection chamber; 15. Liquid outlet; 16. First control valve; 17. Second control valve. Detailed Implementation

[0030] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0032] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0033] This invention first provides a multi-channel microfluidic chip for amplification-free detection, including a microfluidic chip functional layer, wherein the microfluidic chip includes a lysis region, a sample mixing region, and an electrode array region;

[0034] The lysis zone includes a sample inlet and a DNA lysis chamber connected in sequence, and the sample mixing zone includes a sample mixing chamber. The DNA lysis chamber and the sample mixing chamber are connected through a first microchannel.

[0035] The electrode array region includes several reaction detection chambers and detection electrodes. The detection electrodes correspond one-to-one with the reaction detection chambers and are in contact with the internal space of the reaction detection chambers. The detection electrodes are modified with signal amplification probes, and the surface of the detection electrodes is preloaded with Cas12a-crRNA complexes. The reaction detection chambers and the sample mixing chambers are connected through a second microfluidic channel.

[0036] In a preferred embodiment, the sample inlet is provided with a sample inlet valve for controlling the opening and closing of the DNA lysis chamber.

[0037] Preferably, the first microchannel is provided with a first control valve and a second control valve. The washing solution in the DNA lysis chamber is discharged through the first control valve; the liquid is introduced into the sample mixing zone through the cooperation of the first and second control valves.

[0038] Preferably, the sample mixing chamber includes multiple micropillars arranged in an orderly manner, with gaps between adjacent micropillars for the passage of liquid, thereby increasing the complexity of the sample mixing chamber, increasing the probability of collisions between molecules in the liquid, and ensuring thorough mixing of the liquid.

[0039] In a preferred embodiment, the number of reaction detection chambers is 4 to 6, preferably 5. More preferably, each reaction detection chamber is provided with a corresponding detection electrode, and each electrode is preloaded with a Cas12a-crRNA complex, and different targets are detected according to different preloaded Cas12a-crRNA complexes.

[0040] In this invention, the Cas12a-crRNA complex is a composition prepared by mixing Cas12a protein and crRNA, and is used to specifically recognize target DNA.

[0041] Further preferably, the microfluidic chip also includes a liquid outlet, which is connected to one end of the reaction detection chamber. Preferably, the liquid outlet is equipped with an outlet valve.

[0042] In a preferred embodiment, the DNA lysis zone is further provided with a micro heater, which is correspondingly disposed at the bottom of the DNA lysis chamber.

[0043] In one embodiment, the diameter of the injection port is 0.8~1.2 mm, which can be 0.8~1.0 mm, 1.0~1.2 mm, and preferably 1.0 mm.

[0044] Preferably, the diameter of the sample outlet is 0.8~1.2 mm, which can be 0.8~1.0 mm, 1.0~1.2 mm, and preferably 1.0 mm.

[0045] In a preferred embodiment, the DNA lysis chamber, sample mixing chamber, and reaction detection chamber are circular, square, rhomboid, or polygonal; preferably, the DNA chamber, sample mixing chamber, and reaction detection chamber are square; more preferably, the side length of the DNA chamber, sample mixing chamber, and reaction detection chamber is 8-12 mm, which can be 8-10 mm, 10-12 mm, and preferably 10 mm.

[0046] In one specific embodiment, the electrode includes a substrate and a three-electrode system formed on the surface of the substrate. The material of the substrate includes, but is not limited to, at least one of polyimide, polyethylene terephthalate, and polydimethylsiloxane. The material of the three-electrode system includes, but is not limited to, at least one of platinum, gold, silver, copper, chromium, and carbon.

[0047] The three-electrode system includes a working electrode, a reference electrode, and a counter electrode. The working electrode and the counter electrode are electrically connected to external analytical instruments, and the reference electrode provides a potential reference. The working electrode, reference electrode, and counter electrode are all in contact with the sample to be tested within the CRISPR reaction chamber, and the counter electrode and the reference electrode are arranged around the working electrode.

[0048] Preferably, both the working electrode and the counter electrode are PI thin film deposited gold electrodes, and the reference electrode is an Ag / AgCl reference electrode.

[0049] More preferably, the number of detection electrodes is 4 to 6, and each electrode is modified with a signal amplification probe.

[0050] In some embodiments of the present invention, the signal amplification probe is a methylene blue-labeled DNA hairpin probe (MB-DNA); one end of the MB-DNA is labeled with MB, and the other end is fixed to the surface of the working electrode by an Au-S bond.

[0051] Preferably, the nucleotide sequence of the MB-DNA probe is shown in SEQ ID NO.22.

[0052] SEQ ID NO. 22: 5'-SH-(CH2)6-AAAAAAAAAAAAAAAAAAAAA-MB-3'.

[0053] In the Cas12a-crRNA complex, the molar ratio of Cas12a to crRNA is (1~3):(1~4), preferably 2:3.

[0054] In a preferred embodiment, the Cas12a-crRNA complex is a dry powder.

[0055] In a preferred embodiment, the microfluidic chip further includes an insulating layer and a bottom layer, and the functional layer of the microfluidic chip is formed by bonding the insulating layer and the bottom layer together.

[0056] In a preferred embodiment, the microfluidic chip is made of a polymer material, including polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), and polycarbonate. Preferably, the microfluidic chip is made of polydimethylsiloxane.

[0057] In a preferred embodiment, the multi-channel microfluidic chip completes detection via an external power supply and a sample injection system, wherein the external power supply is a battery; the sample injection system includes an injection pump controller, an injection pump execution unit, a syringe, a pipette, etc., for injecting samples and controlling the injection rate.

[0058] The second aspect of this invention provides the use of the above-mentioned multichannel microfluidic chip in the preparation of various drug-resistant Mycobacterium tuberculosis detection products.

[0059] In a preferred embodiment, the product is used to detect the resistance of Mycobacterium tuberculosis to anti-tuberculosis drugs. Preferably, the anti-tuberculosis drugs include, but are not limited to, one or more of rifampin, isoniazid, streptomycin, ethambutol, and fluoroquinolones. More preferably, the product is used to detect drug resistance mutation genes in Mycobacterium tuberculosis, targeting specific drug resistance mutation genes in Mycobacterium tuberculosis, including, but not limited to, one or more of rpoB, katG, embB, gyrA, gyrB, rpsl, and rrs.

[0060] In a preferred embodiment, the Mycobacterium tuberculosis drug resistance mutant gene is selected from any one or more of rpoB, katG, and embB, and the nucleotide sequence of the crRNA used to detect the Mycobacterium tuberculosis drug resistance mutant gene is selected from any one or more of the sequences shown in SEQ ID NO:1~11.

[0061] Preferably, when the Mycobacterium tuberculosis drug resistance mutant gene is rpoB, the nucleotide sequence of the crRNA is selected from any one or more of SEQ ID NO:1 to SEQ ID NO:6;

[0062] Preferably, when the Mycobacterium tuberculosis drug resistance mutant gene is katG, the nucleotide sequence of the crRNA is selected from any one or more of SEQ ID NO:7~SEQ ID NO:8;

[0063] Preferably, when the Mycobacterium tuberculosis drug resistance mutant gene is embB, the nucleotide sequence of the crRNA is selected from any one or more of SEQ ID NO:9 to SEQ ID NO:11.

[0064] The product also includes crRNA for detecting wild-type Mycobacterium tuberculosis, wherein the crRNA for detecting wild-type Mycobacterium tuberculosis is selected from any one or more sequences shown in SEQ ID NO:12~21.

[0065] In a preferred embodiment, the product's crRNA for detecting wild-type Mycobacterium tuberculosis is co-incubated with the crRNA for detecting wild-type Mycobacterium tuberculosis in the reaction detection chamber of a multi-channel microfluidic chip.

[0066] This invention also provides a method for using the above-mentioned multi-channel microfluidic chip, comprising the following steps:

[0067] 1) The DNA to be tested is extracted in the DNA lysis chamber and lysed into single-stranded DNA, and then enters the reaction detection chamber through the sample mixing chamber;

[0068] 2) The Cas12a-crRNA complex was preloaded onto the surface of each detection electrode according to the detection target, mixed with single-stranded DNA, and subjected to a CRISPR reaction to cleave the signal amplification probe modified on the working electrode.

[0069] 3) The electrodes were inactivated by protein inactivation, and the electrodes were scanned using an electrochemical workstation. Differential pulse voltammetry (DPV) was used to detect drug resistance in Mycobacterium tuberculosis.

[0070] In one embodiment, in step 1), DNA extraction is performed using a magnetic bead method, specifically by incubating the sample with functionalized magnetic beads to immobilize the DNA in the sample. Preferably, the incubation time is 8-12 minutes, which can be 8-10 minutes, 10-12 minutes, but is more preferably 10 minutes.

[0071] The pyrolysis temperature is 90~100℃, which can be 90~95℃ or 95~100℃, preferably 95℃. The pyrolysis time is 10~20 minutes, which can be 10~15 minutes or 15~20 minutes, preferably 15 minutes.

[0072] Preferably, after DNA lysis, it needs to undergo thorough denaturation at a temperature of -2 to 2°C, which can be -2 to 0°C, 0 to 2°C, or preferably 0°C. The time for thorough denaturation is 4 to 6 minutes, which can be 4 to 5 minutes, 5 to 6 minutes, or preferably 5 minutes.

[0073] In one embodiment, in step 2), the temperature of the CRISPR reaction is 35~40°C, which can be 35~37°C or 37~40°C, preferably 37°C; the reaction time is 40~50 minutes, which can be 40~45 minutes or 45~50 minutes, preferably 45 minutes.

[0074] Preferably, the target recognition probe includes a Cas12a-crRNA complex, wherein the molar ratio of Cas12a to crRNA in the Cas12a-crRNA complex is (1~3):(1~4), preferably 2:3.

[0075] In a preferred embodiment, the multi-channel microfluidic chip can simultaneously detect multiple drug resistance genes of a drug-resistant Mycobacterium tuberculosis strain, including but not limited to rpoB, katG, embB, gyrA, gyrB, rpsl, and rrs.

[0076] More preferably, the nucleotide sequence of the crRNA is selected from any one or more of the sequences shown in SEQ ID NO:1~11.

[0077] Preferably, when the Mycobacterium tuberculosis drug resistance mutant gene is rpoB, the nucleotide sequence of the crRNA is selected from any one or more of SEQ ID NO:1 to SEQ ID NO:6;

[0078] Preferably, when the Mycobacterium tuberculosis drug resistance mutant gene is katG, the nucleotide sequence of the crRNA is selected from any one or more of SEQ ID NO:7~SEQ ID NO:8;

[0079] Preferably, when the Mycobacterium tuberculosis drug resistance mutant gene is embB, the nucleotide sequence of the crRNA is selected from any one or more of SEQ ID NO:9 to SEQ ID NO:11.

[0080] In one implementation, during step 3), the potential scan range in the differential pulse voltammetry (DPV) detection process is -0.4 to 0.1 V.

[0081] Preferably, the crRNA further includes crRNA for detecting wild-type Mycobacterium tuberculosis, wherein the crRNA for detecting wild-type Mycobacterium tuberculosis is selected from any one or more sequences shown in SEQ ID NO:12~21.

[0082] The crRNA for detecting drug-resistant mutant genes and the crRNA for detecting wild-type Mycobacterium tuberculosis are preloaded on the detection electrode. Dual detection is performed by detecting both the crRNA for drug-resistant mutant genes and the crRNA for wild-type Mycobacterium tuberculosis. The detection of crRNA for wild-type Mycobacterium tuberculosis confirms that the target is Mycobacterium tuberculosis, and the detection of crRNA for drug-resistant mutant genes confirms the corresponding drug-resistant mutant genes, thereby improving the accuracy of detection.

[0083] In some embodiments of the present invention, the signal amplification probe is a methylene blue-labeled DNA hairpin probe (MB-DNA); one end of the MB-DNA is labeled with MB, and the other end is fixed to the surface of the working electrode by an Au-S bond.

[0084] Preferably, the nucleotide sequence of the MB-DNA probe is shown in SEQ ID NO.22.

[0085] In this invention, crRNA and Cas12a protein assemble into a complex. Within the same system, different crRNAs form combined Cas12a-crRNA complexes with Cas12a protein. When the target is present, the Cas12a-crRNA complex specifically recognizes the target through a target recognition probe, forming a Cas12a-crRNA-Target triplet, activating trans-cleavage activity. The activated Cas12a system cleaves the signal amplification probe, subsequently releasing methylene blue. The methylene blue further promotes the methyl bromide redox reaction, enhancing signal transduction, thereby achieving highly sensitive and specific quantitative detection of drug-resistant Mycobacterium tuberculosis. Furthermore, by adding the combined Cas12a-crRNA complex to a CRISPR reaction chamber using a microfluidic chip, the detection of drug-resistant Mycobacterium tuberculosis can be achieved without relying on PAM sites.

[0086] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0087] Example 1 - Fabrication of Microfluidic Chips

[0088] like Figure 1As shown, the microfluidic chip includes a functional layer comprising a lysis zone, a sample mixing zone, and an electrode array zone. The lysis zone includes a sample inlet 11 and a DNA lysis chamber 12 connected sequentially. The sample mixing zone includes a sample mixing chamber 13. The DNA lysis chamber 12 and the sample mixing chamber 13 are connected via a first microchannel, which is equipped with a first control valve and a second control valve. The sample mixing chamber 13 contains multiple orderly arranged micropillars, with gaps between adjacent micropillars for liquid passage. The DNA lysis zone is equipped with a microheater, which is correspondingly positioned at the bottom of the DNA lysis chamber 12. The electrode array... The microfluidic chip comprises five reaction detection chambers 14 and corresponding detection electrodes. The reaction detection chambers 14 are connected to the sample mixing chamber 13 via a second microfluidic channel. The detection electrodes are located below the reaction detection chambers and in contact with their internal space. The detection electrodes are detachably connected to the microfluidic chip. Each detection electrode consists of polyimide and a three-electrode system formed on the polyimide surface. The three-electrode system includes a working electrode, a reference electrode, and a counter electrode. Both the working and counter electrodes are PI thin-film deposited gold electrodes. The reference electrode is an Ag / AgCl reference electrode. The working electrode is modified with an MB-DNA probe (SEQ ID NO. 22: 5'-SH-(CH2)6-AAAAAAAAAAAAAAAAAAAA-MB-3') and 6-mercapto-1-ethanol (MCH). The surface of the three-electrode system is pre-loaded with a Cas12a-crRNA complex. The microfluidic chip also includes an insulating layer and a bottom layer. The functional layer of the microfluidic chip is encapsulated with the insulating layer and the bottom layer through layer-by-layer bonding.

[0089] Fabrication of microfluidic chips:

[0090] a. Use CAD to draw the chip layer sample inlet, DNA lysis chamber, sample mixing chamber, reaction detection chamber, liquid outlet and microchannels, and export it as a GDSII file;

[0091] b. The photolithography master mold is made through steps such as spin coating, pre-baking, exposure, post-baking, development, and silanization;

[0092] c. Use volatile trimethylchlorosilane to hydrophobize the master mold for 5 min;

[0093] d. Mix Sylgard 184 prepolymer and curing agent evenly at a mass ratio of 10:1, place in a vacuum dryer to remove air bubbles, pour into a mold along a glass rod, remove air bubbles again using a vacuum dryer, and place in an 80℃ oven for 4 hours to complete curing and crosslinking.

[0094] e. Use a blade to cut a slit along the inner edge of the mold, carefully peel off the PDMS, and complete the demolding process;

[0095] f. Set the plasma generator parameters to 300 W power and 3 min time for surface plasma bonding;

[0096] g. Preparation of the three-electrode system: A gold layer is deposited on the polyimide film by metal deposition to form a three-electrode system, and silver / silver chloride conductive paste is printed in the reference electrode area.

[0097] h. Modification of the interface-sensitive membrane: MB-DNA probe and 6-mercapto-1-ethanol (MCH) were modified into the working electrode region of the three-electrode system through chemical bonds of thiol and gold, respectively. The specific steps are as follows: 1) Electrochemical probe modification: 45 μL of 5 μM MB-DNA probe (SEQ ID NO:22) was added to a centrifuge tube, the electrode was inserted into the centrifuge tube, inverted, and incubated overnight at 4℃; 2) Electrode sealing: 45 μL of 2 mM MCH solution was added to each centrifuge tube, the above electrode was transferred to this centrifuge tube, and incubated at 37℃ for 1 h.

[0098] i. Preparation of Cas12a-crRNA complex lyophilized powder: Take a PCR tube, add 2 μL of 1 μM Cas12a, 2 μL of 10× Buffer, and 11 μL of ultrapure water to each PCR tube, then add crRNA to the PCR tube, making the combined volume 18 μL. Freeze-dry the combined solution to obtain Cas12a-crRNA complex lyophilized powder. Place the processed lyophilized powder on the surface of a three-electrode system and, according to the nucleic acid target to be detected, place it into the reaction chamber corresponding to the functional layer.

[0099] j. The functional layer, insulating layer, and bottom layer are bonded together layer by layer, and the finished product is packaged.

[0100] Example 2: Design and Validation of crRNA

[0101] The gene information used in this embodiment is as follows:

[0102]

[0103] (1) Design of crRNA: Gene sequencing was performed on non-drug-resistant Mycobacterium tuberculosis (Mycobacterium tuberculosis H37Rv genome sequence in Genbank) and drug-resistant Mycobacterium tuberculosis (rpoB, katG, embB). The sequences of non-drug-resistant Mycobacterium tuberculosis and drug-resistant Mycobacterium tuberculosis (embB) were compared using Snapgene software. crRNAs were designed for mutation sites. Among them, the rifampicin resistance gene rpoB corresponds to 6 crRNAs, the isoniazid resistance gene katG corresponds to 2 crRNAs, and the ethambutol resistance gene embB corresponds to 3 crRNAs. The crRNA sequences and the corresponding mutation sites are shown in Table 1.

[0104] (2) The rationale for using the three crRNAs corresponding to the ethambutol resistance gene embB was verified by a trans-cleavage assay:

[0105] (2.1) Single crRNA verification system: Add 2 μL of 1 μM Cas12a, 3 μL of 1 μM crRNA, 2 μL of 10× Buffer, and 10 μL of ultrapure water to a PCR tube. After incubating at room temperature for 10 min, add 1 μL of 5 μM FQ reporter (5'-FAM-TTATT-BHQ-3') and 2 μL of plasmid obtained by constructing the nucleotide sequences of the three mutation sites of the ethambutol resistance gene embB.

[0106] (2.2) Dual crRNA verification system: Take 3 PCR tubes, add 2 μL of 1 μM Cas12a, 2 μL of 10× Buffer, and 10 μL of ultrapure water to each PCR tube, and then add 3 μL each of 1 μM crRNA-1, 1 μM crRNA-2, and 1 μM crRNA-3 to each of the 3 tubes. Mix well and let stand at room temperature for 10 min. Take 8.5 μL from each PCR tube and combine (crRNA-1 + crRNA-2, crRNA-1 + crRNA-3, crRNA-2 + crRNA-3), with a combined volume of 17 μL per tube. Add 1 μL of 5 μM FQ reporter (5'-FAM-TTATT-BHQ-3') and 2 μL of plasmid obtained by constructing the nucleotide sequences of the three mutation sites of the ethambutol resistance gene embB.

[0107] (2.3) Triple crRNA validation system: Take 3 PCR tubes, add 2 μL of 1 μM Cas12a, 2 μL of 10× Buffer, and 10 μL of ultrapure water to each PCR tube, and then add 3 μL each of 1 μM crRNA-1, 1 μM crRNA-2, and 1 μM crRNA-3 to each of the 3 tubes. Mix well and let stand at room temperature for 10 min. Take 5.66 μL from each PCR tube and combine (crRNA-1 + crRNA-2 + crRNA-3) to make a volume of 17 μL per tube. Add 1 μL of 5 μM FQreporter (5'-FAM-TTATT-BHQ-3') and 2 μL of plasmid obtained by constructing the nucleotide sequences of the three mutation sites of the ethambutol resistance gene embB.

[0108] Place the PCR tubes into a real-time quantitative PCR analyzer, set each cycle to 30 seconds, the number of cycles to 120, and the temperature to 37°C to read the fluorescence. Results are as follows: Figure 3 As shown, a comparison of detection signals validated by single, dual, and triple crRNA revealed that triple crRNA exhibited the fastest Ct value response, followed by dual crRNA, while single crRNA showed the slowest response. This indicates that the triple crRNA system has higher detection efficiency. The comparison diagram of embB drug resistance mutation sites against wild-type Mycobacterium tuberculosis sites is shown below. Figure 2 As shown.

[0109] (3) Verification of the rationality of crRNA by cis-cutting assay: The solution preparation method is consistent with the above (2) steps. In this step, FQ reporter is not added, and ultrapure water is used to make up to 20 μL of system. The above prepared solution is placed in a thermostat or water bath and reacted at 37℃ for 1 hour. 2 μL of RNase A protein (5 mg / mL) is added and the reaction continues for 1 hour. Then, the reaction is carried out at 85℃ for 10 min. The solution is removed and the temperature of the reaction solution is allowed to return to room temperature. The above reaction solution is subjected to 15% PAGE gel running at 110V for 100 min with 1×TAE buffer. Using a 25-bp marker as a reference, the staining method (dye is added later) is used to stain for 10 min to verify that the triple crRNA system has higher efficiency.

[0110] Table 1 crRNA Sequence

[0111]

[0112] Example 3 - A method for rapid detection of multiple drug-resistant Mycobacterium tuberculosis using a microfluidic chip detection platform

[0113] The detection principle of drug-resistant Mycobacterium tuberculosis is as follows: Figure 4 As shown, the specific method is as follows:

[0114] (I) Liquefaction of sputum samples: Using a syringe pump to control the flow rate, open the injection valve and the first control valve, insert the syringe pump connector into the injection port of the microfluidic chip, close the second control valve, control the flow rate at 10 μL / s, introduce the sputum sample, add an equal volume of 4% sodium hydroxide to the DNA lysis zone, let stand at room temperature for 30 minutes, vortex once every 10 minutes, for a total of 3 vortexes. Use a commercially available genomic magnetic bead extraction kit (Shanghai Sangon Biotech Co., Ltd., B518770) to extract Mycobacterium tuberculosis genome, fix the nucleic acid, and pass PBS (pH 7.4, 0.01M) into the DNA lysis zone for washing. Expel the washing solution through the DNA lysis zone control valve, repeat 3 times.

[0115] (ii) Denaturation of the Mycobacterium tuberculosis genome. This denaturation process is carried out in the DNA lysis chamber of the chip. The specific procedure is as follows: the extracted genome is heated at 95°C for 15 min using a micro heater, and then immediately transferred to 0°C and held for 5 min to achieve complete denaturation.

[0116] (III) Preparation of CRISPR-Cas12a reaction solution lyophilized powder

[0117] Take 11 PCR tubes. Add 2 μL of 1 μM Cas12a, 2 μL of 10× Buffer, and 11 μL of ultrapure water to each tube. Then, add 3 μL each of 1 μM crRNA-1, 1 μM crRNA-2, 1 μM crRNA-3, 1 μM crRNA-4, 1 μM crRNA-5, 1 μM crRNA-6, 1 μM crRNA-7, 1 μM crRNA-8, 1 μM crRNA-9, 1 μM crRNA-10, and 1 μM crRNA-11 to each of the 11 tubes. Mix well and incubate at room temperature for 10 min. Dispense the solution from each PCR tube... Equal volumes of solution were taken from each tube and combined (crRNA-1+crRNA-2+crRNA-3+crRNA-4+crRNA-5+crRNA-6, crRNA-7+crRNA-8, crRNA-9+crRNA-10+crRNA-11). crRNA-1+crRNA-2+crRNA-3+crRNA-4+crRNA-5+crRNA-6 detects the rifampicin resistance gene rpoB, crRNA-7+crRNA-8 detects the isoniazid resistance gene katG, and crRNA-9+crRNA-10+crRNA-11 detects the ethambutol resistance gene embB. Each tube contained 18 μL of the combined solution. The combined solution was then freeze-dried at -80°C overnight. After pre-cooling in a freeze dryer for half an hour until the cold trap temperature reached -50°C, the solution was placed back into the freeze dryer. The lyophilized powder was then placed into the corresponding reaction chamber of the chip according to the detected nucleic acid target.

[0118] Take 10 PCR tubes, add 2 μL of 1 μM Cas12a, 2 μL of 10× Buffer, and 11 μL of ultrapure water to each tube. Then, add 3 μL of wild-type corresponding crRNA (SEQ ID NO:12~SEQ ID NO:21) to each of the 10 tubes, mix well, and incubate at room temperature for 10 min. Take liquid from each PCR tube and combine them, resulting in a combined volume of 18 μL per tube. Freeze-dry the combined solution by placing it at -80℃ overnight. After pre-cooling in a freeze dryer for half an hour, and then placing it in the cold trap at -50℃, the solution is ready. Place the processed lyophilized powder into the reaction chambers of the chip. Introduce the wild-type corresponding crRNA for dual detection to confirm that the target is Mycobacterium tuberculosis, and then confirm the corresponding drug resistance mutation gene, thereby improving the detection accuracy.

[0119] SEQ ID NO: 12: 5'-UAAUUUCUACUAAGUGUAGAUGCGCUAGCGUUCUACGAGGCUGU-3';

[0120] SEQ ID NO: 13: 5'--UAAUUUCUACUAAGUGUAGAUCGGUGGAUGACGCCGACGCCACG-3';

[0121] SEQ ID NO: 14: 5'-UAAUUUCUACUAAGUGUAGAUCCGCCGUCCCCACCUCGCCCGCC-3';

[0122] SEQ ID NO: 15: 5'-UAAUUUCUACUAAGUGUAGAUCGCCAGUCCCGCCAGUGCCAGCG-3';

[0123] SEQ ID NO: 16: 5'-UAAUUUCUACUAAGUGUAGAUCGCCGGUACCGCCAACUCCGCCG-3';

[0124] SEQ ID NO: 17: 5'-UAAUUUCUACUAAGUGUAGAUCUGCCGUUGCCGCCGGCGCCGCC-3';

[0125] SEQ ID NO: 18: 5'-UAAUUUCUACUAAGUGUAGAUGCCGAUCUGGAACACCUAUAUCG-3';

[0126] SEQ ID NO: 19: 5'-UAAUUUCUACUAAGUGUAGAUGUGUGUUGAAGCCCGCACCGCAG-3';

[0127] SEQ ID NO:20: 5'-UAAUUUCUACUAAGUGUAGAUGCCGGCGACGCUCCCACCUUGCC-3';

[0128] SEQ ID NO: 21: 5'-UAAUUUCUACUAAGUGUAGAUUGCCGUUAGCGCCGUUGCCGCCG-3'.

[0129] (iv) Electrochemical signal detection, the specific procedure is as follows:

[0130] (1) Electrode incubation: The above CRISPR-Cas12a complex lyophilized powders were pre-loaded onto the surface of each detection electrode according to the drug resistance mutation gene. Buffer was introduced through the second control valve, and the lysed sample was transferred to the detection reaction chamber. The mixture was thoroughly vortexed and mixed evenly, and then directly contacted with the surface of the three-electrode system. The microfluidic chip was placed on a heated plate at 37°C and subjected to a CRISPR reaction for 45 minutes. The combined Cas12a-crRNA complex formed by Cas12a and various crRNAs underwent multiple cleavage reactions.

[0131] (2) Protein inactivation: Add 45 μL of 1 mg / mL proteinase K solution to each reaction chamber and incubate at 37°C for 30 min;

[0132] (3) Sample detection: Using 1×PBS buffer as the base solution, the detection electrode was scanned using an electrochemical workstation, with the potential set from -0.4 V to 0.1 V. The sample was analyzed using the standard curve method to obtain the linear relationship curve between the peak current value and the logarithm of the concentration. Different concentrations of target nucleic acids were set for the three drug-resistant mutant genes katG, embB, and rpoB. For the katG gene, the concentrations included 0, 3×10 -14 Moles per liter, 3 × 10 -12 Moles per liter, 3 × 10 -10 Moles per liter; for the embB gene, including 0, 2.5 × 10 -15 Moles per liter, 2.5 × 10 -13 Moles per liter, 2.5 × 10 -11 Moles per liter; for the rpoB gene, including 0, 2.5 × 10⁻⁶. -17 Moles per liter, 2.5 × 10 -15 Moles per liter, 2.5 × 10 -13 Moles per liter.

[0133] The results are as follows Figures 5-8 As shown, the current signal at the interface for the detection of katG, embB, rpoB, and wild-type Mycobacterium tuberculosis decreases with increasing target concentration. Figures 5-6 ), and also has a certain relationship with the logarithm of concentration, yielding a linear equation ( Figure 7 The detection limits for the three tuberculosis drug resistance genes, katG, embB, and rpoB, reached 3 × 10⁻⁶. -14 2.5×10 -15 2.5×10 -17 moles per liter ( Figure 8 ).like Figure 9 As shown, by detecting the specific interference samples of wild-type and other drug-resistant genes, the specific anti-interference ability of the three Mycobacterium tuberculosis drug resistance genes of this method is significant, and all three Mycobacterium tuberculosis drug resistance genes can be specifically identified.

[0134] In summary, the above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A multi-channel microfluidic chip for amplification-free detection, characterized in that, It includes a microfluidic chip functional layer, which includes a pyrolysis region, a sample mixing region, and an electrode array region; The lysis zone includes a sample inlet and a DNA lysis chamber connected in sequence, and the sample mixing zone includes a sample mixing chamber. The DNA lysis chamber and the sample mixing chamber are connected through a first microchannel. The electrode array region includes several reaction detection chambers and detection electrodes. The detection electrodes correspond one-to-one with the reaction detection chambers and are in contact with the internal space of the reaction detection chambers. The electrodes are modified with signal amplification probes, and the surface of the electrodes is preloaded with Cas12a-crRNA complexes. The reaction detection chambers and the sample mixing chambers are connected through a second microfluidic channel.

2. The multi-channel microfluidic chip as described in claim 1, characterized in that, The number of reaction detection chambers is 4 to 6, and each reaction detection chamber is equipped with a corresponding detection electrode. Each detection electrode is pre-loaded with a Cas12a-crRNA complex for detecting different targets; and / or, the microfluidic chip also includes a liquid outlet, which is connected to the reaction detection chamber; and / or, the DNA lysis region is also equipped with a micro heater, which is correspondingly located at the bottom of the DNA lysis chamber; and / or, the sample mixing chamber includes multiple microcolumns arranged in an orderly manner.

3. The multi-channel microfluidic chip as described in claim 1, characterized in that, The electrode includes a substrate and a three-electrode system formed on the surface of the substrate.

4. The multi-channel microfluidic chip as described in claim 3, characterized in that, The three-electrode system includes a working electrode, a reference electrode, and a counter electrode. Preferably, the working electrode and the counter electrode are both PI thin film deposited gold electrodes, and the reference electrode is an Ag / AgCl reference electrode; and / or, the material of the substrate includes at least one of polyimide, polyethylene terephthalate, and polydimethylsiloxane.

5. The multi-channel microfluidic chip as described in claim 4, characterized in that, The signal amplification probe is a methylene blue-labeled DNA hairpin probe, one end of which is labeled with MB and the other end is fixed to the surface of the working electrode by an Au-S bond. Preferably, the nucleotide sequence of the signal amplification probe is as shown in SEQ ID NO.22; and / or, in the Cas12a-crRNA complex, the molar ratio of Cas12a to crRNA is (1~3):(1~4).

6. The use of the multichannel microfluidic chip as described in any one of claims 1-5 in the preparation of various drug-resistant Mycobacterium tuberculosis detection products.

7. The use as described in claim 6, characterized in that, The product is used to detect the resistance of Mycobacterium tuberculosis to anti-tuberculosis drugs, wherein the anti-tuberculosis drugs are selected from one or more of rifampin, isoniazid, streptomycin, ethambutol, and fluoroquinolones; and / or, the product is used to detect drug resistance mutation genes in Mycobacterium tuberculosis, wherein the drug resistance mutation sites in Mycobacterium tuberculosis are selected from one or more of rpoB, katG, embB, rpsl, and gyrA.

8. The use as described in claim 7, characterized in that, The Mycobacterium tuberculosis drug-resistant mutant gene is selected from any one or more of rpoB, katG, and embB, and the nucleotide sequence of the crRNA is selected from any one or more of the sequences shown in SEQ ID NO:1 to 11; preferably, when the Mycobacterium tuberculosis drug-resistant mutant gene is rpoB, the nucleotide sequence of the crRNA is selected from any one or more of SEQ ID NO:1 to SEQ ID NO:6; when the Mycobacterium tuberculosis drug-resistant mutant gene is katG, the nucleotide sequence of the crRNA is selected from any one or more of SEQ ID NO:7 to SEQ ID NO:8; when the Mycobacterium tuberculosis drug-resistant mutant gene is embB, the nucleotide sequence of the crRNA is selected from any one or more of SEQ ID NO:9 to SEQ ID NO:

11.

9. A method of using the multi-channel microfluidic chip as described in any one of claims 1-5, comprising the following steps: 1) The DNA to be tested is extracted in the DNA lysis chamber and lysed into single-stranded DNA, and then enters the reaction detection chamber through the sample mixing chamber; 2) The Cas12a-crRNA complex was preloaded onto the surface of each electrode according to the detection target, mixed with single-stranded DNA, and subjected to a CRISPR reaction to cleave the signal amplification probe modified on the working electrode. 3) The electrodes were inactivated by protein inactivation, and the electrodes were scanned using an electrochemical workstation. Differential pulse voltammetry (DPV) was used to detect drug resistance in Mycobacterium tuberculosis.

10. The method of use as described in claim 9, characterized in that, It also includes one or more of the following features: a) In step 1), the DNA extraction is performed using the magnetic bead method; b) In step 1), the pyrolysis temperature is 90~100℃ and the time is 10~20 minutes; c) In step 2), the CRISPR reaction temperature is 35~40℃ and the time is 40~50 minutes; d) In step 3), during the differential pulse voltammetry (DPV) detection process, the potential scanning range is -0.4~0.1V.

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