CRISPR (clustered regularly interspaced short palindromic repeats)-based DNA / RNA (deoxyribonucleic acid / ribonucleic acid) synchronous detection method and platform and application of CRISPR-based

Abstract

The invention belongs to the field of analysis and detection, and provides a CRISPR (clustered regularly interspaced short palindromic repeats)-based DNA / RNA (deoxyribonucleic acid / ribonucleic acid) synchronous detection method and a CRISPR-based DNA / RNA synchronous detection platform and application of the CRISPR-based DNA / RNA synchronous detection platform in respiratory tract pathogen multiplex detection.The CRISPR-based DNA / RNA synchronous detection method comprises the steps that corresponding crRNA is designed according to nucleic acid to be detected, the nucleic acid to be detected is mixed and incubated with a Cas12a-crRNA compound or a Cas13a-crRNA compound and then mixed and incubated with MB (at) LP-linker-FB according to the claim 6, and after incubation is finished, DNA / RNA synchronous detection is carried out. And performing magnetic separation, collecting supernate, adding Triton X-100 for cracking MB-coated LP, and detecting peak current generated by MB by adopting DPV, thereby obtaining the MB-coated LP. According to the invention, a general nucleic acid detection strategy with a synergistic effect of a CRISPR / Cas system and a liposome nano container is constructed, and then a parallel loading system of Cas12a and Cas13a is established based on a multi-channel centrifugal microfluidic detection platform, so that multi-linked detection of pathogens with genetic materials, namely DNA and RNA, in a single sample is realized. According to the designed MB-coated LP-linker-FB composite signal carrier, on one hand, the MB packaging efficiency and the release control force are improved, and on the other hand, a highly specific detection mechanism is established through enzyme digestion response.

Description

Technical Field

[0001] The present invention belongs to the field of analysis and detection, and relates to a CRISPR-based DNA / RNA synchronous detection strategy and platform and its application in multiplex detection of respiratory pathogens. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Respiratory infections are a major threat to global public health. From an etiological perspective, respiratory pathogens encompass three broad categories: viruses, bacteria, and atypical pathogens (such as Mycoplasma pneumonia (MP) and Chlamydia). These pathogens exhibit significant diversity in their genetic material types. For example, adenoviruses (ADV) use double-stranded DNA (dsDNA) as their genetic information carrier, while influenza A (H1N1) and respiratory syncytial virus (RSV) rely on single-stranded RNA (ssRNA) for replication and pathogenicity. This diversity in nucleic acid types directly results in a single detection system being unable to comprehensively cover all potential pathogens during mixed infections or cross-transmission, leading to the risk of missed detection. Furthermore, traditional empirical treatment relies on a trial-and-error strategy of using broad-spectrum antibiotics or antiviral drugs, exacerbating the problem of antibiotic overuse and increasing treatment failure rates due to the rise in pathogen resistance. Therefore, the technology of simultaneous detection of DNA and RNA pathogens is a necessary condition for solving the current difficulties in the diagnosis and treatment of respiratory infections and realizing precision medicine.

[0004] The CRISPR / Cas system has the ability to target different nucleic acid types, allowing the same detection steps to be used after target identification, providing a methodological basis for the simultaneous detection of multiple pathogens.

[0005] However, existing platforms mostly rely on fluorescence and colorimetric detection, and face problems such as insufficient sensitivity and strong background interference. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides a multi-channel centrifugal microfluidic detection platform based on Cas12a / Cas13a-crRNA and MB@LP (liposomes encapsulating MB) signal amplification, which can realize the synchronous detection of ADV, MP, RSV and H1N1 under the condition of no nucleic acid amplification. The platform designs eight Cas enzyme parallel reaction chambers, which avoid Cas12a / Cas13a cross interference by spatial separation and centrifugal force timing control; electrochemical sensing technology is adopted, and MB@LP-DNA / RNA-magnetic beads (MB@LP-linker-FB) are used as signal amplification medium. When the target pathogen nucleic acid is present in the sample, the Cas12a / Cas13a-crRNA complex specifically recognizes the target and activates the trans-cleavage activity, accurately cutting off the DNA / RNA linker connecting FB and MB@LP, and MB@LP is freed from FB bondage; after magnetic separation, Triton X-100 is added to crack MB@LP, releasing the high-concentration MB signal molecules encapsulated therein, and detecting the response current of MB on SPCE by DPV, thereby realizing quantitative analysis of the target.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A first aspect of the present invention provides a method for preparing MB@LP-linker-FB, comprising:

[0009] The MB@LP solution and linker were mixed evenly, incubated, and dialyzed to obtain MB@LP-linker;

[0010] The MB@LP-linker was mixed with the streptavidin-modified FB after multiple washings, incubated, the magnetic beads were collected, and washed multiple times to obtain MB@LP-linker-FB.

[0011] The second aspect of the present invention provides MB@LP-linker-FB prepared by the above method.

[0012] The third aspect of the present invention provides a CRISPR-based DNA / RNA simultaneous detection method, comprising:

[0013] Design the corresponding crRNA according to the nucleic acid to be tested, mix the test nucleic acid with the Cas12a-crRNA complex or the Cas13a-crRNA complex, and then mix and incubate it with the MB@LP-linker-FB described in claim 6. After the incubation, collect the supernatant after magnetic separation, add Triton X-100 to lyse MB@LP, and use DPV to detect the peak current generated by MB to obtain.

[0014] The fourth aspect of the present invention provides a platform for simultaneous detection of CRISPR DNA / RNA based on the above method, using a microfluidic channel as the detection platform, a carbon electrode as the working electrode, an Ag / AgCl reference electrode, and a carbon electrode as the counter electrode.

[0015] The fifth aspect of the present invention provides the application of the above-mentioned MB@LP-linker-FB and the above-mentioned platform in the multi-link detection of respiratory pathogens.

[0016] Beneficial effects of the present invention

[0017] (1) The present invention constructs a universal nucleic acid detection strategy that synergizes the CRISPR / Cas system with liposome nanocontainers, and then establishes a parallel loading system for Cas12a and Cas13a based on a multi-channel centrifugal microfluidic detection platform, realizing the multiplex detection of pathogens whose genetic materials are DNA and RNA respectively in a single sample. The designed MB@LP-linker-FB composite signal carrier improves the MB encapsulation efficiency and release control on the one hand, and establishes a highly specific detection mechanism through enzyme cleavage response on the other hand. Based on the two-stage centrifugal control strategy designed by capillary valve effect and graded fluid resistance, combined with the vertical integration structure of radial microfluidic chamber and array electrode, the semi-automatic operation of "centrifugal driven sample distribution → CRISPR target identification → magnetic separation enrichment signal → electrochemical quantitative detection" is realized. After optimizing the preparation of MB@LP-linker-FB, the reaction time of Cas enzyme and substrate, and the rupture time of MB@LP, the detection platform was used to establish the standard curve of characteristic nucleic acids of common respiratory pathogens such as adenovirus, Mycoplasma pneumoniae, respiratory syncytial virus and influenza A (H1N1) virus, with a detection limit of aM level. High selectivity was maintained in a complex matrix containing multiple non-target proteins, and the actual sample test results were consistent with clinical test results. This work provides an integrated solution with high sensitivity and multiplex detection capabilities for rapid on-site diagnosis, while also establishing an efficient technical platform for the identification of mixed infections and accurate diagnosis in resource-limited settings.

[0018] (2) The method of the present invention is simple, practical and easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0020] Figure 1 Schematic diagram of respiratory pathogen detection strategy based on the synergistic effect of Cas12a / Cas13a and MB@LP-linker-FB;

[0021] Figure 2 Schematic diagram of the microfluidic detection platform structure (A) and actual assembly diagram (B), bottom diagram of the electrode base plate (C);

[0022] Figure 3 The speed experiment for sample diversion in the first stage (A), the effect diagram after diversion (B), and the volume of solution diverted to each reaction pool (C); the speed experiment for centrifuging the reaction solution into the detection pool in the second stage (D), the effect diagram after centrifugation (E), and the volume of solution in each detection pool (F);

[0023] Figure 4 Signal responses of MB@LP synthesized by different methods. A: Thin film hydration method (MB solution); B: Thin film hydration method (MB solution containing 0.2 M ammonium sulfate); C: Ammonium sulfate gradient method;

[0024] Figure 5 Signal responses of MB@LP after storage at 4°C for 0, 7, 14, 21, and 28 days;

[0025] Figure 6 TEM images of LP (A), MB@LP (B), MB@LP-linker1 (C), and MB@LP-linker1-FB (D);

[0026] Figure 7 hydration kinetic diameters of LP, MB@LP, MB@LP-linker1, and MB@LP-linker2;

[0027] Figure 8 Zeta potential of LP, MB@LP, MB@LP-linker1, and MB@LP-linker2;

[0028] Figure 9 UV-vis absorption curves of LP, MB solution, MB@LP, MB@LP-linker1, MB@LP-linker2, Triton X-100-cleaved MB@LP-linker1 and MB@LP-linker2, and Triton X-100;

[0029] Figure 10 UV-vis absorption curves of the supernatant of the final wash of MB@LP-linker1-FB and MB@LP-linker2-FB and the MB@LP-linker1-FB and MB@LP-linker2-FB complexes after adding Triton X-100;

[0030] Figure 11(A) Current response of MB@LP-linker-FB prepared with different linker concentrations, (B) FB with different volumes

[0031] Current response of MB@LP-linker-FB prepared under 5 h and overnight incubation;

[0032] Figure 12 (A)Cas12a-crRNA(ADV),(B)ADV,(C)Cas12a-crRNA(ADV)+ADV,(D)

[0033] Cas12a-crRNA(ADV)+ADV+linker1,(E)Cas12a-crRNA(ADV)+linker1,(F)linker1,and(G)Cas12a-crRNA(MP)+linker1,(H)Cas12a-crRNA(MP)+MP+linker1,(I)Cas12a-crRNA(MP)+

[0034] Electropherograms of MP, (J) MP, (K) Cas12a-crRNA (MP), and (M) DNA marker (20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 300, 400, 500 bp);

[0035] Figure 13 (A)Cas13a-crRNA(H1N1),(B)H1N1,(C)Cas13a-crRNA(H1N1)+H1N1,(D)

[0036] Electrophoresis images of Cas13a-crRNA(H1N1)+H1N1+linker2, (E)Cas13a-crRNA(H1N1)+linker2, (F)linker2, and (G)Cas13a-crRNA(RSV)+linker2, (H)Cas13a-crRNA(RSV)+RSV+linker2, (I)Cas13a-crRNA(RSV)+RSV, (J)RSV, (K)Cas13a-crRNA(RSV), and (M) DNA marker (20, 40, 60, 80, 100, 130, 140, 160, 180, 200, 300, 400, 500 bp);

[0037] Figure 14 DPV responses of the sensing strategy to different concentrations of ADV (A) and RSV (B);

[0038] Figure 15 Current response values ​​of the MB@LP-linker1-FB complex before and after the addition of Triton X-100 after incubation in PBS buffer, 1×NEBuffer 2.1, and Cas12a solution for 1 h;

[0039] Figure 16 The effect of the reaction time of Cas12a-crRNA recognizing the target and trans-cleaving the linker on the current signal;

[0040] Figure 17 The time it takes for Triton X-100 to rupture MB@LP;

[0041] Figure 18 DPV responses of the assay to different concentrations of ADV (A), MP (C), RSV (E), and H1N1 (G) (0, 1aM, 10aM, 1fM, 10fM, 100fM, 1pM, 10pM, 100pM, and 1nM); Linear relationships between DPV peak current and the logarithm of ADV (B), MP (D), RSV (F), and H1N1 (H) concentrations;

[0042] Figure 19 Results of five tests of 1 pM ADV (A) and RSV (B) under the same conditions;

[0043] Figure 20 The signals generated by this protocol when detecting 1 pM ADV, MP, RSV, and H1N1 single samples were compared with the signals generated when detecting a mixed sample (1 pM ADV, MP, RSV, H1N1, 1 ng / mL lysozyme and salivary α-amylase);

[0044] Figure 21 Schematic diagram of the detection platform in actual detection (solid color fill represents the experimental group, square fill represents the blank control group);

[0045] Figure 22 The detection scheme responds to DPV in normal human oral swab samples spiked with different concentrations of ADV (A), MP (C), RSV (E), and H1N1 (G) (0, 1aM, 10aM, 1fM, 10fM, 100fM, 1pM, 10pM, 100pM, and 1nM); the linear relationship between DPV peak current and the logarithm of ADV (B), MP (D), RSV (F), and H1N1 (H) concentrations;

[0046] Figure 23 Test results of six actual samples (bar graph: this protocol, left coordinate; circle: PCR, right coordinate). DETAILED DESCRIPTION

[0047] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0048] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. The reagents or raw materials used in the present invention can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in the present invention are used in accordance with conventional methods in the art or according to product specifications. Similarly, unless otherwise specified, the test methods of the present invention are also tested in accordance with conventional methods in the art or the common methods or standards in the industry. In addition, any methods and materials similar to or equivalent to the described contents can be applied to the inventive method. The preferred embodiments and materials described herein are for demonstration purposes only.

[0049] The present invention also provides a method for preparing MB@LP-linker-FB, comprising:

[0050] The MB@LP solution and linker were mixed evenly, incubated, and dialyzed to obtain MB@LP-linker;

[0051] The MB@LP-linker was mixed with the streptavidin-modified FB after multiple washings, incubated, the magnetic beads were collected, and washed multiple times to obtain MB@LP-linker-FB.

[0052] In some embodiments, the preparation method of MB@LP is an ammonium sulfate gradient method or a thin film hydration method.

[0053] In some embodiments, the linker is 5'-Biotin-T (90) -(CH2)6NH2-3', or, 5'-Biotin-T (20) UUUUUT (18) UUUUUT (18) UUUUUT (20) -(CH2)6NH2-3'.

[0054] In some embodiments, the linker concentration is 50-500 nM.

[0055] In some embodiments, the volume ratio of the MB@LP-linker to the FB suspension is 100 μL:15-50 μL;

[0056] In some embodiments, the concentration of the FB suspension is 10-15 mg / mL.

[0057] The present invention provides a CRISPR-based DNA / RNA simultaneous detection method, characterized by comprising:

[0058] Design the corresponding crRNA according to the nucleic acid to be tested, mix the test nucleic acid with the Cas12a-crRNA complex or the Cas13a-crRNA complex, and then mix and incubate it with the MB@LP-linker-FB described in claim 6. After the incubation, collect the supernatant after magnetic separation, add Triton X-100 to lyse MB@LP, and use DPV to detect the peak current generated by MB to obtain.

[0059] In some embodiments, the target is selected from at least one of ADV, MP, MP, RSV, RSV, H1N1, and H1N1.

[0060] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0061] The main reagents and materials used in the present invention are as follows: sodium chloride, Sinopharm Chemical Reagent Co., Ltd.; potassium chloride, Sinopharm Chemical Reagent Co., Ltd.; potassium dihydrogen phosphate, Sinopharm Chemical Reagent Co., Ltd.; disodium hydrogen phosphate dodecahydrate, Sinopharm Chemical Reagent Co., Ltd.; methylene blue (MB), Sinopharm Chemical Reagent Co., Ltd.; chloroform, Yantai Far East Fine Chemical Co., Ltd.; DEPC water, Sangon Biotech (Shanghai) Co., Ltd.; 6× glycerol gel loading buffer, Sangon Biotech (Shanghai) Co., Ltd.; tetramethylethylenediamine, Sangon Biotech (Shanghai) Co., Ltd.; tris(hydroxymethyl)aminomethane, Sangon Biotech (Shanghai) Co., Ltd.; lysozyme, Sangon Biotech (Shanghai) Co., Ltd.; salivary α-amylase, Sangon Biotech (Shanghai) Co., Ltd.; ammonium sulfate, MacLean Biochemical Technology Co., Ltd.; cholesterol, MacLean Biochemical Technology Co., Ltd.; ammonium persulfate, Aladdin (Shanghai) Biochemical Technology Co., Ltd.; DSPE-PEG-NHS, Aladdin (Shanghai) Biochemical Technology Co., Ltd. DPPC, Aladdin (Shanghai) Biochemical Technology Co., Ltd. MD44-30000 dialysis bag, Hunan Yibo Biotechnology Co., Ltd. Streptavidin magnetic beads (FB), Shanghai Beyotime Biotechnology Co., Ltd. TBS buffer, Shanghai Beyotime Biotechnology Co., Ltd. 30% acrylamide solution, Shanghai Beyotime Biotechnology Co., Ltd. 20 bp DNA ladder marker, Takara Biotechnology Co., Ltd. SPCE, Botan Technology (Weihai) Co., Ltd. Cas12a, New England Biolabs Co., Ltd. Cas13a, Shanghai Tolo Biotechnology Co., Ltd.

[0062] Table 1 Oligonucleotide sequences used in the present invention

[0063]

[0064]

[0065] Note: Oligonucleotide sequences (HPLC purified) were purchased from Sangon Biotech (Shanghai) Co., Ltd. In the names of ADV and MP listed in Table 1, TS represents the target strand (the strand that binds to crRNA), and NTS represents the complementary strand of the target strand. The bases marked with "underline" are the complementary parts of crRNA and target, and the bases marked with "bold" are the PAM sequences. (T) x represents x T bases.

[0066] Example 1 Preparation of MB@LP

[0067] In order to obtain MB@LP with high encapsulation efficiency, the following three preparation methods were compared and analyzed.

[0068] Ammonium sulfate gradient method: DPPC (7.3 mg), cholesterol (3.9 mg), and DSPE-PEG-NHS (MW: 5000 Da, 9.8 mg) were dissolved in 4 mL of chloroform in a 50 mL round-bottom flask and sonicated for 10 minutes. The mixture was then evaporated under reduced pressure at 45°C until a thin lipid film formed on the inner wall of the round-bottom flask. Subsequently, 3 mL of 0.2 M ammonium sulfate solution was added for 1.5 hours of hydration. After hydration, the liposomes were sonicated for 30 minutes to adjust the size of the liposomes. The liposomes were then extruded through 0.45 μm and 0.22 μm polycarbonate filters three times each to obtain a liposome solution with uniform particle size. The liposome solution was then dialyzed against 0.9 wt% NaCl overnight using an MD44-30000 dialysis tubing to remove any ammonium sulfate present outside the liposomes. The resulting liposome solution was mixed with 1 mL of a 2 mg / mL MB solution and incubated at 65°C for 2 h. MB was molecularly encapsulated within the liposome's aqueous phase across the phospholipid membrane, yielding MB@LP. Finally, the MB@LP was dialyzed against PBS buffer (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH = 7.4) for 24 h to remove excess MB. The dialyzed MB@LP solution was stored at 4°C for further use.

[0069] Thin film hydration method: When hydrating the lipid film on the inner wall of a round-bottom flask, 2 mL of PBS buffer containing 1 mg / mL MB was added for hydration. MB@LP was extruded through polycarbonate filters (0.45 μm and 0.22 μm) and then dialyzed against PBS buffer for 24 h to obtain MB@LP.

[0070] Thin film hydration method (ammonium sulfate): Hydrate with 2 mL of 0.2 M ammonium sulfate solution containing 1 mg / mL MB. Subsequent procedures are the same as for the thin film hydration method.

[0071] Example 2 Preparation of MB@LP-linker-FB

[0072] 100 nM linker was added to 1 mL of MB@LP solution and incubated at 4°C for 8 hours. The solution was then dialyzed overnight against PBS buffer to obtain MB@LP-linker. 30 μL of 10 mg / mL streptavidin-modified FB was transferred to a 1.5 mL centrifuge tube and separated on a magnetic rack for 1 minute. The supernatant was removed. The FB was then resuspended in 0.5 mL of 1× TBS buffer. The supernatant was removed from the tube on a magnetic rack, completing one wash step. This wash step was repeated twice. After removing the supernatant, the tube was washed three times with PBST (0.05% Tween-20) and three times with PBS buffer. Since the purchased FB and its solution were not RNase-free, the FB was washed twice with 0.5 mL of 0.05 M NaCl solution treated with DEPC water for 2 minutes each, followed by a single wash with 0.5 mL of 0.1 M NaCl treated with DEPC water. After washing, add 100 μL of MB@LP-linker and incubate overnight at 4°C. Separate the tube on a magnetic rack for 1 minute and remove the supernatant. Add 0.5 mL of PBS buffer to the separated magnetic beads and vortex thoroughly to resuspend the beads. Separate the tube on a magnetic rack for 1 minute and remove the supernatant. Repeat the wash cycle two more times to obtain the MB@LP-linker-FB complex.

[0073] Example 3 Fabrication of a microfluidic detection platform

[0074] The microfluidic platform was prepared by molding. In short, the mold pattern was designed using SolidWorks software, and then an acrylic mold was made using a CNC engraving machine. PDMS glue was injected into the mold and demolded after curing. A cutter and puncher were used to prepare a chip of the required size, and then the layers were bonded according to the design of the detection platform. In this work, a screen printed carbon electrode (SPCE) was used. The working electrode (carbon) had a diameter of 5 mm, the reference electrode (Ag / AgCl) and the counter electrode (carbon) were both 1 mm wide, and the overall working area was 10 mm.

[0075] Example 4 Actual sample pretreatment and detection analysis

[0076] The detection process specifically includes: (1) Sample loading stage: Take a certain concentration of the test solution into the sample pool, centrifuge at 600rpm for 45s, and disperse the solution into the corresponding reaction pool through the microchannel; (2) CRISPR reaction stage: Add MB@LP-linker-FB (FB: 0.3mg) and Cas12a / Cas13a-crRNA corresponding to different targets (Cas: 75nM, crRNA: 100nM) to the reaction pool. As the reaction proceeds, the activated trans-cleavage activity of Cas12a / Cas13a-crRNA cuts the linker chain connecting MB@LP and FB, allowing MB@LP to break free from the FB. (3) Signal separation stage: Fix FB with the bottom plate magnet and centrifuge at 800rpm for 90s to drive the free MB@LP into the detection pool; (4) Electrochemical detection stage: Add 10μL of 1% Triton X-100 to the detection pool to cleave MB@LP. Differential pulse voltammetry (DPV) scans were performed in the potential range of -0.5 to 0 V to read the current response values ​​of MB.

[0077] Experimental Example 1 Detection Principle

[0078] The present invention designs a cascade amplification detection strategy based on CRISPR-Cas12a / Cas13a and MB@LP ( Figure 1), for high-sensitivity electrochemical detection of respiratory pathogens. The core design of the detection strategy is as follows: LP coated with MB is prepared by the ammonium sulfate gradient method. The directional coupling of MB@LP and FB is achieved by a bifunctionally modified linker: the affinity reaction between the 5'-end biotin of the linker and the streptavidin on the FB surface, and the covalent binding of the 3'-end NH2 to the NHS group on the LP surface are used to finally form MB@LP-linker-FB. This complex has both magnetic separation function and signal molecule encapsulation characteristics, providing a basis for subsequent target response. When target DNA (such as ADV or MP) is present, the Cas12a-crRNA complex specifically recognizes the target and activates its nonspecific trans-cutting ssDNA activity. The linker1 designed in this strategy is a T-base ssDNA chain, which serves as a substrate for Cas12a trans-cutting. The breakage of linker1 causes MB@LP to dissociate from the FB surface, and the supernatant is collected after magnetic separation. Triton X-100 was added to destroy the MB@LP membrane structure, releasing MB molecules, and the peak current generated by MB was detected by DPV. For RNA viruses (such as RSV and H1N1), the Cas13a system was used for detection. Because RNA is easily degraded by nucleases in the environment, this study designed linker2 as a chimeric DNA / RNA chain: an RNA sequence consisting of three U bases was inserted into the T base backbone (Table 3-2). This design achieves dual functions: the U base provides an RNA-specific recognition site for Cas13a trans-cleavage; the DNA backbone significantly improves the stability of linker2 in complex environments. When the target RNA activates the Cas13a-crRNA trans-cleavage activity, the RNA fragment in linker2 is cut, and MB@LP is freed from the FB bond. Subsequently, the supernatant is taken and Triton X-100 is added to lyse MB@LP and the MB signal is detected by DPV testing.

[0079] Experimental Example 2 Design and Debugging of Centrifugal Microfluidic Detection Platform

[0080] Based on the centrifugal force driven fluid control principle, an eight-channel centrifugal microfluidic detection platform was designed to achieve the simultaneous detection of four pathogens: ADV, MP, RSV and H1N1. Figure 2 The core mechanism of this mechanism (A and B) is the pinning effect of a capillary valve, which means that liquid will not pass through the valve until the centrifugal pressure overcomes the capillary back pressure. When the rotational angular velocity ω is high enough, the centrifugal force overcomes the surface tension of the liquid, allowing it to enter the next chamber.

[0081] The detection platform has a three-layer structure: the electrode base plate integrates 8 groups of evenly arranged SPCEs as detection electrodes; the middle microfluidic layer (thickness 20mm) includes a central sample pool (diameter 10mm), 8 reaction pools (diameter 6mm) and 8 bottomless detection pools (diameter 10mm). Each chamber is connected by a precisely machined radial microchannel, in which the detection pool and the electrode base plate directly constitute an electrolytic cell detection system. Both the sample pool and the reaction pool have a 1mm thick bottom to ensure sealing; the top sealing cover (thickness 3mm) is provided with a sample hole (diameter 2mm) and a vent array (diameter 2mm) to ensure pressure balance during liquid delivery. The liquid in the central sample pool can be transported to each reaction pool and detection pool by centrifugal action, and the distribution of the solution in the reaction pool and detection pool as the detection process progresses can be controlled by centrifugal speed control and capillary fine valve action. A ring-shaped recessed area is set at the corresponding position of the reaction pool at the bottom of the electrode base plate for embedding magnets ( Figure 2 The red area in the middle C) adopts the mortise and tenon joint design in the center (cylindrical hollow protruding structure, Figure 2 The green part in the middle C (with a wall thickness of 3 mm) ensures a secure connection to the centrifuge. The elasticity of the PDMS material ensures the stability of the mosaic structure and improves the mechanical stability of the system.

[0082] By systematically optimizing the channel geometry parameters and valve position of the microfluidic detection platform, an efficient two-stage centrifugal fluid control system was established. In the first stage of fluid control, when the centrifuge was running at 600 rpm / min ( Figure 3 During low-speed operation (A), after 45 seconds of centrifugation, the liquid successfully broke through the precise microchannel connecting the sample pool and the reaction pool (the channel was designed to be 10 mm long and 0.3 mm wide, with a height difference of 1 mm between the bottom of the channel and the bottom of the reaction pool), achieving the initial distribution process of the solution. Figure 3 The actual effect can be observed in Figure B. To verify the distribution uniformity, the experiment was tested with 1mL MB solution. The results are shown in Figure 1. Figure 3 Figure C shows that the relative standard deviation (RSD) of the solution volume of the eight reaction pools is only 0.25%, confirming the reliability of the first-stage channel design. In the second-stage reaction solution transfer process, the channel from the reaction pool to the detection pool is designed to be a more refined long and narrow structure (the channel length is extended to 15mm, the width is reduced to 0.1mm, and the bottom of the channel is raised to a position 2mm away from the bottom of the detection pool). At this time, the rotation speed needs to be increased to 800rpm / min ( Figure 3 D), the solution can enter the detection pool from the reaction pool after centrifugation for 90s ( Figure 3 E). The volume of the detection cell solution is measured as follows Figure 3Figure F shows an RSD of 0.33%. The average volume of 122 μL results in only a 2.5% volume loss compared to the theoretical value of 125 μL. This slight loss is primarily due to the adhesion of the liquid film to the inner wall and residual droplets. However, the relative loss rate decreases further with increasing sample volume. The optimized matching of the two-stage centrifugation parameters and channel dimensions ensures precise timing control, quantitative transfer, and uniform distribution of samples within the microfluidic detection platform, providing a reliable microfluidic control foundation for subsequent practical testing.

[0083] Experimental Example 3 Comparison of Preparation Methods and Stability Study of MB@LP

[0084] In order to obtain MB@LP with high encapsulation efficiency, two methods, passive drug loading and active drug loading, were systematically compared. The passive drug loading method includes two subcategories: using MB aqueous solution or MB solution containing 0.2M ammonium sulfate to hydrate the LP film; the active drug loading method is based on the ammonium sulfate gradient method, which drives MB transmembrane transport by adjusting the pH value inside the LP. Among them, the film hydration method relies on the spontaneous encapsulation of MB during the formation of the lipid bilayer, while the ammonium sulfate gradient method uses the chemical potential energy generated by the difference in ion concentration inside and outside the LP to promote the efficient influx of positively charged MB molecules against the concentration gradient. After dialysis to remove the unencapsulated MB, the MB@LP was taken for DPV testing, and the results showed that ( Figure 4 ) were able to completely remove free MB, confirming the effectiveness of the dialysis process. After adding Triton X-100 to cleave the MB@LP and uniformly diluting it 20-fold, the MB@LP prepared by the ammonium sulfate gradient method exhibited significantly enhanced MB peak current (4.5-fold and 1.6-fold higher than the two passive drug-loading methods, respectively), indicating the highest MB encapsulation efficiency. This demonstrates that the ion gradient-driven active drug-loading mechanism can overcome the concentration limitations of passive diffusion.

[0085] Specifically, (NH4)2SO4 solution is used to hydrate the LP film to prepare a LP with both the inner and outer phases being (NH4)2SO4 solution. The (NH4)2SO4 in the outer phase is then removed by dialysis to establish a transmembrane concentration gradient. This concentration difference becomes the basic mechanism for establishing a pH gradient: Under the dissociation equilibrium, it decomposes into NH3 and H + , because different ions have significant differences in their ability to penetrate the phospholipid bilayer (the order of permeability is:

[159] Neutral NH3 molecules can freely penetrate the lipid membrane and diffuse outward, while H + MB molecules (the aqueous solution is weakly alkaline) enter the inner phase through passive diffusion in this system and become ionic under acidic conditions. The sulfate formed has a very low permeability coefficient to the bilayer, which significantly improves the encapsulation efficiency. It is worth noting that the outward permeation of NH3 molecules not only produces an ion concentration difference and establishes a pH gradient, but also forms a transmembrane potential difference. This electrochemical potential difference provides a driving force for the directional transport and internal enrichment of MB. [160,161] .

[0086] Based on the above results, the ammonium sulfate gradient method was selected for the large-scale preparation of MB@LP. Figure 5 The synthesized MB@LP showed no significant leakage during its 28-day storage period. Even after 28 days of storage, the MB released by cleavage maintained 97.1% of the initial signal, demonstrating that the carrier combines high encapsulation efficiency with excellent long-term stability. This property enables it to meet the dual requirements of detection systems for controlled release of signal molecules and low background interference.

[0087] Experimental Example 4: Characterization of MB@LP-linker-FB

[0088] The morphological characteristics of LP at different modification stages were characterized by transmission electron microscope (TEM). The results showed that the original LP sample ( Figure 6 Middle (A) shows a typical vesicle structure, with the hollow chamber formed by the phospholipid bilayer providing an ideal space for MB enrichment. It is worth noting that after extrusion through a 220nm polycarbonate membrane filter, the diameter measured by electron microscopy is consistent with the theoretical cutoff value of the membrane pore size, indicating that we have obtained LPs of uniform size. After MB loading ( Figure 6 In the MB@LP-linker1 image (B), LP still maintains a complete vesicle morphology and uniform particle size, indicating that MB molecules are enriched in the LP cavity without causing structural rupture. Figure 6 Middle C) No obvious linker1 modification features were observed, which may be due to the dissociation of the short-chain nucleotide structure caused by the high-energy electron beam of TEM. However, it is worth noting that the modified composite system still maintains good monodispersity, its particle size distribution is basically consistent with that of the original MB@LP, and the integrity of the vesicle structure is not significantly affected. This phenomenon shows that although the modification process of linker1 is not directly visible in the TEM image, it indirectly confirms the mildness of the modification process and the retention of the intrinsic properties of the liposomes by maintaining the original particle size uniformity and structural stability of the system. The successful modification of the linker was subsequently confirmed by analyzing the test results of hydration kinetic diameter, Zeta potential and UV-vis.

[0089] To evaluate the structural evolution of MB@LP-linker during assembly, dynamic light scattering (DLS) was used to analyze the hydration kinetic diameters of LP, MB@LP, and MB@LP-linker. Figure 7 As shown, the hydration kinetic diameter of the blank LP was 221 nm, consistent with the theoretical cutoff value of the membrane pore size, indicating that the prepared LP exhibited monodispersity and a controllable particle size distribution. After MB encapsulation, the particle size of the MB@LP increased slightly to 226 nm, confirming that the enrichment of MB molecules did not cause significant deformation or swelling of the lipid bilayer, consistent with the TEM results. Upon covalent conjugation of MB@LP with linker1 / linker2, the hydration kinetic diameter of the complexes significantly increased to 251 nm (MB@LP-linker1, PDI = 0.04) and 255.7 nm (MB@LP-linker2, PDI = 0.07), respectively. This size increase stems from the anchoring of the linkers to the MB@LP surface via the conjugation reaction. Notably, the PDI value after functionalization remained below 0.2, indicating that the conjugation process did not induce liposome aggregation, confirming the effective maintenance of the monodispersity of the complexes through the NHS-NH2 directed conjugation strategy.

[0090] To characterize the assembly process of MB@LP-linker, the dynamic changes of surface charge were tracked by Zeta potential analysis. Figure 8 As shown, the blank LP exhibits a negative potential of -7.6 mV due to the ionization of phosphate groups on the surface of the phospholipid bilayer. When the positively charged MB is encapsulated into the LP, the Zeta potential of MB@LP rises to -0.09 mV, confirming that the MB molecule is successfully encapsulated in the LP. Further analysis of the effect of the modified linker on the Zeta potential revealed that after covalent coupling of MB@LP with the negatively charged linker, the surface potential of the complex decreases (MB@LP-linker1: -3.6 mV; MB@LP-linker2: -3.7 mV). The regular changes in surface charge confirm the successful gradual assembly of the MB@LP-linker complex from a physicochemical perspective.

[0091] To verify the successful encapsulation of MB molecules and whether the modified linker would affect the stability of LP, eight groups of samples were analyzed by UV-vis. Figure 9):LP, MB solution, MB@LP, MB@LP-linker1, MB@LP-linker2, MB@LP-linker1 and MB@LP-linker2 cleaved by Triton X-100, and Triton X-100. Spectral analysis showed that neither blank LP nor Triton X-100 showed characteristic absorption peaks in the visible light region (750-500nm), eliminating the interference of light absorption generated by the two themselves. The free MB solution showed concentration-dependent aggregation behavior: at high concentrations, characteristic peaks of dimers and monomers appeared at 610nm and 665nm, respectively, which is consistent with the inherent optical properties of MB molecules.

[162] It is worth noting that MB@LP exhibits a significant absorption peak at 574nm. This shift phenomenon originates from the aggregation state induced by the high concentration of MB inside the LP. Its red-shifted characteristic peak (compared to the monomer 665nm) can serve as spectroscopic evidence of the successful encapsulation of MB in the LP cavity.

[163] . Further analysis of the effect of linker modification revealed that the spectra of MB@LP-linker1 and MB@LP-linker2 almost completely overlapped with the unmodified MB@LP, confirming that the coupling reaction between the biotin and NH2-modified nucleotide chains and the NHS groups on the LP surface would not destroy the encapsulation state of MB. After a 10-fold dilution of the cleavage-treated samples, it was found that both MB@LP-linker1 and MB@LP-linker2 treated with Triton X-100 reproduced the characteristic absorption peaks of the free MB solution, fully demonstrating the complete release of MB after the rupture of MB@LP. The above experimental results form a closed-loop verification: (1) MB is efficiently encapsulated inside the liposomes; (2) the linker modification process does not cause MB leakage or LP structural damage; (3) the generation of MB signals is strictly dependent on the MB@LP cleavage triggered by Triton X-100.

[0092] The successful preparation of MB@LP-linker-FB was verified by TEM and UV-vis. TEM analysis showed that ( Figure 6 D), and the freely distributed MB@LP-linker1( Figure 6 Different from (C), MB@LP-linker1-FB exhibits a petal-like morphology, and the observed MB@LP is slightly deformed due to the steric effect. Figure 10After multiple washes, no characteristic MB absorption peak was observed after the addition of Triton X-100 to the final wash solution, indicating that the free MB@LP-linker had been removed. However, after the addition of Triton X-100 to MB@LP-linker-FB, a significant absorption signal was detected, confirming that MB@LP-linker was stably anchored to the FB surface through biotin-streptavidin cross-linking. It is worth noting that the MB@LP-linker2-FB system has a slightly lower absorbance, which is related to the fact that the DNA-RNA hybrid chain formed by the inserted RNA fragment in its linker2 is more susceptible to hydrolysis than the DNA chain. For this reason, all buffer solutions need to be pretreated with DEPC water during the preparation process.

[0093] Experimental Example 5 Optimization of Preparation Conditions of MB@LP-linker-FB

[0094] To achieve the best electrochemical response of the MB@LP-linker-FB complex, this section systematically optimized the ratio of linker to FB based on the principle of molecular self-assembly equilibrium. Using linker1 as a model, a gradient experiment of linker concentration (50-500nM) was established under the conditions of controlling the amount of MB@LP (100μL) and FB (0.3mg). The DPV test results ( Figure 11 Middle A) Found: When the linker concentration is 100nM, the current response of MB reaches its maximum value, indicating that the assembly efficiency of the MB@LP-linker-FB complex is optimal at this time. It is worth noting that when the linker concentration exceeds 100nM, the current response decays. This may be because the linker molecules modified too densely on the surface of MB@LP produce steric hindrance due to a certain flexibility. The end of Biotin is blocked by the adjacent linker chain and cannot be fully exposed to bind to the streptavidin on FB. Secondly, each MB@LP carries multiple linker molecules. When these complexes are mixed with FB, multiple linker chains on the same MB@LP will compete for the limited streptavidin sites on FB, resulting in unstable binding, thereby reducing the effective binding efficiency.

[0095] Based on the optimal linker concentration, the effect of FB dosage (15-50 μL) on the system performance was further investigated. 15, 20, 30, 40, and 50 μL of 10 mg / mL FB suspension were taken for thorough washing, and then 100 μL of MB@LP-linker was added for incubation. MB@LP-linker-FB was then thoroughly washed by magnetic separation, and PBS buffer and Triton X-100 were subsequently added to cleave MB@LP, and then the current response value of MB was detected. The experimental results are shown in Figure 2. Figure 11As shown in Figure B, the current response reaches its peak when the amount of FB suspension is 30 μL (corresponding to a FB mass of 0.3 mg), while excess FB causes the current value to decrease. High-density FB forms a dense suspension layer in the solution, hindering the diffusion of MB@LP-linker to the FB surface. Excessive FB also dilutes the local concentration of MB@LP-linker, reducing the effective collision probability and delaying binding efficiency. Furthermore, crosslinking time experiments show that overnight incubation improves the current response compared to 5-h incubation. This is due to the time required for biotin-streptavidin binding to reach thermodynamic equilibrium. In summary, choosing 100 nM linker and 100 μL MB@LP for overnight crosslinking at 4°C, followed by incubation with 30 μL FB (10 mg / mL), maximizes the assembly of the MB@LP-linker-FB complex while balancing steric hindrance, diffusion limitations, and economic costs, laying the foundation for the construction of stable signal transduction units for subsequent biosensing applications.

[0096] Experimental Example 6: Feasibility Verification of Detection Strategy

[0097] To verify the ability of the Cas12a / 13a-mediated sensing strategy to detect multiple respiratory pathogens (ADV, MP, RSV, H1N1), experiments were designed from two aspects: Cas12a / 13a-specific recognition and cleavage and the overall feasibility of the scheme.

[0098] First, the target recognition and trans-cleavage activity of the Cas12a / 13a-crRNA complex were analyzed by PAGE. Figure 12, no band appeared in lane A (Cas12a-crRNA (ADV) complex), indicating that there was no nucleic acid residue in the complex itself; when the ADV target was co-incubated with Cas12a-crRNA (ADV) (lane C), the original ADV characteristic band (lane B) completely disappeared, indicating that crRNA (ADV) successfully guided Cas12a to perform targeted cutting of ADV; the band positions of lane E (Cas12a-crRNA (ADV) + linker1) and lane F (pure linker1) were consistent, indicating that Cas12a did not cut linker1 when there was no target; and the band in lane D (Cas12a-crRNA (ADV) + ADV + linker1) disappeared, confirming that the target ADV activated the trans-cutting activity of Cas12a and completely degraded linker1. For the MP detection system, there is no band in lane K (Cas12a-crRNA (MP) complex), the band in the middle lane is the MP target, and the band disappears in lane I (Cas12a-crRNA (MP) + MP), verifying the specific cutting of MP by Cas12a guided by crRNA (MP). The bands in lane G (Cas12a-crRNA (MP) + linker1) are consistent with those in lane F, and the band in lane H (Cas12a-crRNA (MP) + MP + linker1) disappears, further proving that the target MP can specifically activate Cas12a-crRNA (MP) to cut linker1. This result shows that the designed crRNA can accurately identify ADV and MP targets, and only activates the trans-cutting function of Cas12a when the corresponding target is present, thereby achieving trans-cutting of linker1, providing a molecular recognition basis for the construction of subsequent signal amplification systems.

[0099] Then, the target recognition and non-specific cleavage linker2 function of Cas13a-crRNA for H1N1 and RSV were verified ( Figure 13 ). Lane A (Cas13a-crRNA (H1N1) complex) has no bands, proving that there are no free nucleic acids remaining in the system; Lane B (H1N1 target) shows a specific band, and the band completely disappears in Lane C (Cas13a-crRNA (H1N1) + H1N1), confirming that Cas13a guided by crRNA (H1N1) achieves specific cleavage of H1N1. The band positions of Lane E (Cas13a-crRNA (H1N1) + linker2) and Lane F (pure linker2) are consistent, indicating that Cas13a-crRNA does not cut linker2 when there is no target; while two new bands appear in Lane D (Cas13a-crRNA (H1N1) + H1N1 + linker2) (T) 20 and (T) 18, which is consistent with the designed U base cleavage site product. For the RSV detection system, lane K (Cas13a-crRNA (RSV) complex) has no band, and the band shown in lane J is the RSV target. The band disappears in lane I (Cas13a-crRNA (RSV) + RSV), verifying the specific cleavage of RSV by Cas13a-crRNA (RSV). Lane G (Cas13a-crRNA (RSV) + linker2) is consistent with the band in lane F, and lane H (Cas13a-crRNA (RSV) + RSV + linker2) also produces (T) 20 and (T) 18 The bands showed that the RSV target can activate Cas13a-crRNA (RSV) to cut linker2. This result shows that the designed crRNA can specifically recognize H1N1 and RSV targets and trigger Cas13a to accurately cut the U site in linker2. The length of the cleavage product is consistent with the theoretical design, confirming the specificity of the targeted RNA sensing strategy and the reliability of trans-cleavage. The above two PAGE experiments confirmed at the molecular level that the designed crRNA can accurately guide the Cas protein to recognize specific pathogen nucleic acids and trigger the controlled cleavage of the linker, providing a rigorous molecular biological basis for the subsequent application of the MB@LP-linker-FB signal conversion system.

[0100] The feasibility of the Cas12a / Cas13a system in detecting ADV and RSV was verified by DPV testing ( Figure 14 ). In the blank control group (no target), the characteristic peak of MB was detected near -0.29V. It was preliminarily judged that the signal came from the dissociation of the partially bound unstable MB@LP-linker-FB complex, causing MB@LP to escape into the solution. When 1fM target was added, the peak current was significantly enhanced, and as the target concentration increased to 1pM and 1nM respectively, the current signal showed a concentration-dependent increase. This result indicates that the target successfully activated the trans-cleavage activity of Cas12a / Cas13a, cutting the linker sequence, thereby dissociating MB@LP from FB. This result systematically verified the feasibility of the Cas12a / Cas13a system in target nucleic acid detection from the perspective of electrochemical signal transduction, and laid a methodological foundation for subsequent sample analysis.

[0101] In order to explore the signal source under non-target conditions, three control experiments were designed: the MB@LP-linker1-FB complex was incubated in PBS buffer, 1×NEBuffer 2.1 buffer, and a solution containing 75nM Cas12a protein, and DPV detection was performed after 1 hour. Figure 15The results show that only weak signals were generated in the supernatants of the three systems, indicating that the MB@LP structure remained stable in these three environments and no obvious rupture occurred. However, after adding Triton X-100 to the supernatant of the 1×NEBuffer 2.1 system, the detected current signal increased slightly compared with the PBS group. This may be due to the presence of Mg 2+ The presence of changes the Zeta potential of the lipid membrane surface, causing MB@LP to partially aggregate and induce dissociation. It is worth noting that the presence of Cas12a protein further enhances the signal, and it is speculated that it may interfere with the stability of the complex through nonspecific adsorption, causing MB@LP to dissociate. In view of the nonspecific interference that may be caused by Cas protein, two improvement measures were introduced during the experimental optimization. First, FB was washed three times with 0.05% PBST buffer to remove loosely adsorbed streptavidin, thereby reducing the effect of nonspecific adsorption on the signal. Secondly, the ΔI signal correction strategy (ΔI=I 样本 –I 背景 ) to subtract background signals to improve detection accuracy.

[0102] Experimental Example 7 Optimization of detection conditions

[0103] In order to achieve the best performance of the proposed strategy, the cleavage time of the Cas protein and the liposome rupture time were optimized respectively. In the detection platform, the target recognition and trans-cleavage of the Cas protein are carried out simultaneously in the same reaction system, that is, MB@LP-linker-FB is already present in the reaction solution before the target recognition and cleavage are completed, rather than being added later, so a total cleavage time needs to be optimized. With 1nM ADV as the target, the DPV test results are as follows Figure 16 It shows that as the action time of Cas protein increases, the current signal first rises and then tends to stabilize. When the reaction time reaches 45 minutes, the signal reaches a peak, indicating that the linker sequence has been completely trans-cleaved. This short time is due to the excellent catalytic efficiency of the Cas system. After the Cas12a-crRNA complex binds to the dsDNA activator, its trans-cleavage catalytic efficiency constant k for ssDNA is cat / K m Up to 1.7×10 7 s -1 M -1 , which means that about 17 substrate turnovers can be completed per second

[164] The Cas13a system is more responsive to RNA activators, and its catalytic efficiency k cat / K m 1.09×10 9 s -1 M -1 , which can achieve efficient substrate conversion of 4854 times per second

[165] Therefore, 45 min was determined to be the optimal reaction time for this system.

[0104] Then, the rupture conditions of MB@LP were optimized by controlling the action time of Triton X-100. Figure 17 The DPV signal shows a characteristic pattern of initially increasing rapidly and then leveling off with increasing rupture time. Within the rupture time range of 0-20 minutes, the signal shows a positive correlation with time, but after 20 minutes, the signal reaches a plateau. Therefore, 20 minutes was determined to be the optimal duration for MB@LP rupture. This optimization process ensures sufficient MB release, thereby improving the signal consistency and reproducibility of the detection system.

[0105] Experimental Example 8 Detection Performance Analysis

[0106] Under the optimized experimental conditions, this method successfully achieved high-sensitivity detection of ADV, MP, RSV and H1N1. Figure 18 As shown in Figure 2, the DPV response signal showed a significant positive correlation with the increase of target concentration. In the linear range of 1fM to 100pM, the peak current showed a good linear relationship with the logarithm of the target concentration (R 2 >0.99), as shown in Table 2. The limits of detection (LODs) obtained by further calculation were 65.5aM ADV, 17.7aM MP, 37.1aM RSV, and 69.2aM H1N1, respectively. Compared with other detection methods reported in the literature (Table 3), this protocol showed significant advantages in both detection sensitivity and linear range. The efficient recognition and cleavage capabilities of Cas12a / Cas13a and the signal amplification properties of the MB@LP-linker-FB complex together give this protocol excellent detection performance.

[0107] Table 2 Linear fitting equation and detection limit of detection results

[0108]

[0109] Table 3 Other sensing strategies for detecting respiratory pathogens

[0110]

[0111]

[0112] In addition, to verify the reproducibility of this protocol, 5 independent experiments were performed with 1 pM ADV and RSV under the same experimental conditions. Figure 19As shown in the figure, the calculated RSDs of the DPV signals were 3.54% and 4.52%, respectively, demonstrating the excellent detection repeatability of the system. This result not only highlights the high specificity of Cas proteins but also verifies the stability of MB@LP in signal enrichment and transmission, providing key support for the reliability of subsequent actual sample detection.

[0113] Experimental Example 9 Performance Analysis of Detecting Complex Samples

[0114] To evaluate the selectivity of the proposed method for complex samples, a mixed sample containing 1pM ADV, MP, RSV, H1N1 and common interfering substances in the oral environment (1ng / mL lysozyme, 1ng / mL salivary α-amylase) was prepared to simulate the coexistence of multiple pathogens and biological matrix interference in the real detection environment. The experimental results are shown in Figure 2. Figure 20 The results showed that the DPV detection signal of the mixed interference sample was not significantly different from the signal intensity of the corresponding target alone. This result fully confirms that the crRNA-guided Cas protein can specifically recognize the target nucleic acid sequence to ensure the accuracy of the detection; the scheme has excellent anti-interference ability against non-target interferents (such as lysozyme and amylase) and non-target pathogens, effectively avoiding the generation of false positive signals.

[0115] Experimental Example 10: Detection of actual samples

[0116] In the initial stage of this study, an eight-channel microfluidic detection platform was designed. During the preliminary experiment, it was found that a blank control was needed to achieve signal correction. Therefore, the non-adjacent sample diversion microchannels were selectively sealed with medical-grade epoxy resin glue, and the detection system was optimized into an array structure with 4 groups of experimental units and 4 groups of blank control units alternating. Figure 21 Among them, four experimental units are used to detect ADV, MP, RSV and H1N1, respectively. Each reaction pool is added with the corresponding Cas12a / Cas13a-crRNA detection system and MB@LP-linker-FB. The adjacent blank control unit uses equal concentrations of Cas12a / Cas13a-crRNA, equal amounts of MB@LP-linker-FB and normal human respiratory secretion dilutions. This spatially alternating detection array design can effectively eliminate the inherent background signal interference of the detection strategy by synchronously detecting the electrical signals of the experimental and control groups, thereby improving the reliability of the test results.

[0117] The analytical performance of the sensing strategy in complex biological matrices was systematically evaluated by detecting four pathogens, ADV, MP, RSV, and H1N1, in spiked normal human oral swab samples. Figure 22As shown in the table, quantitative analysis results show that the calibration curves in actual samples are highly consistent with the standard buffer solution system, with LODs ranging from 32.8 to 81.5 aM (Table 4), comparable to the detection level of the standard buffer solution system. These data confirm that the developed sensing system can maintain excellent detection stability in complex environments, indicating that this strategy has important application value in clinical sample point-of-care testing.

[0118] Table 4 Linear fitting equation and detection limit of the test results of oral swab samples of normal subjects

[0119]

[0120]

[0121] After obtaining approval from the Ethics Review Committee, oral swab samples from six patients were collected from Qilu Hospital of Shandong University to verify the detection performance of the detection platform in real clinical samples. The nucleic acid was first treated and extracted using a nucleic acid lysis buffer, and then tested using this detection platform. The test results are as follows: Figure 22 The results showed that RSV was detected positive in samples 1-3; ADV, MP, RSV and H1N1 were all negative in samples 4-6. To further evaluate the accuracy of the test results, the obtained results were compared with the clinical PCR test results. The two test results were consistent, and the electrochemical signal intensity was negatively correlated with the Ct value of PCR detection, with a Pearson correlation coefficient of -0.998. Specifically, when the pathogen load increased, the sensor current response increased, and the corresponding Ct value decreased, which is consistent with the negative correlation between nucleic acid concentration and Ct value in PCR technology. This experiment verified that this detection platform can still achieve accurate detection of respiratory pathogens in the absence of nucleic acid amplification, further demonstrating its application potential in POCT.

[0122] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing MB@LP-linker-FB, characterized in that: include: The MB@LP solution and linker were mixed evenly, incubated, and dialyzed to obtain MB@LP-linker; The MB@LP-linker was mixed with the streptavidin-modified FB after multiple washings, incubated, the magnetic beads were collected, and washed multiple times to obtain MB@LP-linker-FB.

2. The method for preparing MB@LP-linker-FB according to claim 1, wherein: MB@LP was prepared by the ammonium sulfate gradient method or the thin film hydration method.

3. The method for preparing MB@LP-linker-FB according to claim 1, wherein: The linker is 5'-Biotin-T (90) -(CH2)6NH2-3', or, 5'-Biotin-T (20) UUUUUT (18) UUUUUT (18) UUUUUT (20) -(CH2)6NH2-3'.

4. The method for preparing MB@LP-linker-FB according to claim 1, wherein: The linker concentration is 50-500 nM.

5. The method for preparing MB@LP-linker-FB according to claim 1, wherein: The volume ratio of the MB@LP-linker to the FB suspension is 100 μL:15-50 μL; Alternatively, the concentration of the FB suspension is 10-15 mg / mL.

6. MB@LP-linker-FB prepared by the method according to any one of claims 1 to 5.

7. A CRISPR-based DNA / RNA simultaneous detection method, characterized in that: include: Design the corresponding crRNA according to the nucleic acid to be tested, mix the test nucleic acid with the Cas12a-crRNA complex or the Cas13a-crRNA complex, and then mix and incubate it with the MB@LP-linker-FB described in claim 6. After the incubation, collect the supernatant after magnetic separation, add Triton X-100 to lyse MB@LP, and use DPV to detect the peak current generated by MB to obtain.

8. The CRISPR-based DNA / RNA simultaneous detection method according to claim 7, wherein: The target object is selected from at least one of ADV, MP, MP, RSV, RSV, H1N1, and H1N1.

9. A platform for simultaneous detection of CRISPR DNA / RNA based on the method of claim 7 or 8, characterized in that: A microfluidic channel was used as the detection platform, a carbon electrode was used as the working electrode, an Ag / AgCl reference electrode, and a carbon electrode as the counter electrode.

10. Use of the MB@LP-linker-FB described in claim 6 and the platform described in claim 9 in multiplex detection of respiratory pathogens.

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