System for detecting ArsM in sample and detection device and application thereof

Abstract

The invention discloses a system for detecting ArsM in a sample, a detection method, a detection device and application thereof. The detection system of the present invention comprises a clustered regularly spaced short palindromic repeat (CRISPR)-Cas complex comprising a guide RNA targeting ArsM and a CRISPR-related (Cas) protein or a functional fragment thereof; and a horse radish peroxidase (HRP) complex to which a single-stranded DNA (ssDNA) is linked. According to the invention, a cascade amplification detection system based on CRISPR / Cas12a is constructed, and the cascade amplification detection system is further integrated on a multi-channel paper-based microfluidic device to realize on-site high-throughput analysis of nucleic acid molecules.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and specifically relates to a cascade amplification detection system coupling a CRISPR / Cas system and an HRP system, a multi-channel paper-based microfluidic device integrating the cascade amplification detection system, and their applications. Background Art

[0002] Arsenic (As) is a toxic metalloid element ubiquitous in geochemical systems. It can cause a variety of diseases, including skin diseases, cardiovascular disorders, and cancer, and is classified as a Class I carcinogen by the World Health Organization. Rice, due to its potent arsenic accumulation and its prominent position in the diet, is a significant source of human arsenic exposure. Microbial arsenic methylation, widespread in rice paddies, is a key process for converting inorganic arsenic into less toxic organic arsenic, playing a crucial role in arsenic speciation within rice paddies. Studies have shown that, although methylarsenic accounts for a relatively low proportion of soil arsenic, it can contribute to more than half of the arsenic in rice. Arsenic biomethylation in rice paddies is primarily mediated by soil microorganisms, with the As(III) S-adenosylmethionine methyltransferase gene (ArsM) being a key gene regulating this process. Studies have found that the abundance of ArsM in rice paddy soils is significantly positively correlated with methylarsenic concentrations. Therefore, ArsM is an important biomarker of arsenic methylation in rice paddies. Measuring ArsM abundance can provide a valuable reference for assessing arsenic detoxification capacity and, therefore, inform the screening of rice paddy sites for potential arsenic risk.

[0003] Currently, ArsM testing mainly relies on genome sequencing and real-time fluorescence quantitative PCR (qPCR). The former can provide specific ArsM sequence information, providing a basis for PCR primer design and ArsM classification; the latter can obtain quantitative data such as copy number. However, both methods are costly, have long testing cycles, have certain requirements for sample quality, and require ultra-clean working environments, professional personnel, and precision instruments and equipment to complete. Considering that many high-arsenic rice fields are located in less developed countries, they often lack the conditions for conducting such tests, and biological samples are easily degraded during transportation and turnover, resulting in distorted test results. Developing an ArsM field test (POU) method that is simple to operate, low-cost, and does not rely on precision equipment is of great significance for assessing the potential risk of arsenic in rice fields.

[0004] CRISPR is an emerging technology that is expected to lead the next generation of nucleic acid point-of-use (POU) detection. It has attracted widespread attention due to its strong specificity, excellent signal transduction ability and outstanding reaction compatibility. Cas12a is one of the most widely used systems in the CRISPR toolbox in the field of POU detection. However, the existing CRISPR / Cas12a testing methods still seem difficult to apply directly to environmental media. This may be related to clinical testing needs and the characteristics of CRISPR itself. Since the sensitivity of CRISPR is not enough to detect low-concentration samples, in order to improve sensitivity, most studies will first perform pre-amplification to increase the concentration of the target fragment and then perform CRISPR detection. Since samples of different concentrations can reach saturation during the pre-amplification process, although this method effectively improves sensitivity, it often lacks quantitative capabilities. For ArsM, which is widely present in rice field media, qualitative results alone cannot provide assistance for risk assessment. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention constructs a cascade amplification detection system based on CRISPR / Cas12a, and further integrates it into a multi-channel paper-based microfluidic device to achieve on-site high-throughput analysis of nucleic acid molecules.

[0006] One aspect of the present invention provides a system for detecting ArsM in a sample, comprising: a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas complex comprising a guide RNA targeting ArsM and a CRISPR-associated (Cas) protein or a functional fragment thereof; and a horseradish peroxidase (HRP) complex linked to single-stranded DNA (ssDNA).

[0007] In some embodiments, the guide RNA comprises any one of the nucleotide sequences shown in SEQ ID NOs: 2-8.

[0008] In some embodiments, the guide RNA includes at least four of the nucleotide sequences shown in SEQ ID NOs: 2-8.

[0009] In some embodiments, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 2-8.

[0010] In some embodiments, the ssDNA is attached to magnetic beads.

[0011] In some embodiments, the ssDNA has a nucleotide sequence as shown in SEQ ID NO:9.

[0012] In some embodiments, the Cas protein is selected from Cas12.

[0013] Another aspect of the present invention provides a method for detecting ArsM in a sample, comprising the following steps:

[0014] 1) preparing a CRISPR-Cas complex containing a guide RNA targeting ArsM and a Cas protein or a functional fragment thereof;

[0015] 2) preparing an HRP complex linked to ssDNA, wherein the ssDNA is preferably linked to magnetic beads;

[0016] 3) Mixing the premix containing the CRISPR-Cas complex with the HRP complex and the plasmid and reacting them.

[0017] In some embodiments, the method further comprises step 4), performing magnetic separation on the reaction product solution of step 3), and subjecting the supernatant to a color development reaction.

[0018] In some embodiments, in step 3), the reaction time is 35-45 minutes.

[0019] In some embodiments, in step 4), the supernatant is transferred to a TMB solution for color development reaction.

[0020] In some embodiments, the method further comprises detecting the fluorescence intensity of the chromogenic reaction product.

[0021] In some embodiments, fluorescence intensity is detected with excitation light at 565-575 nm and emission light at 580-590 nm.

[0022] Another aspect of the present invention provides a detection device for use in the detection system or the detection method of the present invention, wherein the detection device includes a paper chip and a detection board.

[0023] In some embodiments, the paper chip includes a first area, a second area, a third area, a fourth area, a fifth area and a sixth area having hydrophobic areas and hydrophilic areas; the first area is configured with a plurality of hydrophilic adsorption areas distributed in parallel, and the rest are hydrophobic areas; the second area is configured with a hydrophilic area drainage channel extending from a central hole to both ends and further leading to multiple end points, and the rest are hydrophobic areas; the third area is configured with a plurality of X-shaped hydrophilic area drainage channels distributed in parallel, and the rest are hydrophobic areas; the fourth area is configured with a plurality of hydrophilic loading areas distributed in parallel, and the rest are hydrophobic areas; the fifth area has the same configuration as the third area; the sixth area has the same configuration as the second area; the multiple end points of the hydrophilic area drainage channels of the second area correspond one-to-one with the intersection points of the X-shaped hydrophilic area drainage channels of the third area when superimposed; the multiple end points of the X-shaped hydrophilic area drainage channels of the third area correspond one-to-one with the hydrophilic adsorption areas of the first area when superimposed; the hydrophilic adsorption areas of the first area correspond one-to-one with the hydrophilic loading areas of the fourth area when superimposed.

[0024] In some embodiments, the diameter of the hydrophilic loading zone of the fourth region is smaller than the diameter of the hydrophilic adsorption zone of the first region.

[0025] In some embodiments, the diameter of the hydrophilic adsorption area of ​​the first region is 4-8 mm (eg, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm or any value therebetween).

[0026] In some embodiments, the diameter of the central hole of the hydrophilic zone drainage channel of the second region is 4-8 mm (for example, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm or any value therebetween), and the width of the end point is 1-3 mm (for example, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm or any value therebetween).

[0027] In some embodiments, the width of the X-shaped hydrophilic region drainage channel in the third region is 1-3 mm (eg, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, or any value therebetween).

[0028] In some embodiments, the diameter of the hydrophilic loading zone of the fourth region is 3-5 mm (eg, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, or any value therebetween).

[0029] In some embodiments, the length of the first region, the second region, the third region, the fourth region, the fifth region and / or the sixth region is 60-100 mm (for example, 60 mm, 70 mm, 80 mm, 90 mm, 100 mm or any value therebetween) and the width is 40-50 mm (for example, 40 mm, 42 mm, 44 mm, 46 mm, 48 mm, 50 mm or any value therebetween).

[0030] In some embodiments, the first region, the second region, the third region, the fourth region, the fifth region and / or the sixth region are connected in a set order or are independent of each other.

[0031] In some embodiments, the hydrophobic region is coated with a hydrophobic material.

[0032] In some embodiments, the hydrophobic material comprises paraffin wax.

[0033] In some embodiments, the loading area of ​​the fourth region is provided with an adsorption material for adsorbing nucleic acids.

[0034] In some embodiments, the adsorbent material comprises glass fibers.

[0035] In some embodiments, the detection plate is provided with detection areas corresponding one-to-one to the hydrophilic loading areas of the fourth region of the paper chip.

[0036] In some embodiments, the detection plate is a PMMA plate.

[0037] Another aspect of the present invention provides the use of the detection system, the detection method or the detection device of the present invention in high-throughput detection of ArsM.

[0038] Another aspect of the present invention provides a detection kit comprising the CRISPR-Cas complex and the HRP complex in the detection system of the present invention.

[0039] In some embodiments, the kit further comprises the detection device described in the present invention.

[0040] The present invention introduces HRP enzyme-linked probes into the CRISPR / Cas12a system, successfully realizes cascade amplification, and further improves the sensitivity by nearly an order of magnitude to 16fM, and enables quantitative testing visualized by the naked eye between 16fM and 125pM.

[0041] In order to shorten the detection time and increase the field test throughput, the present invention integrates the cascade amplification detection system into the microfluidic chip and constructs a high-throughput paper-based microfluidic chip that can test 32 samples simultaneously. The spiked test results show that the recovery rate is between 90-110%.

[0042] In addition, to address the problem of insufficient sensitivity of a single CRISPR / Cas12a, the present invention designed seven crRNAs that can specifically recognize ArsM, and through optimized construction of a combined system, successfully reduced the detection limit by 32 times and eliminated background signal interference.

[0043] In summary, this study not only constructed for the first time an ArsM cascade amplification field detection system based on the CRISPR / Cas12a combination system, realizing highly sensitive visual quantitative testing of the ArsM field, but also designed a microfluidic device with a much higher number of channels than previous paper chips, greatly improving the pre-treatment and detection efficiency. At the same time, due to its modular assembly strategy, it provides a highly potential field high-throughput detection platform not only for ArsM detection, but also for other nucleic acid molecular markers in the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 A schematic diagram of the combined CRISPR / Cas12a-HRP assay for ArsM is shown.

[0045] Figure 2 The paper chip design dimensions of the detection device are shown.

[0046] Figure 3 The real-time fluorescence curve of ArsM detection is shown, wherein ag represents the real-time fluorescence curve of ArsM detection of crRNA 1-7, respectively.

[0047] Figure 4 The endpoint fluorescence of ArsM detection using Cas12a-crRNA is shown, wherein ag is the endpoint fluorescence of ArsM detection using crRNA 1-7, respectively.

[0048] Figure 5 The fluorescence signal graph of the detection limit test endpoint of the coupled CRISPR / Cas12a system is shown, where a: detection limit of crRNA1-2 combination; b: detection limit of crRNA1-3 combination; c: detection limit of crRNA1-4 combination; d: detection limit of crRNA1-5 combination; e: detection limit of crRNA1-6 combination; f: detection limit of crRNA1-7 combination.

[0049] Figure 6The real-time fluorescence curves of ArsM detection based on the coupled crRNA system are shown, where af: real-time fluorescence curves of ArsM detection based on crRNA1-2, crRNA1-3, crRNA1-4, crRNA1-5, crRNA1-6, and crRNA1-7 coupling systems, respectively.

[0050] Figure 7 The evaluation, time optimization, detection limit, and concentration-signal intensity linear fitting diagram of the coupled CRISPR / Cas12a-HRP cascade amplification system are shown. a: Schematic diagram of the fluorescence curve of the color reaction in the presence and absence of ArsM and the results of naked eye observation; b: Endpoint fluorescence intensity diagram of low-concentration and high-concentration samples at different reaction times; c: Endpoint fluorescence signal diagram of the detection limit test under 40 minutes; d: ArsM concentration-endpoint fluorescence signal intensity fitting diagram; e: Color development and grayscale value extraction results of different ArsM concentrations; f: ArsM concentration-endpoint grayscale value fitting diagram

[0051] Figure 8 The paper chip is made by printing paraffin wax on filter paper. The black area is the hydrophobic area impregnated with paraffin wax, which is used to confine the fluid, while the white area is the filter paper that is not impregnated with paraffin wax, which is the migration channel of the fluid.

[0052] Figure 9 The results of colorimetric detection of ArsM are shown. The left column shows the negative control, and the seven columns on the right show samples with varying ArsM concentrations ranging from 16 fM to 125 pM. ArsM concentrations increase from left to right, and each column and four chambers represent replicate samples.

[0053] Figure 10 Shows the results of the study on ArsM abundance in paddy soil and its relationship with the soil environment, where a is the test area, b is the test tool, c is the systematic clustering of environmental factors, d is the principal component analysis of environmental factors, e is the linear relationship between pH and ArsM gene abundance, and f is the linear relationship between Se content and ArsM gene abundance. DETAILED DESCRIPTION

[0054] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and examples. The specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention in any way. In addition, descriptions of known structures and techniques are omitted in the following description to avoid unnecessary confusion about the concepts of the present disclosure. Such structures and techniques are also described in many publications.

[0055] The As(III) S-adenosylmethionine methyltransferase gene (ArsM) is a key gene regulating the arsenic biomethylation process. It catalyzes the transfer of a methyl group from S-adenosylmethionine (S-adenosyl-L-methionine) to As(III), forming the intermediate MMAs(III). This intermediate then introduces another methyl group to form DMA(III), which is then converted into the relatively stable DMA(V). DMA(V) can then be further methylated to form volatile trimethylarsenic (TMA) for excretion and detoxification.

[0056] Paper-based microfluidic devices construct capillary-driven microfluidic channels by printing and impregnating hydrophobic paraffin wax in specific patterns on hydrophilic filter paper. This, combined with other techniques such as nucleic acid extraction, ultimately integrates lysate purification, nucleic acid washing, elution, and distribution onto a single sheet of paper, reducing testing costs and simplifying experimental procedures. However, current paper-based sensing devices often have only a few channels. Given the strong spatial heterogeneity of rice field systems, a reasonable evaluation requires sufficient sample volume to reduce errors, and existing devices are unable to meet these requirements. Therefore, designing a high-throughput paper-based microfluidic device can apply paper microfluidic technology to the ArsM field for rapid testing, which is of great significance in simplifying the operational process and shortening the detection time.

[0057] Based on the above, the present invention provides a high-throughput paper-based sensing device based on the cascade amplification of CRISPR coupling system and HRP modified probe, to achieve rapid detection of ArsM in the field. First, 7 crRNAs were designed for the ArsM target sequence (SEQ IN NO: 1), and their detection limit, background signal, etc. were evaluated. Then, the effect of the coupling system on the detection limit was explored by adding crRNA one by one, and the coupling method with the highest sensitivity was selected and optimized to reduce the background signal. Next, the single-stranded DNA probe was modified, and an ssDNA probe (HRP-ssDNA-beads probe) connected with HRP and magnetic beads was constructed to achieve cascade amplification of the detection signal, thereby further improving sensitivity. At the same time, the introduction of this probe also successfully achieved visual reading of the test results. Then, for the problems of more field samples and complicated detection process, a test channel cascade amplification strategy was adopted to construct a high-throughput paper-based microfluidic chip, and the ArsM cascade detection method constructed was successfully integrated into this device, thereby significantly improving the detection flux of the current paper-based microfluidic device. Finally, the present invention applied this device to potentially arsenic-contaminated paddy soil to further verify the reliability of the method and provide data support for local arsenic risk assessment.

[0058] In view of the complex trace environmental biological samples and the poor site detection conditions, the present invention has developed a cascade amplification detection method based on the coupled CRISPR / Cas12a system and HRP enzyme-linked probe. The test principle is as follows Figure 1 As shown. When the target gene is present, the crRNA targeting the specific segment will guide Cas12a to recognize ArsM and perform cis-cleavage on it. Subsequently, the trans-cleavage activity with higher cutting efficiency will be activated, thereby achieving preliminary amplification of the signal while converting the recognition signal into a cleavage signal. Next, multiple crRNAs targeting different segments of the ArsM gene are introduced into the same reaction, and secondary amplification of the signal is achieved through cooperative cleavage between different complexes. Then, an enzyme-linked probe is introduced into the reaction to visualize and amplify the signal again. HRP, which is widely used in immunoassays, was selected as the reporter group due to its strong signal amplification ability and visualization strategy that can be directly observed by the naked eye, and is connected to the magnetic beads via ssDNA. The various Cas12a-crRNA complexes activated by the target then carry out non-specific cleavage of the ssDNA connected to the HRP and magnetic beads, and then the magnetic beads are fixed to the bottom under the action of magnetic force. The HRP separated by trans-cleavage is transferred to the TMB solution, catalyzing the generation of a blue product that can be directly observed by the naked eye, and the colorimetric information can be extracted by ImageJ. When there is no target gene, Cas12a-crRNA is in a silent state and cannot cut the ssDNA connecting HRP and magnetic beads. Because under the action of magnetic force, HRP will precipitate from the solution together with the magnetic beads, and there will be no HRP in the supernatant, so it cannot catalyze the TMB solution to turn blue.

[0059] The method constructed in the present invention has the characteristics of high sensitivity, low background, strong specificity and high throughput, and can provide a highly potential means for rapid screening of ArsM in rice field systems.

[0060] Example

[0061] Materials and Methods

[0062] 1. Reagents

[0063] crRNA was synthesized from Guangzhou Bolais (China), MgCl2, GuSCN, and bovine serum albumin were purchased from Sigma (Germany), NaHCO3 and azido-PEG4-NHS ester were purchased from Aladdin, kanamycin sulfate and PBS buffer were purchased from Solebol, magnetic beads were purchased from Biotech (China), enzyme-free sterile water, HRP, TMB colorimetric reagent, and optical sealing film were purchased from Thermo Fisher Scientific (USA), plasmids were synthesized from GenWeichi (USA), Tris and DNA probes were synthesized from Shanghai Bioengineering (China), plasmid extraction kits were purchased from Tiangen Biotechnology (China), glass fiber and filter paper were purchased from Whatman (USA), paraffin was purchased from Xerox (USA), desalting columns were purchased from Cytiva (USA), ultrafiltration tubes were purchased from Millipore (USA), and various forms of arsenic standard substances were purchased from the National Institute of Metrology, China.

[0064] 2. ArsM Plasmid Preparation

[0065] The ArsM plasmid constructed by Jinweizhi was introduced into competent Escherichia coli DH5α via heat shock transformation. 50 μL of the bacterial culture was then added to LB agar medium containing 50 mg / L kanamycin and cultured overnight at 37°C. Appropriate colonies were then inoculated into monoclonal antibody liquid culture medium and cultured overnight at 200 rpm at 37°C until saturation. Finally, the plasmid was extracted using a plasmid extraction kit, and the DNA concentration was tested using Qubit 4.0. The plasmid was then stored at -80°C until ready for use.

[0066] 3. Construction of Cas12a detection reaction

[0067] 1 μL of Cas12a-crRNA premix containing 1 μM Cas12a (GenBank: UVJ64946.1, GI: 2289390842), 1.25 μM crRNA, and 1X NEB buffer 2.1 was added to 17 μL of 1X NEB buffer 2.1 containing different concentrations of plasmids, and then 2 μL of 5 μM ssDNA probe was added. The reaction solution was then placed in an ABI7500 fluorescence PCR instrument and incubated at 37°C for 60 minutes, with fluorescence data recorded every minute.

[0068] 4. Construction of the coupled crRNA system

[0069] According to the method of "3. Cas12a detection reaction construction", different Cas12a-crRNA premixes were prepared respectively, and then the crRNA premixes to be used in combination were mixed in equal proportions. 1 μL of the mixture was added to 17 μL of 1XNEB buffer 2.1 containing plasmids of different concentrations, and then 2 μL of probe was added. The reaction implementation curve was recorded using ABI7500.

[0070] 5. Optimization of the coupled crRNA system

[0071] Seven crRNA (SEQ ID NOs: 2-8) coupling premixes were prepared according to the method of "4. Construction of coupling crRNA system". Then, 0.4, 0.6, 0.8, 1.0, and 1.2 μL of the premix were added to 17.6, 17.4, 17.2, 17, and 16.8 μL of 1X NEB buffer 2.1 containing different concentrations of plasmids, and then 2 μL of ssDNA probe (5'6-FAM / TTATTATT / 3'BHQ1) was added. The solution was placed in an ABI7500 and incubated for 60 min, and the fluorescence data was recorded once every minute.

[0072] 6. Preparation of HRP-ssDNA-beads

[0073] 2 mg of HRP was dissolved in 1000 μL of NaHCO 3 solution to prepare an HRP solution with a final concentration of 50 μM, and then a 1000-fold excess of azido-PEG4-NHS ester was added to the HRP solution and reacted at room temperature for two hours for azide modification. The modified HRP was removed from the excess azido-PEG4-NHS ester using a desalting column and concentrated by ultrafiltration. 200 μL of 10 μM azide-modified HRP was then taken, and a 2-fold excess of TEG-ssDNA-biotin (SEQ ID NO: 9) was added thereto, incubated in PBS buffer for 15 h, and then the excess DNA was removed by ultrafiltration to obtain HRP-ssDNA-biotin.

[0074] Next, 40 μL of streptavidin-modified magnetic beads were added to 600 μL of enzyme-free sterile PBS buffer containing 0.1% Tween 20. 5 μL of 800 nM HRP-DNA-biotin was then added to the solution. The mixture was incubated at 1500 rpm for 5 minutes, followed by centrifugation at 20,000 rpm for 1 minute, and the supernatant was discarded. Next, the magnetic beads were rinsed at least eight times with 1X PBS containing 0.1% Tween 20 to completely remove any residual HRP. This yielded an enzyme-linked probe (HRP-ssDNA-beads) containing HRP and magnetic beads.

[0075] 7. Construction of crRNA-HRP coupled cascade amplification detection platform

[0076] Prepare 7 crRNA premixes according to the "5. Optimization of the coupled crRNA system" method, then take 5 μL of the premix and an appropriate amount of 1X NEB buffer 2.1 and add them to the magnetic beads obtained in step 6. Each tube is divided into 4 eight-tubes, and then add an appropriate amount of plasmid at different concentrations and dilute to 60 μL with 1X NEB Buffer 2.1. Incubate the reaction at 37 ° C with shaking for 40 minutes, then centrifuge instantly and use a magnet to fix the magnetic beads to the bottom to separate the HRP that has not been cut and released into the supernatant. Then aspirate the supernatant, and then add 30 μL of the supernatant to 30 μL of TMB. After a brief incubation, use a fluorescence photometer to measure its fluorescence intensity with 570 nm excitation light and 585 nm emission light. For the colorimetric method, take a photo with a mobile phone directly after incubation to record the color development results, and then use ImageJ to extract the color development information.

[0077] 8. Preparation of Paper-based Microfluidic Devices

[0078] The paper-based microfluidic device consists of a paper chip and a detection plate. The paper chip was designed by Coredraw, and the specific dimensions are as follows: Figure 2 The paraffin wax was then printed onto the filter paper using a paraffin printer (Xerox Colorqube 8570). The filter paper was then heated at 120°C for 2 minutes using a hot plate to allow the wax to penetrate the paper.

[0079] Among them, the paper chip includes the first, second, third, fourth, fifth and sixth regions with hydrophobic and hydrophilic regions: the first region is equipped with 32 hydrophilic adsorption regions distributed in parallel, and the rest are hydrophobic regions; the second region is equipped with a hydrophilic region drainage channel that leads from the central hole to both ends and further leads to 8 end points, and the rest are hydrophobic regions; the third region is equipped with 8 X-shaped hydrophilic region drainage channels distributed in parallel, and the rest are hydrophobic regions; the fourth region is equipped with 32 hydrophilic loading regions distributed in parallel, and the rest are hydrophobic regions, and the fourth region is equipped with 8 X-shaped hydrophilic region drainage channels distributed in parallel. The loading area is provided with glass fiber for adsorbing nucleic acids; the configuration of the fifth area is the same as that of the third area; the configuration of the sixth area is the same as that of the second area; the 8 end points of the hydrophilic area drainage channel of the second area correspond one-to-one with the intersection points of the 8 X-shaped hydrophilic area drainage channels of the third area when superimposed; the 32 end points of the X-shaped hydrophilic area drainage channels of the third area correspond one-to-one with the 32 hydrophilic adsorption areas of the first area when superimposed; the hydrophilic adsorption areas of the first area correspond one-to-one with the hydrophilic loading areas of the fourth area when superimposed.

[0080] The detection plate (PMMA plate) of the paper-based microfluidic device was designed by RDworks, with a length of 80 mm and a width of 44 mm. The thickness of the plate is 5 mm. The diameter of the reaction chamber is 5 mm, and its distribution is similar to Figure 2The same as the area 1 in the figure. It is designed and made by a laser cutting machine with a cutting speed of 0.48mm / s and a maximum power of 94%.

[0081] 9. Sample collection and testing of geochemical indicators such as arsenic, trace elements, pH, and total organic carbon (TOC)

[0082] Soil samples were collected from Wanshan District, Tongren City, Guizhou Province, and aliquoted and stored according to test parameters. Samples for biological testing were stored in liquid nitrogen, returned to the laboratory, and refrigerated at -80°C until use. Samples for geochemical testing were collected, refrigerated at 4°C, returned to the laboratory, and, after freeze-drying, ground in an agate mortar. Samples for arsenic, trace elements, and TOC testing were ground and passed through a 200-mesh sieve. Samples for pH testing were passed through a 10-mesh sieve.

[0083] Arsenic and trace element analysis was performed using ICP-MS. The sieved soil was first digested using the HNO₃-HF high-temperature, high-pressure, sealed method as follows: 50 mg of soil sample was weighed into a Teflon inner cup, 3 mL of HNO₃ and 0.5 mL of HF were added, and the mixture was placed in a Teflon outer container and incubated at 150°C for 48 hours. Then, 1 mL of H₂O₂ was added and the HF was removed by acidification until nearly dry. 0.2 mL of HNO₃ and 1.8 mL of ultrapure water were added and dissolved at 150°C for 6 hours. Finally, the sample was transferred to a centrifuge tube and the volume was adjusted to 10 mL for analysis.

[0084] Soil pH was measured using a Shanghai Leici PHS-3C portable pH meter. First, 10 g of a 10-mesh soil sample was weighed into a 50 mL centrifuge tube. Then, 25 mL of ultrapure water was added. After oscillating on an oscillator for 10 minutes to thoroughly mix, the sample was allowed to stand until the soil had fully settled. The glass electrode of the pH meter was then inserted into the supernatant and the pH value of the test solution was recorded. TOC and TN tests were performed using an organic element analyzer. Two grams of sieved and dried soil sample was placed in a 15 mL centrifuge tube. An appropriate amount of 2M hydrochloric acid was added. After reacting for 7 hours, the sample was thoroughly washed with ultrapure water to remove excess hydrochloric acid and dried. The sample was then ground, wrapped in aluminum foil, and tested on the analyzer.

[0085] Example 1 Construction and evaluation of Cas12a-crRNA coupling system

[0086] Based on the Cas12a recognition principle and ArsM sequence characteristics, seven targeting crRNAs were first designed (as shown in Table 1 below). Then, according to method 2.3, two-fold stepwise dilution of the gradient concentration plasmid and negative controls were used to verify and evaluate its sensitivity, signal-to-noise ratio, etc. Figure 3 ag- Figure 4As shown in ag, except for crRNA6 which has a certain background signal, the fluorescence values ​​of other crRNAs are negligible when no target is added. In terms of sensitivity, the detection limits of different crRNAs are relatively close, ranging from 3.9 to 7.8 pM. Therefore, the crRNA designed in this embodiment has the characteristics of low background and high sensitivity as a whole, laying a solid foundation for building a combined system.

[0087] According to method 2.4, we further constructed a combination system consisting of six different amounts of crRNA, crRNA1+2 to crRNA1+2+…+6+7, by using the iterative introduction method to determine whether the sensitivity of the CRISPR / Cas12a detection system can be continuously improved by increasing the amount of crRNA. Figure 5 As shown in ab, when 2 or 3 crRNAs are added, the combined use does not improve the sensitivity of the detection. This indicates that the crRNAs involved in the combined use may need to meet certain conditions to improve the sensitivity of the detection reaction, and the background signal in a single system will not be reduced. Figure 5 As shown in Figure c, after further addition of crRNA4, the sensitivity of the combined system was improved, and the detection limit was reduced from 7.8pM to 1.9pM. Figure 5 As shown in df, the introduction of crRNA5, 6, and 7 can reduce the detection limit, and the lowest value of 0.12pM is achieved when all 7 are used together. Compared with the most sensitive crRNA7, its detection limit is increased by 32 times. In addition to sensitivity, the combination can also effectively improve the reaction speed. Figure 3 ag and Figure 6 As shown in af, under high concentration conditions, the combined reaction signal value can reach saturation in a shorter time, while under low concentration conditions, the combined reaction often has a higher rising slope. In addition, it is worth noting that the introduction of crRNA7 can also significantly reduce the background signal, which will undoubtedly greatly improve the testing results of the subsequent enzyme-linked amplification module, because the background signal may also be amplified in subsequent tests.

[0088] Table 1

[0089]

[0090]

[0091] Example 2 Introduction and optimization of enzyme-linked amplification module

[0092] Considering the low abundance of ArsM in the rice field system and the limited on-site detection conditions, this example further introduces an HRP enzyme-linked probe that amplifies the trans-cleavage signal into the detection system based on the above methods 2.6 and 2.7. This not only improves the detection limit, but also enables visual detection by naked eye through color change. To evaluate the feasibility, we first refer to the reaction conditions of the simultaneous use of 7 crRNAs, and detect the 125pM sample and the blank sample respectively, and observe the results using fluorescence and colorimetry. Figure 7 As shown in a, the color of the experimental group is significantly darker than that of the control group, and the fluorescence signal of the experimental group at 575 nm is also significantly stronger than that of the control group, indicating that this method is feasible.

[0093] Since on-site testing generally does not have the conditions for real-time monitoring of reactions, endpoint detection is generally the only option. Reaction time is a key factor in whether endpoint detection can achieve ideal results. If the time is too short, low-concentration samples will not be able to produce an observable signal. If the time is too long, the reaction will reach saturation, which will not further enhance the signal but will extend the test time. Based on this, this embodiment optimizes the reaction time, such as Figure 7 As shown in Figure b, when the reaction time is 30 minutes, the signal of the low-concentration sample is even lower than the background value and no observable signal can be generated, while the signal of the high-concentration sample is only 400,000 after deducting the background. When the reaction time is increased to 40 minutes, the signal of the low-concentration sample increases significantly, even exceeding the fluorescence intensity of the high-concentration sample under 30 minutes. The test effect of the high-concentration sample also improved greatly, from 400,000 to nearly 1.6 million, an increase of more than 200%. However, if the reaction time is further increased, the signal values ​​of both the low-concentration and high-concentration samples will no longer increase, indicating that the reaction has reached saturation. In summary, 40 minutes was selected as the reaction incubation time.

[0094] Next, the detection limit and detection range of the optimized reaction were evaluated, e.g. Figure 7 As shown in c, the fluorescence method can detect samples with concentrations as low as 16fM, which is another 8-fold reduction based on the previous combination. When the FAM-BHQ1 fluorescent probe was used previously, the quantitative relationship between sample concentration and fluorescence intensity was relatively poor, but after adding the HRP cascade amplification system, its fitting R 2 Up to 0.98 (such as Figure 7 d), the reason may be that, on the one hand, the reaction time of the combined system is short, which effectively avoids the annihilation effect of reaction saturation on the concentration effect; on the other hand, after the use of cascade amplification, the difference in ssDNA cleavage rate caused by concentration difference is further amplified, so that the signal difference of samples with different concentrations is fully reflected.

[0095] To facilitate field testing, this embodiment further evaluated the test effect of the colorimetric method, such as Figure 7As shown in Figure ef, the sensitivity of the colorimetric method can also reach 16fM, and as the sample concentration increases, the solution gradually transitions from a faint light blue to a dark blue, showing a good concentration-color effect. After extraction by ImageJ, its grayscale value and concentration fitting R 2 The fluorescence and colorimetric fitting curves both exhibit power functions, demonstrating a cascade amplification effect when combined with HRP. The exponents of the power functions are all less than 1, likely due to the saturation of HRP color development with increasing concentration.

[0096] In general, the introduction of the HRP cascade amplification system further improves the sensitivity of the reaction, enhances the system's response to changes in sample concentration, expands the dynamic range, and provides fluorescence colorimetric dual-channel detection capabilities, enabling visual quantitative analysis with the naked eye, making it adaptable to the needs of different types of site detection.

[0097] Example 3 Design and integration of high-throughput paper-based microfluidic devices

[0098] Although the reaction developed in Example 2 overcomes the need for precision equipment such as thermal cyclers and signal readers for field testing, the cumbersome pretreatment process and low processing throughput still severely restrict its application in large-scale field screening. Therefore, this example introduces a paper-based microfluidic chip to address these pretreatment and throughput issues.

[0099] The design of paper-based microfluidic device is as follows Figure 8 As shown, it consists of two parts: a paper chip and a PMMA plate for incubation reaction. The paper chip consists of a white hydrophilic area and a black hydrophobic area impregnated with paraffin. The liquid will move along the white channel under the restriction of the hydrophobic area and the capillary force. The specific flow path is Figure 3 Previously, most paper-based chips had only 3-5 channels, which could only test a few samples and could not meet the needs of large-scale screening. To address this problem, this embodiment introduces the cascade amplification concept into the paper-based chip design. Figure 3 As shown, after the reagent is added from the central circular hole of area 2 or area 6, it is evenly dispersed to eight end points driven by capillary action. These eight points correspond to the center of the X-shaped channel of area 3 or area 5. Therefore, the solution will be divided into eight parts and then divided into four parts, eventually forming 32 channels.

[0100] When using, Figure 8As shown, first remove the paper chip and fold it according to step 2, so that area 4 is stacked on area 1. Then, the sample liquid to be tested and guanidine isothiocyanate (GuSCN) lysis solution are sequentially added to the glass fiber in area 4. This allows the DNA in the sample liquid to be retained in the glass fiber and separated from other substances, while area 1 absorbs non-nucleic acid substances in the test liquid. After the sample is loaded, fold it according to step 3, so that area 2 is stacked on area 3, and area 3 is stacked on area 4. Add washing solution to the center hole of area 2. After the washing solution (80% ethanol solution) has completely flowed through, repeat the washing process. The washing process should be at least two times, and the number of washes can be increased as appropriate. After washing, unfold each folded area, wait until the glass fiber is completely dry, and fold it according to step 4, so that area 6 is stacked on area 5, and area 5 is stacked on area 4. Add eluent (double-distilled water) to the center hole of area 6. Transfer the DNA to the reaction area of ​​the paper microfluidic plate, add the corresponding reagents, and seal the plate for reaction.

[0101] In order to evaluate the feasibility of the above device, different concentrations of standard plasmids were tested using the paper-based sensor device to test its sensitivity, dynamic range, and background signal. Figure 9 As shown, samples with a concentration of 16fM can produce colorimetric signal changes observable to the naked eye. After extracting the colorimetric information through ImageJ, 16fM-125pM samples can be quantitatively analyzed, indicating that the device has good compatibility with the method developed in this study.

[0102] To further verify the reliability of the device, a soil spike experiment was designed. The test results are shown in Table 2. The recovery rate was between 90% and 110%, which met the test accuracy requirements of site detection and demonstrated its excellent site detection capabilities.

[0103] Table 2. Detection performance of ArsM in paddy soil samples spiked with ArsM

[0104]

[0105] Example 4 Study on the Abundance of ArsM in Paddy Soil and Its Relationship with Soil Environment

[0106] In order to further verify the feasibility of the method of the present invention in actual sample detection, this embodiment selected 16 points in Tongren, Guizhou to carry out field tests, such as Figure 10 a and 10b. The abundance of ArsM in paddy soil in the study area ranged from 1.05×10 6 ~6.49×10 7Next, the samples were transferred to the laboratory for testing of physical and chemical parameters to explore factors that may affect the abundance of ArsM. The specific results are shown in Table 3 below.

[0107] Table 3. Analysis of ArsM abundance and its environmental factors in paddy soil

[0108]

[0109]

[0110] First, we used hierarchical clustering and principal component analysis to identify environmental factors that were strongly correlated with ArsM abundance. Figure 10 c, 10d) showed that each parameter had a large distance from ArsM abundance. It seemed that only pH and Se might have a certain correlation with ArsM abundance. In the principal component analysis, the length of ArsM itself and the projection lengths on PC1 and PC2 were very short, indicating that the two dimensions of principal component analysis could not well indicate the changes in ArsM gene abundance. Next, based on the above results, we selected two environmental factors, pH and Se, for linear and nonlinear correlation analysis. Figure 10 As shown in Figures ef, both had very weak effects on ArsM abundance, with neither reaching a significant correlation level. However, at concentrations of 0-4 ppm, there was a positive correlation between selenium content and ArsM gene abundance, indicating that selenium appears to increase ArsM gene abundance. Because ArsM is a key pathway for microbial arsenic methylation and detoxification, this result suggests that increased selenium content can enhance microbial arsenic detoxification potential.

[0111] In summary, the field work not only verified the accuracy of the high-throughput paper-based sensor device based on the CRISPR cascade amplification system in POU detection, but also discovered the environmental factor affecting the abundance of the ArsM gene - soil selenium through environmental factor coupling analysis. It also provided a new perspective for the study of selenium-arsenic interaction and demonstrated the great potential of this detection platform in rapid field detection and even mechanism research.

[0112] According to the above embodiments, the present invention has for the first time constructed a high-throughput paper-based microfluidic detection device based on coupled CRISPR / Cas12a and HRP cascade amplification, with a sensitivity of up to 16fM, a dynamic range of 5 orders of magnitude (16fM-125pM), extremely low background signal and high throughput that can detect 32 samples simultaneously and can complete the test within 1 hour. In addition, the platform can react under constant temperature conditions, and the results can be directly observed by the naked eye, getting rid of the need for precision equipment such as thermal cyclers, and can therefore be used for field reactions. More importantly, the processing cost of a single microfluidic device is low, which greatly reduces the cost of testing. The constructed method was applied to the arsenic risk investigation of Wanshan rice fields and its relationship with environmental indicators was analyzed. The results showed that the abundance of ArsM at the test point was between 1.05x 10 6 –6.49x 10 7 There was no significant correlation between ArsM and environmental factors, which will provide a reference for future risk assessment based on molecular markers. Because the platform uses modular assembly, it can be used to test other targets by simply replacing crRNA. Therefore, it is not only ArsM, but also other nucleic acid molecular markers with great potential for site detection and risk screening.

[0113] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A system for detecting ArsM in a sample, comprising: A clustered regularly interspaced short palindromic repeats (CRISPR)-Cas complex comprising a guide RNA targeting ArsM and a CRISPR-associated (Cas) protein or a functional fragment thereof; and Horseradish peroxidase (HRP) complex linked to single-stranded DNA (ssDNA).

2. The system according to claim 1, wherein: The guide RNA includes any one of the nucleotide sequences shown in SEQ ID NOs: 2-8, preferably includes at least 4 of the nucleotide sequences shown in SEQ ID NOs: 2-8, and more preferably includes the nucleotide sequences shown in SEQ ID NOs: 2-8.

3. The system according to claim 1, wherein: The ssDNA is connected to the magnetic beads; preferably, the ssDNA has a nucleotide sequence as shown in SEQ ID NO: 9; and / or The Cas protein is selected from Cas12.

4. A method for detecting ArsM in a sample, comprising the following steps: 1) preparing a CRISPR-Cas complex containing a guide RNA targeting ArsM and a Cas protein or a functional fragment thereof; 2) preparing an HRP complex linked to ssDNA, wherein the ssDNA is preferably linked to magnetic beads; 3) mixing the premix containing the CRISPR-Cas complex with the HRP complex and the plasmid and reacting; and preferably, 4) The reaction product solution of step 3) is subjected to magnetic separation, and the supernatant is subjected to a color development reaction.

5. The method according to claim 4, characterized in that In step 3), the reaction time is 35-45 min; and / or In step 4), the supernatant is transferred to TMB solution for color development reaction; Preferably, the method further comprises detecting the fluorescence intensity of the color reaction product. More preferably, the fluorescence intensity is detected with excitation light of 565-575 nm and emission light of 580-590 nm.

6. A detection device for the detection system according to any one of claims 1 to 3 or the detection method according to claim 4 or 5, wherein the detection device comprises a paper chip and a detection plate.

7. The detection device according to claim 6, characterized in that The paper chip comprises a first region, a second region, a third region, a fourth region, a fifth region and a sixth region having a hydrophobic region and a hydrophilic region; The first region is configured with a plurality of parallel distributed hydrophilic adsorption regions, and the rest are hydrophobic regions; The second region is provided with a hydrophilic drainage channel extending from a central hole to both ends and further leading to multiple end points, and the rest is a hydrophobic region; The third region is configured with a plurality of X-shaped hydrophilic drainage channels distributed in parallel, and the rest are hydrophobic regions; The fourth region is configured with a plurality of parallel distributed hydrophilic loading areas, and the rest are hydrophobic areas; The configuration of the fifth area is the same as that of the third area; The sixth area has the same configuration as the second area; The multiple end points of the hydrophilic area drainage channels of the second region correspond one to one with the intersection points of the X-shaped hydrophilic area drainage channels of the third region when superimposed; The multiple end points of the X-shaped hydrophilic area drainage channels of the third region correspond one-to-one with the hydrophilic adsorption areas of the first region when superimposed; The hydrophilic adsorption area of ​​the first region and the hydrophilic loading area of ​​the fourth region correspond one to one when superimposed; Preferably, the diameter of the hydrophilic loading zone of the fourth region is smaller than the diameter of the hydrophilic adsorption zone of the first region; More preferably, the diameter of the hydrophilic adsorption zone of the first region is 4-8 mm; More preferably, the diameter of the central hole of the drainage channel in the hydrophilic area of ​​the second region is 4-8 mm, and the width of the end point is 1-3 mm; More preferably, the width of the X-shaped hydrophilic area drainage channel in the third region is 1-3 mm; More preferably, the diameter of the hydrophilic loading zone of the fourth region is 3-5 mm; More preferably, the first region, the second region, the third region, the fourth region, the fifth region and / or the sixth region have a length of 60-100 mm and a width of 40-50 mm; More preferably, the first area, the second area, the third area, the fourth area, the fifth area and / or the sixth area are connected in a set order or are independent of each other.

8. The detection device according to claim 7, characterized in that The hydrophobic area is coated with a hydrophobic material, preferably, the hydrophobic material comprises paraffin; and / or The loading area of ​​the fourth region is provided with an adsorption material for adsorbing nucleic acid, preferably, the adsorption material comprises glass fiber; and / or The detection plate is provided with detection areas corresponding one to one with the hydrophilic loading areas in the first region of the paper chip. Preferably, the detection plate is a PMMA plate.

9. Use of the detection system according to any one of claims 1 to 3, the detection method according to any one of claims 4 to 6, or the detection device according to claim 7 or 8 in high-throughput detection of ArsM.

10. A detection kit comprising the CRISPR-Cas complex and the HRP complex in the detection system according to any one of claims 1 to 3; Preferably, the kit further comprises the detection device according to any one of claims 7 to 9.

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