CRISPR (clustered regularly interspaced short palindromic repeats) system-based nucleic acid detection platform detection method and application

Abstract

The invention provides a CRISPR (clustered regularly interspaced short palindromic repeats) system-based nucleic acid detection platform, a CRISPR system-based nucleic acid detection method and application, and belongs to the technical field of biological detection, the CRISPR system-based nucleic acid detection platform comprises a biosensor and a Cas12a / crRNA compound, the Cas12a / crRNA compound comprises functional crRNA and a dsDNA activator, the functional crRNA can guide Cas12a to recognize the dsDNA activator and trigger the trans-cleavage activity of the dsDNA activator, and the crRNA can guide Cas12a to recognize the dsDNA activator. The activated Cas12a non-specific trans-cleavage alkaline phosphatase coupled DNA reporter molecule causes the release of alkaline phosphatase to reduce the formation of gold / silver nanoparticles so as to generate an output signal, and by synergistically combining separated crRNA-dependent Cas12a activation and AuNP SPR signal amplification, the Cas12a / AuNP SPR / AuNP SPR / AuNP SPR / AuNP SPR / AuNP SPR / AuNP SPR / AuNP SPR / AuNP SPR / AuNP SPR / AuNP SPR / AuNP SPR / AuNP SPR / AuNP SPR / AuNP SPR / The kit does not need pre-amplification or multiple nucleic acid target detection assisted by an instrument, and is rapid, convenient, portable and low in cost.

Description

Technical Field

[0001] This application relates to the field of biodetection technology, and in particular to a nucleic acid detection platform, detection method and application based on the CRISPR system. Background Technology

[0002] Breast cancer is the most common and leading cause of cancer-related death among women worldwide, and its global incidence continues to rise. Therefore, establishing efficient and accurate detection systems for relevant tumor markers is crucial for monitoring disease progression and assessing treatment effectiveness. MicroRNAs (miRNAs) are a class of non-coding small RNAs, approximately 18-25 nucleotides in length, that play a key role in gene expression regulation. Dysregulation of miRNA expression is closely related to cancer development. Particularly in breast cancer, many miRNAs (such as miR-21, miR-155, miR-10b, miR-373, miR-520c, miR-27a, miR-9, and members of the miR-17-92 cluster (including miR-17-5p, miR-20a-5p, miR-92a-3p, miR-106b-5p, and miR-93-5p) are overexpressed in tumor tissues and can be detected in biological fluids such as serum or plasma, making them promising biomarkers. However, detection relying on single miRNAs is highly susceptible to interference from individual variations such as physiological state and metabolic differences. Therefore, there is an urgent need to develop highly sensitive multiplex miRNA detection methods.

[0003] However, the concentration of miRNAs in bodily fluids can be as low as femtomolar (fM), resulting in weak detection signals that are easily interfered with by background noise. Traditional detection methods (such as RT-qPCR, microarrays, and sequencing technologies) have improved detection sensitivity to some extent, but they are complex to operate and highly dependent on equipment. In recent years, the rapid development of CRISPR (clustered regularly spaced short palindromic repeats) technology has provided a revolutionary tool for nucleic acid detection, offering high specificity for target recognition and editability. CRISPR / Cas12a and CRISPR / Cas13a systems have attracted considerable attention due to their unique trans-cleavage activity. Activated Cas12a can non-specifically cleave single-stranded DNA (ssDNA), while activated Cas13a can non-specifically cleave single-stranded RNA (ssRNA). Therefore, the Cas13 system is commonly used for miRNA detection, while Cas12-based miRNA detection requires a reverse transcription step. CRISPR / Cas systems for miRNA detection face several technical bottlenecks, including low sensitivity, poor stability of RNA reporter molecules, and limitations in multiplexing. The sensitivity of a single CRISPR / Cas system is insufficient for detecting low concentrations of miRNAs (typically in the fM to aM range) in biological samples. To improve sensitivity, CRISPR / Cas systems are often combined with pre-amplification techniques, but this increases operational complexity and may lead to aerosol contamination. Commonly used CRISPR / Cas13a systems rely on RNA reporter probes, but RNA is susceptible to nuclease degradation and has poor stability. Chen et al. proposed a separate CRISPR-Cas12a system, innovatively applying the Cas12a system to miRNA detection. By separating crRNA into scaffold RNA and spacer RNA, target miRNAs can assemble with the scaffold RNA to form functional crRNAs, thereby activating the trans-cleavage activity of Cas12a. However, its detection sensitivity still needs improvement. Furthermore, the multiplexing capability of CRISPR / Cas systems is limited due to non-specific trans-cleavage.

[0004] Therefore, there is an urgent need to develop new CRISPR / Cas-based molecular diagnostic methods that possess high sensitivity, strong stability, and multiplicity. Summary of the Invention

[0005] Therefore, it is necessary to provide a biosensor with high sensitivity, strong stability and multiplexity, a nucleic acid detection platform based on the CRISPR system, preparation method and application to address the technical problem of limited multiplex detection capability of the current CRISPR / Cas system.

[0006] To solve the above problems, this application adopts the following technical solution:

[0007] One objective of this application is to provide a nucleic acid detection platform based on a CRISPR system, comprising a biosensor and a Cas12a / crRNA complex. The biosensor comprises gold / silver bimetallic nanoparticles, and the Cas12a / crRNA complex comprises functional crRNA and a dsDNA activator. The functional crRNA can guide Cas12a to recognize the dsDNA activator and trigger its trans-cleavage activity. The activated Cas12a non-specifically trans-cleaves an alkaline phosphatase-coupled DNA reporter molecule, leading to the release of alkaline phosphatase, thereby reducing the formation of gold / silver nanoparticles and generating an output signal.

[0008] In some embodiments, the Cas12a / crRNA complex includes a scaffold RNA, a spacer RNA, and a dsDNA activator complementary to the spacer RNA, wherein the spacer RNA assembles with the scaffold RNA to form the functional crRNA, and the functional crRNA guides Cas12a to recognize the dsDNA activator.

[0009] In some embodiments, the Cas12a concentration is 50 nM, the scaffold RNA concentration is 100 nM, the dsDNA activator is 500 nM, the FAM-ssDNA-biotin concentration is 10 nM, the alkaline phosphatase-labeled streptavidin concentration is 500 pM, and the optimal length of the spacer RNA is 20 nt.

[0010] In some embodiments, the method for fabricating the biosensor includes the following steps:

[0011] Alkaline phosphatase catalyzes the hydrolysis of p-aminophenyl phosphate to obtain p-aminophenol;

[0012] The p-aminophenol reduces silver ions and deposits metallic silver on the surface of gold nanoparticles to form a biosensor with gold / silver bimetallic nanoparticles.

[0013] In some embodiments, the step of catalyzing the hydrolysis of p-aminophenyl phosphate with alkaline phosphatase to obtain p-aminophenol includes the following steps:

[0014] Alkaline phosphatase-labeled streptavidin is mixed with p-aminophenyl phosphate and subjected to a hydrolysis reaction to produce aminophenol and phosphate. The molar ratio of alkaline phosphatase-labeled streptavidin to p-aminophenyl phosphate is in the range of 1:10. 11 -1:10 9 .

[0015] In some embodiments, the steps of further reducing the p-aminophenol to silver ions and depositing metallic silver on the surface of gold nanoparticles to form a biosensor with a gold / silver bimetallic nanostructure include the following steps:

[0016] The aminophenol, silver ion solution, and gold nanoparticles are mixed, and the aminophenol reduces the silver ions and deposits metallic silver on the surface of the gold nanoparticles to form a biosensor with a gold / silver bimetallic nanostructure. The molar concentration ratio of the aminophenol, the silver ion solution, and the gold nanoparticles is 50:50:1-50.

[0017] In some embodiments, the gold nanoparticles are synthesized using L-reduced glutathione as a stabilizer and ligand, and nicotinamide adenine dinucleotide phosphate as a reducing agent.

[0018] The second objective of this application is to provide a detection method for the nucleic acid detection platform based on the CRISPR system, comprising the following steps:

[0019] The target RNA and scaffold RNA assemble to form a functional crRNA, which can guide Cas12a to recognize the dsDNA activator and trigger its trans-cleavage activity. The activated Cas12a non-specifically trans-cleaves the alkaline phosphatase-coupled DNA reporter molecule, resulting in the release of alkaline phosphatase to generate an output signal.

[0020] In some embodiments, the output signal can be obtained via ultraviolet-visible absorption spectroscopy and by the naked eye.

[0021] The third objective of this application is to provide an application of the aforementioned CRISPR-based nucleic acid detection platform in miRNA detection.

[0022] The present application adopts the above technical solution, and its beneficial effects are as follows:

[0023] This application also provides a nucleic acid detection platform based on a CRISPR system and its preparation method, including the aforementioned biosensor and Cas12a / crRNA complex. The Cas12a / crRNA complex includes functional crRNA and a dsDNA activator. The functional crRNA can guide Cas12a to recognize the dsDNA activator and trigger its trans-cleavage activity. The activated Cas12a non-specifically trans-cleaves the alkaline phosphatase-coupled DNA reporter molecule, resulting in the release of alkaline phosphatase to generate an output signal. The number of intact FAM-ssDNA-biotin captured in the microplate wells coated with streptavidin is reduced, which directly leads to a reduction in the binding of alkaline phosphatase-coupled streptavidin (SA-ALP). The reduction in immobilized SA-ALP reduces the deposition of silver on gold nanoparticles, thereby reducing the formation of gold / silver nanoparticles (Au / AgNPs), causing a significant change in the local dielectric environment, and regulating the SPR properties of the nanoparticles. This modulation manifests as a measurable reduction in optical absorption signal, providing quantitative readings. It exhibits extremely high sensitivity (down to the fM range) and high specificity by synergistically binding dissociative crRNA-dependent Cas12a activation with AuNP SPR signal amplification. By enabling multiplex detection of miRNAs in biological fluids, it effectively reduces the interference of individual biological variability and has great potential in enhancing early detection, prognostic assessment, and personalized treatment monitoring.

[0024] The nucleic acid detection platform based on the CRISPR system provided in this application can be widely used for rapid on-site detection of miRNA, and for multiplex nucleic acid target detection without pre-amplification or instrument assistance. It is fast, convenient, portable and low cost. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments of this application or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a schematic flowchart illustrating the workflow of a nucleic acid detection platform based on the CRISPR system provided in an embodiment of the present invention.

[0027] Figure 2 This provides a schematic diagram of ALP-mediated conversion of gold nanoparticles to gold / silver nanoparticles for embodiments of the present invention.

[0028] Figure 3The diagram illustrates the absorbance and linear relationship of the gold / silver nanoparticle generation system mediated by lysed crRNA activation and SA-ALP at gold nanoparticle concentrations of 40, 60, and 80 nM, as provided in embodiments of the present invention.

[0029] Figure 4 This diagram illustrates the construction and optimization of a CRISPR / Cas12a CRISPR-based nucleic acid detection platform integrating ALP, as provided in an embodiment of the present invention.

[0030] Figure 5 This is a schematic diagram of a CRISPR / Cas12a CRISPR-based nucleic acid detection platform for dual-mode detection of spacer RNA, provided in an embodiment of the present invention.

[0031] Figure 6 This is a schematic diagram illustrating the dual-mode detection of miRNA-106b-5p by the CRISPR / Cas12a CRISPR-based nucleic acid detection platform provided in this embodiment of the invention.

[0032] Figure 7 This is a schematic diagram of the CRISPR / Cas12a dual-mode miRNA-9 detection platform provided in an embodiment of the present invention.

[0033] Figure 8 This is a schematic diagram illustrating the performance evaluation of the CRISPR / Cas12a CRISPR-based nucleic acid detection platform for detecting clinical breast cancer-related miRNAs, as provided in this embodiment of the invention. Detailed Implementation

[0034] The embodiments of this application are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application.

[0035] In the description of this application, it should be understood that the terms "upper", "lower", "horizontal", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application.

[0036] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified.

[0037] In the field of molecular diagnostics, the concentration of miRNAs in body fluids can be as low as the femtomolar (fM) level, resulting in weak detection signals that are easily interfered with by background noise. While traditional detection methods (such as RT-qPCR, microarrays, and sequencing technologies) have improved detection sensitivity to some extent, they are complex to operate and highly dependent on equipment. CRISPR / Cas12a and CRISPR / Cas13a systems have attracted considerable attention due to their unique trans-cleavage activity. Activated Cas12a can non-specifically cleave single-stranded DNA (ssDNA), while activated Cas13a can non-specifically cleave single-stranded RNA (ssRNA). Therefore, the Cas13 system is commonly used for miRNA detection, while Cas12-based miRNA detection requires a reverse transcription step. CRISPR / Cas systems for miRNA detection face several technical bottlenecks, including low sensitivity, poor stability of RNA reporter molecules, and limitations in multiplexing. The sensitivity of a single CRISPR / Cas system cannot meet the detection requirements for low concentrations of miRNAs (typically in the fM to aM range) in biological samples. To improve sensitivity, CRISPR / Cas systems are often combined with pre-amplification techniques, but this increases operational complexity and can lead to aerosol contamination. Therefore, there is an urgent need to develop new CRISPR / Cas-based molecular diagnostic methods that possess high sensitivity, strong stability, and multiplicity.

[0038] Therefore, this application provides a biosensor capable of achieving high sensitivity, strong stability and multiplicity, a nucleic acid detection platform based on the CRISPR system, a preparation method and applications.

[0039] The technical solution of this application will be described in detail below with reference to the embodiments.

[0040] The reagents and instruments involved in this application are as follows:

[0041] In this application, all oligonucleotides used in this study (Table S1) were synthesized by Shanghai Sangon Biotech Co., Ltd. Lba Cas12a (Cpf1) nuclease was purchased from Beijing Solarbio Science & Technology Co., Ltd. L-reduced glutathione (GSH) and reduced coenzyme II (NADPH) were purchased from Shanghai Baisha Biotechnology Co., Ltd. Glutathione reductase (GR) and silver nitrate were purchased from Shanghai Sigma-Aldrich Trading Co., Ltd. Chloroauric acid was purchased from Shanghai Maclean Biotechnology Co., Ltd. Rabbit anti-6-FAM polyclonal antibody and DEPC-treated water were purchased from Shanghai Sangon Biotech Co., Ltd. 4-Aminophenyl phosphate monosodium salt hydrate (p-APP) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Alkaline phosphatase coupled with streptavidin (SA-ALP) was purchased from Shanghai Bio-Sens Biotechnology Co., Ltd. HiPure serum / plasma miRNA medium-volume extraction kit (R4317-02) was purchased from Guangzhou Meiji Biotechnology Co., Ltd. The morphology and X-ray energy-dispersive X-ray spectra of the samples were characterized using transmission electron microscopy (FEI-TALOS-F200X, Thermo Fisher Scientific) and scanning electron microscopy (SU8010, Hitachi). The mass of chemical reagents was weighed using an electronic analytical balance (BSA124S-CW, Sartorius). Hydrodynamic diameter and zeta potential were measured using a Zetasizer Nano ZSE (Malvin). Real-time fluorescence signals were measured using a quantitative polymerase chain reaction (qPCR) detection system (FQD-96A, Hangzhou Bori Biotechnology Co., Ltd.). The absorbance of the samples was measured using an Infinite 200pro (Tecan).

[0042] Table S1 Sequences involved in this application

[0043]

[0044]

[0045] Based on the above reagents and instruments, this application details the specific implementation scheme.

[0046] This embodiment provides a method for preparing a biosensor, including alkaline phosphatase catalyzing the hydrolysis of p-aminophenyl phosphate to obtain p-aminophenol; the p-aminophenol reduces silver ions and deposits metallic silver on the surface of gold nanoparticles to form a biosensor with gold / silver bimetallic nanoparticles.

[0047] In this embodiment, the step of hydrolyzing p-aminophenyl phosphate with alkaline phosphatase to obtain p-aminophenol includes the following steps: mixing alkaline phosphatase-labeled streptavidin with p-aminophenyl phosphate for hydrolysis to generate aminophenol and phosphate, wherein the molar ratio of alkaline phosphatase-labeled streptavidin to p-aminophenyl phosphate is in the range of 1:10. 11 -1:10 9.

[0048] In this embodiment, the step of further reducing silver ions with p-aminophenol and depositing metallic silver on the surface of gold nanoparticles to form a biosensor with a gold / silver bimetallic nanostructure includes the following steps: mixing the aminophenol, silver ion solution and gold nanoparticles; reducing silver ions with aminophenol and depositing metallic silver on the surface of the gold nanoparticles to form a biosensor with a gold / silver bimetallic nanostructure; the molar concentration ratio of the aminophenol, the silver ion solution and the gold nanoparticles is 50:50:1 to 50:500:1.

[0049] In this embodiment, the gold nanoparticles are synthesized using L-reduced glutathione as a stabilizer and ligand, and nicotinamide adenine dinucleotide phosphate as a reducing agent.

[0050] Specifically, 7.5 mg of solid GSH was dissolved in ultrapure water and diluted to 100 μL. The GSH solution (24 μM) was then added to chloroauric acid (1%, 72.8 μM) solution, and the pH was adjusted to 2.5–3.0 with 10 M sodium hydroxide (NaOH). The mixture was transferred to a centrifuge tube and centrifuged at 7000 rpm for 3 minutes to collect the Au(I)-GSH complex. The purified Au(I)-GSH complex was redispersed in 1 mL of sodium hydroxide solution (5 mM). This solution was diluted with 9 mL of ultrapure water and the pH was adjusted to 5.5. Subsequently, 4 mg of NADPH and 2 units of GR were added. Finally, the mixture was stirred at room temperature for 2 hours to obtain gold nanoparticles. The gold nanoparticles were purified using an Amicon ultrafiltration tube (molecular weight cutoff = 30 kDa), and the obtained gold nanoparticles were redispersed in 1 mL of ultrapure water.

[0051] It is understood that the biosensor fabrication method provided in this embodiment utilizes alkaline phosphatase (ALP) to catalyze the hydrolysis of p-aminophenyl phosphate (p-APP), generating p-aminophenol (p-AP) and phosphate. Subsequently, p-AP promotes the reduction and deposition of silver ions (Ag+) on the surface of gold nanoparticles (AuNPs), thereby forming gold-silver bimetallic nanoparticles (Au / AgNPs). This ALP-mediated SPR effect provides a simple and rapid method for ALP detection.

[0052] The specific experimental procedures are as follows: First, silver nitrate (2 mM) and p-APP (8 mM) (both dissolved in diethanolamine buffer) were added to SA-ALP solutions of different concentrations (0, 0.05, 0.1, 0.2, 0.4, 0.5, 0.6, 0.8, 1, 1.5, 2, and 2.5 pM). Then, the mixtures were added to gold nanoparticle solutions with concentrations of 40, 60, and 80 nM, respectively. The total volume of each system was adjusted to 100 μL using diethanolamine buffer. The solutions were incubated at 37 °C and 450 rpm for 30 minutes, and the UV-Vis absorption spectra at wavelengths of 300–550 nm were recorded.

[0053] This embodiment also provides a nucleic acid detection platform based on a CRISPR system, including the biosensor and Cas12a / crRNA complex described in the above embodiment. The Cas12a / crRNA complex includes a functional crRNA and a dsDNA activator. The functional crRNA can guide Cas12a to recognize the dsDNA activator and trigger its trans-cleavage activity. The activated Cas12a non-specifically trans-cleaves the alkaline phosphatase-coupled DNA reporter molecule, resulting in the release of alkaline phosphatase to reduce the formation of gold / silver nanoparticles, thereby generating an output signal.

[0054] Please see Figure 1 This is a schematic diagram of the workflow of the CRISPR-based nucleic acid detection platform provided in this embodiment. It includes a schematic diagram of the workflow of the CRISPR / Cas12a CRISPR-based nucleic acid detection platform, which can be used for the detection of breast cancer-related miRNAs. The target miRNA assembles with the scaffold RNA to form a functional crRNA, initiating the assembly of the Cas12a / crRNA complex. This complex recognizes the dsDNA activator and trans-cleaves the ALP-conjugated DNA reporter molecule, leading to ALP release. Conversely, in samples without target miRNA, Cas12a remains intact, the reporter system is not cleaved, and ALP is not released, thus ALP catalyzes Ag... + Reduction on the surface of gold nanoparticles generates bimetallic gold / silver nanoparticles with unique plasmonic properties. The detection signal is output via a dual-mode readout, including instrumental methods (UV-Vis absorption spectroscopy) and visual detection.

[0055] Furthermore, the Cas12a / crRNA complex includes scaffold RNA, spacer RNA, and a dsDNA activator complementary to the spacer RNA. The spacer RNA and the scaffold RNA assemble to form the functional crRNA, which guides Cas12a to recognize the dsDNA activator.

[0056] Specifically, two complementary single-stranded DNAs (50 μM) were denatured at 95 °C for 10 min, then gradually cooled to 25 °C at a rate of 1 °C / 10 s to form a dsDNA activator. A CRISPR reaction mixture (100 μL) containing different concentrations of target RNA (0, 100 pM, 1 nM, 10 nM, 50 nM), Cas12a (250 nM), scaffold RNA (500 nM), dsDNA activator (500 nM), reporter probe (1000 nM), and 1×NEBuffer r2.1 (diluted to a final concentration of 1× by 10×NEBuffer r2.1) was recorded every 10 s using a qPCR thermal cycler. The signal of the 6-FAM fluorophore (λex = 494 nm, λem = 520 nm) was recorded every 10 s.

[0057] Furthermore, the Cas12a concentration is 50 nM, the scaffold RNA concentration is 100 nM, the dsDNA activator is 500 nM, the FAM-ssDNA-biotin concentration is 10 nM, the alkaline phosphatase-labeled streptavidin (SA-ALP) concentration is 500 pM, and the optimal length of the spacer RNA is 20 nt.

[0058] The detection method of the nucleic acid detection platform based on the CRISPR system provided in the above embodiments of this application includes the following steps:

[0059] The target RNA and scaffold RNA assemble to form a functional crRNA, which can guide Cas12a to recognize the dsDNA activator and trigger its trans-cleavage activity. The activated Cas12a non-specifically trans-cleaves the alkaline phosphatase-coupled DNA reporter molecule, resulting in the release of alkaline phosphatase to generate an output signal.

[0060] In some embodiments, the CRISPR-based nucleic acid detection platform can be used for RNA detection.

[0061] Specifically, first, rabbit anti-6-FAM polyclonal antibody was diluted 1:100 with carbonate buffer (pH = 9.6). 100 μL of antibody was added to each well of a 96-well plate and incubated at 4°C for 12 hours. After incubation, the 96-well plate was washed three times with 300 μL of wash buffer (10×PBST diluted to 1× with ultrapure water, containing 0.05% Tween 20, pH 7.4). Then, 300 μL of 2.5% (w / v) bovine serum albumin (BSA) was added to each well of the 96-well plate. The plate was incubated at 37°C for 2 hours in a microplate shaker, and then washed three times with 300 mL of wash buffer. Next, different concentrations of target RNA (0, 100 fM, 1 pM, 5 pM, 10 pM, 100 pM, 1 nM, 5 nM, 10 nM, 50 nM), Cas12a (50 nM), scaffold RNA (100 nM), dsDNA activator (500 nM), FAM-ssDNA-biotin (10 nM), and 1×NEBuffer r2.1 were added to each well of a 96-well plate. DEPC-treated water was added to adjust the total system volume to 100 μL. The plate was incubated at 37 °C and 450 rpm with continuous shaking for 1 hour. After incubation, each well was washed three times with 300 μL of washing buffer. Then, 100 μL of gold nanoparticle reaction system containing gold nanoparticles (60 nM), silver nitrate (2 mM), and p-APP (8 mM) was added. Finally, diethanolamine buffer was added to adjust the total system volume to 100 μL. The solution was incubated at 37°C and 450 rpm for 30 minutes. The 96-well plate was placed in a microplate reader, and the ultraviolet absorption spectrum at wavelengths of 300-550 nm was scanned.

[0062] In other embodiments, the CRISPR-based nucleic acid detection platform is used for miRNA detection.

[0063] Specifically, to verify the universality of this method, an amplification-free CRISPR sensor was used to detect miRNA-106b-5p (21 nt) and miRNA-9 (24 nt), following the same procedures as described above. Twelve miRNA concentration levels (0, 1 fM, 10 fM, 100 fM, 1 pM, 5 pM, 10 pM, 100 pM, 1 nM, 5 nM, 10 nM, 50 nM) were detected, spanning seven orders of magnitude, to evaluate the sensor's performance over a wide dynamic range. For multiplex detection of miRNAs, breast cancer-related miRNAs (T1: miRNA-21, T2: miRNA-155, T3: miRNA-10b, T4: miRNA-373, T5: miRNA-520c, T6: miRNA-27a, T7: miRNA-9, T8: miRNA-17-5p, T9: miRNA-20a-5p, T10: miRNA-92a-3p, T11: miRNA-106b-5p, and T12: miRNA-93-5p) were selected, and the detection procedure was consistent with the description above. The concentration of all targets was 1 nM. Furthermore, the dsDNA activators corresponding to these 12 target miRNAs and all oligonucleotides are labeled D1-D12 in the figure.

[0064] Furthermore, in the above embodiments, miRNA can be extracted through the following process.

[0065] miRNA was extracted from plasma using the HiPure Serum / Plasma MiRNA Medium-Quantity Extraction Kit (R4317-02). First, 50 μL of proteinase K (20 mg / mL) was added to 1 mL of plasma sample. Then, 1 mL of Buffer SML1 was added. The solution was vortexed for 10 seconds and incubated at room temperature for 20 minutes, inverting the tube twice during this period. Next, 2 mL of Buffer PCI2 was added; the solution became cloudy and yellow insoluble matter appeared. After vortexing for 15 seconds, it was incubated at room temperature for 3 minutes, then centrifuged at 4000-5000g for 10 minutes to remove the flocculent yellow insoluble matter. Third, all supernatant was transferred to a new centrifuge tube. An equal volume of isopropanol was added. The solution was then vortexed for 5 seconds and incubated at room temperature for 3 minutes. Next, 750 μL of the solution was transferred to a HiPure RNA mini-column and centrifuged at 12000g for 30 seconds. This step was repeated until all mixtures were filtered through the column. In addition, add 500 μL of Buffer RWC and centrifuge at 12000g for 30 seconds, then add 500 μL of Buffer RW2 and centrifuge at 12000g for 30 seconds (twice). After a second wash with Buffer RW2, centrifuge the empty column at 12000g for 3 minutes to dry the matrix. Finally, add RNase-free water and incubate at room temperature for 1 minute. Centrifuge to collect the extracted RNA and store at -80℃ to obtain miRNA. The extracted miRNA can be detected using the described CRISPR-based nucleic acid detection platform.

[0066] Furthermore, the embodiments of this application described above will be further explained below with reference to the accompanying drawings and data.

[0067] Please refer to the following: Figure 1 The surface plasmon resonance (SPR) effect of gold nanoparticles mediated by SA-ALP was characterized. SA-ALP catalyzes the hydrolysis of p-APP, and the generated p-AP reduces silver ions (Ag+) to silver atoms (Ag0), which are then deposited on the surface of gold nanoparticles to form gold / silver nanoparticles (Au / AgNPs). The formation of the silver shell alters the dielectric environment around the particles, significantly enhancing the surface plasmon resonance (SPR) effect, manifested as a shift in plasmon resonance wavelength and an increase in absorbance.

[0068] Please see Figure 2This diagram illustrates the ALP-mediated transformation of gold nanoparticles into gold / silver nanoparticles. (A) is a TEM micrograph of the gold nanoparticles; (B) is the corresponding particle size distribution histogram (scale bar: 100 nm); (C) is a scanning electron microscope (SEM) image (scale bar: 100 nm); (D) is the X-ray energy-dispersive spectroscopy (EDS) characterization of the gold nanoparticles; (E) is the SEM image of the gold / silver nanoparticles and (F) is the EDS characterization; (G) is the zeta potential of the gold and gold / silver nanoparticles; (H) is the hydrodynamic diameter analysis; and (I) is the UV-Vis absorption spectrum of the gold and gold / silver nanoparticles, with the characteristic peak at 520 nm corresponding to the formation of gold nanoparticles and the peak at 370 nm indicating the formation of gold / silver nanoparticles.

[0069] TEM analysis ( Figure 2 As shown in Figure A, the synthesized gold nanoparticles possess a uniform spherical morphology and good dispersibility, with an average diameter of 14.85 nm. Figure 2 (B) The morphology and elemental composition of gold nanoparticles and gold / silver nanoparticles were further characterized using SEM-EDS. Consistent with the TEM results, the SEM images ( Figure 2 (C) Confirmed that the gold nanoparticles were uniformly spherical with smooth surfaces. EDS analysis showed a characteristic gold (Au) peak at 2.1 keV, and quantitative analysis indicated a gold content of 100%. Figure 2 (D). In contrast, the morphology and composition of gold / silver nanoparticles changed significantly after silver shell deposition. Figure 2 (E). For example Figure 2 As shown in Figure F, ALP-catalyzed silver deposition leads to particle aggregation and increased surface roughness. The EDS spectrum of the gold / silver nanoparticles shows a characteristic peak for gold (Au) at 2.1 keV and a characteristic peak for silver (Ag) at 3.0 keV. Notably, the quantitative content of gold (Au) is significantly lower than that of pure gold nanoparticles, which is due to the extensive silver (Ag) shell covering the surface of the gold nanoparticles. To further analyze the colloidal stability, size characteristics, and optical properties of gold nanoparticles and gold / silver nanoparticles, their zeta potential, hydrodynamic diameter, and UV-Vis absorption spectra were characterized. Figure 2 As shown in Figure G, the zeta potential of gold nanoparticles is -33.2 mV, indicating significant electrostatic repulsion between particles. In contrast, the zeta potential of gold / silver nanoparticles is more negative (-38.3 mV), suggesting that the formation of the silver shell alters the surface chemistry and may increase the surface charge density. Hydrodynamic diameter measurement ( Figure 2The H-axis (H) shows that the average diameter of the gold nanoparticles is approximately 67.05 nm, with a polydispersity index (PDI) of 0.173, indicating good monodispersity. After the formation of gold / silver nanoparticles, the hydrodynamic diameter increases sharply to approximately 1020.33 nm, with a PDI of 0.292. The significant increase in size and PDI is attributed to silver shell deposition and subsequent nanoparticle aggregation.

[0070] Please see Figure 3 The figures show the absorbance and linear relationship of the gold / silver nanoparticle generation system mediated by crRNA lysis and SA-ALP at gold nanoparticle concentrations of 40, 60, and 80 nM. In the 40 nM gold nanoparticle system: (A) shows the change in the UV-Vis absorption spectrum of gold nanoparticles after ALP-catalyzed pAPP hydrolysis as the SA-ALP concentration increases from 0 to 2.5 pM; (B) shows the linear calibration curve of absorbance at 370 nm versus SA-ALP concentration (0.05–2.5 pM). In the 60 nM gold nanoparticle system: (C) shows the change in the UV-Vis absorption spectrum of gold nanoparticles after ALP-catalyzed pAPP hydrolysis as the SA-ALP concentration increases from 0 to 2.5 pM; (D) shows the linear calibration curve of absorbance at 370 nm versus SA-ALP concentration (0.05–2.5 pM). In the 80 nM gold nanoparticle system: (E) shows the change in the UV-Vis absorption spectrum of gold nanoparticles as the SA-ALP concentration increases from 0 to 2.5 pM after the ALP-catalyzed pAPP hydrolysis reaction; (F) shows the linear calibration curve of absorbance at 370 nm versus SA-ALP concentration (0.05-2.5 pM).

[0071] according to Figure 3 UV-Vis absorption spectroscopy revealed a relatively symmetrical surface plasmon resonance (SPR) peak at 520 nm for the gold nanoparticles. After silver shell formation, the gold / silver nanoparticles exhibited a significant absorption peak at 370 nm. These results collectively confirm the successful formation of a silver shell on the surface of the gold nanoparticles, significantly altering their surface morphology, elemental composition, and optical properties. To evaluate the feasibility of signal amplification using the SA-ALP-mediated SPR effect, different concentrations of SA-ALP (0.05 pM to 2.5 pM) were added to catalyze the conversion of gold nanoparticles to gold / silver nanoparticles. With increasing SA-ALP concentration, the intensity of the characteristic absorption peak at 370 nm (A370) systematically increased, and A370 showed a good linear relationship with SA-ALP concentration.

[0072] Figure 3A system comparison of detection performance using gold nanoparticles at different concentrations (40, 60, and 80 nM) showed that the system constructed using 60 nM gold nanoparticles exhibited the best linear correlation (R² = 0.9905). This optimized system effectively utilizes the SPR effect of the generated gold / silver nanoparticles as a signal amplification strategy for the sensor.

[0073] Furthermore, to verify the concept of activating the CRISPR / Cas12a system by splitting crRNA, this application designed a scaffold RNA, a spacer RNA (derived from crRNA), and a dsDNA activator complementary to the spacer RNA. The spacer RNA and scaffold RNA assemble to form a functional crRNA, which guides Cas12a to recognize the dsDNA activator, thereby triggering its trans-cleavage activity. Subsequently, the activated Cas12a nonspecifically cleaves the free single-stranded DNA (ssDNA) reporter probe in the system, generating a signal output.

[0074] Please see Figure 4 This diagram illustrates the construction and optimization of a CRISPR / Cas12a-based nucleic acid detection platform integrating ALP. (A) shows the feasibility verification of activating the CRISPR system with isolated crRNA. (B) shows the optimization of Cas12a and scaffold RNA concentrations; (C) shows the dsDNA activator concentration; (D) shows the FAM-ssDNA-biotin concentration; (E) shows the SA-ALP concentration; and (F) shows the spacer RNA length. The optimal conditions for Cas12a, scaffold RNA, dsDNA activator, FAM-ssDNA-biotin, and SA-ALP are 50 nM, 100 nM, 500 nM, 10 nM, and 500 pM, respectively, with an optimal spacer RNA length of 20 nt.

[0075] To assess the feasibility of this assembly-dependent activation, different concentrations of spacer RNA (0, 100 pM, 1 nM, 10 nM, 50 nM) were added to the CRISPR / Cas12a system, and the kinetic fluorescence intensity of the reporter molecule was monitored. Figure 4(A). As the RNA concentration in the spacer region increased from 100 pM to 50 nM, a significant increase in fluorescence intensity was observed. These results confirm that the detached crRNA assembly can reconstruct functional crRNA through molecular self-assembly, and the restored functional crRNA effectively activates the trans-cleavage activity of Cas12a, leading to the cleavage of the reporter probe. We combined the SA-ALP-mediated SPR effect of gold nanoparticles with the crRNA-detached CRISPR / Cas12a system. To achieve optimal detection sensitivity of the amplification-free CRISPR / Cas12a biosensor activated by crRNA detachment, the conditions of the crRNA-detached CRISPR / Cas12a system and the SA-ALP-mediated gold nanoparticle SPR system were optimized. Initial optimizations focused on the CRISPR / Cas12a assembly ( Figure 4 (Middle BC). The concentrations of Cas12a and scaffold RNA were evaluated, and the results showed that the SPR peak signal change corresponding to gold / silver nanoparticle formation was greatest at 50 nM Cas12a and 100 nM scaffold RNA. The concentration of dsDNA activator was optimized, and the optimal signal response was found in the range of 250-500 nM; 500 nM was ultimately selected for subsequent experiments. Subsequently, key parameters of the biosensor system itself were optimized. (For example...) Figure 4 As shown in Figure D, tests at different FAM-ssDNA-biotin concentrations revealed that 10 nM showed the best signal change amplitude, increasing by 15.8% and 85.8% compared to 5 nM and 20 nM, respectively. SA-ALP concentration optimization ( Figure 4 The results showed that the signal change was greatest at 500 pM, increasing by 66.6% and 15.4% compared to 100 pM and 1000 pM, respectively. Finally, the lengths of the spacer RNA (18, 20, 22, and 24 nt) were assessed. Figure 4 The signal change was greatest when the F-mode RNA was displayed in the 20nt spacer region.

[0076] In summary, the optimal conditions were determined to be: Cas12a 50nM, scaffold RNA 100nM, dsDNA activator 500nM, FAM-ssDNA-biotin 10nM, SA-ALP 500pM, and the optimal spacer RNA length was 20nt.

[0077] Furthermore, the performance of an amplification-free CRISPR biosensor integrating split crRNA activation with ALP-mediated SPR effects was evaluated under optimized reaction conditions for RNA detection.

[0078] Please see Figure 5This is a schematic diagram illustrating a CRISPR / Cas12a-based nucleic acid detection platform using a separate crRNA activation and ALP integration system for dual-mode detection of spacer RNA. (A) shows different target spacer RNA concentrations (0 to 5 × 10⁻⁶). 7 (fM) shows the UV-Vis absorption spectrum of the detection system. (B) is the absorbance at 370 nm as a function of RNA concentration in the spacer region (0 to 5 × 10⁻⁶). 7 The change in fM). The logarithm of ΔA370 and the RNA concentration in the target spacer region ranged from 100 to 1 × 10⁻⁶. 5 fM and (D) are 1×10 5 Up to 5×10 7 Two linear relationships were established within the fM range. (E) shows a schematic diagram and results of visual detection of RNA in the spacer region. Linear regression analysis of grayscale value and logarithm of RNA concentration in the spacer region: (F) shows the low concentration range (10²-10⁻⁶). 5 fM) and (G) are in the high concentration range (1×10⁻⁶). 5 -5×10 7 fM).

[0079] As the RNA concentration in the spacer region increased from 102 to 5×10 7 The absorbance of the gold / silver nanoparticles at 370 nm decreased from 3.74 ± 0.09 to 0.37 ± 0.09 (fM). Figure 5 (A). There was a statistically significant difference in absorbance between the 100 fM spacer RNA sample and the blank control (P < 0.05). Figure 5 (B) indicates that this biosensor can sensitively detect spacer RNA down to 100 fM. In 10 2 Up to 10 5 Within the range of RNA concentration in the spacer region of fM, ΔA370 showed a good linear relationship with the logarithm of the spacer region RNA concentration. The linear regression equation was Y = 0.1271X - 0.04259(R²). 2 =0.9724), the limit of detection (LOD) is 1.88 fM ( = 0.9724), Figure 5 (C) In 10 5 Up to 5×10 7 Another linear relationship was observed over the extended concentration range of fM (Y = 0.1271X - 5.197, R...). 2 =0.9714)( Figure 5 (D). Furthermore, target quantification can be achieved through visual detection of solution color changes (D). Figure 5 (Middle E). As the RNA concentration in the spacer region increased from 0 to 5 × 10⁻⁶ 7The solution color gradually transitioned from dark brown to light yellow, with decreasing transparency; the color difference was clearly visible to the naked eye. ImageJ software analysis showed a good linear relationship between the grayscale value of the solution and the logarithm of the RNA concentration in the spacer region. Figure 5 China F and Figure 5 As shown in G, the corresponding linear regression equations are Y = 4.414X + 171.2(10) 2 -1×10 5 fM,R 2 =0.9620) and Y = 2.268X + 182.1(10 5 fM-5×10 7 fM,R 2 =0.9616). This amplification-free CRISPR / Cas12a system, which integrates the split crRNA activation and SPR effect, exhibits high sensitivity and a wide linear range for RNA detection. Furthermore, the significant color change induced by the SPR effect of the gold / silver nanoparticles enables reliable visual analysis.

[0080] Furthermore, to evaluate the analytical performance of this method in detecting breast cancer-related miRNAs, synthetic miRNA sequences were used as targets.

[0081] Please see Figure 6 This diagram illustrates the dual-mode detection of miRNA-106b-5p using a CRISPR / Cas12a CRISPR-based nucleic acid detection platform with isolated crRNA activation and ALP integration. (A) shows different target miRNA-106b-5p concentrations (0 to 5 × 10⁻⁶). 7 (a) shows the UV-Vis absorption spectrum of the detection system under fM; (b) shows the absorbance at 370 nm as a function of miRNA-106b-5p concentration (0 to 5 × 10⁻⁶). 7 (fM) represents the change; (C) represents the logarithm of ΔA370 and the target miRNA-106b-5p concentration in the range of 10 to 10. 5 fM and 1×10 5 Up to 5×10 7 Two linear relationships were established within the fM range. (D) shows a schematic diagram and results of visual detection of miRNA-106b-5p. (E) shows the linear regression analysis of gray value and the logarithm of miRNA-106b-5p concentration: low concentration range (10 1 -10 5 fM) and high concentration range (10 5 -5×10 7 fM).

[0082] First, using miRNA-106b-5p (21 nt) as a representative target, its detection capability for miRNAs of different lengths was evaluated. As the concentration of miRNA-106b-5p increased from 10 to 5 × 10⁻⁶, the detection capability was further evaluated. 7 The absorbance at fM, 370 nm, decreased from 3.77 ± 0.05 to 1.14 ± 0.08. Figure 6 (A). There was a statistically significant difference in absorbance between the 10fM miRNA-106b-5p sample and the blank control (P<0.05). Figure 6 (B) indicates sensitivity for detecting miRNA-106b-5p down to 10 fM. (From 10 fM to 1 × 10⁻⁶) 5 Within the range of miRNA-106b-5p concentrations in fM, ΔA370 showed a good linear relationship with the logarithm of miRNA-106b-5p concentration. Figure 6 (C). Linear regression equation (Y = 0.1677X - 0.008026, RC) 2 =0.9700) yielded a detection limit (LOD) of 7.16 fM. At 1×10 5 fM to 5×10 7 Another linear relationship was observed over the extended range of fM (Y = 0.6759X - 2.594, R² = 0.9724). As the concentration of miRNA-106b-5p increased from 0 to 5 × 10⁻⁶, this linear relationship continued. 7 fM, the solution color gradually changes from dark brown to light yellow, the transparency decreases, and the difference is clearly visible to the naked eye. Figure 6 (D). ImageJ analysis of the solution grayscale values ​​showed a good linear relationship with the logarithm of the miRNA concentration: 10 to 10. 5 fM: Y = 3.298X + 184.7(R) 2 =0.9793, Figure 6 (E); 1×10 5 fM to 5×10 7 fM: Y=5.436X+173.1 (R2=0.9645).

[0083] Please see Figure 7 This is a schematic diagram of a CRISPR / Cas12a dual-mode miRNA-9 detection platform for isolated crRNA activation and ALP integration. (A) shows the detection system operating at 0 to 5 × 10⁻⁶ ppm. 7 (A) UV-Vis absorption spectra of target miRNA-9 at different concentrations of fM; (B) shows the absorption spectra of different concentrations (0-5×10) at a wavelength of 370 nm. 7 The absorbance curve of fM and the correlation between the logarithm of the target miRNA-9 concentration and the optical density value (nm). (C) is 10-10 5fM and (D) are 1×10 5 -5×10 7 Two linear relationships were obtained within the range. (E) shows a schematic diagram and results of naked-eye miRNA-9 detection. Bilinear regression analysis of grayscale value and logarithm of miRNA-9 concentration: (F) shows the low concentration range (10) -1 ×10 5 fM) and (G) are in the high concentration range (1×10⁻⁶). 5 -5×10 7 fM).

[0084] In addition, length compatibility was assessed using miRNA-9 (24 nt) as a second target. The miRNA-9 concentration was increased from 10 to 5 × 10⁻⁶. 7 The absorbance at fM, 370 nm, decreased from 2.67 ± 0.02 to 0.90 ± 0.03. Figure 7 (A). There was a statistically significant difference in absorbance between the 10 fM miRNA-9 sample and the blank control (P < 0.05). Figure 7 (B) This study confirmed the sensitivity for detecting miRNA-9 down to 10 fM. (The range is from 10 fM to 1 × 10⁻⁶). 5 Within the fM range, ΔA370 showed a good linear relationship with the logarithm of miRNA-9 concentration. Figure 7 (C). Linear regression equation (Y = 0.08159X - 0.006296, RC) 2 =0.9668) yielded a detection limit (LOD) of 3.34 fM. At 1×10 5 fM to 5×10 7 Another linear relationship is established within the extended range of fM (Y = 0.5301X - 2.312, R). 2 =0.9699)( Figure 7 (Middle D). Similarly, as the miRNA-9 concentration increased from 0 to 5 × 10 7 fM, the solution color gradually changes from dark brown to light yellow. Figure 7 ImageJ grayscale analysis showed a good linear relationship between its value and the logarithm of miRNA-9 concentration: 10 to 10. 5 fM: Y = 3.887X + 184.2(R) 2 =0.9692, Figure 7 (middle F); 1×10 5 fM to 5×10 7 fM: Y = 6.929X + 169.4(R) 2 =0.9723, Figure 7(G). These results demonstrate that the amplification-free CRISPR / Cas12a biosensor activated by dissociated crRNA exhibits excellent detection capabilities for miRNAs of varying lengths. The bilinear range standard curves for miRNA-106b-5p and miRNA-9 detection show that this detection method has good adaptability and outperforms most currently reported miRNA detection platforms.

[0085] Please see Figure 8 This diagram illustrates the performance evaluation of a CRISPR / Cas12a-based nucleic acid detection platform for detecting clinical breast cancer-related miRNAs using a CRISPR system with isolated crRNA activation and ALP integration. (A) compares the sensitivity of the gold nanoparticle-based system and the conventional FAM-ssDNA-BHQ1 probe-based CRISPR / Cas12a system for detecting spacer region RNA. The gold nanoparticle system exhibits significantly enhanced sensitivity (detection limit: 10 fM), superior to the conventional system (detection limit: 1 nM), representing a 100,000-fold increase in sensitivity. (B) evaluates the specificity of various miRNAs using different dsDNA activators. Each dsDNA activator-dissociated crRNA pair generates a strong signal only for its corresponding target, with minimal cross-reactivity. A heatmap of multiplex miRNA detection using clinical samples (breast cancer patients: samples 1-7; healthy individuals: samples 8-10) is provided, and compared with RT-qPCR results. Detection targets: (C) are members of the miR-21 and miR-17-92 clusters (miR-17-5p, miR-20a-5p, miR-92a-3p, miR-106b-5p, miR-93-5p), and (D) are miR-155, miR-10b, miR-373, miR-520c, miR-27a, and miR-9. The thermal map intensity reflects the difference in normalized absorbance at 370 nm.

[0086] The analytical sensitivity of this detection system was further evaluated by comparison with amplified CRISPR biosensors that lack SPR enhancement and are isolated crRNA-activated. Figure 8 (A) For the developed amplification-free CRISPR biosensor utilizing the SPR effect of gold nanoparticles to activate crRNA, the UV-Vis absorbance of gold / silver nanoparticles at 370 nm showed a concentration-dependent response. As the RNA concentration in the spacer region increased from 10 × 10⁻⁶ to 5 × 10⁻⁶, the response increased. 7 The fM and A0 / A ratio (A0 is the absorbance of the blank control at 370 nm, and A is the absorbance of the sample) showed a continuous upward trend. Analysis of the traditional isolated crRNA CRISPR system (without SPR) showed that, by fluorescence intensity measurement, only when the RNA concentration in the spacer region was 1×10⁻⁶6 Statistical significance was only achieved at fM or higher (P<0.0001). In contrast, the SPR-enhanced biosensor exhibited significantly superior performance, achieving statistically significant signal differentiation at ultra-low concentrations down to 10 fM (P<0.05), with a dynamic range spanning six orders of magnitude (10 fM - 5 × 10⁻⁵). 7 fM). Compared to traditional crRNA splitting systems, the sensitivity is improved by 5 orders of magnitude (10 fM vs 1 × 10). 6 fM).

[0087] Furthermore, to systematically evaluate the specificity of this method, this application designed a multiplex detection panel for multiplex miRNA detection. It targets 12 breast cancer-related miRNAs, including miRNA-21 (T1), miRNA-155 (T2), miRNA-10b (T3), miRNA-373 (T4), miRNA-520c (T5), miRNA-27a (T6), miRNA-9 (T7), miRNA-17-5p (T8), miRNA-20a-5p (T9), miRNA-92a-3p (T10), miRNA-106b-5p (T11), and miRNA-93-5p (T12). Specific dsDNA activators (D1-D12) were designed for each miRNA.

[0088] like Figure 8 As shown in Figure B, the heatmap visualization reveals significant differences in signal intensity among different miRNAs. The absorbance values ​​of positive signals (represented by blue blocks) consistently remained below 1.99, while the background signals (near white blocks) remained above 2.90. Each miRNA produced a strong positive signal only in the presence of its homologous dsDNA activator (T1D1–T12D12), showing a highly significant difference compared to the non-target combination (ΔA370 nm > 1.16, P < 0.0001). The signal intensity of the non-target combination remained at baseline noise levels, indicating negligible cross-reactivity between different miRNAs. These results confirm that the detection system can accurately and specifically distinguish all 12 target miRNAs, highlighting its potential as a new technology platform for clinical molecular diagnostics.

[0089] This application provides a CRISPR-based nucleic acid detection platform, including the aforementioned biosensor and Cas12a / crRNA complex, which can be used for sensitive and multiplex detection analysis of breast cancer-related miRNAs. This application integrates three key technologies: isolated crRNA assembly, surface plasmon resonance (SPR) of gold nanoparticles (AuNPs), and microplate-based separation. The target miRNA assembles with the scaffold RNA to form a functional crRNA complex, guiding Cas12a to specifically recognize a double-stranded DNA (dsDNA) activator, thereby triggering its trans-cleavage activity and cleaving the FAM-ssDNA-biotin reporter probe. Therefore, the number of intact FAM-ssDNA-biotin captured in the streptavidin-coated microplate wells is reduced, directly leading to a decrease in the binding of alkaline phosphatase-coupled streptavidin (SA-ALP). The reduction in immobilized SA-ALP reduces silver deposition on the gold nanoparticles, thereby reducing the formation of gold / silver nanoparticles (Au / AgNPs), causing a significant change in the local dielectric environment and modulating the SPR properties of the nanoparticles. This modulation manifests as a measurable reduction in optical absorption signal, providing quantitative readings. Therefore, by synergistically combining isolated crRNA-dependent Cas12a activation with AuNP SPR signal amplification, it exhibits extremely high sensitivity (down to the fM range) and high specificity. Furthermore, due to the varying degrees of Au / AgNP formation and associated SPR shifts, the system can achieve instrument-free semi-quantitative analysis based on visually perceptible color gradient changes. Moreover, utilizing a microplate format, this platform achieves simultaneous specific detection of 12 different breast cancer-related miRNAs in a single assay, overcoming the limitations of single-marker analysis. This integrated, amplification-free CRISPR / Cas12a biosensor provides a powerful new technological approach for breast cancer diagnosis. By enabling multiplex detection of miRNAs in biological fluids, it effectively reduces the interference of individual biological variability and holds great potential for enhancing early detection, prognostic assessment, and personalized treatment monitoring.

[0090] The clinical applicability of this method is verified below with reference to specific embodiments.

[0091] To validate the clinical applicability of this method, a blind assay was performed on plasma miRNAs from 7 breast cancer patients and 3 healthy donors. Plasma samples were processed using the HiPure serum / plasma miRNA intermediate extraction kit according to the manufacturer's key protocol. Twelve miRNAs in these samples were analyzed using this platform and compared with RT-qPCR results. Figure 8(C and D in the original text). Heatmap visualization shows that the detection profiles of miR-21, miR-17-5p, miR-20a-5p, miR-92a-3p, miR-106b-5p, miR-93-5p, miR-373, miR-520c, miR-27a, and miR-9 can effectively distinguish between breast cancer patients and healthy individuals. Notably, negative signals for miR-155, miR-10b, and miR-27a were observed in some cancer patients, which may be attributed to tumor heterogeneity and expression variability. The high concordance with RT-qPCR results further validates the accuracy of our method. This underscores the necessity of multiplex miRNA detection, which overcomes the limitations of single-biomarker analysis and provides comprehensive molecular characterization for precision diagnosis. In summary, this CRISPR / Cas12a-based integrated platform provides a rapid, amplification-free liquid biopsy tool capable of simultaneously quantifying multiple miRNA biomarkers with femtomolar sensitivity, showing great potential in early cancer detection and personalized treatment monitoring.

[0092] In summary, this application provides a nucleic acid detection platform based on the CRISPR system, specifically a novel amplification-free CRISPR / Cas12a biosensor platform activated by isolated crRNA, for high-sensitivity and multiplex detection of breast cancer-related miRNAs. This method integrates isolated crRNA-mediated Cas12a trans-cleavage activation with alkaline phosphatase (ALP)-induced local SPR signal enhancement. Target miRNA assembles with scaffold RNA to form functional crRNA, initiating the assembly of the Cas12a / crRNA complex, recognizing a dsDNA activator, and trans-cleaving the ALP-coupled DNA reporter molecule, leading to ALP release. Conversely, in miRNA-negative samples, Cas12a remains intact, the reporter system is not cleaved, and ALP is not released, thus ALP catalyzes Ag... +Reduction on the surface of gold nanoparticles generates bimetallic gold / silver nanoparticles with unique plasmonic properties. The detection signal is output via a dual-mode readout, including instrumental methods (UV-Vis absorption spectroscopy) and visual detection. Compared to traditional CRISPR / Cas12a systems that use the FAM-ssDNA-BHQ1 reporter molecule to split crRNA, the dual signal amplification strategy, including Cas12a trans-cleavage and ALP-SPR, achieves femtomolar sensitivity for breast cancer-related miRNAs, representing a five-order-of-magnitude improvement in sensitivity. Furthermore, in experiments on a microplate platform, the system successfully achieved specific multiplex detection of 12 breast cancer-related miRNA biomarkers. The portability of this method is demonstrated by combining it with smartphone-based grayscale analysis, enabling quantitative detection of target RNAs without the need for dedicated equipment. This sensor holds significant potential for precision diagnosis of breast cancer, dynamic monitoring of treatment efficacy, and prognostic risk assessment, providing a promising new technological platform for clinical molecular diagnostics.

[0093] It is understood that the technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0094] The above are merely preferred embodiments of this application, and only specifically describe the technical principles of this application. These descriptions are only for explaining the principles of this application and should not be construed as limiting the scope of protection of this application in any way. Based on this explanation, any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application, as well as other specific embodiments of this application that can be conceived by those skilled in the art without creative effort, should be included within the scope of protection of this application.

Claims

1. A nucleic acid detection platform based on the CRISPR system, characterized in that, The invention includes a biosensor and a Cas12a / crRNA complex. The biosensor comprises gold / silver bimetallic nanoparticles, and the Cas12a / crRNA complex comprises functional crRNA and a dsDNA activator. The functional crRNA guides Cas12a to recognize the dsDNA activator and triggers its trans-cleavage activity. The activated Cas12a non-specifically trans-cleaves an alkaline phosphatase-coupled DNA reporter molecule, leading to the release of alkaline phosphatase, thereby reducing the formation of gold / silver nanoparticles and generating an output signal.

2. The nucleic acid detection platform based on the CRISPR system as described in claim 1, characterized in that, The Cas12a / crRNA complex includes a scaffold RNA, a spacer RNA, and a dsDNA activator complementary to the spacer RNA. The spacer RNA and the scaffold RNA assemble to form the functional crRNA, which guides Cas12a to recognize the dsDNA activator.

3. The nucleic acid detection platform based on the CRISPR system as described in claim 2, characterized in that, The concentration of Cas12a is 50 nM, the concentration of the scaffold RNA is 100 nM, the concentration of the dsDNA activator is 500 nM, the concentration of FAM-ssDNA-biotin is 10 nM, the concentration of alkaline phosphatase-labeled streptavidin is 500 pM, and the optimal length of the spacer RNA is 20 nt.

4. The nucleic acid detection platform based on the CRISPR system as described in claim 1, characterized in that, The method for preparing the biosensor includes the following steps: Alkaline phosphatase catalyzes the hydrolysis of p-aminophenyl phosphate to obtain p-aminophenol; The p-aminophenol reduces silver ions and deposits metallic silver on the surface of gold nanoparticles to form a biosensor with gold / silver bimetallic nanoparticles.

5. The nucleic acid detection platform based on the CRISPR system as described in claim 4, characterized in that, The step of catalyzing the hydrolysis of p-aminophenyl phosphate with alkaline phosphatase to obtain p-aminophenol includes the following steps: Alkaline phosphatase-labeled streptavidin is mixed with p-aminophenyl phosphate and subjected to a hydrolysis reaction to produce aminophenol and phosphate. The molar ratio of alkaline phosphatase-labeled streptavidin to p-aminophenyl phosphate is in the range of 1:

10. 11 -1:10 9 .

6. The nucleic acid detection platform based on the CRISPR system as described in claim 5, characterized in that, The step of further reducing silver ions with p-aminophenol and depositing metallic silver on the surface of gold nanoparticles to form a biosensor with a gold / silver bimetallic nanostructure includes the following steps: The aminophenol, silver ion solution, and gold nanoparticles are mixed, and the aminophenol reduces the silver ions and deposits metallic silver on the surface of the gold nanoparticles to form a biosensor with a gold / silver bimetallic nanostructure. The molar concentration ratio of the aminophenol, the silver ion solution, and the gold nanoparticles is 50:50:1 to 50:500:

1.

7. The nucleic acid detection platform based on the CRISPR system as described in claim 4, characterized in that, The gold nanoparticles were synthesized using L-reduced glutathione as a stabilizer and ligand, and nicotinamide adenine dinucleotide phosphate as a reducing agent.

8. A detection method for a nucleic acid detection platform based on the CRISPR system as described in any one of claims 1 to 7, characterized in that, Includes the following steps: The target RNA and scaffold RNA assemble to form a functional crRNA, which can guide Cas12a to recognize the dsDNA activator and trigger its trans-cleavage activity. The activated Cas12a non-specifically trans-cleaves the alkaline phosphatase-coupled DNA reporter molecule, resulting in the release of alkaline phosphatase to generate an output signal.

9. The detection method of the nucleic acid detection platform based on the CRISPR system as described in claim 8, characterized in that, The output signal can be detected by ultraviolet-visible absorption spectroscopy and by the naked eye.

10. The application of the CRISPR-based nucleic acid detection platform as described in claim 1 in miRNA detection.

Images