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

Abstract

The invention provides a biosensor, a nucleic acid detection and typing system based on a CRISPR system, a detection method and application, and belongs to the technical field of biological detection, alkaline phosphatase is adopted for catalyzing hydrolysis of p-aminophenyl phosphate to obtain p-aminophenol, the p-aminophenol is used for reducing silver ions, metal silver is deposited on the surfaces of gold nanoparticles, and the gold nanoparticles can be used for detecting the nucleic acid. Compared with a traditional Cas12a detection method combining ALP mediated p-nitrophenyl phosphate hydrolysis, the CRISPR / Cas12a biosensing strategy combining ALP has the advantages that the sensitivity is improved by 10,000 times, target DNA can be detected through naked eyes, the detection limit is as low as 300 aM, and the potential of no instrument application is shown.

Description

Technical Field

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

[0002] Human papillomavirus (HPV) comprises more than 200 genetically distinct variants, posing a significant global public health challenge. Approximately 40 subtypes exhibit tropism for the anogenital and oropharyngeal epithelium, with 14 high-risk types classified as carcinogenic by the World Health Organization (WHO). As the most common sexually transmitted infection globally, over 80% of adults will be infected with at least one HPV subtype in their lifetime. Although cell-mediated immune responses can clear 90% of infections within two years, persistent infection with high-risk HPV can lead to precancerous lesions and even cancer. HPV testing enables direct identification of the virus, facilitating early intervention before precancerous lesions develop. This approach is a cornerstone of preventative healthcare, providing a reliable and effective means of reducing the risk of HPV-related diseases. Its inclusion in global screening programs has the potential to save numerous lives, highlighting its importance in cancer prevention. Several HPV testing methods have been developed, including DNA microarray technology, real-time quantitative PCR, reverse linear hybridization detection using Sanger sequencing, and next-generation sequencing (NGS). These technologies offer high sensitivity and high throughput, but they also have limitations, such as the risk of cross-contamination, high cost, long testing cycles, and technical complexity in primer design. Despite these challenges, optimized HPV testing remains crucial in achieving the global goal of eliminating cervical cancer, especially in resource-constrained regions.

[0003] Since the discovery of the CRISPR system's ability to target and cleave specific nucleic acids, detection systems based on clustered regularly spaced short palindromic repeats (CRISPR) have become powerful diagnostic tools. CRISPR-associated proteins (Cas), including Cas12, Cas13, and Cas14, have been applied in molecular diagnostics due to their paracleavage activity—that is, their ability to cleave adjacent non-target nucleic acids after recognizing a target. Various CRISPR-based detection methods, such as SHERLOCK, DETECTR, and HOLMES, have integrated pre-amplification technologies such as recombinase polymerase amplification (RPA) and polymerase chain reaction (PCR) into their systems, achieving high sensitivity and portability.

[0004] However, these methods still rely on amplification steps, increasing the risk of aerosol contamination and prolonging detection time. To address these challenges, researchers have developed various amplification-free CRISPR / Cas detection strategies, including electrochemiluminescence graphene field-effect transistors, surface plasmon resonance (SPR), digital detection, and multiplex crRNA-based strategies. These methods achieve rapid, portable, and low-cost detection by eliminating the time-consuming and equipment-dependent amplification process, making them particularly suitable for point-of-care testing (POCT) applications in resource-scarce environments. However, current amplification-free CRISPR / Cas detection systems still require further improvement in reproducibility and stability to enhance their potential for clinical diagnostic applications. Summary of the Invention

[0005] Therefore, it is necessary to provide a biosensor capable of reproducible and highly stable detection, a CRISPR-based nucleic acid detection and typing system, preparation method, and application to address the current technical problems of insufficient reproducibility and stability.

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

[0007] One objective of this application is to provide a method for fabricating a biosensor, comprising the following steps:

[0008] Alkaline phosphatase catalyzes the hydrolysis of p-aminophenyl phosphate to obtain p-aminophenol;

[0009] 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.

[0010] In some embodiments, the step of catalyzing the hydrolysis of p-aminophenyl phosphate with alkaline phosphatase to obtain p-aminophenol includes the following steps:

[0011] 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 .

[0012] 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:

[0013] 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.

[0014] In some embodiments, the gold nanoparticles are synthesized via glutathione-mediated reduction.

[0015] A second objective of this application is to provide a biosensor prepared by any of the preparation methods described herein.

[0016] The third objective of this application is to provide a nucleic acid detection and typing system based on the CRISPR system, including the aforementioned biosensor and Cas12a / crRNA complex.

[0017] In some embodiments, the Cas12a / crRNA complex comprises crRNA, Cas12a, 10×NEBuffer, FAM-ssDNA-biotin probe, and DEPC-treated water, wherein the molar ratio of crRNA:Cas12a:FAM-ssDNA-biotin ranges from 1:1 to 5:1 to 10.

[0018] The fourth objective of this application is to provide a detection method for the CRISPR-based nucleic acid detection and typing system, comprising the following steps:

[0019] Viral DNA is extracted from clinical samples. The Cas12a / crRNA complex recognizes the target DNA and is activated, which then undergoes a paracleavage reaction of alkaline phosphatase-labeled reporter DNA, releasing alkaline phosphatase. The released alkaline phosphatase catalyzes the reduction of silver ions on the surface of gold nanoparticles to generate gold / silver bimetallic nanoparticles.

[0020] In some embodiments, the detection signal can be obtained via ultraviolet-visible absorption spectroscopy and visual inspection.

[0021] The fifth objective of this application is to provide an application of a CRISPR-based nucleic acid detection and typing system in the detection of pathogen variants.

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

[0023] The biosensor and its preparation method provided in this application involve 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. The SPR-mediated optical changes during the formation and aggregation of gold / silver bimetallic nanoparticles are monitored by ultraviolet-visible spectroscopy, indicating that the ALP-mediated SPR effect provides a simple and rapid ALP detection method with ultrasensitive ALP detection capability at sub-pM sensitivity.

[0024] This application also provides a nucleic acid detection and typing system based on the CRISPR system and its preparation method, which combines CRISPR / Cas12a technology with alkaline phosphatase-mediated surface plasmon resonance. This platform recognizes target DNA through the Cas12a-crRNA complex, activates the cleavage of alkaline phosphatase-labeled oligonucleotides in the micropores, thereby releasing alkaline phosphatase. The released alkaline phosphatase catalyzes the reduction of silver ions on the surface of gold nanoparticles to generate gold / silver bimetallic nanoparticles. This application combines an ALP-based CRISPR / Cas12a biosensing strategy, which improves the sensitivity by 10,000 times compared to the traditional Cas12a detection method that combines ALP-mediated hydrolysis of p-nitrophenyl phosphate. This method can detect target DNA with the naked eye, with a detection limit as low as 300 aM, demonstrating the potential for instrument-free applications.

[0025] The nucleic acid detection and typing system based on the CRISPR system provided in this application can be widely used for rapid on-site detection of pathogen variants. It can detect multiplex nucleic acid targets without pre-amplification or instrument assistance, and is fast, convenient, portable and low cost. Attached Figure Description

[0026] 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.

[0027] Figure 1 This is a schematic flowchart of a CRISPR-based nucleic acid detection and typing system for viral DNA detection, provided in an embodiment of the present invention.

[0028] Figure 2 This is a schematic diagram illustrating the ALP-mediated conversion process of AuNPs to Au / AgNPs provided in an embodiment of the present invention.

[0029] Figure 3This diagram illustrates the construction and optimization of an ALP-mediated CRISPR / Cas12a-based nucleic acid detection and typing system provided in an embodiment of the present invention.

[0030] Figure 4 This is a schematic diagram of an ALP-mediated CRISPR / Cas12a CRISPR-based nucleic acid detection and typing system for dual-mode HPV-16 detection provided in an embodiment of the present invention.

[0031] Figure 5 This is a clinical illustration of HPV genotyping based on an ALP-mediated CRISPR / Cas12a CRISPR-based nucleic acid detection and typing system, provided as an embodiment of the present invention. Detailed Implementation

[0032] 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.

[0033] 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.

[0034] 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.

[0035] In the field of molecular diagnostics, various CRISPR-based detection methods, such as SHERLOCK, DETECTR, and HOLMES, have integrated pre-amplification technologies such as recombinase polymerase amplification (RPA) and polymerase chain reaction (PCR) into their systems, achieving high sensitivity and portability. However, these methods still rely on amplification steps, increasing the risk of aerosol contamination and prolonging detection time. To address these challenges, researchers have developed various amplification-free CRISPR / Cas detection strategies, including electrochemiluminescence graphene field-effect transistors, surface plasmon resonance (SPR), digital detection, and multiplex crRNA-based strategies. These methods achieve rapid, portable, and low-cost detection by eliminating the time-consuming and equipment-dependent amplification process, making them particularly suitable for point-of-care testing (POCT) applications in resource-scarce environments. However, current amplification-free CRISPR / Cas detection systems still require further improvement in terms of reproducibility and stability.

[0036] Therefore, this application provides a biosensor capable of reproducible and highly stable detection, a CRISPR-based nucleic acid detection and typing system, a preparation method, and applications. The technical solution of this application will be described in detail below with reference to embodiments.

[0037] The reagents and instruments involved in this application are as follows:

[0038] All oligonucleotides used in this application (see Table S1) were synthesized by Shanghai Sangon Biotech Co., Ltd. Anti-6-FAM rabbit polyclonal antibody and DEPC-treated water were also purchased from Sangon Biotech. LbaCas12a (Cpf1) was purchased from New England Biolabs (Ipswich, Massachusetts, USA). Bovine serum albumin (BSA) and phosphate-buffered saline (PBST) containing Tween-20 were purchased from Beijing Solarbio Science & Technology Co., Ltd. Reduced glutathione (L-Glutathione) and reduced β-nicotinamide adenine dinucleotide tetrasodium phosphate (NADPH) were provided by Shanghai Beyotime Biotechnology Co., Ltd. Glutathione reductase (derived from Saccharomyces cerevisiae) and silver nitrate (AgNO3) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Gold trichloride was purchased from Maclean Biotech Co., Ltd. (Shanghai). Alkaline phosphatase (ALP)-labeled streptavidin was purchased from Beijing Bio-Sen Biotechnology Co., Ltd. p-Aminophenyl phosphate (p-APP) was provided by Shanghai Yuanye Biotechnology Co., Ltd. Nucleic acid extraction kits were purchased from Guangzhou Maijing Biotechnology Co., Ltd. UV-Vis absorption spectra were recorded using a HITACHI 3900H UV spectrophotometer (Japan); hydrodynamic particle size and zeta potential were determined using a Malvern Zetasizer Nano ZSE particle size analyzer (UK); transmission electron microscopy (TEM) images were acquired using a FEITalos F200S transmission electron microscope (USA).

[0039] Table S1

[0040]

[0041]

[0042] Based on the above reagents and instruments, this application details the specific implementation scheme.

[0043] 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.

[0044] 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.

[0045] 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; the aminophenol reducing silver ions 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 50:50:1-50.

[0046] In this embodiment, gold nanoparticles are synthesized via glutathione-mediated reduction.

[0047] Specifically, 7.5 mg of reduced glutathione was dissolved in 100 μL of ultrapure water (resistivity 18.2 MΩ·cm) and mixed under strong magnetic stirring. Then, 1 mL of a 1% (w / w) aqueous solution of chloroauric acid (HAuCl4) was rapidly added, and the pH was adjusted to 2.5–3.0 by adding 10 M NaOH dropwise. The mixture was centrifuged at 7000 rpm for 3 minutes at 4 °C to remove unreacted precursors, and the precipitate was collected.

[0048] The resulting precipitate was dissolved in 1 mL of 5 mM NaOH solution, diluted with 9 mL of ultrapure water, and the pH was adjusted to 5.5 to obtain a gold precursor suspension. Then, 4 mg of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and 2 U of glutathione reductase were added, and the reaction was stirred continuously at room temperature for 120 minutes to initiate the controlled-size nanoparticle growth process.

[0049] After the reaction was complete, the colloidal suspension was purified by washing three times with ultrapure water using a 30 kDa molecular weight cutoff centrifuge filter (Millipore). Finally, the purified AuNPs were redispersed in 1 mL of ultrapure water and stored at 4 °C to obtain gold nanoparticles.

[0050] It is understood that in the biosensor preparation method provided in this embodiment, alkaline phosphatase (ALP) catalyzes the hydrolysis of p-aminophenyl phosphate (p-APP) to generate p-aminophenol (p-AP) and phosphate. Subsequently, p-AP promotes the hydrolysis of silver ions (Ag... + The ALP-mediated SPR effect is reduced and deposited 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.

[0051] The specific experimental steps are as follows: A 100 μL reaction system was constructed, containing 2 mM AgNO3, 8 mM p-APP, 80 nM uNPs, and different concentrations of ALP-labeled streptavidin (SA-ALP, with concentrations of 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0, 1.5, and 2.0 pM). This mixture was incubated at 37°C on a shaker at 370 rpm for 30 minutes.

[0052] After the reaction, the UV-Vis absorption spectrum was recorded, and the absorbance at a wavelength of 370 nm was selected to establish a linear relationship between concentration and absorbance signal.

[0053] This embodiment also provides a nucleic acid detection and typing system based on the CRISPR system, including the biosensor and Cas12a / crRNA complex described in the above embodiment.

[0054] In this embodiment, the Cas12a / crRNA complex includes crRNA, Cas12a, 10×NEBuffer, FAM-ssDNA-biotin probe, and DEPC-treated water, and the molar ratio of crRNA:Cas12a:FAM-ssDNA-biotin ranges from 1:1 to 5:1 to 10.

[0055] This embodiment also provides a detection method for a nucleic acid detection and typing system based on the CRISPR system, including the following steps: extracting viral DNA from a clinical sample; the Cas12a / crRNA complex recognizing the target DNA; the Cas12a / crRNA complex being activated, thereby undergoing a paracleavage reaction of alkaline phosphatase-labeled reporter DNA, releasing alkaline phosphatase; the released alkaline phosphatase catalyzing the reduction of silver ions on the surface of gold nanoparticles to generate gold / silver bimetallic nanoparticles.

[0056] Furthermore, the detection signal can be obtained through ultraviolet-visible absorption spectroscopy and by visual inspection.

[0057] Please see Figure 1This is a schematic flowchart of an ALP-mediated CRISPR / Cas12a-based nucleic acid detection and typing system for viral DNA detection. The platform comprises three main steps: viral DNA extraction, DNA recognition, and an ALP-catalyzed CRISPR / Cas12a activation enzymatic metallization reaction, employing both UV-Vis spectrophotometry and visual observation for dual-mode signal detection. First, viral DNA is extracted from clinical samples using a commercial DNA extraction kit. In the presence of target DNA, the Cas12a / crRNA complex is activated, leading to the paracleavage of ALP-labeled reporter DNA and the release of ALP enzymes. In target-free samples, the reporter system remains intact, preventing ALP release and thus inhibiting the metallization reaction. The released ALP catalyzes the formation of silver ions (Ag). + The gold / silver bimetallic nanoparticles (Au / AgNPs) are reduced on the surface of gold nanoparticles (AuNPs) to generate gold / silver bimetallic nanoparticles (Au / AgNPs) with unique plasmonic properties. The detection signal can be read by ultraviolet-visible absorption spectroscopy and the naked eye. Figure 1 The diagram illustrates the molecular components involved in the reaction.

[0058] This embodiment uses HPV nucleic acid detection as an example to explain the detection steps in detail.

[0059] First, 100 μL of capture antibody (anti-6-FAM rabbit polyclonal antibody, 1:100 dilution) was added to a 96-well plate and incubated overnight at 4°C. Then, the plate was washed three times with PBST (PBS buffer containing 0.05% Tween-20, pH 7.4), and 300 μL of 2.5% (w / v) bovine serum albumin (BSA) solution was added, followed by blocking at 37°C for 2 hours. Next, a 100 μL CRISPR reaction mixture was prepared, consisting of: 10 μL 1 μM crRNA, 5 μL 1 μM Cas12a enzyme, 10 μL 10×NE Buffer, 10 μL 0.1 μM FAM-ssDNA-biotin fluorescent probe, 10 μL of synthesized HPV16 L1 gene fragment, and 55 μL DEPC-treated water. The reaction mixture was incubated at 37°C with shaking at 370 rpm for 1 hour. After incubation, the plate was washed three times with PBST buffer (PBS containing 0.05% Tween-20, pH 7.4). Then, 100 μL of 500 pM streptavidin-labeled alkaline phosphatase (SA-ALP) was added to each well, and the plate was incubated at 37°C for 1 hour. After incubation, the plate was washed three more times to remove unbound enzyme-labeled material.

[0060] Subsequently, 100 μL of a colorimetric reaction mixture was added to the reaction wells. This mixture consisted of the following components: 10 μL of 80 mM p-aminophenyl phosphate (p-APP), 1 μL of 200 mM silver nitrate (AgNO3), 8 μL of 1 μM gold nanoparticles (AuNPs), and 81 μL of diethanolamine buffer (pH 9.8, 0.5 M, containing 1 mM MgSO4). The reaction system was incubated on a shaker at 37 °C and 370 rpm for 30 minutes, allowing ALP to catalyze the hydrolysis of p-APP to generate p-aminophenol (p-AP), which further induced AgNO3 reaction. + Bimetallic Au / AgNPs are formed by reduction deposition on the surface of AuNPs to achieve signal output.

[0061] Finally, the UV-Vis absorption spectra of the samples were recorded using a microplate reader (Infinite F Plex, Tecan, Japan), and quantitative analysis of HPV DNA was achieved using absorbance signals at a wavelength of 370 nm.

[0062] This embodiment also provides the application of the above-mentioned CRISPR-based nucleic acid detection and typing system in the detection of pathogen variants.

[0063] In this embodiment, the pathogen variant includes HPV. For ease of explanation, HPV is used as an example below to describe in detail the application of the CRISPR-based nucleic acid detection and typing system provided in this application in the detection of pathogen variants.

[0064] In this embodiment, the ALP-catalyzed hydrolysis of p-nitrophenyl phosphate (p-NPP) was used as an indicator system to evaluate the sensitivity of the constructed method. The 100 μL CRISPR reaction system included 1 μM crRNA, 1 μM Cas12a, 10×NEBuffer, 0.1 μM FAM-ssDNA-biotin probe, a synthesized HPV16 L1 gene fragment, and DEPC-treated water. This reaction mixture was incubated for 60 minutes at 37°C and 370 rpm in a microplate coated with anti-6-FAM antibody. After incubation, the plate was washed three times with PBST to remove unbound material, and then 500 pM SA-ALP was added, followed by incubation at 37°C for 1 hour. After another wash, 60 μM p-NPP substrate was added, and the plate was incubated at 37°C for 30 minutes. After the reaction was complete, the UV-Vis absorption spectrum was recorded, and the absorbance value at 370 nm was used for sensitivity evaluation. To evaluate the specificity of this biosensor system, L1 gene fragments derived from various HPV subtypes were used for detection, including low-risk types (HPV-6 and HPV-11) and high-risk types (HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58). The synthetic sequences of the L1 gene fragments for each HPV subtype and their corresponding crRNA sequences are detailed in Appendix Table S1. All subtype detections were performed according to the standard procedure for HPV16 detection, and the final concentration of the gene fragment for each HPV subtype was 100 pM.

[0065] The above technical solution will be described in detail below with reference to specific embodiments.

[0066] Example

[0067] In this embodiment, a total of 24 cervical swab samples were collected. Sample collection was conducted at Yixing People's Hospital in Jiangsu Province according to approved ethical protocols. HPV nucleic acid extraction was performed using the HiPure Viral RNA / DNA Extraction Kit (Meiji Biotechnology), strictly following the instructions. The specific procedure is as follows: 200 μL of clinical sample was taken, and 20 μL of proteinase K solution was added, followed by 200 μL of Buffer AL. The mixture was vortexed for 15 seconds and incubated in a 56°C water bath for 10 minutes. Then, 250 μL of anhydrous ethanol was added, and the mixture was thoroughly mixed and allowed to stand at room temperature for 3 minutes. The mixture was transferred to a collection tube with a filter cartridge and centrifuged at 10,000g for 30 seconds.

[0068] The filter cartridge was washed once with 500 μL Buffer VHB and twice with 500 μL Buffer RW2, then centrifuged at 13,000 g for 3 minutes to remove residual ethanol. The cartridge was then transferred to a new 1.5 mL centrifuge tube, and 50 μL of nuclease-free water was added. The tube was equilibrated at room temperature for 2 minutes. Finally, it was centrifuged at 13,000 g for 1 minute to collect the eluent, which was the extracted HPV nucleic acid sample and stored at -80°C for subsequent testing. The extracted HPV nucleic acid sample was tested according to the method described in the section "HPV DNA Detection Based on CRISPR System Using ALP-Mediated CRISPR / Cas12a Nucleic Acid Detection and Typing System". Subtype typing was performed using the corresponding crRNA sequence via the ALP-integrated CRISPR / Cas12a platform.

[0069] ALP-catalyzed conversion of gold nanoparticles (AuNPs) into gold / silver bimetallic nanoparticles (Au / AgNPs) systematically validated the feasibility of using ALP-catalyzed modulation of surface plasmon resonance (SPR) signals of AuNPs as an ultrasensitive signal reporter mechanism. The reaction process mainly includes two key steps: First, alkaline phosphatase (ALP) catalyzes the hydrolysis of p-aminophenyl phosphate (p-APP) to generate p-aminophenol (p-AP) and inorganic phosphate; subsequently, the generated p-AP can further reduce silver ions (Ag... + ), and controlled deposition of metallic silver (Ag) on ​​the surface of gold nanoparticles. 0 This process forms a gold / silver bimetallic nanostructure with modulated SPR properties. The reaction mechanism can be summarized in two stages:

[0070]

[0071] Analysis of ALP-mediated SPR response and optical changes in the formation of gold / silver bimetallic nanoparticles.

[0072] Monitoring SPR-mediated optical changes during the formation and aggregation of gold / silver bimetallic nanoparticles (Au / AgNPs) using ultraviolet-visible spectroscopy (UV-vis).

[0073] Please see Figure 2This diagram illustrates the ALP-mediated transformation of AuNPs to Au / AgNPs. Specifically: (A) is a schematic diagram of ALP-mediated surface plasmon resonance (SPR) shifts during the AuNPs-AgNPs transformation. (B, C) are transmission electron microscopy (TEM) images of AuNPs and Au / AgNPs, respectively, showing the changes in particle size and morphology after the ALP-catalyzed metallization reaction. (D) shows the UV-Vis absorption spectra of AuNPs and their reaction products after ALP-catalyzed p-APP hydrolysis, with the absorption peak at 520 nm corresponding to AuNPs and a new absorption peak at 370 nm indicating the formation of Au / AgNPs. (E) shows the hydrodynamic particle size and zeta potential analysis results of AuNPs before and after the ALP-catalyzed reaction. (F) shows the UV-Vis absorption spectrum of Au / AgNPs formed after ALP-catalyzed p-APP hydrolysis, with the absorption intensity at 370 nm gradually increasing with increasing SA-ALP concentration (0-2.5 pM). (G) The linear calibration curve between absorbance at 370 nm and SA-ALP concentration (0.05-2.0 pM) demonstrates the quantitative detection capability of this platform.

[0074] Experiments show that ALP-mediated plasma modulation provides a rapid and convenient analytical platform for ALP detection, such as... Figure 2 As shown in Figure A. To comprehensively characterize the physical properties of AuNPs and Au / AgNPs, various analytical methods were employed, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and ultraviolet-visible absorption spectroscopy. TEM images showed that the synthesized AuNPs were well dispersed, had a uniform morphology, and exhibited almost no aggregation, proving their successful synthesis. Figure 1 (B). Following the ALP-mediated metallization reaction, the formation of Au / AgNPs was clearly observed, accompanied by changes in particle morphology and significant aggregation. Figure 1 (C). To further verify Ag 0 The hydrodynamic particle size and zeta potential of bare AuNPs and Au / AgNPs after ALP-catalyzed metallization were determined using DLS technology after deposition on the surface of AuNPs. Figure 1 As shown in Figure E, the particle size of AuNPs is approximately 57.07 nm, while that of Au / AgNPs is significantly increased to 1028.07 nm. Furthermore, the zeta potentials of AuNPs and Au / AgNPs are -46.13 mV and -31.90 mV, respectively. The increase in particle size and the change in zeta potential provide strong evidence for the successful ALP-mediated enzymatic metallization reaction. The ALP-mediated changes in the plasma response of AuNPs can be used for the quantitative detection of ALP. Figure 1As shown in Figure D, the synthesized AuNPs exhibit a typical absorption peak at 520 nm, while Au / AgNPs show a new absorption peak at 370 nm after a blue shift. This blue shift is attributed to Ag. 0 The SPR changes induced by deposition on the AuNP surface further confirmed the successful formation of Au / AgNPs. Furthermore, experiments showed that the plasma response of this system was ALP concentration-dependent. Figure 2 As shown in Figure F, the absorption intensity of Au / AgNP at 370 nm gradually increases with increasing ALP concentration. A good linear relationship is observed between absorbance and ALP concentration in the 0-2 pM range, and the fitting equation is: Y = 0.2258X + 0.1089( Figure 1 (As shown in G). This concentration-related SPR modulation mechanism verifies that the system possesses ultrasensitive ALP detection capability with sub-pM sensitivity through plasma signal amplification.

[0075] Furthermore, to evaluate the feasibility of ALP-catalyzed surface plasmon resonance (SPR) effect of gold nanoparticles (AuNPs) as a highly sensitive signal reporter mechanism for the CRISPR / Cas12a system, a bifunctional reporter system was designed through molecular engineering. This system utilizes FAM-ssDNA-biotin as a bifunctional linker, which can bind to both micropores coated with anti-FAM antibodies and SA-ALP, constructing a "micropore-ssDNA-ALP" reporter structure. This structure simultaneously possesses the functions of Cas12atrans substrate cleavage and ALP-mediated SPR signal amplification. When the target dsDNA is recognized by the Cas12a-crRNA complex, Cas12a is activated, initiating efficient trans-cleavage activity, thereby cleaving and disrupting the "micropore-ssDNA-ALP" linker structure. Unreleased ALP then catalyzes the deposition of a silver shell on the surface of the gold nanoparticles, inducing the conversion of AuNPs to Au / AgNPs, further amplifying the SPR signal. To maximize the colorimetric detection sensitivity of dsDNA, two key parameters were systematically optimized: SA-ALP concentration and FAM-ssDNA-biotin loading density.

[0076] Please see Figure 3This diagram shows the construction and optimization of the CRISPR / Cas12a CRISPR-based nucleic acid detection and typing system mediated by ALP. (A) shows the optimization of SA-ALP concentration in the CRISPR-based nucleic acid detection and typing system. The absorbance difference (ΔA370) at 370 nm gradually increases from 2 to 500 pM with SA-ALP concentration, while the signal decreases at 1000 pM. (B) shows the optimization of FAM-ssDNA-biotin concentration, with the signal difference reaching its maximum at 10 nM. (C) shows the optimization of target dsDNA concentration from 0 to 2 × 10⁻⁶ pM. 5 The UV-Vis absorption spectrum of the detection system in the fM range. (D) shows the correlation between the target dsDNA concentration and ΔA370, revealing two linear response intervals: 1-10. 4 fM and 20-200pM.

[0077] like Figure 3 As shown in Figure A, the absorbance difference at 370 nm (ΔA370) exhibits a biphasic response to SA-ALP concentration. Within the range of 2 pM to 500 pM, ΔA370 increases linearly with increasing SA-ALP concentration, reflecting enhanced ALP-driven metal deposition. However, when the concentration exceeds 500 pM, ΔA370 decreases significantly, possibly due to increased background noise from non-specifically adsorbed ALP. Similarly, the concentration optimization results for FAM-ssDNA-biotin also show a saturation trend. Figure 3 (See Figure B). When the concentration reached 10 nM, ΔA370 peaked, after which the signal weakened. These results determined that 500 pM SA-ALP and 10 nM FAM-ssDNA-biotin were the optimal reaction conditions for subsequent experiments. Under optimized conditions, the research team further evaluated the detection performance of the system at different dsDNA concentrations. Figure 3 As shown in Figure C, within the wavelength range of 300-450 nm, the UV-Vis absorption spectrum of Au / AgNPs exhibits a concentration-dependent response to changes in dsDNA concentration; the absorbance intensity gradually decreases with increasing target concentration. Quantitative analysis revealed a good linear relationship between ΔA370 and 10 pM. Figure 3 The fitting equation for the method is Y = 0.06818X + 0.07081, with a detection limit of 0.37 fM, indicating that this method possesses extremely high sensitivity for dsDNA detection. Notably, the system also exhibits excellent dynamic linearity in the 20–200 pM range, with a fitting equation of Y = 3.226X - 13.47 (R²). 2=0.9331). The establishment of these two calibration curves together demonstrates the good adaptability of the detection system, enabling both ultrasensitive detection of low concentrations of dsDNA and reliable quantification across five orders of magnitude. The broad linear range and sub-femtomolar detection limit exhibited by this method make it a highly promising detection platform for broad-spectrum dsDNA analysis in complex biological systems.

[0078] Furthermore, to evaluate the analytical performance of this method in HPV-16 gene detection, this application selected a synthesized HPV-16L1 gene fragment as the detection target.

[0079] Figure 4 This is an ALP-mediated CRISPR / Cas12a CRISPR-based nucleic acid detection and typing system for dual-mode HPV-16 detection. (A) represents the HPV-16 gene concentration range of 0 to 2 × 10⁻⁶. 5 Under fM conditions, the UV-Vis absorption spectrum of the detection system. (B, C) are linear regression analyses between ΔA370 and the logarithm of HPV-16 gene concentration, showing two distinct linear intervals: the low concentration range (1-10). 4 fM) and high concentration range (2×10 4 -2×10 5 (fM). (D) is a schematic diagram of the naked-eye detection principle of HPV-16 gene and the results. (E, F) are linear regression analyses between gray values ​​and the logarithm of HPV-16 gene concentration, respectively, showing two linear response intervals: the low concentration range (1-10). 4 fM) and high concentration range (2×10 4 -2×10 5 fM).

[0080] like Figure 4 As shown in Figure A, as the HPV-16 gene concentration increases from 1 fM to 2 × 10⁻⁶, 5 The absorbance at 370 nm gradually decreases, indicating that the detection system exhibits target-dependent spectral attenuation characteristics. For example... Figure 4 As shown in Figures B and C, this method exhibits a good linear response across two different concentration ranges. In the low concentration range (1-10),... 4 Within fM, ΔA370 was linearly correlated with the logarithm of HPV-16 gene concentration. Figure 4In the case of HPV-16 gene detection system B), the fitting equation was Y = 0.05795X + 0.02109, and the limit of detection (LOD) reached 300 aM (S / N = 3), significantly better than the traditional CRISPR / Cas12a detection system. Furthermore, in the higher concentration range (20-200 pM), the system maintained good linear response, with ΔA370 showing a logarithmic correlation with HPV-16 gene concentration, and the fitting equation was Y = 2.187X - 9.119. Further naked-eye visual detection of the HPV-16 gene was conducted. Figure 4 As shown in Figure D, as the HPV-16 gene concentration increased from 0 fM to 2 × 10⁻⁶, 5 The reaction system, at fM, gradually changed color from brown to yellow, exhibiting an observable concentration-dependent color change. By extracting the grayscale values ​​of the test tube colors through image analysis, the relationship between HPV-16 gene concentration and color signal intensity was established. Figure 4 As shown in E and F of Figure 3, the system again exhibits good linear correlation in both concentration ranges. In the low concentration range (1-10), the system shows good linear correlation. 4 Within the range of fM, the gray value showed a linear relationship with the logarithm of the HPV-16 gene concentration, with the fitted equation being: Y = 2.000X + 2.733, and the detection limit remained at 300 aM (S / N = 3). In the high concentration range (20-200 pM), the gray value also showed a good linear correlation with the concentration, with the fitted equation being: Y = 19.35X - 0.9496.

[0081] Furthermore, to further verify the analytical sensitivity of this detection system, this application compared it with the traditional p-NPP colorimetric method. The p-NPP method is currently the gold standard for quantitative analysis of ALP.

[0082] Please see Figure 5 This study evaluates the clinical performance of an ALP-mediated CRISPR / Cas12a-based CRISPR nucleic acid detection and genotyping system for HPV genotyping. Specifically: (A) compares the sensitivity of the AuNPs system with the traditional p-NPP colorimetric system in HPV-16 genotyping, showing that the AuNPs system significantly improves detection sensitivity within the 10pM to 1fM range. (B) Specificity analysis using crRNAs of different HPV subtypes shows that each crRNA-HPV combination exhibits a significant specific response signal with minimal cross-reactivity. (C) The results of HPV-16 detection in clinical samples demonstrate the ability to distinguish between HPV-16 negative samples (numbered 1-4) and positive samples (numbered 5-12) based on absorbance differences at 370nm. (D) Multiplex detection of different HPV subtypes in clinical samples validates the platform's accurate HPV genotyping detection capability.

[0083] like Figure 5As shown in Figure A, the p-NPP detection method shows almost no response at HPV-16 DNA concentrations less than or equal to 10 pM, indicating its limited sensitivity. In contrast, the AuNP-based detection system exhibits a target concentration-dependent decrease in absorbance at 370 nm, and can still achieve differential signal detection at ultra-low concentrations of 1 fM. Compared to the p-NPP method, this method improves detection sensitivity by 10%. 4 The performance is significantly higher (1.0 fM vs. 10.0 pM), outperforming most previously reported HPV testing platforms. To validate its subtype recognition capability, this application designed a multiplex detection system targeting nine clinically relevant HPV subtypes, including low-risk (HPV-6 and HPV-11) and high-risk (HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58). The crRNAs used in the CRISPR-Cas12a system all target conserved sequences in the HPV L1 capsid gene, ensuring subtype-specific recognition. Figure 5 As shown in Figure B, the heatmap visualization results demonstrate the detection signal intensity corresponding to different HPV subtypes. Each HPV subtype only produces a strong signal in the presence of its matching crRNA, while non-target combinations remain at background levels. These results confirm that the detection system has extremely high specificity and no cross-reactivity among multiple HPV subtypes, indicating that it is highly suitable for high-precision HPV genotyping detection.

[0084] Furthermore, to verify the clinical application potential of this testing platform, this application tested 24 cervical swab clinical samples and compared the results with those from a commercial genotyping kit for confirmation. First, 12 samples were tested using crRNA targeting HPV-16, including 8 HPV16-positive samples and 4 HPV16-negative samples. Figure 5 As shown in Figure C, the absorbance difference at 370 nm for HPV-negative samples was less than 0.1, significantly lower than the positivity threshold, while HPV-positive samples exhibited significantly higher absorbance values ​​(ranging from 0.22 to 0.38). These results indicate that the platform can effectively distinguish between HPV16-positive and negative samples. Furthermore, to evaluate the platform's ability in HPV genotyping, clinical samples infected with multiple HPV subtypes were tested. The heatmap analysis results are shown below. Figure 4 As shown in Figure D, the specific signal spectra corresponding to each HPV subtype are presented. Samples carrying HPV-positive subtypes show strong signals, while samples carrying HPV-negative subtypes maintain signals at baseline levels. It is worth emphasizing that this platform achieved 100% consistency with the genotyping results of commercial reference kits across all detected subtypes, further validating its clinical reliability in accurate genotyping and demonstrating its potential application in cervical cancer prevention and screening.

[0085] This application provides a CRISPR-based nucleic acid detection and typing system that integrates an alkaline phosphatase (ALP) CRISPR / Cas12a diagnostic platform for high-precision human papillomavirus (HPV) genotyping. This CRISPR-based nucleic acid detection and typing system utilizes a dual-enzyme amplification architecture, combining the trans-cleavage activity of Cas12a with the ALP-mediated surface plasmon resonance (SPR) effect of gold nanoparticles (AuNPs), thereby achieving extremely high detection sensitivity. In operation, ALP is immobilized within a microplate via covalent linkage to oligonucleotides. Subsequently, after Cas12a is activated by target DNA, it releases ALP by cleaving ssDNA, establishing a target-response signal transduction pathway. This signal is induced by ALP-catalyzed hydrolysis to aggregate AuNPs, and further amplified by the SPR effect. This amplification-free CRISPR detection method represents a significant breakthrough in the field of molecular diagnostics, possessing both ultra-high sensitivity (detection limit up to 300 aM) and extremely low instrument dependence. Furthermore, this platform can accurately identify the HPV L1 gene region, enabling specific genotyping of the nine HPV subtypes covered by the nine-valent HPV vaccine. In clinical validation using cervical swab samples, the platform's results were completely consistent with standard PCR testing. This CRISPR-based nucleic acid detection and genotyping system achieves ultrasensitive detection through the synergistic integration of enzymatic amplification and nanoparticle plasma effects; it utilizes microplate technology to achieve spatial separation of the reaction system, ensuring the accuracy of genotyping; it employs a high-signal design, eliminating the need for nucleic acid pre-amplification steps; and the ALP-mediated colorimetric reaction gives the system visual detection capabilities, allowing results to be read without instruments.

[0086] This platform offers advantages such as reagent savings and signal visualization, making it highly suitable for point-of-care testing (POCT) for cervical cancer screening in primary healthcare institutions. Research results indicate that this technology not only significantly enhances global HPV surveillance capabilities but also provides a universal platform framework for multiplex pathogen detection.

[0087] 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.

[0088] 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 method for preparing a biosensor, characterized in that, 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.

2. The method for preparing the biosensor as described in claim 1, 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 .

3. The method for preparing a biosensor as described in claim 2, 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.

4. The method for preparing the biosensor as described in claim 1, characterized in that, The gold nanoparticles were synthesized via a glutathione-mediated reduction method.

5. A biosensor, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 4.

6. A nucleic acid detection and typing system based on the CRISPR system, characterized in that, Includes the biosensor and Cas12a / crRNA complex as described in claim 5.

7. The nucleic acid detection and typing system based on the CRISPR system as described in claim 6, characterized in that, The Cas12a / crRNA complex comprises crRNA, Cas12a, 10×NE Buffer, FAM-ssDNA-biotin probe, and DEPC-treated water, wherein the crRNA:Cas12a: The molar concentration ratio of FAM-ssDNA-biotin ranges from 1:1 to 5:1 to 10.

8. A detection method for a nucleic acid detection and typing system based on the CRISPR system as described in claim 6, characterized in that, Includes the following steps: Viral DNA is extracted from clinical samples. The Cas12a / crRNA complex recognizes the target DNA and is activated, which then undergoes a paracleavage reaction of alkaline phosphatase-labeled reporter DNA, releasing alkaline phosphatase. The released alkaline phosphatase catalyzes the reduction of silver ions on the surface of gold nanoparticles to generate gold / silver bimetallic nanoparticles.

9. The detection method of the nucleic acid detection and typing system based on the CRISPR system as described in claim 8, characterized in that, The detection signal can be obtained through ultraviolet-visible absorption spectroscopy and visual inspection.

10. The application of the CRISPR-based nucleic acid detection and typing system as described in claim 8 in the detection of pathogen variants.

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