Fluorescence / sers dual-mode detection system based on rpa amplification combined with crispr / cas12a technology and application thereof

Abstract

This invention discloses a fluorescence / SERS dual-mode detection system based on RPA amplification combined with CRISPR / Cas12a technology. First, silver-coated gold core-shell nanoparticles modified with 4-MBA Raman signal molecules are prepared, and ssDNA1 and ssDNA2 are modified respectively to obtain two SERS nanoprobes. The target DNA is rapidly amplified at isothermal using the RPA system. In the CRISPR / Cas12a system, crRNA specifically recognizes the RPA-amplified target DNA and activates Cas12a, which non-specifically cleaves the linker DNA and the fluorescent reporter probe. In negative samples, the linker DNA is intact, cross-linking the two probes to form aggregates. After centrifugation, the supernatant shows a weak SERS signal and no fluorescence response. In positive samples, the linker is cleaved, the probes disperse, the supernatant shows an enhanced SERS signal, and the reporter probe breaks down, releasing a fluorescent signal. This invention achieves rapid and sensitive detection of viral DNA through rapid RPA amplification and specific recognition by the Cas12a protein, using changes in SERS and fluorescence signals.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a fluorescence / SERS dual-mode detection system based on RPA amplification combined with CRISPR / Cas12a technology and its application in viral DNA detection. Background Technology

[0002] DNA is the genetic material of viruses. Each virus has a unique gene sequence. By detecting viral DNA, its genome can be directly analyzed, thus accurately identifying the virus type and confirming its presence. Viral DNA detection differs from antigen-antibody detection; it does not rely on an immune response and can be performed in the early stages of infection or when the viral load is low, providing continuous monitoring data for more effective control and treatment of viral infections. For example, Epstein-Barr virus (EBV), a member of the gamma herpesvirus family, has been shown to be closely related to the development and progression of nasopharyngeal carcinoma. Circulating EBV DNA in plasma has been established as a key biomarker for nasopharyngeal carcinoma. Currently, qPCR technology is the gold standard for EBV DNA detection, but it has significant limitations: the equipment is expensive, the operation is complex and time-consuming, limiting its application in primary healthcare institutions; and it is prone to non-specific amplification, leading to false positives and misleading clinical diagnosis.

[0003] RPA is an ultrasensitive isothermal amplification technique. Utilizing the synergistic action of recombinases, DNA polymerases, and single-stranded DNA-binding proteins, it can achieve exponential amplification of target DNA sequences within 20 minutes at a constant temperature of 37°C. As a rapid, heat-cycling-independent nucleic acid amplification technique, RPA is becoming a versatile alternative to traditional PCR. However, RPA suffers from some practical drawbacks, such as primer dimer formation and other amplification products. The accumulation of these byproducts increases background noise and can lead to false positives, thus adversely affecting detection accuracy. Notably, the CRISPR / Cas system, with its extremely high specificity, offers a promising technological direction for addressing these issues. As a member of the CRISPR / Cas family, the CRISPR / Cas12a system is widely used for nucleic acid detection. After the target double-stranded DNA (dsDNA) is recognized via crRNA, the activated Cas12a protein performs cis-cleavage of the specific target DNA and initiates non-specific concomitant trans-cleavage of single-stranded DNA (ssDNA). The efficient integration of RPA and the CRISPR / Cas12a system has become one of the key drivers propelling next-generation molecular diagnostics.

[0004] Surface-enhanced Raman spectroscopy (SERS) is a spectroscopic technique based on metal nanostructures. By exciting local surface plasmon resonances on metal surfaces, this technique can significantly enhance the Raman scattering signals of biomolecules (such as proteins, DNA / RNA) adsorbed on metal surfaces, ultimately achieving ultrasensitive detection at the single-molecule level. Summary of the Invention

[0005] The present invention aims to provide a fluorescence / SERS dual-mode detection system based on RPA amplification combined with CRISPR / Cas12a technology and its application in viral DNA detection.

[0006] The technical solution adopted in this invention is as follows:

[0007] This invention first provides a fluorescence / SERS dual-mode detection system based on RPA amplification combined with CRISPR / Cas12a technology, which includes:

[0008] (1) Two nucleic acid-functionalized SERS nanoprobes: The SERS nanoprobes are core-shell structured silver-coated gold nanoparticles, internally modified with 4-MBA Raman signal molecules, and surface modified with ssDNA1 and ssDNA2 respectively; one end of ssDNA1 and ssDNA2 is modified with thiol groups to fix them on the surface of the silver-coated gold nanoparticles, and the bases at the other end can be linked to Linker DNA through complementary pairing.

[0009] (2) RPA amplification system: lyophilized particles containing recombinase, polymerase, single-chain binding protein and a large number of free bases, magnesium acetate solution to provide activity for the reaction, and upstream and downstream primers corresponding to and amplifying the target sequence;

[0010] (3) CRISPR cleavage system: includes Cas12a protein for non-specific cleavage of surrounding single-stranded DNA, crRNA for specific recognition and binding of target DNA, Linker DNA for linking SERS nanoprobes, and fluorescent reporter probes labeled with fluorescent group FAM and quenching group BHQ1 at both ends, respectively.

[0011] Furthermore, the Raman signal molecule 4-MBA at 1075 cm⁻¹ -1 There are obvious Raman characteristic peaks at this point.

[0012] Further, the sequence of ssDNA1 is: 5'-TCACAGATGCGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-SH-3', and the sequence of ssDNA2 is: 5'-SH-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCACCGAGCACGA-3'.

[0013] Furthermore, the sequence of the upstream primer is: 5'-CCTTCTCAGTCCAGCGCGTTTACGTAAGCC-3'; and the sequence of the downstream primer is: 5'-CTAGGGAGAGGTAGAAGACCCCCTCTTACA-3'.

[0014] Furthermore, the crRNA sequence is: 5'-UAAUUUCUACUAAGUGUAGAUUGUGGACUCCUGGCGCUCUGAUG-3'.

[0015] Furthermore, the sequence of the Linker DNA is: 5'-ACGCATCTGTGATCGTGCTCGGTG-3.

[0016] Furthermore, the sequence of the fluorescent reporter probe is: 5'-FAM-TTTTTTTTTTTTTTT-BHQ1-3'.

[0017] The present invention also provides a method for constructing a dual-mode detection system, which includes the following steps:

[0018] S1: Preparation of gold nanoparticle solution by seed growth method;

[0019] S2: The Raman signal molecule 4-MBA with Raman characteristic peaks was modified on the surface of gold nanoparticles. Then, AgNO3 was reduced to silver layer through reduction reaction to cover the surface of gold nanoparticles, forming core-shell structured silver-coated gold nanoparticles. Finally, thiolized ssDNA1 and ssDNA2 were anchored to the silver shell surface by freezing method to obtain two nucleic acid functionalized SERS nanoprobes.

[0020] S3: Extract total DNA from the sample and then use RPA to amplify the target DNA using upstream and downstream primers;

[0021] S4: Mix Cas12a protein and crRNA to form a Cas12a-crRNA binary complex. Design the sequence on the crRNA to specifically recognize the target DNA. Then add the target DNA after RPA amplification. When the crRNA recognizes a large amount of target DNA, the cleavage function of Cas12a protein is activated and begins to continuously and non-specifically cleave the fluorescent reporter probe and Linker DNA.

[0022] S5: Detect the fluorescence signal in the reaction system and obtain the fluorescence detection results;

[0023] S6: The CRISPR cleavage system after the reaction was completed was mixed with two SERS nanoprobes and incubated. After thorough centrifugation, the SERS signal intensity of the supernatant was measured by Raman spectroscopy. In the negative sample, the linker DNA was intact, and the two probes were cross-linked to form aggregates. After centrifugation, the aggregates precipitated, and the SERS signal in the supernatant was weak and there was no fluorescence signal. In the positive sample, the linker was cleaved, the probes were dispersed, the SERS signal in the supernatant was enhanced, and the fluorescent reporter probe broke and generated a fluorescence signal.

[0024] Further, in step S1, the formula for the seed growth method is: 100 mL chloroauric acid solution and 1.5 mL sodium citrate solution; the concentration of the chloroauric acid solution is 1% w / w, and the concentration of the sodium citrate solution is 1% w / w.

[0025] Further, in step S2, the formulation of the core-shell structured silver-coated gold nanoparticles is as follows: 10 mL gold nanoparticle solution, 15 μL 4-MBA, 200 μL sodium citrate solution, 100 μL sodium hydroxide solution, 1 mL AgNO3 solution, and 1 mL ascorbic acid solution; the concentration of the 4-MBA solution is 1 mM, the concentration of the sodium citrate solution is 1% w / w, the concentration of the sodium hydroxide solution is 100 mM, the concentration of the AgNO3 solution is 1 mM, and the concentration of the ascorbic acid solution is 10 mM; the volume ratio of ssDNA1 or ssDNA2 to the silver-coated gold nanoparticles is 12:400.

[0026] Further, in step S3, the total volume of the RPA amplification system is 10 μL, the volume of the extracted DNA is 4 μL, and the reaction temperature is 37℃.

[0027] Further, in step S4, the molar ratio of Cas12a protein to crRNA is 1:2, and the reaction temperature of the CRISPR cleavage system is 37°C.

[0028] Further, in step S6, the mixed incubation conditions are incubation at 37°C for 20 minutes; the conditions for sufficient centrifugation are centrifugation for 2 minutes under a relative centrifugal force of 900.

[0029] This invention employs the above technical solution, synthesizing gold nanoparticles via seed growth, then sequentially modifying the nanoparticle surface with Raman signal molecules and a silver layer to form silver-coated gold nanoparticles. Next, ssDNA1 and ssDNA2 are modified onto the nanoparticle surface to obtain nucleic acid-functionalized SERS nanoprobes. A recombinant polymerase amplification (RPA) system, under the synergistic action of recombinase, DNA polymerase, and single-stranded DNA-binding proteins, can achieve exponential rapid amplification of the target DNA sequence under isothermal conditions. A CRISPR / Cas12a cleavage system, if crRNA recognizes the RPA-amplified target DNA, activates the Cas12a protein to non-specifically cleave the linker DNA used to connect the SERS nanoprobes and the fluorescent reporter probes labeled with the fluorescent group FAM and the quencher group BHQ1 at both ends, respectively. Finally, fluorescence / SERS dual-mode detection is performed; when the linker DNA and the fluorescent reporter probe are not cleaved, the linker DNA... DNA links the SERS nanoprobes to form nanoparticle aggregates. After slight centrifugation, the nanoparticle aggregates settle at the bottom, resulting in a very weak SERS signal detected in the supernatant, and the system does not generate a fluorescence signal. Conversely, if the unaggregated SERS nanoprobes are evenly distributed, the supernatant contains a strong SERS signal, and the fluorescent reporter probe breaks down and generates a fluorescence signal.

[0030] The beneficial effects of this invention are as follows:

[0031] (1) This invention employs two SERS nanoprobes with different nucleic acid sequences, ssDNA1 and ssDNA2, respectively modified. The two probes can form nanoaggregates through base complementarity under the action of Linker DNA. When the target viral DNA is present, the CRISPR / Cas12a system is activated and cuts the Linker DNA, preventing aggregate formation. The change in aggregation or dispersion state achieves the weakening or strengthening of the SERS signal, thereby completing the detection of viral DNA.

[0032] (2) The detection limit of the SERS detection mode for target EBV DNA is as low as 0.5 copies / μL, which is an improvement over the traditional qPCR detection method;

[0033] (3) The fluorescence detection mode has a faster reaction speed, which significantly shortens the detection time compared with the traditional qPCR detection method;

[0034] (4) Preliminary qualitative testing can be completed within 30 minutes, and accurate quantitative testing can be completed within 1 hour;

[0035] (5) This invention achieves rapid and sensitive detection of viral DNA by using RPA rapid amplification and specific recognition of Cas12a protein, and by using changes in SERS and fluorescence signals. The SERS detection mode and the fluorescence detection mode verify and complement each other. The fluorescence signal monitors the trans-cleavage process of Cas12a in real time, while the SERS signal provides molecular fingerprint information with low background noise, which improves the accuracy of detection.

[0036] (6) No complex and expensive large temperature control instruments are required, making it suitable for clinical point-of-care testing scenarios. Attached Figure Description

[0037] Figure 1 This is a schematic diagram illustrating the working principle of the fluorescence / SERS dual-mode detection based on RPA-CRISPR / Cas12a. (A) Isothermal amplification of target DNA by RPA and recognition and cleavage by CRISPR / Cas12a; (B) Rapid fluorescence detection; (C) High-sensitivity SERS detection; (D) qPCR detection verification.

[0038] Figure 2 Characterization of SERS nanoprobes. (a) Preparation of SERS nanoprobes; (b) UV-Vis absorption spectrum; (c) Zeta potential distribution; (d) Particle size distribution; (e) Raman spectrum of nanoparticles; (f) Transmission electron microscopy image of SERS nanoprobes; (g) Elemental distribution map of Au@MBA@Ag nanoparticles obtained by energy dispersive spectroscopy.

[0039] Figure 3 This involves optimizing the reaction conditions. (a) Fluorescence curves of different RPA amplification primer pairs; (b) Fluorescence saturation rates at different RPA amplification times; (c) Fluorescence curves of samples without RPA amplification; (d) Raman spectra obtained using different volumes of Cas12a protein; (e) 1075 cm⁻¹ -1 (f) Relationship between peak intensity and Cas12a protein volume; (g) Comparison of simulated positive and negative samples at different centrifugation rates; (e) Raman spectra of nanoparticles at different DNA strand volumes; (f) 1075 cm⁻¹ -1 (f) Relationship between peak intensity and DNA strand volume; (f) Comparison of simulated positive and negative samples under different incubation times.

[0040] Figure 4 This is a system performance evaluation. (a) Feasibility analysis results of agarose gel electrophoresis; (b) Feasibility of the SERS detection system; (c) Reproducibility of different batches of SERS nanoprobes; (d) SERS spectra at different EBV DNA concentrations; (e) 1075 cm⁻¹ -1(f) Linear relationship between peak intensity and different EBV DNA concentrations; (g) Specificity of the SERS detection system; (h) Fluorescence curves at different EBV DNA concentrations; (f) Linear relationship between fluorescence saturation rate and different EBV DNA concentrations; (i) Specificity of the fluorescence detection system.

[0041] Figure 5 This is the application of a dual-mode detection system in nasopharyngeal carcinoma clinical samples. (a) Raw fluorescence detection data of all clinical samples; (b) Fluorescence detection thermograms of all clinical samples; (c) SERS detection of all clinical samples at 1075 cm⁻¹. -1 (d) Peak intensity map of all clinical samples; (e) Raw qPCR data map of all clinical samples; (f) qPCR data map of all clinical samples. Detailed Implementation

[0042] To enable those skilled in the art to more clearly understand the technical solutions of this application, the technical solutions of this application will be described in detail below with reference to specific embodiments. Obviously, the embodiments described below are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] The working principle of the dual-mode detection system of this invention is as follows: Figure 1As shown. (A) RPA isothermal amplification of target DNA and CRISPR recognition and cleavage. A rapid RPA reaction was performed using a standard plasmid or total DNA extracted from nasopharyngeal carcinoma plasma samples as a template. The reaction was carried out at a constant temperature of 37°C through the coordinated action of recombinase, polymerase, and single-stranded binding protein. Subsequently, Cas12a protein was mixed with crRNA to form a Cas12a-crRNA binary complex. Then, the RPA-amplified target DNA was added. In the presence of the target DNA, crRNA guided the target DNA to bind to the Cas12a protein, forming a Cas12a-crRNA-target DNA ternary complex. When the system reached its operating temperature of 37°C, the CRISPR system was activated and began cleaving the surrounding single-stranded DNA. (B) Fluorescence detection principle. After the CRISPR system started the reaction, a fluorescent probe was added. The fluorescent probe was labeled with the fluorescent group FAM and the quenching group BHQ1 at both ends and kept in close proximity by a single-stranded DNA linker. In this state, the fluorescence of FAM was efficiently quenched by BHQ1, so the fluorescence signal could not be detected. Once the Cas12a protein cleaves the ssDNA, FAM and BHQ1 are spatially separated, and FAM begins to emit fluorescence, which can be monitored in real time using a fluorescence spectrometer. (C) SERS Detection Principle. The designed linker DNA hybridizes with two SERS nanoprobes, functionalized ssDNA1 and ssDNA2, through complementary base pairing, forming nanoparticle aggregates. After mild centrifugation, these aggregates precipitate at the bottom of the test tube, leaving a supernatant almost devoid of nanoparticles, producing a weak SERS signal. Conversely, in the presence of the target DNA, the Cas12a protein cleaves the linker DNA, preventing aggregate formation. After centrifugation, the SERS nanoprobes are uniformly dispersed in the solution, producing a strong SERS signal in the supernatant. (D) qPCR Detection Validation. The results of fluorescence and SERS detection were validated by comparing with those of conventional qPCR detection.

[0044] Example 1: Preparation and Characterization of SERS Nanoprobes

[0045] The preparation process of the SERS nanoprobe in this embodiment is as follows: Figure 2 As shown in a, the specific steps include:

[0046] S1: Preparation of gold nanoparticles (Au NPs)

[0047] 100 mL of HAuCl4 solution (1% w / w) was vigorously stirred and heated. After the solution boiled, 1.5 mL of sodium citrate solution (1% w / w) was quickly added. The solution turned dark red and was heated and stirred for 15 minutes to obtain Au NPs.

[0048] S2: Preparation of core-shell nanoparticles with Raman signals

[0049] 15 μL of 4-MBA (1 mM) was added to 10 mL of prepared Au NPs and the mixture was vigorously sonicated at room temperature for 30 minutes. The Raman reporter molecule 4-MBA was immobilized on the surface of the gold nanoparticles via Au-S bonds. After centrifugation, the supernatant was removed, and the mixture was resuspended in ultrapure water to a final volume of 10 mL, forming Au@4-MBA NPs. Next, under vigorous magnetic stirring, 200 μL of sodium citrate solution (1% w / w), 100 μL of NaOH solution (100 mM), and 1 mL of AgNO3 solution (1 mM) were added sequentially, followed by the slow addition of 1 mL of ascorbic acid solution (10 mM). Under the action of ascorbic acid, silver ions in silver nitrate were reduced, forming a uniform silver shell encapsulating the Au@4-MBA core. The solution changed from red to orange. After stirring for another 15 minutes, the mixture was centrifuged to remove the supernatant, and the mixture was resuspended in ultrapure water to a final volume of 10 mL, yielding stable core-shell structures Au@4-MBA@Ag NPs.

[0050] S3: Preparation of Nucleic Acid Functionalized Nanoparticles

[0051] Take 400 μL of Au@4-MBA@Ag NPs and add 12 μL of ssDNA1. Mix well and freeze the solution at -20°C for 30 minutes. Then thaw at room temperature, centrifuge to remove the supernatant to remove excess ssDNA1, and resuspend in ultrapure water to 400 μL. Finally, centrifuge the solution again and resuspend in 400 μL of PBS (1 mM) to obtain Au@4-MBA@Ag@ssDNA1 NPs, i.e., SERS nanoprobes.

[0052] Take 400 μL of Au@4-MBA@Ag NPs and add 12 μL of ssDNA2. Mix well and freeze the solution at -20°C for 30 minutes. Then thaw at room temperature, centrifuge to remove the supernatant to remove excess ssDNA2, and resuspend in ultrapure water to 400 μL. Finally, centrifuge the solution again and resuspend in 400 μL of PBS (1 mM) to obtain Au@4-MBA@Ag@ssDNA2 NPs, i.e., SERS nanoprobes.

[0053] exist Figure 2 In this context, both Au@4-MBA@Ag@ssDNA1 NPs and Au@4-MBA@Ag@ssDNA2 NPs are denoted as Au@MBA@Ag@DNA NPs.

[0054] To further verify the successful construction of SERS nanoprobes, we systematically characterized the obtained products. The synthesized Au NPs had an average particle size of approximately 37 nm, and the maximum absorption peak was located at 527 nm. Figure 2 As shown in b. After 4-MBA is modified onto the AuNPs surface, as shown... Figure 2 As shown in Figure c, the surface potential increased from −30 mV to −24 mV, the particle size increased by approximately 6 nm, and a significant Raman signal appeared, indicating that 4-MBA molecules were successfully adsorbed onto the gold surface via thiol groups. Subsequently, through the reduction reaction of silver nitrate and ascorbic acid, a silver layer uniformly coated the nanoparticle surface, increasing the particle size from 43 nm to 68 nm. The estimated thickness of the silver layer is approximately 12.5 nm. Figure 2 As shown in d. Simultaneously, the maximum absorption peak blue-shifts from 527 nm to 509 nm, and a characteristic absorption peak of silver appears at 369 nm, as shown in d. Figure 2 As shown in b. TEM and elemental mapping further verified the core-shell structure of Au@MBA@Ag NPs: the TEM image shows a light-colored silver shell surrounding the gold core, as shown in b. Figure 2 As shown in f, the elemental distribution map shows that silver is distributed around gold, as... Figure 2 As shown in g, this confirms the successful synthesis of the core-shell structure. Due to the significantly enhanced local electromagnetic field caused by the surface plasmon coupling effect between the gold core and the silver shell, the Raman signal is also amplified, as shown in g. Figure 2 As shown in e. Finally, ssDNA was modified on the Au@MBA@Ag surface to form Au@MBA@Ag@DNA NPs, increasing the particle size by approximately 11 nm. Due to the overall negative charge of the ssDNA molecule, the surface potential decreased from −23 mV to −28 mV; furthermore, the dense DNA layer weakened the Raman signal. When the SERS nanoprobes hybridized with linkerDNA, they formed nanoparticle aggregates, such as... Figure 2 As shown in d, its average particle size is approximately 267 nm.

[0055] The ssDNA sequences involved in this example are ssDNA1: 5'-TCACAGATGCGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-SH-3', ssDNA2: 5'-SH-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCACCGAGCACGA-3'.

[0056] Example 2

[0057] A method for constructing a fluorescence / SERS dual-mode detection system based on RPA amplification combined with CRISPR / Cas12a technology includes the following steps:

[0058] S1: Sample amplification and CRISPR system identification

[0059] S1-1: If the sample is a real clinical sample, it needs to be collected and extracted. First, collect 200 μL of the patient's plasma or serum sample, and then extract total DNA from the collected sample using a DNA extraction kit. If the sample is a standard plasmid sample, no processing is required, and it can be used directly.

[0060] S1-2: RPA amplification of the S1-1 sample. First, add a lyophilized particle containing recombinase, polymerase, single-stranded binding protein, and a large number of free bases to the total reaction tube. Then add 13 μL of DEPC-treated water, followed by 1 μL of upstream and downstream primers (10 μM). After mixing, divide the solution from the total reaction tube into three equal tubes. Add 4 μL of standard plasmid or extracted DNA sample to each tube. Finally, add 1 μL of magnesium acetate solution to activate the reaction. After mixing, incubate at 37°C for 10 minutes.

[0061] S1-3: CRISPR System Recognition. First, the Cas12a protein was mixed with the designed crRNA at a 1:2 molar ratio. Then, 5 μL of 10× reaction buffer was added, followed by DEPC-treated water to bring the reaction volume to 40 μL. Finally, 5 μL of the RPA amplification product from S1-2 was added, and the reaction was initiated at 37°C. When the crRNA recognized the target DNA, the Cas12a protein was activated, triggering its cis and trans cleavage capabilities.

[0062] S2: Fluorescence detection system

[0063] Before initiating the CRISPR system reaction in S1-3, add 2 μL of fluorescent probe. The fluorescent probe is labeled with a fluorophore FAM and a fluorescence quencher BHQ1 at both ends, connected by a single-stranded DNA linker. In this state, the fluorescence of FAM is effectively quenched by BHQ1, so the system does not emit light. When the Cas12a protein is triggered to perform trans-cleavage, the ssDNA is cleaved, the fluorophore FAM and the fluorescence quencher BHQ1 spatially separate, and the energy is no longer quenched. FAM then begins to fluoresce, which can be monitored in real time using a thermostatic fluorescence detector.

[0064] S3: SERS detection system

[0065] S3-1: Add 2 μL of Linker DNA before starting the CRISPR system reaction in S1-3. The Linker DNA can link SERS nanoprobes modified with ssDNA1 and ssDNA2 respectively (Au@4-MBA@Ag@ssDNA1NPs, Au@4-MBA@Ag@ssDNA2NPs) through base complementary pairing to form nanoparticle aggregates.

[0066] S3-2: Take 100 μL each of the SERS nanoprobes linked to ssDNA1 and ssDNA2, mix thoroughly with the CRISPR system, then add 2 μL of NaCl solution (1 mM) to promote base pairing between ssDNAs. Finally, incubate the entire system at 37°C for 20 minutes. Then, place the centrifuge tubes in a handheld centrifuge and centrifuge for two minutes to separate the nanoparticle aggregates and free SERS nanoprobes. After centrifugation, take 100 μL of the supernatant and acquire the SERS signal using a handheld RMS 1000 Raman spectrometer. If the linker DNA is not cleaved, the SERS nanoprobes will extensively interlink and form nanoparticle aggregates, which will deposit at the bottom of the centrifuge tube after centrifugation. In this case, the supernatant contains almost no nanoparticles, resulting in a very weak SERS signal. Conversely, when the target DNA is present, the linker DNA is cleaved by the Cas12a protein and cannot form aggregates. Even after centrifugation, the SERS nanoprobes remain uniformly dispersed in the solution, thus generating a strong SERS signal in the supernatant.

[0067] S4: qPCR detection verification

[0068] The S1-1 sample was simultaneously subjected to qPCR detection. Then, the results of the fluorescence detection system and the SERS detection system were compared with the results of the traditional qPCR detection to verify the results of the fluorescence and SERS detection.

[0069] The primer sequences involved in this embodiment are: upstream primer: 5'-CCTTCTCAGTCCAGCGCGTTTACGTAAGCC-3', downstream primer: 5'-CTAGGGAGAGGTAGAAGACCCCCTCTTACA-3'.

[0070] The crRNA sequence involved in this embodiment is: 5'-UAAUUUCUACUAAGUGUAGAUUGUGGACUCCUGGCGCUCUGAUG-3'.

[0071] The Linker DNA sequence involved in this embodiment is: 5'-ACGCATCTGTGATCGTGCTCGGTG-3'.

[0072] The fluorescent reporter probe sequence involved in this embodiment is: 5'-FAM-TTTTTTTTTTTTTTT-BHQ1-3'.

[0073] In this embodiment, the qPCR primers used are: upstream primer: 5'-GTAAGCCAGACAGCAGCCAA-3', and downstream primer: 5'-GGGCTAGGGAGAGGTAGAAGA-3'.

[0074] Example 3

[0075] To achieve optimal performance of the RPA-CRISPR / Cas12a dual-mode detection system, the present invention optimizes several experimental conditions.

[0076] The RPA reaction is performed at 37°C, and selecting appropriate primer pairs is crucial for ensuring efficient amplification. This invention designs four primer pairs: primer pair 1 (F1 and R1), primer pair 2 (F2 and R1), primer pair 3 (F1 and R2), and primer pair 4 (F2 and R2), aiming to maximize amplification efficiency while minimizing base mismatches. The performance of each primer pair was evaluated using a fluorescence detection system, and the results showed that primer pair 4 had the highest amplification efficiency. Figure 3 As shown in figure a. To ensure detection sensitivity while accelerating detection speed, we further evaluated the impact of amplification time on detection performance. For example... Figure 3 As shown in b, when the amplification time exceeded 20 minutes, the system reached a sensitivity of 1 copies / μL, and the fluorescence intensity saturated; at 10 minutes, the sensitivity remained unchanged, but the signal did not reach saturation; at 5 minutes, the sensitivity dropped to 100 copies / μL. Therefore, an appropriate amplification time should be selected to balance detection sensitivity and detection speed. To verify the necessity of RPA amplification, we performed fluorescence detection on unamplified samples, and the results showed that fluorescence was only detected at concentrations higher than 10... 8 It can be detected at copies / μL, such as Figure 3 As shown in c, this fully demonstrates that RPA amplification significantly improves detection sensitivity.

[0077] To further improve system performance, we optimized the amount of Cas12a protein, the nanoprobe linker volume, the centrifugation rate, and the incubation time to determine the optimal reaction conditions. The results showed that the SERS signal reached its maximum when 4 μL of 10 nM Cas12a protein was added, indicating the strongest cleavage activity under these conditions. Therefore, 4 μL was selected as the optimal amount for subsequent experiments. Figure 3As shown in the diagram. To obtain stable SERS nanoprobes, the volume of DNA added during cryopreservation was also optimized. In the absence of DNA, the nanoparticles exhibited significant aggregation after cryopreservation, leading to a significant decrease in the SERS signal. The signal gradually recovered as the DNA volume increased. The SERS signal enhancement was most pronounced at a DNA volume of 6 μL, while at a DNA volume of 10 μL, the signal level approached that of the un-cryopreserved probe, as shown in the diagram. Figure 3 As shown in gh. Furthermore, to effectively distinguish nanoparticle aggregates formed by linker DNA hybridization from unlinked probes, we optimized the centrifugation rate. The results showed that lower centrifugation rates were insufficient to precipitate the aggregates, while excessively high centrifugation rates led to the precipitation of unlinked probes. Optimal separation was achieved at 900 RCF, as shown in gh. Figure 3 As shown in f. Finally, the incubation time for complementary hybridization between ssDNA1, ssDNA2, and the linker DNA was optimized. The results showed that an incubation time of 20 minutes produced the most complete complementary pairing reaction, thus obtaining the highest signal, as shown in f. Figure 3 As shown in i.

[0078] The primer sequences involved in this embodiment are F1: 5'-TCGCGTTGCTAGGCCACCTTCTCAGTCCAG-3', F2: 5'-CCTTCTCAGTCCAGCGCGTTTACGTAAGCC-3', R1: 5'-AAGTTAGAAACTGGGCCCGAGTCCTTGGA-3', and R2: 5'-CTAGGGAGAGGTAGAAGACCCCCTCTTACA-3'.

[0079] Example 4

[0080] Performance evaluation of the dual-mode detection system.

[0081] To verify the feasibility of the CRISPR system in this experiment, agarose gel electrophoresis and a SERS detection system were used for validation. Agarose gel electrophoresis verified the cis-cleavage activity of the CRISPR system on the target DNA. Using a standard EBV DNA plasmid as the target DNA, due to its circular structure and approximately 3 kb size, a bright band migrating below 3 kb was observed in the first sample channel, while no band was detected in the second sample channel without the plasmid. When crRNA or Cas12a was omitted in the third and fourth sample channels, respectively, the migration position of the target DNA did not change, indicating that cis-cleavage was not activated. When the target DNA, crRNA, and Cas12a were present simultaneously, the CRISPR system was activated, triggering cis-cleavage of the plasmid at the recognition site, converting the circular plasmid into a linear form. Because the circular plasmid migrates faster than the linear plasmid, a weak band appeared at approximately 3 kb in the fifth sample channel. Figure 4 As shown in Figure a. These results indicate that cis-cleavage by the CRISPR system is activated only in the co-existence of target DNA, crRNA, and Cas12a, accompanied by activation of trans-cleavage activity. In the SERS detection system, the absence of Cas12a, crRNA, target DNA, or the presence of only linker DNA resulted in a low-intensity SERS signal, indicating that the SERS nanoprobes were linked by the linker DNA and deposited at the bottom. Only when Cas12a, crRNA, target DNA, and linker DNA were all present were they cleaved by the CRISPR system, leaving the unlinked SERS nanoprobes in the supernatant, thus generating a strong SERS signal, as shown in Figure a. Figure 4 As shown in b. These results demonstrate the necessary conditions and feasibility of the proposed system. Meanwhile, the reproducibility of the nanoparticles is also a key factor affecting the preparation process. This invention prepared 10 SERS nanoprobes at different times and batches. The Raman signals of these nanoprobes showed only slight variations, with a relative standard deviation of 6.77%, demonstrating the high reproducibility of the nanoprobe preparation process of this invention. Figure 4 As shown in c. Furthermore, specificity is crucial for evaluating the performance of the dual-modal detection system. In addition to the target EBV DNA, this invention also uses various DNA virus sequences as interfering substances, including IAV, HPV16, HPV18, and CD2V and P72 from African swine fever. Clearly, the SERS and fluorescence detection systems only exhibit strong Raman or fluorescence signals in the presence of EBV DNA, while for other interfering virus sequences, only minor differences were observed compared to the control group, such as... Figure 4As shown in f and i, this demonstrates that the dual-modal system of the present invention has high specificity for the target. Sensitivity is a key indicator for evaluating system performance; therefore, the present invention used standard EBV DNA samples of different concentrations to evaluate the performance of the dual-modal detection system. In the SERS detection system, a higher EBV DNA concentration corresponds to a stronger SERS signal, indicating that the SERS signal in the supernatant is positively correlated with the EBV DNA concentration, such as... Figure 4 As shown in d, the SERS signal intensity increases logarithmically with increasing EBV DNA concentration, exhibiting a strong linear relationship. 2 =0.971, the standard curve is Y=773.48X+2515.74, as... Figure 4 As shown in e. The linear detection range is from 0.5 copies / μL to 10. 5 0.5 copies / μL is the minimum detection concentration for the SERS detection system. In the fluorescence detection system, since fluorescence tends to saturate when the RPA time exceeds 10 minutes, this invention adjusts the RPA amplification time to optimize the system's fluorescence response range. When the RPA amplification time is 7 minutes, the fluorescence signal gradually increases with increasing reaction time, and the amplification rate accelerates with increasing EBV DNA concentration. Figure 4 As shown in g. 50 copies / μL is the minimum detectable concentration of the system, and the system is in operation at 10... 2 copies / μL to 10 5 A strong linear relationship exists within the concentration range of copies / μL. 2 =0.946, the standard curve is Y=0.122X-0.258, as... Figure 4 As shown in h.

[0082] Example 5

[0083] This embodiment aims to further explore the performance of a dual-mode detection system for clinical samples. Targeting EBV DNA, a biomarker associated with nasopharyngeal carcinoma, a total of 25 clinical plasma samples were tested, including 17 nasopharyngeal carcinoma patients (positive samples) and 8 healthy individuals (negative samples).

[0084] All plasma samples underwent DNA extraction and were subsequently analyzed using three different methods: qPCR, fluorescence detection, and SERS. Results obtained via qPCR were used as a reference standard to evaluate and compare the accuracy of the proposed dual-mode detection system. All samples were analyzed using the dual-mode detection system; in the fluorescence detection system, no fluorescence signal was detected in negative samples, while most positive samples showed saturated fluorescence signals, such as... Figure 5As shown in figure a. In the SERS detection system, all negative samples showed low-intensity SERS signals, while most positive samples showed strong SERS signals, such as... Figure 5 As shown in c. To more clearly analyze the results of clinical samples, fluorescence and SERS detection data are visualized using a heatmap, with fluorescence intensity as shown in Figure c. Figure 5 As shown in b, the strength of the SERS signal is as follows: Figure 5 As shown in d. Subsequently, in the "gold standard" qPCR test, the CT values ​​of all NPC patient samples ranged from 25 to 35, while no CT values ​​were detected in samples from healthy individuals, as shown in d. Figure 5 As shown in e. To more intuitively display the results, a heatmap of qPCR CT values ​​was also generated, as shown in e. Figure 5 As shown in f. These results indicate that the accuracy of fluorescence detection is 92%, and the accuracy of SERS detection is 96%. In summary, compared with the "gold standard" qPCR, the dual-modal detection system of this invention has significant advantages and feasibility in EBV DNA detection, and can provide rapid, reliable, and convenient molecular evidence for the diagnosis and prognosis of nasopharyngeal carcinoma.

Claims

1. A fluorescence / SERS dual-mode detection system based on RPA amplification combined with CRISPR / Cas12a technology, characterized in that, It includes: (1) Two nucleic acid-functionalized SERS nanoprobes: The SERS nanoprobes are core-shell structured silver-coated gold nanoparticles, internally modified with 4-MBA Raman signal molecules, and respectively modified with ssDNA1 or ssDNA2 on the surface; one end of ssDNA1 and ssDNA2 is modified with thiol groups to fix them on the surface of the silver-coated gold nanoparticles, and the bases at the other end can be linked to LinkerDNA through complementary pairing. (2) RPA amplification system: lyophilized particles containing recombinase, polymerase, single-chain binding protein and free bases, magnesium acetate solution, upstream primer and downstream primer; (3) CRISPR cleavage system: includes Cas12a protein for non-specific cleavage of surrounding single-stranded DNA, crRNA for specific recognition and binding of target DNA, Linker DNA for linking SERS nanoprobes, and fluorescent reporter probes labeled with fluorescent group FAM and quenching group BHQ1 at both ends, respectively.

2. The fluorescence / SERS dual-mode detection system based on RPA amplification combined with CRISPR / Cas12a technology according to claim 1, characterized in that, The sequence of ssDNA1 is: 5'-TCACAGATGGCGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-SH-3', and the sequence of ssDNA2 is: 5'-SH-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCACCGAGCACGA-3'.

3. The fluorescence / SERS dual-mode detection system based on RPA amplification combined with CRISPR / Cas12a technology according to claim 1, characterized in that, The sequence of the upstream primer is: 5'-CCTTCTCAGTCCAGCGCGTTTACGTAAGCC-3'; the sequence of the downstream primer is: 5'-CTAGGGAGAGGTAGAAGACCCCCTCTTACA-3'.

4. The fluorescence / SERS dual-mode detection system based on RPA amplification combined with CRISPR / Cas12a technology according to claim 1, characterized in that, The crRNA sequence is: 5'-UAAUUUCUACUAAGUGUAGAUUGUGGACUCCUGGCGCUCUGAUG-3'.

5. The fluorescence / SERS dual-mode detection system based on RPA amplification combined with CRISPR / Cas12a technology according to claim 1, characterized in that, The sequence of the linker DNA is: 5'-ACGCATCTGTGATCGTGCTCGGTG-3'; the sequence of the fluorescent reporter probe is: 5'-FAM-TTTTTTTTTTTTTTT-BHQ1-3'.

6. A method for constructing a dual-mode detection system as described in any one of claims 1 to 5, characterized in that, It includes the following steps: S1: Preparation of gold nanoparticle solution by seed growth method; S2: The Raman signal molecule 4-MBA with Raman characteristic peaks was modified on the surface of gold nanoparticles. Then, AgNO3 was reduced to silver layer through reduction reaction to cover the surface of gold nanoparticles, forming core-shell structured silver-coated gold nanoparticles. Finally, thiolized ssDNA1 and ssDNA2 were anchored to the silver shell surface by freezing method to obtain two nucleic acid functionalized SERS nanoprobes. S3: Extract total DNA from the sample and then use RPA to amplify the target DNA using upstream and downstream primers; S4: Mix Cas12a protein and crRNA to form a Cas12a-crRNA binary complex, then add the target DNA after RPA amplification. When crRNA recognizes the target DNA, the cleavage function of Cas12a protein is activated, and it begins to continuously and non-specifically cleave the fluorescent reporter probe and Linker DNA. S5: Detect the fluorescence signal in the reaction system and obtain the fluorescence detection results; S6: The CRISPR cutting system after the reaction was completed was mixed with two SERS nanoprobes and incubated. After centrifugation, the SERS signal intensity of the supernatant was measured by Raman spectroscopy.

7. The method for constructing a dual-mode detection system according to claim 6, characterized in that, In step S2, the volume ratio of ssDNA1 or ssDNA2 to silver-coated gold nanoparticles is 12:400; the reaction temperature in step S3 is 37°C; the molar ratio of Cas12a protein to crRNA in step S4 is 1:2, and the reaction temperature of the CRISPR cleavage system is 37°C; the incubation conditions after mixing in step S6 are 37°C for 20 minutes; and the centrifugation conditions are centrifugation at a relative centrifugal force of 900 for 2 minutes.

8. The method for constructing a dual-mode detection system according to claim 6, characterized in that, In step S6, the Linker DNA in the negative sample is intact, crosslinking the two probes to form aggregates, which precipitate after centrifugation. The SERS signal in the supernatant is weak and there is no fluorescent signal. In the positive sample, the Linker is cleaved, the probes are dispersed, the SERS signal in the supernatant is enhanced, and the fluorescent reporter probe breaks and generates a fluorescent signal.

9. The application of a dual-mode detection system as described in any one of claims 1 to 5 or the method as described in claim 6 in viral DNA detection.

10. The application according to claim 9, characterized in that: The viral DNA mentioned includes, but is not limited to, EBV DNA.

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