SERS biosensor system based on CRISPR / Cas12a and recombinase polymerase amplification and application of SERS biosensor system in virus DNA detection

Abstract

The invention discloses an SERS (Surface Enhanced Raman Scattering) biosensor system based on CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas12a and recombinase polymerase amplification and application of the SERS biosensor system The preparation method comprises the following steps: preparing silver-coated gold nanoparticles with Raman signal molecules by a seed synthesis method, and modifying the surfaces of the nanoparticles with polythymosinic acid of which two ends are modified with biotin and sulfydryl respectively to obtain DNA functionalized silver-coated gold nanoparticles; a CRISPR / Cas12a detection system is constructed, a sample or the sample subjected to RPA amplification is added into a reaction system, if the sample contains target DNA, CRISPR / Cas12a can cut DNA on the surfaces of silver-coated gold nanoparticles, generated free biotin modified DNA is competitively combined with subsequently added streptavidin modified magnetic beads, so that capture of the DNA functionalized silver-coated gold nanoparticles by the magnetic beads is obviously reduced, and the detection result shows that the detection result is accurate. Further, the SERS signal in the supernate is basically kept unchanged. On the contrary, if the sample does not contain the target DNA, the signal in the supernate is obviously reduced. And sensitive detection of virus DNA is realized based on Raman signal change.

Description

Technical Field

[0001] The present invention belongs to the field of detection technology, and specifically relates to a SERS biosensor system based on CRISPR / Cas12a and recombinase polymerase amplification and its application in viral DNA detection. Background Art

[0002] The prevention and diagnosis of diseases induced by viral infections cannot be separated from the accurate detection of viral DNA, such as HPV viral DNA and cervical cancer, EBV viral DNA and nasopharyngeal carcinoma. The polymerase chain reaction (PCR) and its derivative technologies have become the "gold standard" for detecting many viral DNAs with their high sensitivity and specificity. PCR technology can accurately detect trace amounts of viral DNA, so it is widely used in clinical screening for diseases. However, although PCR testing has obvious technical advantages, its application faces many challenges. First of all, PCR instruments are usually expensive and bulky, complicated to operate, and require a professional laboratory environment.

[0003] In recent years, CRISPR / Cas12a has shown great application potential in disease diagnosis and biosensing. The system consists of CRISPR and Cas12a proteins, which can accurately target exogenous nucleic acids through RNA guidance and use their endonuclease activity to specifically degrade target DNA. In addition to being able to accurately cis-cut target DNA, it can also non-specifically cut surrounding ssDNA molecules. This "side effect" provides a new detection strategy and opens up new possibilities for the development of efficient biosensing platforms.

[0004] Surface enhanced Raman scattering (SERS) is an advanced spectral analysis technique that combines Raman spectroscopy and nanotechnology, which can significantly improve the sensitivity of Raman signals. When the target molecule is placed in a "hot spot" area formed by nanoparticles or nanomaterials (such as silver, gold nanoparticles, and graphene oxide), the local surface plasmon resonance effect and the chemical enhancement effect caused by charge transfer will greatly enhance the weak Raman signal. This effect enables SERS technology to show ultra-high sensitivity in the detection of biological single molecules (such as proteins, lipids, and DNA / RNA). At present, many DNA detection strategies based on the combination of CRISPR / Cas12a and SERS have been developed and have demonstrated extremely high detection sensitivity. Summary of the invention

[0005] The object of the present invention is to provide a SERS biosensor system based on CRISPR / Cas12a and recombinase polymerase amplification and its application in viral DNA detection.

[0006] To achieve the above purpose, the present invention adopts the following technical scheme: silver-coated gold nanoparticles with Raman signal molecules are prepared by seed synthesis method, and polythymidine (polyT) with biotin and thiol groups at both ends of a certain length is modified on the surface of the nanoparticles to obtain DNA-functionalized silver-coated gold nanoparticles; CRISPR / Cas12a detection system is constructed, and the sample or the sample amplified by RPA is added to the reaction system. If the sample contains target DNA, CRISPR / Cas12a will cut the DNA on the surface of the silver-coated gold nanoparticles, and the free biotin-modified DNA produced will compete with the streptavidin-modified magnetic beads added later, thereby causing the capture efficiency of DNA-functionalized silver-coated gold nanoparticles by magnetic beads to be greatly reduced, thereby making the SERS signal in the supernatant remain unchanged. On the contrary, if the sample does not contain target DNA, the signal in the supernatant is significantly reduced. Sensitive detection of viral DNA is achieved based on Raman signal changes.

[0007] The method for constructing a SERS biosensor system for ultrasensitive detection of viral DNA based on CRISPR / Cas12a and recombinase polymerase amplification (RPA) comprises the following steps:

[0008] 1) Preparation of DNA-functionalized silver-coated gold nanoparticles (Biotin-SNA)

[0009] 4-Mercaptobenzoic acid (MBA) was added to gold nanoparticles (Au NPs) and stirred at room temperature, and then centrifuged and resuspended to form Au@MBANPs. Then, sodium citrate solution, sodium hydroxide solution, ascorbic acid solution and silver nitrate solution were added in sequence, and then centrifuged and resuspended to form Au@MBA@Ag NPs.

[0010] The DNA chains modified at both ends were mixed with Au@MBA@Ag NPs at a volume ratio of 1:19-21, frozen at -20°C and then thawed at room temperature, centrifuged and resuspended in DEPC water to form DNA-functionalized silver-coated gold nanoparticles Biotin-SNA;

[0011] 2) Construction of viral DNA detection system

[0012] The sample is lysed to release DNA, and then RPA is used for target pre-amplification. The Cas12a enzyme solution is incubated with crRNA to form a Cas12a-crRNA complex that specifically recognizes the target DNA;

[0013] The above-mentioned Biotin-SNA and Cas12a-crRNA complex was added to the RPA reaction tube, and then the DNA sample was added and incubated in a 37°C constant temperature incubator. After the reaction, streptavidin-modified magnetic beads (SAV-MB, 10 mg / ml) were added to capture for 5 to 7 minutes, and then the magnetic beads were separated with a magnet, and finally the SERS signal of the supernatant was measured.

[0014] If the DNA sample does not contain the target DNA, the trans-cleavage activity of the Cas12a enzyme will not be activated, and Biotin-SNA will bind to the streptavidin-modified magnetic beads, thereby significantly reducing the SERS signal in the supernatant; conversely, if the DNA sample contains the target DNA, the Cas12a-crRNA complex will form a Cas12a-crRNA-dsDNA complex with the target DNA, thereby activating the trans-cleavage activity of the Cas12a enzyme and cutting the DNA on the surface of Biotin-SNA. At this time, the free Biotin-DNA will compete for binding to the subsequently added SAV-MB, thereby greatly reducing the capture efficiency of Biotin-SNA by SAV-MB, thereby keeping the SERS signal in the supernatant basically unchanged.

[0015] Furthermore, the DNA chain sequence and double-end modification groups described in step 1) are as follows: 5'-Biotin-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-SH-3'.

[0016] Furthermore, the volume ratio of Au NPs to MBA in step 1) is 990-1010:1.

[0017] Furthermore, the concentration of the sodium citrate solution in step 1) is 0.95-1.05% w / w, the concentration of the sodium hydroxide solution is 95-105 mM, the concentration of the ascorbic acid solution is 9.5-10.5 mM, the concentration of the silver nitrate solution is 0.95-1.05 mM, and the volume ratio of Au@MBANPs, sodium citrate solution, sodium hydroxide solution, ascorbic acid solution and silver nitrate solution is 100:2:1:10:10.

[0018] Furthermore, the molar ratio of the Cas12a enzyme solution to crRNA in step 2) is 1:2.

[0019] Furthermore, the DNA sample added in step 2) is a non-RPA amplified DNA sample, and the incubation time is 90 to 100 minutes.

[0020] Furthermore, the DNA sample added in step 2) is a DNA sample for RPA amplification, and the incubation time is 25 to 30 minutes. The steps of RPA amplification of DNA samples are as follows: 25 μL 2× Reaction buffer I is added to each RPA reaction tube, 2 μL upstream primer and downstream primer (10 μM) are added respectively, and 6 μL H 2 O, divided into 5 tubes, added 2.5 μL of extracted DNA sample to each tube, and finally added 0.5 μL of magnesium acetate solution (250 mM) to mix, and placed at 37°C for reaction for 20 minutes.

[0021] The present invention adopts the above technical scheme to prepare Biotin-SNA with MBA Raman signal, designs specific crRNA according to the detection object, forms a Cas12a-crRNA complex with Cas12a enzyme, constructs a detection system, and finally uses SAV-MB to capture the reacted Biotin-SNA to achieve sensitive detection of viral DNA.

[0022] The working principle of the SERS biosensor system based on CRISPR / Cas12a and recombinase polymerase amplification described in the present invention is as follows: Figure 1 ) is: (1) RPA amplification can replicate a large amount of target DNA in a short time at 37°C, reduce the time required for subsequent detection, and further improve the detection sensitivity. (2) The prepared Biotin-SNA is added to the reaction tube together with the Cas12a-crRNA complex to construct a detection system, and then the DNA sample is added. If the DNA sample does not contain the target DNA, the trans-cleavage activity of the Cas12a enzyme will not be activated, and the Biotin-SNA will bind to the streptavidin-modified magnetic beads (SAV-MB), thereby significantly reducing the SERS signal in the supernatant. On the contrary, if the DNA sample contains the target DNA, the Cas12a-crRNA complex will form a Cas12a-crRNA-dsDNA complex with the target DNA, thereby activating the trans-cleavage activity of the Cas12a enzyme and cutting the ssDNA on the surface of the Biotin-SNA. At this time, the free Biotin-DNA will compete with the subsequently added SAV-MB, resulting in a greatly reduced capture efficiency of the Biotin-SNA by the SAV-MB, thereby keeping the SERS signal in the supernatant unchanged.

[0023] The beneficial effects of the present invention are: 1. Biotin-SNA with MBA Raman signal is prepared in a short time by freeze-thaw method, which greatly improves the preparation efficiency. 2. By optimizing the length of double-end modified DNA, the amount of double-end modified DNA used is greatly reduced while ensuring the stability of Biotin-SNA in the enzyme cutting system, thereby reducing the preparation cost of SNA. 3. The free Biotin-DNA after cutting will continue to play a role in competitive binding, thereby improving the sensitivity of detection to a certain extent. 4. Through RPA pre-amplification, a large amount of target DNA is replicated in the early stage, which reduces the time required for subsequent detection and further improves the detection sensitivity. 5. All steps are carried out at 37°C, and the detection can be completed within 50 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the preparation scheme and working principle diagram of SERS biosensor for viral DNA detection.

[0025] Figure 2 Characterization of SERS probe. (a) Energy dispersive spectroscopy elemental mapping, HAADF (High Angle Angular Dark Field-Scanning Transmission Electron Microscopy) and BF (Bright-field) TEM images of Au (red), S (blue) and Ag (green) in Au@MBA@Ag NP. (b) Zeta potential image.

[0026] Figure 3 The reaction conditions are optimized. (a) SERS spectra of SAV-MB at different dosages and the supernatant after full capture. (b) SERS intensity of Cas12a enzyme at different concentrations and the supernatant after capture.

[0027] Figure 4 The effect of different concentrations of free Biotin-DNA on the signal intensity of the supernatant. (a) Color comparison of the supernatant after SAV-MB capture in the presence of different concentrations of Biotin-DNA. (b) SERS signal before and after adding SAV-MB in the presence of different concentrations of Biotin-DNA.

[0028] Figure 5 It is a SERS biosensor for HPV-DNA detection; (a) SERS intensity 3D bar graph for HPV16 L1 gene at different reaction times and different concentrations; (b) SERS spectrum and intensity bar graph for HPV16 L1 gene at different concentrations; (c) SERS spectrum and intensity bar graph for HPV18 L1 gene at different concentrations.

[0029] Figure 6 TEM images of Biotin-SNA captured by SAV-MB and the corresponding supernatant images after reaction with different target DNA concentrations.

[0030] Figure 7 RPA amplification at 10 4 , 10 3 , 10 2 Agarose gel electrophoresis images of HPV16 and HPV18 target gene amplification at concentrations of 10 and 1aM. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments. Obviously, the embodiments described below are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1

[0033] Preparation of DNA-functionalized silver-coated gold nanoparticles (Biotin-SNA)

[0034] Au NPs and MBA (1mM) were mixed at a volume ratio of 1000:1 and stirred at room temperature for 2 hours. The supernatant was removed by centrifugation and resuspended to the original volume to form Au@MBANPs. After sodium citrate solution (1% w / w), sodium hydroxide solution (100mM) and ascorbic acid solution (10mM) were added in sequence, silver nitrate solution (1mM) was slowly added dropwise. The volume ratio of Au@MBANPs, sodium citrate solution, sodium hydroxide solution, ascorbic acid solution and silver nitrate solution was 100:2:1:10:10. The solution changed from purple-red to orange. After stirring for 15 minutes, it was centrifuged and resuspended to form Au@MBA@Ag NPs. The double-end modified DNA (5′-Biotin-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-SH-3′) was mixed with Au@MBA@Ag NPs at a volume ratio of 1:20, then frozen at −20 °C for 20 min and thawed at room temperature, centrifuged and resuspended in DEPC water to form Biotin-SNA.

[0035] Au@MBA@Ag NPs were characterized by transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM). Figure 2As shown in (a), Au@MBA@Ag NPs are uniform in size and have a distinct core-shell structure. The zeta potential of the nanoparticles in each synthesis process was detected, as shown in Figure 2 As shown in (b), after binding to DNA, the surface charge of the nanoparticles dropped to -53.9 mV, indicating that DNA successfully bound to the surface of Au@MBA@Ag NPs to form Biotin-SNA.

[0036] Example 2

[0037] Realize the establishment of a detection system for HPV16 and HPV18 DNA detection.

[0038] In this embodiment, the L1 gene fragments of HPV16 and HPV18 are used as the detection objects, and the L1 gene standard sequence is designed. The crRNA16 for identifying the L1 gene of the two viruses is 5'-UAAUUUCUACUAAGUGUAGAUUGAAGUAGAUAUGGCAGCAC-3' and the crRNA18 sequence is 5'-UAAUU UCUACUAAGUGUAGAUACAAUAUGUGCUUCUACACAG-3'. The upstream primer F used for RPA amplification is 5'-CCCAAATATTCAATAAACCTTATTGGTTACA-3', and the downstream primer R is 5'-ACTGAAAAATAAACTGCAAATCATATTCCTC-3'.

[0039] (1) Explore the optimal enzyme concentration of the detection system. Mix different concentrations of Cas12a enzyme and crRNA and add them to 90 μL of the reaction system containing Biotin-SNA. Finally, add 10 μL of target DNA (1 μM) and react at 37°C for 60 min. After the reaction, add 15 μL of SAV-MB (10 mg / mL) magnetic beads for adsorption for 10 min, and then separate and detect the Raman signal of the supernatant with a magnet. The results are as follows Figure 3 (b) When the enzyme dosage is 40mM, the reaction effect reaches saturation, and further increasing the enzyme concentration does not significantly increase the reaction rate.

[0040] (2) Explore the optimal amount of SAV-MB magnetic beads. Premix the Cas12a enzyme and crRNA at a molar ratio of 1:2 and add them to 90 μL of the reaction system containing Biotin-SNA. Finally, add 10 μL of target DNA (1 μM) and react at 37°C for 60 min. After the reaction, add different volumes of SAV-MB (10 mg / mL) magnetic beads for adsorption for 5 min, and then separate and detect the Raman signal of the supernatant with a magnet. The results are as follows Figure 3 (a) When the amount of enzyme used is 15 μL, the adsorption is basically complete. When the amount used exceeds 15 μL, the Raman signal does not decrease significantly.

[0041] (3) Evaluate the effect of free Biotin-DNA on the capture of Biotin-SNA by SAV-MB. Different concentrations of Biotin-DNA solution were added to the Biotin-SNA solution. Free Biotin-DNA greatly reduced the capture of Biotin-SNA by competitive binding. Figure 4 (a) Figure 4 (b). Even at a concentration of 250 pM, free biotin-DNA produced a detectable signal difference compared to the blank control. After removing the biotin-DNA, the capture of biotin-SNA was restored. Figure 4 (a) Figure 4 (b) These findings confirm that free Biotin-DNA competes with Biotin-SNA for binding to SAV-MB.

[0042] Example 3

[0043] Exploring optimal reaction time

[0044] Based on the optimized conditions of Example 2, HPV16 was used as the optimized object, the concentration of the added target DNA was changed, and the reaction time was set to 30 minutes, 60 minutes, 90 minutes and 120 minutes, and the Raman signal intensity after the reaction of the target DNA at different concentrations and different reaction times was obtained, as shown in FIG. Figure 5 As shown in (a), the detection limit was the lowest when the reaction time was 90 minutes, and the detection limit did not decrease significantly when the reaction time was 120 minutes, indicating that the reaction time of 90 minutes was the best.

[0045] Establish the standard working curve of HPV16 and HPV18 L1 genes.

[0046] On the basis of the optimized conditions of Example 2, the concentration of the added target DNA was changed to obtain the Raman signal intensity after the reaction at different concentrations of target DNA, and the standard curves of HPV16 and HPV18 L1 gene standards were established respectively.

[0047] Add different concentrations (1μM-10aM) of target DNA to the reaction system and incubate it at 37°C in a constant temperature incubator. After the reaction, add SAV-MB (10mg / ml) to capture for 5 minutes, separate the magnetic beads with a magnet, take the supernatant and add it to a cuvette, and collect the corresponding spectrum using a portable Raman spectrometer. Establish the standard working curve of HPV16 and HPV18 L1 gene standards.

[0048] In the TEM image ( Figure 6), we observed the effect of target DNA concentration on the capture efficiency of Biotin-SNA and the change in supernatant color. The diameter of SAV-MB is about 300nm. When 1μM target DNA is added, the Biotin on the surface of Biotin-SNA is completely cleaved, resulting in the inability of SAV-MB to capture; when 10pM target DNA is added, the Biotin on the surface of Biotin-SNA is only partially cleaved, and SAV-MB can still partially capture; in the absence of target DNA, the Biotin on the surface of Biotin-SNA is not cleaved and is almost completely captured by SAV-MB.

[0049] We used HPV16 and HPV18L1 gene standards at different concentrations (1μM to 10aM) to evaluate the detection performance of the SERS sensing platform. Figure 5 (b) and (c) show representative SERS spectra collected with different concentrations of standard HPV16 and HPV18 DNA, respectively, and the average SERS intensity plots. The higher target concentration is at 1076 cm -1 The higher SERS intensity value was generated at the supernatant, indicating that the number of SERS probes remaining in the supernatant was positively correlated with the target DNA concentration. The detection limit (LOD) of the sensor in standard HPV16 and HPV18 DNA detection reached 0.347fM and 0.738fM, respectively. The curves are: Y = 0.1024X + 4.59901, R 2 =0.90317. Y=0.11903X+5.06286,R 2 =0.9506

[0050] Example 4

[0051] RPA pre-amplifies HPV16 and HPV18 L1 genes, shortening the reaction time and improving detection sensitivity.

[0052] We added lower concentrations of HPV16 and HPV18 L1 genes (10fM to 1aM) to the RPA system for pre-amplification to shorten the reaction time required for subsequent low-concentration targets.

[0053] Add 25 μL 2× Reaction buffer to each microsphere reaction tube, add 2 μL upstream primer and downstream primer (10 μM) respectively, and add 9 μL H 2 O, divided into 2 tubes. Add 5 μL of extracted DNA sample or standard plasmid sample to each tube, and finally add 1 μL of MgOAC to mix, and place at 37℃ for 20 minutes.

[0054] The results are as follows Figure 7Agarose gel electrophoresis showed that the designed RPA primers could sensitively amplify HPV16 and HPV18 L1 genes at extremely low concentrations.

Claims

1. A method for constructing a SERS biosensor system based on CRISPR / Cas12a and recombinase polymerase amplification, characterized in that: The following steps are involved: 1) Preparation of Biotin-SNA MBA was added to Au NPs and stirred at room temperature, and then centrifuged and resuspended to form Au@MBA NPs. Then, sodium citrate solution, sodium hydroxide solution, ascorbic acid solution and silver nitrate solution were added in sequence, and then centrifuged and resuspended to form Au@MBA@Ag NPs. The DNA chains modified at both ends were mixed with Au@MBA@Ag NPs at a volume ratio of 1:19-21, frozen at -20°C and then thawed at room temperature, centrifuged and resuspended in DEPC water to form DNA-functionalized silver-coated gold nanoparticles Biotin-SNA; 2) Construction of viral DNA detection system The Cas12a enzyme solution is incubated with crRNA to form a Cas12a-crRNA complex that specifically recognizes the target DNA; The above-mentioned Biotin-SNA and Cas12a-crRNA complex was added to the RPA reaction tube, and then the DNA sample was added and incubated in a 37°C constant temperature incubator. After the reaction, streptavidin-modified magnetic beads were added for capture for 5 to 7 minutes, and then the magnetic beads were separated with a magnet, and finally the SERS signal of the supernatant was measured.

2. The method for constructing a SERS biosensor system based on CRISPR / Cas12a and recombinase polymerase amplification according to claim 1, characterized in that: If the DNA sample does not contain the target DNA, the trans-cleavage activity of the Cas12a enzyme will not be activated, and Biotin-SNA will bind to the streptavidin-modified magnetic beads, which will significantly reduce the SERS signal in the supernatant; On the contrary, if the DNA sample contains target DNA, the Cas12a-crRNA complex will form a Cas12a-crRNA-dsDNA complex with the target DNA, thereby activating the trans-cleavage activity of the Cas12a enzyme and cutting the DNA on the surface of Biotin-SNA. At this time, the free Biotin-DNA will compete for binding to the subsequently added streptavidin-modified magnetic beads, resulting in a greatly reduced capture efficiency of Biotin-SNA by streptavidin-modified magnetic beads, thereby keeping the SERS signal in the supernatant unchanged.

3. The method for constructing a SERS biosensor system based on CRISPR / Cas12a and recombinase polymerase amplification according to claim 1, characterized in that: The DNA chain sequence and double-end modification groups described in step 1) are as follows: 5'-Biotin-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTT-SH-3'.

4. The method for constructing a SERS biosensor system based on CRISPR / Cas12a and recombinase polymerase amplification according to claim 1, characterized in that: The volume ratio of Au NPs to MBA in step 1) is 990-1010:

1.

5. The method for constructing a SERS biosensor system based on CRISPR / Cas12a and recombinase polymerase amplification according to claim 1, characterized in that: The concentration of the sodium citrate solution in step 1) is 0.95-1.05% w / w, the concentration of the sodium hydroxide solution is 95-105 mM, the concentration of the ascorbic acid solution is 9.5-10.5 mM, the concentration of the silver nitrate solution is 0.95-1.05 mM, and the volume ratio of Au@MBA NPs, sodium citrate solution, sodium hydroxide solution, ascorbic acid solution and silver nitrate solution is 100:2:1:10:

10.

6. The method for constructing a SERS biosensor system based on CRISPR / Cas12a and recombinase polymerase amplification according to claim 1, characterized in that: The molar ratio of the Cas12a enzyme solution to crRNA in step 2) is 1:

2.

7. The method for constructing a SERS biosensor system based on CRISPR / Cas12a and recombinase polymerase amplification according to claim 1, characterized in that: The DNA sample added in step 2) is a non-RPA amplified DNA sample, and the incubation time is 90 to 100 minutes.

8. Application of the SERS biosensor system based on CRISPR / Cas12a and recombinase polymerase amplification constructed according to the method described in any one of claims 1 to 7 in viral DNA detection.

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