A double-stranded walking type DNA nanomachine and a preparation method and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHONGQING UNIV OF TECH
- Filing Date
- 2026-04-30
- Publication Date
- 2026-07-24
Smart Images
Figure CN122445772A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of fluorescence analysis and detection technology, and in particular to a double-stranded walking DNA nanomachine, its preparation method, and its application. Background Technology
[0002] Circulating tumor DNA (ctDNA) is a cell-free DNA fragment released into the bloodstream from tumor cells. It reflects the genetic characteristics and dynamic changes of tumors and is an important liquid biopsy biomarker for early lung cancer screening, efficacy evaluation, and recurrence monitoring. However, ctDNA is present in extremely low concentrations in the blood and is subject to complex background interference, placing extremely high demands on the sensitivity and specificity of detection methods.
[0003] The CRISPR / Cas12a system has shown broad application prospects in the field of nucleic acid biosensing due to its efficient target recognition capability and strong trans-cleavage activity. This system activates the trans-cleavage activity of Cas12a after crRNA recognizes the target, enabling non-specific cleavage of single-stranded DNA substrates. It boasts advantages such as simple operation and mild reaction conditions. To further improve system sensitivity, researchers combined CRISPR / Cas12a with nanomaterials. By co-immobilizing high-density single-stranded DNA probes and Cas12a on the surface of gold nanoparticles (AuNP), a three-dimensional DNA nanomachine was constructed, effectively improving the contact efficiency between Cas12a and the substrate. In existing studies, the walking arms used to immobilize the Cas12a-crRNA complex generally employ single-stranded DNA structures, such as single-stranded walking nanomachines based on direct RNA activation strategies and nanomachines that incorporate rolling circle amplification products as walking arm supports, all of which have improved detection performance to some extent.
[0004] However, the aforementioned single-stranded walking structure has inherent defects. After Cas12a is activated by the target, its trans-cleavage activity does not have sequence selectivity and will inevitably degrade its own single-stranded walking arm, causing the Cas12a-crRNA complex to fall off the nanomachine, resulting in a decrease in overall structural stability and limited signal amplification efficiency.
[0005] Therefore, designing a walking arm structure that can resist Cas12a trans-cutting, while maintaining the structural integrity of the nanomachines, to improve the stability and sensitivity of ctDNA detection, is one of the key issues that urgently need to be addressed. Summary of the Invention
[0006] In view of this, the present invention provides a double-stranded walking DNA nanomachine for ctDNA fluorescence detection, its preparation method, and its application. By introducing a double-stranded DNA structure into the walking arm design to replace the traditional single-stranded walking arm, the present invention constructs a double-stranded walking three-dimensional DNA CRISPR / Cas12a nanomachine, effectively resisting Cas12a trans-cleavage, maintaining the structural integrity and functional stability of the nanomachine, thereby significantly improving the detection sensitivity and specificity of lung cancer ctDNA, and showing good clinical application prospects.
[0007] The first aspect of this invention is to provide a double-stranded walking DNA nanomachine for ctDNA fluorescence detection, comprising: Gold nanoparticles (AuNP) carriers with a diameter of 20-27 nm; The anchor probe has the following nucleic acid sequence: 5'-GCAGAGAGACAAGTTACA-3' (SEQ ID NO: 1); The 3' end of the sequence is connected to a six-carbon alkane thiol modification group (5'-GCAGAGAGACAAGTTACA-C6SH-3'); the anchor probe forms an Au-S covalent bond with the surface of the gold nanoparticles through the 3' terminal thiol group, and is immobilized on the 3' terminal thiol modification of the gold nanoparticle surface; The FAM fluorescent reporter probe, immobilized on the surface of the gold nanoparticles, has the following nucleic acid sequence: 5'-TTTTTTTTTTTTTTTTATTATTATTATTATT-3' (SEQ ID NO: 2); The 5' end of the sequence is connected to a six-carbon alkane thiol modification group, which is used to immobilize the gold nanoparticles on the surface via Au-S covalent bonds; the 3' end of the sequence is connected to a FAM fluorescent group (5'-HS-C6-TTTTTTTTTTTTTTTATTATTATTATTATT-FAM-3'), which is used for the generation and detection of fluorescence signals. The dual-chain walking arm (dsArm) is formed by hybridizing arm-1 and arm-2; The nucleic acid sequence of arm-1 is as follows: 5'-TTGTGCTTGAAGTGGTACGGGTGTGTTTGTGTGTTTGTGT-3' (SEQ ID NO: 3); The nucleic acid sequence of arm-2 is as follows: 5'-TGTAACTTGTCTCTCTGCCACACAAACACACAAACACA-3' (SEQ ID NO: 4); A bifunctional crRNA, with a 20 nt extension at its 5' end, binds to arm-1 of the dsArm to achieve localization and immobilization of the crRNA-Cas12a complex on the nanomachine; the 3' end recognition sequence guides the Cas12a protein to recognize the PAM sequence and target sequence of the target ctDNA. The Cas12a protein forms a ribonucleoprotein complex with crRNA, which is then loaded onto the DNA nanomachine. The complete nucleic acid sequence of the crRNA is: 5'-CCGUACCACUUCAAGCACAAUAAUUUCUACUAAGUUGUAGAUGCCAGCCCAAAAUCUGUGAU-3' (SEQ ID NO:5).
[0008] Preferably, the gold nanoparticles are prepared by chemical reduction.
[0009] Preferably, the molar ratio of the gold nanoparticle surface anchor probe to the FAM fluorescent reporter probe is 1:25.
[0010] A second aspect of the present invention is to provide a method for preparing the above-mentioned double-stranded walking DNA nanomachine, comprising the following steps: S1: The thiol-modified anchor probe and the thiol-modified FAM fluorescent reporter probe were mixed with the gold nanoparticle solution and incubated at 37°C for 1 h. S2: NaCl solution is gradually added to the above mixed system in a concentration gradient of 50 mM, so that the final concentration gradient of NaCl in the system increases to 300 mM, and salt aging treatment is carried out. S3: Continue incubation at room temperature for at least 6 hours; S4: Add washing buffer (20mM Tris-HCl, 200mM NaCl, 0.1% Tween 20) to the system, centrifuge at 13,000 r / min for 20 min, discard the supernatant, and redisperse the resulting precipitate in storage buffer (10mM Tris-HCl, 100mM NaCl) to obtain AuNP-anchor / FAM; S5: Mix arm-1 and arm-2 at a molar ratio of 1:1 and incubate at 37°C for 30 min to allow the two strands to fully hybridize and form a double-stranded walking arm dsArm; S6: Mix the AuNP-anchor / FAM with dsArm and incubate at 37°C for 30 min to immobilize dsArm onto the AuNP surface through complementary hybridization of arm-2 and the anchor probe; then add crRNA and Cas12a protein sequentially and continue incubating at 37°C for 30 min to complete the final assembly of the double-stranded walking DNA nanomachine.
[0011] Preferably, the method for preparing the thiol-modified anchor probe is as follows: The TCEP solution was mixed with the anchor probe solution containing disulfide bonds at a molar ratio of 500:1 and incubated at 37°C for 30 min to reduce the disulfide bonds in the chain and obtain the thiol-modified anchor probe.
[0012] Preferably, the method for preparing the thiol-modified FAM fluorescent reporter probe is as follows: The TCEP solution was mixed with the FAM fluorescent reporter probe solution containing disulfide bonds at a molar ratio of 500:1 and incubated at 37°C for 30 min to reduce the disulfide bonds to thiol groups, thus obtaining the thiol-modified FAM fluorescent reporter probe.
[0013] This invention reduces disulfide bonds to thiol groups, enabling the nucleic acid probe to stably bind to gold nanoparticles via Au-S covalent bonds.
[0014] A third aspect of the present invention is to provide a method for fluorescence detection of ctDNA using the above-mentioned double-stranded walking DNA nanomachine, comprising the following steps: adding the sample to be tested into a detection system containing the DNA nanomachine and incubating at 37°C for 60 min; exciting at an excitation wavelength of 485 nm and detecting the fluorescence intensity (F) at an emission wavelength of 528 nm; calculating the content of ctDNA in the sample to be tested based on the linear relationship between fluorescence intensity and ctDNA concentration (linear range 0.5 pM-100 pM, detection limit LOD of 0.19 pM).
[0015] This method can distinguish target ctDNA from single-base mismatch sequences, and measures the fluorescence response to single-base mismatch sequences. Not exceeding 0.012 times the target ctDNA.
[0016] The sample to be tested is a human serum sample, and the final serum sample concentration in the detection system is 10%.
[0017] A fourth aspect of this invention is to provide the application of the above-described fluorescence detection method in the preparation of a lung cancer ctDNA detection kit.
[0018] Compared with the prior art, the beneficial technical effects of the present invention are as follows: 1. This invention uses a double-stranded DNA structure to replace the traditional single-stranded walking arm, constructing a double-stranded walking 3D DNACRISPR / Cas12a nanomachine. The double-stranded walking arm (dsArm) is naturally resistant to trans-cleavage of Cas12a, effectively avoiding the degradation of the walking arm itself after Cas12a activation, fundamentally solving the problems of poor stability and low sensitivity of existing single-stranded walking nanomachines.
[0019] 2. This invention maintains the stable anchoring of the Cas12a-crRNA complex on the AuNP surface through a double-stranded walking arm, enabling Cas12a to continuously maintain its efficient trans-cleavage capability of the FAM fluorescent probe during detection. This results in higher signal amplification efficiency and detection sensitivity compared to single-stranded walking nanomachines. In the detection method of this invention, the fluorescence signal intensity exhibits a good linear relationship with the logarithm of the target analyte concentration within the range of 0.5-100 pM. r =0.991), and the lower limit of the linear range is 0.5 pM, indicating that the method of the present invention has a wide linear range and high sensitivity.
[0020] 3. The fluorescent biosensor constructed in this invention has a good ability to distinguish between target ctDNA and sequences containing different mutation sites, and has high specificity for single base mismatch recognition, making it suitable for the accurate detection of lung cancer-related ctDNA.
[0021] 4. The method of this invention has a standardized operation process and stable system assembly. The constructed nanomachines can still maintain excellent detection performance and good recovery rate in actual serum sample matrices, indicating that the method has strong anti-interference ability and has the potential to be translated into clinical applications.
[0022] 5. The detection method of this invention is based on fluorescence signal readout, requires no complex instruments, is compatible with conventional ELISA readers, and has a simple and fast detection process, making it suitable for the development and widespread application of lung cancer ctDNA detection kits. Attached Figure Description
[0023] The present invention will be further described below with reference to the accompanying drawings.
[0024] Figure 1 This is a schematic diagram of the structure of the double-stranded walking DNA nanomachine and a schematic diagram of the working principle of ctDNA detection of the present invention; Figure 2 This is a transmission electron microscope (TEM) characterization image of the double-stranded walking DNA nanomachine of the present invention. Figure 3 The figures show a comparison of the fluorescence response curves of the fluorescent biosensor constructed from the double-stranded / single-stranded walking 3D DNA CRISPR / Cas12a nanomachines in Example 2 of this invention to standard solutions of target analytes at concentrations of 1 nM, 5 nM, and 10 nM, respectively; where (a) is the fluorescence response curve of the double-stranded walking 3D DNA CRISPR / Cas12a nanomachines fluorescent biosensor; and (b) is the fluorescence response curve of the single-stranded walking 3D DNA CRISPR / Cas12a nanomachines fluorescent biosensor. Figure 4The results of the fluorescence biosensor constructed based on the double-stranded walking 3D DNA CRISPR / Cas12a nanomachine in Example 3 of this invention are for the detection of standard solutions of target analytes at different concentrations; where (a) is the fluorescence response curve of the target analytes at different concentrations; and (b) is the standard curve of fluorescence signal response intensity value versus the logarithm of target analyte concentration. Figure 5 This is a bar chart showing the selective detection of lung cancer ctDNA and sequences containing different mutation sites by the double-stranded walking 3D DNA CRISPR / Cas12a nanomachine fluorescent biosensor in Example 4 of the present invention. Figure 6 This is the detection result of standard solutions of target analytes of different concentrations in Comparative Example 1 of this invention, using conventional fluorescence method (without DNA nanomachines). Detailed Implementation
[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0026] Example 1: Fabrication of DNA Nanomachines 1.1 Synthesis of gold nanoparticles (AuNP) Weigh 0.0885 g of trisodium citrate solid granules and mix with 10 mL of deionized water. Stir until homogeneous to obtain 3.43 × 10⁻⁶ ppm. -2 Prepare a mol / L trisodium citrate solution; add 2.44 mL of 5 mM chloroauric acid solution and 40 mL of deionized water to a beaker, place it on a magnetic stirrer, heat to 75°C at 600 r / min and hold for 2 min, then quickly add 10 mL of the prepared 3.43 × 10⁻⁶ mol / L trisodium citrate solution. -2 A mol / L trisodium citrate solution was continuously heated until the solution color changed from pale yellow to grayish blue and then to wine red. Once the liquid color was constant, heating was stopped and stirring was continued for 5 minutes. After cooling to room temperature, gold nanoparticles with an average diameter of 20 nm were obtained.
[0027] 1.2 Preparation of Functionalized Nanoparticles AuNP-anchor / FAM The TCEP solution was mixed with the anchor probe solution containing disulfide bonds at a molar ratio of 500:1 and incubated at 37°C for 30 min to reduce the disulfide bonds in the chain and obtain the thiol-modified anchor probe. The TCEP solution was mixed with the FAM fluorescent reporter probe solution containing disulfide bonds at a molar ratio of 500:1 and incubated at 37°C for 30 min to reduce the disulfide bonds to thiol groups, thus obtaining the thiol-modified FAM fluorescent reporter probe. Add 2 μL of thiol-modified anchor probe (final concentration 1 μM) and 25 μL of FAM fluorescent reporter probe (final concentration 2 μM) to 50 μL of AuNP solution (final concentration 1 nM), mix well and incubate at 37 °C for 1 h. Subsequently, NaCl solution was gradually added to the above mixture at a concentration gradient of 50 mM, so that the final NaCl concentration gradient in the system increased to 300 mM, and salt aging treatment was carried out. The mixture was then incubated at room temperature for 12 h. After salt aging, wash buffer (20 mM Tris-HCl, 200 mM NaCl, 0.1% Tween 20) was added to the system, centrifuged at 13000 r / min for 20 min, the supernatant was discarded, and the resulting precipitate was redispersed in storage buffer (10 mM Tris-HCl, 100 mM NaCl) to obtain AuNP-anchor / FAM, which was stored at 4℃ in the dark for later use.
[0028] 1.3 Fabrication of the Dual-Chain Walking Arm (dsArm) Mix arm-1 (2 μM, 2 μL) and arm-2 (2 μM, 2 μL) at a molar ratio of 1:1 and incubate in a 37°C water bath for 30 min to allow the two strands to fully hybridize and form a double-stranded walking arm dsArm for later use.
[0029] 1.4 Assembly of DNA Nanomachines AuNP-anchor / FAM (30 μL) was mixed with dsArm (2 μL) and incubated at 37°C for 30 min, allowing dsArm to immobilize onto the AuNP surface via complementary hybridization between arm-2 and the anchor probe. Subsequently, crRNA (1 μM, 6 μL) and Cas12a protein (0.5 μM, 6 μL) were added sequentially, and incubation continued at 37°C for another 30 min to complete the final assembly of the double-stranded walking DNA nanomachine (e.g., Figure 1 As shown), this yields a double-stranded walking 3D DNA CRISPR / Cas12a nanomachine ( Figure 2 ).
[0030] Example 2: Sensitivity and stability of a fluorescent biosensor constructed from a double-stranded walking 3D DNA CRISPR / Cas12a nanomachine. To investigate the sensitivity and stability of the fluorescent biosensor constructed using this double-stranded walking 3D DNA CRISPR / Cas12a nanomachine, we compared it with a fluorescent biosensor constructed using a single-stranded walking 3D DNA CRISPR / Cas12a nanomachine. The specific methods are as follows: Fluorescent biosensors constructed from double-stranded 3D DNA CRISPR / Cas12a nanomachines (prepared in Example 1) and single-stranded 3D DNA CRISPR / Cas12a nanomachines were mixed with target analytes under identical conditions and added to 96-well ELISA plates. The ELISA reader parameters were set as follows: detection temperature 37℃, detection time 60 min, and detection interval 1 min, for a total of 60 detections. The 96-well ELISA plates were placed in the ELISA reader for reaction, and the fluorescence signal was monitored in real time. The excitation wavelength of the FAM fluorescent group was 485 nm, and the emission wavelength range was 528 nm. This study used three different concentrations of target analytes: 1 nM, 5 nM, and 10 nM for detection.
[0031] The fluorescent biosensor constructed from the single-stranded walking 3D DNA CRISPR / Cas12a nanomachines differs from that in Example 1 in that the double-stranded walking arm (dsArm) is replaced with a single-stranded walking arm (single-Arm), the sequence of which is: 5'-TTGTGCTTGAAGTGGTACGGTTTTTTTTTTTTTTTTTTTTTTTTTGTAACTTGTCTCTCTGC-3' (SEQ ID NO: 6), wherein the 3' end of the sequence is connected to a hexacarbon alkane thiol modification group (5'-TTGTGCTTGAAGTGGTACGGTTTTTTTTTTTTTTTTTTTTTTTGTAACTTGTCTCTCTGC-C6 SH-3').
[0032] The fluorescence response intensity and reaction time of the fluorescent biosensor constructed using a double-stranded walking 3D DNA CRISPR / Cas12a nanomachine to detect the target analyte are shown in the figure. Figure 3 (a).
[0033] The fluorescence response intensity and reaction time of the fluorescent biosensor constructed using a single-stranded walking 3D DNA CRISPR / Cas12a nanomachine to detect the target analyte are shown in the figure. Figure 3 (b).
[0034] Example 3: Detection performance and concentration response characteristics of a fluorescent biosensor constructed from a double-stranded walking 3D DNA CRISPR / Cas12a nanomachine. To investigate the detection performance and concentration response characteristics of a fluorescent biosensor constructed from a double-stranded walking 3D DNA CRISPR / Cas12a nanomachine, real-time fluorescence monitoring of standard solutions of target analytes at different concentrations was performed. The specific methods are as follows: The fluorescent biosensor constructed from the double-stranded walking 3D DNA CRISPR / Cas12a nanomachines of Example 1 was mixed with different concentrations of target substances in a 96-well ELISA plate. The plate was then placed in an ELISA reader and reacted at 37°C for 60 min, with real-time monitoring of fluorescence signal changes. The excitation wavelength of the FAM fluorescent group was 485 nm, and the emission wavelength range was 528 nm.
[0035] The constructed fluorescent biosensor's signal detection is based on the fluorescence properties of FAM fluorescent probes immobilized on the surface of gold nanoparticles. With increasing target concentration, the CRISPR / Cas12a system is activated for trans-cleavage, causing the FAM probes on the AuNP surface to be cleaved and released, moving away from the AuNPs which exhibit fluorescence quenching effects. This results in an enhancement of the fluorescence signal in the system, and this change is proportional to the logarithm of the added target concentration. A plot of the measured change in fluorescence signal response against the logarithm of the target standard solution concentration is shown. Figure 4 (a) shows the fluorescence emission spectrum at the corresponding concentration. Figure 4 (b) is the corresponding standard curve, with a linear response range of 0.5 pM-100 pM.
[0036] Example 4: Selective detection of a fluorescent biosensor constructed from a double-stranded walking 3D DNA CRISPR / Cas12a nanomachine. To further evaluate the selectivity of this fluorescent biosensor and its application potential in clinical samples, a fluorescent biosensor constructed using the double-stranded walking 3D DNA CRISPR / Cas12a nanomachines of Example 1 was used to target analytes containing different mutation sites. 4. Mutant target 9. Mutant target 14. Mutant target 4 / 9, mutant target 4 / 14, mutant target (9 / 14) and the target ctDNA were specifically detected. The specific steps are as follows: The constructed fluorescent biosensor was combined with 10 nM lung cancer ctDNA and a 10 nM single-mutant target. 4. Mutant target 9. Mutant target 14) and 10 nM double mutation site target (mutanttarget) 4 / 9, mutant target 4 / 14, mutant target (9 / 14) After mixing separately, the samples were added to 96-well microplates. The microplates were placed in a microplate reader and incubated at 37°C for 60 min, with real-time monitoring of fluorescence signal changes. The excitation wavelength of the FAM fluorescent group was 485 nm, and the emission wavelength range was 528 nm.
[0037] The fluorescence signal intensity gain at the reaction equilibrium stage and 5 min before and after equilibrium was quantitatively analyzed, and its mean and standard deviation were calculated. The fluorescence response intensity of 10 nM lung cancer ctDNA was used as a control. The results are presented in bar chart format. See below for details. Figure 5 .
[0038] Example 5: Application of a fluorescent biosensor constructed from a double-stranded walking 3D DNA CRISPR / Cas12a nanomachine in serum ctDNA detection. Based on the excellent stability of the fluorescent biosensor constructed using a double-stranded walking 3D DNA CRISPR / Cas12a nanomachine, we explored its practical application in serum ctDNA samples. Using a spiked recovery method, ctDNA standards at concentrations of 10 pM, 100 pM, and 10000 pM were added to human serum to prepare simulated clinical samples. Subsequently, the samples were mixed with the fluorescent biosensor constructed using the double-stranded walking 3D DNA CRISPR / Cas12a nanomachine and added to a 96-well ELISA plate. The plate was incubated at 37 °C for 60 min using an ELISA reader, and changes in fluorescence signal were monitored in real time.
[0039] Based on the real-time monitoring fluorescence kinetic curve, the fluorescence response values of samples at each concentration were extracted. By comparing the standard curve of fluorescence signal response intensity value with the logarithm of ctDNA concentration, the recovery rate of the double-stranded walking 3D DNACRISPR / Cas12a nanomachine fluorescent biosensor in a system containing 10% human serum was calculated according to the spiking amount and detection value. The specific results are shown in Table 1.
[0040] Table 1. Recovery rates of different concentrations of ctDNA in serum systems using a double-stranded walking 3D DNA CRISPR / Cas12a nanomachine fluorescent biosensor.
[0041] The average recovery rate in human serum matrix in this embodiment was 102.5%, indicating that the method of the present invention has good accuracy and anti-interference ability in actual sample detection.
[0042] Comparative Example 1: Conventional fluorescence method (without DNA nanomachines) The difference from Example 1 is that traditional fluorescent probes are used to directly detect ctDNA, without constructing DNA nanomachines, while the other detection conditions are the same.
[0043] Testing revealed that, compared to double-stranded and single-stranded 3D DNA CRISPR nanomachines, conventional fluorescence methods showed a continuous upward trend in fluorescence signal over time, with higher target concentrations resulting in higher final fluorescence intensity. However, the signal rise rate was slow, and the signal response was weak and detection sensitivity insufficient at low concentrations. Figure 6 ).
[0044] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.
Claims
1. A double-stranded walking DNA nanomachine for ctDNA fluorescence detection, characterized in that, include: Gold nanoparticle carriers, wherein the diameter of the carriers is 20-27 nm; The anchor probe has a nucleic acid sequence as shown in SEQ ID NO:1, and the 3' end of the sequence is connected to a six-carbon alkane thiol modification group; The FAM fluorescent reporter probe is immobilized on the surface of the gold nanoparticles. Its nucleic acid sequence is shown in SEQ ID NO:
2. The 5' end of the sequence is connected to a six-carbon alkane thiol modification group, and the 3' end of the sequence is connected to a FAM fluorescent group. The dual-chain walking arm is formed by hybridization of arm-1 and arm-2; The nucleic acid sequence of arm-1 is shown in SEQ ID NO:3; The nucleic acid sequence of arm-2 is shown in SEQ ID NO:4; The Cas12a protein forms a ribonucleoprotein complex with crRNA, which is then loaded onto the DNA nanomachine. The complete nucleic acid sequence of the crRNA is shown in SEQ ID NO:
5.
2. The double-stranded walking DNA nanomachine for ctDNA fluorescence detection according to claim 1, characterized in that, The molar ratio of the gold nanoparticle surface anchor probe to the FAM fluorescent reporter probe is 1:
25.
3. The method for preparing the double-stranded walking DNA nanomachine as described in claim 1 or 2, characterized in that, Includes the following steps: S1: Mix the thiol-modified anchor probe and the thiol-modified FAM fluorescent reporter probe with the gold nanoparticle solution and incubate; S2: NaCl solution is gradually added to the above mixed system in a concentration gradient of 50 mM, so that the final concentration gradient of NaCl in the system increases to 300 mM, and salt aging treatment is carried out. S3: Continue incubation at room temperature; S4: Add washing buffer to the system, centrifuge, discard the supernatant, and redisperse the resulting precipitate in storage buffer to obtain AuNP-anchor / FAM; S5: Mix arm-1 and arm-2, incubate, and form a double-chain walking arm dsArm; S6: Mix the AuNP-anchor / FAM with the dsArm, incubate once, then add crRNA and Cas12a protein in sequence, incubate a second time, and complete the final assembly of the double-stranded walking DNA nanomachine.
4. The preparation method according to claim 3, characterized in that, In step S1, the incubation temperature is 37°C and the incubation time is 1 hour.
5. The preparation method according to claim 3, characterized in that, In step S3, the incubation temperature is room temperature, and the incubation time is >6 h.
6. The preparation method according to claim 3, characterized in that, In step S4, the washing buffer comprises: 20 mM Tris-HCl, 200 mM NaCl, and 0.1% Tween 20; the storage buffer comprises: 10 mM Tris-HCl and 100 mM NaCl.
7. The preparation method according to claim 3, characterized in that, In step S5, the molar ratio of arm-1 to arm-2 is 1:1; the incubation temperature is 37°C and the incubation time is 30 min.
8. The preparation method according to claim 3, characterized in that, In step S6, the temperature for the first incubation is 37°C and the incubation time is 30 min; the temperature for the second incubation is 37°C and the incubation time is 30 min.
9. A method for fluorescent detection of ctDNA using the double-stranded walking DNA nanomachine as described in claim 1 or 2, characterized in that, Includes the following steps: The sample to be tested was added to the detection system containing the DNA nanomachine and incubated at 37°C for 60 min. The sample was excited at an excitation wavelength of 485 nm, and the fluorescence intensity at an emission wavelength of 528 nm was detected. Based on the linear relationship between fluorescence intensity and ctDNA concentration, the content of ctDNA in the sample was calculated.
10. The application of the fluorescent detection method for ctDNA according to claim 9 in the preparation of a lung cancer ctDNA detection kit.