A nucleic acid detection system based on DNA tetrahedron loaded DNAzyme cascade CRISPR / Cas12a, and a preparation method and application thereof

Abstract

The application discloses a nucleic acid detection system based on DNA tetrahedron loaded DNAzyme cascade CRISPR / Cas12a, a preparation method and application thereof, and specifically, a deoxyribozyme DNAzyme with a circular protruding DNA shielding structure is designed by taking a DNA tetrahedron framework structure as a core skeleton for the first time, a catalytic signal transduction module of the DNAzyme is orderly integrated with a CRISPR / cas12a signal amplification system, and a novel fluorescent biosensor with high sensitivity and high specificity is constructed.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection technology, and relates to a nucleic acid detection system based on DNA tetrahedron loaded with DNAzyme cascade CRISPR / Cas12a, its preparation method and application. Background Technology

[0002] Prostate cancer (PCa) is the second most common malignant tumor among men worldwide, and accurate diagnosis and disease monitoring are crucial for improving patient prognosis. Although screening for prostate-specific antigen (PSA) has improved early detection rates to some extent, its diagnostic specificity is insufficient, and elevated PSA levels can occur in healthy individuals.

[0003] MicroRNAs (miRNAs) are a class of endogenous, single-stranded non-coding RNA molecules, 19-22 nucleotides in length. Due to their high stability in body fluids, tissue-specific expression, and close correlation with tumor development and progression, they have become highly promising biomarkers for liquid biopsies. miR-141, in particular, has been shown to exhibit the greatest difference between prostate cancer patients and healthy controls. Its expression level is closely related to tumor invasiveness, castration resistance, and bone metastasis, demonstrating its great potential as a diagnostic and prognostic biomarker for prostate cancer. However, its low abundance and easy degradation place higher demands on detection techniques. Although PCR and isothermal amplification techniques have improved detection sensitivity and speed, they still suffer from complex operation, reliance on specialized equipment, and detection times of 2-4 hours, making it difficult to meet the needs of rapid clinical diagnosis.

[0004] Due to its flexible programmability, high chemical stability, and low cost, DNAzyme has been used as an analytical module in the field of nucleic acid analysis in many related studies. However, its poor resistance to nuclease degradation under physiological conditions and lack of targeting have seriously restricted its practical application in the fields of nucleic acid analysis and gene therapy.

[0005] The CRISPR / Cas system has been widely used in pathogen detection due to its high specificity and efficient nucleic acid cleavage capability, but it still faces challenges such as reliance on nucleic acid amplification, off-target risks, and complex sample processing. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a nucleic acid detection system based on DNA tetrahedron loaded with DNAzyme cascade CRISPR / Cas12a, its preparation method and application.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] 1. A nucleic acid detection system based on DNA tetrahedral loaded DNAzyme cascade CRISPR / Cas12a, comprising three parts: tetrahedral nanostructure TDN-DNAzyme, magnetic beads MB-TS, and a CRISPR / Cas12a complex. The tetrahedral nanostructure TDN-DNAzyme is obtained by assembling single-stranded DNA T1, T2, T3, and T4 with deoxyribonucleotide sequences as shown in SEQ ID NO. 1–4 and the optimal single-stranded DNAzyme T30 as shown in SEQ ID NO. 7. The magnetic beads MB-TS are obtained by mixing streptavidin magnetic beads MB with activated parental strand TS with deoxyribonucleotide sequences as shown in SEQ ID NO. 9. The CRISPR / Cas12a complex is obtained by pre-incubating Cas12a and crRNA.

[0009] 2. The aforementioned method for preparing a nucleic acid detection system based on DNA tetrahedron-loaded DNAzyme cascade CRISPR / Cas12a includes:

[0010] (A) Preparation of tetrahedral nanostructure TDN-DNAzyme: First, the single-stranded DNA T1, T2, T3, T4 with deoxyribonucleotide sequences as shown in SEQ ID NO.1~4 are assembled with the single-stranded DNAzyme T30 shown in SEQ ID NO.7 to obtain a tetrahedral nanostructure TDN-DNAzyme with four DNAzyme arms;

[0011] (B) Preparation of magnetic beads MB-TS: MB-TS were prepared by mixing streptavidin magnetic beads MB and deoxyribonucleotide sequences as shown in SEQ ID NO.9 to activate the parental TS.

[0012] (C) Preparation of CRISPR / Cas12a complex: Cas12a was pre-incubated with the single-stranded crRNA shown in SEQ ID NO.17 to form CRISPR / Cas12a complex.

[0013] Preferably, in step (A), the molar ratio of T1, T2, T3, T4, and T30 is 1:1:1:1:4; the assembly conditions are: 95℃ for 5 min; 65℃ for 30 min; 50℃ for 30 min; 42℃ for 30 min; 37℃ for 30 min; and 22℃ for 30 min.

[0014] Preferably, the TDN-DNAzyme obtained in step (A) is stored at 4°C.

[0015] Preferably, in step (B), MB and TS are prepared into 5.0 mg / mL MB solution and 5 μM TS solution, respectively, and mixed at a volume ratio of 1:2.5. The mixture is reacted at room temperature for 30 minutes, magnetically separated, and washed to obtain MB-TS.

[0016] Preferably, the MB-TS obtained in step (B) is resuspended in PBST buffer for later use.

[0017] Preferably, in step (C), the pre-incubation conditions are: incubation at 37°C for 0.5 hours.

[0018] 3. Application of the aforementioned nucleic acid detection system in the preparation of a fluorescent sensor for detecting prostate cancer.

[0019] Preferably, the detection target is miR-141, whose ribonucleotide sequence is shown in SEQ ID NO.10.

[0020] 4. Application of the aforementioned nucleic acid detection system in the preparation of prostate cancer detection kits.

[0021] Preferably, the detection target is miR-141, whose ribonucleotide sequence is shown in SEQ ID NO.10.

[0022] 5. A method for detecting prostate cancer-related miR-141 based on the aforementioned nucleic acid detection system for non-diagnostic purposes, comprising first mixing TDN-DNAzyme, MB-TS, and miR-141 in a solution containing Mg 2+ The mixture was incubated in the system, the parental strand TS was cleaved and activated, the daughter strand AS was released, the supernatant was collected by magnetic separation, and CRISPR / Cas12a complex, fluorescent probe and buffer were added. The fluorescence value was detected by real-time quantitative PCR, and a standard curve was constructed by the relationship between the fluorescence value and the miR-141 concentration. Then the test sample was used instead of miR-141 for detection to obtain the corresponding fluorescence value. The concentration of miR-141 in the test sample was calculated according to the standard curve.

[0023] Preferably, 3 μl of TDN-DNAzyme, 1 μl of MB-TS, and 2 μl of miR-141 are mixed first, and then 6 μl of 100 mg is added. 2+ The solution and 8 μl of double-distilled water were added to a total volume of 20 μl. After incubation, the supernatant was collected by magnetic separation. 14 μl of the supernatant was aspirated and 2 μl of the supernatant was added to the supernatant. The supernatant was then added to the supernatant, along with 2 μl of CRISPR / Cas12a complex [Cas12a protein (1 μL, 0.5 μM), Cas12a protein crRNA (1 μL, 0.5 μM)], 2 μL of fluorescent probe (2 μL, 2 μM), and 2 μL of NEB buffer 2 (10×). The fluorescence value was monitored using a qPCR instrument.

[0024] The beneficial effects of this invention are as follows:

[0025] This invention provides a nucleic acid detection system based on DNA tetrahedron loaded with DNAzyme cascaded CRISPR / Cas12a, its preparation method, and its application. Specifically, this invention utilizes the DNA tetrahedral framework structure as the core backbone for the first time, designs a deoxyribozyme DNAzyme with a ring-shaped protruding DNA shielding structure, and orderly integrates the catalytic signal transduction module of the DNAzyme with the CRISPR / Cas12a signal amplification system to construct a novel, highly sensitive, and highly specific fluorescent biosensor.

[0026] In this invention, the DNA framework structure serves as a nanoscale carrier. DNAzymes, through their precise spatial arrangement, construct high-performance DNA nanomachines, enabling the protection and co-localization of deoxyribozymes, reducing interfacial crowding effects, increasing local concentration, and improving hybridization efficiency. This provides an ideal interface and carrier for the detection of low-abundance targets in complex biological samples. Cascading this with the excellent amplification capabilities of the CRISPR / Cas12a system enables ultrasensitive detection of the low-abundance biomarker miR-141. This invention, by combining target recognition with signal amplification via a dual-enzyme system cascade, constructs a logically rigorous and functionally synergistic nanobiosensor, providing a powerful analytical tool for the early diagnosis, progression monitoring, and personalized treatment of prostate cancer.

[0027] The working principle of this invention is as follows:

[0028] First, DNA tetrahedra assemble with DNAzymes to form TDN-DNAzymes. In the presence of the target miR-141, miR-141 undergoes base pairing with the spacer sequence in the catalytic core sequence of the TDN-DNAzyme, shortening the distance between the two catalytic core sequences and forming a complete catalytic pocket to unlock the catalytic activity of the TDN-DNAzyme. 2+ With the assistance of a cyclic cleavage, the activation parent chain TS loaded on the magnetic beads is released. Then, the supernatant is collected by magnetic separation. The free activator chain AS can activate the trans-cleavage activity of CRISPR / Cas12a, cleaving the fluorescent quenching chain to spatially separate the two modified groups and generate a huge fluorescent signal, so as to realize the detection of the target miR-141.

[0029] The specific advantages of this invention are as follows:

[0030] 1. This invention is the first to combine DNAzyme-coupled DNA tetrahedrons with a CRISPR / Cas12a dual-enzyme cascade system, achieving highly specific and sensitive detection of miRNAs. The CRISPR / Cas12a system significantly enhances the detection signal, overcoming the low digestion efficiency of existing DNAzyme systems. Furthermore, the flexible programmability of DNAzyme compensates for the lack of modularity in the CRISPR / Cas12a system.

[0031] 2. This invention, through the design of "structure response + functional coupling", triggers the cyclic release of the activation strand after the DNAzyme recognizes the target, activating the trans-cleavage activity of the CRISPR / Cas12a system. Cas12a further cleaves the fluorescent probe, realizing two-stage enzyme-linked signal amplification, significantly improving detection sensitivity, and meeting the detection needs of low-abundance miRNAs.

[0032] 3. The system of this invention has good biocompatibility and modular design. It can be adapted to a variety of other targets by changing the target binding sequence in the DNAzyme. It has versatility and rapid deployment capability and is suitable for clinical point diagnosis scenarios. Attached Figure Description

[0033] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0034] Figure 1 This is a schematic diagram of the detection principle of the nucleic acid detection system based on DNA tetrahedron loaded with DNAzyme cascade CRISPR / Cas12a according to the present invention. In this diagram, A represents the construction of the DNA tetrahedron and B represents the detection principle.

[0035] Figure 2 This is a PAGE image of the TDN-DNAzyme assembly of the present invention.

[0036] Figure 3 This is an AFM image of the TDN-DNAzyme assembly of the present invention.

[0037] Figure 4 This is a fluorescence kinetic curve of the DNAzyme and TDN-DNAzyme system of the present invention.

[0038] Figure 5 This is a fluorescence kinetics curve of CRISPR / cas12a trans-cleavage in this invention.

[0039] Figure 6 This is a curve showing the presence or absence of a target in the TDN-DNAzyme-CRISPR / cas12a sensor of this invention.

[0040] Figure 7Screening for trans-cutting time in CRISPR / Cas12a systems.

[0041] Figure 8 Mg 2+ Concentration screening.

[0042] Figure 9 Screening for single-stranded DNA molecules of different lengths.

[0043] Figure 10 Screening for incubation time between TDN-DNAzyme and MB-DNA.

[0044] Figure 11 This is a fluorescence kinetics curve for the sensitivity of this invention.

[0045] Figure 12 This is a diagram showing the specific fluorescence signal output of this invention.

[0046] Figure 13 This is a bar chart comparing the repeatability of the present invention.

[0047] Figure 14 This is a bar chart comparing the stability of the present invention.

[0048] Figure 15 This is a bar chart of the spiked recovery experiment of the present invention.

[0049] Figure 16 This is a heatmap of the clinical sample testing results of the present invention.

[0050] Figure 17 This is a box diagram of the clinical sample testing results of the present invention.

[0051] Figure 18 This is the ROC curve of the clinical sample test results of the present invention. Detailed Implementation

[0052] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0053] 1. Preparation of MB-TS

[0054] First, 10 μL (10 mg / mL) of streptavidin magnetic beads (MB) (purchased from MCE) was washed five times with 500 μL of washing buffer (10 mM Tris-HCl, 1 mM EDTA, 1 M NaCl, 0.05% Tween-20, pH 8.0). The washed MB was then resuspended in 10 μL of Tris-HCl buffer (10 mM Tris-HCl, 1 mM EDTA, 0.1 M NaCl, 0.05% Tween-20, pH 8.0) to a final concentration of 5.0 mg / mL. The lyophilized TS powder was dissolved to a 5 μM concentration in TE buffer (10 mM Tris and 0.1 mM EDTA, pH 8.0), and then reacted with a mixture of 10 μL MB, 25 μL TS, and 65 μL Tris-HCl buffer at room temperature for 30 minutes. After magnetic separation, the mixture was washed three times with 50 μL of washing buffer to obtain MB-TS. MB-TS was then resuspended in 50 μL PBST buffer (containing 0.05% Tween-20) for later use (1 mg / ml).

[0055] 2. Preparation and characterization of TDN-DNAzyme nanostructures

[0056] First, each single-stranded DNA was dissolved to 10 μM and mixed at an equimolar concentration in TM buffer (10 mM Tris-HCl, 0.1 mM EDTA, 10 mM MgCl2, pH 8.0). Then, a four-fold molar ratio of DNAzyme was added to form a tetrahedral nanostructure with a final concentration of 1 μM. The prepared sample was then placed in a PCR instrument and incubated at 95℃ for 5 min; 65℃ for 30 min; 50℃ for 30 min; 42℃ for 30 min; 37℃ for 30 min; 22℃ for 30 min; and stored at 4℃ to assemble an inactive TDN-DNAzyme (I-TDN-DNAzyme). This inactivated TDN-DNAzyme was then characterized by PAGE and AFM.

[0057] 3. Verification of the enzyme digestion activity of TDN-DNAzyme

[0058] Mix 1 μL of I-TDN-DNAzyme probe (0.2 μM) with miR-141, add 2 μL of FAM / BHQ1-labeled substrate strand TS (2 μM), and replenish with reaction buffer (10 mM Tris-HCl, 0.1 mM EDTA, 20 mM MgCl2, pH 8.0) to a total volume of 20 μL. Incubate at 37°C for 1 h. During this process, the target binds and reconstitutes the active conformation of the DNAzyme, forming an active TDN-DNAzyme (A-TDN-DNAzyme).

[0059] A-TDN-DNAzyme in Mg 2+ With the aid of assisted specific cleavage of the rA site on the substrate chain TS, FAM is separated from BHQ1, generating a fluorescent signal. After the reaction is completed, the fluorescence value is monitored on a real-time quantitative PCR instrument to obtain the fluorescence kinetic curve of the entire cleavage process.

[0060] 4. Verification of CRISPR-Cas12a trans-cleavage activity

[0061] 1 μL of Cas12a (0.5 μM) was pre-incubated with 1 μL of crRNA (0.5 μM) for 0.5 h to form a CRISPR-Cas12a complex. Then, 2 μL of FQ fluorescence quenching strand, 5 μL of single-stranded activated DNA (AS) were added, and reaction buffer (10 mM Tris-HCl, 0.1 mM EDTA, 20 mM MgCl2, pH 8.0) was added to bring the total volume to 20 μL. The mixture was then incubated at 37 °C for 1.5 h on a real-time quantitative PCR instrument, and the fluorescence kinetics curve of the entire cleavage process was acquired.

[0062] 5. Sensor assembly and testing

[0063] Add 3 μL (0.2 μM) of TDN-DNAzyme assembly, 1 μL (1 mg / mL) of MB-TS, and 2 μL of target RNA to an enzyme-free EP tube, and then... 2+ The mixture was incubated at 37°C for 1.5 h to cleave the TS strand on the magnetic beads, releasing the activator strand AS (SEQ ID NO. 11). The supernatant was collected by magnetic separation, and 2 μL of [Cas12a protein (1 μL, 0.5 μM), Cas12a protein crRNA (1 μL, 0.5 μM)] pre-incubated at 37°C, 2 μL of fluorescent probe (2 μL, 2 μM), and 2 μL of NEB buffer 2 (10×) were added. The fluorescence value was monitored using a qPCR instrument.

[0064] The fluorescent probe sequence (FQ Probe) is: FAM-TTATT-BHQ1.

[0065] Figure 1This is a schematic diagram illustrating the detection principle of the nucleic acid detection system based on DNA tetrahedron-loaded DNAzyme cascade CRISPR / Cas12a according to the present invention. First, the DNA tetrahedron and DNAzyme are assembled to form a TDN-DNAzyme. In the presence of the target miR-141, it undergoes base complementarity pairing with the spacer sequence (orange-yellow fragment) in the catalytic core sequence (green fragment) of the TDN-DNAzyme, shortening the distance between the two catalytic core sequences and forming a complete catalytic pocket to unlock the catalytic activity of the TDN-DNAzyme. 2+ With the assistance of a cyclic cleavage, the activation parent chain TS loaded on the magnetic beads is released. Then, the supernatant is collected by magnetic separation. The free activator chain AS can activate the trans-cleavage activity of CRISPR / Cas12a, cleaving the fluorescent quenching chain to spatially separate the two modified groups and generate a huge fluorescent signal, so as to realize the detection of the target miR-141.

[0066] To verify the assembly efficiency of the TDN-DNAzyme, analysis was performed using 5% non-denaturing polyacrylamide gel electrophoresis. Figure 2 As shown, lanes 1-6 are arranged from left to right. Lane 1 is the marker. As the four strands are assembled sequentially (lanes 2-5), the migration rate of the electrophoretic bands gradually decreases, indicating that the molecular weight of the complex gradually increases, thus confirming the stepwise construction of the tetrahedral framework. Lane 6 corresponds to the complete TDN-DNAzyme structure, and its migration rate is the slowest, indicating that the DNAzyme and TDN have successfully combined and assembled into a stable TDN-DNAzyme complex.

[0067] To further confirm the assembly morphology, the prepared sample was characterized using atomic force microscopy. Figure 3 The data shows that most of the assembled particles in the field of view are of relatively uniform size, with a visible tetrahedral framework structure outline and a height of approximately 2.1 nm. A small amount of particle aggregation was observed in some areas, which may be related to the concentration effect during the sample drying process.

[0068] To verify the cleavage activity of the TDN-DNAzyme, the applicant modified TS with a fluorescent quenching group and detected its fluorescence signal intensity. Figure 4 The results showed that, compared with the control group without the target, the fluorescence signal in the TDN-DNAzyme and DNAzyme systems was significantly enhanced over time after the target was added, demonstrating the high specificity of the two systems for target recognition. It is worth noting that the former system reached fluorescence signal equilibrium in about 60 minutes, while the latter required about 90 minutes.

[0069] The above results indicate that the introduction of the DNA tetrahedral structure enhances the recognition, response, and catalytic ability of DNAzymes to target molecules. This may be because the DNA tetrahedron effectively organizes DNAzymes in three-dimensional space, resulting in a significant increase in the local concentration of DNAzymes and reducing the time and probability required for random collisions between free DNAzyme components.

[0070] The applicant verified the trans-cleavage activity of CRISPR / Cas12a using fluorescence kinetics curves, such as... Figure 5 As shown, only the system containing the activator chain AS exhibited a significant fluorescence signal, indicating that the CRISPR / Cas12a designed by the applicant has excellent trans-cleavage activity.

[0071] To verify the fluorescence response performance of the constructed biosensor, such as... Figure 6 As shown, the detection system containing the target can generate a significantly enhanced fluorescence signal, while the control group without the target only shows a relatively weak background signal, demonstrating the good feasibility of the novel TDN-DNAzyme-CRISPR / Cas12a fluorescence sensor for detecting miR-141.

[0072] To achieve optimal detection performance, the applicant systematically optimized key experimental parameters. First, based on feasibility experiments, it was observed that the background signal was not negligible. To reduce the background signal and achieve the maximum signal-to-noise ratio, the applicant optimized the inverse cutting time of the CRISPR / Cas 12a system, such as... Figure 7 As shown, 30 min was selected as the subsequent reaction time for the CRISPR / Cas12a system.

[0073] Secondly, from a theoretical perspective, Mg 2+ As an essential cofactor for the catalytic activity of TDN-DNAzyme in the system, the concentration of Mg2+ directly affects the catalytic efficiency and is an important parameter affecting the performance of this fluorescence sensor. To explore the optimal working concentration of Mg2+, its effect on the sensor's detection efficiency was investigated within the concentration range of 15 mM to 60 mM. Figure 8 As shown, when Mg 2+ The TDN-DNAzyme-CRISPR / Cas12a sensor achieved optimal detection efficiency and the highest signal-to-noise ratio at a concentration of 30 mM. Therefore, 30 mM was chosen as the concentration for Mg. 2+ The optimal concentration is used for subsequent experiments.

[0074] In the optimization experiment of the swing arm, the applicant designed a series of swing arms of different lengths (single-stranded DNA zyme T15, T30, T45, and T60, with sequences shown in SEQ ID NO. 5-8, respectively) for experimental verification. As shown in the figure, with the increase of length, the signal intensity and signal-to-noise ratio gradually increased, and the T30 swing arm reached the highest signal-to-background ratio. Figure 9 Therefore, the free arm length of the DNAzyme ligation to TDN was selected to be 30 nt. Furthermore, the incubation time between TDN-DNAzyme and MB-DNA was optimized to 1.5 hours. Figure 10 ).

[0075] Under optimal experimental conditions, the applicant evaluated the biosensor's detection sensitivity for miR-141. Real-time fluorescence kinetics curves ( Figure 11 The results showed that as the miR-141 concentration increased (0, 0.5, 1, 5, 10, 50, 100, 500, 1000 pM, corresponding to...), the results indicated that... Figure 11 (From bottom to top, the measured fluorescence intensity gradually increases, showing a positive correlation.)

[0076] Figure 12 This is a specific fluorescence signal output diagram of the present invention. In order to evaluate the specificity of the prepared fluorescent biosensor, the applicant tested the sensor's response to miR-141 and several different interfering miRNAs (miR-21, miR-122, miR-128, miR-3124, miR-4527, whose ribonucleotide sequences are shown in SEQ ID NO.12-16, respectively).

[0077] like Figure 12 As shown, only the experimental group with the target added produced a significant fluorescence signal, while other interfering sequences, even at high concentrations (10 times the target concentration), had significantly lower fluorescence signals than the target experimental group, indicating that the fluorescent biosensor has ultra-high specificity in detecting miR-141.

[0078] Reproducibility and stability are two crucial indicators for the practical application of fluorescent biosensors. To evaluate the reproducibility of this sensor, the applicant tested the same batch of biosensors (within the detection range) and different batches of biosensors (between the detection range) under the same conditions. Figure 13 As shown, the intra-batch relative standard deviation (RSD) was 3.30%, while the inter-batch RSD was 3.49%, indicating that the biosensor has good repeatability. Subsequently, the applicant stored the prepared biosensor at 4°C for 0, 1, 3, 5, and 7 days, respectively, and used it to detect the same concentration of miR-141 to evaluate the sensor's storage stability. The experimental results... Figure 14As can be seen, the fluorescence signal did not change significantly after one week of storage (RSD=4.35%), indicating that the sensor has good long-term stability.

[0079] To verify the sensor's applicability in complex biological matrices such as serum, the applicant used a spiked recovery method to detect miR-141 in human serum samples. Serum samples from healthy volunteers were diluted 10-fold with PBS (1×, pH 7.4), and a series of sample solutions of different concentrations were prepared by adding miR-141 standards to the serum samples. These solutions were then analyzed according to the optimized detection protocol. Results are as follows: Figure 15 As shown, the measured results were not significantly different from the actual miR-141 levels.

[0080] Subsequently, the applicant used a fluorescence sensor to detect the expression level of miR-141 in 20 clinical serum samples (10 PCa patients and 10 healthy individuals), and the results were as follows. Figure 16 As shown, normalizing the detected fluorescence signal intensity into a heatmap clearly shows that prostate cancer patients have higher miR-141 expression levels. A t-test (p < 0.001) confirmed this difference was significant. Figure 17 This allows for the differentiation between patients and healthy individuals. Furthermore, serum miR-141 levels are analyzed based on receiver operating characteristic (ROC) curves (…). Figure 18 The highest accuracy (AUC = 1.00) was achieved when distinguishing prostate cancer patients from healthy individuals. These findings are consistent with previous studies, showing that cancer patients have higher levels of miR-141 expression, validating the sensitivity and specificity of the TDN-DNAzyme-CRISPR / Cas12a fluorescence detection system in clinical applications.

[0081] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A nucleic acid detection system based on DNA tetrahedron-loaded DNAzyme cascade CRISPR / Cas12a, characterized in that, It consists of three parts: a tetrahedral nanostructure TDN-DNAzyme, magnetic beads MB-TS, and a CRISPR / Cas12a complex. The tetrahedral nanostructure TDN-DNAzyme is obtained by assembling single-stranded DNA T1, T2, T3, and T4 with deoxyribonucleotide sequences as shown in SEQ ID NO. 1-4 and single-stranded DNAzyme T30 as shown in SEQ ID NO.

7. The magnetic beads MB-TS are obtained by mixing streptavidin magnetic beads MB and activated parental strand TS with deoxyribonucleotide sequences as shown in SEQ ID NO.

9. The CRISPR / Cas12a complex is obtained by pre-incubating Cas12a and crRNA.

2. The preparation method of the nucleic acid detection system based on DNA tetrahedron-loaded DNAzyme cascade CRISPR / Cas12a as described in claim 1, comprising: (A) Preparation of tetrahedral nanostructure TDN-DNAzyme: First, the single-stranded DNA T1, T2, T3, T4 with deoxyribonucleotide sequences as shown in SEQ ID NO.1~4 are assembled with the single-stranded DNAzyme T30 shown in SEQ ID NO.7 to obtain a tetrahedral nanostructure TDN-DNAzyme with four DNAzyme arms; (B) Preparation of magnetic beads MB-TS: MB-TS were prepared by mixing streptavidin magnetic beads MB and deoxyribonucleotide sequences as shown in SEQ ID NO.9 to activate the parental TS. (C) Preparation of CRISPR / Cas12a complex: Cas12a was pre-incubated with the single-stranded crRNA shown in SEQ ID NO.17 to form CRISPR / Cas12a complex.

3. The preparation method according to claim 2, characterized in that, In step (A), the molar ratio of T1, T2, T3, T4, and T30 is 1:1:1:1:4; the assembly conditions are: 95℃ for 5 min; 65℃ for 30 min; 50℃ for 30 min; 42℃ for 30 min; 37℃ for 30 min; and 22℃ for 30 min.

4. The preparation method according to claim 2, characterized in that, In step (B), MB and TS were prepared into 5.0 mg / mL MB solution and 5 μM TS solution, respectively, and mixed at a volume ratio of 1:2.

5. The mixture was reacted at room temperature for 30 minutes, magnetically separated, and washed to obtain MB-TS.

5. The preparation method according to claim 2, characterized in that, The MB-TS obtained in step (B) is resuspended in PBST buffer for later use.

6. The preparation method according to claim 2, characterized in that, In step (C), the pre-incubation conditions are: incubation at 37℃ for 0.5h.

7. The application of the nucleic acid detection system of claim 1 in the preparation of a fluorescent sensor for detecting prostate cancer.

8. The application of the nucleic acid detection system according to claim 1 in the preparation of a prostate cancer detection kit.

9. The application according to claims 7 and 8, characterized in that, The detection target was miR-141, whose ribonucleotide sequence is shown in SEQ ID NO.

10.

10. A method for detecting prostate cancer-related miR-141 based on the aforementioned nucleic acid detection system for non-diagnostic purposes, characterized in that, First, mix TDN-DNAzyme, MB-TS, and miR-141 in a solution containing Mg. 2+ The mixture was incubated in the system, the parental strand TS was cleaved and activated, the daughter strand AS was released, the supernatant was collected by magnetic separation, and CRISPR / Cas12a complex, fluorescent probe and buffer were added. The fluorescence value was detected by real-time quantitative PCR, and a standard curve was constructed by the relationship between the fluorescence value and the miR-141 concentration. Then the test sample was used instead of miR-141 for detection to obtain the corresponding fluorescence value. The concentration of miR-141 in the test sample was calculated according to the standard curve.

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