Non-enzymatic nucleic acid detection method based on single molecule mooring

Abstract

The invention relates to the technical field of nucleic acid detection, and discloses an enzyme-free nucleic acid detection method based on single-molecule tethering, which comprises the following steps: synthesizing a single-molecule tethering probe for specifically recognizing target RNA (Ribonucleic Acid), and modifying the single-molecule tethering probe in a detection chip through combination of biotin and streptavidin; a recognition probe capable of being specifically combined with target RNA is modified on PS carboxyl microspheres through covalent combination of amino and carboxyl, and functional microspheres are prepared; adding the functionalized microspheres into a to-be-detected sample, performing room-temperature rotary incubation, introducing into a detection chip, then acquiring an image, and reading a nucleic acid detection result. According to the non-enzymatic nucleic acid detection method based on the single-molecule tethering, the target gene is enriched through the functionalized PS microspheres and introduced into the detection chip of the modified single-molecule tethering probe, after the target gene is combined with the probe, a biological signal is converted into a visual microsphere displacement signal, and then the target gene is detected through the visual microsphere displacement signal. The visual detection of the single nucleic acid molecule is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and particularly relates to an enzyme-free nucleic acid detection method based on single-molecule tethering. Background Art

[0002] Nucleic acids (DNA and RNA) play important roles in life activities such as storage and transmission of genetic information and protein synthesis. Through nucleic acid detection, pathogen screening and clinical diagnosis can be achieved. Nucleic acid amplification technology is the main nucleic acid detection means at present, including temperature-variable amplification and isothermal amplification. Among them, polymerase chain reaction (PCR) is the most widely used method at present. Through multiple rounds of cyclic reactions, PCR can exponentially amplify the target nucleic acid, and the reaction time is usually 1.5 to 2 hours. As a reliable nucleic acid detection means, PCR has developed a series of derivative technologies: such as quantitative real-time PCR (qPCR), reverse transcription-PCR (RT-PCR), digital PCR, etc. With the increase in the application scenarios of nucleic acid detection, the high dependence of PCR on biological enzymes and precise temperature control instruments limits its further development. Although isothermal amplification means can fill the gap in application scenarios lacking temperature-variable equipment, the harsh storage conditions of biological enzymes are still a severe challenge for nucleic acid detection in underdeveloped regions. In addition, temperature-variable and isothermal amplification methods relying on biological enzymes usually require on-site preparation of reaction reagents, which further increases the operation difficulty of nucleic acid detection technology.

[0003] Cancer screening is a typical case of the application of nucleic acid testing in clinical diagnosis. A number of early cancer screening products based on PCR and next-generation sequencing (NGS) technologies have been launched. Most of them adopt the method of liquid biopsy, extracting tumor-related information by detecting cell-free nucleic acids (cf-NAs) present in body fluids such as an individual's blood, urine, and cerebrospinal fluid, to achieve early cancer screening, diagnosis, and prognosis analysis. However, due to cost issues, the prices of the above-mentioned early cancer screening products greatly limit their clinical applications. Currently, the mainstream methods for early cancer screening in clinics are still endoscopy or imaging examinations, and these methods cannot diagnose tumors with small volumes (diameter less than 5 mm) or non-tumor-forming cases, resulting in the missed diagnosis of early tumors. In addition, the above methods also have limitations such as high invasiveness and low specificity. According to the statistical data of the World Health Organization (WHO), the number of newly added and death cases of cancer globally shows an increasing trend year by year. Therefore, developing a reliable and economical early cancer screening technology can significantly improve the survival rate of patients and reduce unnecessary social medical expenses. Summary of the Invention

[0004] The object of the present invention is to provide a single-molecule tethered enzyme-free nucleic acid detection method. The target gene is enriched by functionalized PS microspheres and introduced into a detection chip modified with a single-molecule tethered probe. After the target gene binds to the probe, the biological signal is converted into a visualizable microsphere displacement signal, realizing the visual detection of single nucleic acid molecules. This detection method replaces the traditional biochemical amplification reaction with physical amplification, greatly reducing the dependence on complex reagents, and the sensitivity can reach 1 GC / μL at the same time.

[0005] To achieve the above object, the present invention provides a single-molecule tethered enzyme-free nucleic acid detection method, including the following steps:

[0006] Step 1: Synthesize a single-molecule tethered probe that specifically recognizes the target RNA, and modify it in the detection chip through the binding of biotin and streptavidin;

[0007] Step 2: Modify the recognition probe that can specifically bind to the target RNA on the PS carboxyl microspheres through the covalent binding of amino and carboxyl groups to prepare functionalized microspheres;

[0008] Step 3: Add the functionalized microspheres obtained in Step 2 to the sample to be tested, incubate at room temperature with rotation for 30 min, introduce them into the detection chip obtained in Step 1, and then collect images and read the nucleic acid detection results.

[0009] Furthermore, in step 1, the single-molecule tethered probe includes a Duplex 1 recognition element, a Duplex 2 anchoring element, an Oligo-s anchoring element, and a probe backbone.

[0010] Furthermore, the synthesis method of the single-molecule tethered probe is as follows:

[0011] 1) Design primers containing EcoRⅠ restriction enzyme sites and HindⅢ restriction enzyme sites respectively with a DNA template of 2000bp - 12000bp in length for the PCR synthesis of the probe backbone;

[0012] 2) Use EcoRⅠ and HindⅢ restriction endonucleases to double-digest the probe backbone in step 1) to generate sticky ends corresponding to the Duplex 1 recognition element and the Duplex 2 anchoring element at its 3' end and 5' end respectively;

[0013] 3) Connect the Duplex 1 recognition element, the Duplex 2 anchoring element, the Oligo-s anchoring element, and the probe backbone with sticky ends through T4 ligase to synthesize a complete single-molecule tethered probe.

[0014] Restriction endonucleases include but are not limited to EcoRⅠ and HindⅢ, and EcoK I, BamH I, Hind II, Xba I, NotI, EcoP15 I, Mva1269 I, Cfr10 I, etc. can all be used for double digestion to generate a probe backbone with specific sticky ends.

[0015] Among them, the PCR reaction system is shown in Table 1, the double-digestion reaction system is shown in Table 2, and the T4 ligation reaction system is shown in Table 3.

[0016] Table 1 PCR reaction system

[0017] Reagent Volume Taq DNA Polymerase 1 μL 10X mix buffer 2 μL dNTPs (2.5 mM Each) 4 μL Long strand-F 2 μL Long strand-R 2 μL <![CDATA[DNA template / ddH2O]]> 1 μL <![CDATA[ddH2O]]> 8 μL Total 20 μL

[0018] Table 2 Double-digestion reaction system

[0019] Reagent Volume PCR product 20 μL Hind III (3000 U) 3 μL EcoR I (3000 U) 3 μL 10X M buffer 3 μL <![CDATA[ddH2O]]> 1 μL Total 30 μL

[0020] Table 3 T4 ligation reaction system

[0021]

[0022] Furthermore, in step 2, the recognition probe is synthesized from two partially complementary ssDNAs, namely Oligo-beads modified with amino groups and Oligo-neck for capturing the target RNA.

[0023] Further, in step 2, the functionalized microspheres are one of polystyrene microspheres, polyethylene (PE) microspheres, polypropylene (PP) microspheres, polylactic acid (PLA) microspheres, polyetheretherketone (PEEK) microspheres, silica microspheres, polymer microspheres (such as polyurethane, polymethacrylate), polyamide (nylon) microspheres, carbon black microspheres, biodegradable polymer microspheres (such as polyhydroxyalkanoates), magnetic microspheres, polycarbonate (PC) microspheres. The concentration of the functionalized microspheres is 1-50 mg / mL, and the particle size of the functionalized microspheres is 0.5 μm - 10 μm.

[0024] Further, in step 3, the detection chip is a PDMS microfluidic chip, 300 μm wide, 300 μm high, 12 mm long, and is provided with a detection window.

[0025] Further, the detection process is as follows:

[0026] Add 10 μL of functionalized microspheres and 1 μL of the sample solution to be tested into 1 mL of TE buffer. After rotating and incubating at room temperature for 30 min, introduce it into the detection chip at a flow rate of 5 μL / min; then connect PBS solution filtered through a 0.22 μm filter at both ends of the chip and flush the chip channel unidirectionally at a flow rate of 20 μL / min for 5 min to remove free microspheres; then collect images of 10 detection windows, and observe the displacement of the functionalized microspheres under the action of the single-molecule tethered probe as they move with the fluid through a microscope (or a camera or mobile phone equipped with a macro lens); superimpose the images taken in two fluid directions in Photoshop software, adjust the transparency of the first layer to 50%, and observe that the microspheres swing in situ along the fluid movement direction, with a swing amplitude of about 1 μm; calculate the number of microspheres through image recognition and conduct qualitative and quantitative analysis of the detection results.

[0027] The present invention also provides the application of the above-described enzyme-free nucleic acid detection method based on single-molecule tethering in detecting prostate cancer marker genes.

[0028] Further, the prostate cancer marker genes are PCA 3 gene, DLX 1 gene, HOXC 6 gene, KFBP 5 gene or ERG gene.

[0029] The present invention also provides a nucleic acid detection device for the above-described enzyme-free nucleic acid detection method based on single-molecule tethering, comprising: functionalized microspheres for binding and enriching target genes and a detection chip based on PDMS microfluidic technology, wherein the detection chip is loaded with single-molecule tethered probes.

[0030] The advantages and positive effects of the enzyme-free nucleic acid detection method based on single-molecule tethering described in the present invention are:

[0031] 1. The present invention enriches the target RNA through functionalized PS microspheres, and then introduces the microspheres into a detection chip modified with single-molecule tethered probes. The microspheres carrying the target gene will be captured by the probes, converting the biological signal into a visual signal of the microspheres, thereby completing the detection. The detection process uses physical amplification instead of the biochemical amplification used in traditional nucleic acid detection, amplifying the RNA biological signal into the displacement signal of the microspheres in the chip through angular momentum, realizing the visual detection of single nucleic acid molecules with a sensitivity of up to 1 GC / μL.

[0032] 2. The detection method described in the present invention reduces the dependence on complex biochemical reagents, thus providing a brand-new idea for nucleic acid detection and playing a prominent role in the field of early cancer screening.

[0033] 3. By adjusting the single-molecule tethered probes and recognition probes, the present invention can replace the corresponding detection sites, thereby being able to detect different target genes and having a wide range of applications.

[0034] The technical solution of the present invention will be further described in detail below with reference to the drawings and embodiments. Description of the Drawings

[0035] Figure 1 Schematic diagram of the structure of the single-molecule tethered probe in the embodiment of the present invention;

[0036] Figure 2 Verification of the surface functionalization of PS microspheres in the embodiment of the present invention;

[0037] Figure 3 Verification of the probe backbone PCR in the embodiment of the present invention;

[0038] Figure 4 Verification of the synthesis PCR of the single-molecule tethered probe in the embodiment of the present invention;

[0039] Figure 5 Structural diagram of the single-molecule tethered detection chip in the embodiment of the present invention;

[0040] Figure 6 Workflow diagram of the single-molecule tethered nucleic acid detection system in the embodiment of the present invention;

[0041] Figure 7 Verification result of the feasibility of the single-molecule tethered nucleic acid detection system in the embodiment of the present invention, where A is the result of the NC group, B is the result of the PC group, and C is the enlarged view of the red box area in B;

[0042] Figure 8 Verification result of the specificity of the single-molecule tethered nucleic acid detection system in the embodiment of the present invention, where A is the total number of microspheres in each experimental group, and B is the number of microspheres in the 1-10 detection windows in each experimental group;

[0043] Figure 9 This is the sensitivity verification result of the single-molecule tethered nucleic acid detection system in the embodiment of the present invention, where A is the total number of microspheres in each gradient concentration experimental group, and B is the number of microspheres in detection windows 1–10 among them. Detailed implementation manners

[0044] The technical solutions of the present invention will be further described below through the accompanying drawings and embodiments. Unless otherwise defined, the technical terms or scientific terms used in the present invention should have the ordinary meaning understood by those of ordinary skill in the art to which the present invention belongs. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0045] Unless otherwise defined or explained, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the method of the present invention.

[0046] An enzyme-free nucleic acid detection method based on single-molecule tethering includes the following steps:

[0047] Step 1: Synthesize a single-molecule tethered probe that specifically recognizes the target RNA, and modify it in the detection chip through the binding of biotin and streptavidin;

[0048] Step 2: Modify the recognition probe that can specifically bind to the target RNA on the PS carboxyl microspheres through the covalent binding of amino and carboxyl groups to prepare functionalized microspheres;

[0049] Step 3: Add the functionalized microspheres obtained in Step 2 to the sample to be tested, incubate at room temperature with rotation for 30 min, introduce them into the detection chip obtained in Step 1, then collect images and read the nucleic acid detection results.

[0050] Both the single-molecule tethered probe and the recognition probe are specially designed for the specific recognition of the prostate cancer marker gene PCA 3. By programming the sequences of the Duplex 1 recognition element and the Oligo-neck, the corresponding detection sites can be changed.

[0051] The following example uses PCA3 lncRNA (GI: 858438211) as the target gene to illustrate the construction process of the single-molecule tethered enzyme-free nucleic acid detection platform.

[0052] Example 1

[0053] 1. Design of PCA3 primers and probes:

[0054] According to the structure of the single-molecule tethered probe and the sequence of the target gene, four elements were designed respectively: Duplex 1 recognition element, Duplex 2 anchoring element, Oligo-s anchoring element, and probe backbone. Among them, the length of the probe backbone is 4495 bp, which was synthesized by PCR using the gene number NZ_JAACYX010000069.1(13,153..17,667) as a template to design primers. It should be noted that the upstream and downstream primers of the probe backbone need to contain EcoRⅠ restriction site (GAATTC) and HindⅢ restriction site (AAGCTT) respectively. In addition, in order to ensure the structural integrity of the assembled single-molecule tethered probe, corresponding primers also need to be designed for verification. All oligonucleotide chains were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0055] Restriction enzymes include but are not limited to EcoRⅠ and HindⅢ, and EcoK I, BamH I, Hind II, Xba I, NotI, EcoP15 I, Mva1269 I, Cfr10 I, etc. can all be used for double enzyme digestion to generate a probe backbone with specific sticky ends. The finally synthesized primer and probe sequences are shown in Table 4.

[0056] Table 4 Primer and Probe Sequences of Single-Molecule Tethered Nucleic Acid Detection System (PCA3)

[0057]

[0058]

[0059] 2. Construction of single-molecule tethered nucleic acid detection system:

[0060] The structural schematic diagram of the single-molecule tethered nucleic acid detection system is as Figure 1 shown, which is divided into three components from top to bottom: functionalized PS microspheres, target gene (Target), and single-molecule tethered probe.

[0061] 3. Surface functionalization of PS microspheres:

[0062] The microsphere product code used in the present invention is YP001, and it is a 1-μm carboxylated microsphere purchased from Shanghai Yiyuan Biotechnology Co., Ltd.

[0063] The surface of the microspheres is modified with a dimer of Oligo-beads and Oligo-neck. The length of Oligo-beads is 30 nt, and the 3'-end is modified with an amino group. The length of Oligo-neck is 60 nt, and 30 nt of it is completely complementary to Oligo-beads. That is, Oligo-beads and Oligo-neck can spontaneously assemble into a stable dimer structure in solution. The assembly efficiency can be further improved by using an assembly program of 90 °C for 10 min and 25 °C for 20 min.

[0064] The modification of the assembled dimer of Oligo-beads and Oligo-neck is completed through the amidation reaction of amino and carboxyl groups to achieve the surface functionalization of PS microspheres. An agarose gel electrophoresis experiment is carried out to verify the nucleic acid modification on the surface of PS microspheres. The modification results are as Figure 2 shown. Three laboratory groups are set up. Among them, Oligo-beads is the nucleic acid solution used for modification and serves as the standard control group. Unmodified is the supernatant taken after simply mixing and incubating the Oligo-beads solution with PS microspheres. Modified is the supernatant taken after modifying the Oligo-beads solution and PS microspheres through the amidation reaction of amino and carboxyl groups. The concentrations of the nucleic acid solutions used in the above three experimental groups are the same. The electrophoresis results show that only the Modified group does not show the Oligo-beads band, indicating that Oligo-beads have been successfully modified on the PS microspheres. The functionalized microspheres can be stored in a 4 °C refrigerator for future use.

[0065] 4. Synthesis of single-molecule tethered probes:

[0066] The single-molecule tethered probe consists of four elements: a Duplex 1 recognition element, a Duplex 2 anchoring element, an Oligo-s anchoring element, and a probe backbone. The specific structure is as Figure 1 shown. The probe backbone is a double-stranded DNA with a length of 4495 bp. The EcoRⅠ restriction site is connected to the Duplex 1 recognition element, and the HindⅢ restriction site is connected to the Duplex 2 anchoring element. The other end of the Duplex 2 anchoring element is connected to Oligo-s, and thus the synthesis of the single-molecule tethered probe can be completed. All the above connections are completed by T4 ligase.

[0067] The Duplex components are all composed of two single-stranded DNAs. The Duplex 1 recognition component is composed of Duplex 11 and Duplex 12. Duplex 11 is a single-stranded DNA with a length of 31 nt, and its 5'-end is phosphorylated for T4 ligation. Duplex 12 is a single-stranded DNA with a length of 57 nt, and 27 nt of it is completely complementary to Duplex 11, that is, Duplex 11 and Duplex 12 can spontaneously assemble into a stable Duplex 1 recognition component in solution. The Duplex 2 anchoring component is composed of Duplex 21 and Duplex 22. Similarly, 27 nt of them is completely complementary, enabling them to spontaneously assemble into a stable Duplex 2 anchoring component in solution. The above assembly process using the assembly program of 90 °C for 10 min and 25 °C for 20 min can further improve the assembly efficiency. The final concentration of the Duplex component is 50 μM.

[0068] The probe backbone was synthesized by PCR using the gene number NZ_JAACYX010000069.1 (13,153..17,667) as a template and Longstrand-F and Long strand-R as primers. The Taq DNA polymerase used in this step has the product number SL2600 and is purchased from Beijing Coolaber Technology Co., Ltd. The PCR product was verified by agarose gel electrophoresis, and the PCR verification result of the probe backbone is as Figure 3 shown. Two experimental groups, PC and NC, were set up. Among them, the PC group used the NZ_JAACYX010000069.1 gene as a template, and the NC group added ddH2O. And three parallel control groups were set up for each of the two experimental groups. The results showed that the PC group all had a target band with a length of 4495 bp, while the NC group was blank, indicating that the Long strand-F and Long strand-R primer pair could specifically amplify the target band.

[0069] The obtained DNA long chain also needs to be ligated with the Duplex 1 recognition component, the Duplex 2 anchoring component, and the Oligo-s anchoring component through T4 ligase. Therefore, EcoRⅠ restriction sites (GAATTC) and HindⅢ restriction sites (AAGCTT) were added to Long strand-F and Long strand-R respectively, and sticky ends were created at both ends of the DNA long chain by double enzyme digestion. The T4 ligase used in this step has the product number 2011A, the EcoRⅠ restriction endonuclease has the product number 1040S, and the HindⅢ restriction endonuclease has the product number 1060S, all of which are purchased from Baeyer Biotechnology (Beijing) Co., Ltd. PCR, double enzyme digestion, and T4 ligation were all carried out according to the steps of the product instructions, and the final volume of the single-molecule tethered probe obtained was 40 μL.

[0070] The single-molecule tethered probe synthesized by the action of T4 ligase is about 100 bp longer than the probe backbone. A pair of primers, PCA3Ligation-F and PCA3Ligation-R, were designed in the extended part for PCR verification of the single-molecule tethered probe targeting PCA3. The PCR products were subjected to agarose gel electrophoresis, and the results are as Figure 4 shown. Two groups of negative controls (NC) were set up, namely NC-ddH2O with ddH2O as the template and NC-Ori-DNA with the probe backbone as the template. In addition, a positive control (PC) group with the single-molecule tethered probe as the template was set up. All the above experimental groups were set with three parallel controls. The results showed that only the PC group obtained amplification products. The NC-Ori-DNA group had a very faint band at the 4495 bp position, which was the diluted probe backbone and was observed to be slightly shorter than the amplification products of the single-molecule tethered probe. The above results indicate the successful synthesis of the single-molecule tethered long chain. The single-molecule tethered long chain can be stored in a 4°C refrigerator for future use.

[0071] 5. Fabrication of the single-molecule tethered detection chip:

[0072] The single-molecule tethered detection chip is a PDMS microfluidic chip prepared by standard soft lithography. The structure is as Figure 5 shown. The chip is 300 μm wide, 300 μm high, and 12 mm long, and 10 detection windows are set for image acquisition.

[0073] The prepared chip was plasma-cleaned and bonded, and streptavidin was modified on the chip surface. The product number of the streptavidin used was P5087, purchased from Shanghai Beyotime Biotechnology Co., Ltd. Then, 20 μL of the single-molecule tethered probe was added to 1 mL of TE buffer, mixed well and centrifuged, and then introduced into the chip at a flow rate of 10 μL / min. Then, 3% BSA in PBST (0.1% Tween-20) was introduced to block for 30 min. After rinsing with TE for 10 min, it can be used for detection, or the chip inlet and outlet can be sealed with polyimide tape (to prevent solution evaporation) and stored in a 4°C refrigerator for future use.

[0074] 6. Workflow of the single-molecule tethered nucleic acid detection system:

[0075] The working principle of the single-molecule tethered nucleic acid detection system is as Figure 6As shown in the figure, after collecting the samples, the prepared functionalized microspheres are used to enrich the target genes in the samples. The specific operation steps are as follows: Take 10 μL of microspheres and add them to the sample, incubate at room temperature with rotation for 30 min, centrifuge at 13000 rpm / min for 10 min, discard the supernatant, add 1 mL of TE buffer to resuspend the microspheres, and introduce them into the detection chip at a flow rate of 5 μL / min. Finally, the number of microspheres captured in the detection chip can be observed through an optical magnification device such as a microscope, and images can also be collected and analyzed using image recognition software to export the detection results.

[0076] Example 2

[0077] 1. Feasibility verification of the single-molecule tethered nucleic acid detection system:

[0078] Based on the principles of DNA probe technology and microfluidics technology, a label-free nucleic acid detection platform based on single-molecule tethering was developed. This platform uses physical amplification instead of the biochemical amplification used in traditional nucleic acid detection, and amplifies the RNA biological signal into the displacement signal of microspheres in the chip through angular momentum to achieve the visualization detection of single nucleic acid molecules. Using the prostate cancer marker gene PCA3 lncRNA (GI: 858438211) as the target gene, the feasibility of the single-molecule tethered nucleic acid detection platform was verified.

[0079] Prepare a PCA3 lncRNA solution with a concentration of 6e+7 GC / μL, then take 1 μL and add it to 1 mL of TE buffer, and then add 10 μL of functionalized microspheres and mix well to form the PC group. In addition, according to the above steps, ddH2O is used instead of the PCA3 lncRNA solution as the NC group. Rotate and incubate the two experimental groups at room temperature for 30 min, introduce them into the detection chip at a flow rate of 5 μL / min, then use ddH2O to rinse the chip channels at a flow rate of 5 μL / min to remove free microspheres, and then observe the chip and collect images using a microscope. The results are as Figure 7 shown. There are almost no microspheres retained in the detection chip of the NC group, while in the PC group, a large number of microspheres are captured under the combined action of the single-molecule tethered probe and the target gene. The results show that in the single-molecule tethered nucleic acid detection system, qualitative and even quantitative analysis of the target gene can be carried out by counting the retained microspheres in the chip.

[0080] 2. Specificity verification of the single-molecule tethered nucleic acid detection system:

[0081] To ensure that the single-molecule tethered nucleic acid detection system can be used for target detection in complex environments and better applied to early cancer screening and other scenarios, 6 experimental groups were set up for specificity verification. Among them, PCA3 lncRNA and ddH2O were still used as the PC and NC groups. The other 4 groups were other biomarker genes of prostate cancer, namely: DLX 1 (GI: 1745), HOXC 6 (GI: 3223), KFBP 5 (GI: 297179221), ERG (GI: 113204731).

[0082] The nucleic acids of each group above were formulated into a solution with a concentration of 6e+8 GC / μL. Take 1 μL and add it to 1 mL of TE buffer, then add 10 μL of functionalized microspheres and mix well. In addition, according to the above steps, ddH2O was used to replace the nucleic acid solution as the NC group. All experimental groups were incubated by rotation at room temperature for 30 min, passed through the detection chip at a flow rate of 5 μL / min, and then the chip channels were rinsed with ddH2O at a flow rate of 5 μL / min to remove free microspheres. After that, the chip was observed under a microscope and images were collected (n = 3). The results are as Figure 8 shown. A is the total number of microspheres in each experimental group, and B is the number of microspheres in the 1–10 detection windows in each experimental group. Except for the PCA3 experimental group showing significant positive signals, the number of microspheres read in other experimental groups was close to that of the NC group. The results indicate that the single-molecule tethered nucleic acid detection system has good specificity and can meet the actual application requirements.

[0083] 3. Sensitivity verification of the single-molecule tethered nucleic acid detection system:

[0084] Single-molecule tethering is a novel enzyme-free nucleic acid detection method that uses angular momentum amplification instead of polymerase chain reaction amplification. The unique physical amplification method enables the single-molecule tethering reaction system to achieve visual detection of single nucleic acid molecules. For this reason, a group of sensitivity verification experiments was set up, using PCA3 lncRNA with different gradient concentrations as the target gene to test the LOD (limit of detection) of the single-molecule tethered nucleic acid detection system.

[0085] PCA3 lncRNA solutions with concentrations ranging from 6e-1 GC / μL to 6e+5 GC / μL were prepared. Take 1 μL of each and add it to 1 mL of TE buffer, then add 10 μL of functionalized microspheres and mix well. In addition, according to the above steps, ddH2O was used to replace the nucleic acid solution as the NC group. All experimental groups were incubated by rotation at room temperature for 30 min, passed through the detection chip at a flow rate of 5 μL / min, and then the chip channels were rinsed with ddH2O at a flow rate of 5 μL / min to remove free microspheres. After that, the chip was observed under a microscope and images were collected (n = 3). The results are as Figure 9As shown, A is the total number of microspheres in each gradient concentration experimental group, and B is the number of microspheres in detection windows 1-10. Except that the number of microspheres in the experimental group with a concentration of 6e-1 GC / μL was close to that of the NC group and the detection result was negative, the detection results of the experimental groups with concentrations of 6e+0 GC / μL - 6e+5 GC / μL were all positive, and the number of microspheres showed a significant positive correlation with the concentration gradient. The results indicate that the single-molecule tethered nucleic acid detection system, as an enzyme-free nucleic acid detection technology, has extremely high sensitivity, and its lowest detection limit (LOD) can reach 1 GC / μL.

[0086] This invention proves that the single-molecule tethered nucleic acid detection system has achieved a breakthrough in sensitivity up to 1 GC / μL under enzyme-free conditions, which is of great significance in the field of biosensing. First of all, the high sensitivity of this technology provides important support for early disease diagnosis, making it possible to detect low-abundance target molecules, thereby improving the accuracy and reliability of detection. Secondly, its enzyme-free property means the simplification of the detection process and the reduction of costs, reducing the dependence on expensive reagents and being suitable for application in resource-limited environments. The expansion of this technology can be widely applied in fields such as clinical diagnosis, environmental monitoring, and food safety detection, and is expected to promote the further development of biological detection technologies. At the same time, this system also provides new ideas for the development of future portable and point-of-care testing devices, promoting the realization of true on-site rapid detection.

[0087] Therefore, the present invention adopts the above-mentioned enzyme-free nucleic acid detection method based on single-molecule tethering. The target gene is enriched by functionalized PS microspheres and introduced into a detection chip modified with single-molecule tethered probes. After the target gene binds to the probe, the biological signal is converted into a visualizable microsphere displacement signal, realizing the visual detection of single nucleic acid molecules. This detection method replaces the traditional biochemical amplification reaction with physical amplification, greatly reducing the dependence on complex reagents, and at the same time, the sensitivity can reach 1 GC / μL.

[0088] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions of the present invention or make equivalent replacements, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. An enzyme-free nucleic acid detection method based on single molecule tethering, characterized in that, It includes the following steps: Step 1: Synthesize a single-molecule tethered probe that specifically recognizes the target RNA, and modify it in the detection chip through the binding of biotin and streptavidin; Step 2: Modify the recognition probe that can specifically bind to the target RNA on the PS carboxyl microspheres through the covalent binding of amino and carboxyl groups to prepare functionalized microspheres; Step 3: Add the functionalized microspheres obtained in Step 2 to the sample to be tested, incubate at room temperature with rotation for 30 min, introduce them into the detection chip obtained in Step 1, then collect images and read the nucleic acid detection results.

2. The enzyme-free nucleic acid detection method based on single molecule tethering according to claim 1, wherein: The single-molecule tethered probe in Step 1 includes a Duplex 1 recognition element, a Duplex 2 anchoring element, an Oligo-s anchoring element, and a probe backbone.

3. The enzyme-free nucleic acid detection method based on single-molecule tethering according to claim 2, wherein, The synthesis method of the single-molecule tethered probe is as follows: 1) Design primers containing EcoRⅠ restriction sites and HindⅢ restriction sites respectively with a DNA template of 2000bp - 12000bp in length for the PCR synthesis of the probe backbone; 2) Use EcoRⅠ and HindⅢ restriction endonucleases to perform double enzyme digestion on the probe backbone in Step 1) to generate sticky ends corresponding to the Duplex 1 recognition element and the Duplex 2 anchoring element at its 3' end and 5' end respectively; 3) Connect the Duplex 1 recognition element, the Duplex 2 anchoring element, the Oligo-s anchoring element, and the probe backbone with sticky ends through T4 ligase to synthesize a complete single-molecule tethered probe.

4. A method for enzyme-free nucleic acid detection based on single molecule tethering according to claim 1, characterized in that: The recognition probe in Step 2 is synthesized from two partially complementary ssDNAs, namely Oligo-beads modified with amino groups and Oligo-neck for capturing the target RNA.

5. A method for enzyme-free nucleic acid detection based on single-molecule tethering according to claim 1, characterized in that: The functionalized microspheres in Step 2 are one of polystyrene microspheres, polyethylene microspheres, polypropylene microspheres, polylactic acid microspheres, polyetheretherketone microspheres, silica microspheres, polymer microspheres, polyamide microspheres, carbon black microspheres, biodegradable polymer microspheres, magnetic microspheres, polycarbonate microspheres. The concentration of the functionalized microspheres is 1 - 50 mg / mL, and the particle size of the functionalized microspheres is 0.5 μm - 10 μm.

6. The method for enzyme-free nucleic acid detection based on single-molecule tethering according to claim 1, wherein: The detection chip in Step 3 is a PDMS microfluidic chip, 300 μm wide, 300 μm high, 12 mm long, and is provided with a detection window.

7. A method for enzyme-free nucleic acid detection based on single-molecule tethering according to claim 1, characterized in that, The detection process is as follows: Add the functionalized microspheres and the sample solution to be tested into TE buffer, incubate at room temperature with rotation for 30 min, then introduce them into the detection chip at a flow rate of 5 μL / min; Then connect PBS solution filtered through a 0.22 μm filter at both ends of the chip and flush the chip channel unidirectionally at a flow rate of 20 μL / min for 5 min to remove free microspheres; Then collect images of the detection window, and observe the displacement of the functionalized microspheres under the action of the single-molecule tethered probe as they move with the fluid through a microscope; Overlay the images taken in two fluid directions in Photoshop software, adjust the transparency of the first layer to 50%, and observe that the microspheres swing in place along with the fluid movement direction, with a swing amplitude of 0.8 - 1.2 μm; Calculate the number of microspheres through image recognition and perform qualitative and quantitative analysis on the detection results.

8. Use of a single-molecule tethering-based enzyme-free nucleic acid detection method according to any one of claims 1-7 in detecting prostate cancer marker genes.

9. The application according to claim 8, wherein: The prostate cancer marker genes are PCA 3 gene, DLX 1 gene, HOXC 6 gene, KFBP 5 gene or ERG gene.

10. A nucleic acid detection device for a single-molecule tethering-based enzyme-free nucleic acid detection method according to any one of claims 1-7, characterized in that, Comprising: Functionalized microspheres for binding and enriching target genes and a detection chip based on PDMS microfluidic technology, wherein a single-molecule tethering probe is loaded in the detection chip.

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