WO2024032828A2 - 一种基于dna纳米机器的核酸检测方法、试剂盒与生物传感器 - Google Patents

一种基于dna纳米机器的核酸检测方法、试剂盒与生物传感器 Download PDF

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WO2024032828A2
WO2024032828A2 PCT/CN2023/127710 CN2023127710W WO2024032828A2 WO 2024032828 A2 WO2024032828 A2 WO 2024032828A2 CN 2023127710 W CN2023127710 W CN 2023127710W WO 2024032828 A2 WO2024032828 A2 WO 2024032828A2
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chain
reaction
dnazyme
sequence
target gene
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WO2024032828A3 (zh
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孟凡明
田博
李婷婷
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Central South University
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Definitions

  • the invention belongs to the field of molecular biology detection technology, and specifically relates to a nucleic acid detection method, a kit and a biosensor based on DNA nanomachines.
  • Malaria is a vector-borne infectious disease caused by protozoan parasites of the genus Plasmodium and is transmitted through the bite of infected female Anopheles mosquitoes. It is one of the three major health threats in the world along with tuberculosis and AIDS.
  • the WHO's "World Malaria Report 2021" shows that there will be approximately 247 million new cases of malaria globally in 2021, and the number of deaths will be approximately 619,000.
  • the WHO announced that China has obtained malaria-free certification, which means that China has eliminated the occurrence of local malaria.
  • the number of people entering and leaving the country has increased, and the number of imported malaria cases remains high.
  • Nucleic acid detection is still an important detection strategy for diagnosing diseases. Ultra-sensitive detection of specific DNA or RNA sequences is of great significance to genetic research, diagnosis and personalized medicine. Among them, PCR technology is still the most popular for amplifying specific DNA. Amplification techniques for fragmentation and detection of low-abundance nucleic acids. However, PCR requires precise control of temperature cycling for successful DNA amplification, and the resulting instrument limitations have hindered its wider application. At the same time, various isothermal amplification technologies are rapidly developed and applied. Among them, the rolling circle amplification (RCA) method proposed in the 1990s is an isothermal amplification method with the advantages of simplicity, versatility and robustness. Enzymatic nucleic acid amplification methods.
  • RCA rolling circle amplification
  • Nicking-enhanced rolling circle amplification (Nicking-enhanced RCA, NickRCA) is introduced at the same time as rolling circle amplification
  • the enzyme cleavage site creates a nick on the product chain and serves as the starting site for the polymerase.
  • Functional nucleic acid is a DNA or RNA molecule isolated from a DNA library using in vitro screening technology. It is a nucleic acid sequence with specific recognition of target substances and catalytic functions. Types include aptamers, deoxyribonucleases (DNAzyme), G-quadruplexes and other DNA assemblies, etc. Among them, DNAzyme is a DNA single-chain enzyme with catalytic activity. Compared with protein enzymes, DNAzyme has the advantages of simple structure, stable activity and low synthesis cost. It has been widely used in biosensors, bioimaging, environmental monitoring, and nanomachines. and other fields. DNAzyme with RNA cleavage activity has high catalytic efficiency and strong specific recognition ability under the action of metal ions.
  • DNA nanomachines are molecular machines with specific functions by integrating functional nucleic acids (aptamers, DNAzymes, etc.), relying on programmed sequence-specific interactions of DNA chains to perform tasks according to molecular or environmental signals.
  • POCT point-of-care testing
  • MNP magnetic nanoparticles
  • IVD in vitro diagnostics
  • MNP magnetic nanoparticles
  • MNP magnetic nanoparticles
  • IVD in vitro diagnostics
  • MNP magnetic nanoparticles
  • the magnetism of MNP makes it very easy to enrich and separate, and it can move or rotate directionally under the guidance of an external magnetic field.
  • Superparametric MNPs can only be magnetized when subjected to an external magnetic field and the magnetization axis is in the same direction as the magnetic field.
  • DNAzyme is connected to MNP assembly, and through isothermal amplification reaction, directional conduction and amplification of detection signals can be achieved. Combined with magnetic biosensors, rapid real-time detection of pathogens can be completed under limited conditions.
  • One of the purposes of the present invention is to provide a nucleic acid detection method based on DNA nanomachines, which can perform on-site rapid and instant detection in an environment with backward economy, limited sanitary conditions and lack of medical resources;
  • the second/third purpose of the present invention is to provide A kit/biosensor using the nucleic acid detection method has the advantage of being integrated and portable;
  • the fourth/fifth/sixth object of the present invention is to provide a kit/biosensor for detecting malaria parasites and a method of using it, which is suitable for It has a wide range and is of great significance for malaria prevention and control.
  • a nucleic acid detection method based on DNA nanomachines including the following steps:
  • the padlock probe has a phosphorylated 5' end and is provided with a target gene Recognition region, primer recognition region and endonuclease recognition region.
  • the target gene recognition region is located at both ends of the padlock probe and is complementary to the target gene sequence to be tested.
  • the endonuclease cleavage site of the endonuclease recognition region is A.
  • the sequence of the protection chain is the same as the partial region of the padlock probe, and has a chemically blocked 3'end;
  • the DNAzyme chain is complementary to the substrate chain with a single RNA site, and the sequence of the partial region of the DNAzyme chain is the same as that of the padlock probe.
  • the sequence of the target gene recognition region in the padlock probe is the same; the DNAzyme chain and the substrate chain have biotinylated 3'ends;
  • S5 take the ligase reaction product and nanocomplex system solution, add metal ion solution, DNA polymerase, endonuclease, T4 polynucleotide kinase and protective chain, mix, and perform nick-enhanced rolling circle expansion. amplification reaction, DNAzyme endonuclease reaction, and strand displacement reaction to obtain the final reaction system;
  • S6 use sensor detection on the final reaction system to achieve qualitative analysis and/or quantitative analysis of the target gene to be tested.
  • NickRCA is used to amplify the target gene sequence
  • DNAzyme is used to build a nanomachine.
  • the amplification product combines with it to change the DNAzyme conformation.
  • the enzyme activity is activated to cleave the substrate chain to achieve directional amplification of the signal.
  • the target gene sequence and the padlock probe match each other at both ends.
  • a methylation-protected enzyme cutting site is introduced inside the padlock probe ( AGCT), after the primer is combined with the padlock probe, it triggers isothermal nucleic acid amplification NickRCA under the action of polymerase and endonuclease, and obtains a single-stranded amplification product; during this process, a protective chain is added to assist the endonuclease to fully cut and generate more
  • the short-chain products are simultaneously bound to the 3' end of the amplification product to prevent the single-strand amplification product from being shredded by the possible exonuclease activity of the added DNA polymerase; the ends of the DNAzyme chain and substrate chain are biotinylated, Combined with nanomarkers, a nanocomplex is formed.
  • the amplification product is complementary to the nanocomplex, mediating its conformational change. Under the action of metal ions, it stimulates enzyme activity and identifies the substrate.
  • RNA is cut, and the substrate chain is divided into two parts, producing a cleavage product of 2'(3')-cyclic phosphate and 5'-hydroxyl end, carrying the 5'-hydroxyl group of the nanomarker
  • the terminal cleavage product breaks away from the complex and is free in the system.
  • the 2'(3')-cyclic phosphate cleavage product generates a 3'hydroxy product under the action of T4 polynucleotide kinase, which is then corrected and extended by DNA polymerase.
  • the amplification product is released and re-enters the system for recycling.
  • the sensor is used to detect the parameter indicators of the markers in the system required to achieve qualitative and quantitative analysis of the target gene in the solution to be tested.
  • step S4 the molar ratio of DNAzyme chain and substrate chain in the nanocomplex is (1.5 ⁇ 1):1, Preferably 1:1.
  • the nanomarker includes one or more of magnetic nanoparticles, gold nanoparticles, and fluorescent groups.
  • DNAzymes use metal ions as coenzymes and rely on metal ions to stimulate their activity to cause catalytic reactions.
  • step S4 the DNAzyme is a Mg 2+ -dependent DNAzyme, and the metal ion solution is a Mg 2+ solution.
  • the DNAzyme is 8-17 DNAzyme.
  • the senor includes one or more of an opto-magnetic end-point phase difference detection system, an electron paramagnetic resonance spectrometer, an ultraviolet-visible spectrophotometer, and a fluorescence microscope.
  • the DNA polymerase is phi29 DNA polymerase; the endonuclease is a restriction endonuclease or a nicking endonuclease.
  • a kit including reaction solution a, reaction solution b, and reaction solution c; wherein, the reaction solution a includes a padlock probe, a primer, and a DNA ligase; the reaction solution b includes a DNAzyme chain, a substrate chain, A nanocomplex formed by combining nanomarkers; the reaction solution c includes T4 polynucleotide kinase, DNA polymerase, endonuclease, protective chain and metal ion solution; a padlock probe is designed according to the target gene sequence to be tested and DNAzyme chain, and correspondingly designed primers, protection strands and substrate chains; the padlock probe has a phosphorylated 5' end, and is provided with a primer recognition region, an endonuclease recognition region, and at both ends are provided with to-be-detected
  • the target gene recognition region is complementary to the target gene sequence, and the endonuclease cleavage site of the endonuclease recognition region is methylated; the
  • the above kit is used to detect Plasmodium, wherein the sequence of the padlock probe is as shown in SEQ ID NO: 1, the sequence of the primer is as shown in SEQ ID NO: 3, and the sequence of the protective chain is as shown in SEQ ID NO: 1. As shown in SEQ ID NO:4, the sequence of the DNAzyme chain is shown in SEQ ID NO:5, and the sequence of the substrate chain is shown in SEQ ID NO:6.
  • a biosensor includes the above-mentioned kit.
  • a biosensor for detecting Plasmodium includes the above-mentioned kit for detecting Plasmodium.
  • a method for detecting Plasmodium using the above-mentioned Plasmodium detection kit or biosensor including the following steps:
  • S3 use sensor detection for the final reaction system to achieve qualitative analysis and/or quantitative analysis of the target gene to be tested;
  • sequence of the target gene is shown in SEQ ID NO: 2.
  • the nanomarker is a magnetic nanoparticle (MNP); the sensor is an opto-magnetic end-point phase difference sensor.
  • MNP magnetic nanoparticle
  • Its mechanism of action is to use the optical occlusion changes caused by the periodic rotation of MNP under the action of an external alternating magnetic field to detect the substance to be measured combined with MNP.
  • the presence of target molecules causes the MNP to combine to form different sizes and shapes.
  • the target molecule concentration is further determined based on the physical properties of the aggregates and their rotational dynamics in response to external oscillating magnetic fields.
  • the main reason for the periodic rotation of MNP is Brownian relaxation.
  • the applied magnetic field is a sinusoidal magnetic field
  • the MNP or MNP combination is magnetized, and the MNP magnetization axis points in the direction of the magnetic field; when the magnetic field disappears, the magnetization disappears, and the MNP or MNP combination returns to random orientation.
  • the movement process of MNP from magnetization to magnetization disappearance is called Brownian relaxation, and the time taken is called Brownian relaxation time, and Brownian relaxation time is proportional to the hydrodynamic volume of MNP. Therefore, substances bound to MNP are detected.
  • the presence of target gene molecules causes MNPs to aggregate.
  • the DNAzyme chain modified with MNP hybridizes with the substrate chain modified with MNP. Due to the principle of minimum energy, MNP spontaneously aggregates to form an assembly.
  • the overall hydrodynamic volume of MNP increases, and the Brownian relaxation time Increase; when the target gene exists, it triggers the ligase reaction and NickRCA reaction, amplifies the target gene signal, and obtains the NickRCA product-mediated nanocomplex bound to MNP ( MD&S ), changes the conformation of the complex, and DNAzyme chains are formed with the assistance of Mg 2+ RNA cleavage activity
  • MD&S NickRCA product-mediated nanocomplex bound to MNP
  • DNAzyme chains are formed with the assistance of Mg 2+ RNA cleavage activity
  • the substrate chain is cut off, the end modified with MNP is free in the system, and the hydrodynamic volume decreases.
  • MNP is used as a marker, and the signal changes are measured based on the hydrodynamic volume changes of MNP before and after the reaction for qualitative and quantitative analysis.
  • the detection limit can reach the fM level.
  • the detection method provided by the present invention has low requirements for sample pretreatment during the detection process (human tissue samples do not contain magnetism), and can be detected in almost any body fluids and samples, eliminating potential loss of samples and speeding up analysis.
  • the kit or biosensor provided by the present invention can be used independently or as an auxiliary detection of RT-PCR technology, greatly reducing the detection cost during a major epidemic outbreak and reducing the financial burden on underdeveloped areas.
  • the detection method, kit or biosensor provided by the present invention combines Nick RCA amplification technology (NickRCA) with the RNA cleavage activity of DNAzyme for the first time to detect Plasmodium conserved region sequences, which greatly improves the efficiency of DNAzyme.
  • the efficiency of the selected target gene sequence is widely present in various Plasmodium species, which improves the applicable scope of malaria detection. Compared with RT-PCR and other existing malaria detection methods, this method is highly integrated, low-cost and time-consuming. It is short, has relatively low technical requirements for operators, has strong specificity and high accuracy, and can be suitable for on-site detection under limited environmental conditions.
  • the proposed biosensing system is constructed by replacing the target gene and its related sequence target gene binding region, which is expected to be completed under limited conditions. Rapid real-time detection of multiple pathogens is of great significance for the prevention and control of widely spread dangerous pathogens.
  • Figure 1 is a schematic diagram of the principle of a magnetic biosensor based on DNA nanomachines in one embodiment.
  • Figure 2 is a diagram showing the verification results of molecular amplification electrophoresis in one embodiment.
  • Figure 3 is an electrophoresis verification result of the binding product of NickRCA product and DNA nanomachine in one embodiment.
  • Figure 4 is a graph showing the hydrodynamic volume results of using dynamic light scattering to detect related MNP assembly structures in one embodiment.
  • Figure 5 is an atomic force microscope verification result of a related MNP assembly structure in one embodiment.
  • Figure 6 is a graph showing the detection results based on the opto-magnetic end point phase difference sensor in one embodiment.
  • Plasmodium pathogen nucleic acid detection sites are generally located on 18S rRNA, but because the mitochondrial genome has a higher copy number than 18S rRNA. Therefore, this detection target screened and compared a total of 151 existing Plasmodium mitochondrial gene (mtDNA) sequences, conducted conservation analysis, and obtained a highly conserved target gene sequence containing 36 nt. The sequence information is shown in Table 1.
  • the total length of the padlock probe sequence is 78 nt. Since the double-stranded DNA torque is approximately 10.44 bp/turn, in order to reduce the difficulty of strand displacement of the polymerase during the amplification process, the length of the padlock probe should be an integral multiple of the unnatural DNA torque. There are 18nt at both ends of the padlock probe that are complementary to the target sequence, and a restriction endonuclease site is introduced into it. The cutting base is modified with methylation to prevent itself from being cut, and the 5' end of the padlock probe Phosphorylation is required to meet the working conditions of ligase.
  • the sequence is calculated through the online nucleic acid secondary structure prediction tool NUPACK software to calculate the binding ability and prediction of the padlock probe and the target gene sequence. Measure the secondary structure of the padlock probe and try to reduce the stability of irrelevant secondary structures (such as intramolecular hairpin structures, intermolecular dimers) while ensuring binding force to improve amplification efficiency.
  • the sequence information of the padlock probe is shown in Table 1.
  • the primer sequence matches the padlock probe sequence and is distinguished from the target gene sequence and the protective strand.
  • the sequence information of the protective chain is shown in Table 1.
  • the full length of the protective chain is 25 nt, which is completely complementary to the NickRCA product and the 3' end is chemically blocked.
  • RO has two main functions: RO introduces a restriction enzyme site near the 5' end position, which can recognize the RCA product.
  • the 3' end of the NickRCA product generated after cutting matches the RO, so it will not be digested by the polymerase.
  • the DNA nanomachine integrates the DNAzyme chain and the corresponding substrate chain, specifically:
  • the DNAzyme chain (hereinafter referred to as "D chain") is verified by electrophoresis and the 8-17 DNAzyme with RNA cleavage activity is selected.
  • the designed D chain has a total length of 130nt. From both ends to the middle, the D chain is: substrate chain binding arm region, enzyme active center, padlock probe same region and random sequence. The 3' end of the D chain is modified with biotin. See Table 1 for sequence information.
  • S chain Both ends of the designed substrate chain (hereinafter referred to as "S chain”) match the D chain, and the middle region is equipped with a cleavage site matching the D chain.
  • the 3' end of the S chain is modified with biotin, S
  • the chain sequence refers to the 8-17 DNAzyme binding chain in the literature. The sequence information is shown in Table 1.
  • the padlock probe in Table 1 contains a methylated C (bold and underlined); the substrate chain contains a cleavage site a (rA, bolded and underlined).
  • the 8-17 DNAzyme in Table 1 was synthesized by Beijing Qingke Biotechnology Co., Ltd., and the remaining sequences were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for synthesis.
  • the nucleic acid molecules involved in this example are as described in Example 1. Agarose electrophoresis is used to verify each reaction product and verify the generation of expected nucleic acid fragments. It should be noted that the target gene-containing solution in this example is a standard solution. Directly initiates rolling circle amplification without adding primers. The following are the main reagents and equipment involved:
  • TE buffer Beijing Solebao Technology, T1120
  • 10 ⁇ TBE buffer Beijing Prilite Gene Technology Co., Ltd., B1111-500mL
  • TAE buffer Thermo Fisher, B1110
  • Tris- HcL Beijing Prilite Gene Technology Co., Ltd., B1011), 1M
  • zinc sulfate heptahydrate Shanghai Huzhao, 7446-20-0
  • magnesium sulfate New England BioLabs, M0374S
  • 100mM bovine serum albumin (BSA) ) (Beijing Solebao Technology, 109Z054)
  • Agarose gel Bourgos, Spain, BY-R0100
  • SYBR Gold Thermo Fisher, S11494
  • 6 ⁇ Loading dye Haunan Aikerui Bioengineering Co., Ltd., AG11902
  • DNA Marker Thermo Fisher, SM0371
  • phi29 DNA polymerase is a polymerase with high processivity (higher than 70kb) and can achieve efficient isothermal DNA amplification. It has strong strand displacement activity and is a commonly used tool enzyme for RCA reactions. phi29 DNA polymerase also has 3’ ⁇ 5’ exonuclease (proofreading) activity that preferentially acts on single-stranded DNA or RNA; AluI restriction endonuclease recognizes specific sites in the double strand for cleavage.
  • the migration rate of circular DNA structures in agarose gel is slower than that of linear DNA structures of the same length, so electrophoresis can be used to qualitatively verify whether the padlock probe is connected into a circle by ligase.
  • the rolling circle amplification (RCA) product has a complex structure, large molecular weight, and extremely low migration rate in agarose gel. It is used to qualitatively verify whether the reaction amplification and hybridization reaction are successfully designed.
  • Ligase reaction (20nM): Mix 5 ⁇ L ligase buffer (10 ⁇ ), 1 ⁇ L padlock probe (1 ⁇ M), 3 ⁇ L target gene (1 ⁇ M), 33.5 ⁇ L water, 5 ⁇ L bovine serum albumin (2mg/mL) Mix with 2.5 ⁇ L Ampligase DNA ligase (5U/ ⁇ L) and react at 50°C for 15 minutes.
  • (2) RCA reaction Mix 6 ⁇ L ligase reaction product (1nM), 3 ⁇ L polymerase buffer (10 ⁇ ), 3 ⁇ L bovine serum albumin (2 mg/mL), 1 ⁇ L deoxynucleotide dNTP (10mM), and 16 ⁇ L water, Mix 1 ⁇ L phi29 DNA polymerase (10U/ ⁇ L) and react at 37°C for 60 minutes.
  • Electrophoresis verification agarose gel electrophoresis (2.5%, 1 ⁇ TAE buffer), mix 15 ⁇ L of sample to be analyzed with 1 ⁇ L SYBR Gold and 3 ⁇ L loading buffer, and perform gel electrophoresis at room temperature (100V, 45 minutes) and capture the image.
  • the samples to be analyzed include:
  • Lane 4 RCA reaction product triggered by 200pM ligase reaction product
  • the electrophoresis results are shown in Figure 2.
  • the analysis shows that except for the shortest chain in lane 3, which is the excess target chain, the other bands are above the padlock probe chain, proving that the ligase reaction is proceeding normally; in lane 4, the ultra-long RCA product is produced; in lane 5, A smear-like product appears, but there are more long-chain products than short-chain products, and the NickRCA product is generated; a smear-like product appears in lane 6, and multiple significantly shorter products are produced compared with lane 5, proving that the protective chain can assist cleavage to generate more short chain products.
  • the type 8-17 DNAzyme strand (D strand) and the corresponding substrate strand (S strand) were selected.
  • the D strand has the same region as the padlock probe, allowing it to match the NickRCA product.
  • the catalytic active center of the D chain is formed. Under the action of Mg 2+ , the D chain cleaves the RNA site in the S chain to produce 2'(3')-cyclic phosphate and 5'- Cleavage product of the hydroxyl terminus.
  • T4 polynucleotide kinase can convert 2'(3')-cyclophosphate into The acid is converted to the 3'hydroxyl group.
  • phi29 DNA polymerase has 3'-5' exonuclease (proofreading) activity that preferentially acts on single-stranded DNA or RNA, so phi29 DNA polymerase preferentially recognizes and converts it into 3' by T4 polynucleotide kinase
  • the mismatched base of the hydroxyl group is corrected and extended to displace the NickRCA chain bound to the D chain.
  • the electrophoresis verification experimental protocol is as follows:
  • Ligase reaction (20nM): Mix 10 ⁇ L ligase buffer (10 ⁇ ), 2 ⁇ L padlock probe (1 ⁇ M), 6 ⁇ L target gene (1 ⁇ M), 67 ⁇ L water, 10 ⁇ L bovine serum albumin (2mg/mL) and Mix 5 ⁇ L Ampligase DNA ligase (5U/ ⁇ L) and react at 50°C for 15 minutes.
  • System 1 Mix 1.2 ⁇ L ligase reaction product (5nM), 3 ⁇ L polymerase buffer (10 ⁇ ), 3 ⁇ L bovine serum albumin (2mg/mL), 2 ⁇ L deoxynucleotide dNTP (10mM), 10.8 ⁇ L water, 2 ⁇ L Mix phi29 DNA polymerase (10 U/ ⁇ L), add 2 ⁇ L AluI restriction enzyme (10 U/mL) and 6 ⁇ L protective strand (20 ⁇ M), and react at 37°C for 120 minutes. Take 15 ⁇ L of the mixed solution and add 6 ⁇ L of MgSO 4 (50 mM).
  • System 3 React the remaining mixed solution of the above system 2 at 80°C for 20 minutes to inactivate phi 29 DNA polymerase and AluI restriction endonuclease, then add 24 ⁇ L of MgSO 4 (50 mM), 12 ⁇ L of Mix D&S complex (5 ⁇ M); separate three mixed solutions (3.A, 3.B, 3.C) of 24 ⁇ L each from system 3. middle:
  • System 3.A Add 6 ⁇ L water to system 3.A and react at 37°C for 120 minutes;
  • System 3.B Add 3 ⁇ L T4 polynucleotide kinase buffer (10 ⁇ ) and 1 ⁇ L water to system 3.B. After reacting at 37°C for 60 minutes, add 1 ⁇ L phi29 DNA polymerase (10U/ ⁇ L) and 1 ⁇ L T4. Polynucleotide kinase (10U/ ⁇ L), mix thoroughly and then react at 37°C for 60 minutes;
  • System 4 Add 15 ⁇ L of the mixed solution taken out of the above system 2 to 6 ⁇ L of MgSO 4 (50 mM) and mix, add 3 ⁇ L of D&S complex (5 ⁇ M), react at 37°C for 60 minutes, and then add 1 ⁇ L of water and 3 ⁇ L of T4 polynucleotide kinase. Mix buffer (10 ⁇ ) and 1 ⁇ L T4 polynucleotide kinase (10 U/ ⁇ L) thoroughly, and react at 37°C for 60 minutes.
  • Electrophoresis verification agarose gel electrophoresis (2.5%, 1 ⁇ TBE buffer), mix 15 ⁇ L of the sample to be analyzed with 1 ⁇ L SYBR Gold and 3 ⁇ L loading buffer, and perform gel electrophoresis at room temperature (100V, 45 minutes) and capture the image.
  • the samples to be analyzed include:
  • Lane 4 System 1 product triggered by 200 pM target gene.
  • Lane 5 System 3.A product triggered by 200pM target gene
  • Lane 6 System 3.B product triggered by 200pM target gene
  • Lane 7 System 3.C product triggered by 200pM target gene
  • Lane 8 System 4 product triggered by 200 pM target gene.
  • Lane 9 System 5 product triggered by 200 pM target gene.
  • the S chain can be cut and extended after correction.
  • the corresponding band in lane 5 is divided into two (c position) in lane 6, which are strand displacement.
  • magnetic nanoparticles (BNF-Starch-1mL (Micromod, 10-19-102)) were selected as nanomarkers; nucleic acid sequences were used as MNP scaffolds, and MNP assemblies were formed through nucleic acid sequences.
  • the D chain and S chain in the DNA nanomachine are each modified with biotin at their 3' ends, so that MNP is modified on the D chain and S chain respectively, and D&S is formed through complementary base pairing of the two chains. complex, further forming MNP assembly.
  • the MNPs were modified with a streptavidin loading of 0.2 times, that is, 40 nucleic acid strands were modified on each MNP.
  • System 1 Take 20 ⁇ L of MNP (10 mg/mL) and magnetically separate the free streptavidin in the primary elution system;
  • System 3 MNP combined with S chain (M S ). Mix 18 ⁇ L S chain (0.2 ⁇ M) and 22 ⁇ L water, heat the metal bath at 60°C for 10 minutes to open the secondary structure of the DNA chain, and quickly cool down for 5 minutes. Add 10 ⁇ L of system 1 solution and react at 37°C for 30 minutes. The streptavidin on the MNP specifically binds to the modified biotin on the S chain. Finally, add Tris-Hcl buffer to 100 ⁇ L to obtain the MS system solution.
  • the BeNano 90 nanoparticle size analyzer (Bettersize Instruments, Dandong, China) was used to verify the hydrodynamic volume of the MNP assembly before and after the reaction, including MD , MNP in the system after the 3.6pM target gene initiated amplification reaction and destructuring, and MD &S , and related parameters. :
  • the laser light source wavelength is 671nm
  • the detection angle is 90°
  • the detection temperature is 25°C.
  • Detection instrument Asylum Research MFP-3D BIO biological atomic force microscope (Oxford Instruments plc, A-bingdon); parameters: Probe: AC160TSR3 silicon probe, aluminum reflective coating, tip radius: 7nm, scanning area of 5 ⁇ 5 ⁇ m 2 (resolution The rate is 256 ⁇ 256) and the scanning speed is about 1Hz.
  • AFM image processing used Gwyddion 2.43 software.
  • the selected malaria conserved region was used as the target gene, rolling circle amplification was performed, and then reacted with the M D&S assembly prefabricated in Example 3 above, and the optical and magnetic end-point phase difference sensor was used to verify the molecular amplification system. Sensitivity and qualitative and quantitative analysis of amplification products. The specific experimental steps are as follows:
  • Ligase reaction (20nM): Mix 10 ⁇ L ligase buffer (10 ⁇ ), 2 ⁇ L padlock probe (1 ⁇ M), 6 ⁇ L target gene (1 ⁇ M), 67 ⁇ L water, 10 ⁇ L bovine serum albumin (2mg/mL) and Mix 5 ⁇ L Ampligase DNA ligase (5U/ ⁇ L) and react at 50°C for 15 minutes.
  • Amplification reaction and M D&S solution 30 ⁇ L ligase reaction product (0.5fM-1000fM), 6 ⁇ L polymerase buffer (10 ⁇ ), 6 ⁇ L bovine serum albumin (2mg/mL), 3 ⁇ L deoxynucleotides dNTP (10mM), 3 ⁇ L MgSO 4 (100mM), 3 ⁇ L M D&S assembly (5 ⁇ M), 1 ⁇ L phi29 DNA polymerase (10U/ ⁇ L), 1 ⁇ L AluI restriction endonuclease (10U/ ⁇ L), 3 ⁇ L T4 poly Mix the polynucleotide kinase buffer (10 ⁇ ), 1 ⁇ L T4 polynucleotide kinase (10 U/ ⁇ L), and 6 ⁇ L protective strand (20 ⁇ M) thoroughly, and incubate with rotating magnet at 37°C for 75 minutes.
  • Target gene detection analysis Use an opto-magnetic end-point phase difference sensor to verify the sensitivity of the molecular amplification system.
  • the opto-magnetic detection parameters 1mT magnetic field, 450nm wavelength light source, detection at room temperature. The results are shown in Figure 6.
  • the detection involves 10 Group of samples, including blank control group, different target genome groups with concentrations of 0.5fM-1000fM (respectively 0.5fM, 1fM, 3.2fM, 10fM, 32fM, 50fM, 100fM, 320fM, 1000fM), 10 groups of samples only have ligase reaction
  • the relative phase difference of the opto-magnetic signal is selected as the difference between the average of the 0.5Hz-10.7Hz phase signal and the blank.
  • the sensitivity of this molecular amplification system can reach fM level.
  • the qualitative verification of the amplification product was verified by electrophoresis; the quantitative verification was performed by calculating the opto-magnetic detection phase (0.5Hz-10.7Hz) caused by the hydrodynamic volume after M D&S destructure triggered by the target concentration of 0.5fM-1000fM ) for quantification.
  • the concentrations of enzymes, probes, primers, and protective chains involved in the above examples are all used in excess to ensure maximum reaction efficiency. If the concentration is lower, the reaction efficiency will be reduced.
  • the reaction time of each step is the shortest time to obtain the best signal, and is determined by the reaction efficiency. If the reaction efficiency is low, the same reaction result can generally be achieved by extending the reaction time.
  • the reaction temperature of each step depends on the optimal working temperature of the enzyme, which is generally 37°C.

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Abstract

本发明公开了一种基于DNA纳米机器的核酸检测方法及其在试剂盒或生物传感器中的应用。所述检测方法根据所述待测靶基因序列设计挂锁探针和DNAzyme链，并对应设计引物、保护链与底物链，通过NickRCA技术实现靶基因序列的扩增放大，利用DNAzyme构建DNA纳米机器，扩增产物与DNA纳米机器结合改变DNAzyme构象，在金属离子作用下，激活酶活性切割底物链实现信号的定向放大，在反应体系中添加纳米标记物作信号参照物，利用传感器检测实现对待测靶基因的定性和/或定量分析。本发明具有集成化、便携性、低成本等优势，可适用于条件有限的环境下完成现场快速即时检测，对于疾病防控具有重要意义。

Description

一种基于DNA纳米机器的核酸检测方法、试剂盒与生物传感器 技术领域
本发明属于分子生物学检测技术领域，具体涉及一种基于DNA纳米机器的核酸检测方法、试剂盒与生物传感器。
背景技术
疟疾是疟原虫属原生动物寄生虫引起的虫媒传染病，通过被感染的雌性按蚊叮咬传播，与结核病、艾滋病并列为世界三大主要健康威胁。WHO《2021年世界疟疾报告》中显示：2021年全球疟疾的新发病例约为2.47亿例，死亡人数约为61.9万人。2021年世卫组织宣布中国获得无疟疾认证，意味着中国已消除本土疟疾的发生，但随着经济全球化，国际间的合作增多，出入境人员随之增多，输入性疟疾病例数量居高不下，死亡病例时有发生，疟疾的防治形势依然不容乐观。临床确诊疟疾感染通常采用外周血涂片染色镜检法，为疟原虫感染阳性诊断金标准，但该方法最佳检验时期为患者高热发生时，且对检验人员经验性和专业性要求较高，早期感染和血液中疟原虫数量较少时易发生漏检而产生假阴性；免疫学检验方法存在假阳性率高，检测窗口期受限等缺点；使用聚合酶链式反应检测疟疾，需要精确温控，对检测设备和实验室硬件有一定要求。由于疟疾感染通常发生在经济落后、卫生条件有限且医疗资源匮乏的环境下，因此，开发出便携、准确且性价比高且适配移动电源的高灵敏度分子诊断工具对于疟疾早期发现及规范治疗具有重要临床意义。
核酸检测仍是诊断疾病的重要检测策略，实现特定DNA或RNA序列的超灵敏检测对遗传研究、诊断和个性化医疗具有重要意义，其中，PCR技术依旧是最流行的用于扩增特定的DNA片段和检测低丰度核酸的扩增技术。然而，PCR需要精确控制温度循环才能成功进行DNA扩增，由此产生的仪器限制已经阻碍其进行更广泛的应用。同时，各种等温扩增技术在快速发展并应用，其中，20世纪90年代提出的滚环扩增(Rolling circle amplification，RCA)方法是一种具有简单、多用途和鲁棒性等优点的等温酶促核酸扩增方法。切刻增强的滚环扩增(Nicking-enhanced RCA，NickRCA)即在滚换扩增的同时引入 酶切位点，在产物链上制造切口并作为聚合酶的起始位点，此过程中多个聚合酶在同一模板上同时工作，利用聚合酶的链置换特性将旧链置换并进行新一轮扩增，得到周期性重复的产物，产物长度为模板长度的(n-1)倍(n∈Z+，且n>＝2)。
功能核酸是一种利用体外筛选技术从DNA文库中分离而得的DNA或RNA分子，是具有特异性识别靶标物质和催化功能的核酸序列，种类包括适配体、脱氧核糖核酸酶(DNAzyme)、G-四链体以及其他DNA组装体等。其中，DNAzyme是一种具有催化活性的DNA单链酶，相比于蛋白质酶，DNAzyme具有结构简单、活性稳定且合成成本低等优势，已广泛应用于生物传感器、生物成像、环境监测、纳米机器等领域。具有RNA切割活性的DNAzyme在金属离子作用下，催化效率高、特异性识别能力强，其结构包括一个高度保守的催化核心及两条序列可编辑的结合臂，通过两条结合臂特异性识别底物链，并催化底物链中间的核糖核苷酸磷酸二酯键断裂。而DNA纳米机器是一种通过整合功能核酸(适配体、DNAzyme等)特定功能的分子机器，依赖于DNA链的程序化序列特异性相互作用，根据分子或环境信号执行任务。
传统意义上的医学检测技术是指在具有自动化技术的集中临床实验室中进行，以低成本进行大量检测。而在分散的和资源有限的环境下，传统的分析方法受限制，因此，床旁检测(point-of-care testing，POCT)作为一种替代的医疗检测形式出现，是未来医疗检测发展的主要方向之一，有望在可及性、速度以及结果的准确性方面获得重大突破，且不需要复杂的实验室基础设施及专门的实验室人员。但POCT也具有易用性、检测时间短、灵敏度高和成本低等各种要求，在大多数情况下，单一功能材料的生物传感器难以满足。这也推动当前体外诊断(In vitro diagnostics，IVD)技术、纳米技术领域的飞速发展，例如磁性纳米颗粒(magnetic nanoparticle，MNP)，兼具有磁性粒子和纳米粒子特性，可以在磁场中产生磁响应。MNP具有优越的可操作性、生物兼容性、信号对比度(常见生物样本无磁性信号干扰，将MNP作为标记物或造影剂可获得极佳的信噪比)、穿透性与稳定性。MNP所具有的磁性使其十分容易富集与分离，能在外部磁场的引导下进行定向移动或旋转。 超顺MNP只有在受到外部磁场作用时才能被磁化且磁化轴与磁场同向，在没有磁场的情况下不会保留剩余磁化。超顺MNP的物理化学稳定性不会影响生物相互作用，使得体外和体内的检测和磁操作成为可能，功能化的超顺MNP广泛用于生物医学应用。
发明内容
发明人通过深入研究后，认识到等温扩增技术结合DNA纳米机器如脱氧核酶(DNAzyme)与纳米技术在核酸检测领域的可操作性，利用纳米技术如磁性纳米颗粒(MNP)的低背景噪声优势，使得DNAzyme连接MNP组装，通过等温扩增反应，可实现对检测信号的定向传导和放大，再结合磁学生物传感器可在有限条件下完成病原体的快速实时检测。
本发明目的之一旨在提供一种基于DNA纳米机器的核酸检测方法，可在经济落后、卫生条件有限且医疗资源匮乏的环境下进行现场快速即时检测；本发明的目的之二/三在于提供一种应用所述核酸检测方法的试剂盒/生物传感器，具有集成便携的优点；本发明的目的之四/五/六在于提供一种检测疟原虫的试剂盒/生物传感器及其使用方法，适用范围广，对于疟疾防控具有重要意义。
上述目的通过以下技术方案实现：
一种基于DNA纳米机器的核酸检测方法，包括以下步骤：
S1，筛选待测靶基因；
S2，根据所述待测靶基因序列设计挂锁探针和DNAzyme链，并对应设计引物、保护链与底物链；其中，所述挂锁探针具有磷酸化的5'末端，且设有靶基因识别区、引物识别区和内切酶识别区，所述靶基因识别区设于挂锁探针两端，与待测靶基因序列互补，所述内切酶识别区的内切酶切割位点甲基化；所述保护链与挂锁探针部分区域的序列相同，且具有化学封闭的3'末端；所述DNAzyme链与带有单RNA位点的底物链互补配对，DNAzyme链部分区域序列与挂锁探针中靶基因识别区序列相同；所述DNAzyme链与底物链具有生物素化的3'末端；
S3，将所述待测品溶液与挂锁探针、引物、DNA连接酶混合进行连接酶反应，得连接酶反应产物；
S4，制备纳米复合体体系溶液；所述纳米复合体由DNAzyme链、底物链、纳米标记物结合生成；
S5，取所述连接酶反应产物、纳米复合体体系溶液，加入金属离子溶液、DNA聚合酶、内切酶、T4多聚核苷酸激酶和保护链，混合，进行切刻增强的滚环扩增反应、DNAzyme内切酶反应、链置换反应，得到终反应体系；
S6，对终反应体系使用传感器检测，实现对待测靶基因的定性分析和/或定量分析。
在上述技术方案中，通过NickRCA实现靶基因序列扩增放大，利用DNAzyme构建纳米机器，扩增产物与其结合改变DNAzyme构象，在金属离子作用下，激活酶活性切割底物链实现信号的定向放大。具体地，靶基因序列与挂锁探针两端相互匹配，两者杂交后在连接酶作用下进行连接酶反应，形成环状模板；挂锁探针内部引入被甲基化保护的酶切位点(AGCT)，引物与挂锁探针结合后在聚合酶和内切酶作用下引发等温核酸扩增NickRCA，得到单链扩增产物；此过程中加入保护链，辅助内切酶充分切割生成更多的短链产物的同时结合在扩增产物的3'端，防止单链扩增产物被加入的DNA聚合酶可能具有的核酸外切酶活性校正切碎；DNAzyme链、底物链末端生物素化，结合纳米标记物形成纳米复合体，由于DNAzyme链部分区域序列与挂锁探针相同，扩增产物与纳米复合体互补结合，介导其构象发生变化，在金属离子作用下，激发酶活性，识别底物链中的特定位点，实行RNA切割，将底物链分成两个部分，产生2'(3')-环磷酸和5'-羟基末端的切割产物，携带纳米标记物的5'-羟基末端切割产物脱离复合体，游离在体系中，2'(3')-环磷酸切割产物在T4多聚核苷酸激酶作用下，生成3'羟基产物后在DNA聚合酶校正与延伸作用下进行链置换反应，扩增产物释放，再次进入体系内循环利用，反应终止后，利用传感器检测所需要的体系中标记物的参数指标，实现对待测品溶液中靶基因的定性定量分析。
进一步地，步骤S4中，所述纳米复合体中DNAzyme链与底物链的摩尔比为(1.5～1):1， 优选为1:1。
进一步地，步骤S4中，所述纳米标记物包括磁纳米颗粒、纳米金、荧光基团中的一种或几种。
大部分DNAzyme都以金属离子为辅酶，依赖于金属离子激发其活性而发生催化反应。
进一步地，步骤S4中DNAzyme为Mg2+依赖的DNAzyme，所述金属离子溶液为Mg2+溶液。
优选的，所述DNAzyme为8-17DNAzyme。
进一步地，步骤S6中，所述传感器包括光磁终点相差检测系统、电子顺磁共振波谱仪、紫外-可见分光光度计、荧光显微镜中的一种或几种。
进一步地，所述DNA聚合酶为phi29 DNA聚合酶；所述内切酶为限制性内切酶或切刻内切酶。
一种试剂盒，包括反应液a、反应液b、反应液c；其中，所述反应液a包括挂锁探针、引物、DNA连接酶；所述反应液b包括由DNAzyme链、底物链、纳米标记物结合形成的纳米复合体；所述反应液c包括T4多聚核苷酸激酶、DNA聚合酶、内切酶、保护链与金属离子溶液；根据待测靶基因序列设计挂锁探针和DNAzyme链，并对应设计引物、保护链与底物链；所述挂锁探针具有磷酸化的5'末端，且设有引物识别区、内切酶识别区，以及在两端设有与待测靶基因序列互补的靶基因识别区，所述内切酶识别区的内切酶切割位点甲基化；所述保护链与挂锁探针部分区域的序列相同，且具有化学封闭的3'末端；所述DNAzyme链与带有单RNA位点的底物链互补配对，DNAzyme链部分区域序列与挂锁探针中靶基因识别区序列相同；所述DNAzyme链与底物链具有生物素化的3'末端。
进一步地，上述试剂盒用于检测疟原虫，其中，所述挂锁探针的序列如SEQ ID NO:1所示，所述引物的序列如SEQ ID NO:3所示，所述保护链的序列如SEQ ID NO:4所示，所述DNAzyme链的序列如SEQ ID NO:5所示，所述底物链的序列如SEQ ID NO:6所示。
一种生物传感器，包括上述试剂盒。
一种检测疟原虫的生物传感器，包括上述检测疟原虫的试剂盒。
一种利用上述检测疟原虫的试剂盒或生物传感器检测疟原虫的方法，包括以下步骤：
S1，将含有靶基因的待测品溶液与反应液a混合，进行连接酶反应，得连接酶反应产物；
S2，取所述连接酶反应产物与所述反应液b、反应液c混合，进行切刻增强的滚环扩增反应、DNAzyme内切酶反应、链置换反应，得到终反应体系；
S3，对终反应体系使用传感器检测，实现对待测靶基因的定性分析和/或定量分析；
其中，所述靶基因的序列如SEQ ID NO:2所示。
进一步地，在本发明的一个具体实施例中，所述纳米标记物为磁纳米颗粒(MNP)；所述传感器为光磁终点相差传感器。
其作用机理是利用MNP在外加交变磁场的作用下进行周期性旋转产生的光学遮挡变化，对与MNP结合的待测物质进行检测,目标分子的存在使MNP结合在一起形成不同大小和形状的聚集体，根据聚合体的物理特性以及它们对外部振荡磁场响应的旋转动力学，进一步确定目标分子浓度。
MNP的周期性旋转的主要原因为布朗弛豫。进行光磁检测时，由于施加磁场为正弦磁场，磁场存在时，MNP或MNP结合物发生磁化，MNP磁化轴指向磁场方向；磁场消失，磁化消失，MNP或MNP结合物恢复随机指向。由磁化到磁化消失MNP的运动过程称为布朗弛豫，经历时间称为布朗弛豫时间，而布朗弛豫时间与MNP的流体力学体积成正比。因此对与MNP结合的物质进行检测，靶基因分子的存在使MNP聚集，通过MNP的流体力学体积变化影响的布朗弛豫，进一步实现对靶基因分子的定量。在本发明的一个实施例中，修饰有MNP的DNAzyme链与修饰有MNP的底物链杂交，由于能量最低原理，MNP自发聚集，形成组装体，MNP整体流体力学体积增大，布朗弛豫时间增加；靶基因存在时，引发连接酶反应和NickRCA反应，放大靶基因信号，得到NickRCA产物介导结合有MNP的纳米复合体(MD&S)，改变复合体构象，在Mg2+辅助下DNAzyme链的RNA切割活性 激活，底物链被切断，修饰有MNP的一端游离在体系内，流体力学体积减小。在此过程中，以MNP为标记物，根据反应前后MNP流体力学体积变化，测得信号变化进行定性定量分析，检测限可达fM级别。
现有实验室疟疾诊断主要分为病原学检测、免疫学检测以及分子生物学检测三类。但由于疟疾的广泛传播多发生于经济落后、卫生条件有限的环境中，现有实验室检测手段难以大规模满足当地显示要求。与现有技术相比，本发明提出的一种基于DNA纳米机器的核酸检测方法与应用，具有以下有益效果：
(1)本发明提供的检测方法在检测过程对样品的预处理要求低(人类组织样本不含磁性)，几乎可以在任何体液和样品中进行检测，消除样品潜在的损失，加快分析的速度。
(2)本发明提供的试剂盒或生物传感器可以实现独立使用，或用作RT-PCR技术的辅助检测，极大减轻疫情大爆发时的检测成本，减轻欠发达地区的财政负担。
(3)本发明提供的检测方法或试剂盒或生物传感器首次将切刻滚环扩增技术(NickRCA)与DNAzyme的RNA切割活性相结合用于检测疟原虫保守区域序列，极大的提高了DNAzyme的效率，选取的靶基因序列广泛存在于各疟原虫种属，提高了疟疾检测的适用范围，相较于RT-PCR以及现有其他疟疾检测方法，该方法集成化高、成本低、耗时短、对操作人员技术要求相对较低，特异性强，准确度高，可适用于有限环境条件下的现场检测。
(4)构建以DNAzyme为核心的纳米机器，使用可适配移动电源的光磁传感模式以MNP作为标记物检测疟原虫病原体，适用于医疗条件匮乏、卫生环境有限的疟疾多发地区，极大的降低检测成本，实现疟疾的快速、实时、灵敏的POC检测。
(5)依靠本发明提供的检测方法或试剂盒或生物传感器所蕴含的分子机制，通过替换靶基因及其相关序列靶基因结合区进行生物传感体系的拟搭建，预期可在有限条件下完成多种病原体的快速实时检测，对于广泛传播的危险病原体的防控具有重要意义。
说明书附图
图1为一个实施例中基于DNA纳米机器的磁学生物传感器的原理示意图。
图2为一个实施例中分子扩增电泳验证结果图。
图3为一个实施例中NickRCA产物与DNA纳米机器的结合产物的电泳验证结果图。
图4为一个实施例中使用动态光散射检测相关MNP组装结构的流体力学体积结果图。
图5为一个实施例中相关MNP组装结构的原子力显微镜验证结果图
图6为一个实施例中基于光磁终点相差传感器的检测结果图。
具体实施方式
下面结合附图和实施例对本发明做进一步的详细说明，以令本领域技术人员参照说明书文字能够据以实施。除非另有定义，实施例中所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。如无特别说明，所使用的方法均为本领域常规的方法；所使用的药剂均可通过商业途径或已知公开的方法制备得到。
以疟原虫病原体的检测为例，对本发明做进一步的详细说明。
实施例1检测疟原虫的生物传感器中的序列设计
1.1靶基因序列的筛选
疟原虫病原体核酸检测位点一般位于18S rRNA上，但由于线粒体基因组比18S rRNA有更高的拷贝数。因此本次检测靶标通过对已有疟原虫线粒体基因(mtDNA)序列共151条进行筛选比对，进行保守性分析，得到高度保守的包含36nt的靶基因序列，序列信息见表1。
1.2挂锁探针的设计
挂锁探针序列总长度78nt，由于双链DNA扭矩约为10.44bp/turn，为降低扩增过程中聚合酶的链置换难度，挂锁探针的长度应为非自然状态DNA扭矩的整数倍。挂锁探针两端分别有18nt与靶序列互补匹配，并在其中引入限制性内切酶位点，切刻碱基用甲基化修饰，以防止其本身被切断，且挂锁探针5'末端需磷酸化以满足连接酶工作条件。序列通过在线核酸二级结构预测工具NUPACK软件计算挂锁探针与靶基因序列的结合力、预 测挂锁探针的二级结构，在保证结合力的前提下尽量降低无关二级结构(如分子内发卡结构，分子间二聚体)的稳定性，以提高扩增效率。挂锁探针的序列信息见表1。
1.3引物的设计
根据上述挂锁探针序列设计引物序列(见表1)。引物序列与挂锁探针序列匹配，并与靶基因序列以及保护链区分。
1.4保护链(RO)的设计
保护链(RO)序列信息见表1，所述保护链全长25nt，与NickRCA产物完全互补，3'末端化学封闭。RO的主要功能有两个：RO靠近5'末端位置处引入限制性酶切位点，可识别RCA产物，切刻后生成的NickRCA产物3'末端与RO匹配，因此不会被聚合酶消化。
1.5 DNA纳米机器的设计
DNA纳米机器整合了DNAzyme链与对应的底物链，具体为：
(1)DNAzyme链(以下简称“D链”)经过电泳验证后选择具有RNA切割活性的8-17DNAzyme。所设计D链全长130nt，D链从两端到中间依次为：底物链结合臂区域、酶活性中心、挂锁探针相同区域以及随机序列，在D链的3’端修饰有生物素，序列信息见表1。
(2)设计的底物链(以下简称“S链”)的两端与D链匹配，中间区域设有与D链匹配的切割位点，在S链的3’端修饰有生物素，S链序列引用文献中的8-17DNAzyme结合链，序列信息见表1。
表1涉及的DNA序列

表1中挂锁探针含有一个甲基化的C(加粗并下划线)；底物链中含有一个切割位点a(rA，加粗并下划线)。
表1中的8-17DNAzyme由北京擎科生物科技股份有限公司合成，其余序列送至生工生物工程(上海)股份有限公司合成。
实施例2电泳验证分子产物
本实施例中涉及的核酸分子如实施例1所述，使用琼脂糖电泳验证各个反应产物，验证预期核酸片段生成，需要说明的是，本实施例中的含靶基因溶液为标准品溶液，可以直接引发滚环扩增，无需添加引物。以下为主要涉及的试剂和设备：
(1)试剂：TE缓冲液(北京索莱宝科技，T1120)；10×TBE缓冲液(北京普利莱基因技术有限公司，B1111-500mL)；TAE缓冲液(Thermo Fisher，B1110)；Tris-HcL(北京普利莱基因技术有限公司，B1011)，1M；七水合硫酸锌(上海沪试，7446-20-0)；硫酸镁(New England BioLabs，M0374S)，100mM；牛血清白蛋白(BSA)(北京索莱宝科技，109Z054)；琼脂糖凝胶(Burgos,Spain，BY-R0100)；SYBR Gold(Thermo Fisher，S11494)；6×Loading染料(湖南艾科瑞生物工程有限公司，AG11902)；DNA Marker(Thermo Fisher，SM0371)；GL DNA Marker 5000(湖南艾科瑞生物工程有限公司，AG11906)；dNTP混合物(北京索莱宝科技，PC2200)，10mM；DNA连接酶配套试剂：10×Ampligase DNA连 接酶缓冲液、Ampligase DNA连接酶(5U/μL，Biosearch technologiesr，A32750)；DNA聚合酶配套试剂：10×聚合酶缓冲液、phi29 DNA聚合酶(10U/μL，Thermo Fisher，EP0094)；DNA激酶配套试剂：10×多聚核苷酸激酶缓冲液、T4多聚核苷酸激酶(New England BioLabs，M0201V)，10U/μL；AluI限制性内切酶(New England BioLabs，R0137S)，10U/μL。
上述分子试剂中，phi29 DNA聚合酶是一种具有高持续合成能力的聚合酶(高于70kb)且可实现高效等温DNA扩增，具有强大的链置换活性，是进行RCA反应的常用工具酶。phi29 DNA聚合酶还具有优先作用于单链DNA或RNA的3’→5’核酸外切酶(校正读码)活性；AluI限制性内切酶识别双链中特定位点进行切割。
(2)设备：凝胶成像系统(广州BIO-OI生物技术有限公司，OI100)；金属浴恒温器(江苏无锡耐思生物科技有限公司，HS11000)；电泳仪(北京君意东方电泳设备有限公司，JY600C)。
2.1电泳验证分子扩增产物：
环状DNA结构在琼脂糖胶中的迁移速率较同长度的线性DNA结构更慢，故可用电泳定性验证挂锁探针是否被连接酶连接成环。滚环扩增(RCA)产物结构复杂、分子量大，在琼脂糖胶中迁移速率极低，用于定性验证反应扩增和杂交反应是否设计成功。
(1)连接酶反应(20nM)：将5μL连接酶缓冲液(10×)、1μL挂锁探针(1μM)、3μL靶基因(1μM)、33.5μL水、5μL牛血清白蛋白(2mg/mL)与2.5μL Ampligase DNA连接酶(5U/μL)混合，50℃反应15分钟。
(2)RCA反应：将6μL连接酶反应产物(1nM)、3μL聚合酶缓冲液(10×)、3μL牛血清白蛋白(2mg/mL)、1μL脱氧核苷酸dNTP(10mM)、16μL水，1μL phi29 DNA聚合酶(10U/μL)进行混合，37℃反应60分钟。
(3)NickRCA反应：将6μL连接酶反应产物(1nM)、3μL聚合酶缓冲液(10×)、3μL牛血清白蛋白(2mg/mL)、1μL脱氧核苷酸dNTP(10mM)、15μL水、1μL phi29 DNA聚合酶(10U/μL)进行混合，1μL AluI限制性内切酶(10U/μL)，37℃反应60分钟。
(4)NickRCA与保护链反应：将6μL连接酶反应产物(1nM)、3μL聚合酶缓冲液(10×)、3μL牛血清白蛋白(2mg/mL)、1μL脱氧核苷酸dNTP(10mM)、9μL水、1μL phi29 DNA聚合酶(10U/μL)进行混合，加入1μL AluI限制性内切酶(10U/μL)、6μL保护链(20μM)，37℃反应60分钟。
(5)电泳验证：琼脂糖凝胶电泳(2.5％，1×TAE buffer)，取15μL待分析的样品与1μL SYBR Gold和3μL上样缓冲液混合后，室温下进行凝胶电泳(100V，45分钟)并拍摄图像。所述待分析的样品包括：
(a)泳道1：4μM靶基因链；
(b)泳道2：5μM挂锁探针链；
(c)泳道3：250nM上述连接酶反应产物；
(d)泳道4：200pM连接酶反应产物引发的RCA反应产物；
(e)泳道5：200pM连接酶反应产物引发的NickRCA反应产物；
(f)泳道6：200pM连接酶反应产物引发的保护链辅助的NickRCA反应产物；
(g)泳道M：Maker。
电泳结果见图2，分析可得，泳道3中除最短链为过量靶标链，其余条带均在挂锁探针链上方，证明连接酶反应正常进行；泳道4中超长RCA产物产生；泳道5中出现涂抹状产物，但是长链产物多于短链产物，NickRCA产物生成；泳道6中出现涂抹状产物，且与泳道5相比产生多条明显较短产物，证明保护链可以辅助切割生成更多的短链产物。
2.2电泳验证NickRCA产物与DNA纳米机器的结合反应
如前所述，选择8-17型DNAzyme链(D链)和对应底物链(S链)，D链具有与挂锁探针的相同区域，使其可以与NickRCA产物匹配。制备D链与S链的复合体D&S，将其与NickRCA产物加入同一反应体系中反应。其中，D&S复合体与NickRCA产物结合后，D链催化活性中心形成，在Mg2+作用下，D链切割S链中RNA位点后，产生2’(3’)-环磷酸和5’-羟基末端的切割产物。经文献调研，T4多聚核苷酸激酶可以将2’(3’)-环磷 酸转化为3’羟基。phi29 DNA聚合酶具有优先作用于单链DNA或RNA的3’-5’核酸外切酶(校正读码)活性，因此phi 29 DNA聚合酶优先识别经T4多聚核苷酸激酶转化为3’羟基的错配碱基，校正后进行延伸，将结合在D链上的NickRCA链置换下来。
电泳验证实验方案如下：
(1)连接酶反应(20nM)：将10μL连接酶缓冲液(10×)、2μL挂锁探针(1μM)、6μL靶基因(1μM)、67μL水、10μL牛血清白蛋白(2mg/mL)与5μL Ampligase DNA连接酶(5U/μL)混合，50℃反应15分钟。
(2)D&S复合体合成：将18μL D链(20μM)、12μL S链(20μM)、18μL水混合，90℃反应10分钟，打开DNA链二级结构，然后每隔10分钟降低10℃，降低至37℃，使二者充分结合，形成D&S复合体，其中D链与S链的比例为3：2，保证体系中不存在游离的S链。
(3)NickRCA反应
体系1：将1.2μL连接酶反应产物(5nM)、3μL聚合酶缓冲液(10×)、3μL牛血清白蛋白(2mg/mL)、2μL脱氧核苷酸dNTP(10mM)、10.8μL水、2μL phi29 DNA聚合酶(10U/μL)进行混合，加入2μL AluI限制性内切酶(10U/mL)与6μL保护链(20μM)，37℃反应120分钟。取出15μL混合溶液加入6μL的MgSO4(50mM)。
体系2：将3μL连接酶反应产物(5nM)、7.5μL聚合酶缓冲液(10×)、7.5μL牛血清白蛋白(2mg/mL)、5μL脱氧核苷酸dNTP(10mM)、27μL水、5μL phi29 DNA聚合酶(10U/μL)进行混合，加入5μL AluI限制性内切酶(10U/mL)与15μL保护链(20μM)，37℃反应120分钟。取出15μL混合溶液备用。
(4)NickRCA与D&S复合体链置换反应(两步法酶灭活)
体系3：将上述体系2剩余的混合溶液在80℃下反应20分钟，以将phi 29 DNA聚合酶与AluI限制性内切酶进行灭活处理，然后加入24μL的MgSO4(50mM)、12μL的D&S复合体(5μM)混合；从体系3中分出来三份各24μL的混合溶液(3.A、3.B、3.C)，其 中：
体系3.A：往体系3.A中加入6μL水，37℃反应120分钟；
体系3.B：往体系3.B中加入3μL T4多聚核苷酸激酶缓冲液(10×)、1μL水，37℃反应60分钟后加入1μL phi29 DNA聚合酶(10U/μL)、1μL T4多聚核苷酸激酶(10U/μL)，充分混匀后37℃再反应60分钟；
体系3.C：往体系3.C中加入3μL T4多聚核苷酸激酶缓冲液(10×)、1μL水、1μL phi29 DNA聚合酶(10U/μL)、1μL T4多聚核苷酸激酶(10U/μL)，37℃反应120分钟。
(5)NickRCA与D&S复合体链置换反应(两步法不灭活酶)
体系4：将上述体系2取出的15μL混合溶液加入6μL的MgSO4(50mM)混合，加入3μL的D&S复合体(5μM)，37℃反应60分钟后加入1μL水、3μL T4多聚核苷酸激酶缓冲液(10×)、1μL T4多聚核苷酸激酶(10U/μL)充分混合，37℃反应60分钟。
(6)NickRCA与D&S复合体链置换反应(一步法)
体系5：将3μL连接酶反应产物(5nM)、7.5μL聚合酶缓冲液(10×)、7.5μL牛血清白蛋白(2mg/mL)、5μL脱氧核苷酸dNTP(10mM)、27μL水，5μL phi29 DNA聚合酶(10U/μL)进行混合，加入5μL AluI限制性内切酶(10U/μL)，15μL保护链(20μM)、9.5μL的MgSO4(50mM)、7.5μL的D&S复合体(5μM)充分混合。取出26μL加入3μL T4多聚核苷酸激酶缓冲液(10×)、1μL T4多聚核苷酸激酶(10U/μL)充分混合，37℃反应120分钟。
(7)电泳验证：琼脂糖凝胶电泳(2.5％，1×TBE buffer)，取15μL待分析的样品与1μL SYBR Gold和3μL上样缓冲液混合后，室温下进行凝胶电泳(100V，45分钟)并拍摄图像。所述待分析的样品包括：
(a)Maker，置于泳道M；
(b)泳道1：1μM S链；
(c)泳道2：1μM D链；
(d)泳道3：1μM上述D&S复合体；
(e)泳道4：200pM靶基因引发的体系1产物。
(f)泳道5：200pM靶基因引发的体系3.A产物；
(g)泳道6：200pM靶基因引发的体系3.B产物；
(h)泳道7：200pM靶基因引发的体系3.C产物；
(i)泳道8：200pM靶基因引发的体系4产物。
(j)泳道9：200pM靶基因引发的体系5产物。
电泳验证结果见图3。分析可得：泳道3中产物为泳道1和泳道2的3：2结合产物；泳道4中出现涂抹状产物，证明NickRCA反应成功进行；泳道5为NickRCA反应产物与D&S复合体结合产物，与泳道4中产物条带相比，该泳道中对应位置有产物消失(泳道4中a位置)，且出现新条带(b位置)，因此证明D&S复合体与NickRCA产物结合；泳道6中在泳道5的基础上加入phi29 DNA聚合酶和T4多聚核苷酸激酶，可在切断S链，校正后进行延伸，泳道5中对应条带在泳道6中分成两条(c位置)，分别是链置换产物D+D’与NickRCA产物与D&S复合体的结合产物，证明DNAzyme正常工作，链置换反应发生；泳道7同泳道6；泳道8为两步法酶不灭活，NickRCA中的限制性内切酶一直在工作，因此洞口中超长链产物较少，其次泳道中出现D+D’条带，证明DNAzyme正常工作，链置换反应发生；泳道9为一步反应，出现D+D’，证明DNAzyme正常工作，链置换反应发生。
实施例3MNP组装体(MD&S)制备及表征
在本实施例中，选用磁性纳米颗粒(BNF-Starch-1mL(Micromod，10-19-102))作为纳米标记物；利用核酸序列作为MNP的支架，通过核酸序列形成MNP组装体。如前所述，DNA纳米机器中的D链与S链在其3’端各修饰有生物素，从而将MNP分别修饰在D链与S链上，通过两条链的碱基互补配对形成D&S复合体，进一步形成MNP组装体。按照MNP产品说明书中计算，以链霉亲和素载量0.2倍即每个MNP上修饰40条核酸链，对MNP进行修饰。
3.1MD&S的制备
具体实验步骤如下：
(1)体系一：取20μL的MNP(10mg/mL)，磁分离一次洗脱体系游离链霉亲和素；
(2)体系二：MNP与D链结合(MD)。18μL D链(0.2μM)、22μL Tris-Hcl缓冲液(50mM，PH＝8.0)混合，金属浴锅60℃加热10分钟打开DNA链二级结构，迅速降温5分钟。加入10uL体系一溶液，37℃反应30分钟，MNP上的链霉亲和素与D链上修饰的生物素特异性结合，最后补充Tris-Hcl缓冲液至100μL，得MD体系溶液。
(3)体系三：MNP与S链结合(MS)。18μL S链(0.2μM)、22μL水混合，金属浴锅60℃加热10分钟打开DNA链二级结构，迅速降温5分钟。加入10μL体系一容溶液，37℃反应30分钟，MNP上的链霉亲和素与S链上修饰的生物素特异性结合，最后补充Tris-Hcl缓冲液至100μL，得MS体系溶液。
(3)将MD体系溶液与MS体系溶液混合，在-20℃冰冻60分钟、室温下(25℃)化冻10分钟，然后25℃旋转磁孵育60分钟，使MD与MS充分结合，形成MNP组装体MD&S，于4℃保存48小时后可以使用。
3.2MD&S的表征
使用动态光散射(Dynamic light scattering，DLS)技术与原子力显微镜(Atomic Force Microscope，AFM)进行验证。
(1)DLS验证
使用BeNano 90纳米粒度分析仪(Bettersize Instruments，丹东，中国)分别验证反应前后MNP组装体流体力学体积，包括MD、3.6pM靶基因引发扩增反应解结构后体系内MNP以及MD&S，相关参数：激光器光源波长671nm，检测角度为90°，检测温度：25℃。
检测结果如图4所示：形成的MD&S有小部分未聚集，聚集部分流体力学体积增大，约为1000nm；3.6pM靶标引发扩增反应解结构后MNP流体力学体积减小，约为254nm。
(2)原子力显微镜验证
检测仪器：Asylum Research MFP-3D BIO生物型原子力显微镜(Oxford Instruments plc,A-bingdon)；参数：探头：AC160TSR3硅探头，铝反射涂层，针尖半径：7nm，扫描面积为5×5μm2(分辨率为256×256)，扫描速度约为1Hz。AFM图像处理使用Gwyddion 2.43软件。
如图5所示：分别是(a)MD、(b)MD&S、(c)扩增反应解结构后体系内的游离磁珠，由图可见，(b)MNP大量聚集，MD&S组装体形成；(c)3.6pM靶标引发扩增反应将MD&S组装体结构打开。
实施例4光磁终点相差检测验证
如前所述，以筛选出的疟疾保守区作为靶基因，进行滚环扩增后与如上述实施例3中预制好的MD&S组装体进行反应，使用光磁终点相差传感器验证分子扩增体系的灵敏度以及对扩增产物进行定性定量分析。具体实验步骤如下：
(1)连接酶反应(20nM)：将10μL连接酶缓冲液(10×)、2μL挂锁探针(1μM)、6μL靶基因(1μM)、67μL水、10μL牛血清白蛋白(2mg/mL)与5μL Ampligase DNA连接酶(5U/μL)混合，50℃反应15分钟。
(2)如前述3.1步骤一致，制备MD&S组装体。
(3)扩增反应及MD&S解结构：30μL连接酶反应产物(0.5fM-1000fM)、6μL聚合酶缓冲液(10×)、6μL牛血清白蛋白(2mg/mL)、3μL脱氧核苷酸dNTP(10mM)、3μL的MgSO4(100mM)、3μL的MD&S组装体(5μM)、1μL phi29 DNA聚合酶(10U/μL)、1μL AluI限制性内切酶(10U/μL)、3μL T4多聚核苷酸激酶缓冲液(10×)、1μL T4多聚核苷酸激酶(10U/μL)、6μL保护链(20μM)充分混合，37℃旋转磁孵育75分钟。
(4)靶基因检测分析：使用光磁终点相差传感器验证分子扩增体系的灵敏度，其中光磁检测参数：1mT磁场，450nm波长光源，室温下检测，结果如图6所示，该检测涉及10组样品，包括空白对照组、浓度为0.5fM-1000fM的不同靶基因组(分别是0.5fM、1fM、3.2fM、10fM、32fM、50fM、100fM、320fM、1000fM)，10组样品仅连接酶反 应产物浓度差别，光磁信号相对相位差选取0.5Hz-10.7Hz相位信号的平均与空白的差值。该分子扩增体系灵敏度可达fM级别。
在上述实施例中，扩增产物的定性验证通过电泳验证；定量验证通过计算0.5fM-1000fM浓度的靶标引发的MD&S解结构后因流体力学体积导致的光磁检测相位(0.5Hz-10.7Hz)的相位平均值进行定量。
进一步说明，上述实施例中涉及到的酶、探针、引物、保护链的浓度均为过量使用以保证最大反应效率，若浓度较低会降低反应效率。各步骤反应时间为获得最佳信号的最短时间，由反应效率决定，若反应效率较低，一般可通过延长反应时间达到同样反应结果。各步骤反应温度取决于酶的最适工作温度，一般为37℃。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，但只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。以上所述实施例仅表达了本发明的几种实施方式，但并不能理解为对本发明专利范围的限制，本发明的范围由所附权利要求而不是上述说明限定，它完全可以被适用于各种适合本发明的领域，对于熟悉本领域的技术人员而言，根据本专利文件公开的内容很容易地实现其他实施和修改，因此凡未脱离本发明所为的等效实施与改变，落在权利要求的等同要件的含义和范围内的所有变化，均落入本发明的保护范围内。

Claims (10)

  1. 一种基于DNA纳米机器的核酸检测方法，其特征在于，包括以下步骤：
    S1，筛选待测靶基因；
    S2，根据所述待测靶基因序列设计挂锁探针和DNAzyme链，并对应设计引物、保护链与底物链；其中，所述挂锁探针具有磷酸化的5'末端，且设有靶基因识别区、引物识别区和内切酶识别区，所述靶基因识别区设于挂锁探针两端，与待测靶基因序列互补，所述内切酶识别区的内切酶切割位点甲基化；所述保护链与挂锁探针部分区域的序列相同，且具有化学封闭的3'末端；所述DNAzyme链与带有单RNA位点的底物链互补配对，DNAzyme链部分区域序列与挂锁探针中靶基因识别区序列相同；所述DNAzyme链与底物链具有生物素化的3'末端；
    S3，将所述待测品溶液与挂锁探针、引物、DNA连接酶混合进行连接酶反应，得连接酶反应产物；
    S4，制备纳米复合体体系溶液；所述纳米复合体由DNAzyme链、底物链、纳米标记物结合生成；
    S5，取所述连接酶反应产物、纳米复合体体系溶液，加入金属离子溶液、DNA聚合酶、内切酶、T4多聚核苷酸激酶和保护链，混合，进行切刻增强的滚环扩增反应、DNAzyme内切酶反应、链置换反应，得到终反应体系；
    S6，对终反应体系使用传感器检测，实现对待测靶基因的定性分析和/或定量分析。
  2. 根据权利要求1所述的检测方法，其特征在于，步骤S4中，所述纳米复合体中DNAzyme链与底物链的摩尔比为(1.5～1):1，优选为1:1。
  3. 根据权利要求1所述的检测方法，其特征在于，步骤S4中，所述纳米标记物包括磁纳米颗粒、纳米金、荧光基团中的一种或几种。
  4. 根据权利要求1-3任一项所述的检测方法，其特征在于，步骤S6中，所述传感器包括光磁终点相差检测系统、电子顺磁共振波谱仪、紫外-可见分光光度计、荧光显微镜中的一种或几种。
  5. 一种试剂盒，其特征在于，包括反应液a、反应液b、反应液c；
    其中，所述反应液a包括挂锁探针、引物、DNA连接酶；所述反应液b包括由DNAzyme链、底物链、纳米标记物结合形成的纳米复合体；所述反应液c包括T4多聚核苷酸激酶、DNA聚合酶、内切酶、保护链与金属离子溶液；
    根据待测靶基因序列设计挂锁探针和DNAzyme链，并对应设计引物、保护链与底物链；所述挂锁探针具有磷酸化的5'末端，且设有引物识别区、内切酶识别区，以及在两端设有与待测靶基因序列互补的靶基因识别区，所述内切酶识别区的内切酶切割位点甲基化；所述保护链与挂锁探针部分区域的序列相同，且具有化学封闭的3'末端；
    所述DNAzyme链与带有单RNA位点的底物链互补配对，DNAzyme链部分区域序列与挂锁探针中靶基因识别区序列相同；所述DNAzyme链与底物链具有生物素化的3'末端。
  6. 根据权利要求5所述的试剂盒，其特征在于，所述挂锁探针的序列如SEQ ID NO:1所示，所述引物的序列如SEQ ID NO:3所示，所述保护链的序列如SEQ ID NO:4所示，所述DNAzyme链的序列如SEQ ID NO:5所示，所述底物链的序列如SEQ ID NO:6所示，所述试剂盒用于检测疟原虫。
  7. 一种生物传感器，其特征在于，包括如权利要求5所述的试剂盒。
  8. 一种检测疟原虫的生物传感器，其特征在于，包括如权利要求6所述的试剂盒。
  9. 一种利用如权利要求6所述的试剂盒或如权利要求8所述的生物传感器检测疟原虫的方法，其特征在于，包括以下步骤：
    S1，将含有靶基因的待测品溶液与反应液a混合进行连接酶反应，得连接酶反应产物；
    S2，取所述连接酶反应产物与所述反应液b、反应液c混合，进行切刻增强的滚环扩增反应、DNAzyme内切酶反应、链置换反应，得到终反应体系；
    S3，对终反应体系使用传感器检测，实现对待测靶基因的定性分析和/或定量分析；
    其中，所述靶基因的序列如SEQ ID NO:2所示。
  10. 根据权利要求9所述的方法，其特征在于，所述纳米标记物为磁纳米颗粒；所述传感器为光磁终点相差传感器。
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