Multi-foot DNA walker based on framework DNA functional material as well as detection method and application of multi-foot DNA walker

Abstract

The invention discloses a multi-legged DNA walker based on a framework DNA functional material as well as a detection method and application of the multi-legged DNA walker. According to the multi-legged DNA walker constructed based on the framework DNA functional material, the detection sensitivity of a VP sensing method is improved, the dynamic efficiency of the DNA walker is improved through a specific DNA tetrahedral structure, fluorescence signal output is achieved in combination with a G quadruplex / NMM system, and a biological signal of VP is converted into a detectable fluorescence signal; meanwhile, a fluorescence sensing detection method based on a multi-legged DNA walker is established, rapid, sensitive, high-specificity and enzyme entropy driving-free detection of VP is achieved, a good linear relation is shown in the range of 102-107 CFU / mL, the detection limit is as low as 23 CFU / mL, good sensitivity and specificity are achieved, and the method can be used for detection in different sample environments and has good application prospects. And more new products and methods are provided for trace or trace detection and analysis of VP.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biological detection, and more specifically, relates to a multi-legged DNA walker based on framework DNA functional materials and a detection method and application thereof. Background Art

[0002] Vibrio parahemolyticus (VP) is a halophilic bacterium and a foodborne pathogen. It mainly comes from shrimps and crabs in seafood. It is easy to cause food poisoning after consumption. Therefore, the detection of VP in seafood is particularly important. However, due to the presence of a large number of interfering components such as miscellaneous bacteria and environmental eDNA in the matrix of VP detection (such as marine ranch aquaculture water or infected aquatic animals), the traditional VP analysis method has poor detection specificity and there are false positives or false negatives. Therefore, constructing a new sensing method with high sensitivity, good specificity and convenient operation is one of the important ways to achieve effective detection of VP.

[0003] The classic DNA walker functional material consists of three parts: the walking track (Track), the DNA walking chain (Walker) that can bind to the track, and the fuel chain (Fuel) that drives it off the track. Driven by the Fuel, the Walker converts chemical energy into kinetic energy, prompting the Walker to move autonomously and asymptotically along the preset Track to achieve signal cascade amplification. The walking efficiency of the DNA walker is the key to determining the amplification performance of the DNA walker. In order to obtain better kinetic efficiency, DNA walkers have evolved from the initial one-dimensional (1D) DNA walker to the two-dimensional planar (2D) DNA walker and the current three-dimensional (3D) DNA walker. Among them, the 3D DNA walker with a fixed Walker and multiple Tracks modified on the same nanoparticle has attracted much attention because it increases the local concentration of the Track in the solution and reduces the risk of the Walker leaving the Track, achieving controllable migration of DNA and significantly improving the walking efficiency. However, the limited length of the fixed Walker, the slow action of the single-legged DNA Walker, and the poor reaction kinetics affect the further development of the walking efficiency of the 3D DNA walker.

[0004] The framework DNA functional materials assembled based on structural DNA nanotechnology provide new possibilities and directions for the development of 3D DNA walkers. However, 3D DNA walkers are rarely used for VP detection. In order to overcome the poor specificity of existing VP detection methods and the problems of false positives or false negatives, it is necessary to provide more effective methods and means to achieve rapid, sensitive, specific, and enzyme-free detection of VP, and provide new methods for micro or trace analysis of VP. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing VP detection method and to provide a multi-legged DNA walker based on framework DNA functional materials and a detection method and application thereof.

[0006] The purpose of the present invention is to provide a multi-legged DNA walker based on framework DNA functional materials.

[0007] Another object of the present invention is to provide a method for preparing a multi-legged DNA walker.

[0008] Another object of the present invention is to provide applications of a multi-legged DNA walker.

[0009] Another object of the present invention is to provide a biosensor product.

[0010] Another object of the present invention is to provide a fluorescence sensing detection method based on a multi-legged DNA walker.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention provides a multi-legged DNA walker based on a framework DNA functional material, comprising a tetrahedral framework DNA functional material, a nano-based DNA functional material, a fuel chain GF, and a fluorescent material; the tetrahedral framework DNA functional material contains a Vibrio parahaemolyticus aptamer and four scaffold chains; the nano-based DNA functional material is a track chain modified with nanoparticles; the sequences of the Vibrio parahaemolyticus aptamer, the four scaffold chains, the track chain, and the fuel chain GF are shown in SEQ ID NO.1-7 in sequence.

[0013] The multi-legged DNA walker provided by the present invention is designed based on the specific affinity between the aptamer and the target and the principle of complementary base pairing to obtain a tetrahedral framework DNA functional material with a free short nucleotide sequence at the vertex, comprising a Vibrio parahaemolyticus aptamer and four scaffold chains (including Walker) to form a closed DNA tetrahedron structure (TDNA). In the presence of the target Vibrio parahaemolyticus, the aptamer is detached from the TDNA and the DNA walker is triggered. Through the use of the nano-based DNA functional material, the Walker is driven by the fuel chain to walk on the track chain, and then the signal triggering and migration amplification of the multi-legged DNA walker and the assembly signal output of the G quadruplex are used to achieve rapid, sensitive and specific detection of the target VP.

[0014] In addition, the multi-legged DNA walker provided by the present invention is not limited by fixed sites, and can also reduce the occurrence of derailment events of Walker / Track during the replacement process with Fuel, effectively improving the power of the 3D DNA walker. By utilizing the structural characteristics of DNA tetrahedron design controllable, simple manufacturing, and predictable spatial conformation, combined with the advantages of strong load capacity and wider moving space of the 3D DNA walker's continuous and controllable movement on the 3D surface, an entropy-driven multi-legged DNA walker is constructed, without the need for enzyme assistance, using the Walker released by the dissociation of the reaction trigger element to activate the DNA hybridization-dissociation reaction, so that it moves autonomously along the predetermined track, realizing the controllable migration of DNA, and converting and amplifying the ability to perceive low-level targets. Finally, G quadruplex DNA functional materials and fluorescent materials are used as signal output modules to generate strong fluorescence, converting the biological signals of VP into detectable fluorescent signals, providing a basis for the final construction of a paper-based POCT platform for on-site detection of VP marine ranches.

[0015] Furthermore, the 5' end of the track chain is modified with a dithiol group.

[0016] Preferably, the nanoparticles are gold nanoparticles (AuNPs).

[0017] Preferably, the fluorescent material mainly reacts with the G quadruplex to generate a fluorescence enhancement signal, and thus other specific fluorescent materials in the art may be used, such as N-methylmorpholine (NMM) or 3,3′,5,5′-tetramethylbenzidine (TMB).

[0018] More preferably, the fluorescent material is N-methylmorpholine (NMM).

[0019] When the multi-legged DNA walker provided by the present invention is used, when VP exists in the sample, the aptamer can specifically recognize and bind to VP, promote the transformation of TDNA to TDN, and release free Walker. Subsequently, based on intramolecular interaction, the free Walker on TDN acts as a trigger element, and hybridizes with the hairpin Track on AT to achieve the initial assembly of the DNA walker (formation of Walker / Track complex). The fuel chain (GF) is used to replace the Walker on the Track by the GF through the chain displacement mediated by Toehold. The Walker is freed again and binds to other Tracks on AuNPs to achieve the controllable movement of the DNA Walker on AuNPs and generate the Track / GF structure. The formation of the Track / GF structure pair releases the previously closed G quadruplex structure, self-assembles to form a G quadruplex DNA functional material, specifically binds to the free fluorescent material in the solution, induces the fluorescence enhancement of the fluorescent material, realizes the fluorescence opening, and converts the biological signal of VP into a fluorescent signal for output. When VP is absent, the blocking effect of the aptamer on TDNA cannot be relieved, DNAWalker cannot be activated, the hairpin AT and hairpin GF structures remain stable and cannot bind to each other, and the free NMM fluorescence signal is weak or no fluorescence signal is generated.

[0020] The present invention provides a method for preparing a multi-legged DNA walker, comprising the following steps:

[0021] S1. Preparation of nano-based DNA functional materials using nanoparticles and track chains;

[0022] S2. Preparation of tetrahedral framework DNA functional materials: adding the Vibrio parahaemolyticus aptamer and four scaffold chains into a buffer solution and mixing them, and obtaining a TDNA structural material with target-specific activation characteristics by a rapid annealing method;

[0023] S3. Mix the nano-based DNA functional material of S1 and the tetrahedral framework DNA functional material of S2 with the fuel chain GF and the fluorescent material, and react in the dark to obtain a multi-legged DNA walker.

[0024] Preferably, the molar ratio of the nanomaterial to the track chain in step S1 is 1:30-50; and the molar ratio of the Vibrio parahaemolyticus aptamer to the four scaffold chains in step S2 is 4:1-2.

[0025] More preferably, the molar ratio of the nanomaterial to the track chain is 1:40; the molar ratio of the Vibrio parahaemolyticus aptamer to the four scaffold chains is 4:1, wherein one scaffold chain corresponds to one aptamer, and the molar ratio of the aptamer to one scaffold chain is 1:1.

[0026] Preferably, in step S3, the molar ratio of the nano-based DNA functional material, the tetrahedral framework DNA functional material, the fuel chain GF and the fluorescent material is 20-30:1-2:90-110:5000-6000.

[0027] More preferably, the molar ratio of the nano-based DNA functional material, the tetrahedral framework DNA functional material, the fuel chain GF and the fluorescent material is 30:1:100:6000.

[0028] Preferably, the reaction time in step S3 is 1 to 2 hours.

[0029] More preferably, the fluorescent material is NMM with a concentration of 6 to 108 μmol / L; and the concentration of the track chain AT is 1 to 2 nmol / L.

[0030] More preferably, the concentration of NMM is 8 μmol / L; the concentration of AT is 1.5 nmol / L.

[0031] In particular, during the detection reaction, the cation concentration (K + ), stabilize the structure of the G quadruplex, further enhance the fluorescence performance after the G quadruplex / NMM combination, and preferably regulate the cationic K in the reaction system + The concentration is 20~40mmol / L.

[0032] The present invention provides the use of a multi-legged DNA walker in detecting Vibrio parahaemolyticus for non-disease diagnosis and treatment purposes, or in preparing a Vibrio parahaemolyticus detection product.

[0033] The present invention provides a biosensor product, comprising the multi-legged DNA walker.

[0034] At the same time, the present invention uses a multi-legged DNA walker to perform biosensor detection on VP, showing that the VP concentration is 10 2 ~10 7 The method has good detection performance in the range of CFU / mL, with an LOD of 23CFU / mL. The spike recovery experiment of VP proves that the method has practical application value, providing a new, highly sensitive, specific, enzyme-free, fast and accurate detection method for the detection of low-concentration VP.

[0035] The present invention also provides a fluorescence sensing detection method based on a multi-legged DNA walker, and uses the multi-legged DNA walker based on a framework DNA functional material to detect Vibrio parahaemolyticus.

[0036] The present invention has the following beneficial effects:

[0037] The present invention is based on DNA nanotechnology, designs and prepares a variety of DNA functional materials, utilizes the specific recognition of VP targets by aptamers, and constructs a multi-legged DNA walker that can detect VP quickly, sensitively and specifically through the signal triggering and migration amplification of the multi-legged DNA walker and the assembly signal output of the G quadruplex, thereby realizing the detection of the target VP. When the target VP is not present, the migration of the multi-legged DNA walker cannot be triggered, the fuel GF still maintains a stable hairpin structure, and no detection signal is generated. When the target VP is present, the multi-legged DNA walker triggers migration, the GF hairpin opens, and the G quadruplex specifically binds to the NMM to achieve the enhancement of the fluorescence signal. At the same time, a method for fluorescent sensing detection of VP based on a multi-legged DNA walker is provided, and the VP concentration is 10 2 ~10 7 The method has good detection performance in the range of CFU / mL, with an LOD of 23CFU / mL. The spike recovery experiment of VP proves that the method has practical application value, providing a new, highly sensitive, specific, enzyme-free, fast and accurate detection method for the detection of low-concentration VP. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the principle of the multi-legged DNA walker sensing method based on framework DNA functional materials.

[0039] Figure 2 (A) UV characterization before and after AuNPs modification (B) Zeta potential characterization before and after AuNPs modification (C) Hydrodynamic diameter characterization before AuNPs modification (D) Hydrodynamic diameter characterization after AuNPs modification (E) DNA tetrahedral structure characterization (Lane 1: TDN; Lane 2: TDNA; Lane 3: W1; Lane 4: W2; Lane 5: W3; Lane 6: W4).

[0040] Figure 3 For feasibility analysis, (A) fluorescence spectrum changes of the detection method under different conditions and (B) PAGE characterization results (in the figure (a) VP+TDNA+AT+GF+NMM (b) TDNA+AT+GF+NMM (c) NMM; lane 1: VP+TDNA; lane 2: VP+TDNA+AT; lane 3: TDNA+AT; lane 4: AT+GF; lane 5: TDNA+AT+GF; lane 6: A(unmodified)+GF; lane 7: Walker+A(unmodified)+GF).

[0041] Figure 4 This is a diagram showing the results of thermodynamic data for simulating strand displacement of a DNA walker.

[0042] Figure 5This is a graph of the test results for condition optimization analysis (A reaction time, B reaction system, C fluorescent material concentration, D orbital chain concentration).

[0043] Figure 6 Sensitivity analysis of the detection method based on multi-legged DNA walkers (A) Changes in fluorescence intensity of the multi-legged walker detection method under different VP concentrations (B) Linear relationship between fluorescence intensity at 617nm and VP concentration.

[0044] Figure 7 This is a graph showing the results of specificity analysis.

[0045] Figure 8 Detection and analysis of VP infected samples (A) Changes in fluorescence intensity of colonized samples (B) Results of plate culture of colonized samples. DETAILED DESCRIPTION

[0046] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0047] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0048] Example 1 DNA sequence design of DNA biosensor

[0049] The present invention is based on DNA nanotechnology and takes Vibrio parahaemolyticus as the detection target. A variety of DNA biosensor functional materials are designed and prepared: Vibrio parahaemolyticus aptamers, tetrahedral framework DNA functional materials, AuNPs-based DNA functional materials, and fuel chain GF for specific recognition. The designed sequences are sequenced and analyzed using NUPACK software to analyze the complementary pairing relationship between Walker, Track and GF and these DNA chains, and sequences with higher base hybridization pairing stability are selected. The specific design method is as follows.

[0050] 1. Selection of aptamers for Vibrio parahaemolyticus (VP)

[0051] Aptamer (Aptamer, A) is a short single-stranded nucleic acid sequence obtained by exponential enrichment ligand system evolution technology (Systematic Evolution of Ligands by Exponential Enrichment, SELEX), which can form a high-order structure through three-dimensional interactions (hydrophobic interaction, van der Waals force, electrostatic interaction, etc.) to specifically bind to the target, forming an aptamer-target complex, which has high specificity and affinity for the target. The existing aptamer sequence that has been verified to have specific recognition and binding to Vibrio parahaemolyticus is used as the recognition element of the constructed sensing method, and its sequence is: 5'-TCTAAAAATGGGCAAAGAAACAGTGACTCGTTGAGATACT-3'. Complementary sequences are further designed based on this target sequence.

[0052] 2. Design of framework DNA functional materials (DNA tetrahedron structure, TDN)

[0053] The framework DNA functional material consists of four complementary single strands (W1, W2, W3, W4) and an aptamer sequence, wherein the four complementary single strands all contain ①Walker sequence + ②scaffold sequence as a tetrahedral framework, and one end of the four scaffold sequences carries the same Walker sequence. The design and selection of the specific sequences are as follows:

[0054] (1) Walker sequence

[0055] a. Number of bases: When the number of bases involved in hybridization is greater than 12, the stability of hybridization can be guaranteed. In order to ensure the stable binding of the aptamer sequence and the Walker sequence and to smoothly dissociate the double helix structure in the presence of the target, 20 complementary bases of the aptamer sequence are selected as the Walker sequence according to the aptamer sequence to ensure the stable hybridization of the Walker and the aptamer.

[0056] b. Sequence composition: Based on the principle of base complementary pairing, the sequence can be combined with the target aptamer sequence (5'-TCTAAA AATG GGCAAAGAAACAGTGA CTCGTTGAGATACT-3') underlined part specifically binds to achieve the formation of a partial double helix structure.

[0057] (2) Scaffold sequence

[0058] a. Number of bases: When the number of bases involved in hybridization is greater than 12, the stability of hybridization can be guaranteed. Based on the stability of the triangular structure, a triangular pyramid tetrahedron structure is selected as the framework DNA structure, and 16 base sequences are selected as the edges of the tetrahedron structure. The base complementarity of the middle three edges of the four scaffold sequences is used to realize the construction of the tetrahedron structure, thereby ensuring the stable formation of the framework DNA functional nucleic acid.

[0059] b. Sequence composition: Based on the principle of complementary base pairing, the sequence can be partially complementary and hybridized with the sequence in different scaffold sequences. The sequence in W1 (5'-AGTAAGCATTCAGACTT-3') can be complementary paired with the sequence in W4 (5'-AAGTCTGAATGCTTACT-3'), the sequence in W1 (5'-ACCTGTGAGTTACGATC-3') can be complementary paired with the sequence in W2 (5'-GATCGTAACTCACAGGT-3'), the sequence in W1 (5'-CTTGTCGCTAAGACGAG-3') can be complementary paired with the sequence in W3 (5'-CTCGTCTTAGCGA The sequence in W2 (5'-TGACGTGTACACTGATA-3') can be complementary to the sequence in W3 (5'-TATCAGTGTACACGTCA-3'), the sequence in W2 (5'-AGTCGATCTGGACAGCA-3') can be complementary to the sequence in W4 (5'-TGCTGTCCAGATCGACT-3'); the sequence in W3 (5'-AGTACCTCGTCCATGTC-3') can be complementary to the sequence in W4 (5'-GACATGGACGAGGTACT-3'), the specific sequences and their pairings are as follows:

[0060]

[0061] In the above sequence structure, through the principle of complementary base pairing, these DNA chains can be complementary to each other to form a tetrahedral structure (TDN) with a free Walker, and the partial complementarity between the Walker sequence and the aptamer sequence is used to form a closed DNA tetrahedral structure (TDNA), which can prevent the nonspecific intermolecular interaction between TDN and AT and avoid the triggering of the DNA walker in the absence of a target. In the presence of the target Vibrio parahaemolyticus, the aptamer detaches from the TDNA and the DNA walker is triggered.

[0062] 3. Design of AuNPs-based DNA functional materials (AT)

[0063] AT is composed of a track chain sequence (Track) in a DNA walker modified on gold nanoparticles. Its Track sequence is mainly composed of ① dithiol modification, ② isolation sequence, ③ complementary sequence and ④ recognition start sequence, among which ③ complementary sequence and ④ recognition start sequence constitute ⑤ migration start sequence. The design and selection of specific sequences are as follows:

[0064] (1) Isolation sequence

[0065] a. Number of bases: After the oligonucleotide chain is modified on the gold nanoparticles, a steric hindrance effect will occur, which will affect the hybridization efficiency and thus the efficiency of the experimental method. In order to reduce the steric hindrance effect and obtain the optimal binding efficiency, 12 bases are selected as the isolation sequence to ensure the smooth occurrence of the reaction.

[0066] b. Sequence composition: To reduce the effect of the isolation sequence on the reaction, a sequence that is completely non-complementary to the reaction sequence (5'-TTT TTT TTACGA-3') was selected to ensure that it is in a single-stranded form.

[0067] (2) Complementary sequence

[0068] a. Number of bases: If the complementary sequence is too short, it cannot play a protective role and Track can be opened when the target is not present. If it is too long, the hairpin structure may be too stable and the free Walker cannot open the hairpin Track, thereby failing to trigger the migration of the multi-legged DNA walker. According to the recognition start sequence, 15 bases can protect and ensure that the Track hairpin structure exists stably in the absence of a target and can be opened by the free Walker when the target is present.

[0069] b. Sequence composition: Based on the principle of base complementary pairing, it can be combined with the sequence (5'-TTT TTT TTACGACTC TCACTGTTT CTT GAATGG GCA AAG AAACAG TGA GAG -3') to form a hairpin structure of Track, allowing it to exist stably in a free state.

[0070] (3) Identify the startup sequence

[0071] a. Number of bases: When the number of bases involved in hybridization is greater than 12, the stability of hybridization can be guaranteed. In order to trigger smoothly and open the Track hairpin structure, 20 bases are selected as the DNA walker recognition start sequence according to the free Walker sequence to ensure stable hybridization between the recognition start sequence and Walker and the opening of the hairpin Track.

[0072] b. Sequence composition: Based on the principle of complementary base pairing, the sequence can bind completely and specifically to the free Walker sequence (5'-TCACTGTTTCTTTGCCCATT-3'), thereby opening the Track hairpin structure and releasing the migration initiation sequence.

[0073] (4) Migration startup sequence

[0074] a. Number of bases: When the number of bases involved in hybridization is greater than 12, the stability of hybridization can be guaranteed. When the base binding force between Track and fuel chain GF is greater than that between Track and Walker, and there is a Toehold sequence, it can induce the dissociation of the Track / Walker double helix structure and the formation of the Track / GF double helix structure. Therefore, 32 bases are selected as the migration initiation sequence. When part of its sequence is free, it can act as a Toehold to induce the formation of the Track / GF double helix structure and re-release the free Walker, ensuring the stable hybridization binding between Track and GF, as well as the release and recycling of Walker, to achieve the migration movement of Walker.

[0075] b. Sequence composition: Based on the principle of base complementary pairing, the sequence can be combined with the fuel chain GF (5'-GGGTTTTGGGTTTTGGGTTTTGGG CTG TTT CTT TGC CCATTC AAG AAA CAG TGA GA The underlined part in the sequence AACA GCCCAAAAC C-3') specifically binds to form the Track / GF double helix structure and releases free Walker.

[0076] The specific design of the Track sequence is as follows:

[0077]

[0078] Among them, ① dithiol modification (-NH2), ② isolation sequence (number of bases: 12; sequence composition: 5'-TTT TTTTTACGA-3'), ③ complementary sequence (number of bases: 14; sequence composition: 5'-CTC TCA CTG TTT CTT-3'), ④ recognition start sequence (number of bases: 20; sequence composition: 5'-AA TGG GCAAAG AAA CAG TGA-3') and ⑤ migration start sequence (number of bases: 32; sequence composition: 5'-TC TCA CTG TTT CTT GAA TGG GCA AAG AAA CAG-3').

[0079] In the above sequence structure, the double thiol modification can modify the track chain on the gold nanoparticles by forming a gold-thiol bond between the thiol group and the gold nanoparticles; and by adding the isolation sequence, the influence of steric hindrance on the constructed system is reduced, and the complementary sequence partially binds to the recognition start sequence, so that Track forms a hairpin structure, and then the free Walker binds to the recognition start sequence to open the hairpin structure of Track, release the migration start sequence, promote the subsequent binding of the fuel chain (GF) and Track, and realize the migration walking of the framework DNA functional nucleic acid on the surface of the gold nanoparticles.

[0080] 4. Design of the fuel chain (GF)

[0081] The fuel chain (G4-Fuel) is a hairpin probe, which consists of ①G quadruplex sequence, ②migration sequence and ③complementary sequence. After the formation of G quadruplex, it can bind to porphyrin dye, enhance its fluorescence and realize signal output. The design and selection of specific sequences are as follows:

[0082] (1) G-quadruplex sequence

[0083] a. Number of bases: G-quadruplex is an atypical DNA secondary structure, which is formed by a DNA or RNA sequence rich in guanine (base G). The structural characteristics of the G-quadruplex are determined by two factors: one is that multiple tetrads rich in base G are formed by Hoogsteen hydrogen bonds; the other is that the secondary structure of the DNA sequence is folded through the auxiliary coordination of cations. In order to ensure the self-assembly of the G-quadruplex, 24 bases were selected as the G-quadruplex sequence.

[0084] b. Sequence composition: Since the structure of the G-quadruplex is polymorphic, the sequence (5′-GGG TTT TGG GTT TTG GGT TTT GGG-3′) is mainly used as the G-quadruplex sequence in the present invention.

[0085] (2) Migration sequence

[0086] a. Number of bases: When the number of bases involved in hybridization is greater than 12, the stability of hybridization can be guaranteed, and the base binding force between Track and the fuel chain GF is the largest. Therefore, according to the migration initiation sequence in Track, 32 bases are selected as the sequence, which can protect and ensure that the GF hairpin structure exists stably in the absence of a target and can be opened when the target is present.

[0087] b. Sequence composition: According to the principle of complementary base pairing, this sequence can hybridize and complement with the migration initiation sequence in Track (5'-TC TCA CTG TTT CTT GAA TGG GCA AAG AAA CAG TGA-3'), forming a double-helix DNA region that can be bound by Track / GF, inducing sustainable migration and signal output of the DNA walker.

[0088] (3) Complementary sequence

[0089] a. Number of bases: If the complementary sequence is too short, it cannot play a protective role and the GF can be opened even when the target does not exist. If it is too long, the hairpin structure may be too stable and the Toehold in the free migration initiation sequence in the Track sequence cannot open the hairpin GF, thereby failing to trigger the migration and signal output of the multi-legged DNA walker. Therefore, 16 bases are selected as the complementary sequence. In order to ensure that the G quadruplex can only output signals when the GF hairpin is opened, part of the G quadruplex sequence is selected as the stem of the hairpin structure so that the self-assembly of the G quadruplex can be completed when the hairpin structure is opened.

[0090] b. Sequence composition: Based on the principle of base complementary pairing, the sequence can be combined with the sequence (5'-GGGTTTTGGGTTTTGGGTTTTGGG CTG TTT CTT TGC CCA TTC AAG AAA CAG TGA GAA ACA GCC CAAAAC 3') specifically binds to the underlined part to achieve the formation of the GF hairpin structure, enabling it to exist stably in a free state and preventing the self-assembly of the G quadruplex in a target-free state.

[0091] The specific design of the GF sequence is as follows:

[0092]

[0093] Among them, ①H1 start complementary sequence (number of bases: 21; sequence composition: 5'-TAC GCT TAG GAC CTGGTG TGT-3'), ②pyrimidine-rich sequence (number of bases: 14; sequence composition: 5'-CAC CCA ACA CAC CA-3'), and ③H2 open sequence (number of bases: 21; sequence composition: 5'-CAC CCA ACA CAC CAG GTC CTA-3').

[0094] In the above sequence structure, the complementary sequence partially complementarily binds to the migration sequence and the G-quadruplex sequence, so that the GF forms a hairpin structure. After that, some bases in the migration initiation sequence in Track are released, acting as a toehold to induce the GF to bind to Track / Walker, open the GF hairpin structure, form a Track / GF double helix structure, release Walker, realize the migration of the DNA walker and realize signal output through the self-assembly of the G-quadruplex.

[0095] In summary, the DNA sequence of the DNA biosensor provided by the present invention is shown in Table 1, and the reaction principle involved is as follows Figure 1 As shown, based on the specific affinity between the aptamer and the target, a DNA sequence that is partially complementary to the VP aptamer is selected as the Walker of the DNA walker. The aptamer is used as a blocking sequence. The double helix structure (AW) formed by the binding of the aptamer and the Walker blocks the initiation of the DNA walker. Combined with the sequence design of the DNA tetrahedron, a TDN structure with four free Walkers is designed and assembled as a DNA nanoelement that triggers the initiation of the DNA walker. According to the sequence of the Walker, a Track hairpin that specifically binds to it is designed and modified on AuNPs. The constructed DNA functional material (AT) realizes the effect of Track in increasing the local solution concentration. When the aptamer binds to the free sequence of TDN, the closed DNA tetrahedron structure formed can prevent the nonspecific intermolecular interaction between TDN and AT, avoiding the triggering of the DNA walker in the absence of a target.

[0096] When VP exists, the aptamer can specifically recognize and bind to VP, prompting the transformation of TDNA to TDN and releasing free Walker. Subsequently, based on intramolecular interactions, the free Walker on TDN acts as a trigger element, hybridizing with the hairpin Track on AT to achieve the initial assembly of the DNA walker (formation of Walker / Track complex). The hairpin (GF) with a closed G quadruplex sequence is used as a fuel chain, and the Walker on Track is replaced by GF through Toehold-mediated chain displacement. The Walker is free again and binds to other Tracks on AuNPs, achieving controllable movement of DNA Walker on AuNPs and generating Track / GF structure. The formation of the Track / GF structure pair releases the previously closed G quadruplex structure, self-assembles to form a G quadruplex DNA functional material, and specifically binds to the free NMM in the solution, inducing the fluorescence enhancement of NMM, achieving fluorescence onset, and converting the biological signal of VP into a fluorescent signal for output. When VP is absent, the blocking effect of the aptamer on TDNA cannot be relieved, DNA Walker cannot be initiated, the hairpin AT and hairpin GF structures remain stable and cannot bind on their own, and the free NMM fluorescence signal is weak or no fluorescence signal is generated.

[0097] Table 1 Sequence Listing

[0098]

[0099] Example 2 Preparation of DNA Biosensor

[0100] 1. Preparation of gold nanoparticles (AuNPs)

[0101] AuNPs were prepared by sodium citrate reduction method: First, all glassware required in the preparation process were soaked in aqua regia (HNO3: HCl = 1: 3) to remove excess impurities, washed with ultrapure water after removal, and dried naturally after removing the residual aqua regia. The dissolved HAuCl4·3H2O was placed in a three-necked flask and heated to boil, and sodium citrate solution was added under stirring. The solution was heated and stirred continuously until the color of the solution changed from yellow to wine red, and the mixture was stirred and heated for 15 minutes. The heating was stopped and the stirring state was maintained until the solution naturally cooled to room temperature. The cooled mixture was filtered with a micromembrane with a pore size of 0.22 μm to obtain AuNPs, which were stored at 4°C for later use.

[0102] 2. Preparation of AuNPs-based DNA functional materials (AuNPs-Track, AT)

[0103] First, a thiol-modified DNA of the Track sequence was reduced with TCEP solution at a molar ratio of 1:1666 at room temperature in the dark for 2 hours to reduce the effect of disulfide bonds on the subsequent modification process. The TCEP-treated DNA solution was mixed with AuNPs at a molar ratio of 20:1 and incubated at room temperature. Subsequently, NaCl solution was added to the mixture of DNA and AuNPs four times within 44 hours until the final concentration of NaCl was 0.3 mol / L. Finally, the mixed solution was centrifuged at 12000r / min for 30 minutes, and the supernatant was removed and resuspended in PBS buffer solution. This process was repeated 3 times to remove unattached DNA. The final centrifuged substrate was resuspended in 600μL of PBS solution to complete the preparation of AT, and the obtained AT was stored at 4°C for use.

[0104] 3. Preparation of aptamer-blocked tetrahedral DNA structure (TDNA)

[0105] The framework DNA functional material constructed in Example 1 has a target recognition and activation function. The free Walker of TDN is blocked by an aptamer, and the initial assembly of the DNA walker is achieved through the combination of Walker and Track. Using the rapid annealing method, W1, W2, W3, W4 and the aptamer are added to the TM buffer solution at a molar concentration ratio of 1:1:1:1:4, and the mixed solution is placed in a water bath heating pot and heated to 95°C, kept for 15 minutes, then taken out, cooled to 4°C and kept for 2 hours to obtain a TDNA structure with target-specific activation characteristics, and stored at 4°C for use.

[0106] 4. Multi-legged DNA walker

[0107] The preparation was carried out in a one-step method: AT was placed in a water bath and slowly heated to 95°C before use, and maintained for 15 minutes to ensure the formation of the AT hairpin structure. 30 nmol / L TDNA, 1 nmol / L AT, 100 nmol / L GF and 6 μmol / L N-methylmorpholine (NMM) were added to a PBS buffer solution and mixed thoroughly to obtain a reaction solution.

[0108] Detection method: Add VP to the reaction solution prepared above, keep the total volume at 600 μL, and react at room temperature in the dark for 2 hours. Finally, measure the fluorescence spectrum of the solution at 600-650 nm under an excitation wavelength of 389 nm.

[0109] Example 3 Characterization of DNA functional materials

[0110] The prepared AuNPs-based DNA functional materials and DNA tetrahedron structures were characterized by UV spectrophotometer and polyacrylamide gel electrophoresis (PAGE, 8%), respectively.

[0111] The results are as follows Figure 2 As shown in A, the UV spectrum of AuNPs modified with DNA shows an obvious red shift compared with pure AuNPs, which indicates the successful construction of AuNPs-based DNA functional materials. The red shift is caused by the increase in the particle size of AuNPs due to DNA modification on the surface of AuNPs.

[0112] The AuNPs samples before and after modification were further characterized by hydrodynamic diameter and Zeta, and the changes in particle size and potential of the AuNPs samples before and after modification were analyzed.

[0113] The results are as follows Figure 2 BD, AuNPs prepared by citrate reduction method have citrate ions attached to their surface, which plays a role in stabilizing dispersion, so the pure AuNPs before modification are negatively charged. Since DNA has negative charge, when AuNPs are modified with DNA, their potential will be lower, which is consistent with the Zeta potential characterization results ( Figure 2 B). The hydrodynamic particle size of AuNPs samples before and after modification was characterized by DLS ( Figure 2 CD) shows that the particle size of the modified AuNPs sample increases significantly, which proves the successful preparation of AuNPs-based DNA functional materials.

[0114] Finally, combined with the results of polyacrylamide gel electrophoresis Figure 2 Lanes 1-6 in E all showed clear single bands, indicating the successful synthesis of tetrahedral DNA structure (TDN).

[0115] Example 4 Feasibility Analysis and Optimization

[0116] 1. Influence of fluorescent dyes

[0117] To verify the feasibility of the sensing method for detecting multi-legged DNA walkers, the fluorescence signals generated by the multi-legged DNA walkers were detected under different conditions. The specific conditions are: (a) VP+TDNA+AT+GF+NMM (b) TDNA+AT+GF+NMM (c) NMM.

[0118] The results are as follows Figure 3As shown in A, in condition c, when only NMM dye is present, the fluorescence signal of the solution is very weak. This signal is generated by the weak fluorescence of NMM as a fluorescent dye. In condition b, when there is no target VP in the reaction solution, the fluorescence signal is also relatively weak and does not change significantly. In condition a, when VP is present, the fluorescence signal of the solution changes significantly, which indicates that the addition of VP triggers the signal recognition element, prompting the transformation of TDNA to TDN, releasing free Walker, activating the multi-legged DNA walker, and forming a G quadruplex / NMM complex, which significantly enhances the fluorescence of NMM. The feasibility of the detection method can be verified by the change of the fluorescence signal.

[0119] To further clarify the cause of the fluorescence change, the reactions under different conditions were centrifuged, and the AuNPs and the DNA loaded thereon were deposited at the bottom by centrifugation. The remaining reaction products in the supernatant were taken and verified and analyzed by PAGE.

[0120] Results from Figure 3 As shown in B, two high molecular weight bands with differences can be observed in lane 1, namely TDNA and TDN bands, which indicates that in the presence of VP, the aptamer can specifically recognize and bind to VP, prompting the conversion of TDNA to TDN. The lighter color of the TDN band in lane 2 indicates that TDN will react with Track on AT, and in the absence of GF, it will be fixed on AT and unable to migrate. Under centrifugation, AT will deposit the TDN fixed on it together, so the TDN band becomes lighter. Lanes 3, 4, and 5 all show that in the absence of VP, TDNA and GF do not react with Track on AT. In addition, by comparing lanes 6 and 7, the premise of using A and GF for reaction is that free Walker is required to start. The feasibility of the reaction operation of the detection method can be verified by electrophoresis.

[0121] 2. Analysis of thermodynamic changes

[0122] From the perspective of thermodynamics, the thermodynamic changes during the migration of DNA walkers in the system are analyzed.

[0123] The simulation diagram of the reaction sequence hybridization relationship is as follows Figure 4 As shown, when the reaction system only has GF and AT ( Figure 4 A), the overall pairing probability of the reaction system is low, and GF and AT tend to maintain their own hairpin structure. That is, when no VP is added, the system is in a balanced steady state, and the AT and GF in the system cannot hybridize, presenting a stable single-stranded hairpin structure. When Walker is added, the system reaches a steady state, the pairing probability of the GF hairpin structure decreases, and the probability of base pairing with the AT chain increases ( Figure 4 B).

[0124] At the same time, the equilibrium concentration of the system reaction in this method is analyzed and Gibbs free energy is introduced to calculate the change of the standard free energy of the DNA walker during the reaction. ΔG is used to represent the change of the thermodynamic standard free energy difference of the reaction. The reaction process can be expressed as:

[0125] W+AT+GF=W+W / AT+AT / GF

[0126] W / AT represents the double helix DNA structure formed by the combination of W and AT, which induces the opening of AT's hairpin structure, reduces the probability of AT's base pairing, and prompts the AT free end to open the GF's hairpin structure; AT / GF represents the double helix structure formed by the hybridization of the track chain Track and the driving fuel chain GF, which displaces Walker and drives the multi-legged DNA Walker to migrate. Thus, the calculation formula of ΔG can be obtained:

[0127] ΔG=GW / AT+GW+GAT / GF-GW-GAT-GGF

[0128] The calculated ΔG=-39.98kcal / moL, the more negative the ΔG value is, the easier it is for the reaction self-assembly process to form, proving that the DNA walker has feasible thermodynamic conditions for the migration reaction.

[0129] 3. Condition optimization

[0130] In order to obtain the best detection performance, the reaction time, reaction system, fluorescent material concentration, and track chain concentration, which are factors affecting the walking of the DNA walker, were optimized, and the detection performance under different reaction conditions was measured. The optimization results are presented by the values ​​of two signal output modes, where Change of Fluore scence intensity represents the difference in fluorescence signal at 617nm between the sample detection group with VP added and the blank detection group without VP during the fluorescence detection process (N=3, mean ± SD, *p<0.05 is a significant difference, **p<0.01 is an extremely significant difference, ns is no significant difference).

[0131] The results of the effects of different reaction times on the fluorescence enhancement of G-quadruplex / NMM are shown in Figure 5 As shown in A, the fluorescence change shows a growing trend with the change of time. After 2 hours, the fluorescence change reaches a peak. However, as time continues to increase, the fluorescence change shows a downward trend, which means that when the reaction time is 2 hours, the migration rate of the multi-legged walker just reaches the maximum. After the reaction time exceeds 2 hours, the background interference of the reaction gradually increases, causing the fluorescence change value to show a downward trend. Therefore, 2 hours is selected as the optimal reaction time to achieve the best fluorescence gain.

[0132] The presence of cations is an important condition for the assembly of G quadruplexes. Its presence can stabilize the structure of G quadruplexes. + ) on the fluorescence enhancement performance of G-quadruplex / NMM. Figure 5 As shown in Figure B, when adding 0-40mmol / L KCl, the signal response difference of the solution increases first and then tends to be flat. When the KCl concentration is 30mmol / L, the fluorescence enhancement activity of G quadruplex / NMM reaches the maximum, and there is no obvious difference in the fluorescence change of the reaction under 40mmol / KCl concentration and 30mmol / L KCl concentration. Therefore, 30mmol / L was selected as the optimal reaction concentration of KCl to complete the stable assembly of G quadruplex.

[0133] The results of fluorescent material concentration determination are as follows Figure 5 As shown in Figure C, when the concentration of the added NMM reaches 8μmol / L, the change in fluorescence intensity reaches equilibrium, and the increase in NMM concentration does not further produce greater fluorescence changes. This indicates that when the concentration of NMM is 8μmol / L, the binding of NMM to the G quadruplex in the reaction reaches saturation. Therefore, 8μmol / L was selected as the optimal NMM concentration for the reaction. AT, as the track chain in the multi-legged walker, is the key to the migration of the multi-legged walker. The concentration of AT will affect the migration efficiency of the walker.

[0134] The results of AT concentration optimization are as follows Figure 5 As shown in D, it can be seen that the fluorescence change value of the reaction increases continuously with the increase of AT concentration, reaching the maximum when the AT concentration is 1.5nmol / L, but when the AT concentration is greater than 1.5nmol / L, the fluorescence change value shows a downward trend, which indicates that when the AT concentration is 1.5nmol / L, the migration rate of the multi-legged walker reaches saturation. When the AT concentration is greater than 1.5nmol / L, the blank group without VP triggering serves as background interference, and its fluorescence intensity is enhanced, causing the fluorescence change value to show a downward trend.

[0135] In summary, the optimal system reaction time is 2h, the KCl concentration is 30mmol / L, the NMM concentration is 8μmol / L, and the AT concentration is 1.5nmol / L.

[0136] 4. Sensitivity analysis

[0137] The multi-legged DNA walker is used to detect Vibrio parahaemolyticus. After VP specifically binds to its aptamer, the multi-legged Walker is released to induce the walking of the DNA walker to achieve fluorescence enhancement. Therefore, the concentration of VP is related to the fluorescence enhancement performance. Under the above-mentioned optimal conditions, the VP detection method in Example 2 is used to quantitatively analyze Vibrio parahaemolyticus of different concentrations.

[0138] The results are as follows Figure 6 As shown, in 10 2 ~10 7 Within the concentration range of CFU / mL, the change in fluorescence intensity is proportional to the concentration of VP, and there is a good linear relationship. The linear regression equation is Y=2.741*Log[C]-2.946 (C: VP concentration; Y: fluorescence intensity change, ΔF=F / F0-1, R 2 =0.99), and the detection limit was 23 CFU / mL (RSD=5.6%).

[0139] 5. Specificity analysis

[0140] To further verify the specific selectivity of the detection method, Vibrio alginolyticus (VA) and Vibrio harveyi (VH) were selected as the interference factor group, and the interference samples were detected using the multi-legged DNA walker through the sensing method of Example 2. At the same time, a mixed sample of VP, VA and VH was set up as a control group to compare with the detection results of VP.

[0141] Test results such as Figure 7 As shown in the figure, the fluorescence intensity of the VP sample group is significantly higher than that of the interference factor group, indicating that the constructed sensing method has good selectivity for VP, while in the mixed sample group, the DNA sensing method can still achieve specific selectivity for VP and show strong fluorescence in the presence of VA and VH interference. These results show that the DNA sensing method based on DNA functional materials can effectively identify targets and quantify target signals.

[0142] Example 5 Detection of actual samples

[0143] The shrimp samples colonized with VP were selected to simulate the actual marine ranch test samples. VP was injected into the shrimp through a syringe for colonization, and the colonized shrimp was cultured in simulated seawater for 24 hours. Part of the hepatopancreas of the shrimp infected with VP was selected as the test object, 25g of the sample was taken, and 3% sodium chloride alkaline peptone water was added as a solution to prepare a 1:10 sample homogenate.

[0144] The detection method is divided into two parts: ① Actual sample detection: A multi-legged DNA walker is used to detect a 1:10 sample homogenate according to the detection method in Example 2, and at the same time, the Vibrio parahaemolyticus detection method specified in the national standard GB 4789.7-2013 is used to perform qualitative detection using the plate culture method.

[0145] ② Spike recovery method: Collect seawater samples from Zhanjiang Bay, filter them, and mark them to evaluate the applicability of the constructed sensing method. In addition, take 25g of shrimp hepatopancreas sample without VP colonization and stir it with 225mL sterile PBS solution to form shrimp meat homogenate. Irradiate the seawater sample and the prepared shrimp meat homogenate with ultraviolet light to achieve simple sterilization. Take different concentrations of VP standard solution and add it to a certain volume of seawater sample and shrimp meat sample, and the final concentration is 10 3 -10 4 CFU / mL, prepare the spiked sample for subsequent measurement. Afterwards, remove impurities by using a filter membrane to obtain the VP spiked mixture solution.

[0146] Test results such as Figure 8 As shown, the multi-legged DNA walker showed a significant fluorescence enhancement effect on shrimp samples ( Figure 8 A), indicating that the shrimp sample has been infected with VP, which is consistent with the method of testing Vibrio parahaemolyticus in the national food safety standard. Figure 8 It can be clearly seen in B that blue-green colonies appear in the TCBS medium with screening performance for VP, proving the presence of VP in the sample. Therefore, it is shown that the detection results of the multi-legged DNA walker constructed in the present invention are consistent with the actual sample detection results, showing good VP detection specificity.

[0147] To further verify the sensitivity of this method for VP detection in complex environments, different concentrations (10 3 ~10 4 CFU / mL) of VP samples were added to the sterilized shrimp meat sample homogenate and seawater samples, and the spike recovery method was used for actual detection and quantitative analysis.

[0148] The test results are shown in Table 2. The average recovery rate of the multi-legged DNA walker detection method constructed based on DNA functional materials is between 94% and 98%, and the RSD is between 1.01% and 3.07%. Among them, the average recovery rate of shrimp meat samples is generally lower than that of seawater samples. The reason may be that during the filtration process, some VP is still attached to the shrimp meat homogenate, resulting in a lower overall VP concentration. However, the constructed method can still maintain a high and stable detection performance.

[0149] Table 2 Actual sample test results (n=3, mean±SD)

[0150]

[0151] In summary, the present invention is based on DNA nanotechnology, designs and prepares a variety of DNA functional materials, utilizes the specific recognition of VP targets by aptamers, and constructs a multi-legged DNA walker that can detect VP quickly, sensitively, and specifically through the signal triggering and migration amplification of the multi-legged DNA walker and the assembly signal output of the G quadruplex, thereby realizing the detection of the target VP. When the target VP does not exist, the migration of the multi-legged DNA walker cannot be triggered, the fuel GF still maintains a stable hairpin structure, and no detection signal is generated. When the target VP is present, the multi-legged DNA walker triggers migration, the GF hairpin opens, and the G quadruplex specifically binds to the NMM to achieve enhancement of the fluorescence signal. At the same time, the present invention also provides a method for fluorescent sensing detection of VP based on a multi-legged DNA walker, which can detect VP at a concentration of 10 2 ~10 7 The method has good detection performance in the range of CFU / mL, with an LOD of 23CFU / mL. The spike recovery experiment of VP proves that the method has practical application value, providing a new, highly sensitive, specific, enzyme-free, fast and accurate detection method for the detection of low-concentration VP.

[0152] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A multi-legged DNA walker based on framework DNA functional materials, characterized in that: Including tetrahedral framework DNA functional materials, nano-based DNA functional materials, fuel chain GF, fluorescent materials; The tetrahedral framework DNA functional material contains a Vibrio parahaemolyticus aptamer and four scaffold chains; The nano-based DNA functional material is a track chain modified with nano-materials; The sequences of the Vibrio parahaemolyticus aptamer, four scaffold chains, track chain and fuel chain GF are shown in SEQ ID NO.1-7 respectively.

2. The multi-legged DNA walker according to claim 1, characterized in that: The 5' end of the track chain is modified with a dithiol group.

3. The multi-legged DNA walker according to claim 1, characterized in that: The nano material is gold nanoparticles.

4. The multi-legged DNA walker according to claim 1, characterized in that: The fluorescent material is N-methylmorpholine or 3,3',5,5'-tetramethylbenzidine.

5. The method for preparing the multi-legged DNA walker according to any one of claims 1 to 4, characterized in that: The following steps are included: S1. Preparation of nano-based DNA functional materials using nanomaterials and track chains; S2. Preparation of tetrahedral framework DNA functional materials: adding the Vibrio parahaemolyticus aptamer and four scaffold chains into a buffer solution and mixing them, and obtaining a TDNA structural material with target-specific activation characteristics by a rapid annealing method; S3. Mix the nano-based DNA functional material of S1 and the tetrahedral framework DNA functional material of S2 with the fuel chain GF and the fluorescent material, and react in the dark to obtain a multi-legged DNA walker.

6. The preparation method according to claim 5, characterized in that: The molar ratio of the nanomaterial to the track chain in step S1 is 1:30-50; the molar ratio of the Vibrio parahaemolyticus aptamer to the four scaffold chains in step S2 is 4:1-2.

7. The preparation method according to claim 5, characterized in that: In step S3, the molar ratio of the nano-based DNA functional material, the tetrahedral framework DNA functional material, the fuel chain GF and the fluorescent material is 20-30:1-2:90-110:5000-6000.

8. Use of the multi-legged DNA walker according to any one of claims 1 to 4 in detecting Vibrio parahaemolyticus for purposes other than disease diagnosis and treatment, or in the preparation of a Vibrio parahaemolyticus detection product.

9. A biosensor product, characterized in that: A multi-legged DNA walker comprising any one of claims 1 to 4.

10. A fluorescence sensing detection method based on a multi-legged DNA walker, characterized in that: Vibrio parahaemolyticus is detected using the multi-legged DNA walker described in any one of claims 1 to 4 or the product described in claim 9.

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