DNA (deoxyribonucleic acid) functional material based on Toehold-mediated strand displacement reaction as well as detection method and application of DNA functional material

Abstract

The invention discloses a DNA (deoxyribonucleic acid) functional material based on Toehold mediated strand displacement reaction as well as a detection method and application of the DNA functional material. According to the DNA functional material based on Toehold mediated strand displacement reaction, a TSMD / HCR dual-signal cascade amplification mechanism is introduced, effective and rapid amplification is carried out, sensitive detection of low-concentration VP is achieved, a good linear relation is achieved within the VP concentration range of 10 <-10 > CFU / mL, and the detection limit is as low as 1.8 CFU / mL; meanwhile, a novel multi-signal output sensing method is constructed by combining the characteristics of peroxidase-like and taking a DNA functional material as a signal output substrate, the method can realize visual detection on the VP on a culture site and can also realize accurate quantitative detection on the VP in a laboratory, and the method also has a multi-signal self-calibration function, ensures the accuracy of an analysis result, and has a good application prospect. Diversified detection of vibrio parahaemolyticus is realized, and a new thought is provided for diversified detection of VP.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biological detection, and more specifically, relates to a DNA functional material based on Toehold-mediated strand displacement reaction and a detection method and application thereof. Background Art

[0002] At present, most of the detection methods for Vibrio parahemolyticus (VP) are based on a single signal feedback mechanism and lack a self-calibration mode, which leads to insufficient accuracy and precision. In addition, the signal output of the existing detection methods mainly relies on large-scale instruments and equipment, which cannot meet the needs of rapid on-site detection in marine ranches. At the same time, the existing VP rapid detection technologies all have the problem of low sensitivity. In the case of low concentration or trace VP (such as aquaculture seawater), VP needs to be concentrated, which is cumbersome and easy to lose, resulting in inaccurate analysis results. Therefore, it is urgent to establish a rapid detection technology to improve the detection sensitivity and specificity of VP, which is not limited to the signal output mode of large-scale equipment, can be simply operated, easy to carry, and used in different detection environments to achieve accurate and sensitive detection of VP. It is also for the multi-scenario and diversified detection needs of Vibrio, which is of great significance to the prevention and control of marine ranch diseases.

[0003] Toehold-mediated strand displacement reaction (TMSD) is an efficient, enzyme-free amplification method that can achieve exponential amplification of single strands within 15 minutes. In the prior art, label-free fluorescent biosensors based on target-induced TMSDA-triggered G4 dimer / ThT luminescence can be used for the detection of ochratoxin A (OTA), which has the advantages of simplicity, low cost, and easy operation, but is rarely used in the detection and analysis of VP. Therefore, it is necessary to provide more new methods for the efficient, sensitive, and specific detection of VP, and to improve the accuracy of its detection, so as to realize the trace detection of VP with multiple signal outputs. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the problems and defects of the existing VP detection being not sensitive and accurate enough and being limited by the output signal of large equipment, and to provide a DNA functional material based on Toehold-mediated strand displacement reaction and its detection method and application.

[0005] The purpose of the present invention is to provide a DNA functional material based on Toehold-mediated strand displacement reaction.

[0006] Another object of the present invention is to provide a method for preparing a DNA functional material based on a Toehold-mediated strand displacement reaction.

[0007] Another object of the present invention is to provide an application of DNA functional materials based on Toehold-mediated strand displacement reaction.

[0008] Another object of the present invention is to provide a biosensor.

[0009] Another object of the present invention is to provide a method for detecting Vibrio parahaemolyticus for non-disease diagnosis purposes.

[0010] Another object of the present invention is to provide a Vibrio parahaemolyticus detection kit.

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

[0012] The present invention provides a DNA functional material based on Toehold-mediated strand displacement reaction, comprising a VP aptamer, a complementary strand, a capture probe, a signal strand, a fuel strand, a molecular beacon, a hairpin probe H1-G4a, a hairpin probe H2 and an oligonucleotide strand; the sequences of the VP aptamer, the complementary strand, the capture probe, the signal strand, the fuel strand, the molecular beacon, the hairpin probe H1-G4a, the hairpin probe H2 and the oligonucleotide strand are shown in SEQ ID NO.1-9, respectively.

[0013] The present invention uses Vibrio parahaemolyticus as the detection target, and the designed DNA functional material introduces the TSMD / HCR dual signal cascade amplification technology based on the Toehold-mediated chain displacement reaction, and uses the biological recognition element as a trigger to induce the TSMD cyclic reaction loop. In addition, the TSMD reaction product is used as the trigger element of the downstream HCR to trigger the second signal amplification, thereby achieving rapid amplification of low-concentration VP signals and improving the detection sensitivity. Finally, the triple helix DNA technology is introduced, and the oligonucleotide chain (TFO-G4b) with homologous polypyrimidine or homologous polypurine can be specifically recognized through the Hoogsteen (or reverse Hoogsteen) bond and combined with the HCR amplification product (dsDNA) to form a triple helix structure, which can quickly, accurately, sensitively, specifically and efficiently detect VP, and the detection sensitivity is 10-10 6 It has a good linear relationship within the VP concentration range of CFU / mL, with a detection limit as low as 1.8CFU / mL. It can also be combined with fluorescent materials to output multiple detection signals.

[0014] In the DNA functional material, the aptamer (A) and the complementary chain (C) specifically bind to form an AC double helix structure as a system reaction trigger element. When the detection target VP exists, the AC chain is disconnected, releasing the complementary chain C, which is recognized and bound by the capture probe (P) to form a partial double helix DNA, initiating the subsequent dual signal cascade amplification. By driving the addition of the DNA functional material fuel chain and signal chain (FS), the DNA motor (TSMD) is triggered, inducing the formation of double helix DNA (AF), the release of C and the signal chain (Signal), and C then opens P, cyclically triggering the reaction, achieving the first target signal amplification, and promoting the massive release of Signal. The free Signal can bind to the molecular beacon (MB) in the solution, open the hairpin structure of MB, expose the sequence complementary to H1-G4a, induce the opening of H1-G4a hairpin, and the opened H1-G4a hairpin can expose the free sequence complementary to H2, induce the opening of H2 hairpin, and the free sequence of the opened H2 hairpin has a sequence that can be partially complementary to the H1-G4a hairpin, continue to open the H1-G4a hairpin, and repeat the cycle, thereby triggering HCR cascade signal amplification and realizing the second signal amplification. Based on the dual signal cascade amplification reaction, accurate and specific detection of trace targets can be achieved.

[0015] When the target does not exist, the double helix structure of AC is stable and signal initiation cannot be achieved, triggering the opening of the capture probe P, which in turn makes it impossible to release the signal on FS and initiate the HCR cascade amplification. The spatial distance between H1-G4a and TFO-G4b is too far to assemble a complete G quadruplex, and no detectable results are produced.

[0016] The present invention provides a method for preparing a DNA functional material, which comprises adding a VP aptamer, a complementary strand, a capture probe, a signal strand, a fuel strand, a molecular beacon, a hairpin probe H1-G4a, a hairpin probe H2 and an oligonucleotide strand into a buffer solution and mixing them thoroughly.

[0017] Preferably, the VP aptamer, complementary strand, capture probe, signal strand, fuel strand, molecular beacon, hairpin probe H1-G4a, and hairpin probe H2 are incubated at 90-98° for 10-15 min before use.

[0018] Furthermore, the molar ratio of the VP aptamer, complementary strand, capture probe, signal strand, fuel strand, molecular beacon, hairpin probe H1-G4a, hairpin probe H2 and oligonucleotide strand is (1-3): (1-3): (4-6): (4-6): (4-6): (4-6): (15-25): (15-20): (20-30).

[0019] Preferably, the molar ratio of the VP aptamer, complementary strand, capture probe, signal strand, fuel strand, molecular beacon, hairpin probe H1-G4a, hairpin probe H2 and oligonucleotide strand is 2:2:5:5:5:5:20:20:25.

[0020] The present invention provides application of DNA functional material based on Toehold-mediated strand displacement reaction in detecting Vibrio parahaemolyticus for non-disease diagnosis purpose.

[0021] The present invention provides application of a DNA functional material based on a Toehold-mediated strand displacement reaction in preparing a Vibrio parahaemolyticus detection product.

[0022] The present invention provides a biosensor comprising the above DNA functional material.

[0023] Preferably, the biosensor further contains hemin, ABTS and H2O2.

[0024] The DNA functional material provided by the present invention has the characteristic of binding to G quadruplex / Hemin-like peroxidase. The provided biosensor uses DNA functional material as a signal output substrate, and can realize a diversified signal output detection method integrating three signal output combinations of ultraviolet + colorimetric + intelligent electronic device (such as mobile phone) analysis. Signal output mode: The G quadruplex structure in the DNA functional material can specifically bind to hemin (Hemin) to form a G quadruplex / Hemin complex with peroxidase-like activity, which can catalyze the ABTS / H2O2 system reaction, so that the color of the reaction solution changes from colorless to green, and the first signal output mode is generated, that is, a colorimetric signal that can be distinguished by the naked eye. The color generated can also be directly analyzed by an intelligent electronic device, such as the ARGB value data possessed by a mobile phone, to trigger the second signal output mode, and can perform portable portable signal analysis. In addition, the third signal output mode can be realized by ultraviolet analysis of the solution after the reaction, verifying the test results, and further calibrating. At the same time, the biosensor introduced the dual signal cascade amplification mechanism of TSMD / HCR and the signal output mechanism of triple-helix DNA to realize the construction of a sensitive, specific, multi-scenario, and multi-signal output detection method for VP.

[0025] Therefore, when the biosensor is used, Hemin needs to be mixed with DNA functional materials and Hemin should be added to the reaction system before ABTS and H2O2.

[0026] In particular, during the detection reaction, by adding spermine to the system, the stalk can be used to maintain the stability of the triple helix structure; and by adjusting the K +, which can further stabilize the structure of the G quadruplex and enhance the catalytic performance of the G quadruplex / Hemin.

[0027] Therefore, the biosensor also contains spermine and K + .

[0028] Preferably, K in the reaction system + The concentration is 10-20mmol / L, the pH is 5-6, the concentration of Hemin is 1.5-2.5μmol / L, the reaction time of the system is 1.5-2.5h, and the concentrations of ABTS and H2O2 are both 1.5-2.5mmol / L.

[0029] The present invention provides a method for detecting Vibrio parahaemolyticus for non-disease diagnosis purposes, and adopts the above-mentioned biosensor to perform qualitative and / or quantitative detection on Vibrio parahaemolyticus.

[0030] The invention also provides a Vibrio parahaemolyticus detection kit, which contains the above biosensor.

[0031] The present invention has the following beneficial effects:

[0032] The present invention provides a DNA functional material based on Toehold-mediated strand displacement reaction, introduces TSMD / HCR dual signal cascade amplification mechanism, effectively and rapidly amplifies the target signal, realizes sensitive detection of low concentration or even trace VP, and performs VP detection in 10-10 6 It has a good linear relationship within the VP concentration range of 1.2 CFU / mL, and the detection limit is as low as 1.8 CFU / mL; at the same time, using DNA functional materials as signal output substrates, combined with the characteristics of G quadruplex / Hemin peroxidase, it specifically captures the TSMD / HCR dual signal cascade amplification products to form a triple helix DNA structure to complete signal output. It has a self-calibration function for multiple signals to ensure the accuracy of the analysis results, and can detect VP quickly, accurately, sensitively, specifically and efficiently.

[0033] Its signal output is achieved through the self-assembly of G quadruplexes and the enzyme-like properties of the G quadruplex / Hemin complex, realizing three signal output modes: colorimetric, ultraviolet, and smart electronic device (such as mobile phone) analysis. It provides new ideas for multi-scenario and diversified detection of VP, promotes the further application of DNA functional materials in VP instant detection technology, provides a portable signal output mode, and lays a foundation for the subsequent establishment of a portable paper-based POCT platform for VP ocean ranches. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram of a new sensing method for DNA functional materials based on Toehold-mediated strand displacement reaction and a diversified detection method.

[0035] Figure 2 It is a feasibility analysis result diagram (A is UV signal output analysis; B is colorimetric and mobile phone intelligent signal output analysis; C is circular dichroism characterization; D is PAGE characterization, among which lane 1: CT+P+FS+MB+HI-G4a+H2+TFO-G4b; lane 2: VP+CT+P+FS+MB+HI-G4a+H2+TFO-G4b; lane 3: CT+P+FS+MB; lane 4: VP+CT+P+FS+MB; lane 5: S+MB+HI-G4a+H2+TFO-G4b; lane 6: MB+HI-G4a+H2+TFO-G4b; lane 7: FS+MB+HI-G4a+H2+TFO-G4b; lane 8: FS; E is ABTS / H2O2 reaction kinetic characterization).

[0036] Figure 3 It is the result of optimization analysis of detection conditions (A is the reaction system; B is the solution pH; C is the Hemin concentration; D is the reaction time; F is the ABTS concentration; E is the H2O2 concentration).

[0037] Figure 4 It is a graph of the detection sensitivity analysis results (A is the relationship between the ultraviolet absorption signal and the VP concentration; B is the linear relationship between the ultraviolet absorption value at 420nm and the concentration; C is the relationship between the colorimetric signal and the VP concentration; D is the linear relationship between the G value and the concentration).

[0038] Figure 5 It is a graph of specificity analysis results.

[0039] Figure 6 It is a graph of the analysis results of the actual detection sample signal output performance (A is colorimetric signal analysis; B is ultraviolet signal analysis; C is G value intelligent analysis; D is plate culture sample detection). DETAILED DESCRIPTION

[0040] 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.

[0041] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0042] Example 1 DNA sequence design of biosensor

[0043] Taking Vibrio parahaemolyticus as the detection target, we designed the Vibrio parahaemolyticus aptamer, AC, P, FS, MB, H1-G4a, H2, TFO-G4b and other biosensing DNA functional materials of DNA biosensor. The specific design method is as follows:

[0044] 1. Selection of Vibrio parahaemolyticus aptamers (A)

[0045] Aptamer is a short single-stranded nucleic acid sequence obtained by exponential enrichment (Systematic Evolution of Ligands by Exponential Enrichment, SELEX) technology. It 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, and its sequence is: 5'-TCTAAAAATGGGCAAAGAAACAGTGACTCGTTGAGATACT-3'. Complementary sequences are further designed based on this target sequence.

[0046] 2. Design of aptamer complementary chain (C)

[0047] The complementary strand of the aptamer is used as the trigger sequence of the system, and is mainly composed of a C sequence complementary to the aptamer. The specific design method is as follows:

[0048] 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 to the C sequence and to smoothly dissociate from the double helix structure in the presence of the target, 20 complementary bases of the aptamer sequence are selected as the C sequence according to the aptamer sequence to ensure the stable hybridization of C and the aptamer.

[0049] 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.

[0050] The specifically designed complementary chain (C) sequence is: 5'-CGA G TC ACT GTT TCT TTG CCC AT -3' (the underlined part is complementary to the aptamer). Through the principle of complementary base pairing, the C sequence and the partial complementarity of the aptamer sequence form a double helix DNA structure that can prevent the interaction between C and the capture probe chain (P), avoiding nonspecific initiation of the reaction in the absence of Vibrio parahaemolyticus. In the presence of Vibrio parahaemolyticus, the aptamer / C double helix structure dissociates by utilizing the specific recognition and binding of Vibrio parahaemolyticus, thereby triggering the subsequent reaction.

[0051] 3. Design of capture probe chain (P)

[0052] The capture probe chain is the capture chain of the aptamer complementary chain C, which is mainly composed of ①Toehold complementary sequence, ②Hairpin complementary sequence and ③C sequence capture sequence, among which ①Toehold complementary sequence, ②Hairpin complementary sequence and ③C sequence capture sequence constitute ④F complementary sequence. The specific design method is as follows:

[0053] (1) Toehold complementary sequence

[0054] a. Number of bases: When the number of hybridization bases of the toehold sequence that triggers the reaction is greater than 6, it can promote the occurrence of toehold-mediated strand displacement reaction. According to the number of toehold complementary bases of the F sequence and the complementary sequence of its own hairpin structure, 8 bases are selected as the toehold complementary sequence to ensure that the hairpin exists stably in the absence of the C sequence. The toehold-mediated strand displacement reaction will occur only in the presence of the C sequence.

[0055] b. Sequence composition: According to the principle of base complementary pairing, the sequence can specifically bind to the underlined part of the F complementary sequence (5'-TCA CTG TTTCTT TGC CCA TTC AAG AAA CAG TGA GAG TCG TAA-3'), thereby achieving a toehold-mediated strand displacement reaction when the P hairpin is open.

[0056] (2) Hairpin complementary sequence

[0057] a. Number of bases: If the complementary sequence is too short, it cannot play a protective role. P can be opened even when the complementary chain C does not exist. If it is too long, the hairpin structure may be too stable, and the complementary chain C cannot open the hairpin P, and thus cannot be released. According to the C sequence capture sequence, 15 bases can protect and ensure that the P hairpin structure exists stably in the absence of a target, and when the target is present, the free C can open the hairpin structure.

[0058] b. Sequence composition: Based on the principle of base complementary pairing, it can be combined with the P sequence (5'-TTA CGA CTC TCA CTGTTT CTT GAA TGG GCA AAG AAA CAG TGA GAG -3') underlined part specifically binds to form the P hairpin structure.

[0059] (3) C sequence capture sequence

[0060] 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, the P hairpin structure is opened. According to the number of bases in the C sequence, 20 bases are selected as the C sequence capture sequence to ensure that P can successfully recognize the C sequence and stably hybridize with C.

[0061] b. Sequence composition: Based on the principle of base complementary pairing, the sequence can be combined with the target C sequence (5'-CGA G TC ACT GTT TCT TTG CCC ATT -3') sequence to specifically bind to the underlined portion, opening the P hairpin structure and forming a partial double-helix DNA structure CP.

[0062] (4) F complementary sequence

[0063] a. Number of bases: When the number of bases involved in hybridization is greater than 12, the stability of hybridization can be guaranteed. If the number of hybridization bases of P and F is too short, the chain displacement reaction cannot be triggered, the first signal amplification reaction cannot be cycled, and the HCR reaction cannot be triggered. If it is too long, the combination of P and F may trigger HCR non-specifically in the absence of a target, generating a false positive signal. According to the number of bases and binding sites of C and S, 51 bases are selected as the F complementary sequence to ensure that only the CP formed by P opened by C can undergo a chain displacement reaction with FS.

[0064] b. Sequence composition: Based on the principle of complementary base pairing, it can specifically bind to the fuel F sequence (5'-TCA CTG TTT CTT TGCCCA TTC AAG AAA CAG TGA GAG TCG TAA-3') to achieve the strand displacement reaction of TSMD.

[0065] The specific design of the P sequence is as follows:

[0066]

[0067] Among them, ② is partially complementary to ③ sequences, making P a hairpin structure that exists stably in the solution, and ② can specifically recognize and bind to the complementary chain (C) sequence, opening the hairpin structure and completely releasing ①. Under the action of ①, ④ promotes the newly formed CP double helix structure to undergo a chain displacement reaction with the driver DNA (FS), forming a FP double helix structure and releasing the complementary chain C and the signal chain S, inducing the cyclic occurrence of the first signal amplification TSMD reaction and the triggering of the second signal amplification HCR.

[0068] 4. Design of fuel chain (F)

[0069] The fuel chain (Fuel) is an important element for the TSMD reaction. It consists of a preamble sequence, ①S complementary sequence and ②Toehold sequence. The fuel chain is ③P complementary sequence. The specific design method is as follows:

[0070] (1) S complementary 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 successfully form a double-helix DNA structure and ensure that the driver DNA (FS) formed does not react non-specifically with the P hairpin, 20 bases are selected as the S complementary sequence according to the sequence of the signal chain (S) to ensure that F can successfully and stably hybridize with S.

[0072] b. Sequence composition: Based on the principle of complementary base pairing, the sequence can specifically bind to the signal chain S (5'-C TCA CTG TTT CTTGAA TGG G-3') sequence to achieve the formation of the FS double-helix DNA structure.

[0073] (2) Toehold sequence

[0074] a. Number of bases: When the number of hybridization bases of the toehold sequence that triggers the reaction is greater than 6, it can serve as a trigger element to trigger the occurrence of a strand displacement reaction. Based on the number of complementary bases of the F sequence, the position of the C complementary sequence, the complementary position and number of bases of the S complementary sequence, 8 bases are selected as the toehold sequence to ensure that the P hairpin and FS exist stably in the absence of the C sequence. Toehold-mediated strand displacement reaction will occur only in the presence of the C sequence.

[0075] b. Sequence composition: According to the principle of base complementary pairing, it can be combined with (5'- TTACGACT C TCA CTG TTTCTT GAATGG GCA AAG AAA CAG TGA GAG-3') The underlined part specifically binds, and in the presence of CP, CP will undergo a toehold-mediated strand displacement reaction with FS.

[0076] (3) P complementary sequence

[0077] a. Number of bases: When the number of bases involved in hybridization is greater than 12, the stability of hybridization can be guaranteed. If the number of hybridization bases of P and F is too short, the chain displacement reaction cannot be triggered, the first signal amplification reaction cannot be cycled, and the HCR reaction cannot be triggered. If it is too long, the combination of P and F may trigger HCR non-specifically in the absence of a target, generating a false positive signal. According to the number of bases and binding sites of C and S, 51 bases are selected as the P complementary sequence to ensure that only CP can undergo chain displacement reaction with FS.

[0078] b. Sequence composition: Based on the principle of complementary base pairing, it can specifically bind to the capture probe P (5'-TTA CGA CTC TCA CTGTTT CTT GAA TGG GCA AAG AAA CAG TGA GAG-3') to achieve the strand displacement reaction of TSMD.

[0079] The specific design of F sequence is as follows:

[0080]

[0081] Among them, ① will complementarily bind with the signal chain (S) to form a partially complementary double-helix DNA structure, which exists stably in a target-free solution. When the CP double-helix DNA structure exists, ② will bind to the Toehold complementary sequence on P and undergo a chain displacement reaction under the action of ③, ensuring the dissociation of the complementary chain C from the signal chain S, thereby realizing the cyclic occurrence of the first signal amplification TSMD reaction and the triggering of the second signal amplification HCR.

[0082] 5. Design of signal chain (S)

[0083] The signal chain (Signal) consists of a short deoxynucleotide sequence, which is the amplification product of the first signal amplification mechanism (TSMD). After TSMD is triggered, S will be separated from the driver DNA (FS) and can serve as the trigger element of the next signal amplification mechanism (HCR), activating the second signal amplification and achieving ultra-sensitive detection of the target. The specific design method is as follows:

[0084] 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 smoothly form a double-helix DNA structure and ensure that the formed driver DNA (FS) does not react non-specifically with the P hairpin, 20 bases are selected as the S complementary sequence according to the sequence and base binding position of the fuel F to ensure that F can successfully and stably hybridize with S and that the formed FS double-helix structure does not react non-specifically with the free P hairpin.

[0085] b. Sequence composition: Based on the principle of complementary base pairing, the sequence can specifically bind to the underlined part of the molecular beacon MB sequence (5'-AAG GAG AGAGAA GAA TGG TCA AAT C CCA TTC AAG AAA CAG TGA G AT TTG ACC AT-3') to achieve the formation of a partial double helix structure.

[0086] The specific S sequence is: 5'-C TCA CTG TTT CTT GAA TGG G-3'.

[0087] 6. Design of Molecular Beacon (MB)

[0088] Molecular Beacon (MB) is a molecular beacon, which consists of ①HCR initiation sequence, ②S capture sequence and ③ complementary sequence. The specific design method is as follows:

[0089] (1) HCR startup sequence

[0090] b. 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 successfully trigger HCR and open the H1-G4a hairpin structure, 24 bases are selected as the HCR start sequence based on the number of bases in the H1-G4a partial complementary sequence, signal chain S sequence, and Toehold sequence to ensure stable hybridization between the start sequence and H1-G4a.

[0091] b. Sequence composition: Based on the principle of complementary base pairing, the sequence can be combined with ③(5'-AAG GAGAGA GAA GAA TGG TCA AAT C CCA TTC AAG AAA CAG TGA in the same chain MB G AT TTG ACC AT -3') underlined sequence specifically complementary binding, forming part of the stem in the hairpin MB, and can be combined with the ① (5'- ATT TGA CCA TTC TTC TCT CTC CTT GCA CTA AAG GAG AGA GAA GAA TG GGA TGG GCGGG-3') underlined sequence specific complementary hybridization ensures the strong complementary double-stranded structure of H1-G4a, initiates triggering HCR, and realizes signal cascade amplification.

[0092] (2) S capture sequence

[0093] a. Number of bases: When the number of bases involved in hybridization is greater than 12, the stability of hybridization can be guaranteed. According to the number of bases in the signal chain S, 20 bases are selected to form the S capture sequence in MB, which can ensure the stable hybridization binding between MB and S and the opening of the hairpin MB.

[0094] b. Sequence composition: Based on the principle of complementary base pairing, it can specifically bind to the signal chain S (5'-C TCA CTG TTT CTT GAATGG G-3') to achieve specific capture of S.

[0095] (3) Complementary sequence

[0096] a. Number of bases: If the complementary sequence is too short, it cannot play a protective role and can non-specifically open MB when the S chain is not present. If it is too long, S may not be able to open the hairpin MB, and thus fail to trigger HCR. According to the HCR initiation sequence and S capture sequence, 14 bases can protect and ensure that the MB hairpin structure exists stably in a free state and induce the detachment of S when the target is present, and the free S opens the hairpin structure.

[0097] b. Sequence composition: Based on the principle of complementary base pairing, it can specifically bind to the underlined part of the HCR promoter sequence (5'-AAG GAG AGA GAAGAA TGG TCA AAT C CCA TTC AAG AAA CAG TGA G AT TTG ACC AT-3') to achieve the formation of the MB hairpin structure.

[0098] The specific design of MB sequence is as follows:

[0099]

[0100] Among them, ① it acts as a start-up sequence to trigger HCR and realize signal cascade amplification through the HCR reaction; ② it specifically captures S, opens the hairpin MB, releases the HCR start-up sequence, and promotes the subsequent HCR cascade signal amplification reaction; ③ it specifically binds to the HCR start-up sequence and the S capture sequence partially complementarily to form hairpin MB, thereby reducing non-target erroneous opening of the hairpin structure.

[0101] 7. Design of Hairpin probe 1-G4a (H1-G4a)

[0102] H1-G4a is a hairpin probe, which consists of ①HCR promoter complementary sequence, ④H2 promoter sequence containing ②purine-rich sequence, and ③split G-quadruplex G4a sequence. The specific design method is as follows:

[0103] (1) HCR starts complementary sequence

[0104] 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 formation of the hairpin structure of H1-G4a, sufficient bases are required as complementary bases of the stem of the hairpin, and at the same time, a certain number of bases are required as toeholds to bind to the HCR promoter sequence to trigger toehold-mediated chain displacement. Therefore, 24 bases are selected as the HCR promoter complementary sequence of H1-G4a to hybridize complementary to the HCR promoter sequence.

[0105] b. Sequence composition: Based on the principle of complementary base pairing, (5'-ATT TGA CCA TTC TTC TCT CTC CTTGCA CTA AAG GAG AGA GAA GAA TG GGA TGG GCGGG-3') The underlined part can be combined with (5'-ATT TGA CCA TTC TTC TCT CTC CTT GCA CTA AAG GAG AGA GAA GAA TG G The underlined part in ① is complementary to the stem of the hairpin structure, enabling it to exist stably in the free state, while the bold part in ① serves as the toehold of the hairpin. When the HCR initiator chain is in the free state, the toehold sequence hybridizes with it, thereby triggering toehold-mediated chain displacement, allowing the H1-G4a hairpin structure to open.

[0106] (2) Purine-rich sequence

[0107] a. Number of bases: The homopolypurine sequence composed of 14 bases can combine with the purine-rich chain region in the double-helix DNA to form triple-helix DNA. When the number of bases involved in the hybridization is greater than 12, the stability of the hybridization can be guaranteed. Selecting 14 bases to form the purine-rich sequence of H1-G4a can ensure the stable hybridization and binding between the purine-rich sequence of H1-G4a and the pyrimidine-rich sequence of H2, and can be recognized and bound by the homopolypyrimidine sequence to form a triple helix.

[0108] b. Sequence composition: According to the principle of base complementary pairing, this sequence can hybridize and complement with the pyrimidine-rich sequence in H2 (5'-TTCTTC TCT CTC CTT-3'), and according to the pairing mode of CG*C and TA*T, it is hybridized and complemented by the homopolypyrimidine sequence (5'- TTC CTC TCT CTT CT TGGGT-3') binds in parallel to form triple helical DNA.

[0109] (3) Splitting the G-quadruplex G4a sequence

[0110] a. Number of bases: Based on the structural characteristics and self-assembly properties of G quadruplexes, in order to achieve better target specificity, the G quadruplexes are split with three G (guanine) bases as a tetrad. In order to achieve the formation of a triple helical DNA structure to trigger the release of the signal, three tetrads, i.e. 13 bases, are selected as the split G quadruplex G4a sequence to ensure that the signal is triggered on the basis of the formation of a specific triple helical DNA structure.

[0111] b. Sequence composition: The sequence 5'-TGGGATGGGCGGGTGGGT-3' was selected as the G quadruplex sequence of the present invention. Based on the assembly of the G quadruplex, (5'-TG GGA TGG GCGGG-3') was selected as the split G quadruplex G4a sequence.

[0112] (4)H2 startup sequence

[0113] a. Number of bases: When the number of bases involved in hybridization is greater than 12, the stability of hybridization can be guaranteed. To ensure the formation of the H1-G4a hairpin structure and serve as the opening sequence of the H2 hairpin, 21 bases are selected as the H2 start sequence.

[0114] b. Sequence composition: Based on the principle of base complementary pairing, (5'-GCA CTA AAG GAG AGA GAA GAA -3') The underlined part can be combined with (5'-ATT TGA CCA TTC TTC TCT CTC CTT -3') to form the stem of the hairpin structure, allowing it to exist stably in the free state. At the same time, the free state ④ can serve as a start sequence and interact with (5'-AAG GAG AGA GAA GAA TGG TCA AAT in H2 TTC TTC TCT CTC CTT TAG TGC -3') and the underlined part hybridizes to open the H2 hairpin structure.

[0115] The specific design of the H1-G4a sequence is as follows:

[0116]

[0117] Among them, ① binds to the HCR start sequence, the H1-G4a hairpin structure opens, and the H2 opening sequence is released, prompting the H2 hairpin to open; ② during the HCR process, it is complementary to the pyrimidine sequence in the other chain to form double-helix DNA, and is recognized and bound by the homopolypyrimidine sequence to form triple-helix DNA; ④ is complementary to part ① in H1-G4a to form a hairpin structure, ensuring that H1-G4a exists in a hairpin structure when free, and the H1-G4a hairpin structure is opened by SMB. When ④ is in a free state, it can serve as a start sequence to open the H2 hairpin structure.

[0118] 8. Design of Hairpin probe 2 (H2)

[0119] Hairpin probe 2 (H2) is a hairpin probe, which consists of ①H1-G4a priming sequence and ③H2 priming complementary sequence containing ②double helical purine chain. The specific design method is as follows:

[0120] (1)H1-G4a promoter sequence

[0121] 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 formation of the hairpin structure of H2, sufficient bases are required as complementary bases of the stem of the hairpin. At the same time, a certain number of bases are required as toeholds to bind to the HCR initiation complementary sequence in H1-G4a to trigger toehold-mediated chain displacement. Therefore, 24 bases are selected as the H1-G4a initiation sequence.

[0122] b. Sequence composition: Based on the principle of base complementary pairing, (5'-AAG GAG AGA GAA GAA The underlined part of H2 can be combined with the ③ part (5'-TTC TTC TCT CTC CTT The underlined part of ① is complementary to that of ① in H1-G4a, forming the stem of the hairpin structure, enabling it to exist stably in the free state, while the bold part of ① (5'-AAG GAGAGA GAA GAA TGG TCA AAT-3') serves as the loop of the hairpin and also has a toehold effect. When ① is in the free state, the toehold sequence will hybridize complementary to ① in H1-G4a, thereby triggering toehold-mediated chain displacement, allowing the H1-G4a hairpin structure to open.

[0123] (2) Double helix pyrimidine chain

[0124] a. Number of bases: The homopolypyrimidine sequence composed of 14 bases can combine with the purine-rich chain region in the double-helix DNA to form a triple-helix DNA. When the number of bases involved in the hybridization is greater than 12, the stability of the hybridization can be guaranteed. Selecting 14 bases to form the pyrimidine-rich sequence of H2 can ensure the stable hybridization and binding between the purine-rich sequence of H1-G4a and the pyrimidine-rich sequence of H2, and can be recognized and combined in parallel by the homopolypyrimidine sequence to form a triple helix.

[0125] b. Sequence composition: According to the base pairing principle, it hybridizes and pairs with the purine-rich sequence (5'-AAG GAG AGA GAAGAA-3') in H1-G4a to form a double-helix DNA region that can be bound by the homopolypyrimidine sequence.

[0126] (3) H2 initiates complementary sequence

[0127] 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 formation of the hairpin structure of H2, sufficient bases are required as complementary bases of the stem of the hairpin, and at the same time, a certain number of bases are required as toeholds to bind to the H2 start sequence in H1-G4a to trigger toehold-mediated chain displacement. Therefore, 21 bases are selected as the start complementary sequence of H2 to hybridize complementary to the H2 start sequence in H1.

[0128] b. Sequence composition: Based on the principle of complementary base pairing, ③ (5'-TTC TTC TCT CTC CTT TAGTGC-3') can complement ④ (5'-GCA CTA AAG GAG AGA GAA GAA-3') in H1-G4a, open the hairpin structure of H2, release the ① sequence, and induce HCR cascade amplification.

[0129] The specific H2 sequence design is as follows:

[0130]

[0131] Among them, ① bind to the HCR promoter complementary sequence in H1-G4a, the H1-G4a hairpin structure opens, the H2 promoter sequence is released, the H2 hairpin opens, and then triggers the HCR signal cascade reaction; ② in the HCR process, it is complementary to the purine sequence in the other chain to form double-helix DNA, forming a double-helix DNA region that can be bound by the homopolypyrimidine sequence; ③ bind to the H2 promoter sequence in H1-G4a, the H2 hairpin structure opens, the H1-G4a promoter sequence is released, and the H1-G4a hairpin opens.

[0132] 9. Design of oligonucleotide chain (TFO-G4b)

[0133] The oligonucleotide chain (TFO-G4b) consists of ① a homopolypyrimidine sequence and ② a split G-quadruplex G4b sequence; the oligonucleotide chain can be combined with the amplified product dsDNA to form a triple-stranded DNA. The formation of the triple helix DNA shortens the distance between the split G-quadruplexes in space, so that G4b and G4a on H1-G4a self-assemble into a complete G-quadruplex structure. The structural characteristics of the G-quadruplex are used as a signal output method. The specific design method is as follows:

[0134] (1) Homopolypyrimidine sequences

[0135] a. Number of bases: The homopolypyrimidine oligonucleotide sequence and length require at least 12-14 uninterrupted pyrimidines to be sufficient to bind to dsDNA to form triple helix DNA. Therefore, 14 bases are selected to form the homopolypyrimidine sequence to ensure the stable binding of the triple-stranded DNA.

[0136] b. Sequence composition: The triple-stranded DNA composed of TA*T and CG*C has good stability, so C and T are selected as the constituent bases, based on the Hoogsteen bond and the purine chain in dsDNA (5'-ATT TGA CCA TTC TTC TCT CTCCTT GCA CTA AAG GAG AGA GAA GAA The underlined part of TG GGA TGG GCGGG-3') binds in a forward and parallel direction to form a triplex DNA.

[0137] (2) Splitting the G-quadruplex G4b sequence

[0138] a. Number of bases: Based on the structural characteristics and self-assembly properties of G quadruplexes, in order to achieve better target specificity, the G quadruplexes are split with three G (guanine) bases as a tetrad. In order to trigger the release of the signal for the formation of a triple helical DNA structure, a tetrad, i.e., 5 bases, is selected as the split G quadruplex G4b sequence to ensure that the signal is triggered on the basis of the formation of a specific triple helical DNA structure.

[0139] b. Sequence composition: The sequence 5'-TGGGATGGGCGGGTGGGT-3' was selected as the G quadruplex sequence of the present invention. Based on the assembly of the G quadruplex, (5'-TGGGT-3') was selected as the split G quadruplex G4b sequence.

[0140] The specific TFO-G4b sequence design is as follows:

[0141]

[0142] In summary, the sequence designed by the present invention is shown in Table 1. According to the specific affinity of the aptamer (A) to VP, a complementary chain (C) that specifically binds to the VP aptamer is designed to form an AC double helix structure as a system reaction trigger element. When the target VP exists, the aptamer has a stronger molecular interaction with VP than with the complementary chain, thereby causing the AC chain to break and release the complementary chain C. C can be recognized and bound by the capture probe (P) to form a partial double helix DNA, which initiates the subsequent dual signal cascade amplification. By driving the addition of DNA functional material (FS), the DNA motor (TSMD) is triggered to induce the formation of double helix DNA (AF), the release of C and the signal chain (Signal), and the release of C can open P again, triggering the reaction in a cycle, achieving the first target signal amplification, and promoting the massive release of Signal. The free Signal can bind to the molecular beacon (MB) in the solution, open the hairpin structure of MB, and expose the sequence complementary to H1-G4a. This sequence can further induce the opening of the H1-G4a hairpin. The opened H1-G4a hairpin can expose the free sequence complementary to H2, inducing the opening of the H2 hairpin. The free sequence of the opened H2 hairpin has a sequence that can be partially complementary to the H1-G4a hairpin, and the H1-G4a hairpin continues to open. The cycle repeats, triggering the HCR cascade signal amplification and achieving the second signal amplification. The specific reaction principle is as follows: Figure 1 As shown, based on the dual signal cascade amplification reaction, accurate and specific detection of trace targets can be achieved.

[0143] In addition, the introduction of oligonucleotide chains (TFO-G4b) promotes the formation of triple helical DNA, and the spatial proximity of TFO-G4b and H1-G4a can induce the assembly of G quadruplex sequences. The G quadruplex structure can specifically bind to hemin (Hemin) to form a G quadruplex / Hemin complex with peroxidase-like activity, which can catalyze the ABTS / H2O2 system reaction, causing the color of the reaction solution to change from colorless to green, generating the first signal output mode, that is, a colorimetric signal that can be discerned by the naked eye. The generated color can be recognized by intelligent electronic devices, and ARGB value data analysis can be performed to trigger the second signal output mode, that is, portable signal analysis.

[0144] In addition, the third signal output method can be used to further verify the test results by ultraviolet analysis of the solution after the reaction, that is, further calibration of laboratory precision instruments. When the target does not exist, the double helix structure of AC is stable, and the signal cannot be initiated, triggering the opening of the capture probe P, which in turn makes it impossible to release the signal on FS, and the HCR cascade amplification cannot be initiated. The spatial distance between H1-G4a and TFO-G4b is too far, and a complete G quadruplex cannot be assembled. None of the three signal output methods produce detectable results.

[0145] Table 1 Sequence Listing

[0146]

[0147] Example 2 Preparation and detection method of biosensor DNA functional material

[0148] 1. Preparation of DNA functional materials

[0149] First, the fuel chain (F), signal chain (S), aptamer (A) and complementary chain (C) were added to the PBS buffer solution at a molar concentration ratio of 1:1 and mixed evenly to obtain AC functional material with target recognition initiation and FS functional material with target driving effect. Then, AC, capture probe (P), FS, MB, H1-G4a and H2 were placed in a water bath and slowly heated to 95°C and maintained for 15 min to ensure the formation of DNA double helix structure and hairpin structure.

[0150] Take 20nmol / L AC, 50nmol / L LP, 50nmol / L DS, 50nmol / L MB, 200nmol / L H1-G4a, 200nmol / L H2, 250nmol / L TFO-G4b and 1.5μmol / L Hemin, add them into PBS buffer solution and mix them thoroughly to obtain DNA functional material.

[0151] 2. Detection of target DNA

[0152] During the test, the sample containing VP was added to the mixture of the above DNA functional materials and reacted at room temperature for 2 hours to perform a double cascade signal amplification reaction. Then, 2mmol / L ABTS and 2mmol / L H2O2 were added to the reaction mixture to keep the total volume at 300μL, and the test analysis was performed after the reaction for 15 minutes.

[0153] Analysis: (1) Directly observe the color change of the solution, and after it gradually changes from colorless to green, conduct a qualitative analysis of VP; (2) Use electronic equipment to collect the ARGB value signal of the solution color, conduct quantitative analysis of the color depth, and conduct a preliminary semi-quantitative analysis of the concentration; (3) Then use a UV spectrophotometer to record the wavelength change of the solution at 400-450nm for accurate quantitative analysis; a triple signal output mode can be achieved to meet the diverse detection needs of VP in different situations.

[0154] 3. Smart device analysis application

[0155] After the test is completed, you can use smart electronic equipment to collect information about the solution color change during the test, and select the G value from the ARGB value for analysis. If you use a mobile phone camera to record the solution color, it is recommended to do it in a dark environment to reduce the impact of other light sources on the shooting environment.

[0156] The experiments of the present invention are all set up in the dark box of the ultraviolet analyzer, and the light source in the dark box is used to ensure that the shooting environment is a unified light source to improve the credibility and accuracy of the photos. Then, the reaction solution in the dark box is photographed through the external hole. The obtained photos are uniformly obtained by using the downloaded APP "color picker" to obtain the ARGB value corresponding to the sample, and the G value is selected for analysis.

[0157] Example 3 Feasibility Analysis of Biosensing DNA Functional Material Sensing Detection

[0158] 1. Feasibility analysis

[0159] By using the detection method of Example 2, under four different conditions (where the stability of the triple helix structure is maintained by the addition of spermine), the changes in the UV signal, color change, and detection performance of the smartphone signal were set. The feasibility of the reaction was verified by UV, colorimetric, and G value changes, and the reasons for the generation of readable signals were observed and analyzed by circular dichroism (CD) and PAGE to verify the feasibility of the diversified signal output method based on dual signal cascade amplification.

[0160] Four different conditions: (a) VP+CT+P+FS+MB+HI-G4a+H2+TFO-G4b+spermine+Hemin+ABTS+H2O2; (b) CT+P+FS+MB+HI-G4a+H2+TFO-G4b+spermine+Hemin+ABTS+H2O2; (c) VP+CT+P+FS+MB+HI-G4a+H2+spermine+Hemin+ABTS+H2O2; (d) only Hemin+ABTS+H2O2.

[0161] The results are as follows Figure 2As shown in the figure, when there is no target VP (condition b) and only the reaction substrate of Hemin / ABTS / H2O2 exists (condition d), the three signals are very weak and the difference is not big. This shows that in the absence of VP, the dual signal cascade amplification technology cannot be triggered, the split G quadruplex cannot be assembled, and only the reaction substrate slowly undergoes redox reaction to produce extremely low weak signals ( Figure 2 A).

[0162] When VP is present (condition a), VP binds to the aptamer, starts the reaction trigger element, induces the reaction, and amplifies the double signal cascade. A large number of complete G quadruplexes are assembled in a short time, which accelerates the reduction efficiency of the ABTS / H2O2 system, significantly increases the UV signal, and produces an obvious color change. The color of the solution changes from colorless to green ( Figure 2 B). At the same time, the G value also decreased significantly. When VP was present but TFO-G4b was absent (condition c), the signals generated by the reaction were very weak, similar to the signal output phenomena of conditions b and d. This indicates that the signal output was achieved through the G quadruplex self-assembly of the triple helical DNA structure formed by the specific binding of TFO-G4b to the dual signal cascade amplification product dsDNA.

[0163] In order to further verify that the three signal outputs are achieved through the assembly of G-quadruplexes through the formation of triple helical DNA structures, CD and PAGE were used to characterize the TSMD, HCR and TSMD / HCR hybridization processes and triple helical DNA structures. The results are shown in Figure 2As shown in Figures C and D. When there is no VP in the reaction, TSMD signal amplification cannot be triggered (lane 3), the signal chain S in FS cannot be released, and the start of the second HCR signal amplification cannot be triggered (lanes 6-7), that is, when VP does not exist, the dual cascade amplification reaction cannot be triggered (lane 1), triple helical DNA cannot be formed, and the self-assembly of G quadruplexes is limited; this is also confirmed in the CD figure. When VP does not exist, only a slight fluctuation of the negative peak at 250nm and the positive peak at 280nm can be observed in the system. This is the characteristic peak of the double helical DNA structure of A, FS, MB, H1 and H2 in the system (condition b). Compared with lane 3, when VP exists, the appearance of new bands can be clearly observed in lane 4, which proves that VP can trigger the TSMD reaction as a signal initiation element, and the product signal chain S after the TSMD reaction is triggered can be used as a triggering element for the next level of HCR signal amplification. In lane 5, the extended dsDNA amplification product band can be clearly observed, proving the triggering of the HCR process. That is, after adding VP, VP can trigger the TSMD / HCR dual cascade signal amplification reaction (lane 2), and from the CD spectrum, in addition to the obvious changes in the characteristic peaks of dsDNA at 250nm and the positive peak at 280nm, a new negative peak can be observed at 210nm (condition a), which is the characteristic peak of triple helix DNA, indicating the formation of triple helix DNA. The formation of triple helix DNA structure can also be observed from the CD spectrum of condition c. When TFO-G4b is missing in the reaction system, only the characteristic peak of dsDNA can be observed, and the characteristic peak of triple helix DNA does not appear at 210nm, that is, triple helix DNA cannot be formed.

[0164] In addition, two groups of reactions (conditions a and b) were set up, an experimental group with or without VP added and a control group, to clarify the catalytic effect of the enzyme-like properties of G-quadruplex / Hemin on the ABTS / H2O2 system reaction. Under the same conditions, the same concentration of ABTS and H2O2 were added at the same time, and the real-time UV signals of the two groups of reactions were monitored. The results are shown in Figure 2. Figure 2 E shows that compared with the blank group without VP, the UV signal of the experimental group with VP added was significantly higher than that of the blank group. After 400s of reaction, the UV signal tended to be flat and remained unchanged, which means that after 400s, ABTS has been completely reduced to ABTS by H2O2. + The UV signal of the blank group still showed an increasing trend after 900s of reaction. It can be seen that the G quadruplex / Hemin produced by the reaction has a strong catalytic effect, which can accelerate the reaction of the ABTS / H2O2 system and detect a signal output phenomenon with obvious differences in a short time.

[0165] In summary, the TSMD / HCR dual signal cascade amplification mechanism specifically recognizes VP, and triple signal output is achieved only in the presence of VP. The triple signal output through UV, colorimetry, and mobile phone G value analysis requires the use of triple helix DNA specific formation to complete the self-assembly of the G quadruplex structure. The formation of the triple helix DNA structure requires TFO-G4b to specifically recognize the HCR amplification product dsDNA, indicating that HCR / triple helix DNA is the second signal test for TSMD signal amplification, verifying the specific recognition of the target by the constructed dual signal cascade amplification / multiple signal output detection method.

[0166] 2. Condition Optimization

[0167] In order to obtain the best detection performance, the factors affecting the dual signal cascade amplification method and signal output results were optimized: reaction system, solution pH, Hemin concentration, reaction time, and ABTS and H2O2 concentrations. The optimization results are presented by the numerical values ​​of the two signal output modes. Change of absorbances and ΔG represent the difference in absorbance and G value in color recognition during UV detection between the sample detection group with added target and the blank detection group without target, respectively (N=3, mean ± SD, *p<0.05 is a significant difference, **p<0.01 is a very significant difference, ns is no significant difference).

[0168] K in the reaction system + The presence of can participate in the assembly of split G quadruplexes and stabilize the structure of G quadruplexes, while different K + The results of the effect of concentration on the catalytic performance of G-quadruplex / Hemin are as follows Figure 3 As shown in A, when adding 0-20mmol / L KCl, the signal response difference of the solution increases first and then tends to be flat. When the KCl concentration is 15mmol / L, the catalytic activity of G quadruplex / Hemin reaches the maximum. Therefore, 15mmol / L is selected as the optimal reaction concentration of KCl to complete the stable assembly of G quadruplex.

[0169] The pH of the solution affects the efficiency of triple helix DNA generation, and the effects of different pH on reaction performance are shown in the following table. Figure 3As shown in Figure B, as the pH changes from 4 to 8, the reaction signal tends to increase first and then decrease. At a pH of 6, the signal output difference reaches a peak. This shows that when the pH of the reaction solution is 6, the overall solution is in the direction of promoting the production of triple helical DNA structure, the triple helical DNA structure is stably formed, and the assembly of split G quadruplexes is induced, so that the signal output reaches the optimal level. When the pH is greater than 6, the structure of the triple helical DNA occurs in the direction of inhibiting production, and the assembly of split G quadruplexes is inhibited, so that the signal cannot be output. Therefore, the optimal pH value of this detection method is 6.

[0170] The results of optimizing Hemin concentration are as follows: Figure 3 As shown in Figure C, with the increase of Hemin concentration, the signal output changes first increase and then stabilize. When the concentration of Hemin reaches 1.5 μmol / L, the catalytic activity of Hemin / G quadruplex reaches the maximum, and the best signal response difference is obtained. With the continued increase of Hemin concentration, the signal response difference does not produce significant differences. Therefore, 1.5 μmol / L Hemin concentration is selected as the optimal reaction concentration.

[0171] In order to achieve the best reaction effect, the system reaction time is optimized and adjusted within the range of 0.5h-2.5h. The results are as follows Figure 3 As shown in D, it can be seen that the difference between the UV signal and the G value increases with time, and the growth of the difference between the two signals gradually slows down after 1.5 hours. This shows that after the reaction time is 1.5 hours, the signal amplification efficiency gradually slows down. When the reaction time reaches 2 hours and 2.5 hours, there is no obvious difference in the changes of the two signals, but when the reaction time is 2.5 hours, the signal difference decreases, which indicates that when the reaction time is 2.5 hours, the dual signal cascade amplification is gradually slowing down, while the blank background interference is increasing. Therefore, 2 hours is selected as the optimal reaction time, at which time the signal output performance of the reaction system reaches the maximum and the best signal feedback is obtained.

[0172] The results of the effects of ABTS and H2O2 concentrations on signal output are shown in the figure below. Figure 3As shown in E and F, the difference between the UV signal and the G value increases with the increase of the ABTS concentration when the concentration of ABTS and H2O2 is within the range of 0.5-3mmol / L, and increases first and then decreases with the increase of the H2O2 concentration. When the concentration of ABTS is 2mmol / L, the signal response difference reaches the highest. When the ABTS concentration continues to increase, there is no significant change compared with the signal response of 2mmol / L, which indicates that under the same reaction conditions, the ABTS concentration of 2mmol / L is sufficient to be fully catalyzed by the G quadruplex produced by the reaction. When the concentration of H2O2 is 2mmol / L, the highest signal response difference is obtained. When the concentration of H2O2 continues to increase, the signal response difference gradually decreases, which indicates that under the condition of high concentration of H2O2, the substrate catalytic performance of the ABTS / H2O2 system will increase, thereby expanding the background interference and reducing the difference. Therefore, considering the cost and signal response, ABTS and H2O2 with a concentration of 2mmol / L can fully react and catalyze to obtain the best signal feedback.

[0173] In summary, after condition optimization, the best KCl concentration for the detection reaction was 15 mmol / L, pH was 6, the concentration of Hemin was 1.5 μmol / L, the reaction time of the system was 2 h, and the concentrations of ABTS and H2O2 were both 2 mmol / L.

[0174] 3. Sensitivity analysis

[0175] Under the above optimal conditions, a dual signal cascade amplification / triple helix DNA / diverse signal output VP detection method was constructed to detect 10-10 6 The VP with CFU / mL concentration was detected and the detection sensitivity of the method was analyzed. According to the signal output mechanism, the VP concentration could be detected and quantitatively analyzed from the changes in UV signal and G value.

[0176] The results are as follows Figure 4 As shown, at a UV wavelength of 420 nm, the UV absorption value of the reaction is 10 1 -10 6 In the range of VP concentration of CFU / mL, it was linearly related to VP concentration, and the linear regression equation was Y=0.09561*Log[C]+0.1892 (C: VP concentration; Y: UV absorption value, R 2 =0.99), and the detection limit was 1.8 CFU / mL (RSD = 3.7%). The colorimetric results gradually deepened with the increase of concentration. When the G value analysis of the colorimetric results was performed by a smartphone, the G value was 10 1 -10 6The VP concentration range of CFU / mL showed a good linear relationship with the VP concentration, and the linear regression equation was Y=-12.96*Log[C]+159.8 (C: VP concentration; Y: G value, R 2 =0.99), and the detection limit was 2.9 CFU / mL (RSD = 2.3%). It can be seen that the UV signal and the colorimetric signal are proportional to the concentration of VP, and the G value signal is inversely proportional to the concentration of VP, and there is a good linear relationship. This shows that the smooth occurrence of the TSMD / HCR / triple helix DNA reaction promotes the smooth assembly of the G quadruplex, and the UV signal, the colorimetric signal, and the G value analysis generated by the colorimetric signal all show regular signal feedback.

[0177] In summary, the colorimetric qualitative results and the two analytical quantitative results all show a certain linear relationship with the VP concentration, and the detection limit of the UV analysis result is similar to the detection limit obtained by the mobile phone G-value intelligent analysis. To a certain extent, the two signal outputs can not only provide accurate quantitative analysis of VP concentration, but also achieve the effect of data calibration, thereby increasing the credibility and accuracy of the detection.

[0178] 3. Specificity analysis

[0179] In order to further test the specificity of this method, Vibrio alginolyticus (VA) and Vibrio harveyi (VH) were selected as interference factor groups for control experiments, and the anti-interference ability of the constructed dual signal cascade amplification / multi-signal output detection method was analyzed through mixed VP, VA, and VH sample detection experiments.

[0180] Test results such as Figure 5 As shown in the figure, in the system with VP added, its UV absorption signal and G value analysis signal are significantly different from the detection results of VH and VA, indicating that this sensing method has good selectivity. For the detection of mixed samples of VH, VA and VP, its UV signal and G value are not significantly different from the detection results of VP samples, confirming that this method can be used for specific detection of VP in complex matrices and has good anti-interference ability.

[0181] Example 4 Detection of actual samples

[0182] Different samples were tested qualitatively and quantitatively. The VP-colonized shrimp samples were used to prepare the sample experimental group, which was tested using the constructed method (condition c). The actual detection performance was verified by preparing the experimental control group with direct addition of VP (condition b) and the sterilized shrimp samples as the blank experimental control group (condition a), and compared with the biochemical culture method in the national standard GB 4789.7-2013. The specific conditions were set as follows:

[0183] (a) Blank+AC+P+FS+MB+H1-G4a+H2+TFO-G4b+Hemin+ABTS+H2O2; (b) VP+AC+P+FS+MB+H1-G4a+H2+ TFO-G4b+Hemin+ABTS+H2O2; (c) Sample+AC+P+FS+MB+H1-G4a+H2+TFO-G4b+Hemin+ABTS+H2O2.

[0184] VP colonization shrimp 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 VP-infected shrimp 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.

[0185] 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.

[0186] Test results such as Figure 6 As shown, the three signal output results of the Sample group detected by the method provided by the present invention were not significantly different from those of the experimental group, but were significantly different from those of the blank control group, showing good detection specificity and sensitivity. In addition, it can be observed in the national standard biochemical culture determination results that VP is contained in the VP-colonized shrimp samples, which is in line with the actual detection situation and can meet the needs of on-site detection in marine ranches.

[0187] The sterilized seawater samples and shrimp meat samples were further taken for spike recovery analysis. The results are shown in Table 2. The average recovery level of the dual signal cascade amplification / multi-signal output detection method provided by the present invention is between 92.3% and 95%, and the RSD is between 2.81% and 4.45%. This shows that the DNA sensing method constructed by the present invention is suitable for the effective detection of actual marine ranch aquaculture samples, and the detection performance has good stability. It shows that the method constructed by the present invention has good qualitative and accurate quantitative performance in the actual detection of VP, and can provide a high-sensitivity detection application of multiple signal output results for marine ranch VP.

[0188] Table 2 Actual sample test results (n=3, mean±SD)

[0189]

[0190] In summary, the present invention provides a DNA functional material based on Toehold-mediated strand displacement reaction, introduces TSMD / HCR dual signal cascade amplification mechanism, effectively and rapidly amplifies the target signal, realizes sensitive detection of low concentration or even trace VP, and can detect VP at 10-10 6 CFU / mL VP concentration range has a good linear relationship, and the detection limit is as low as 1.8CFU / mL. At the same time, using DNA functional materials as signal output substrates, combined with the characteristics of G quadruplex / Hemin peroxidase, the TSMD / HCR dual signal cascade amplification products are specifically captured to form a triple helix DNA structure to complete the signal output. Finally, through the self-assembly of G quadruplexes and the enzyme-like properties of G quadruplex / Hemin complexes, three signal output modes of colorimetry, ultraviolet, and smart electronic devices (such as mobile phones) analysis are realized. This method has the self-calibration function of multiple signals to ensure the accuracy of the analysis results. It can detect VP quickly, accurately, sensitively, specifically, and efficiently, and provides a new idea for the multi-scenario and diversified detection of VP. It can realize visual detection of VP at the breeding site and accurate quantitative detection of VP in the laboratory, which promotes the further application of DNA functional materials in VP instant detection technology, provides a portable signal output mode, and provides a basis for the subsequent establishment of a portable paper-based POCT platform for VP marine ranches.

[0191] 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 DNA functional material based on Toehold-mediated strand displacement reaction, characterized in that: It contains VP aptamer, complementary strand, capture probe, signal strand, fuel strand, molecular beacon, hairpin probe H1-G4a, hairpin probe H2 and oligonucleotide strand; The sequences of the VP aptamer, complementary strand, capture probe, signal strand, fuel strand, molecular beacon, hairpin probe H1-G4a, hairpin probe H2 and oligonucleotide strand are shown in SEQ ID NO.1-9, respectively.

2. The method for preparing the DNA functional material according to claim 1, characterized in that: The VP aptamer, complementary strand, capture probe, signal strand, fuel strand, molecular beacon, hairpin probe H1-G4a, hairpin probe H2 and oligonucleotide strand are added to a buffer solution and mixed thoroughly.

3. The preparation method according to claim 2, characterized in that: The VP aptamer, complementary strand, capture probe, signal strand, fuel strand, molecular beacon, hairpin probe H1-G4a, and hairpin probe H2 need to be incubated at 90-98° for 10-15 min before use.

4. The preparation method according to claim 2, characterized in that: The molar ratio of the VP aptamer, complementary chain, capture probe, signal chain, fuel chain, molecular beacon, hairpin probe H1-G4a, hairpin probe H2 and oligonucleotide chain is (1-3): (1-3): (4-6): (4-6): (4-6): (4-6): (15-25): (15-20): (20-30).

5. Use of the DNA functional material according to claim 1 in detecting Vibrio parahaemolyticus for non-disease diagnosis purposes.

6. Use of the DNA functional material according to claim 1 in the preparation of Vibrio parahaemolyticus detection products.

7. A biosensor, characterized in that: Containing the DNA functional material according to claim 1.

8. The biosensor according to claim 7, characterized in that: It also contains hemin, ABTS and H2O2.

9. A method for detecting Vibrio parahaemolyticus for non-disease diagnosis purposes, characterized in that: The biosensor according to claim 7 or 8 is used to perform qualitative and / or quantitative detection of Vibrio parahaemolyticus.

10. A Vibrio parahaemolyticus detection kit, characterized in that: A biosensor comprising the biosensor according to claim 7 or 8.

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