CN109321635B - Nucleic acid detection method based on multiple hybrid chain reactions and application - Google Patents

Abstract

The invention discloses a nucleic acid detection method based on multiple hybrid chain reactions and application thereof, belonging to the technical field of biological detection. The detection method comprises the following steps: pretreating a sample to be detected into a solution containing target DNA, adding corresponding three hairpin probes H1, H2 and H3 into the solution, identifying and opening H1 by the target DNA, triggering H1 and H2 to generate hybrid chain reaction in a single direction or two directions, triggering and opening H3 by a loop sequence after the H2 is opened to form a plurality of hybrid chain products, capturing the plurality of hybrid chain products on an interface in a multi-valence manner by the hybridization of the opened H3 and the probe on the interface, and analyzing a signal label on the interface to realize the quantitative detection of the target DNA. In the detection method provided by the invention, the hybridization chain reaction is carried out in solution, so that the hybridization efficiency is obviously improved, and then the reaction product is captured on the interface efficiently in a multivalent capture mode, so that the ultrasensitive nucleic acid detection is realized; the designed signal probe has universality, and the detection cost is greatly saved.

Description

Nucleic acid detection method based on multiple hybrid chain reactions and application

Technical Field

The invention relates to the technical field of biological detection, in particular to a nucleic acid detection method based on multi-branch hybrid chain reaction and application thereof to accurate analysis and detection of drugs/biological small molecules, DNA, RNA, antigens/antibodies, enzymes, exosomes and cells in liquid biopsy.

Background

Liquid biopsy in accurate medical analysis is one of the current popular detection techniques, and changes in specific DNA, RNA, drugs/biomolecules, proteins, exosomes and cell contents in serum, plasma or blood can be used for early disease screening and diagnosis, disease postoperative recurrence judgment and drug-induced level monitoring. The current gene sequencing is successfully applied to early cancer screening, and the probability of cancer of people is predicted by detecting gene mutation, so that the method has high accuracy, but the current detection cost of the technology is still high, and the technology is not beneficial to wide popularization and use, so that the development of a simple, low-cost and high-accuracy sensor is urgently needed.

With the continuous development of DNA nanotechnology, various DNA nanostructure materials have been introduced for detecting disease-related DNA, RNA, etc. in human peripheral blood, and due to the generally low content of target molecules in human peripheral blood, Rolling Circle Amplification (RCA) (Lizardi p.m., Huang x.x., Zhu z.r., Ward p.b., Thomas d.c., Ward d.c., nat. genetics, 1998,19,225-232.) and Hybridization Chain Reaction (HCR) (Dirks r.m., Pierce n.a., p.natl.ad.sci.u.s.a., 2004, 152101, 15275-78.) are commonly used for amplifying output detection signals and detecting various biological target molecules. The HCR does not need extra polymerase compared with the RCA, and the reaction can be carried out at room temperature, and the product is a double-stranded long-chain structure with a repeating unit.

Patent document CN 106093438A discloses a portable method for detecting vascular endothelial growth factor by hybrid chain reaction, which is to form an antibody-protein-aptamer sandwich structure by VEGF, an aptamer and an antibody on a polyethylene micropore, include an 18-base extension sequence at the end of the aptamer, and then use a synthesized two-end sucrose transferase labeled auxiliary probe, where the two-end enzyme labeled probe uses the 18-base extension sequence as a template to initiate chain hybrid amplification reaction, so as to fix a large amount of sucrose transferase on the micropore, and these enzymes catalyze sucrose to convert into glucose, and use a glucometer to indicate the concentration of the generated glucose, thereby measuring the content of vascular endothelial growth factor in the target to be analyzed.

The hybrid chain reaction has been widely applied to the detection of various biological target molecules, and the main bottleneck defects existing at present have the following points:

HCR has higher hybridization efficiency in solution environment compared with interface, however, the signal output sensitivity of the common signal output method in solution, such as fluorescence, color development, chemiluminescence and the like, is poorer than that of the interface technology; the technology such as electrochemistry, interface fluorescence enhancement, Raman spectroscopy, surface plasmon resonance and the like with relatively higher signal output sensitivity needs HCR to be carried out at the interface, and the hybridization efficiency of HCR is reduced.

The hairpin probes used in HCR are not universal and vary with the assay subject.

And c, the stability of the hairpin probe used in the HCR is changed along with the change of a detection object, and when the stability of the hairpin structure is poor, the hybrid chain reaction is easily caused by the hairpin structure, so that the signal-to-noise ratio in the detection process is reduced.

d. Sensors that interface to initiate the hybridization chain reaction are generally more sensitive, but often require multiple steps for target detection.

e. The effective detection concentration range of the target cannot be regulated.

The currently developed methods using hybrid chain reaction combined with enzyme catalysis can realize the detection sensitivity of hundreds or even dozens of copies of DNA and RNA, however, the methods are complicated, and multi-step treatment is required to realize the detection of target molecules, so that the methods are limited to the ideal detection environment of a laboratory and are difficult to be applied to clinical detection.

Disclosure of Invention

The invention aims to provide a nucleic acid detection method based on multiple hybridization chain reactions, which utilizes three hairpin probes to carry out one-way or two-way hybridization chain reactions on target DNA to form multiple hybridization chain reaction products, and then utilizes interface capture and signal amplification output to further realize the detection of target molecules so as to solve the partial defects of the traditional hybridization chain reactions in target analysis, such as low detection sensitivity, poor hybridization efficiency, complicated experimental steps, no universality of detection probes, uncontrollable dynamic detection range and the like.

In order to achieve the purpose, the invention adopts the following technical scheme:

a nucleic acid detection method based on multi-branch hybridization chain reaction comprises the following steps:

(1) pretreating a sample to be detected to obtain a solution containing target DNA, wherein the target DNA comprises a sequence a;

(2) synthesizing a hairpin probe: designing hairpin probes H1, H2 and H3 according to a target DNA sequence, wherein H1 comprises three parts, namely H1b, H1a 'and H1c, a partial sequence of H1b and a partial sequence of H1c are complementary to form a double strand to serve as a stem part of a hairpin structure of H1, and H1 a' is a complementary sequence of a sequence; h2 includes three parts of H2b ', H2d and H2 c', wherein a partial sequence of H2b 'and a partial sequence of H2 c' are complementary to form a double strand serving as a stem of a hairpin structure of H2, H2b 'is complementary to H1b, and H2 c' is complementary to H1 c; h3 includes four parts H3e, H3f, H3e ', H3g, where H3e is complementary to H3 e' to form a double strand as the stem of the hairpin structure of H3, H3f forms the loop of the hairpin structure, H3g is the single-stranded sticky end of the 3 'end of H3, and H3 e' and H3g are complementary to H2 d;

or H1 includes two parts, A and B, where the partial sequence of A and the partial sequence of B are complementary into double strand as the stem of the hairpin structure of H1, A is the complementary sequence of a sequence; h2 includes three parts, A ', H2d and B', wherein the partial sequence of A 'is complementary to the partial sequence of B' to form a double strand as the stem of hairpin structure of H2, A 'is complementary to A, B' is complementary to B; h3 is designed as above;

the H2 or H3 is modified with a signal label;

(3) nucleic acid detection based on multiple hybrid chain reactions: adding H1, H2 and H3 into a solution containing target DNA, mixing, reacting at room temperature to form a reaction product of a plurality of hybrid chains, placing a carrier with a DNA probe on a gold interface, wherein the carrier is self-assembled with the DNA probe and is arranged in the reaction solution, the DNA probe has a sequence which is complementary and matched with partial sequences of H3f and H3e in H3, and the DNA probe is hybridized with H3 to capture the reaction product of the plurality of hybrid chains on the gold interface; and then analyzing the signal mark change on the gold interface to determine the content of the target DNA in the solution, and further calculating the content of the target object in the sample to be detected.

The principle of the detection method of the invention is as follows:

in the presence of a target DNA, the hairpin H1 can be recognized and opened (the ring part base of H1 or the base in the sticky end direction can be used as a target specificity recognition site according to the design), so that the hybridization chain reaction is initiated bidirectionally or unidirectionally, after H2 is opened, the ring part base is changed into a single straight chain, H3 can be opened continuously, the hybridization chain straight chain product is changed into a multi-branch long chain product, and the HCR product can be captured at an interface efficiently by hybridizing the multi-branch single chain and a probe assembled at any interface. Modifying a fluorescent signal mark or an electrochemical signal mark on each hairpin probe, wherein the signal increment of the interface is positively correlated with the concentration of a target, and judging the concentration of the nucleic acid according to the fluorescent intensity or the electrochemical signal. And (3) realizing the detection of the concentration of the unknown sample target by constructing a standard detection curve.

As the hybridization chain reaction is carried out in a solution system, the hybridization efficiency is obviously improved, the hybridization reaction product is efficiently captured on an interface in a multivalent capture mode, and the ultrasensitive nucleic acid detection is realized by analyzing the signal mark change.

Aiming at the design of the hairpin probe, the invention provides two schemes, namely: a sequence complementary to the target DNA is inserted in the middle of the conventional hairpin structure H1, and a base sequence other than the complementary strand of the target DNA is inserted in the middle of the conventional hairpin structure H2, the base sequence being complementary to H3. Specifically, the method comprises the following steps: h1 includes three parts of H1b, H1a ', H1c, H1 a' is a complementary sequence of a sequence, H1b and H1c are not completely complementary paired, H1b includes b1 and b2, H1c includes c1 and c2, b2 and c2 are complementary to form a double strand as a stem of a hairpin structure of H1, H1a 'and c1 serve as a loop of the hairpin structure, and b2 is shorter than H1 a'; h2 includes three parts of H2b ', H2d and H2c ', H2b ' includes b2 ' and b1 ', H2c ' includes c2 ' and c1 ', b2 ' is complementary to c2 ' to form a double strand as the stem of the hairpin of H2, b1 ' and H2d as the loop of the hairpin, H2b ' is complementary to H1b, and H2c ' is complementary to H1 c; h3 includes four portions H3e, H3f, H3e ', H3g, H3 e' and H3g are complementary to H2 d.

The target DNA is complementary with H1 a' in a loop sequence of H1, H1b, H1c, H1b and H1c exposing a stem part after H1 is opened respectively initiate HCR reaction, and bidirectional HCR reaction is realized. After the hairpin structure of H2 is changed into a straight chain, H2d in the middle can open H3 through the toehold chain substitution hybridization to form a multi-branched hybrid chain reaction product. By adopting the design of the scheme, when different target nucleic acid sequences are detected, only the recognition sequence H1 a' in H1 needs to be changed, H2 and H3 can be used universally, a signal mark is modified in H2 or H3, and the detection cost can be greatly saved by the common use of a signal probe.

Scheme II: with the conventional hairpin structure H1, H2 is a base sequence inserted in the middle of the conventional hairpin structure except the complementary strand of the target DNA, and is complementary to H3. Specifically, the method comprises the following steps: h1 includes two parts A and B, A includes A1 and A2, B includes B1 and B2, A2 and B2 are complementary to form double chain as the stem of H1 hairpin structure, B1 is used as the loop of hairpin structure, A is complementary with a sequence; h2 includes three parts, A ', H2d and B ', A ' includes A2 ' and A1 ', B ' includes B2 ' and B1 ', A2 ' and B2 ' are complementary to form a double chain as the stem of the hairpin structure of H2, A1 ' and H2d serve as the loop of the hairpin structure, A ' is complementary to A, B ' is complementary to B; the design of H3 is the same as above.

The target DNA is complementary with a stem sequence A of H1, and after H1 is opened, the B at the other end is exposed to initiate HCR reaction, so that unidirectional HCR reaction is realized. By adopting the design of the scheme, when different target nucleic acid sequences are detected, the recognition sequences in H1 and H2 need to be changed, and H3 can be used universally.

The design of H3 is the same in the two schemes, H3 e' and H3g in H3 and H2d in H2 are complementarily hybridized, and H3f and H3e are complementarily paired with DNA probes on an interface, so that the interface capture of the hybridization chain reaction product is realized.

Since H3 e' and H3g in H3 are complementary to H2d, the sequence of H3e is identical to the partial sequence of loop sequence H2d of H2, therefore, the DNA probe is likely to be hybridized with H2 in a complementary way, in order to reduce the probability that the DNA probe opens the hairpin structure of H2, the DNA probe is designed to be matched with the partial sequence of H3e in a complementary way, and the bases of loop sequence H3f of H3 are increased, so that the binding energy of the DNA probe and H3 is ensured to be greater than that of the DNA probe and H2.

Based on the design of the hairpin probe, the detection method provided by the invention can be carried out in a reaction system, and the identification of the target, the interface multivalent capture and the signal amplification output can be realized only by one step. The present study shows that the three hairpin probes can maintain self-stability in the hybridization solution in the absence of target DNA.

The sample to be detected is DNA, RNA, medicine/biological small molecule, antigen/antibody, enzyme, exosome or tumor cell. The detection method provided by the invention can detect other biological target molecules besides DNA and RNA, and in the step (1), target DNA corresponding to the content of the target molecules is released through the pretreatment of aptamer or DNase. The skilled person can select a corresponding pretreatment method according to a specific target molecule, for example, after an aptamer or a double-stranded probe partially complementary to the target molecule is bound to the target molecule, the released complementary strand is the target DNA for initiating a hybridization chain reaction.

The detection method has no specific requirement on the buffer system of the hybridization solution, and generally adopts high-salt, neutral and slightly alkaline buffer conditions which are favorable for hybridization reaction. Preferably, in step (1), the buffer system of the target DNA-containing solution is SPSC solution. The SPSC solution consists of: 50mM Na₂HSO₄/NaH₂SO₄，1M NaCl，pH 7.5。

In step (2), the hairpin probes H1, H2, H3 can be DNA, RNA, Peptide Nucleic Acid (PNA) or Locked Nucleic Acid (LNA).

The DNA probe may also be a peptide nucleic acid or a locked nucleic acid.

Research shows that increasing the number of bases for stem hybridization in H1 can improve the stability of hairpin structure, reduce the ability of target nucleic acid to recognize and open hairpin structure, and reduce the activity of H1 participating in hybridization chain reaction, so that the dynamic detection range of target nucleic acid can be adjusted to high concentration, and only higher concentration can generate equivalent signal change.

When an auxiliary strand which can be complementary with the stem of H1 is added into the detection system, the auxiliary target nucleic acid can be used for opening the H1 hairpin structure, the recognition activity of H1 and the activity participating in the hybridization chain reaction are increased, so that the dynamic detection range of the nucleic acid can be adjusted to a low concentration, and less target nucleic acid is needed for generating equivalent signal change.

Therefore, the ability of H1 to bind to target DNA can be precisely regulated by changing the free energy of H1 or adding an auxiliary chain, so that the dynamic detection range of the DNA can be regulated.

Those skilled in the art can perform design operations such as substitution, deletion, or addition of bases on the H1, H2, and H3 sequences as appropriate, and achieve the above-described multi-branch and single/double-directional hybridization chain reaction equally. Preferably, in step (2), the sequences H1b, H1 a' and H2d are all 18-28bp in length. This length range is suitable in view of cost and stability.

More preferably, the length of H1B, H1a ' and H2d sequences is 24bp, wherein B1, c1, B1 ', c1 ', A1, B1, A1 ' and B1 ' are 6bp, and B2, c2, B2 ', c2 ', A2, B2, A2 ' and B2 ' are 18 bp. H3 consists of two 18 base stem H3e, H3 e', 8 base loop H3f and 6 base sticky end H3 g. The corresponding DNA probe consisted of 20 bases, 8 of which were complementary to H3f, and the remaining 12 were complementary to H3 e.

Preferably, the signal label is biotin or a fluorophore.

Preferably, the working concentration of H1, H2, H3 is 1 nM-100. mu.M.

When biotin (biotin) is marked on H2 or H3, the hybrid chain reaction is finished, excessive horseradish peroxidase modified streptavidin (SA-HRP) is added into the reaction solution to be combined with the biotin in the multi-hybrid chain reaction product, a gold electrode with a DNA tetrahedron top probe self-assembled on the interface is placed in the reaction solution to capture the multi-hybrid chain reaction product, then the gold electrode is taken out and fully cleaned and is placed in a TMB substrate solution containing hydrogen peroxide, and the catalytic reduction current generated by the reduction of the TMB catalyzed by the horseradish peroxidase is detected. The catalytic reduction current is positively correlated with the quantity of horseradish peroxidase on an electrode interface, and simultaneously the quantity of horseradish peroxidase is positively correlated with the target DNA, so that the target DNA in a solution to be detected can be quantitatively analyzed by detecting the change of the catalytic reduction current. Specifically, a standard curve is drawn, and then quantitative analysis is performed according to the standard curve.

Preferably, 100nM of H1, H2-biotin and H3 are added to 100. mu.L of SPSC solution containing the target DNA, and the reaction is carried out at room temperature for 2.5 hours, wherein the concentration of SA-HRP in the reaction system is 2.5. mu.g/mL. Under the above conditions, the best signal-to-noise ratio can be obtained.

When FAM-dabcyl fluorescence quenching group is marked on H2 or H3, when target DNA does not exist in the solution, the distance between the fluorescence and the quenching group is about 2nm, the fluorescence emitted by FAM is quenched in a short distance, when the target DNA is added to trigger multiple hybridization chain reactions, the distance between FAM and dabcyl is increased to about 10.2nm, the fluorescence emitted by FAM is obviously enhanced, and the target DNA is detected through a fluorescence switch.

Preferably, 50nM of H1, H2-biotin and H3 are added to 100. mu.L of SPSC solution containing the target DNA, and the reaction is carried out at room temperature for 2.5 hours. Under the above conditions, the hybridization reaction obtains a fluorescence intensity almost close to saturation.

In step (3), the DNA probe and the reaction solution are incubated at room temperature for 30min to obtain the best signal-to-noise ratio.

The invention also aims to provide a biological target molecule detection sensor based on multi-branch hybridization chain reaction, wherein the biological target molecule is pretreated to obtain target DNA containing a sequence a, the detection kit comprises hairpin probes H1, H2 and H3, H1 comprises three parts, namely H1b, H1a 'and H1c, wherein the partial sequence of H1b is complementary to the partial sequence of H1c to form a double strand serving as a stem part of a hairpin structure of H1, and H1 a' is a complementary sequence of the sequence a; h2 includes three parts of H2b ', H2d and H2 c', wherein a partial sequence of H2b 'and a partial sequence of H2 c' are complementary to form a double strand serving as a stem of a hairpin structure of H2, H2b 'is complementary to H1b, and H2 c' is complementary to H1 c; h3 includes four parts, H3e, H3f, H3e 'and H3g, where H3e is complementary to H3 e' to form a double strand as the stem of the hairpin structure of H3, H3f forms the loop of the hairpin structure, H3g is the 3 'single-stranded sticky end of H3, and H3 e' and H3g are complementary to H2 d;

or H1 includes two parts, A and B, where the partial sequence of A and the partial sequence of B are complementary into double strand as the stem of the hairpin structure of H1, A is the complementary sequence of a sequence; h2 includes three parts, A ', H2d and B', wherein the partial sequence of A 'is complementary to the partial sequence of B' to form a double strand serving as the stem of hairpin structure of H2, A 'is complementary to A, and B' is complementary to B; h3 is designed as above;

the 5' end of H2 or H3 is marked with biotin or a fluorescent group;

also included are electrodes or chips with self-assembled DNA probes on the gold interface, which have sequences that are complementary paired to partial sequences of H3f and H3e of H3.

The multiple hybrid chain products formed by the hairpin probes H1, H2 and H3 provided by the invention are captured to any sensing interface through hybrid multivalence, so that the hybrid chain reaction technology provided by the invention can be applied to other sensing fields besides the electrochemical sensor and the fluorescence sensor, such as: SPR, microbalance, surface fluorescence enhancement, Raman and other interface sensing fields.

Preferably, the kit further comprises an auxiliary strand for assisting the target DNA in opening the hairpin structure of H1, wherein the 3' end of H1 has a single-stranded sticky end H1x, and the auxiliary strand is complementary with partial sequences of H1x and H1 c.

The buffer system of the detection sensor adopts SPSC solution.

Hairpin probes H1, H2, H3 can be DNA, RNA, Peptide Nucleic Acid (PNA), or Locked Nucleic Acid (LNA). The DNA probe may also be a peptide nucleic acid or a locked nucleic acid.

Specifically, when the target DNA is Exon-5 in the p53 gene, the sequence of H1 is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3, the sequence of H2 is shown as SEQ ID NO.4, and the sequence of H3 is shown as SEQ ID NO. 5;

when the target DNA is Exon-6 in the p53 gene, the sequence of H1 is shown as SEQ ID NO.6, the sequence of H2 is shown as SEQ ID NO.4, and the sequence of H3 is shown as SEQ ID NO. 5.

When different DNA dynamic concentration ranges are detected, the stability of H1 is changed by using an auxiliary chain, and the sequence of the auxiliary chain is shown as SEQ ID NO.7 or SEQ ID NO. 8.

The invention has the following beneficial effects:

(1) in the detection method provided by the invention, the hybridization chain reaction is carried out in solution, so that the hybridization efficiency is obviously improved; by designing three hairpin probes, a plurality of hybrid chain reaction products are formed, the reaction products are efficiently captured on an interface in a multivalent capture mode, and ultrasensitive nucleic acid detection is realized by analyzing signal markers.

The steps can realize target identification, interface multivalent capture and signal method output only by one step, the defect of complicated steps of the traditional method is overcome, and the detection sensor based on the technology has market commercialization prospect.

(2) The signal probe designed by the invention has universality, and the detection cost is greatly saved.

(3) By adjusting the stability of the hairpin structure of H1, the affinity of H1 for recognizing the target can be improved or weakened, thereby realizing the adjustment of the dynamic range of nucleic acid detection.

Drawings

Fig. 1 shows the free energy of five H1 structures designed using NUPACK software simulation.

FIG. 2 shows the results of two-way chain hybridization in example 1, in which (A) a schematic diagram of the reaction of two-way chain hybridization; (B) 5H 1 structures are respectively verified with the target DNA recognition ability through agarose gel electrophoresis; (C) identifying 5H 1 structures with target DNA and initiating two-way hybridization chain reaction agarose gel electrophoresis verification; (D) and (3) verifying the two-way hybridization chain reaction mechanism by agarose gel electrophoresis.

FIG. 3 shows the fluorescence intensity dynamics of 5H 1 structures for recognizing 0 and 100nM target DNA (A) H1-i2 structure; (B) h1-i1 structure; (C) h1-0 structure; (D) h1-a1 structure; (E) h1-a2 structure.

FIG. 4 is a schematic diagram of a bidirectional chain hybridization reaction, (A) a schematic diagram of the 3 'end direction and the 5' end direction of the bidirectional chain hybridization reaction and a verification of the fluorescence intensity variation; (B)3 'end direction and 5' end direction hybridization chain reaction gel electrophoresis verification; (C) identifying target DNA by a one-step method of two-way hybridization chain reaction, carrying out multivalent capture and fluorescence imaging on gold interface hybridization chain products, and comparing the interface fluorescence intensity of 0nM target DNA and 100nM target DNA.

FIG. 5 shows the detection of target DNA by the two-way hybridization chain reaction fluorescence switch effect, and (A) the detection of DNA at a concentration of between 0.5pM and 50. mu.M in solution by the two-way hybridization chain reaction fluorescence switch effect, with the fluorescence intensity being positively correlated with the nucleic acid concentration. The dynamic range of DNA detection can be regulated and controlled by different H1 structures, and the detection sensitivity is about 5 orders of magnitude regulation and control; (B) the percent fluorescence enhancement is positively correlated with nucleic acid concentration.

FIG. 6 is a graph of the cyclic voltammograms for the electrochemical catalytic detection of target DNA, (A) for the electrochemical catalytic detection of 0 and 10nM target DNA; (B) i-t curves for electrochemical catalytic detection of 0 and 10nM target DNA.

FIG. 7 is the concentration optimization of SA-HRP with prebinding of biotin in the chain hybridization reaction product.

FIG. 8 shows that the target DNA is detected electrochemically by 5H 1 structures, (A) the H1-i2 structure is positively correlated with the stable catalytic current value when being used for electrochemically detecting the target DNA between 1nM and 10 μ M; (B) when the H1-i1 structure is used for electrochemically detecting target DNA between 100pM and 1 mu M, the structure is positively correlated with the stable catalytic current value; (C) when the H1-0 structure is used for electrochemically detecting target DNA between 1pM and 100nM, the structure is positively correlated with the stable catalytic current value; (D) when the H1-a1 structure is used for electrochemically detecting target DNA between 100fM-10nM, the structure is positively correlated with the stable catalytic current value; (E) when the H1-a2 structure is used for electrochemically detecting target DNA between 10fM-1nM, the structure is positively correlated with the stable catalytic current value; taking the measured standard curve as an example, after hill curve fitting, the conversion relation between the concentration and the signal is as follows: y110.32 + (12132.78X)^0.32)/(1.29+X^0.32) Wherein X is the DNA concentration in units of fM and Y is the measured electrocatalytic stability current value in units of nA; (F) the 5H 1 structures detect the corresponding detection limit change of the target DNA through bidirectional hybridization chain reaction and simultaneously detect the corresponding stable catalytic current value when 1nM Exon-5.

FIG. 9 shows agarose gel electrophoresis verification of two-way hybridization chain reaction and one-way hybridization chain reaction.

FIG. 10 shows that 3H 1 structures are respectively identified with target DNA through sticky ends and subjected to agarose gel electrophoresis verification of one-way hybridization chain reaction.

FIG. 11 shows that different H1 structures catalyze the detection of target DNA by bidirectional or unidirectional hybridization chain reaction, (A) 5H 1 structures electrochemically catalyze the detection of target DNA in different dynamic ranges by bidirectional hybridization chain reaction; (B) 3H 1 structures are respectively identified with the target DNA through sticky ends and trigger one-way hybridization chain type reverse electrochemical catalysis to detect the target DNA in different dynamic ranges; (C) the stable catalytic current values corresponding to the background and detection limit concentrations in the step (A); (D) the values of the stable catalytic current corresponding to the background and detection limit concentrations in (B) are shown.

FIG. 12 shows the electrochemical detection of Exon-5 and Exon-6 in 75% serum using H1-a2 structure (A) 7 samples at different concentrations of Exon-5 and Exon-6 at 100pM, 200pM, 500pM and 1000 pM; (B) standard curves for Exon-5 and Exon-6 were detected in 75% serum.

Detailed Description

The features and advantages of the present invention are further illustrated by the following examples, which are provided merely to illustrate the process of the present invention and are not intended to limit the remainder of the disclosure in any way.

Example 1

Nucleic acid detection based on bidirectional chain type hybridization reaction and fluorescence switch effect

1. Designing a hairpin probe: on the basis of a classical hybridization chain reaction sequence, a related hairpin probe is designed according to a detected target DNA (a DNA sequence Exon-5 in a p53 gene) and is combined with NUPACK software, and a related nucleic acid sequence is synthesized and modified by the committee biological engineering corporation (Shanghai).

The hairpin probe concentration was accurately quantified using an ultraviolet spectrophotometer using SPSC buffer (50mM Na)₂HSO₄/NaH₂SO₄1M NaCl, pH 7.5) to 5 μ M, and rapidly cooling to 4 ℃ in PCR at 95 ℃ for 2min to form a stable hairpin structure.

DNA probes designed in Table 1

Note: a20, SH-B20, SH-C20 and SH-D20 are used for synthesizing DNA tetrahedral probes assembled on electrodes or gold sheet interfaces to capture HCR products; single-stranded probes for detection are also possible.

H1(Exon-5) -inhibitor-1, H1(Exon-5) -inhibitor-2 and H1(Exon-5) form hairpin structures H1-i1, H1-i2 and H1-0 respectively; the Activator-1 added in H1(Exon-5) is H1-a1, and the Activator-2 added in H1(Exon-5) is H1-a 2.

FIG. 1 analyzes the free energy of different H1 hairpin structures by NUPACK software, and when the number of base pairs of stems is increased, the free energy of stem-loop structures is reduced, the stability of stem-loops is increased, and the recognition affinity of the stem-loop structures and targets is reduced. When the auxiliary strand is added to hybridize with H1 through the sticky end, the stability of the H1 stem is reduced, and the binding ability to the target can be improved.

FIG. 2(A) is a schematic diagram of a bidirectional chain hybridization reaction. The hybridization efficiency of different H1 hairpin structures (1. mu.M) and nucleic acid (200nM) at the same concentration was verified by gel electrophoresis, and the results are shown in FIG. 2(B), where the yield of H1-a2 hybridized with DNA is highest, indicating that the recognition activity of H1-a2 is strongest and the recognition activity of H1-i2 is lowest. Similarly, the efficiency of the two-way chain hybridization reaction initiated by different H1 is different (FIG. 2(C)), when no priming DNA exists, the three hairpin structures can maintain the stability of the three hairpin structures in the hybridization solution, and after the target DNA is added, the hybridization chain product with large molecular weight is generated. FIG. 2(C) further verifies the stability of the hairpin structure itself by gel electrophoresis.

FIG. 3 further examines the two-way chain hybridization reaction triggered by different H1 structures by monitoring the fluorescence intensity change in real time. The 100. mu.L SPSC solution contains 50nM of H1, FAM-H2-dabcyl and H3, the hairpin structure remains stable when no target nucleic acid is present in the solution, the distance between the quenching and fluorescent group on H2 is about 2nM, the fluorescence emitted by FAM is quenched in a short distance, and the fluorescence intensity remains substantially stable with time. When 100nM Exon-5 was added, the two-way chain hybridization reaction initiated, quenched and increased the distance of the fluorophore, and fluorescence gradually increased over time. The different H1 structures have different abilities to recognize nucleic acids and different activities involved in HCR reactions, so the rate of change of fluorescence is fastest in the H1-a2 system with the best recognition activity.

FIG. 4(A) verifies the 3 'and 5' direction of the chain hybridization reactions, and after H1 recognizes the target DNA (DNA 1 in Table 1), the 3 'direction of the hybridization chain reaction is blocked when 3' termination is added; upon addition of 5 'termination (sequence identical to DNA 2), the hybridization chain reaction in the 5' direction was blocked. The hybridization products are respectively marked as 5 'trigger and 3' trigger, 100nM 5 'trigger or 3' trigger is added into SPSC solution containing 50nM H1, H2 and H3, and the change of fluorescence intensity shows that 5 'trigger and 3' trigger can initiate chain hybridization reaction in each direction, thereby verifying that H1 can initiate two-way hybridization chain reaction after being opened by loop sequence.

FIG. 4(B) further verifies the formation of 5 ' and 3 ' triggers and 3 ' direction strand hybridization reactions by gel electrophoresis.

Fig. 4(C) uses fluorescence imaging to verify one-step implementation of target recognition, multivalent capture and signal amplification output. 100nM target DNA1 (the total volume is 100 muL) is added into 100nM probe solutions of H1, H2 and H3, after a bidirectional chain type hybridization reaction is initiated, each chain type product is provided with a plurality of single-stranded structures and can be hybridized with a tetrahedral top probe assembled on a gold sheet interface, so that the chain type hybridization products are efficiently hybridized on the gold interface through polyvalent capture, a fluorescence microscope is used for imaging the gold sheet to output corresponding fluorescence signals, and the higher the concentration of the target substance is, the stronger the fluorescence intensity of the gold interface is. In FIG. 4(C), the fluorescence imaging results of 0nM and 100nM Exon-5 respectively trigger the two-way chain hybridization reaction, and the fluorescence intensity of the gold interface containing 100nM target is significantly increased. The method is only exemplary in detection and verification, and the detection performance of nucleic acid is not studied in detail by the method. If the fluorescence-enhanced gold chip and the microarray spotting technology are combined, the sensitivity of nucleic acid detection can be further improved, and high-throughput analysis and test can be realized.

10nM H1, FAM-H2-dabcyl and H3 were mixed with different concentrations of Exon-5 (0-50. mu.M) in SPSC buffer solution, incubated at room temperature for 2.5H, and the fluorescence intensities (excitation voltage 800V, excitation slit 5nM, emission slit 5nM, excitation wavelength 492nM, wavelength scanning range 502-700nM) were measured for each concentration of target DNA using a fluorescence spectrometer. FIG. 5 shows that different H1 structures initiate bidirectional chain hybridization to detect Exon-5, and different H1 recognition activities can regulate and control the detection dynamic range, namely H1-i2(10 nM-50. mu.M), H1-i1(1 nM-50. mu.M), H1-0(50 pM-50. mu.M), H1-a1(5 pM-50. mu.M), and H1-a2(0.5 pM-50. mu.M).

Example 2

Nucleic acid detection based on bidirectional chain type hybridization reaction and electrochemical catalysis

Designing a hairpin probe: the modification of the fluorescent group on H2 is changed into the modification of biotin, and the rest sequences are kept consistent.

100 mu L of SPSC solution contains 100nM H1, H2-biotin and H3, the two-way hybridization chain reaction is initiated in the presence of target DNA (DNA 1 in Table 1), after 2.5 hours, excessive horseradish peroxidase modified streptavidin is added into the solution and can be combined with the biotin on the two-way hybridization chain reaction product, the complex is captured at an electrode interface in a multivalent way by hybridization of a plurality of H3 and DNA tetrahedron top probes self-assembled at a gold interface, the electrode is placed in TMB substrate solution, and in the presence of hydrogen peroxide, the horseradish peroxidase catalyzes TMB reduction to generate catalytic reduction current. The catalytic reduction current is positively correlated with the quantity of horseradish peroxidase on an electrode interface, and simultaneously, the quantity of horseradish peroxidase is positively correlated with nucleic acid molecules, so that the target nucleic acid is sensitively analyzed by amplifying an electric signal through the catalytic reduction current.

As can be seen from FIG. 6, in the cyclic voltammetry curves of 10nM target DNA, there was a distinct reduction peak (FIG. 6(A)) compared to the absence of target DNA, and the steady catalytic reduction current (6841nA) was much greater than the background signal (110.6nA) (FIG. 6 (B)).

The concentration of SA-HRP when the HCR reaction solution was pre-conjugated with SA-HRP was optimized during the detection, and as a result, the best signal-to-noise ratio was obtained when the concentration was 2.5. mu.g/mL (FIG. 7).

H1 with different activities can detect different dynamic concentration ranges of DNA, H1-i2 can detect 1 nM-10. mu.M (FIG. 8(A)), H1-i1 can detect 100 pM-10. mu.M (FIG. 8(B)), H1-0 can detect 1 pM-1. mu.M (FIG. 8(C)), H1-a1 can detect 100fM-100nM (FIG. 8(D)), and H1-a2 can detect 10fM-10nM (FIG. 8 (E)). FIG. 8(F) shows that the five structures of H1 have different minimum detection limits, and the catalytic reduction current is gradually increased with the increase of the activity of H1 in the presence of the same 1nM target DNA.

Example 3

Nucleic acid detection based on one-way chain type hybridization reaction and electrochemical catalysis

The hairpin probes H1, H2 and H3 are also used to change the target DNA to DNA2, which can open H1 by a chain substitution reaction with the stem of H1.

The target nucleic acid DNA2 initiates H1 and H2 one-way chain type hybridization reaction through sticky end chain substitution hybridization opening on H1, opened H2 can also form a multi-branch hybridization chain type long-chain product through chain substitution opening H3, and the electrochemical detection process is basically consistent with that of the detection DNA 1.

FIG. 9 verifies by agarose gel electrophoresis that the target DNA1 and DNA2 can prime the hybridization chain reaction bi-directionally and uni-directionally, respectively.

FIG. 10 verifies by agarose gel electrophoresis that DNA2 triggers the different active H1 hybridization chain reaction in one direction.

The different dynamic ranges of the two-way hybridization chain reaction and the one-way hybridization chain reaction were summarized for detection of DNA1 (FIGS. 11(A) and 11(C)) and DNA2 (FIGS. 11(B) and 11(D)), respectively. H1 with different activities can detect different dynamic concentration ranges of DNA2 respectively, H1-i2 can detect 1nM-10 muM, H1-i1 can detect 1pM-1 muM, and H1-0 can detect 1fM-1 nM.

Example 4

Nucleic acid detection based on bidirectional chain type hybridization reaction and electrochemical catalysis in serum

When DNA is detected in serum, the efficiency of the hybridization chain reaction is reduced due to interference of a large number of biomolecules other than non-targets, and the reduced concentration of salt ions required for hybridization. To increase the efficiency of the hybridization chain reaction in serum, 25. mu.L of 2XSPSC buffer was added to 75. mu.L of serum. The serum contains a large amount of albumin which can play a role in blocking and preventing horseradish peroxidase from being adsorbed on an electrode interface, so that the detection sensitivity of detecting DNA1 in 75% of serum is equivalent to that in a buffer solution.

FIG. 12(A) shows the results of H1-a2 as a nucleic acid recognition structure in 75% serum for electrochemical detection of 7 samples with different concentrations of Exon-5 and Exon-6, the concentrations being 100pM, 200pM, 500pM and 1000pM, respectively, the results of detection being substantially consistent with the actual concentration of added nucleic acid, indicating that there is good detection accuracy in complex environments.

Standard curves for detection of Exon-5 and Exon-6 in 75% serum were constructed, ranging from 10fM to 10nM (FIG. 12 (B)).

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Claims (9)

1. A biological target molecule detection sensor based on multi-branch hybridization chain reaction, the biological target molecule is pretreated to obtain target DNA containing a sequence a, the biological target molecule detection sensor is characterized in that the biological target molecule detection sensor comprises hairpin probes H1, H2 and H3,

h1 includes three parts of H1b, H1a ', H1c, H1 a' is a complementary sequence of a sequence, H1b includes b1 and b2, H1c includes c1 and c2, b2 and c2 are complementary to form a double strand as a stem of a hairpin structure of H1, H1a 'and c1 serve as loop parts of the hairpin structure, and b2 is shorter than H1 a';

h2 includes three parts of H2b ', H2d and H2c ', H2b ' includes b2 ' and b1 ', H2c ' includes c2 ' and c1 ', b2 ' is complementary to c2 ' to form a double strand as the stem of the hairpin structure of H2, b1 ' and H2d are the loop of the hairpin structure, H2b ' is complementary to H1b, H2c ' is complementary to H1c, and H2d is a base sequence other than the complementary strand of the target DNA;

h3 includes four parts H3e, H3f, H3e ', H3g, where H3e is complementary to H3 e' to form a double strand as the stem of the hairpin structure of H3, H3f forms the loop of the hairpin structure, H3g is the single-stranded sticky end of the 3 'end of H3, and H3 e' and H3g are complementary to H2 d;

the length of the H1b, H1 a' and H2d sequences is 18-28 bp; the H2 or H3 is modified with a signal label;

the gold-contained electrode or chip is self-assembled with DNA probe with the sequence complementary and matched with the partial sequence of H3f and H3e of H3;

the nucleic acid detection method using the biological target molecule detection sensor comprises the following steps:

(1) pretreating a sample to be detected to obtain a solution containing target DNA, wherein the target DNA comprises a sequence a;

(2) synthesizing the hairpin probe;

(3) nucleic acid detection based on multiple hybrid chain reactions: adding H1, H2 and H3 into a solution containing target DNA, mixing, reacting at room temperature to form a reaction product of multiple hybrid chains, placing a carrier with a self-assembled DNA probe on a gold interface into the reaction solution, and capturing the reaction product of the multiple hybrid chains on the gold interface by hybridizing the DNA probe with H3; and then analyzing the signal mark change on the gold interface to determine the content of the target DNA in the solution, and further calculating the content of the target object in the sample to be detected.

2. The biological target molecule detection sensor of claim 1, wherein the sample to be detected is DNA, RNA, drug/small biological molecule, antigen/antibody, enzyme, exosome or tumor cell.

3. The sensor for detecting biological target molecules according to claim 1, wherein in the step (1), the buffer system of the solution containing the target DNA is SPSC solution.

4. The biological target molecule detection sensor of claim 1, wherein the lengths of the H1b, H1a ' and H2d sequences are 24bp, wherein b1, c1, b1 ', c1 ' are 6bp, and b2, c2, b2 ', c2 ' are 18 bp; h3 consists of two 18 base stem H3e, H3 e', 8 base loop H3f and 6 base sticky end H3 g.

5. The biological target molecule detection sensor of claim 1, wherein the 5' end of H2 or H3 is labeled with biotin or a fluorophore.

6. The biological target molecule detection sensor of claim 1, wherein the working concentration of H1, H2, H3 is 1nM-100 μ Μ.

7. The biological target molecule detection sensor of claim 1, further comprising an auxiliary strand for assisting the target DNA in opening the hairpin structure of H1, wherein the 3' end of H1 has a single-stranded sticky end H1x, and the auxiliary strand is complementary to partial sequences of H1x and H1 c.

8. The biological target molecule detection sensor of claim 1, wherein the DNA probe is a locked nucleic acid probe or a peptide nucleic acid probe.

9. The biological target molecule detection sensor of claim 1, wherein when the target DNA is Exon-5 in p53 gene, the sequence of H1 is shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3, the sequence of H2 is shown as SEQ ID No.4, and the sequence of H3 is shown as SEQ ID No. 5;

when the target DNA is Exon-6 in the p53 gene, the sequence of H1 is shown as SEQ ID NO.6, the sequence of H2 is shown as SEQ ID NO.4, and the sequence of H3 is shown as SEQ ID NO. 5.

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