Nucleic acid detection sensor based on S9.6 antibody bivalent capture

Abstract

The invention discloses a nucleic acid detection sensor based on S9.6 antibody bivalent capture, and belongs to the technical field of biological detection. The sensor comprises hairpin probes H0, H1 and H2, a single-chain probe I and an electrode or a chip which is self-assembled with an S9.6 antibody, the single-chain probe I is used for initiating H1 and H2 to generate a hybridization chain reaction so as to prepare a signal amplification material, in the presence of target nucleic acid, the hairpin probe H0 is recognized and opened and is hybridized with the prepared signal amplification material so as to form a compound, the compound contains two sections of DNA / RNA double strands, the DNA / RNA double strands are specifically recognized by an S9.6 antibody so as to be captured on a sensing interface, and the DNA / RNA double strands are captured by the S9.6 antibody. And determining the content of target nucleic acid by analyzing the signal change on the sensing interface. The method is suitable for any single-stranded nucleic acid detection, the detection process is simple, convenient and rapid, and high-sensitivity detection performance is obtained.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a nucleic acid detection sensor based on S9.6 antibody divalent capture and its use in accurate analysis and detection of DNA and RNA. Background Art

[0002] Liquid biopsy is one of the most popular detection technologies currently. It detects specific DNA and RNA changes in body fluids such as blood, saliva, and urine. It can be used for early disease screening and diagnosis, postoperative disease recurrence judgment, and pharmacokinetic level monitoring. Currently, gene sequencing has been successfully applied to early cancer screening. By detecting gene mutations, the probability of an individual suffering from cancer is predicted. This method has a high accuracy rate. However, the detection cost of this technology is high, which is not conducive to its widespread promotion and use. Therefore, there is an urgent need for the development of simple, low-cost, fast, and highly accurate sensors.

[0003] With the continuous development of DNA nanotechnology, various DNA technologies have emerged for detecting disease-related nucleic acids in human body fluids. Since the target molecule content in human body fluids is generally low, the sensors currently developed often use DNA amplification technology, such as hybridization chain reaction (Dirks RM, Pierce NA, Triggered amplification by hybridization chain reaction. P. Natl. Acad. Sci. USA, 2004, 101, 15275-15278) to amplify the output detection signal.

[0004] Hybridization chain reaction has been widely used in the detection of various biological target molecules. The main bottleneck defects currently exist are as follows: a. Although the HCR hybridization efficiency in homogeneous solutions is high, the signal output methods of homogeneous sensors, such as fluorescence, colorimetry, chemiluminescence, etc., require signal molecules to diffuse to the interface, and their sensitivity is relatively poor compared to heterogeneous sensors; heterogeneous sensors also require HCR to be carried out at the interface, which weakens the reaction efficiency of HCR. b. The hairpin probes used in HCR are not universal and change with the change of the detection object. c. The stability of the hairpin probes used in HCR changes with the change of the detection object. When the hairpin structure is less stable, it is easy to trigger a hybridization chain reaction by itself, reducing the signal-to-noise ratio during the detection process. d. Heterogeneous sensors are generally more sensitive, but usually require multiple steps for target detection. e. Current HCR-related sensors are difficult to achieve rapid and sensitive detection at the same time.

[0005] DNA-RNA hybrid-specific monoclonal antibody S9.6 can recognize DNA / RNA hybrid double strands. Using the specific binding ability of S9.6 antibody to DNA / RNA double strands, S9.6 antibody is often used for RNA detection (Mo Wang, et al. Label-free, Ultrasensitive and Electrochemical Immunosensing Platform for microRNA Detection Using Anti-DNA: RNA Hybrid Antibody and Enzymatic Signal Amplification. Electrochimica Acta, 2015, 165, 130-135). However, the types of sensors developed based on S9.6 antibody are limited, and there are no research reports on combining it with signal amplification reactions (such as HCR) to improve the sensitivity of target detection. Summary of the invention

[0006] The purpose of the present invention is to provide a nucleic acid detection sensor based on S9.6 antibody divalent capture, combining the S9.6 antibody capture technology with the hybridization chain reaction to achieve high-sensitivity detection of target nucleic acids, so as to solve the defects of traditional hybridization chain reaction in nucleic acid analysis, such as low detection sensitivity, poor hybridization efficiency, cumbersome and time-consuming experimental steps, and the inability of detection probes to be universal.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The present invention provides a nucleic acid detection sensor based on the bivalent capture of S9.6 antibody, wherein the nucleic acid is a target RNA or DNA containing a sequence obtained after pretreatment of the sample, and the sensor comprises hairpin probes H0, H1 and H2, a single-stranded probe I, and an electrode or chip self-assembled with S9.6 antibody;

[0009] The H0 includes three parts: H0a', H0b and H0c, wherein H0a' is a complementary sequence of the a sequence. When the a sequence is RNA, H0a' and H0b are DNA sequences, and H0c is a DNA or RNA sequence. When the a sequence is DNA, part of the sequence in H0a' of H0 is an RNA sequence, and the rest is a DNA sequence. The partial sequence of H0a' is complementary to H0c to form a double strand as the stem of the H0 hairpin structure, and H0b is the loop of the H0 hairpin structure.

[0010] The I includes two parts, Id and Ic', Ic' is the complementary sequence of H0c, when H0c is a DNA sequence, Ic' is an RNA sequence; when H0c is an RNA sequence, Ic' is a DNA sequence; Id, H1 and H2 are all DNA sequences, and Id is used to trigger a hybridization chain reaction between H1 and H2;

[0011] The H1 and / or H2 are modified with a signal tag.

[0012] The detection principle of the biosensor provided by the present invention is as follows:

[0013] In the presence of the target nucleic acid, the card probe H0 can be identified and opened. After H0 is opened, the stem H0c is exposed, which can hybridize with the Ic' of the initiator chain I (initiator). The Id of the single-stranded probe I triggers H1 and H2 to undergo a hybridization chain reaction to form a "target nucleic acid / H0 / pHCR" complex. The present invention uses probe sequence design to allow the target nucleic acid to hybridize with H0 to form a DNA / RNA double strand, and H0c and Ic' to hybridize to form another DNA / RNA double strand. Therefore, the "target nucleic acid / H0 / pHCR" complex contains two DNA / RNA double strands. Through the specific recognition ability of S9.6 and DNA / RNA double strands, the "target nucleic acid / H0 / pHCR" complex is efficiently captured on the sensing interface. The electrochemical signal group is modified on H1 and / or H2, and the signal increment of the sensing interface is positively correlated with the target nucleic acid concentration, and the nucleic acid concentration is judged based on the electrochemical signal. By constructing a standard detection curve, the target nucleic acid content detection of unknown samples is achieved.

[0014] Studies have shown that compared with the "target nucleic acid / H0 / pHCR" complex containing only one DNA / RNA double strand, the present invention contains two DNA / RNA double strands, which makes it more efficient in being captured at the electrode interface.

[0015] In the present invention, H0a', H0c and Ic' partial sequences are designed according to the corresponding sequences (a sequences) of the target DNA and RNA, and H0b can be universal. The hairpin probe H0 and the single-stranded probe I can be DNA, RNA or DNA / RNA mixed sequences, and the specific sequence design is based on the fact that the "target nucleic acid / H0 / pHCR" complex generated by the final reaction contains two DNA / RNA double strands.

[0016] Specifically, when the target nucleic acid is RNA, the hairpin structure rH0 (H0 for RNA detection) has two functions, one is to recognize and hybridize with the target RNA through a chain displacement reaction, and the other is to hybridize with the single-stranded probe I. The above two hybridization reactions form two DNA / RNA double strands. Specifically: rH0 includes three parts, rH0a', rH0b, and rH0c, from the 5' end to the 3' end, wherein rH0a' is a complementary sequence of the a sequence, rH0b does not hybridize with other sequences, and acts as a gap to enhance the bivalent capture affinity, and rH0c is complementary to the partial sequence of rH0a'. After hybridization, rH0 forms a stable hairpin structure. rH0a' and rH0b are DNA sequences, and rH0c is a DNA or RNA sequence. When rH0c is a DNA sequence, Ic' is required to be an RNA sequence, and when rH0c is an RNA sequence, Ic' is required to be a DNA sequence.

[0017] The target RNA is complementary to H0a' in rH0. After opening rH0, rH0c in the stem is exposed. rH0c hybridizes with Ic' of single-stranded probe I. Id of single-stranded probe I triggers a hybridization chain reaction between H1 and H2 to form a "target nucleic acid / H0 / pHCR" complex containing two DNA / RNA double strands, which can be specifically bound by S9.6 antibody and captured on the sensing interface.

[0018] When the target nucleic acid is DNA, the function of the hairpin structure dH0 (H0 for DNA detection) is basically the same as that of RNA detection. Specifically: dH0 includes three parts, dH0a', dH0b, and dH0c, in sequence, wherein dH0a' is a complementary sequence to the a sequence, dH0b does not hybridize with other sequences, and acts as a gap to enhance the bivalent capture affinity, dH0c is complementary to a partial sequence of dH0a', and after hybridization, dH0 forms a stable hairpin structure. In order to form two DNA / RNA double strands, the toehold end of dH0a' is modified with 3-20 bases of RNA, the specific amount is related to the DNA sequence, the remaining part of dH0a' is a DNA sequence, dH0b and dH0c are both DNA sequences, and Ic' is required to be an RNA sequence at this time.

[0019] The target DNA is complementary to dH0a'. After opening dH0, dH0c at the other end is exposed. The released dH0c hybridizes with dIc' to form a DNA / dH0 / pHCR complex. The DNA / dH0 / pHCR complex contains two DNA / RNA double-stranded segments, which can be specifically bound by the S9.6 antibody and captured on the sensing interface.

[0020] Those skilled in the art can perform design operations such as replacement, deletion or addition of bases on the H0 and I sequences according to specific circumstances to achieve the above-mentioned hybridization chain reaction.

[0021] In the present invention, H0a' and H0c sequences are related to the length of the target nucleic acid sequence. Preferably, the length of H0a' sequence is 20-30 nt. When the a sequence is DNA, 3-20 bases at the 5' end (toehold part) of H0a' are RNA; the length of H0c sequence is 5-6 nt less than H0a'.

[0022] More preferably, the toehold portion in H0a' is 5-7 bases in length.

[0023] Considering factors such as cost and detection performance, it is more appropriate for H0b to be in the range of 6-50 bases in length. Studies have shown that H0b is the gap between the two DNA / RNA double strands in the DNA / dH0 / pHCR complex, and the size of the gap affects the efficiency of the complex capturing the electrode interface. Preferably, the length of H0b is 15-25 bases. Preferably, the H0b sequence can be continuous T.

[0024] Preferably, the length of the Ic' sequence is 2-10 nt shorter than that of H0c; and the length of the Id sequence is 15-50 nt.

[0025] More preferably, the length of the H0a' sequence is 22 nt, the length of the H0c sequence is 17 nt, the length of the Ic' sequence is 15 nt, and the length of the Id sequence is 21 nt.

[0026] As a specific embodiment of the present invention, when the target RNA is miR-629, the sequence of H0 is as shown in any one of SEQ ID NO.2-7, I is composed of Id and Ic', and the sequence of Ic' is as shown in SEQ ID NO.9;

[0027] When the target DNA is a KRAS gene fragment with a nucleotide sequence as shown in SEQ ID NO.12, H0 consists of an RNA sequence and a DNA sequence, the RNA sequence is shown in SEQ ID NO.13, the DNA sequence is shown in SEQ ID NO.14, I consists of Id and Ic', and the Ic' sequence is shown in SEQ ID NO.15.

[0028] In the present invention, there is no fixed requirement for the sequence design of Id, H1 and H2, which is subject to the realization of hybridization chain reaction. For different detection objects, the sequence design of Id, H1 and H2 can be the same to achieve universality.

[0029] Specifically, H1 includes three parts, H1d', H1e and H1f, H1d' is the complementary sequence of Id, and the partial sequence of H1d' is complementary to H1f to form a double strand as the stem of the H1 hairpin structure, and H1e is the loop of the H1 hairpin structure.

[0030] H2 includes three parts, H2g, H2h and H2i. The partial sequences of H2g and H2i are complementary to form a double strand as the stem of the H2 hairpin structure. H2h serves as the loop of the H2 hairpin structure. H2i is complementary to H1e and H1f. H2h and H2g are complementary to H1d'.

[0031] In the presence of single-stranded probe I, Id is complementary to H1d', the hairpin structure of H1 is opened, exposing H1e and H1f, and complements H2i to open the hairpin probe H2, exposing H2h and H2g, which further recognize and open the hairpin structure of H1 to trigger a hybridization chain reaction.

[0032] Preferably, the sequence lengths of H1d' and H2i are 16-28 nt, H1f is 5-8 bases shorter than H1d', and H2g is 5-6 bases shorter than H2i; and the sequence lengths of H1e and H2h are 5-8 nt.

[0033] More preferably, the sequence length of H1d' and H2i is 21 nt, the sequence length of H1f and H2g is 16 nt, and the sequence length of H1e and H2h is 5 nt.

[0034] As a specific embodiment of the present invention, the Id sequence is shown as SEQ ID NO.8; the nucleotide sequence of H1 is shown as SEQ ID NO.10; and the nucleotide sequence of H2 is shown as SEQ ID NO.11.

[0035] In the present invention, Id, H1 and H2 can be mixed in a solution system to generate a hybridization chain reaction to pre-prepare a hybridization chain reaction product pHCR with a single strand of Ic'. H1 and / or H2 are modified with a signal marker, and as the hybridization chain reaction proceeds, more signal markers are enriched in the reaction product to obtain a signal amplification material.

[0036] Preferably, the 5' end of H1 and / or H2 is labeled with biotin, but the present invention is not limited thereto, and other electrochemical signal labels may also be used.

[0037] In the present invention, all hybridization reactions are carried out in a solution system, which significantly improves the target nucleic acid recognition and HCR hybridization efficiency. The "target nucleic acid / H0 / pHCR" complex is then efficiently captured on the interface through a bivalent capture method, and ultra-sensitive nucleic acid detection is achieved by analyzing the changes in signal markers.

[0038] Preferably, the method for preparing an electrode or chip self-assembled with S9.6 antibodies comprises: dropping a S9.6 antibody solution onto the surface of a printed carbon electrode, and incubating at room temperature for more than 5 hours to allow the S9.6 antibodies to self-assemble on the electrode surface.

[0039] When biotin is labeled on pHCR, an excess of horseradish peroxidase-modified streptavidin (SA-HRP) is incubated at the interface of the sensor electrode to bind to the biotin in the "target nucleic acid / H0 / pHCR" complex, and then the sensor electrode is fully cleaned and placed in a TMB substrate solution containing hydrogen peroxide to detect the catalytic reduction current generated by horseradish peroxidase-catalyzed TMB reduction. The catalytic reduction current is positively correlated with the amount of horseradish peroxidase at the electrode interface, and the amount of horseradish peroxidase is positively correlated with the target nucleic acid, so the target nucleic acid in the test solution can be quantitatively analyzed by detecting the change in the catalytic reduction current. Specifically, a standard curve is drawn, and then quantitative analysis is performed based on the standard curve.

[0040] The present invention also provides a nucleic acid detection method using the nucleic acid detection sensor, comprising the following steps:

[0041] (1) Preparing a hybridization chain reaction product: mixing the hairpin probes H1 and H2 and the single-stranded probe I in a buffer system to perform a hybridization chain reaction to prepare a hybridization chain reaction product pHCR;

[0042] (2) Nucleic acid detection based on S9.6 antibody bivalent capture: H0 is added to the pretreated nucleic acid sample solution to form target nucleic acid / H0 through hybridization reaction, and then the pre-prepared pHCR is added to form a target nucleic acid / H0 / pHCR complex through hybridization reaction. Then, the electrode or chip self-assembled with S9.6 antibody is placed in the reaction solution, and the complex is captured to the electrode interface through the specific binding of S9.6 antibody to DNA / RNA double strands. Finally, the signal changes on the interface are analyzed to determine and calculate the content of target nucleic acid in the solution.

[0043] Based on the hairpin probe design of the present invention, the nucleic acid detection method can be carried out in a reaction system, and target recognition, interface multivalent capture and signal amplification output can be achieved in just one step. The present invention shows that when there is no target nucleic acid, no H0 / pHCR complex will be generated to trigger background signals.

[0044] There is no specific requirement for the buffer system of the hybridization solution in the detection method, which is usually a high-salt, neutral and slightly alkaline buffer condition, which is conducive to the hybridization reaction. Preferably, the buffer system is a SPSC solution.

[0045] The composition of SPSC solution is: 50 mM Na2HSO4 / NaH2SO4, 1 M NaCl, pH 7.5.

[0046] Preferably, in step (1), the ratio of the single-stranded probe I to the hairpin probes H1 and H2 is 1:5-50:5-50. Studies have shown that the larger the molecular weight of the pHCR product, the better the signal amplification effect. More preferably, the ratio of the single-stranded probe I to the hairpin probes H1 and H2 is 1:10-15:10-15.

[0047] Preferably, in step (2), the working concentrations of H0 and pHCR are 1 nM-100 μM. More preferably, the working concentration of H0 is 500 nM, and the working concentration of pHCR is 100 nM based on the single-stranded probe I.

[0048] Preferably, in step (2), the hybridization reaction conditions are 25-30°C for 2-5 min; and the S9.6 antibody reaction conditions are room temperature for 30-60 min.

[0049] More preferably, HO with a final concentration of 500 nM is added to 50 μL of SPSC solution containing the target nucleic acid, and the mixture is reacted at 30°C for 5 minutes, followed by the addition of pHCR with a final concentration of 100 nM (in terms of I concentration), and the mixture is reacted at 30°C for 2 minutes. 10 μL of the above reaction solution is placed on the sensing interface and incubated at room temperature for 30 minutes. When biotin is used as the signal marker, 8 μL of 1 mg / mL SA-HRP is finally placed on the sensing electrode interface and incubated at room temperature for 4 minutes. Under the above conditions, the best signal-to-noise ratio can be obtained.

[0050] The "target nucleic acid / H0 / pHCR" complex formed by the hairpin probe H0 and the prefabricated pHCR amplification material provided by the present invention is captured to any sensing interface through divalent capture. Therefore, in addition to the above-mentioned electrochemical sensor, the S9.6 divalent capture strategy proposed in the present invention can also be applied to other sensing fields, such as: SPR, microbalance, surface fluorescence enhancement, Raman and other interface sensing fields.

[0051] The present invention has the following beneficial effects:

[0052] (1) The nucleic acid detection sensor provided by the present invention utilizes the high affinity and specific binding ability of the S9.6 antibody and the DNA / RNA double strand, and can efficiently capture the complex generated by the target nucleic acid on the sensing interface through a bivalent capture method, and realize ultra-sensitive nucleic acid detection by analyzing the signal label. Compared with the complex containing only one DNA / RNA double strand, the present invention contains two DNA / RNA double strands, which makes it more efficient to be captured at the electrode interface.

[0053] (2) In the process of nucleic acid detection using the nucleic acid detection sensor of the present invention, only one or two steps are required to realize nucleic acid recognition, interface capture and signal amplification output, and the detection can be completed within 41 minutes. It has great advantages among sensors of the same type, solves the defects of the traditional method with complicated steps, and has market commercialization prospects.

[0054] (3) The signal amplification probe designed by the present invention is universal and greatly saves detection costs. The amplification reaction of the pre-prepared signal amplification product is carried out in a solution, which not only significantly improves the hybridization efficiency, but also saves nucleic acid detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Schematic diagram of the principle of detecting miR-629 based on the bivalent capture of S9.6 antibody in Example 1, wherein (A) is the self-assembled S9.6 antibody on the printed carbon electrode; (B) is the pre-prepared hybridization chain reaction product; and (C) is the miR-629 detection process.

[0056] Figure 2 Gel electrophoresis was used to verify the feasibility of miR-629 detection.

[0057] Figure 3 Gel electrophoresis was used to investigate the effects of different I:H1:H2 ratios on HCR products.

[0058] Figure 4 The effect of different I:H1:H2 ratios on miR-629 detection was investigated for electrochemical detection.

[0059] Figure 5 The number of bases in the H0 loop affects the capture of "miR-629 / rH0 / pHCR".

[0060] Figure 6 To verify the time required for each step of miR-629 detection, (A) is the hybridization time of miR-629 and rH0; (B) is the hybridization time of miR-629 / rH0 and HCR product; (C) is the capture time of "miR-629 / rH0 / pHCR" complex at the electrode interface; (D) is the SA-HRP incubation time.

[0061] Figure 7 The electrochemical detection results of miR-629, where (A) is the corresponding it curve when electrochemically detecting miR-629 between 10aM-10nM; (B) is the positive correlation with the stable catalytic current value when electrochemically detecting miR-629 between 10aM-10nM.

[0062] Figure 8 Schematic diagram of the principle of detecting KRAS gene fragments based on bivalent capture of S9.6 antibody in Example 2.

[0063] Fig. 9 The feasibility of KRAS gene fragment detection was verified by gel electrophoresis.

[0064] Fig.10 The results of electrochemical detection of KRAS gene fragments, where (A) is the corresponding it curve when electrochemically detecting KRAS gene fragments between 100aM-10nM; (B) is the positive correlation with the stable catalytic current value when electrochemically detecting KRAS gene fragments between 100aM-10nM. DETAILED DESCRIPTION

[0065] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0066] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0067] The printed carbon electrodes used in the following examples were purchased from Shenzhen Xinshuang Sensor Co., Ltd., and the S9.6 antibody and SA-HRP were purchased from the French company Antibody System.

[0068] The nucleotide sequence is from 5' to 3' from left to right.

[0069] Example 1: Detection of miR-629 based on bivalent capture of S9.6 antibody

[0070] 1. Card issuing probe design

[0071] The relevant hairpin probes were designed according to the detection target miR-629. There is no requirement for the design of hairpin probes for HCR. The trigger sequence adds a sequence for hybridization to form a DNA / RNA double strand. The sequence is related to the target miR-629. The sequence is shown in Table 1. All sequences were commissioned to Sangon Biotech Co., Ltd. (Shanghai) for synthesis and modification.

[0072] Table 1. Designed DNA / RNA probes

[0073]

[0074] 2. Preparation of hairpin probe

[0075] The concentration of hairpin probes was accurately quantified using a UV spectrophotometer, and all hairpin probes were diluted to 5 μM with SPSC buffer (50 mM Na2HSO4 / NaH2SO4, 1 M NaCl, pH 7.5), placed in a PCR instrument at 95 °C for 2 min, and quickly cooled to 4 °C to form a stable hairpin structure.

[0076] 3. Self-assembly of S9.6 antibody on printed carbon electrode

[0077] like Figure 1 As shown in (A), 10 μL of S9.6 antibody with a concentration of 100 μg / mL was dropped onto the carbon electrode interface and incubated at room temperature overnight.

[0078] 4. Prefabricated hybridization chain reaction product (pHCR)

[0079] like Figure 1 As shown in (B), the preparation process is no different from the traditional hybridization chain reaction, only an RNA sequence is added at the initiator sequence for hybridization to form a DNA / RNA double strand. Detailed synthesis process of pHCR: 100 μL 2 μM H1, 100 μL 2 μM H2 and 100 μL 2 μM rI are placed in a PCR instrument at 95 degrees Celsius for 5 minutes, then cooled to room temperature, H1 and H2 form a stem-loop structure, 100 μL 2 μM H1, 100 μL 2 μM H2 and 10 μL 2 μM rI are mixed evenly and reacted at 30 degrees Celsius for 2 hours to obtain pHCR products.

[0080] 5. miR-629 detection

[0081] rH0-15 (pre-annealed to form a stem-loop structure) was added to a solution containing miR-629 (final concentration of 100aM-10nM), mixed, reacted at 30°C for 5 minutes, then pre-prepared pHCR was added, and the reaction was continued at 30°C for 2 minutes (at this time, the concentration of rH0-15 in the reaction solution was 500nM, the concentrations of H1 and H2 were 1μM, and the concentration of rI was 100nM). The "miR-629 / rH0 / pHCR complex" was formed through recognition hybridization, and then 10 μl of the above reaction solution was placed on the S9.6 antibody-assembled printed carbon electrode interface and incubated at room temperature for 30 minutes. The S9.6 antibody specifically bound to the DNA / RNA double strand and was captured to the electrode interface. Then 10 μl of 1 μg / ml SA-HRP was drop-coated on the electrode interface and incubated at room temperature for 4 minutes. After washing, the change in the chronoamperometric signal on the electrode surface was analyzed to determine and calculate the content of the target nucleic acid in the solution.

[0082] The principle of the above detection process is as follows Figure 1As shown in (C), rH0 and miR-629 open the stem-loop structure through chain displacement hybridization reaction, and the opened stem sequence continues to hybridize with pHCR. rH0 is a DNA sequence, and the remaining unhybridized sequence at rI on miR-629 and pHCR is an RNA sequence. Therefore, the formed "miR-629 / rH0 / pHCR" contains two DNA / RNA double strands. The "miR-629 / rH0 / pHCR" is captured on the electrode chip interface by the specific recognition of the DNA / RNA double strand by the S9.6 antibody. pHCR is modified with biotin and can be connected to SA-HRP. The electrochemical signal is output through a portable electrochemical workstation and Bluetooth connection. When miR-629 is not present, rH0 and pHCR will not hybridize, so the output electrochemical signal is weak, and the electrochemical signal is positively correlated with the miR-629 concentration.

[0083] 6. Gel electrophoresis to verify the feasibility of miR-629 detection

[0084] like Figure 2 As shown, the following settings are respectively set: Lane 1, 1μM miR-629+1μM rH0-15; Lane 2, 1μM rH0-15; Lane 3, 1μM rI+1μM rH0-15; Lane 4, 1μM miR-629+1μM rI+1μM rH0-15; Lane 5, 1μM pHCR+1μM rH0-15; Lane 6, 1μM miR-629+1μM pHCR+1μM rH0-15; Lane 7, 1μM pHCR; Lane 8, DNA marker. The products of the corresponding lanes were obtained by incubating the corresponding probes at 30 degrees Celsius for 30 minutes, and the buffer solution was SPSC.

[0085] The results showed that when miR-629 was present, "miR-629 / rH0 / rI" (lane 4) and "miR-629 / rH0 / pHCR" (lane 6) could be formed, and when miR-629 was not present, no new hybridization products were formed.

[0086] 7. Verify the effect of different I:H1:H2 ratios on HCR products by gel electrophoresis

[0087] like Figure 3As shown, the settings are as follows: Lane 1, DNA marker; Lane 2, 0.2μM rTrigger-15r+1μM H1+1μM H2 (1:5:5); Lane 3, 0.2μM rTrigger-15r+1.5μM H1+1.5μM H2 (1:7.5:7.5); Lane 4, 0.2μM rTrigger-15r+2μM H1+2μM H2 (1:10:10); Lane 5, 0.2μM rTrigger-15r+2.5μM H1+2.5μM H2 (1:12.5:12.5); Lane 6, 0.2μM rTrigger-15r+3μM H1+3μM H2 (1:15:15). H1, H2 and rI in different proportions were mixed evenly and reacted at 30 degrees Celsius for 2 hours to obtain the corresponding pHCR product.

[0088] The results showed that as the I:H1:H2 ratio increased, the proportion of HCR products larger than 1500 base pairs increased, and the larger the molecular weight of the HCR product, the better the signal amplification effect.

[0089] 8. Verify the effect of different I:H1:H2 ratios on miR-629 detection signal by electrochemical detection

[0090] This experiment fixed the concentration of the target miR-629 and investigated the effect of different H / I ratios of pre-prepared pHCR on the detection results. The specific method is: add rH0-15 to the solution containing miR-629 (10nM), mix well and react at 30°C for 5 minutes, then add different H / I ratios of pre-prepared pHCR (1:5:5-1:15:15), continue to react at 30°C for 2 minutes, and form a "miR-629 / rH0 / pHCR complex" through recognition and hybridization. Then, 10 microliters of the above reaction solution is placed on the interface of the S9.6 antibody-assembled printed carbon electrode, incubated at room temperature for 30 minutes, and captured to the electrode interface through the specific binding of the S9.6 antibody to the DNA / RNA double strand, and then 10 microliters of 1 microgram / ml SA-HRP is dripped on the electrode interface, incubated at room temperature for 4 minutes, and the chronoamperometric signal on the electrode surface is analyzed after washing.

[0091] The results are as follows Figure 4 As shown in the figure, as the I:H1:H2 ratio increases, the electrochemical signal measured at the same concentration of miR-629 gradually increases. Since the molecular weight of the HCR product has little effect on the background, the increase in the I:H1:H2 ratio is beneficial to the detection of miR-629, but increasing the concentrations of H1 and H2 will also increase the detection cost (both H1 and H2 are modified with biotin, which is one of the main cost sources of this detection method).

[0092] 9. Electrochemical detection verifies the effect of different numbers of bases in the rH0 loop on the capture of the “miR-629 / rH0 / pHCR” complex at the electrode interface

[0093] This experiment fixed the concentration of the target miR-629 and investigated the effect of different rings in rH0 on the detection results. The specific method is: add rH0 with different ring lengths (rH0-5, rH0-10, rH0-15, rH0-20, rH0-25, rH0-30) to the solution containing miR-629 (10nM), mix well and react at 30°C for 5 minutes, then add pHCR (1:10:10), continue to react at 30°C for 2 minutes, and form a "miR-629 / rH0 / pHCR complex" through recognition and hybridization. Then, 10 microliters of the above reaction solution is placed on the interface of the S9.6 antibody-assembled printed carbon electrode, incubated at room temperature for 30 minutes, and captured to the electrode interface through the specific binding of the S9.6 antibody to the DNA / RNA double strand, and then 10 microliters of 1 microgram / ml SA-HRP is dripped on the electrode interface, incubated at room temperature for 4 minutes, and the chronoamperometric signal on the electrode surface is analyzed after washing.

[0094] In addition, when the Ic' sequence is an RNA sequence, the miR-629 / rH0 / rI complex contains two DNA / RNA double strands, and when the Ic' sequence is a DNA sequence, the miR-629 / rH0 / I complex contains one DNA / RNA double strand.

[0095] The results are as follows Figure 5 As shown in the figure, the bases in the loop of rH0 play the role of the gap between the two DNA / RNA double strands in the "miR-629 / rH0 / pHCR" complex. The size of the gap affects the capture of the "miR-629 / rH0 / pHCR" complex at the electrode interface. From the electrochemical detection results of miR-629 at the same concentration, it can be seen that when the number of bases in the loop of rH0 is greater than 15 bases, the capture efficiency of the "miR-629 / rH0 / pHCR" complex at the electrode interface is maximized.

[0096] In addition, the experimental results also show that the "miR-629 / rH0 / pHCR" complex containing two DNA / RNA double strands has a better capture efficiency at the electrode interface than the complex containing only one DNA / RNA double strand.

[0097] 10. Investigate the time required for each step of the electrochemical detection process of miR-629

[0098] Figure 6(A) is the hybridization time of miR-629 and rH0. From the results, we can know that 10nM miR-629 and 500nM rH0 can be hybridized in 2 minutes. However, in order to ensure the complete hybridization of low-concentration miR-629 and rH0, this hybridization time is fixed to 5 minutes for the detection of different concentrations of miR-629.

[0099] Figure 6 (B) is the hybridization time of miR-629 / rH0 and HCR products. From the results, it can be seen that 10nM miR-629 / rH0 and 100nM pHCR can be hybridized in 2 minutes.

[0100] Figure 6 (C) is the investigation of the capture time of the "miR-629 / rH0 / pHCR" complex at the electrode interface. From the results, it can be seen that the capture of the "miR-629 / rH0 / pHCR" complex at the electrode interface reaches saturation in 30 minutes.

[0101] Figure 6 (D) is the SA-HRP incubation time investigation. From the results, it can be seen that the connection of SA-HRP at the electrode interface can be completed in 4 minutes.

[0102] 11. Electrochemical detection of miRNA at different concentrations

[0103] In SPSC buffer solution, different concentrations of miR-629 (10aM-10nM) were hybridized with 500nM rH0, and the stem sequence opened by rH0 continued to hybridize with 100nM pHCR to form a "miR-629 / rH0 / pHCR" complex. The "miR-629 / rH0 / pHCR" complex was captured on the electrode chip interface through the S9.6 antibody that specifically recognized the DNA / RNA double strands. pHCR was modified with biotin and could be connected to SA-HRP. The electrochemical signal was output through a portable electrochemical workstation and Bluetooth connection.

[0104] Figure 7 (A) is the it curve when detecting miR-629 at 10aM-10nM. Figure 7 (B) shows that electrochemical detection of miR-629 between 10aM and 10nM is positively correlated with the stable catalytic current value.

[0105] Example 2: Detection of KRAS gene fragments based on bivalent capture of S9.6 antibody

[0106] 1. Design of hairpin probe: dH0 is designed based on a KRAS gene fragment. Since the detection object is DNA, in order to form a DNA / RNA double strand when the KRAS gene fragment is recognized and hybridized, 10 bases of RNA are modified at the 5' end of dH0. The hybridization chain reaction stem loop probe remains unchanged, and the trigger sequence extends a section that changes due to different detection sequences, but it is also RNA modification. The sequence is shown in Table 2.

[0107] Table 2

[0108]

[0109] 2. Preparation of hairpin probes, self-assembly of S9.6 antibodies on printed carbon electrodes, and prefabricated hybridization chain reaction products are the same as in Example 1.

[0110] 3. Detection of KRAS gene fragments

[0111] Add dH0 to the solution containing KRAS fragment (1fM-10nM), mix well and react at 30°C for 5 minutes, then add pHCR (1:10:10, the trigger sequence is changed to dI), and continue to react at 30°C for 2 minutes to form a "KRAS / dH0 / pHCR complex" through recognition hybridization. Then, 10 μl of the above reaction solution is placed on the S9.6 antibody-assembled printed carbon electrode interface and incubated at room temperature for 30 minutes. The S9.6 antibody specifically binds to the DNA / RNA double strand and is captured to the electrode interface. Then, 10 μl of 1 μg / ml SA-HRP is drop-coated on the electrode interface and incubated at room temperature for 4 minutes. After washing, the chronoamperometric signal on the electrode surface is analyzed.

[0112] The mechanism of the above detection process is as follows Figure 8 As shown, when the target KRAS gene fragment exists, dH0 and KRAS gene open the stem-loop structure through chain substitution hybridization reaction, and the opened stem sequence continues to hybridize with pHCR. The 5' end of dH0 is an RNA sequence, the 3' end is a DNA sequence, the KRAS gene is a DNA sequence, and the remaining unhybridized sequence at dI on pHCR is an RNA sequence. Therefore, the formed "KRAS / dH0 / pHCR" contains two DNA / RNA double strands. The "KRAS / dH0 / pHCR" is captured on the electrode chip interface through the specific recognition of the DNA / RNA double strand by the S9.6 antibody. The pHCR is modified with biotin and can be connected to SA-HRP. The electrochemical signal is output through the portable electrochemical workstation and Bluetooth connection. When there is no KRAS gene, dH0 and pHCR will not hybridize, so the output electrochemical signal is weak, and the electrochemical signal is positively correlated with the KRAS gene concentration.

[0113] 4. Gel electrophoresis to verify the feasibility of KRAS gene detection

[0114] like Fig. 9 As shown, the following settings were set: Lane 1, 1μM KRAS+1μM dH0; Lane 2, 1μM dH0; Lane 3, 1μM dI+1μM dH0; Lane 4, 1μM KRAS+1μM dI+1μM dH0; Lane 5, 1μM pHCR+1μM dH0; Lane 6, 1μM KRAS+1μM pHCR+1μM dH0; Lane 7, 1μM pHCR; Lane 8, DNA marker. The products of the corresponding lanes were obtained by incubating the corresponding probes at 30 degrees Celsius for 30 minutes, and the buffer solution was SPSC.

[0115] The results showed that when the KRAS gene was present, "KRAS / dH0 / dI" (lane 4) and "KRAS / dH0 / pHCR" (lane 6) could be formed, and when the KRAS gene was not present, no new hybridization products were formed.

[0116] 5. Electrochemical detection of KRAS gene at different concentrations

[0117] In SPSC buffer solution, different concentrations of KRAS gene (100aM-10nM) were hybridized with 500nM dH0, and the stem sequence opened by dH0 continued to hybridize with 100nM pHCR to form a "KRAS / dH0 / pHCR" complex. The "KRAS / dH0 / pHCR" complex was captured on the electrode chip interface through the S9.6 antibody that specifically recognized the DNA / RNA double strands. pHCR was modified with biotin and could be connected to SA-HRP. The electrochemical signal was output through a portable electrochemical workstation and Bluetooth connection.

[0118] Fig.10 (A) is the it curve for 100aM-10nM KRAS gene detection. Figure 18 (B) shows that the electrochemical detection of KRAS gene between 100aM-10nM is positively correlated with the stable catalytic current value.

Claims

1. A nucleic acid detection sensor based on S9.6 antibody bivalent capture, wherein the nucleic acid is a target RNA or DNA containing a sequence obtained after pretreatment of the sample, characterized in that: The sensor includes hairpin probes H0, H1 and H2, a single-stranded probe I, and an electrode or chip self-assembled with S9.6 antibodies; The H0 includes three parts: H0a', H0b and H0c, wherein H0a' is a complementary sequence of the a sequence. When the a sequence is RNA, H0a' and H0b are DNA sequences, and H0c is a DNA or RNA sequence. When the a sequence is DNA, part of the sequence in H0a' of H0 is an RNA sequence, and the rest is a DNA sequence. The partial sequence of H0a' is complementary to H0c to form a double strand as the stem of the H0 hairpin structure, and H0b is the loop of the H0 hairpin structure. The I includes two parts, Id and Ic', Ic' is the complementary sequence of H0c, when H0c is a DNA sequence, Ic' is an RNA sequence; when H0c is an RNA sequence, Ic' is a DNA sequence; Id, H1 and H2 are all DNA sequences, and Id is used to trigger a hybridization chain reaction between H1 and H2; The H1 and / or H2 are modified with a signal tag.

2. The nucleic acid detection sensor based on the bivalent capture of S9.6 antibody according to claim 1, characterized in that: The length of H0a' sequence is 20-30nt. When the a sequence is DNA, the 5' end 3-20 bases of H0a' are RNA; the length of H0c sequence is 5-6nt less than H0a'; the length of H0b sequence is 6-50nt.

3. The nucleic acid detection sensor based on the bivalent capture of S9.6 antibody according to claim 2, characterized in that: The length of H0b sequence is 15-25 nt.

4. The nucleic acid detection sensor based on bivalent capture of S9.6 antibody according to claim 1, characterized in that: The length of Ic' sequence is 2-10 nt shorter than that of H0c; the length of Id sequence is 15-50 nt.

5. The nucleic acid detection sensor based on the bivalent capture of S9.6 antibody according to claim 1, characterized in that: The 5' end of H1 and / or H2 is labeled with biotin.

6. The nucleic acid detection sensor based on the bivalent capture of S9.6 antibody according to claim 1, characterized in that: The method for preparing an electrode or chip self-assembled with S9.6 antibody comprises: dropping a S9.6 antibody solution onto the surface of a printed carbon electrode, and incubating at room temperature for more than 5 hours to allow the S9.6 antibody to self-assemble on the electrode surface.

7. The nucleic acid detection sensor based on bivalent capture of S9.6 antibody according to claim 1, characterized in that: The nucleic acid detection method using the nucleic acid detection sensor comprises the following steps: (1) Preparing a hybridization chain reaction product: mixing the hairpin probes H1 and H2 and the single-stranded probe I in a buffer system to perform a hybridization chain reaction to prepare a hybridization chain reaction product pHCR; (2) Nucleic acid detection based on S9.6 antibody bivalent capture: H0 is added to the pretreated nucleic acid sample solution to form target nucleic acid / H0 through hybridization reaction, and then the pre-prepared pHCR is added to form a target nucleic acid / H0 / pHCR complex through hybridization reaction. Then, the electrode or chip self-assembled with S9.6 antibody is placed in the reaction solution, and the complex is captured to the electrode interface through the specific binding of S9.6 antibody to DNA / RNA double strands. Finally, the signal changes on the interface are analyzed to determine and calculate the content of target nucleic acid in the solution.

8. The nucleic acid detection sensor based on the bivalent capture of S9.6 antibody according to claim 7, characterized in that: In step (1), the ratio of the single-stranded probe I to the hairpin probes H1 and H2 is 1:5-50:5-50.

9. The nucleic acid detection sensor based on the bivalent capture of S9.6 antibody according to claim 7, characterized in that: In step (2), the hybridization reaction conditions are 25-30°C for 2-5 min; the S9.6 antibody reaction time is 30-60 min.

10. The nucleic acid detection sensor based on the bivalent capture of S9.6 antibody according to claim 7, characterized in that: When the target RNA is miR-629, the sequence of H0 is as shown in any one of SEQ ID NO.2-7, I is composed of Id and Ic', the sequence of Id is as shown in SEQ ID NO.8, and the sequence of Ic' is as shown in SEQ ID NO.9; When the target DNA is a KRAS gene fragment with a nucleotide sequence as shown in SEQ ID NO.12, H0 consists of an RNA sequence and a DNA sequence, the RNA sequence is shown in SEQ ID NO.13, the DNA sequence is shown in SEQ ID NO.14, I consists of Id and Ic', the Id sequence is shown in SEQ ID NO.8, and the Ic' sequence is shown in SEQ ID NO.15; The sequence of H1 is shown in SEQ ID NO.10; the sequence of H2 is shown in SEQ ID NO.11.

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