阅读说明：本技术 一种检测miRNA-141的生物传感器及其制备方法与应用 (Biosensor for detecting miRNA-141 and preparation method and application thereof ) 是由 王玉 孙文玉 刘素 黄加栋 王业茹 江龙 张曼茹 李莎莎 张雪 于 2020-04-09 设计创作，主要内容包括：本发明涉及生物传感器技术领域,特别涉及基于荧光强度变化与链置换反应与杂交链式反应辅助的循环放大的生物传感器。为了解决以上现有技术中检测miRNA-141的方法特异性和灵敏度都比较低、成本高的问题。一种基于荧光检测miRNA-141的生物传感器,将荧光强度变化与链置换反应与杂交链式反应辅助的循环放大相结合,均相反应混合液。制备方法：合成银簇；miRNA-141、S4、S、AgNCs-DNA、H1、H2在均相体系中混合反应。利用链置换反应辅助的循环放大,实现Trigger的循环利用,起到了信号放大的作用。(The invention relates to the technical field of biosensors, in particular to a biosensor based on fluorescence intensity change and auxiliary cyclic amplification of strand displacement reaction and hybridization chain reaction. The method aims to solve the problems of low specificity and sensitivity and high cost of the method for detecting miRNA-141 in the prior art. A biosensor for detecting miRNA-141 based on fluorescence combines fluorescence intensity change with strand displacement reaction and hybrid chain reaction assisted cyclic amplification to perform homogeneous reaction on mixed liquid. The preparation method comprises the following steps: synthesizing silver clusters; miRNA-141, S4, S, AgNCs-DNA, H1 and H2 are mixed and reacted in a homogeneous system. And the cyclic utilization of the Trigger is realized by using the chain displacement reaction-assisted cyclic amplification, and the signal amplification effect is achieved.)

1. A biosensor for detecting miRNA-141 is characterized by comprising a composite probe S, AgNCs-DNA, miRNA-141, S4, H1, H2 and buffer;

the composite probe S is formed by hybridizing S3, S1 and S2 into a double strand;

the base sequences used were:

the H1 base sequence is shown in SEQ No. 1; specifically 5-GTCTTG-GTTAGATCGCCAAGGTTTCTGTAGCTACGA CGATCAGACCTTGGCGAGTCGTAGCTACAGAAACCTTGGCGA-3’；

The H2 base sequence is shown in SEQ No. 2; specifically 5-ACCTTGGCGA GTCGTAGCTACAGAAACCTTGGCGATC TAACTTCTGTAGCTACGACTCGCCAAGGT-CTGATCCAAGAC-3’

The base sequence of AgNCs-DNA is shown in SEQ No. 3; specifically 5-CCCACCCACCCGCCCAAGTCTTGTCGCCAAGGTCAAGAC-BHQ1-3’

The base sequence of S2 is shown in SEQ No. 4; specifically 5-GTCGTAGCTACAGAAACCTTGGCGATCTAAC-3’

The base sequence of S3 is shown as SEQ No. 5; specifically 5-gatcgccaaggt-ttctgtagctacgacgggat-ccatctttaccagacagtgtta-3’

The base sequence of S1 is shown in SEQ No. 6; specifically 5'-tctggtaaagatggatccc-3'

The base sequence of S4 is shown in SEQ No. 7; specifically 5'-tctggtaaagatggatcccgtcgtagctacagaaaccttggcgatc-3'

The miRNA-141 base sequence is shown as SEQ No. 8; specifically 5'-uaacacugucugguaaagaugg-3';

the 3' of the AgNCs-DNA is connected with-BHQ 1.

2. The method for preparing a biosensor in accordance with claim 1, comprising the steps of:

(1) preparation of a composite probe S: s3 is hybridized with S1 and S2 to form a double strand in advance to form a composite probe S;

(2) preparing nano silver cluster AgNCs by using hairpin AgNCs-DNA chain:

(3) mixing miRNA-141, S4, S, AgNCs-DNA, H1, H2, buffer and water, and reacting;

(4) and (3) adding the nano silver cluster AgNCs-DNA prepared in the step (2) and carrying out fluorescence detection.

3. The method according to claim 2, wherein the step (1) comprises the steps of: s3, S1, S2, buffer and water are mixed uniformly, reacted for 3min at 95 ℃, and then cooled to room temperature slowly.

4. The preparation method according to claim 2, wherein the step (2) of preparing the nano silver clusters AgNCs comprises the following steps: adding H2 and AgNO into PB buffer solution₃Mixing, standing at 4 deg.C for 15-30min, and adding prepared NaHPO₄Shaking for 1min, and standing at 4 deg.C for more than 4 hr.

5. The preparation method according to claim 2, wherein the step (3) is carried out by: s, S4, H1, H2, buffer, miRNA-141 and water are mixed uniformly and reacted for 1.2H at 37 ℃.

6. Use of the biosensor prepared by the method of claim 2 to detect miRNA-122 in non-disease diagnosis.

Technical Field

The invention belongs to the technical field of biosensors, and relates to a biosensor for detecting miRNA-141, a preparation method and application thereof, in particular to a biosensor for detecting miRNA-141 based on double signal cyclic amplification of strand displacement reaction and hybridization chain reaction and silver cluster fluorescence intensity change, and a preparation method and application thereof.

Background

MicroRNAs (miRNAs) are an endogenous, regulatory-functional, non-coding RNA species found in eukaryotes, and are about 20-25 nucleotides in size. Most miRNAs in human body inhibit the translation of miRNA through the complementary complete or incomplete pairing form of the base of the non-transcription region at the 3' end of the target gene miRNAs, induce the cutting degradation of miRNA, further influence the growth and development of human body from cells to individuals at various levels, and simultaneously participate in various disease processes including tumor. miRNA-141, the nucleic acid strand base order of which is 5'-UAACACUGUCUGGUAAAGAUGG-3'. The research shows that the abnormal expression of miRNA-141 is closely related to cell proliferation, apoptosis, differentiation, ontogeny, and prostate cancer development, invasion and metastasis. The method provides a new way for searching biomarkers of prostate cancer and prostatic hyperplasia.

The currently reported detection methods of MiRNA mainly comprise quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Northern blotting method, clone sequencing method, real-time fluorescent quantitative PCR and the like, but the real-time fluorescent quantitative PCR has higher requirements on temperature and instruments and has high requirements on professional technology; the Northern blotting method is time-consuming, complex in process and low in detection sensitivity, and the cloning sequencing method is time-consuming and labor-consuming. Therefore, it is urgently needed to establish a rapid, accurate, sensitive and high-specificity detection method for detecting miRNA-141.

Disclosure of Invention

In order to solve the problems of low specificity and sensitivity, high cost and long detection period of the method for detecting miRNA-122 in the prior art, the invention provides a biosensor for detecting miRNA-141 based on dual signal cyclic amplification of strand displacement reaction and hybridization chain reaction and silver cluster fluorescence intensity change, which has high specificity and sensitivity, low cost and high detection speed, and also provides a preparation method and application of the biosensor for detecting miRNA-141 based on dual signal cyclic amplification of strand displacement reaction and hybridization chain reaction and silver cluster fluorescence intensity change.

The invention is obtained by the following steps:

a biosensor for detecting miRNA-141 comprises a composite probe S, AgNCs-DNA, miRNA-141, S4, H1, H2 and buffer;

the composite probe S is formed by hybridizing S3, S1 and S2 into a double strand;

the base sequences used were:

the H1 base sequence is shown in SEQ No. 1; specifically 5-GTCTTG-GTTAGATCGCCAAGGTTTCTGTAGCTACGA CGATCAGACCTTGGCGAGTCGTAGCTACAGAAACCTTGGCGA-3’；

The H2 base sequence is shown in SEQ No. 2; specifically 5-ACCTTGGCGA GTCGTAGCTACAGAAACCTTGGCGATC TAACTTCTGTAGCTACGACTCGCCAAGGT-CTGATCCAAGAC-3’

The base sequence of AgNCs-DNA is shown in SEQ No. 3; specifically 5-CCCACCCACCCGCCCAAGTCTTGTCGCCAAGGTCAAGAC-BHQ1-3’

The base sequence of S2 is shown in SEQ No. 4; specifically 5-GTCGTAGCTACAGAAACCTTGGCGATCTAAC-3’

The base sequence of S3 is shown as SEQ No. 5; specifically 5-GATCGCCAAGGT-TTCTGTAGCTACGACGGGAT-CCATCTTTACCAGACAGTGTTA-3’

The base sequence of S1 is shown in SEQ No. 6; specifically 5'-TCTGGTAAAGATGGATCCC-3'

The base sequence of S4 is shown in SEQ No. 7; specifically 5'-TCTGGTAAAGATGGATCCCGTCGTAGCTACAGAAACCTTGGCGATC-3'

The miRNA-141 base sequence is shown as SEQ No. 8; specifically 5'-UAACACUGUCUGGUAAAGAUGG-3';

the 3' of the AgNCs-DNA is connected with-BHQ 1.

The preparation method of the biosensor comprises the following steps:

(1) preparation of a composite probe S: s3 is hybridized with S1 and S2 to form a double strand in advance to form a composite probe S;

(2) preparing nano silver cluster AgNCs by using hairpin AgNCs-DNA chain:

(3) mixing miRNA-141, S4, S, AgNCs-DNA, H1, H2, buffer and water, and reacting;

(4) and (3) adding the nano silver cluster AgNCs-DNA prepared in the step (2) and carrying out fluorescence detection.

The process of the step (1) is as follows: s3, S1, S2, buffer and water are mixed uniformly, reacted for 3min at 95 ℃, and then cooled to room temperature slowly.

The preparation method of the nano silver cluster AgNCs in the step (2) comprises the following steps: adding H2 and AgNO into PB buffer solution₃Mixing, standing at 4 deg.C for 15-30min, and adding prepared NaHPO₄Shaking for 1min, and standing at 4 deg.C for more than 4 hr.

The process of the step (3) is as follows: s, S4, H1, H2, buffer, miRNA-141 and water are mixed uniformly and reacted for 1.2H at 37 ℃.

The biosensor prepared by the method is applied to detecting miRNA-122 in non-disease diagnosis.

In the invention, 8 DNA chains are used in total, and the sequences are respectively as follows:

H1:5’-GTCTTG-GTTAGATCGCCAAGGTTTCTGTAGCTACGACGATCAGACCTTGGCGAGTCGTAGCTACAGAAACCTTGGCGA-3’

H2:5’-ACCTTGGCGA GTCGTAGCTACAGAAACCTTGGCGATCTAACTTCTGTAGCTACGACTCGCCAAGGT-CTGATCCAAGAC-3’

AgNCs-DNA:5’-CCCACCCACCCGCCCAAGTCTTGTCGCCAAGGTCAAGAC-BHQ1-3’

S2:5’-GTCGTAGCTACAGAAACCTTGGCGATCTAAC-3’

S3:5’-GATCGCCAAGGT-TTCTGTAGCTACGACGGGAT-CCATCTTTACCAGACAGTGTTA-3’

S1: 5’-TCTGGTAAAGATGGATCCC-3’

S4:5’-TCTGGTAAAGATGGATCCCGTCGTAGCTACAGAAACCTTGGCGATC-3’

miRNA-141: 5’-UAACACUGUCUGGUAAAGAUGG-3’

wherein the bold part of S3 is complementarily paired with S1, the italic part of S3 is complementarily paired with the italic part of S2, and S3 forms a hybrid double strand with S1 and S2. In the presence of the target miRNA-141, a toehold end-mediated strand displacement amplification reaction occurs, and S1 is displaced from S3. S4 continues to carry out strand displacement amplification reaction by the product of the reaction process, and S2 and miRNA-141 are displaced from S3 to form S3 and S4 hybrid double strands. S2 continues the next round of amplification as a trigger chain. S2 complementarily paired with the italic part of the H1 strand, hairpin probe H1 opened, the bold part of H1 complementarily paired with the bold part of H2, hairpin probe H2 opened, the italic part of H2 complementarily paired with the italic part of H1, Hybrid Chain Reaction (HCR) performed, H1 and H2 dotted underline failed to participate in complementary pairing, and the dotted underline could open the AgNCs-DNA hairpin, allowing separation of the 5 'silver cluster and the 3' quencher, thereby generating a fluorescent signal. The AgNCs-DNA strand italics part is the silver cluster sequence.

The detection mode of the invention is to detect miRNA-141 by fluorescence, and the fluorescence of the AgNCs-DNA synthesized in the system is quenched by BHQ at the 3' end under the condition of not adding the target miRNA-141. After the target miRNA-141 is added, a S2 chain is generated through a chain displacement amplification reaction, an H1 is opened by an S2 chain, an H2 is opened by an H1 chain, and a hybridization chain reaction is carried out. The purple filled-in portion of H1, H2 will open the AgNCs-DNA strand, which separates from the quenching group BHQ, thereby generating a fluorescent signal.

The invention generates an S2 chain based on the chain displacement reaction to carry out subsequent reaction, and constructs the fluorescent biosensor by using a hybridization chain reaction assisted circulation amplification and a fluorescence method. The sensor has the advantages of high detection speed, low detection limit, high sensitivity and the like, can make up for the defects and shortcomings of the existing miRNA-141 detection method, and realizes quick and accurate quantitative detection of the miRNA-141.

The invention has the beneficial effects that:

1. ultrasensitive detection

The double-circulation amplification of the target miRNA-141 is realized by using a strand displacement amplification reaction and a hybridization chain reaction; the molecular beacon is opened by utilizing the product of the hybridization chain reaction to generate a fluorescence signal, and the hybridization chain reaction is an amplification mode, so that the fluorescence signal is very strong, the amplification of a detection signal is realized, the detection method is simple and convenient to operate, the result is obvious, the detection period is short, the detection sensitivity is improved, and the ultrasensitive detection on the target miRNA is realized;

2. the reaction condition is mild, and the reaction speed is high

The sensor has mild reaction conditions and high reaction speed; the main detection process is realized in a homogeneous phase, so that the reaction speed is improved, the complexity of operation is reduced, and the rapid, simple and sensitive detection of the target object is realized;

3. industrialization

The preparation method is simple, has stable performance, and is suitable for the detection of a tumor marker miRNA in the field of medical health, laying a foundation for the treatment of subsequent tumors and the practical application of biosensor industrialization; the process for manufacturing the biosensor has low cost and is suitable for the requirement of low price in industrialization.

Drawings

FIG. 1 is a schematic diagram of the experiment;

FIG. 2 is a calibration curve of the sensor detection of example 1;

FIG. 3 is a graph showing the time-optimized detection results of example 2;

FIG. 4 is a graph showing the result of the optimized detection of the concentration of AgNCs-DNA in example 3;

FIG. 5 is a graph showing the results of concentration optimization of S4 in example 4.

Detailed Description

The present invention is further illustrated by the following specific examples.

A preparation method of a biosensor for detecting miRNA-141 comprises the following steps:

(1) s3 is hybridized with S1 and S2 to form a double strand in advance to form a composite probe S;

(2) preparing nano silver cluster AgNCs by using hairpin AgNCs-DNA chain:

(3) mixing miRNA-141, S4, S, AgNCs-DNA, H1 and H2 in a homogeneous system for reaction.

The preparation operation steps of the AgNCs silver cluster in the step (2) are as follows:

1. preparing a PB buffer solution (the concentration is 20 mM), wherein the PB buffer solution is composed of disodium hydrogen phosphate and sodium dihydrogen phosphate, 0.7163g of disodium hydrogen phosphate and 0.3120g of sodium dihydrogen phosphate are respectively weighed to prepare 100m L solutions, then, a part of disodium hydrogen phosphate and a part of sodium dihydrogen phosphate are mixed, and the pH value of the mixed solution is adjusted to 6.5 for later use.

2. Preparation of AgNO₃The concentration is 2mM, the volume is 1m L₃Is easy to decompose when exposed to light, and is prepared on site₃The centrifuge tube is wrapped with tinfoil paper.

3. A1M L centrifuge tube was added 76. mu. L PB (20 mM), 15. mu. L hairpin 2 (100. mu.M), and 4.5. mu. L AgNO₃(2 mM), shake for 1min, and place in a refrigerator at 4 deg.C for 15-30 min. During this period, NaHPO was formulated_4，The concentration is 2mM, the volume is 1m L₄It is prepared on site, and is easily decomposed by heating, and is prepared by using ice water of 0 deg.C.

4. After removal from the freezer, 4.5. mu. L NaHPO was added₄(2 mM) in the reaction system, shaking for 1min, and placing in a refrigerator at 4 ℃ for more than 4 h.

5. 30ul of prepared AgNCs are put into a centrifuge tube, 120ul of ultrapure water is added into the centrifuge tube and mixed evenly, 150ul of solution is taken into a micro cuvette by a pipette, the micro cuvette is scanned by a fluorescence analyzer, and the emission peak is measured to be 625nm by using exciting light 560nm, thereby proving that the AgNCs are synthesized successfully.

The procedure of the step (1) was to mix well the S3 chain (1. mu.M) of 2. mu. L and the S1 chain (1. mu.M) of 2. mu. L, the S2 chain (1. mu.M) of 2. mu. L, the buffer (50mM) of 4. mu. L, and the water of 6. mu. L, react at 95 ℃ for 3min, and then slowly cool to room temperature.

The procedure of the step (3) was to mix the reaction product 2. mu. L, the S4 strand (1. mu.M) of 2. mu. L, the H1 strand (1. mu.M) of 2. mu. L, the H2 strand (1. mu.M) of 2. mu. L, the buffer (50mM) of 4. mu. L, the miRNA-141 of 1. mu. L and ultrapure water uniformly, react at 37 ℃ for 1.2H, add the synthesized AgNCs-DNA 2. mu. L (1.0. mu.M) to the reaction product, and then detect it with a fluorometer.

The detection mode of the invention is to detect miRNA-141 by fluorescence, and the fluorescence of the AgNCs-DNA synthesized in the system is quenched by BHQ at the 3' end under the condition of not adding the target miRNA-141. After the target miRNA-141 is added, a S2 chain is generated through a chain displacement amplification reaction, an H1 is opened by an S2 chain, an H2 is opened by an H1 chain, and a hybridization chain reaction is carried out. The purple filled-in portion of H1, H2 will open the AgNCs-DNA strand, which separates from the quenching group BHQ, thereby generating a fluorescent signal.

The invention generates an S2 chain based on the chain displacement reaction to carry out subsequent reaction, and constructs the fluorescent biosensor by using a hybridization chain reaction assisted circulation amplification and a fluorescence method. The sensor has the advantages of high detection speed, low detection limit, high sensitivity and the like, can make up for the defects and shortcomings of the existing miRNA-141 detection method, and realizes quick and accurate quantitative detection of the miRNA-141.