CN110564817A - light-up silver cluster probe-based fluorescence biosensor and application thereof in miR-122 detection - Google Patents
light-up silver cluster probe-based fluorescence biosensor and application thereof in miR-122 detection Download PDFInfo
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Abstract
The invention relates to the technical field of biosensors, in particular to a light-up silver cluster probe-based fluorescence biosensor and a preparation method thereof, and also relates to a G-rich base enhanced silver nano cluster method. The detection mode of the invention is to detect miR-122 through the generation of a fluorescence signal, the hybridization of miR-122 and a complementary chain thereof triggers the change of a three-way structure, a toehold end is exposed, a silver cluster probe hybridizes with a GrHP hairpin through the toehold end, thereby triggering a chain displacement reaction, a G-rich sequence is close to the silver cluster part, and the fluorescence intensity of the silver cluster is enhanced. The sensor has the advantages of high efficiency, high specificity, simple and convenient operation, economy and no mark, does not need enzyme in a system, can make up the defects and shortcomings of the existing miR-122 detection method, and realizes quick and accurate quantitative detection and early diagnosis of related diseases.
Description
Technical Field
The invention relates to the technical field of biosensors, in particular to a light-up silver cluster probe-based fluorescence biosensor and a preparation method thereof, and also relates to a G-rich base enhanced silver nano cluster method.
Background
MicroRNAs (MiRNAs) are a group of small, non-coding RNAs that are expressed in a variety of tissues and organs and are involved in most physiological processes. Dysregulation of mirnas can lead to the development of several diseases, including cardiovascular disease, parkinson's disease, and various cancers. Among numerous miRNAs, miR-122 is closely related to the occurrence of liver diseases, and miR-122 shows high expression in the liver of normal people, but the expression level is reduced in the liver of liver cancer patients. In addition, miR-122 is also associated with chronic hepatitis C virus and drug-induced liver injury. Therefore, sensitive detection of miR-122 is very important for studying pathogenesis of liver diseases and early diagnosis of liver cancer.
the miR-122 detection technologies reported at present comprise an electrochemical method, a colorimetric method, PCR, surface plasmon resonance and the like, and some of the methods have the problems of complex operation, high cost, unstable signals, poor reproducibility and the like. Therefore, a technology which is simple to operate, efficient, good in reproducibility and reliable needs to be constructed for detecting miR-122 at present.
Disclosure of Invention
Aiming at the lack of a technology which is simple to operate and low in cost at present, the invention provides a fluorescence biosensor which is used for detecting miR-122 and is based on a three-way initiated toehold strand displacement reaction and G-rich sequence enhanced silver cluster luminescence, the experiment mainly takes DNA as a template to synthesize silver nano clusters for generating fluorescence signals, and the G-rich sequence is caused to be close to silver cluster fluorescent groups by means of the toehold mediated strand displacement reaction so as to enhance the fluorescence intensity to realize the detection of miR-122. The invention can realize the rapid migration of DNA chains by utilizing the toehold mediated chain displacement reaction, obviously improve the DNA hybridization rate and is more beneficial to the detection of miR-122 in an actual sample.
The invention is obtained by the following steps:
the light-up silver cluster probe-based fluorescence biosensor comprises the following raw materials: IS chain, GrHP probe, BS chain, silver cluster, buffer solution and miR-122;
the GrHP base series is shown as SEQ No. 1;
the IS base series IS shown as SEQ No. 2;
The BS base series is shown as SEQ No. 3;
the base series of AgNC-SP is shown as SEQ No. 4;
The base series of the miR-122 is shown in SEQ No. 5.
The preparation method of the fluorescence biosensor comprises the following steps:
(1) Preparing silver nanoclusters;
(2) and the silver nanoclusters and the three-way structure in the homogeneous phase are subjected to a chain displacement reaction.
the process of the step (1) is as follows:
A mixed solution of 15. mu.L, 100. mu.M AgNC-SP and 4.5. mu.L, 2mM silver nitrate was added to 76. mu.L, 20mM, pH6.5 phosphate buffer and reacted at 4 ℃ for 15 min; and then adding 4.5 mu L of 2mM sodium borohydride into the system, and standing in the dark for more than 6 hours to obtain the silver nanocluster.
The process of the step (2) is as follows:
adding an IS chain, a GrHP probe, a chain and the silver nanocluster in the step (1) into a buffer solution, reacting for half an hour at 37 ℃ to form a three-way structure, and then adding miR-122 into a three-way system for reaction; and finally, adding ultrapure water into the reaction system, mixing, and detecting by using a fluorescence spectrophotometer.
preferably, the process of step (2) is:
Adding 3 mu L of 5 mu M IS chain, 3 mu L of 5 mu M GrHP probe, 3 mu L of 5 mu M BS chain and 3 mu L of 5 mu M silver nanocluster obtained in the step (1) into 1 Xbuffer solution, reacting for half an hour at 37 ℃ to form a three-way structure, and then adding miR-122 into the three-way system to react for 30 min; finally, adding 50 mu L of ultrapure water into the reaction system, mixing and detecting by using a fluorescence spectrophotometer; fluorescence spectrum measurement conditions: the excitation wavelength was 565nm, and the excitation and emission slits were 10 nm.
the buffer solution is as follows: 50mM NaCl, 10mM Tris-acetate, 10mM MgCl2，100μg·mL-1 BSA；25℃，pH7.9。
The fluorescent biosensor is applied to miR-122 detection.
In the invention, 5 DNA chains are used in total, and the sequences are respectively as follows:
GrHP: GCA ACG GGT GGG GTG GGG TGG GGC ACC CGT TGC CAA TGG TGT TTG TT G GCG CCG GCT TTC T
IS:GTG CGA ATTCAA ACA CCA TTG TCA CAC TCC A
BS: TTT AGA AAG CCG GCG CC T TCG CAC TTT
AgNC-SP: CAC CAT TGG CAA CGG GTGCCC TTA ATC CCC
miR-122: UGG AGU GUG ACA AUG GUG UUU G
In the GrHP hairpin structure, the G-rich sequences (G-rich) are shown in bold, and these G-rich bases significantly enhance the fluorescence intensity of the silver clusters. The bold underlined bases in the GrHP structure and the bold underlined bases in the IS strand are complementary pairings, and the obliquely underlined bases in the BS strand are also complementary. In IS chainCAA ACA CCA TTG TCA CAC TCC A base and target miR-122 are completely complementary and paired. Bolded bases in IS chain for blocking toeh in GrHPan old moiety and a strand displacement moiety. In addition, the bolded bases in the IS strand are complementarily paired with the bolded bases in the BS strand. The bold bases in the AgNC-SP strand serve as templates for the synthesis of silver clusters, and the italic bases in the AgNC-SP strand and the italic bases in the GrHP strand are complementary.
The system comprises two prepared probes: one is a three-way probe; the other is a DNA-AgNCs signal probe (AgNC-SP). The AgNC-SP probe contains a DNA template synthesizing silver nanoclusters and a tail sequence complementary to a hairpin containing G-rich (GrHP). The three-way structure (TWJ) IS composed of three strands, GrHP, an inhibitor strand IS and a base strand BS, and comprises a 10-base toehold portion designed to ensure the successful strand displacement reaction. The GrHP structure is composed of a part containing a G-rich sequence at the 3' end, a sequence capable of being complementary with the tail part of the AgNC-SP chain and a part complementary with the BS chain. The IS chain comprises a sequence which can be completely complementary with the miR-122, and a part which can be complementary with the substrate chain IS arranged at the 5' end. The BS chain is used as a support for supporting the stable existence of the TWJ structure, and the design can avoid background signals generated by self-assembly between the AgNC-SP and the GrHP chain.
In the absence of the target miR-122 in the system, because both the toehold part and the chain migration part in GrHP are blocked by the IS chain, AgNC-SP can not replace the IS chain from the GrHP chain, so that only the weak fluorescence intensity of the silver cluster can be detected. When miR-122 exists in the system, the miR-122 performs strand hybridization and displacement by taking the base exposed at the 3' end in the IS strand as a toehold to form a miR-122/IS hybrid double strand. Since the IS strand IS displaced by miR-122, the originally blocked toehold part on the GrHP strand IS exposed, so that the AgNC-SP probe can be used as a foothold through the exposed toehold part, IS combined and undergoes strand migration until the part of the GrHP hairpin hybridization IS completely opened, and an AgNC-SP/GrHP hybrid double strand IS formed. At this time, since the silver cluster portion was close to the G-rich portion in GrHP after hybridization of AgNC-SP and GrHP, the fluorescence intensity of the silver cluster was significantly enhanced by G-rich. Therefore, the label-free fluorescent system designed by us can be used for detection of miR-122 and related clinical application.
in the invention, the detection of miR-122 is realized in a homogeneous solution, and the fluorescence intensity of the silver cluster is enhanced through a three-way initiated toehold mediated strand displacement reaction and a G-rich sequence so as to generate a strong fluorescence signal, thereby realizing the detection of miR-122 and having simpler and time-saving detection steps.
the invention has the beneficial effects that:
1. The miR-122 and a complementary strand thereof are subjected to base complementary pairing to initiate subsequent reaction, and the method has the characteristic of high specificity;
2. The invention can realize the orderly assembly of DNA chains by means of a three-way structure, and can skillfully perform the migration of the DNA chains by means of the toehold mediated chain displacement reaction, thereby accelerating the speed of the DNA chain migration;
3. the invention obviously enhances the silver cluster luminescence by means of the G-rich sequence so as to improve the signal-to-noise ratio;
4. the sensor has mild reaction conditions and high reaction speed.
5. The main processes of the detection principle of the invention are realized in homogeneous phase, thus improving the reaction speed, reducing the complexity of operation and realizing the rapid, simple and high signal-to-noise ratio detection of the target;
6. The preparation method is simple, has stable performance, and is suitable for the detection of miR-122 in the field of medical health, laying a foundation for the treatment of subsequent tumors and the practical application of biosensor industrialization;
7. 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 this experiment.
FIG. 2 is a fluorescence spectrum of a feasibility study of example 1; curve a represents the fluorescence intensity in the presence of miR-122, curve b represents the fluorescence intensity of a blank sample, curve c represents the fluorescence intensity when miR-141 replaces miR-122, curve d represents the fluorescence intensity when miR-20a replaces miR-122, and the miR-122 concentrations are all 200 nM.
FIG. 3 is a calibration curve of the sensor detection of example 2;
FIG. 4 is a linear relationship of concentration detected by the sensor of example 2.
Detailed Description
The present invention is further illustrated by the following specific examples.
The preparation method of the biosensor comprises the following steps:
(1) Preparing silver nanoclusters;
(2) Carrying out a chain displacement reaction on the silver nanoclusters and the three-way structure in the homogeneous phase to form an AgNC-SP/GrHP hybrid structure;
in the preparation method, the preparation of the silver nanocluster comprises the following steps:
The preparation of the silver nanoclusters is prepared according to a sodium borohydride reduction method reported in the literature. A mixed solution of DNA template strand AgNC-SP (15. mu.L, 100. mu.M) for synthesizing silver clusters and silver nitrate (4.5. mu.L, 2 mM) was added to 76. mu.L of phosphate buffer (20 mM, pH 6.5) and reacted at 4 ℃ for 15 min. Thereafter, 4.5. mu.L of 2mM sodium borohydride was added to the system and left in the dark for more than 6 hours to form stable silver nanoclusters.
in the preparation method, the silver nanoclusters and the three-way structure in the homogeneous phase undergo a strand displacement reaction to form an AgNC-SP/GrHP hybrid structure:
IS strands (3. mu.L, 5. mu.M), GrHP probes (3. mu.L, 5. mu.M), BS strands (3. mu.L, 5. mu.M) and previously synthesized silver clusters (3. mu.L, 5. mu.M) were added to 1 Xbuffer (50 mM NaCl, 10mM Tris-acetate, 10mM MgCl2，100 μg•mL-1BSA (pH 7.9@25 ℃) at 37 ℃ for half an hour to form a three-way structure, and then adding 3 mu L of miR-122 with different concentrations into the three-way system to react for 30 min. Thereafter, 50. mu.L of ultrapure water was added to the reaction system, and the mixture was mixed and detected by a fluorescence spectrophotometer. Fluorescence spectrum measurement conditions: the excitation wavelength was 565nm, and the excitation and emission slits were 10 nm.
the detection mode of the invention is to detect miR-122 through the generation of a fluorescence signal, the hybridization of miR-122 and a complementary chain thereof triggers the change of a three-way structure, a toehold end is exposed, a silver cluster probe hybridizes with a GrHP hairpin through the toehold end, thereby triggering a chain displacement reaction, a G-rich sequence is close to the silver cluster part, and the fluorescence intensity of the silver cluster is enhanced. The sensor has the advantages of high efficiency, high specificity, simple and convenient operation, economy and no mark, does not need enzyme in a system, can make up the defects and shortcomings of the existing miR-122 detection method, and realizes quick and accurate quantitative detection and early diagnosis of related diseases.
Example 1
in the preparation method, the preparation of the silver nanocluster comprises the following steps:
the preparation of the silver nanoclusters is prepared according to a sodium borohydride reduction method reported in the literature. A mixed solution of DNA template strand AgNC-SP (15. mu.L, 100. mu.M) for synthesizing silver clusters and silver nitrate (4.5. mu.L, 2 mM) was added to 76. mu.L of phosphate buffer (20 mM, pH 6.5) and reacted at 4 ℃ for 15 min. Thereafter, 4.5. mu.L of 2mM sodium borohydride was added to the system and left in the dark for more than 6 hours to form stable silver nanoclusters.
the silver nanoclusters are synthesized, and the main steps of the reaction process in the homogeneous solution are as follows:
IS strands (3. mu.L, 5. mu.M), GrHP probes (3. mu.L, 5. mu.M), BS strands (3. mu.L, 5. mu.M) and previously synthesized silver clusters (3. mu.L, 5. mu.M) were added to 1 Xbuffer (50 mM NaCl, 10mM Tris-acetate, 10mM MgCl2，100 μg•mL-1BSA (pH 7.9@25 ℃) at 37 ℃ for half an hour to form a three-way structure, and then adding 3 mu L of 200nM miR-122 into the three-way system to react for 30min to obtain a curve a. In the three-way structure, a curve obtained without adding miR-122 is b, and miR-122 is replaced by miR-141 and miR-20a to obtain curves c and d respectively. Finally, 50. mu.L of ultrapure water was added to the reaction system, and the mixture was mixed and examined with a fluorescence spectrophotometer. Fluorescence spectrum measurement conditions: the excitation wavelength was 565nm, and the excitation and emission slits were 10 nm.
According to detection, as shown in FIG. 2, the detected fluorescence intensity is strong in the presence of the target miR-122 (curve a), the fluorescence intensity is low in the absence of the target (curve b), and the target miR-122 is replaced by miR-141 and miR-20a, and the fluorescence intensities are low (curves c and d).
example 2
In the preparation method, the preparation of the silver nanocluster comprises the following steps:
The nanoclusters are prepared according to a sodium borohydride reduction method reported in the literature. A mixed solution of DNA template strand AgNC-SP (15. mu.L, 100. mu.M) for synthesizing silver clusters and silver nitrate (4.5. mu.L, 2 mM) was added to 76. mu.L of phosphate buffer (20 mM, pH 6.5) and reacted at 4 ℃ for 15 min. Thereafter, 4.5. mu.L of 2mM sodium borohydride was added to the system and left in the dark for more than 6 hours to form stable silver nanoclusters.
The silver nanoclusters are synthesized, and the main steps of the reaction process in the homogeneous solution are as follows:
IS strands (3. mu.L, 5. mu.M), GrHP probes (3. mu.L, 5. mu.M), BS strands (3. mu.L, 5. mu.M) and previously synthesized silver clusters (3. mu.L, 5. mu.M) were added to 1 Xbuffer (50 mM NaCl, 10mM Tris-acetate, 10mM MgCl2，100 μg•mL-1BSA (pH 7.9@25 ℃) at 37 ℃ for half an hour to form a three-way structure, and then 3. mu.L of different concentrations of miR-122 (0.1 nM, 0.5nM, 1nM, 5nM, 10nM, 50nM, 100nM, 200 nM) was added to the three-way system for reaction for 30 min. Thereafter, 50. mu.L of ultrapure water was added to the reaction system, and the mixture was mixed and detected by a fluorescence spectrophotometer. Fluorescence spectrum measurement conditions: the excitation wavelength was 565nm, and the excitation and emission slits were 10 nm.
As detected, as shown in FIG. 3, the fluorescence signal intensity is gradually increased with the increase of the concentration of miR-122. Furthermore, as shown in FIG. 4, the log of miR-122 concentration is linear with fluorescence intensity.
the above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the embodiments, and any other changes, modifications, combinations, substitutions and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.
Sequence listing
<110> university of Jinan
<120> light-up silver cluster probe-based fluorescence biosensor and application thereof in miR-122 detection
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Claims (7)
1. The light-up silver cluster probe-based fluorescence biosensor is characterized by comprising the following raw materials: IS chain, GrHP probe, BS chain, silver cluster, buffer solution and miR-122;
the GrHP base series is shown as SEQ No. 1;
The IS base series IS shown as SEQ No. 2;
The BS base series is shown as SEQ No. 3;
the base series of AgNC-SP is shown as SEQ No. 4;
The base series of the miR-122 is shown in SEQ No. 5.
2. The method of making a fluorescent biosensor as claimed in claim 1, comprising the steps of:
(1) Preparing silver nanoclusters;
(2) And the silver nanoclusters and the three-way structure in the homogeneous phase are subjected to a chain displacement reaction.
3. the method according to claim 2, wherein the step (1) comprises the steps of:
A mixed solution of 15. mu.L, 100. mu.M AgNC-SP and 4.5. mu.L, 2mM silver nitrate was added to 76. mu.L, 20mM, pH6.5 phosphate buffer and reacted at 4 ℃ for 15 min; and then adding 4.5 mu L of 2mM sodium borohydride into the system, and standing in the dark for more than 6 hours to obtain the silver nanocluster.
4. The method according to claim 2, wherein the step (2) comprises the steps of:
adding an IS chain, a GrHP probe, a BS chain and the silver nanoclusters in the step (1) into a buffer solution, reacting for half an hour at 37 ℃ to form a three-way structure, and then adding miR-122 into a three-way system for reaction; and finally, adding ultrapure water into the reaction system, mixing, and detecting by using a fluorescence spectrophotometer.
5. the method according to claim 2 or 4, wherein the step (2) comprises the steps of:
adding the IS chain, the GrHP probe, the BS chain and the silver nanoclusters in the step (1) into a 1 x buffer solution, reacting for half an hour at 37 ℃ to form a three-way structure, and then adding miR-122 into a three-way system to react for 30 min; finally, adding 50 mu L of ultrapure water into the reaction system, mixing and detecting by using a fluorescence spectrophotometer; fluorescence spectrum measurement conditions: the excitation wavelength was 565nm, and the excitation and emission slits were 10 nm.
6. The method according to claim 2, wherein the buffer solution is: 50mM NaCl, 10mM Tris-acetate, 10mM MgCl2，100μg·mL-1 BSA；25℃，pH7.9。
7. The use of the fluorescent biosensor of claim 1 in the detection of miR-122.
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| CN113398135A (en) * | 2021-06-07 | 2021-09-17 | 中国药科大学 | Nano system for HILI (hypohalite) in-situ detection and drug release |
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| CN110004213A (en) * | 2019-03-04 | 2019-07-12 | 山东师范大学 | The method for mediating strand replacement reaction to cause rolling circle amplification and FRET detection miRNA based on Toehold |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110004213A (en) * | 2019-03-04 | 2019-07-12 | 山东师范大学 | The method for mediating strand replacement reaction to cause rolling circle amplification and FRET detection miRNA based on Toehold |
Non-Patent Citations (1)
| Title |
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| XUE ZHANG等: "DNA three-way junction-actuated strand displacement for miRNA detection using a fluorescence light-up Ag nanocluster probe", 《ANALYST》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113398135A (en) * | 2021-06-07 | 2021-09-17 | 中国药科大学 | Nano system for HILI (hypohalite) in-situ detection and drug release |
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