CN108663354A - One kind is based on DNA- silver nanoclusters structure Electrochemiluminescsensor sensor, preparation and its application - Google Patents
One kind is based on DNA- silver nanoclusters structure Electrochemiluminescsensor sensor, preparation and its application Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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- G—PHYSICS
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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Abstract
The present invention provides one kind based on DNA silver nanoclusters structure Electrochemiluminescsensor sensor, preparation and its application, the sensitivity detection to miRNA 21 may be implemented, by the way that object MiRNA is added, it is opened with the hair clip DNA HP1 of object miRNA number of base complementary pairings and is combined with object, formation object hybridizes chain with hair clip DNA H1's, after addition with the more hair clip DNA HP2 of hair clip DNA HP1 base pair complementarities, object miRNA gradually is deprived of, and participation recycles next time.Therefore the addition of miRNA can cause this catalysis hair clip self assembly amplification system, utilize using the electrochemiluminescence signal detection object of the DNA silver nanoclusters formed as template.
Description
Technical field
The invention belongs to biosensor technology fields, and in particular to one kind building electroluminescent chemistry based on DNA- silver nanoclusters
The sensitivity detection to miRNA-21 may be implemented in luminescence sensor, preparation and its application.
Background technology
MicroRNAs (miRNAs) is that small molecule non-encoding ribonucleic acid (RNA) is found in eukaryocyte about
There are 19-25 nucleotide, it plays key effect in eucaryote.They serve as posttranscriptional gene expression regulation person.It is more next
More evidences shows that the unconventionality expression of miRNAs is related with many diseases, including malignant tumour, cancer and nerve degeneration.
The expression of miRNAs can be used as the biomarker of cancer and the diagnosing and treating of other diseases.With embryology and oncology base
The rapid development of plinth research, important mechanisms one of of the miRNAs gene table science of heredity receive more and more attention.
Detection for miRNAs has had many traditional detection methods to be used to detect miRNAs, including
Northern hybridization, real-time PCR, genetic chip etc..However, these methods have some intrinsic limitations.
Northern crossover operations are complicated and need radioactive label, not only result in serious pollution, and sensitivity is low.real-
Although time PCR and biochip technology high sensitivity, required instrument and expensive reagents, therefore limit method
Be widely used.Especially biochip technology, almost few users can afford.
For the disadvantage for overcoming these conventional methods intrinsic, some low costs, highly sensitive detection method have been developed
Being imperative.There is provided simple, low detection limit and selective method come detect that miRNAs ten divides must
It wants.
Invention content
It is passed based on DNA- silver nanoclusters structure electrogenerated chemiluminescence in order to solve the above technical problems, the present invention provides one kind
Sensor and preparation method thereof is template using DNA double chain, and silver nanoparticle is synthesized to the reduction of silver nitrate using sodium borohydride
Cluster, and acted on using the complementary pairing of base between DNA and miRNA sequence sequence, construct a catalysis hair clip self assembly
Amplification system.By the way that object MiRNA is added, beaten with the hair clip DNA HP1 of object miRNA number of base complementary pairings
It opens and is combined with object, formation object hybridizes chain with hair clip DNA H1's, matches with hair clip DNA HP1 base complementrities being added
After more hair clip DNA HP2, object miRNA gradually is deprived of, and participation recycles next time.Therefore miRNA
Addition can cause this catalysis hair clip self assembly amplification system, utilize using the electroluminescent chemistry of the DNA silver nanoclusters formed as template
Luminous signal detects object.
Detecting miRNAs's based on DNA- silver nanoclusters structure Electrochemiluminescsensor sensor the present invention also provides a kind of
Using.
Specific technical solution of the present invention is as follows：
A kind of preparation method building Electrochemiluminescsensor sensor based on DNA- silver nanoclusters provided by the invention, including
Following steps：
(1), the DNAS1 of 2.5OD, DNA HP1, DNA HP2 and miRNA-21 sequence are dissolved in buffer solution respectively,
Obtain DNA S1 buffer solutions, DNA HP1 buffer solutions, DNA HP2 buffer solutions and miRNA-21 buffer solutions；
(2), the glass-carbon electrode after polishing treatment is immersed in chlorauric acid solution, carries out electro-deposition, taken out, cleaning obtains
The glass-carbon electrode of gold nanoparticle is modified；
(3), the glass-carbon electrode for having modified gold nanoparticle that step (2) obtains is immersed in the buffer solution of DNAS1,
Culture, then takes out, and cleans, has been modified the glass-carbon electrode of single stranded DNA S1；
(4), the glass-carbon electrode for having modified single stranded DNA S1 that step (3) obtains is immersed in 6- sulfydryl -1- hexanol solution,
Culture, cleaning；
(5), miRNA-21 buffer solutions and DNA HP1, DNA HP2 buffer solutions are mixed, hybridization is prepared into DNA
HP1- HP2 solution；
(6) the 6- sulfydryls -1- hexanols/DNAS1 glass-carbon electrodes modified obtained in step (4) are immersed in step (5)
The DNA HP1-HP2 solution arrived, culture, then takes out, and cleans, has been modified the glass-carbon electrode of DNA HP1-HP2；
(7) glass-carbon electrode for having modified DNA HP1-HP2 that step (6) obtains is immersed in silver nitrate solution, culture, then
By electrode immerse sodium borohydride solution in, culture, then take out, cleaning to get.
Specifically, step (1) is that respectively DNAS1, DNA HP1 of 2.5OD that will be bought and DNA HP2 sequences difference are molten
Solution respectively obtains a concentration of 100 μM of DNA S1 buffer solutions, a concentration of 100 μM of DNA HP1 in phosphate buffer solution
Buffer solution and a concentration of 100 μM of DNA HP2 buffer solutions；The miRNA-21 of 2.5OD is configured to 20 μM slow with DEPC water
Solution is rushed, is saved backup at 4 DEG C；Above-mentioned solution obtained is according to using needs to be diluted.
Further, DNAS1 sequences are in step (1)：5'-SH-GGTTGCTATATCG-3’；
DNA HP1 sequences are：
5'-CCCCCCCCCCCCTCAACATCAGTCTGATAAGCTACCGTCTTGAGCTAGCTTATCAGACTGCGATAT
AGCAACC-3；
DNA HP2 sequences are：5'-TAAGCTAGCTCAAGACGGTAGCTTATCAGACTGCCGTCTTGAGCCCCCCCCCCCCC；
MiRNA-21 sequences are：5'-UAGCUUAUCAGACUGAUGUUGA-3’.
Further, the pH of phosphate buffer solution is 7.4 in step (1), a concentration of 0.1M.
Specifically, step (2) is：Glass-carbon electrode after polishing treatment is immersed into the gold chloride that 2mL mass fractions are 0.1%
In solution, electro-deposition is carried out, electro-deposition current potential is -0.2V, and electrodeposition time is 100 seconds.The gold nanoparticle of acquisition has very
Good bio-compatibility can be applied to biosensor in conjunction with a plurality of designed probe, therefore by the gold nano grain of preparation
Detection have good amplification.
Further, in step (2), the specific processing method of glass-carbon electrode after the polishing treatment is：Glass-carbon electrode is first
It is processed by shot blasting successively with the aluminium powder of 0.3 and 0.5mm, then is sequentially placed into volume ratio HNO3:H2O=1:1 solution, ethyl alcohol are molten
In liquid and ultra-pure water, ultrasonic cleaning is carried out, the time of ultrasonic cleaning is respectively 3~5min.
Further, step (3) is specially：The glass-carbon electrode for having modified gold nanoparticle is immersed to the slow of 5 μM of DNAS1
It rushes in solution after cultivating 10-12h and takes out, cleaned with phosphate buffer solution.The DNA probe S1 of absorption is removed without with this.
Further, it is cultivated described in step (4), specifically：1-1.5h is cultivated at 37 DEG C -40 DEG C.6- sulfydryls -1- oneself
Alcohol is used for enclosed-electrode surface-active site, avoids non-specific adsorption.
Further, step (5) is specifically：By the DNA HP1 buffer solutions of 5 μM of 40 μ L, the DNA HP2 of 5 μM of 40 μ L
Buffer solution and the mixing of miRNA-21 buffer solutions hybridize 2h at 37 DEG C and obtain DNA HP1-HP2 solution；
Further, it is cultivated described in step (6), specifically：1.5-2h is cultivated at 37 DEG C.
Further, step (7) is specifically：The glass-carbon electrode for having modified DNA HP1-HP2 is immersed into 120 μ L, 30 μM of nitre
In sour silver solution, it is protected from light culture 0.5-1h at 4 DEG C, then immerse in 120 μ L, 30 μM of sodium borohydride solutions, training is protected from light at 4 DEG C
Support 2-2.5h, using sodium borohydride reproducibility synthesize using DNA as the silver nanoclusters of template, then take out, cleaning to get.
It is provided by the invention a kind of based on DNA- silver nanoclusters structure Electrochemiluminescsensor sensor, using above method system
It is standby to obtain.
A kind of answering based on DNA- silver nanoclusters structure Electrochemiluminescsensor sensor detection miRNAs provided by the invention
With specific detection method is：
A, the DNAS1 of 2.5OD, DNA HP1, DNA HP2 and miRNA-21 sequence are dissolved in respectively in buffer solution, are obtained
To DNA S1 buffer solutions, DNA HP1 buffer solutions, DNA HP2 buffer solutions and miRNA-21 buffer solutions；
B, the glass-carbon electrode after polishing treatment is immersed in chlorauric acid solution, carries out electro-deposition, taken out, cleaning is repaiied
The glass-carbon electrode of gold nanoparticle is adornd；
C, the glass-carbon electrode for having modified gold nanoparticle that step b is obtained is immersed in the buffer solution of DNAS1, is trained
It supports, then takes out, clean, modified the glass-carbon electrode of single stranded DNA S1；
D, the glass-carbon electrode for having modified single stranded DNA S1 that step c is obtained is immersed in 6- sulfydryl -1- hexanol solution, culture,
Cleaning；
E, the miRNA-21 buffer solutions and DNA HP1 of various concentration, DNA HP2 buffer solutions are mixed, is hybridized, prepared
At the DNA HP1-HP2 solution of various concentration；
F, 6- sulfydryls -1- hexanols/DNAS1 glass-carbon electrodes of having modified that step d is obtained are immersed into the difference obtained in step e
The DNA HP1-HP2 solution of concentration, culture, then takes out, and cleans, has been modified the glass-carbon electrode of DNA HP1-HP2；
G, the glass-carbon electrode for having modified DNA HP1-HP2 that step f is obtained is immersed in silver nitrate solution, culture, then will
Electrode immerse sodium borohydride solution in, culture, then take out, cleaning to get；
H, glass-carbon electrode that step g is obtained to silver nanoclusters immerses the phosphate buffer PBS containing potassium peroxydisulfate
In (0.1M), it is detected with electroluminescent chemiluminescence method；It is corresponded to according to silver nanoclusters prepared by the miRNA-21 of various concentration
Luminous intensity, build the linear relationship of signal strength and miRNA-21 concentration, detection of the realization to miRNA-21.
Specifically, step a is to dissolve respectively DNAS1, DNA HP1 of 2.5OD and the DNA HP2 sequences of purchase respectively
It is slow that a concentration of 100 μM of DNA S1 buffer solutions, a concentration of 100 μM of DNA HP1 are respectively obtained in phosphate buffer solution
Rush solution and a concentration of 100 μM of DNA HP2 buffer solutions；The miRNA-21 of 2.5OD is configured to 20 μM of buffering with DEPC water
Solution saves backup at 4 DEG C；Above-mentioned solution obtained is according to using needs to be diluted.
Further, DNAS1 sequences are in step a：5'-SH-GGTTGCTATATCG-3’；
DNA HP1 sequences are：
5'-CCCCCCCCCCCCTCAACATCAGTCTGATAAGCTACCGTCTTGAGCTAGCTTATCAGACTGCGATAT
AGCAACC-3；
DNA HP2 sequences are：5'-TAAGCTAGCTCAAGACGGTAGCTTATCAGACTGCCGTCTTGAGCCCCCCCCCCCCC；
MiRNA-21 sequences are：5'-UAGCUUAUCAGACUGAUGUUGA-3’.
Further, the pH of phosphate buffer solution is 7.4 in step a, a concentration of 0.1M.
Specifically, step b is：It is molten that glass-carbon electrode after polishing treatment is immersed into the gold chloride that 2mL mass fractions are 0.1%
In liquid, electro-deposition is carried out, electro-deposition current potential is -0.2V, and electrodeposition time is 100 seconds.The gold nanoparticle of acquisition has fine
Bio-compatibility, biosensor can be applied in conjunction with a plurality of designed probe, therefore by the gold nano grain of preparation
Detection has good amplification.
Further, in step c, the specific processing method of glass-carbon electrode after the polishing treatment is：Glass-carbon electrode first according to
The secondary aluminium powder with 0.3 and 0.5mm is processed by shot blasting, then is sequentially placed into volume ratio HNO3:H2O=1:1 solution, ethanol solution
In ultra-pure water, ultrasonic cleaning is carried out, the time of ultrasonic cleaning is respectively 3~5min.
Further, step c is specially：The glass-carbon electrode for having modified gold nanoparticle is immersed to the buffering of 5 μM of DNAS1
It is taken out after cultivating 10-12h in solution, is cleaned with phosphate buffer solution, the DNA probe S1 of absorption is removed without with this.
Further, it is cultivated described in step d, specifically：1-1.5h, 6- sulfydryl -1- hexanols are cultivated at 37 DEG C -40 DEG C
For enclosed-electrode surface-active site, non-specific adsorption is avoided.
Further, step e is specifically：The DNA HP2 of the DNA HP1 buffer solutions of 5 μM of 40 μ L, 5 μM of 40 μ L are delayed
The miRNA-21 buffer solutions mixing for rushing solution and 10 μ L various concentrations, 2h, object miRNA-21 and its portion are hybridized at 37 DEG C
Divide the DNA HP1 of base pair complementarity to combine and form hybridization chain, the DNA HP2 with the more base pairings of DNA HP1 chains will
MiRNA-21, which is deprived, gets off to be formed DNA HP1-HP2, is deprived of the miRNA-21 to get off and participates in next cyclic process, because
This obtains the DNA HP1-HP2 solution of various concentration by catalysis hair clip self assembly amplification process；
MiRNA-21 buffer concentrations described in step e are respectively 10-3,10-2,10-1,1,10,102,103With 104fM。
Further, it is cultivated described in step f, specifically：2h is cultivated at 37 DEG C.
Further, step g is specifically：The glass-carbon electrode for having modified DNA HP1-HP2 is immersed into 120 μ L, 30 μM of nitric acid
In silver-colored solution, it is protected from light culture 0.5-1h at 4 DEG C, then immerse in 120 μ L, 30 μM of sodium borohydride solutions, culture is protected from light at 4 DEG C
2-2.5h, using sodium borohydride reproducibility synthesize using DNA as the silver nanoclusters of template, then take out, cleaning to get.
Step h is specially：
The 0.1MPBS phosphate that the glass-carbon electrode immersion 3mL that step g is obtained to silver nanoclusters contains 0.5M potassium peroxydisulfates delays
In fliud flushing, photomultiplier high pressure is set as 800V, and scanning voltage range is 0V to -1.6V, carries out electroluminescent chemistry at room temperature
Shine detection, is added according to the miRNA-21 of various concentration, causes catalysis hair clip self assembly amplification system, generates various concentration
DNA HP1-HP2, the DNA HP1-HP2 of various concentration form the silver nanoclusters (such as Figure 1B) of various concentration, different silver nanoparticles
Cluster corresponds to different luminous intensities, builds the linear relationship of signal strength and miRNA-21 concentration, realizes the inspection to miRNA-21
It surveys.
The PH of phosphate buffer solution used is 6.0-8.5 in this experiment, and optimal pH is 7.4.
It can be generated and be reacted with coreagent potassium peroxydisulfate as the silver nanoclusters that template is formed using DNA, reaction process is：
(1)AgNCs +e–→AgNCs–·(2)S2O82 –+e–→SO4 2–+SO4 –·(3)AgNCs–·+SO4 –·→AgNCs*+SO4 2–(4)
AgNCs*→AgNCs+hν.By this process, the optical signal of silver nanoclusters is obtained, and the amount of signal strength and silver nanoclusters has
It closes, that is, related with the concentration of double-stranded DNA HP1-HP2, it is also just indirectly related with the concentration of miRNA-21.With miRNA-
The electrochemiluminescence signal of the increase of 21 concentration, silver nanoclusters enhances therewith, constructs signal strength and miRNA-21 concentration
Linear relationship, realize detection to miRNA-21.Therefore, this sensor can quantify the miRNA-21 of various concentration
Detection.
Compared with prior art, the preparation method of this biosensor is synthesized simply using silver nanoclusters, and energy consumption is low, at
This low, good biocompatibility, by DNA, silver nanoclusters are associated, and then using DNA base complementary pairing principle construction at one
The gold nanoparticle of a catalysis hair clip self assembly amplification system, Simultaneous Electrodeposition can adsorb more DNAS1, and DNAS1 can inhale
Attached more DNA HP1-HP2, form more silver nanoclusters, therefore gold nanoparticle also being had to silver nanoclusters
Further amplification, therefore more silver nanoclusters with electrogenerated chemiluminescence performance can be prepared, convenient for instrument to light
The collection of signal.Using the electrochemiluminescence signal of silver nanoclusters, the linear relationship of structure and miRNA-21 concentration is realized
Detection to miRNA-21.Therefore there is high sensitivity, detection to limit the detection of miRNA-21, low, selectivity is good, stability is good
The characteristics of.
Description of the drawings
Figure 1A is based on the schematic diagram that DNA is the structure Electrochemiluminescsensor sensor process that template forms silver nanoclusters；
Figure 1B is the schematic diagram that miRNA-21 is detected based on this Electrochemiluminescsensor sensor；
Fig. 2A is the electrogenerated chemiluminescence inspection that miRNA-21 feasibility Experiments are detected based on this Electrochemiluminescsensor sensor
It surveys；
Fig. 2 B are the cyclic voltammetry detection that miRNA-21 feasibility Experiments are detected based on this Electrochemiluminescsensor sensor；
A is bare glassy carbon electrode；
B is the glass-carbon electrode of gold nanoparticle/silver nanoclusters modification；
C is the gold nanoparticle/silver nanoclusters glass-carbon electrode for introducing object；
Fig. 3 A are the cyclic voltammogram of each step of modified electrode；
A is bare glassy carbon electrode；
B is the glass-carbon electrode of gold nanoparticle modification；
C is the glass-carbon electrode of single stranded DNA S1 modifications；
D is the glass-carbon electrode with 6- sulfydryl -1- hexanol solution treatments；
E is the glass-carbon electrode with object miRNA-21 solution treatments；
Fig. 3 B are the impedance diagram of each step of modified electrode；
A is bare glassy carbon electrode；
B is the glass-carbon electrode of gold nanoparticle modification；
C is the glass-carbon electrode of single stranded DNA S1 modifications；
D is the glass-carbon electrode with 6- sulfydryl -1- hexanol solution treatments；
E is the glass-carbon electrode with object miRNA-21 solution treatments；
Influence diagram of the concentration to this experiment that Fig. 4 A are DNAH1 and DNAH2.
Fig. 4 B are the optimization figure of phosphate buffer solution PH；
Fig. 4 C are the optimization figure for being catalyzed hair clip self assembly incubation time；
Fig. 4 D are the optimization figure for being catalyzed hair clip self assembly cultivation temperature；
It is that template forms silver nanoclusters structure Electrochemiluminescsensor sensor to various concentration miRNA- that Fig. 5, which is based on DNA,
21 luminous intensity figure；
A is 10-3FM, b 10-2FM, c 10-1FM 1, d 1fM, e 10fM, f 102FM, g 103FM, h 104
fM；
Fig. 6 standard curves of the sensor to various concentration miRNA-21 thus；
Fig. 7 A are the corresponding electrogenerated chemiluminescence intensity map of disturbance object；
A is blank, and b miRNA-155, c miRNA-101, d are single base mispairing miRNA-21,
E is miRNA-21, f be mixture include miRNA-155, miRNA-101, single base mispairing miRNA-21 and
miRNA-21；
Fig. 7 B thus sensor to detect object 1fM miRNA-21 stability diagram.
Specific implementation mode
Embodiment 1
A kind of application detecting miRNAs based on DNA- silver nanoclusters structure Electrochemiluminescsensor sensor, specific detection side
Method is：
(1), it is 7.4 a concentration of the DNAS1 of the 2.5OD of purchase, DNA HP1, DNA HP2 sequences to be dissolved in pH respectively
In 0.1M phosphate buffer solutions, DNA S1 buffer solutions, a concentration of 100 μM of DNA HP1 for respectively obtaining 100 μM of concentration are slow
Rush solution and a concentration of 100 μM of DNA HP2 buffer solutions；The miRNA-21 of 2.5OD is configured to 20 μM slow with DEPC water
Solution is rushed, is saved backup at 4 DEG C；Above-mentioned solution obtained is according to using needs to be diluted.
Wherein DNAS1 sequences：5'-SH-GGTTGCTATATCG-3 ', DNA HP1 sequences：
5'-CCCCCCCCCCCCTCAACATCAGTCTGATAAGCTACCGTCTTGAGCTAGCTTATCAGACTGCGATAT
AGCAACC-3’
DNA HP2 sequences：
5'-TAAGCTAGCTCAAGACGGTAGCTTATCAGACTGCCGTCTTGAGCCCCCCCCCCCCC
MiRNA-21 sequences：5'-UAGCUUAUCAGACUGAUGUUGA-3’
(2), glass-carbon electrode is first processed by shot blasting with the aluminium powder of 0.3 and 0.5mm successively, then is sequentially placed into volume ratio
HNO3: H2O=1:In 1 solution, ethanol solution and ultra-pure water, ultrasonic cleaning is carried out, time of ultrasonic cleaning is respectively 3~
5min immerses the glass-carbon electrode after polishing treatment in the chlorauric acid solution that 2mL mass fractions are 0.1%, carries out electro-deposition,
Electro-deposition current potential is -0.2V, and electrodeposition time is 100 seconds, obtains the glass-carbon electrode of gold nanoparticle modification；
(3), the glass-carbon electrode for having modified gold nanoparticle is immersed in the buffer solution containing 5 μM of DNAS1, room temperature
Lower culture 12h makes DNAS1 be attached to electrode surface by golden sulfide linkage；
(4), the glass-carbon electrode for having modified single stranded DNA S1 that step (3) obtains is immersed in 10mM 6- sulfydryl -1- hexanols
In solution, 1h is cultivated at 37 DEG C, 6- sulfydryl -1- hexanols are used for enclosed-electrode surface-active site, avoid non-specific adsorption；
(5), 40 μ L, 5 μM of DNA HP1 buffer solutions, 40 μ L, 5 μM of DNA HP2 buffer solutions and 10 μ L are a concentration of
10-3, 10-2,10-1,1,10,102,103, 104The buffer solution of fM miRNA-21 mixes respectively, and 2h objects are hybridized at 37 DEG C
MiRNA-21 is combined to form hybridization chain with the H1 of its number of base complementary pairing, and the H2 with the more base pairings of H1 chains will
MiRNA-21, which is deprived, gets off to be formed DNA HP1-HP2, is deprived of the miRNA-21 to get off and participates in next cyclic process, because
This obtains various concentration DNA HP1-HP2 solution by catalysis hair clip self assembly amplification process；
(6), the 6- sulfydryls -1- hexanols/DNAS1 glass-carbon electrodes modified obtained in step (4) are immersed in step (5)
To DNA HP1-HP2 solution in, 2h is cultivated at 37 DEG C, to modified the glass-carbon electrode of DNA HP1-HP2；
(7), the glass-carbon electrode for having modified DNA HP1-HP2 is immersed in 120 μ L, 30 μM of silver nitrate solutions, is kept away at 4 DEG C
Optical culture 0.5-1h, then immerse in 120 μ L, 30 μM of sodium borohydride solutions and be protected from light culture 2-2.5h at 4 DEG C, utilize sodium borohydride
Reproducibility synthesize using DNA as the silver nanoclusters of template；
(8), the phosphate that the glass-carbon electrode immersion 3mL that step (7) is obtained to silver nanoclusters contains 0.5M potassium peroxydisulfates is slow
In fliud flushing PBS (0.1M, PH7.4), photomultiplier high pressure is set as 800V, and scanning voltage range is 0V to -1.6V, in room
Temperature is lower to carry out electrochemiluminescdetection detection, obtains the corresponding signal strength of miRNA-21 concentration of various concentration, such as Fig. 5；Structure
The linear relationship of signal strength and miRNA-21 concentration, such as Fig. 6 realize the detection to miRNA-21 using this linear relationship.
Embodiment 2
The feasibility amplification detection that miRNA-21 is detected based on DNA- silver nanoclusters structure Electrochemiluminescsensor sensor is ground
Study carefully：
A, the bare glassy carbon electrode of polishing treatment, without subsequent processing；
B, the bare glassy carbon electrode of polishing treatment is immersed in chlorauric acid solution and carries out electro-deposition acquisition modification gold nanoparticle
Glass-carbon electrode, then be immersed in the buffer solution containing 5 μM of DNAS1, cultivate 10-12h at room temperature, then by this electrode
It is immersed in 10mM 6- sulfydryl -1- hexanol solution, 1h is cultivated at 37 DEG C, the electrode of acquisition is then immersed in 5 μM of DNA
In the buffer solution of HP1-HP2, it is molten that the glass-carbon electrode for having modified DNA HP1-HP2 is finally immersed into 120 μ L, 30 μM of silver nitrates
In liquid, it is protected from light culture 1h at 4 DEG C, then immerses in 120 μ L, 30 μM of sodium borohydride solutions and is protected from light culture 2h at 4 DEG C, utilizes boron
The reproducibility of sodium hydride is synthesized using DNA as the silver nanoclusters (such as Figure 1A) of template；
C, the assembling process (such as Figure 1B) of electrode, specific operation process is similar to step b, by 6- sulfydryl -1- hexanol solution
The glass-carbon electrode of processing is immersed in containing 104FM objects miRNA-21, the mixing of 5 μM of DNA HP1 and 5 μM of DNA HP2 are molten
In liquid, object causes catalysis hair clip self assembly amplification system, obtains DNA HP1-HP2, has modified the glass of DNA HP1-HP2
Carbon electrode passes through the processing of identical with step b silver nitrate and sodium borohydride, obtains causing the silver nanoclusters formed by object；
Electrochemiluminescdetection detection (such as Fig. 2A) and cycle volt will be carried out according to above-mentioned a, the electrode that tri- kinds of methods of b, c obtain
Peace method detects (such as Fig. 2 B), this design of the results show obtained can effectively amplification detection object miRNA-21.
In assembling process, electrode surface uses cyclic voltammetry (Fig. 3 A) and Electrode with Electrochemical Impedance Spectroscopy characterization (Fig. 3 B), card respectively
Bright assembling process is successful.
Embodiment 3
MiRNA-21 optimal conditions are detected based on DNA- silver nanoclusters structure Electrochemiluminescsensor sensor：
The Electrochemiluminescsensor sensor detection detection miRNA-21 prepared according to embodiment 1, changes DNA HP1 and DNA
The concentration of HP2 is all 1,2,3,4,5,6,7 μM (DNA HP1 and DNA HP2 concentration is identical), and controlling the concentration of miRNA-21 is
1fM, other conditions are identical.Detection forms the electrochemiluminescence signal of silver nanoclusters, as a result such as Fig. 4 A, shows 5 μM as experiment
Optium concentration；
According to embodiment 1 prepare Electrochemiluminescsensor sensor detection detection miRNA-21, change solution PH be 6,
6.5,7,7.4,7.5,8 and 8.5, the concentration for controlling miRNA-21 is 1fM, and other conditions are identical.Detection forms silver nanoclusters
Electrochemiluminescence signal show that the best pH value of solution is 7.4 as a result such as Fig. 4 B；
The Electrochemiluminescsensor sensor detection detection miRNA-21 prepared according to embodiment 1,
Change catalysis hair clip self assembly amplification process incubation time be 0.5,1,1.5,2,2.5,3h, control miRNA-21
Concentration be 1fM, other conditions are identical.Detection forms the electrochemiluminescence signal of silver nanoclusters, as a result such as Fig. 4 C, shows
2h is the best incubation time for being catalyzed hair clip self assembly amplification process；
The Electrochemiluminescsensor sensor detection detection miRNA-21 prepared according to embodiment 1, changes catalysis hair clip from group
The cultivation temperature for filling amplification process is 20,25,30,35,37,40,45 DEG C, and the concentration for controlling miRNA-21 is 1fM, other
Part is identical.Detection forms the electrochemiluminescence signal of silver nanoclusters, as a result such as Fig. 4 D, shows that 37 DEG C are catalysis hair clip self assemblies
The optimum culturing temperature of amplification process.
Embodiment 4
Under the optimal experiment condition explored in embodiment 3, examined according to Electrochemiluminescsensor sensor prepared by embodiment 1
MiRNA-21 is surveyed, the concentration for changing miRNA-21 is respectively 10-3,10-2,10-1,1,10,102,103, 104FM, detection are opposite
The electrogenerated chemiluminescence intensity for the silver nanoclusters that should be obtained builds linear relationship, realizes the detection to miRNA-21.
Embodiment 5
Under the optimal experiment condition explored in embodiment 3, examined according to Electrochemiluminescsensor sensor prepared by embodiment 1
The method for surveying miRNA-21, miRNA-155, miRNA-101, single base mispairing miRNA- are replaced with by miRNA-21 respectively
The mixture of 21 chaff interferents and miRNA-21 and chaff interferent, the electrogenerated chemiluminescence for detecting the silver nanoclusters of corresponding acquisition are strong
Degree illustrates that this Electrochemiluminescsensor sensor has selectivity well such as Fig. 7 A；In addition, when object is 1fM, work as group
After loading electrode is persistently scanned 15 cycles, experimental result such as Fig. 7 B, relative standard deviation is 1.61%, shows that this is electroluminescent
Chemiluminescence sensor has good stability.
The testing result of sensor prepared using the present invention compared with prior art, detection range, detection limit comparison：Knot
Fruit such as the following table 1：
Table 1：
| Method | Detection range | Detection limit | Bibliography |
| Electrochemiluminescsensor sensor | 100aM-100pM | 22aM | Chen et al.,2016 |
| Electrochemiluminescsensor sensor | 1.0fM-1.0nM | 0.5fM | Feng et al.,2016 |
| Electrochemical sensor | 25fM-300fM | 8.2fM | Wang et al.,2015 |
| Fluorescent optical sensor | 1fM-10pM | 0.6fM | Dong et al.,2014 |
| Fluorescent optical sensor | 10fM-1nM | 10fM | Duan et al.,2013 |
| Electrochemiluminescsensor sensor | 1aM-10pM | 1aM | The application |
The application detection range is wide, 1aM-10pM, and detection is limited to 1aM, and detection method is simple, high sensitivity, and stability is good.
Claims (10)
1. a kind of preparation method building Electrochemiluminescsensor sensor based on DNA- silver nanoclusters, which is characterized in that the system
Preparation Method includes the following steps：
(1), the DNAS1 of 2.5OD, DNA HP1, DNA HP2 and miRNA-21 sequence are dissolved in buffer solution respectively, are obtained
DNA S1 buffer solutions, DNA HP1 buffer solutions, DNA HP2 buffer solutions and miRNA-21 buffer solutions；
(2), the glass-carbon electrode after polishing treatment is immersed in chlorauric acid solution, carries out electro-deposition, taken out, cleaning is modified
The glass-carbon electrode of gold nanoparticle；
(3), the glass-carbon electrode for having modified gold nanoparticle that step (2) obtains is immersed in the buffer solution of DNAS1, is trained
It supports, then takes out, clean, modified the glass-carbon electrode of single stranded DNA S1；
(4), the glass-carbon electrode for having modified single stranded DNA S1 that step (3) obtains is immersed in 6- sulfydryl -1- hexanol solution, culture,
Cleaning；
(5), miRNA-21 buffer solutions and DNA HP1, DNA HP2 buffer solutions are mixed, hybridization is prepared into DNA HP1-
HP2 solution；
(6) the 6- sulfydryls -1- hexanols/DNAS1 glass-carbon electrodes modified obtained in step (4) are immersed and is obtained in step (5)
DNA HP1-HP2 solution, culture, then takes out, and cleans, has been modified the glass-carbon electrode of DNA HP1-HP2；
(7) glass-carbon electrode for having modified DNA HP1-HP2 that step (6) obtains is immersed in silver nitrate solution, culture, then will be electric
Pole immerse sodium borohydride solution in, culture, then take out, cleaning to get.
2. preparation method according to claim 1, which is characterized in that DNAS1 sequences are in step (1)：5'-SH-
GGTTGCTATATCG-3’；
DNA HP1 sequences are：
5'-CCCCCCCCCCCCTCAACATCAGTCTGATAAGCTACCGTCTTGAGCTAGCTTATCAGACTGCGATATAGCA
ACC-3；
DNA HP2 sequences are：5'-TAAGCTAGCTCAAGACGGTAGCTTATCAGACTGCCGTCTTGAGCCCCCCCCCCCCC；
MiRNA-21 sequences are：5'-UAGCUUAUCAGACUGAUGUUGA-3’.
3. preparation method according to claim 1 or 2, which is characterized in that step (3) is specially：Gold nano will have been modified
It is taken out after culture 10-12h in the buffer solution of the DNAS1 of 5 μM of the glass-carbon electrode immersion of particle, it is clear with phosphate buffer solution
It washes.
4. according to claim 1-3 any one of them preparation methods, which is characterized in that step is cultivated described in (4), specifically
1-1.5h is cultivated at 37 DEG C -40 DEG C.
5. according to claim 1-4 any one of them preparation methods, which is characterized in that step is cultivated described in (6), specifically
It is：1.5-2h is cultivated at 37 DEG C.
6. according to claim 1-5 any one of them preparation methods, which is characterized in that step (7) is specifically：It will modification
The glass-carbon electrode of DNA HP1-HP2 immerses in 120 μ L, 30 μM of silver nitrate solutions, culture 0.5-1h is protected from light at 4 DEG C, then immerse
In 120 30 μM of μ L sodium borohydride solutions, it is protected from light culture 2-2.5 hours at 4 DEG C, is synthesized with DNA using the reproducibility of sodium borohydride
For the silver nanoclusters of template, then take out, cleaning to get.
7. a kind of any one of claim 1-6 do prepare based on DNA- silver nanoclusters build Electrochemiluminescsensor sensor.
8. detecting answering for miRNAs based on DNA- silver nanoclusters structure Electrochemiluminescsensor sensor described in a kind of claim 7
With.
9. application according to claim 8, which is characterized in that the detection method is：
A, the DNAS1 of 2.5OD, DNA HP1, DNA HP2 and miRNA-21 sequence are dissolved in respectively in buffer solution, are obtained
DNA S1 buffer solutions, DNA HP1 buffer solutions, DNA HP2 buffer solutions and miRNA-21 buffer solutions；
B, the glass-carbon electrode after polishing treatment is immersed in chlorauric acid solution, carries out electro-deposition, taken out, cleaning is modified
The glass-carbon electrode of gold nanoparticle；
C, the glass-carbon electrode for having modified gold nanoparticle that step b is obtained is immersed in the buffer solution of DNAS1, is cultivated, so
After take out, clean, modified the glass-carbon electrode of single stranded DNA S1；
D, the glass-carbon electrode for having modified single stranded DNA S1 that step c is obtained is immersed in 6- sulfydryl -1- hexanol solution, culture, clearly
It washes；
E, the miRNA-21 buffer solutions and DNA HP1 of various concentration, DNA HP2 buffer solutions are mixed, hybridization is prepared into not
With the DNA HP1-HP2 solution of concentration；
F, 6- sulfydryls -1- hexanols/DNAS1 glass-carbon electrodes of having modified that step d is obtained are immersed into the various concentration obtained in step e
DNA HP1-HP2 solution, culture, then take out, clean, modified the glass-carbon electrode of DNA HP1-HP2；
G, the glass-carbon electrode for having modified DNA HP1-HP2 that step f is obtained is immersed in silver nitrate solution, culture, then by electrode
Immerse sodium borohydride solution in, culture, then take out, cleaning to get；
H, glass-carbon electrode that step g is obtained to silver nanoclusters immerses in the phosphate buffer PBS (0.1M) containing potassium peroxydisulfate,
It is detected with electroluminescent chemiluminescence method；The silver nanoclusters prepared according to the miRNA-21 of various concentration are corresponding luminous strong
Degree builds the linear relationship of signal strength and miRNA-21 concentration, realizes the detection to miRNA-21.
10. application according to claim 9, which is characterized in that miRNA-21 buffer concentrations described in step e are distinguished
It is 10-3,10-2,10-1,1,10,102,103With 104fM。
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