CN105838790B - A kind of silver nanoclusters sensor and preparation method thereof and the application in detection viral gene - Google Patents
A kind of silver nanoclusters sensor and preparation method thereof and the application in detection viral gene Download PDFInfo
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- CN105838790B CN105838790B CN201610248826.9A CN201610248826A CN105838790B CN 105838790 B CN105838790 B CN 105838790B CN 201610248826 A CN201610248826 A CN 201610248826A CN 105838790 B CN105838790 B CN 105838790B
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
Abstract
The present invention provides a kind of silver nanoclusters sensor and preparation method thereof and the application in measurement viral gene, which is DNA fluorescent optical sensor, and the DNA is molecular beacon type multifunctional dna sequence, has quencher its 3' is terminal modified.Preparation method includes design multifunctional dna sequence, forms molecular beacon type DNA sequence dna and prepare three steps of silver nanoclusters sensor.The sensor that the present invention constructs can detect a variety of viral genes simultaneously, effectively make up the defect of term single gene testing result inaccuracy.
Description
Technical field
The invention belongs to chemical fields, and in particular to a kind of silver nanoclusters sensor and preparation method thereof and in detection virus
Application in gene.
Background technique
It is frequently necessary to detect various viral genes in clinical disease diagnosis, and often there are multiple gene hypotypes in virus, it is existing
Some methods usually once only detect a kind of gene, need the result in conjunction with repeated detection to carry out clinical diagnosis, increase behaviour
The step of making, and repeated detection can produce bigger effect the accuracy of diagnosis.
Biosensor is that most one kind is reported in current biosensor, wherein many due to fluorescence analysis
Advantage and keep the application of biological sensor the most extensive.Biological sensor be by probe carry out fluorescent marker or with
Fluorescent material combines, and when probe is acted on target substance, fluorescence signal changes, and identification information is converted to detectable
Fluorescence signal, to realize the analysis to target substance.In recent years, the content that makes internal disorder or usurp of grinding of biological sensor is mainly concentrated to spy
The presence of the target analytes such as sequencing column, protein, drug, small molecule, inorganic ions and content carry out qualitative and quantitative analysis.
The molecular level aggregation that silver nanoclusters are made of several to dozens of silver atoms.Silver nanoclusters are as a kind of novel
Fluorescent nano material has unique optical property, all has and is widely applied in biochemical sensitive and bio-imaging field.Currently,
The preparation method of silver nanoclusters is mainly by restoring silver-colored presoma using oligonucleotide as stabilizer.Wherein, with phonetic rich in born of the same parents
The oligonucleotides of pyridine base is that the silver nanoclusters of templated synthesis have good water-soluble, biocompatibility and stablize the excellent of fluorescence
Point.
Summary of the invention
The technical issues of solving the problems, such as: present invention aim to address term single gene detection inaccuracy, propose a kind of sharp
Type silver nanoclusters sensor living, the sensor can detect a variety of viral genes simultaneously.
Technical solution:
A kind of silver nanoclusters sensor, the sensor are DNA fluorescent optical sensor, and the DNA is that molecular beacon type is multi-functional
DNA sequence dna has quencher its 3' is terminal modified.
The partial size of the silver nanoclusters sensor is 1~2nm.
The preparation method of the silver nanoclusters sensor, comprising the following steps:
Step 1, multifunctional dna sequence is designed, which is followed successively by the mould of silver nanoclusters from the end 5' to the end 3'
Plate sequence, identification sequence and its partial complementarity sequence have quencher 3' is end modified；
Step 2, molecular beacon type DNA sequence dna is formed, the multifunctional dna sequence of step 1 is subjected to thermal denaturation and is annealed, i.e.,
；
Step 3, silver nanoclusters sensor is prepared, using step 2 gained molecular beacon type DNA sequence dna as stabilizer, successively
Silver nitrate is added and reduction reaction occurs for sodium borohydride, obtains silver nanoclusters sensor.
Further, multifunctional dna sequence in step 3, silver nitrate, sodium borohydride molar ratio be 1: 6: 6.
The silver nanoclusters sensor detects the application in different virus gene at the same time.
The silver nanoclusters sensor detects the application in different influenza virus sub-strain genes at the same time.
The utility model has the advantages that
1. the signal ability quilt that activated form silver nanoclusters sensor of the invention only after identifying determinand, extinguishes originally
Activation, so that activated form fluorescence detection is carried out, to remove washing step from and improve the sensitivity of detection；
2. activated form silver nanoclusters sensor of the invention can detect a variety of targets such as gene order, small molecule simultaneously
Substance and large biological molecule substance；
3. activated form silver nanoclusters sensor of the invention can be promoted and be applied to cell imaging and living imaging field.
Detailed description of the invention
Fig. 1 is the more targets of silver nanoclusters sensor while detection schematic diagram in embodiment 1；
Fig. 2 is the fluorescence spectra of polychrome silver nanoclusters sensor in embodiment 1；
Fig. 3 be added in polychrome silver nanoclusters sensor in embodiment 1 two kinds of avian influenza virus subtype gene H1N1 and
Fluorescence emission spectrogram of compound after H5N1.
Specific embodiment
Following embodiment further illustrates the contents of the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case where spirit of that invention and essence, to modification made by the method for the present invention, step or condition and replaces, belong to the present invention
Range.Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.
A kind of silver nanoclusters sensor, the sensor are DNA fluorescent optical sensor, and the DNA is that molecular beacon type is multi-functional
DNA sequence dna has quencher its 3' is terminal modified.Two kinds of virus subtype bases are detected simultaneously using silver nanoclusters sensor of the invention
The process of cause is as shown in Figure 1, its principle are as follows: design multifunctional dna sequence, the end 5' to the end 3' is followed successively by silver nanoclusters template sequence
Column, identification sequence and partial complementarity sequence, by thermal denaturation and anneal, and hairpin structure DNA sequence dna are formed, as stabilizer, base
In the reaction of sodium borohydride reduction silver nitrate, activated form molecular beacon type silver nanoclusters are synthesized.By changing template sequence, in conjunction with
Different identification sequences prepares the molecular beacon type polychrome silver nanoclusters with different targetings.Multifunctional dna sequence used
As shown in SEQ ID NO.1 and SEQ ID NO.2, be prepared respectively H1N1 targeted molecular beacon type green silver nanoclusters and
H5N1 targets red silver nanoclusters.When target gene is not present, silver nanoclusters are mutually leaned on quencher BHQ in molecular beacon type
Closely, fluorescence resonance energy transfer, fluorescent quenching occurs；When there are a kind of target H1N1 or H5N1, due to target gene and pass
The identification sequence of the molecular beacon ring-shaped area of sensor is specifically bound, and forms double-stranded DNA, is opened hairpin structure, is made Yin Na
Rice cluster activates a kind of fluorescence signal of color silver nanoclusters far from fluorescent quenching molecule, realizes the highly sensitive inspection to term single gene
It surveys；And when existing simultaneously two kinds of target genes, the fluorescence signal of two kinds of color silver nanoclusters is activated, to realize more target bases
Because detecting simultaneously.
Embodiment 1
The preparation of solution used:
0.1052g DisodiumHydrogen Citrate and 0.47056g trisodium citrate are weighed, is settled to 100mL with ultrapure water, is adjusted
PH value of solution is 7 to get 20 mM citric acid solutions.By 0.1698g AgNO3Be dissolved in 100mL ultrapure water be configured to it is dense
Degree is the AgNO of 10mM3Working solution；Weigh 0.0378g NaBH4Being dissolved in and being configured to concentration in 100mL water is 10mM's
NaBH4Working solution；DNA chain is dissolved in citric acid solution (20 mM, pH7), 100 μM of stock solutions are configured to.
The preparation of silver nanoclusters sensor:
Step 1, multifunctional dna sequence design
Multifunctional dna sequence includes three parts, as shown in Figure 1, being followed successively by the template sequence of silver nanoclusters from the end 5' to the end 3'
Arrange (function fragment I), identification sequence (function fragment II), the 6 base sequence (functions with identification sequence 5' terminal sequence partial complementarity
Segment III), 3' is end modified quencher BHQ1 molecule.Particular sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2
Step 2, molecular beacon type DNA sequence dna is prepared
500 μ L citric acid solutions (20mM, pH7) are added in 1.5mL specification centrifuge tube, take 90 μ L multi-functional
DNA sequence dna (100 μM) is uniformly mixed, is put into 95 °C of water-baths and is allowed to be denaturalized 10 minutes, be quickly put into and be allowed to keep in ice-water bath
1 hour, since specific hybrid occurs for identification sequence and partial complementarity sequence, molecular beacon type DNA was formed under the above conditions
Sequence, the end 5' are silver nanoclusters template sequence, and the end 3' is quencher BHQ1 molecule.
Step 3, silver nanoclusters sensor is prepared
It using two kinds of hair clip type DNA sequence dnas of formation as stabilizer, is put into ice-water bath and is kept for 10 minutes, be added
5.4 μ L silver nitrate working solutions, which shake 2 minutes, is allowed to uniformly mixed, is protected from light to be put into ice-water bath and continue ice bath 15 minutes；It is added
The freshly prepared sodium borohydride working solution of 5.4 μ L quickly mixes and is allowed to react 5 minutes, is protected from light and is put into the preservation of 4 °C of refrigerators and is allowed to
Overnight, activated form molecular beacon type silver nanoclusters are obtained.Reactant molar ratio DNA profiling in experiment: silver nitrate: sodium borohydride=
1 : 6 : 6。
In order to determine the excitation wavelength and launch wavelength of activated form silver nanoclusters sensor, with unmodified quencher BHQ's
Molecular beacon type multifunctional dna sequence is prepared according to same process as stabilizer and is always on type molecular beacon type silver nanoparticle
Cluster.Characterize the silver nanoclusters fluorescence synthesized: the excitation wavelength of green silver nanoclusters is 445 nm, and launch wavelength is 520 nm；
The excitation wavelength of red silver nanoclusters is 525 nm, and launch wavelength is 595 nm.
The molecular beacon type green silver nanoclusters and red silver nanoclusters solution, mixing for respectively taking 100 uL to be prepared are equal
It is even, 500 nM H1N1(SEQ ID NO.3 are added) or H5N1 gene order (SEQ ID NO.4), it is sufficiently mixed uniformly at least
5 minutes, set excitation wavelength as 445 nm, monitor its fluorescence intensity at 520 nm wavelength, or set excitation wavelength as
525 nm measure its fluorescence intensity at 595 nm wavelength.As shown in Figure 2, when target gene not being added, 520nm and 595nm
Almost without fluorescence signal at wavelength；As shown in figure 3, after 500 nMH1N1 and H5N1 are added, the fluorescence signal significantly activated out,
The two intensity shows detection while the method established can effectively realize two kinds of viral genes, is expected to answer in the same order of magnitude
Use clinical detection.
Polychrome silver nanoclusters of the invention can be extended to different genes while detect, and provide for clinical gene diagnosis new
Method.
Sequence table
<110>Yancheng Institute Of Technology
<120>a kind of silver nanoclusters sensor and preparation method thereof and the application in detection viral gene
<130> 123
<160> 4
<170> PatentIn version 3.3
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<212> DNA
<213>artificial sequence
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ccctttaacc ccttcttcat cgagagtgta gtcggaagaa 40
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<213>artificial sequence
<400> 2
cctccttcct ccagactctt gagttctcag tatggagtct 40
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<212> DNA
<213> Influenza virus
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catactgaga actcaagagt ct 22
Claims (1)
1. a kind of activated form fluorescence silver nanoclusters sensor, and be applied to more target genes and detect simultaneously, which is that DNA is glimmering
Optical sensor, it is characterised in that: the DNA is molecular beacon type multifunctional dna sequence, is followed successively by silver nanoparticle from the end 5' to the end 3'
The template sequence of cluster is known as function fragment I, complementary series or identification sequence and is known as function fragment II and identification sequence 5' terminal sequence
6 base sequences of partial complementarity are known as function fragment III, and 3' is terminal modified quencher；Multifunctional dna be SEQ ID NO.1 and
Nucleotide sequence shown in SEQ ID NO.2, the silver nanoclusters prepared using SEQ ID NO.1 or SEQ ID NO.2 as template
With strong fluorescence signal；When target gene is not present, silver nanoclusters are close with quencher, and fluorescence resonance energy occurs and turns
It moves, fluorescent quenching；When there are a kind of target, since the identification sequence of target gene and ring-shaped area is specifically bound, hair
Clamping structure is opened, and silver nanoclusters activate a kind of fluorescence signal of color silver nanoclusters far from quencher, and fluorescence enhancement detection is single
Gene；Emit green fluorescence using SEQ ID NO.1 as the silver nanoclusters of template, using SEQ ID NO.2 as the Yin Na of template
Rice cluster emits red fluorescence, and two kinds of color fluorescence intensity are in the same order of magnitude, when there are two kinds of targets, while activating two kinds of face
The fluorescence signal of color silver nanoclusters, fluorescence-activation detect more target genes simultaneously；
The preparation method of above-mentioned activated form fluorescence silver nanoclusters sensor, comprising the following steps:
Step 1, multifunctional dna sequence is designed, which is followed successively by the template sequence of silver nanoclusters from the end 5' to the end 3'
Column, identification sequence and its partial complementarity sequence have quencher 3' is end modified；
Step 2, molecular beacon type DNA sequence dna is formed, the multifunctional dna sequence of step 1 is subjected to thermal denaturation and is annealed, 1.5
The citric acid solution that 500 μ L, 20 mM pH is 7 is added in mL specification centrifuge tube, takes 90 μ L, 100 μM of multifunctional dnas
Sequence is uniformly mixed, is put into 95 °C of water-baths and is allowed to be denaturalized 10 minutes, be quickly put into ice-water bath and kept for 1 hour；
Step 3, silver nanoclusters sensor is prepared, using step 2 gained molecular beacon type DNA sequence dna as stabilizer, is put into ice water
It is kept for 10 minutes in bath, sequentially adds silver nitrate and reduction reaction occurs for sodium borohydride, 5.4 μ L silver nitrate working solutions are added
Concussion 2 minutes is uniformly mixed, is protected from light to be put into ice-water bath and continue ice bath 15 minutes；The 5.4 freshly prepared hydroborations of μ L are added
Sodium working solution quickly mixes, and reacts 5 minutes, is protected from light and is put into 4 °C of refrigerators preservations overnight, obtains activated form molecular beacon type silver
Nano-cluster sensor；Prepare the multifunctional dna sequence of silver nanoclusters sensor, silver nitrate, sodium borohydride molar ratio be 1: 6:
6；Molecular beacon type silver nanoclusters are prepared, described its partial size of silver nanoclusters sensor is 1~2 nm；Green silver nanoclusters
Excitation wavelength is 445 nm, and launch wavelength is 520 nm；The excitation wavelength of red silver nanoclusters is 525 nm, and launch wavelength is
595 nm, and two kinds of color fluorescence intensity are in the same order of magnitude.
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| CN105928917B (en) * | 2016-04-20 | 2019-05-17 | 盐城工学院 | A kind of silver nanoclusters sensor and its preparation method and application |
| CN110684865A (en) * | 2019-09-27 | 2020-01-14 | 湘潭大学 | Method for detecting H5N1 by using molybdenum disulfide nanoprobe based on catalytic hairpin self-assembly |
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| CN103008682A (en) * | 2012-12-29 | 2013-04-03 | 湖南科技大学 | Method for synthesizing fluorescent silver nano clusters by taking general DNA (Deoxyribose Nucleic Acid) as stabilizer |
| CN105928917A (en) * | 2016-04-20 | 2016-09-07 | 盐城工学院 | Silver nanocluster sensor, and preparation method and application thereof |
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| CN101603077A (en) * | 2008-06-11 | 2009-12-16 | 北京大学 | The method of a kind of general molecular beacon nucleic acid probe and detection DNA thereof |
| CN103008682A (en) * | 2012-12-29 | 2013-04-03 | 湖南科技大学 | Method for synthesizing fluorescent silver nano clusters by taking general DNA (Deoxyribose Nucleic Acid) as stabilizer |
| CN105928917A (en) * | 2016-04-20 | 2016-09-07 | 盐城工学院 | Silver nanocluster sensor, and preparation method and application thereof |
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