CN109777860A - One kind being used for Zn2+The functional nucleic acid biosensor of quantitative detection - Google Patents
One kind being used for Zn2+The functional nucleic acid biosensor of quantitative detection Download PDFInfo
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- CN109777860A CN109777860A CN201910087970.2A CN201910087970A CN109777860A CN 109777860 A CN109777860 A CN 109777860A CN 201910087970 A CN201910087970 A CN 201910087970A CN 109777860 A CN109777860 A CN 109777860A
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Abstract
The present invention relates to a kind of zinc ion (Zn2+) detection method, by Zn2+Dependence cleavage reaction is combined with hybridization chain reaction (HCR) and strand displacement amplification (SDA) reaction, constructs a kind of hypersensitive visual biosensor detection Zn based on the cascade amplification of three step functional nucleic acid of constant temperature2+, to detect Zn2+Provide simple, quick, overdelicate tool.
Description
Technical field
The invention mainly relates to zinc ion detection technique fields, in particular to a kind of to be used for Zn2+The constant temperature grade of quantitative detection
Join the visualization function nucleic acid biosensor of amplification.
Background technique
Zinc is the essential trace elements of the human body, is played an important role in various physiological processes.Zinc mainly pass through food and
Water intake lacks or excessively can all influence human health.The diseases such as Parkinson's disease, Alzheimer's disease and apoplexy are and people
Internal Zn2+Horizontal abnormality it is related.In addition, internal excessive Zn2+Level may cause cell viability increase.Therefore, one is developed
Zn in the accurate detection actual sample of kind2+The technology of content is most important.
Traditional Zn2+Detection method includes inductively coupled plasma atomic emission (ICP-AES), inductively coupled plasma
Constitution composes (ICP-MS), atomic absorption spectrum (AAS) etc..Although these methods have highly sensitive and specificity, they are high
Degree depend on precision instrument, and sample detection pretreatment and continuous mode it is all troublesome.In addition, the installation of instrument and equipment
The requirement of condition needed for environment and maintenance requirement is very high, and needs well-trained professional to operate these specific purpose tools.
Therefore, these methods are not suitable for quickly detection.
In recent years, with the fast development of molecular biotechnology, the nucleic acid molecules such as metal with special biological
Ion dependence cutting deoxyribozyme (cDNA enzyme) and metal ion aptamers gradually appear.So far, it is known that DNA enzymatic packet
Include Zn2+, Cu2+, Pb2+And Hg2+Dependent DNA enzyme.Most of DNA enzymatics just will have catalysis to live with the help of metal cofactor
Property, some of them are highly selective and high efficiency.In addition, DNA enzymatic stablize it is good and at low cost.These special natures make DNA enzymatic exist
It is widely applied in biosensor, such as: biological sensor, colorimetric bio sensor and electrochemica biological sensor.
HCR is a kind of novel beyond body nucleic acid isothermal amplification technique.This method does not need the participation of enzyme and the variation of temperature.
The presence of nucleic acid triggering can stimulate two kinds of nucleic acid hair clip alternately to open, all hairs being continued until in buffer
Until folder all exhausts.The length dna nano wire containing notch is generated by self-assembling reaction, it is final to realize signal amplification.HCR technology
It is widely used in being related to detecting nucleic acid molecules, protein, cell, ochratoxin A (OTA) and various other small molecules at present
During.HCR detection technique has that temperature is constant, easy to operate, experimental cost is low, specific height, good biocompatibility etc.
Advantage.In addition, nucleic acid hair clip and triggering are easy to modify.Although existing using HCR technology and quantifying heavy metal ion such as Pb2+With
Hg2+Research, but about Zn2+The specific research of ion detection is rarely reported.
1992, the researcher from the U.S. research center Becton Dickinson described a kind of body for being known as SDA
Outer isothermal amplification method.This technology is assisted by nickase, and nickase is activated by specific primer and generates single-stranded DNA product.
During SDA, researcher is usually in two terminal modified particular sequences of target sequence to realize that restriction endonuclease identifies.?
In the reaction process, endonuclease is in the end of its recognition site cutting target sequence, and then archaeal dna polymerase extends 3 ' ends and lacks
Mouth simultaneously replaces DNA chain, and a large amount of single stranded DNA is formed after cyclic amplification, realizes the amplification of target signal.The technical operation is simple,
Testing time is short, sensitivity and specificity are high, good compatibility.
As specific function nucleic acid, the sequence rich in guanine (G) can when combining with hemin (Hemin)
It is formed co-ordination complex (G4DNAzyme), with peroxidase catalytic activity.The catalytic activity of the compound is than independent
About 10 times of Hemin high, the significant catalytic performance for improving Hemin itself.G4DNAzyme is widely used in catalysis H2O2It adjusts
Redox reaction.For example, it can be catalyzed H2O2By the colourless bis- (3- ethyl benzo thiazole phenanthroline -6- sulfonic acid of 2,2'- azine group -
Salt) (ABTS2-) it is oxidized to green free radical ABTS-.The colour developing principle is simple, quickly and does not need enzymatic, answers extensively
Building for colorimetric bio sensor.
Summary of the invention
The present invention is quasi- to develop a kind of Zn2+Quick and super sensitivity detection method, establish and be based on Zn2+Dependence cleavage reaction,
The hypersensitive for hybridizing three step functional nucleic acid of the constant temperature cascade amplification of chain reaction (HCR) and strand displacement amplification (SDA) reaction is visual
Change biosensor.HCR trigger sequence is inserted into Zn2+Dependence is cut in the substrate chain of deoxyribozyme (17E), and by G- tetra-
5 ' ends of serobila complementary series and restriction enzyme site sequence insertion HCR hair clip.Zn is added in buffer2+, catalysis 17E cutting substrate
Chain release triggering, triggering HCR reaction form the DNA nanowire structure for having branch.Then, amplification enzyme and restriction endonuclease is added
SDA reaction is carried out, DNA nano wire is separated into DNA structure largely with two-way amplification ability immediately, eventually forms a large amount of
Tetra- serobila sequence of G- simultaneously forms a large amount of G4 DNAzyme in conjunction with Hemin.After chromogenic substrate is added, G4 DNAzyme high efficiency
It is catalyzed H2O2By colourless ABTS2-It is oxidized to green free radical ABTS-, to realize Zn2+Visual retrieval.To detect Zn2+
Provide simple, quick, overdelicate tool.
According to the original series of 17E ribozyme, HCR trigger sequence is inserted into the substrate chain of 17E ribozyme, and by tetra- chain of G-
The substrate chain (S-HCR) and HCR hair clip of 5 ' end design 17E ribozymes of body complementary series and restriction enzyme site sequence insertion HCR hair clip
(Y-H1 and Y-H2) sequence.All primers are synthesized by Ningbo of Zhejiang health shellfish biochemistry Co., Ltd.
1 zinc ion dependence of table cuts ribozyme and hair fastener sequence
Cleavage reaction buffer
It weighs 1.7532g NaCl (300mM) and 0.9532g HEPES (40mM) is dissolved in the ultrapure water of 100mL, and adjust
PH to 7.5 is configured to cutting buffer (300mM NaCl, 40mM HEPES, pH 7.5), and 4 DEG C save backup.
17E and substrate chain dissolve and hybridize
Zn will be housed2+Dependence cuts ribozyme chain, the test tube of substrate chain powder is put into supercentrifuge, 4 DEG C, 13400
× g takes out addition distilled water dissolution sequence to 10 μM of concentration after being centrifuged 10min, and 4 DEG C save backup.The substrate of 100 μ L is taken respectively
Chain (10 μM), in (10 μM) of the catalyzing enzyme chain 800 μ L cutting buffers of addition of 100 μ L, then 95 DEG C of heating 5min are slowly dropped to
Room temperature is configured to 1 μM of Zn2+Dependence cuts ribozyme solution (17ES-HCR), and 4 DEG C save backup.
Zn2+Dependence cleavage reaction and HCR reaction
2 Zn of table2+Cleavage reaction and HCR reaction system
SDA reaction
3 SDA reaction system of table
SDA response procedures are as follows: 5min makes enzyme-deactivating at 55 DEG C of amplification 20min, 95 DEG C, then 4 DEG C of holding 5min.
Chromogenic reaction and uv-visible absorption spectra analysis
4 ABTS coloring reaction system of table
Compared with prior art, the invention has the benefit that
(1) it develops based on Zn2+Three step functional nucleic acid constant temperature of dependence cleavage reaction, HCR reaction and SDA cascade amplification
Biosensor.
(2) three-step reaction in the present invention can be completed at a constant temperature, and signal amplification can be completed at a constant temperature.
(3) the present invention is based on HCR-SDA reactions to generate a large amount of G4 DNAzyme, realizes visual signal output.
(4) rapid detection method of the invention does not need large-scale instrument and equipment and professional operator, detects sensitive
Degree height, high specificity, detection limit can be down to 1.075pM.
(5) present invention has versatility, can be by Zn2+Dependence cutting deoxyribozyme replaces with other metal ions specificity
Deoxyribozyme is cut, realizes the detection of plurality of target substance.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 HCR triggers result；
Fig. 2 feasibility analysis result figure；
Fig. 3 biosensor reaction system optimization；(A) SDA proliferation time is to OD415nmInfluence；(B) Bst.DNA polymerize
Enzyme concentration is to OD415nmInfluence；(C) Nt.Bst NBI nicking enzyme concentration is to OD415nmInfluence；(D) Hemin concentration is to OD415nm
Influence；
The standard curve when range of linearity of Fig. 4 zinc ion concentration is 10pM to 100nM.
Specific embodiment
The invention discloses one kind to be used for Zn2+The visualization function biological nucleic acid sensing of the constant temperature cascade amplification of quantitative detection
Device, those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that all
Similar replacement and change is apparent to those skilled in the art, they are considered as being included in the present invention.This
The method of invention and application are described by preferred embodiment, and related personnel can obviously not depart from the present invention
Hold, in spirit and scope to method described herein and application is modified or appropriate changes and combinations, carrys out implementation and application sheet
Inventive technique.
All raw and auxiliary materials, reagent and the instrument selected in the present invention, equipment are all well known in the art selection, but unlimited
Implementation of the invention is made, other some reagents well known in the art and equipment are applied both to the reality of following implementation of the present invention
It applies.
Below with reference to embodiment, the present invention is further explained:
Embodiment one:
According to the original series of 17E ribozyme, HCR trigger sequence is inserted into the substrate chain of 17E ribozyme, and by tetra- chain of G-
The substrate chain (S-HCR) and HCR hair clip of 5 ' end design 17E ribozymes of body complementary series and restriction enzyme site sequence insertion HCR hair clip
(Y-H1 and Y-H2) sequence.All primers are synthesized by Ningbo of Zhejiang health shellfish biochemistry Co., Ltd.
1 zinc ion dependence of table cuts ribozyme and hair fastener sequence
Cleavage reaction buffer
It weighs 1.7532g NaCl (300mM) and 0.9532g HEPES (40mM) is dissolved in the ultrapure water of 100mL, and adjust
PH to 7.5 is configured to cutting buffer (300mM NaCl, 40mM HEPES, pH 7.5), and 4 DEG C save backup.
17E and substrate chain dissolve and hybridize
Zn will be housed2+Dependence cuts ribozyme chain, the test tube of substrate chain powder is put into supercentrifuge, 4 DEG C, 13400
× g takes out addition distilled water dissolution sequence to 10 μM of concentration after being centrifuged 10min, and 4 DEG C save backup.The substrate of 100 μ L is taken respectively
Chain (10 μM), in (10 μM) of the catalyzing enzyme chain 800 μ L cutting buffers of addition of 100 μ L, then 95 DEG C of heating 5min are slowly dropped to
Room temperature is configured to 1 μM of Zn2+Dependence cuts ribozyme solution (17ES-HCR), and 4 DEG C save backup.
Zn2+Dependence cleavage reaction and HCR reaction
2 Zn of table2+Cleavage reaction and HCR reaction system
SDA reaction
3 SDA reaction system of table
SDA response procedures are as follows: 5min makes enzyme-deactivating at 55 DEG C of amplification 20min, 95 DEG C, then 4 DEG C of holding 5min.
Chromogenic reaction and uv-visible absorption spectra analysis
4 ABTS coloring reaction system of table
The verifying of two detection architecture of embodiment optimizes
The present invention first carries out Zn2+After dependence cleavage reaction, then carry out HCR reaction, each component concentration ratio in HCR reaction
Are as follows: S-HCR:Y-H1:Y-H2=100nM:500nM:500nM.After the HCR of 37 DEG C, 60min reaction, then carry out agarose
Gel electrophoresis analysis.The DNA band for obtaining processing group has apparent disperse compared to control group, it was demonstrated that the ignitionability of HCR,
As shown in Figure 1.
The feasibility of detection architecture is verified
The present invention is provided with 5 processing groups: (1) 17ES-HCR；(2)17ES-HCR+100μM Zn2+；(3)Y-H1+Y-H2；
(4)17ES-HCR+Y-H1+Y-H2；(5)17ES-HCR+100μM Zn2++Y-H1+Y-H2.First carry out Zn2+Dependence cutting is anti-
After should reacting with HCR, HCR reaction product is taken to carry out SDA, then SDA reaction product is carried out chromogenic reaction and ultraviolet-ray visible absorbing
Spectrum analysis.By comparing the difference of the absorption spectrum absorbance of different disposal, the light absorption value for obtaining processing 5 is apparently higher than other
Processing group, as shown in Figure 2.
The optimization of detection architecture reaction condition
With 1mM Zn2+17 ES-HCR of catalysis cutting 100nM carries out HCR reaction, hair fastener concentration after cleavage reaction
Ratio is Y-H1:Y-H2=500nM:500nM.It takes HCR reaction product to carry out SDA, SDA reaction product is finally taken develop the color instead
It answers and uv-visible absorption spectra is analyzed.In order to improve the sensitivity of the visual biosensor, expanded by comparing SDA
Time, Bst.DNA polymerase concentration, Nt.Bst NBI nicking enzyme concentration and Hemin concentration are systematically analyzed.As a result it proves
SDA proliferation time is best proliferation time (Fig. 3 A) when being 20min, and the concentration of Bst.DNA polymerase senses when being 0.08U/ μ L
Device performance is more preferable (Fig. 3 B), the concentration of Nt.Bst NBI nicking enzyme OD in 0.2U/ μ L415nmReach maximum (Fig. 3 C), in addition, 4 μ
The Hemin of M is its optium concentration (Fig. 3 D).
Detection architecture sensitivity verifying
In order to evaluate the sensitivity of the visual biosensor, measurement contains various concentration under optimum experimental condition
Zn2+The Δ OD of standard solution (0~10 μM)415nmValue, each concentration three parallel.In the reaction system, the Δ of experimental group
OD415nmWith Zn2+The increase of concentration and increase, it is seen that green it is also deeper and deeper.Zn2+When concentration is 10pM to 100nM, institute
It is linear for mapping, and dependent equation are as follows: Δ OD415nm=0.1411lg [C (pM)] -0.0209, coefficient R2For
0.9968, it is suitble to quantitative detection.
Certain embodiment of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the present invention should be with appended claims.
Sequence table
<110>China Agricultural University
<120>a kind of to be used for Zn2+The functional nucleic acid biosensor of quantitative detection
<130> MP1901895Z
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 68
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 1
ggccaaacag ttttaaacac tgactcacta traggaagag atttctgagc ttcggattct 60
gtttggcc 68
<210> 2
<211> 81
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 2
cccatcccgc ccaacccttt agtagactca ggccaaacag aatccgaagc tcagaccctg 60
ctgagcttcg gattctgtaa a 81
<210> 3
<211> 80
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 3
cccatcccgc ccaacccttt actagactcc tgagcttcgg attctgtttg gcctttacag 60
aatccgaagc tcagcagggt 80
Claims (7)
1. one kind is used for Zn2+The visualization function nucleic acid biosensor of the constant temperature cascade amplification of quantitative detection, which is characterized in that
By Zn2+Dependence cleavage reaction is combined with hybridization chain reaction (HCR) and strand displacement amplification (SDA) reaction, and HCR is triggered sequence
Column are inserted into Zn2+Dependence is cut in the substrate chain of deoxyribozyme (17E), and by tetra- serobila complementary series of G- and restriction enzyme site sequence
5 ' ends of column insertion HCR hair clip, are added Zn in buffer2+, catalysis 17E cutting substrate chain release triggering is sub, and triggering HCR is anti-
Answer, form the DNA nanowire structure for having branch, then, amplification enzyme and restriction endonuclease is added and carries out SDA reaction, DNA nano wire with
It is separated into DNA structure largely with two-way amplification ability, eventually form a large amount of tetra- serobila sequence of G- and is tied with Hemin
Conjunction forms a large amount of G4 DNAzyme.
2. functional nucleic acid biosensor described in claim 1 is realizing Zn2+Visual retrieval application, feature exists
In after chromogenic substrate is added, G4 DNAzyme high efficiency is catalyzed H2O2By colourless ABTS2-Chromogenic substrate is oxidized to oneself of green
By base ABTS-, Zn is realized according to the depth of color2+Visual retrieval.
3. visualization function nucleic acid biosensor according to claim 1, which is characterized in that the substrate chain of 17E ribozyme
Sequence is as shown in SEQ ID NO.1.
4. visualization function nucleic acid biosensor according to claim 1, which is characterized in that HCR hairpin such as SEQ
Shown in NO.2~3 ID.
5. visualization function nucleic acid biosensor according to claim 1, which is characterized in that cleavage reaction buffer is
300mM NaCl, 40mM HEPES, pH 7.5.
6. visualization function nucleic acid biosensor according to claim 1, which is characterized in that first carry out Zn2+Dependence
After cleavage reaction, then HCR reaction is carried out, each component concentration ratio in HCR reaction are as follows: S-HCR:Y-H1:Y-H2=100nM:
500nM:500nM。
7. visualization function nucleic acid biosensor according to claim 1, which is characterized in that SDA response procedures are as follows:
55 DEG C of amplification 20min, 5min makes enzyme-deactivating at 95 DEG C, then 4 DEG C of holding 5min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113088518A (en) * | 2021-03-29 | 2021-07-09 | 中国药科大学 | Functional dendritic DNA, nano sensing probe based on dendritic DNA and application of nano sensing probe |
| CN113354812A (en) * | 2021-06-01 | 2021-09-07 | 重庆大学 | Novel semiquinone free radical nano material and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107966438A (en) * | 2017-10-27 | 2018-04-27 | 中国农业大学 | A kind of sensor of resistance to high salt of functional nucleic acid based on zinc and its application |
| CN108841937A (en) * | 2018-06-20 | 2018-11-20 | 中国农业大学 | It is general to separate ultrafast amplification magnesium, zinc cutting-type functional nucleic acid visible detection method |
| CN108949931A (en) * | 2018-06-20 | 2018-12-07 | 中国农业大学 | A kind of general ultrafast amplification visible sensor of partition of zinc ion cutting-type |
-
2019
- 2019-01-29 CN CN201910087970.2A patent/CN109777860A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107966438A (en) * | 2017-10-27 | 2018-04-27 | 中国农业大学 | A kind of sensor of resistance to high salt of functional nucleic acid based on zinc and its application |
| CN108841937A (en) * | 2018-06-20 | 2018-11-20 | 中国农业大学 | It is general to separate ultrafast amplification magnesium, zinc cutting-type functional nucleic acid visible detection method |
| CN108949931A (en) * | 2018-06-20 | 2018-12-07 | 中国农业大学 | A kind of general ultrafast amplification visible sensor of partition of zinc ion cutting-type |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113088518A (en) * | 2021-03-29 | 2021-07-09 | 中国药科大学 | Functional dendritic DNA, nano sensing probe based on dendritic DNA and application of nano sensing probe |
| CN113088518B (en) * | 2021-03-29 | 2024-05-17 | 中国药科大学 | Functional dendritic DNA, nano sensing probe based on dendritic DNA and application of nano sensing probe |
| CN113354812A (en) * | 2021-06-01 | 2021-09-07 | 重庆大学 | Novel semiquinone free radical nano material and preparation method and application thereof |
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Application publication date: 20190521 |