CN108333155A - A kind of biosensor of fluoroscopic examination mercury ion - Google Patents
A kind of biosensor of fluoroscopic examination mercury ion Download PDFInfo
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- CN108333155A CN108333155A CN201810029658.3A CN201810029658A CN108333155A CN 108333155 A CN108333155 A CN 108333155A CN 201810029658 A CN201810029658 A CN 201810029658A CN 108333155 A CN108333155 A CN 108333155A
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- dna
- biosensor
- silver
- concentration
- fluorescence intensity
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
Abstract
The present invention provides a kind of biosensors of fluoroscopic examination mercury ion, including following component：Silver-colored cluster containing DNA and the DNA that G tetrads can be formed, potassium ion and ferroheme.The biosensor detection reaction condition of the present invention is mild, improves reaction speed, reduces the complexity of operation, realizes quick, simple, the sensitive detection of object；The preparation method of the biosensor is simple, and performance is stablized, reproducible, the practical application of detection and biosensor industrialization suitable for food security and Mercury in Water Body ion of silver-colored cluster；The process costs for making the biosensor are low, the inexpensive requirement suitable for industrialization.
Description
Technical field
The present invention relates to a kind of biosensors of fluoroscopic examination mercury ion, belong to biosensor technology field.
Background technology
Metal mercury poisoning is often caused in the form of mercury vapour.Since mercury vapour has the diffusivity of height and larger liposoluble
Property, alveolar is entered by respiratory tract, through blood circulating to whole body.After mercury metal in blood enters brain tissue, it is oxidized to
Mercury ion gradually accumulates in brain tissue, reaches a certain amount, will be damaged to brain tissue.Another part mercury ion
It is transferred to kidney.Therefore, chronic mercury poisoning clinical manifestation is mainly neurological symptom, such as headache, dizzy, extremity numbness and pain
Bitterly, muscular tremor, ataxia etc..Easy excitement is a kind of special state of mind of chronic mercury poisoning, shows as emotional, mouth
Eat, be timid, anxiety, uneasiness, absent-minded, failure of memory, depression etc..
The detection method for the mercury ion reported at present includes atomic emission spectrometry, atomic absorption spectrography (AAS), spectrophotometric
Often there is expensive equipment, analytical cycle length, sample pretreatment in method, Hydride Generation-atomic Fluorescence Spectrometry etc., these methods
Complicated, the problems such as testing cost is expensive, the requirement of the convenient, fast of mercury ion detecting, sensitivity etc. has been difficult to adapt to it.
Therefore, be badly in need of establishing at present it is a kind of quickly, accurately, sensitive and high specific detection method detects the residual of mercury ion.
Invention content
Method specificity for detection mercury ion in the prior art and sensitivity is all relatively low, of high cost, detection cycle
Long problem, the present invention provide a species specificity and high sensitivity, at low cost, detection speed it is fast silver is quenched based on G- tetrads
Cluster fluorescence detects the biosensor of mercury ion.
To achieve the above object, the present invention adopts the following technical scheme that.
A kind of biosensor of fluoroscopic examination mercury ion, including following component：
a）Silver-colored cluster containing DNA（D1-AgNCs）；
And b）The DNA of G- tetrads can be formed（D2）, potassium ion（K+）And ferroheme；
The DNA of the silver cluster（D1）Sequence is as shown in SEQ No.1；The DNA for forming G- tetrads（D2）Sequence such as SEQ
Shown in No.2；
The D2 passes through potassium ion（K+）The DNA of G- tetrads is formed with ferroheme.
The K+It is provided by buffer solution；The buffer solution is preferably phosphate buffered saline（PBS）.
The silver-colored cluster containing DNA is obtained using following preparation method：By buffer solution, the solution of D1, AgNO3Mixing is placed on
15-30min at 4 DEG C；Then cold NaHBO is added4Solution, 4 DEG C stand 4h or more to get.
Described D1, AgNO3With NaHBO4Molar ratio be 1:6:6.
The buffer solution is phosphate buffer（PB）.
A method of using above-mentioned biosensor fluoroscopic examination mercury ion, include the following steps：
（1）The fluorescence intensity of D1-AgNCs is measured, centrifuge tube then is added in D1-AgNCs and D2,5 × buffer solution, ferroheme
In, then it is separately added into the Hg of series concentration2+Solution and prepare liquid shake 30s, are put into 37 DEG C of water-bath and react 1h.
（2）By step（1）The mixed solution of gained, measures fluorescence intensity respectively；Make Hg2+Standard of the concentration to fluorescence intensity
Curve calculates regression equation；Its Hg is calculated according to the fluorescence intensity of prepare liquid2+Concentration.
Final concentration of 0.4-1.4 μM of the D1-AgNCs, preferably 0.4-1.0 μM.
A concentration of 0.4-1.4 μM of the D2, preferably 0.4-1.0 μM.
The operation principle of this biosensor：
A part is can be assembled into the sequence of silver-colored cluster AgNCs in D1, in remaining sequence a part be rich in T bases, a part with
Partial sequence base complementrity in D2, a part is rich in T bases in D2, and a part is the sequence that can form G- tetrads rich in G
Row.In no object（Hg+）When, two chains of D1 and D2 although have a partial complementarity sequence but the hybridization temperature of two chains compared with
It is low, it cannot hybridize together.When there is object Hg2+In the presence of, it, will shape because two chains of D1 and D2 have the sequence rich in T
At T-Hg2+- T structures are to steadily hybridize 2 chains together.The silver-colored cluster of D1 chains synthesis, D2 chains form G- tetrads, without mesh
Mark object Hg2+In the presence of, D1-AgNCs has stronger fluorescence；As object Hg2+In the presence of, D1 is hybridized with D2, the G- tetrads of D2
Physical efficiency falls the fluorescence of D1-AgNCs to quenching, and object Hg is realized by the variation of fluorescence intensity2+Detection.
The present invention has the following advantages：
The main process of the biosensor detection of the present invention is realized in homogeneous, and reaction condition is mild, is improved anti-
Speed is answered, the complexity of operation is reduced, realizes the quick of object, it is simply, sensitive to detect；The biosensor
Preparation method is simple, and performance is stablized, silver-colored cluster it is reproducible, be suitable for detection and the biology of food security and Mercury in Water Body ion
The practical application of sensor industrialization；The process costs for making the biosensor are low, the inexpensive requirement suitable for industrialization.
Description of the drawings
Fig. 1 is the operation principle schematic diagram of this biosensor；
Fig. 2 is influences of the various concentration D1 to detection mercury ion；
Fig. 3 is influences of the various concentration D2 to detection mercury ion；
Fig. 4 is the fluorescence intensity of series concentration mercury ion；
Fig. 5 is the standard curve for detecting mercury ion.
Specific implementation mode
With reference to embodiment and attached drawing, the present invention will be further described, but the present invention is not limited by following embodiments
System.
In embodiment, 5 × PBS buffer solution contains：Na2HPO4(100mM), NaH2PO4 (100mM), NaCl (700 mM),
MgCl2(5 mM), pH value 7.4.
5 × PBS buffer solution of configuration is both needed to carry out high-temperature sterilization processing with ultra-pure water used.Specific method is, by 5 ×
PBS and ultra-pure water are individually positioned in different conical flasks, are then sealed with masking foil and newspaper.In high-pressure sterilizing pot
Sterilize 20 min at a temperature of 120 DEG C.
The preparation of 1 D1-AgNCs of embodiment.
Configure PB buffer solutions（A concentration of 20mM）, PB buffer solutions are made of disodium hydrogen phosphate and sodium dihydrogen phosphate,
The disodium hydrogen phosphate of 0.7163g and the sodium dihydrogen phosphate of 0.3120g are weighed respectively, are respectively made into 100ml solution, are then taken a part
Disodium hydrogen phosphate is mixed with a part of sodium dihydrogen phosphate, be mixed solution pH value be adjusted to 6.5 then it is spare.
Prepare AgNO3A concentration of 2mM, volume 1mL, AgNO3Light-exposed easy decomposition, matching while using configure AgNO3Shi Xianyong
Masking foil wraps up centrifuge tube.
The centrifuge tube of 1mL is taken, the PB of 76 μ L is added（20mM）, 15 μ L D1 are added（Final concentration of 15 μM）, 4.5 μ L are added
AgNO3（2mM）, 1min is shaken, 4 DEG C of refrigerator 30min are put in.NaHBO is prepared during this period4A concentration of 2mM, volume 1mL,
NaHBO4Matching while using is heated and is easily decomposed, prepared with 0 DEG C of ice water.
After being taken out in refrigerator, 4.5 μ L NaHBO are added4（2mM）In reaction system, 1min is shaken, 4 DEG C of refrigerators are placed on
4h or more obtains DNA-AgNCs solution.
Influences of the 2 various concentration D1 of embodiment to detection mercury ion.
（1）D1-AgNCs solution in embodiment 1 is diluted, final concentration is respectively 0.4 μM, 0.6 μM, 0.8 μM, 1.0 μ
M, 1.2 μM, 1.4 μM, then fluorescence intensity is measured respectively, it takes 2 μ L to be added in 7 centrifuge tubes respectively, 2 μ L is then added in each tube
15 μM of D2 chains, 5 × PBS of 8 μ L, 2 μ L ferrohemes（1μM）, it is eventually adding 2 μ L Hg2+Solution（3μM）, 30s is shaken, is put into 37
DEG C water-bath in react 1h.
（2）After 1 h, mixed solution is taken out from water-bath, then surveys its fluorescence intensity.
Make Fig. 2 using fluorescence intensity as ordinate with a concentration of abscissas of D1.As seen from the figure, the fluorescence signal detected is strong
Degree is as the concentration of D1 increases in 0.4-1.4 μM of section and reduces, and after concentration is more than 1.0 μM, fluorescence intensity tends to be steady
It is fixed.
Influences of the 3 various concentration D2 of embodiment to detection mercury ion.
（1）The fluorescence intensity for measuring D1-AgNCs samples S4 in embodiment 1 takes a concentration of 15 μM of S4 of 2 μ L to be added 7 respectively
In branch centrifuge tube, it is then respectively adding the D2 chains of 2 μ L various concentrations（Final concentration is respectively 0.4 μM, 0.6 μM, 0.8 μM, 1.0 μM,
1.2 μM, 1.4 μM）, each tube adds 5 × PBS of 8 μ L, 2 μ L ferrohemes again（1μM）, finally by 2 μ L Hg2+Solution（3μM）It is added
In centrifuge tube, 30s is shaken, is put into 37 DEG C of water-bath and reacts 1h.
（2）Mixed solution is taken out after 1 h from water-bath, then surveys its fluorescence intensity.
Make Fig. 3 using fluorescence intensity as ordinate with a concentration of abscissa of D2 chains.As seen from the figure, the fluorescence signal detected
Intensity is as the concentration of D2 chains increases in 0.4-1.4 μM of section and reduces, and after concentration is more than 1.0 μM, fluorescence intensity tends to
Stablize.
Detection of the embodiment 4 to mercury ion.
（1）The fluorescence intensity for measuring D1-AgNCs samples S4 in embodiment 1 takes a concentration of 15 μM of S4 of 2 μ L to be added 8 fragmented
In heart pipe, the D2 chains of a concentration of 15 μM of 2 μ L, 5 × PBS of 8 μ L, 2 μ L ferrohemes are then added in each tube（1μM）, then distinguish
The Hg of 2 μ L series concentrations is added2+Solution（1nM, 5nM, 10nM, 50nM, 70nM, 100nM, 200nM, 1 μM, 2 μM, 3 μM, 5
μM）And prepare liquid, 30s is shaken, is put into 37 DEG C of water-bath and reacts 1h.
（2）After 1 h, mixed solution is taken out from water-bath, then surveys its fluorescence intensity, as shown in Figure 4；Make in solution
Hg2+For concentration to the standard curve of fluorescence intensity, it is Y=- 222*logC+191 to calculate regression equation as shown in Figure 5, and wherein Y is glimmering
Luminous intensity, C Hg2+Concentration, related coefficient 0.96324；It is corresponding that its can be calculated according to the fluorescence intensity for measuring prepare liquid
Hg2+Concentration.
<110>University Of Ji'nan
<120>A kind of biosensor of fluoroscopic examination mercury ion
<130> 20180112
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> D1
<400> 1
cctccttcct cccttttttt agcagc 26
<210> 2
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> D2
<400> 2
gctgcttttt tttgggtagg gcgggttggg 30
Claims (6)
1. a kind of biosensor of fluoroscopic examination mercury ion, which is characterized in that including following component：
a）Silver-colored cluster containing DNA；
And b）The DNA of G- tetrads, potassium ion and ferroheme can be formed；
The DNA sequence dna of the silver cluster is as shown in SEQ No.1；The DNA sequence dna for forming G- tetrads such as SEQ No.2 institutes
Show.
2. biosensor according to claim 1, which is characterized in that the silver-colored cluster containing DNA is obtained using following preparation method
：By buffer solution, DNA solution, AgNO3Mixing is placed on 15-30min at 4 DEG C；Then cold NaHBO is added4Solution, 4 DEG C of standings
4h or more to get.
3. biosensor according to claim 2, which is characterized in that DNA, AgNO3With NaHBO4Molar ratio be 1:6:
6。
4. a kind of method using biosensor fluoroscopic examination mercury ion as described in claim 1, which is characterized in that including
Following steps：
（1）Measure the fluorescence intensity of the silver-colored cluster containing DNA, then by containing DNA silver-colored cluster and can be formed the DNA of G- tetrads, 5 × it is slow
Fliud flushing, ferroheme are added in centrifuge tube, then are separately added into the Hg of series concentration2+Solution and prepare liquid shake 30s, are put into 37 DEG C
Water-bath in react 1h；
（2）By step（1）The mixed solution of gained, measures fluorescence intensity respectively；Make Hg2+Concentration is bent to the standard of fluorescence intensity
Line calculates regression equation；Its Hg is calculated according to the fluorescence intensity of prepare liquid2+Concentration.
5. according to the method described in claim 4, it is characterized in that, final concentration of 0.4-1.4 μM of the silver-colored cluster containing DNA.
6. according to the method described in claim 4, it is characterized in that, a concentration of 0.4-1.4 μ of the DNA of G- tetrads can be formed
M。
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Cited By (3)
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| CN110452810A (en) * | 2019-08-15 | 2019-11-15 | 济南大学 | A kind of biosensor and its preparation method and application detecting MicroRNA |
| CN110632300A (en) * | 2019-09-20 | 2019-12-31 | 济南大学 | Aptamer-based biosensor for detecting salmonella and preparation method and application thereof |
| CN112304913A (en) * | 2020-10-22 | 2021-02-02 | 济南大学 | Hg detection method2+Fluorescent biosensor and preparation method and application thereof |
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Application publication date: 20180727 |