CN108359714A - A kind of biosensor of detection mercury ion - Google Patents
A kind of biosensor of detection mercury ion Download PDFInfo
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- CN108359714A CN108359714A CN201810072952.2A CN201810072952A CN108359714A CN 108359714 A CN108359714 A CN 108359714A CN 201810072952 A CN201810072952 A CN 201810072952A CN 108359714 A CN108359714 A CN 108359714A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q1/6825—Nucleic acid detection involving sensors
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
Abstract
The present invention provides a kind of biosensors detecting mercury ion based on neighbouring surface hybridization analysis and rolling circle amplification, including hybridization system, amplification system and fluorescence system, and the concentration of mercury ion is measured using fluoroscopic examination.The high sensitivity of this biosensor detection, specificity are good；Reaction condition is mild, and reaction speed is fast；Its detection method is easy to operate, detection cycle is short；This biosensor preparation method is simple, and performance is stablized, reproducible, the Hg suitable for food security and water body of fluoroscopic examination2+Detection and biosensor industrialization practical application.
Description
Technical field
The present invention relates to a kind of biosensors detecting mercury ion based on aptamer, and in particular to one kind is based on neighbour
The biosensor of near surface hybridization analysis and rolling circle amplification detection mercury ion, belongs to biosensor technology field.
Background technology
Mercury is a kind of toxic element, cannot be biodegradable, and can be enriched with by food chain by endanger human health, especially
Have human nerve, kidney, liver etc. compared with macrolesion, cognitive disorder and dyskinesia can be caused.And recently as industry
Development, mercury ion pollution are on the rise, and huge harm is caused to the mankind and environment.The improvement of mercury ion pollution is very tired
Difficulty should prevent trouble before it happens.Therefore very urgent to the monitoring in real time of mercury ion, to environmental protection, health medical treatment, Food Monitoring etc.
Field has great significance.Meanwhile Hg is detected to fast accurate2+Method more stringent requirements are proposed.
The detection method for the mercury ion reported at present includes atomic emission spectrometry, atomic absorption spectrography (AAS), inductive coupling
Often there is expensive equipment, analytical cycle length, specimen preprocessing in plasma mass spectroscopic methodology, electrochemical method etc., these methods
The problems such as reason is complicated, testing cost is expensive, the requirement for the convenient, fast of mercury ion detecting, sensitivity etc. has been difficult to
It adapts to.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
It stays.Aptamer has good selectivity for sensor, can form special construction, such as T with specific metal ion
Base energy and Hg2+Form T-Hg2+The mismatch structures of-T can accurately measure metal ion, simultaneously using this special construction
It can also avoid the generation of interference.
Invention content
In order to which the method specificity and sensitivity that solve to detect mercury ion in the prior art are all relatively low, detection time length
Problem, present invention aims at provide a species specificity and high sensitivity, detection speed it is fast based on neighbouring surface hybridization analysis
The biosensor of mercury ion is detected with rolling circle amplification.
Another object of the present invention is to provide a kind of application of above-mentioned biosensor in detecting mercury ion.
To achieve the above object, the present invention adopts the following technical scheme that.
A kind of biosensor detecting mercury ion based on neighbouring surface hybridization analysis and rolling circle amplification, including：
a）Hybridization system, including T4 DNA ligases, sequence S1 as shown in SEQ No. 1, SEQ No. 2 and SEQ No. 3,
The few chain DNAs of S2 two and Padlock probes；
b）Amplification system：Including sequence primer as shown in SEQ No. 4（Primer）, phi29 polymerases and dNTP；
c）Fluorescence system：Endonuclease IV and fluorescence probe, the fluorescence probe sequence is as shown in SEQ No. 5, including one
A abasic site（AP site）, fluorophor and corresponding fluorescent quenching group are modified respectively before the site and after adjacent base.
The fluorophor of the fluorescence probe and corresponding fluorescent quenching group are preferably 6- Fluoresceincarboxylic acids（FAM）And 4-
(4 '-dimethylaminoazobenzene base) benzoic acid（Dabcyl）.
The T4 DNA ligases are in component a）In concentration be preferably 4 × 10-2 U/mL- 8×10-2 U/mL。
S1, S2 widow chain DNA is in component a）In concentration be preferably 1 μM -10 μM.
The Padlock probes are in component a）In concentration be preferably 10 μM -15 μM.
The primer in component b）In concentration be preferably 10 μM -15 μM.
The phi29 polymerases are in component b）In concentration be preferably 1 × 10-3 U/mL-2×10-3 U/mL。
The dNTP is in component b）In concentration be preferably 1 mM-, 5 mM.
The endonuclease IV is in component c）In concentration be preferably 1 × 10-3 U/mL- 1.5×10-3 U/mL。
The fluorescence probe is in component c）In concentration be preferably 15 μM -20 μM.
A method of mercury ion being detected using above-mentioned biosensor, is included the following steps：
（1）Will, the solution of S1, S2 and padlock probe, T4 DNA ligase solution, 10 × T4 DNA ligase buffer solutions,
Aqua sterilisa is added in EP pipes, adds testing sample solution or serial mercury ion standard solution reaction；
（2）By the solution of Primer, phi29 polymerase and dNTP, 10 × Phi29 DNA polymerase buffer liquid step is added
（1）EP pipes in react；
（3）To step（2）EP pipes in fluorescence probe solution and endonuclease IV solution reactions is added；
（4）By step（3）In reaction solution carry out fluoroscopic examination, according to the fluorescence intensity of mercury ion standard solution make standard song
Line obtains regression equation, calculates the ion concentration of mercury in sample to be tested.
The step（1）Middle reaction temperature is 16 DEG C -25 DEG C, and the reaction time is 16 h-24 h.
The step（2）With（3）Middle reaction temperature is 37 DEG C, and the reaction time is 2 h.
The step（4）The condition of fluoroscopic examination is：Excitation wavelength 486nm, launch wavelength 518nm, detection range 450
nm-530 nm。
In the above method, a concentration of 1 μM -10 μM of preferred mercury ion detecting.
The T4 DNA ligases buffer solution and Phi29 DNA polymerase buffer liquid are T4 DNA ligases and Phi29
The common buffer solution that archaeal dna polymerase is reacted can be configured voluntarily or commercially available.In one embodiment of the invention,
10 × T4 DNA ligase buffer solutions contain 50 mM Tris-HCl, 10 mM MgCl2, 10 mM dithiothreitol (DTT)s（DTT）, 1
MM atriphos（ATP）, pH 7.5；10 × Phi29 DNA polymerase buffer liquid contain 50 mM Tris-HCl, 10 mM
MgCl2, 10 mM (NH4)2SO4, 4 mM dithiothreitol (DTT)s（DTT）, pH 7.5.
The present invention has the following advantages：
Neighbouring surface hybridization analysis principle, which is utilized, in this biosensor realizes specific binding with object；Rolling is utilized
Ring amplifying technique realizes the amplification of signal；The specific cleavage site of endonuclease IV is utilized（AP site）, realize that positioning is cut
It cuts；By changing fluorophor（FAM）With quencher（Dabcyl）Distance generates fluorescence signal；Utilize T-Hg2+The cycle of-T
And the complementary site of multiple signal probes, secondary cycle amplification is realized, fluorescence signal is exaggerated, improves the sensitive of detection
Degree is realized to object Hg2+Ultrasensitiveness detection；The reaction condition of this biosensor is mild, and reaction speed is fast；Due to making
With fluorescence method, detection method is easy to operate, detection cycle is short；The main process of testing principle is realized in homogeneous,
Reaction speed is improved, the complexity of operation is reduced, realizes the quick of object, it is simply, sensitive to detect；This biology
Transducer production method is simple, and performance is stablized, reproducible, the Hg suitable for food security and water body of fluoroscopic examination2+Inspection
Survey the practical application with biosensor industrialization.
Description of the drawings
Fig. 1 is fluorescence intensity with endonuclease IV enzyme amount change curves；
Fig. 2 is fluorescence intensity with padlock concentration and probe concentration change curves；
Fig. 3 is fluorescence intensity with primer concentration curve figures；
Fig. 4 is fluorescence intensity with fluorescence probe concentration curve figure；
Fig. 5 is the standard curve that biosensor detects mercury ion；
Fig. 6 is the principle schematic that biosensor detects 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.
10 in embodiment × T4 DNA ligase buffer solutions contain 50 mM Tris-HCl, 10 mM MgCl2, 10 mM bis-
Sulphur threitol（DTT）, 1 mM atriphos（ATP）, pH 7.5；10 × Phi29 DNA polymerase buffer liquid contain 50 mM
Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM dithiothreitol (DTT)s（DTT）, pH 7.5.
Influences of the 1 various concentration endonuclease IV of embodiment to fluorescence intensity.
（1）By 8 μ L aqua sterilisas, 2 μ L 10 × T4 DNA ligase buffer solutions（50 mM Tris-HCl, 10 mM
MgCl2, 10 mM dithiothreitol (DTT)s（DTT）, 1 mM atriphos（ATP）, pH 7.5）, 2 μ L S1 chains（5 μM）、2 μL
S2 chains（5 μM）, 2 μ L Hg2+Solution（10 μM）, 2 μ L padlock probes（10 μM）, 2 μ L T4 DNA ligases（4×
10-2U/mL）, it is added in the EP pipes of preprepared sterilizing, constitutes 20 μ L systems.30 s are shaken, 20 at 16 DEG C
h；
（2）2 μ L primer are added into the EP pipes（10 μM）, 2 μ L phi29 archaeal dna polymerases（1×10-3U/mL）、2
μ L 10 × phi29 DNA polymerase buffers liquid, 2 μ L dNTP（1 mM）, 30 s are shaken, react 2 h under the conditions of 37 DEG C；
（3）1 μ L fluorescence probes are added into the EP pipes after above-mentioned reaction（15 μM）, 2 μ L endonuclease IV（Concentration point
It Wei not 10 U/mL, 50 U/mL, 100 U/mL, 200 U/mL, 300 U/mL）30s is shaken, 2 h in 37 DEG C of insulating box are put into；
（4）Solution after the 10 above-mentioned reactions of μ L is diluted to 70 μ L, fluorescence is detected at 518 nm with luminoscope.Luminoscope swashs
Hair wavelength is set as 486 nm, and launch wavelength is 518 nm, 450 nm-530 nm of detection range, reads fluorescence signal variation, inspection
Survey object.
With a concentration of abscissas of endonuclease IV, using fluorescence intensity as ordinate, make fluorescence intensity with endonuclease
IV enzyme amount change curves, as shown in Figure 1.It can be seen from the figure that with the increase of endonuclease IV amounts, fluorescence intensity is not
Disconnected enhancing, after enzyme amount reaches 200 U/mL, fluorescence intensity is basically unchanged.
Influence of the 2 various concentration padlock probes of embodiment to fluorescence intensity.
（1）By 8 μ L aqua sterilisas, 2 μ L 10 × T4 DNA ligase buffer solutions（50 mM Tris-HCl, 10 mM
MgCl2, 10 mM dithiothreitol (DTT)s（DTT）, 1 mM atriphos（ATP）, pH 7.5）, 2 μ L S1 chains（5 μM）、2 μL
S2 chains（5 μM）, 2 μ L Hg2+Solution（10 μM）, 2 μ L padlock probes（Concentration is respectively 1 μM, 2.5 μM, 5 μM, 10
μM、15 μM）, 2 μ L T4 DNA ligases（4×10-2U/mL）, it is added in the EP pipes of preprepared sterilizing, constitutes
20 μ L systems.30 s are shaken, 20 h at 16 DEG C；
（2）2 μ L primer are added into the EP pipes（10 μM）, 2 μ L phi29 archaeal dna polymerases（1×10-3U/mL）、2
μ L 10 × phi29 DNA polymerase buffers liquid, 2 μ L dNTP（1 mM）, 30 s are shaken, react 2 h under the conditions of 37 DEG C；
（3）Into the EP pipes after above-mentioned reaction plus 1 μ L enter fluorescence probe（15 μM）, 2 μ L endonuclease IV（200 U/
mL）30 s are shaken, is put into 37 DEG C of insulating box and reacts 2 h；
（4）Solution after the 10 above-mentioned reactions of μ L is diluted to 70 μ L, fluorescence is detected at 518 nm with luminoscope.Luminoscope
Excitation wavelength is set as 486 nm, and launch wavelength is 518 nm, 450 nm-530 nm of detection range, reads fluorescence signal variation,
Detect object.
Using padlock concentration and probe concentrations as abscissa, using fluorescence intensity as ordinate, make fluorescence intensity with padlock probes
Concentration curve figure, as shown in Figure 2.It can be seen from the figure that with the increase of padlock concentration and probe concentrations, fluorescence intensity is not
Disconnected enhancing, fluorescence intensity is basically unchanged after concentration reaches 10 μM.
Influence of the 3 difference primer concentration of embodiment to fluorescence intensity.
（1）By 8 μ L aqua sterilisas, 2 μ L 10 × T4 DNA ligase buffer solutions（50 mM Tris-HCl, 10 mM
MgCl2, 10 mM dithiothreitol (DTT)s（DTT）, 1 mM atriphos（ATP）, pH 7.5）, 2 μ L S1 chains（5 μM）、2 μL
S2 chains（5 μM）, 2 μ L Hg2+Solution（10 μM）, 2 μ L padlock probes（10 μM）, 2 μ L T4 DNA ligases（4×
10-2U/mL）, it is added in the EP pipes of preprepared sterilizing, constitutes 20 μ L systems.30 s are shaken, 20 at 16 DEG C
h；
（2）2 μ L primer are added into the EP pipes（Concentration is respectively 1 μM, 2 μM, 5 μM, 10 μM, 15 μM）、2 μL
Phi29 archaeal dna polymerases（1×10-3U/mL）, 2 μ L 10 × phi29 DNA polymerase buffers liquid, 2 μ L dNTP（1
mM）, 30 s are shaken, react 2 h under the conditions of 37 DEG C；
（3）Into the EP pipes after above-mentioned reaction plus 1 μ L enter fluorescence probe（15 μM）, 2 μ L endonuclease IV（200 U/
mL）30 s are shaken, is put into 37 DEG C of insulating box and reacts 2 h；
（4）Solution after the 10 above-mentioned reactions of μ L is diluted to 70 μ L, fluorescence is detected at 518 nm with luminoscope.Luminoscope
Excitation wavelength is set as 486 nm, and launch wavelength is 518 nm, 450 nm-530 nm of detection range, reads fluorescence signal variation,
Detect object.
With a concentration of abscissas of primer, using fluorescence intensity as ordinate, makees fluorescence intensity and change song with primer concentration
Line chart, as shown in Figure 3.It can be seen from the figure that with the increase of primer concentration, fluorescence intensity constantly enhances, and concentration reaches
Fluorescence intensity is basically unchanged after 10 μM.
Influence of the different fluorescence probe concentration of embodiment 4 to fluorescence intensity.
（1）By 8 μ L aqua sterilisas, 2 μ L 10 × T4 DNA ligase buffer solutions（50 mM Tris-HCl, 10 mM
MgCl2, 10 mM dithiothreitol (DTT)s（DTT）, 1 mM atriphos（ATP）, pH 7.5）, 2 μ L S1 chains（5 μM）、2 μL
S2 chains（5 μM）, 2 μ L Hg2+Solution（10 μM）, 2 μ L padlock probes（10 μM）, 2 μ L T4 DNA ligases（4×
10-2U/mL）, it is added in the EP pipes of preprepared sterilizing, constitutes 20 μ L systems.30 s are shaken, 20 at 16 DEG C
h；
（2）2 μ L primer are added into the EP pipes（10 μM）, 2 μ L phi29 archaeal dna polymerases（1×10-3U/mL）、2
μ L 10 × phi29 DNA polymerase buffers liquid, 2 μ L dNTP（1 mM）, 30 s are shaken, react 2 h under the conditions of 37 DEG C；
（3）Into the EP pipes after above-mentioned reaction plus 1 μ L enter fluorescence probe（Concentration is respectively 2 μM, 2.5 μM, 5 μM, 10 μM,
15 μM、20 μM）, 2 μ L endonuclease IV（200 U/mL）30 s are shaken, is put into 37 DEG C of insulating box and reacts 2 h；
（4）Solution after the 10 above-mentioned reactions of μ L is diluted to 70 μ L, fluorescence is detected at 518 nm with luminoscope.Luminoscope
Excitation wavelength is set as 486 nm, and launch wavelength is 518 nm, 450 nm-530 nm of detection range, reads fluorescence signal variation,
Detect object.
With a concentration of abscissa of fluorescence probe, using fluorescence intensity as ordinate, makees fluorescence intensity and become with fluorescence probe concentration
Change curve graph, as shown in Figure 4.It can be seen from the figure that with the increase of fluorescence probe concentration, fluorescence intensity constantly enhances, dense
Fluorescence intensity is basically unchanged after degree reaches 15 μM.
The measurement of 5 ion concentration of mercury of embodiment.
（1）By 8 μ L aqua sterilisas, 2 μ L 10 × T4 DNA ligase buffer solutions（50 mM Tris-HCl, 10 mM
MgCl2, 10 mM dithiothreitol (DTT)s（DTT）, 1 mM atriphos（ATP）, pH 7.5）, 2 μ L S1 chains（5 μM）、2 μL
S2 chains（5 μM）, 2 μ L Hg2+Solution（10 μM）, 2 μ L padlock probes（10 μM）, 2 μ L T4 DNA ligases（4×
10-2U/mL）, it is added in the EP pipes of preprepared sterilizing, constitutes 20 μ L systems.30 s are shaken, 20 at 16 DEG C
h；
（2）2 μ L primer are added into the EP pipes（10 μM）, 2 μ L phi29 archaeal dna polymerases（1×10-3U/mL）、2
μ L 10 × phi29 DNA polymerase buffers liquid, 2 μ L dNTP（1 mM）, 30 s are shaken, react 2 h under the conditions of 37 DEG C；
（3）Into the EP pipes after above-mentioned reaction plus 1 μ L enter fluorescence probe（15 μM）, 2 μ L endonuclease IV（200 U/
mL）30 s are shaken, is put into 37 DEG C of insulating box and reacts 2 h；
（4）Solution after the 10 above-mentioned reactions of μ L is diluted to 70 μ L, fluorescence is detected at 518 nm with luminoscope.Luminoscope
Excitation wavelength is set as 486 nm, and launch wavelength is 518 nm, 450 nm-530 nm of detection range, reads fluorescence signal variation,
Detect object.
Prepare liquid and the fluorescence intensity of mercury ion standard solution are as shown in table 1, using ion concentration of mercury as abscissa, with fluorescence
Intensity is ordinate, does standard curve, as shown in figure 5, it is F=175.54+29.77 × C to calculate regression equationHg2+(μM), it is related
Coefficient is 0.9836, calculates to obtain a concentration of 14.60 μM of mercury ion in prepare liquid.
The fluorescence intensity of table 1 prepare liquid and mercury ion standard solution
| Hg2+Concentration（μM） | Fluorescence intensity |
| 1 | 140 |
| 2 | 260 |
| 5 | 350 |
| 10 | 480 |
| Prepare liquid | 610 |
The operation principle of this biological sensor is as shown in Figure 6：
（1）Neighbouring surface hybridization analysis：Work as Hg2+In the presence of, S1 and S2 is due to being rich in T bases, it will be able to form T-Hg2+- T's
Mismatch structures.Make the S1 that cannot be combined originally with padlock-probe both ends and S2 due to close to each other, generation neighbouring surface hybridization point
Analysis reaction, does not form T-Hg2+Two parts sequence of-T can be combined with two sections of sequences in padlock-probe respectively, when there is T4
In the presence of DNA ligase, it will be able to make padlock-probe end connection cyclization, prepare to carry out rolling circle amplification in next step.
（2）Rolling circle amplification reacts：Under the action of phi29 polymerases and dNTP, using primer as primer, with padlock
The ring that probe connects into is the extension that template carries out DNA.Meanwhile the long-chain that rolling circle amplification extends can make S1-Hg2+- S2 is tied
Structure is detached with circular DNA.Meanwhile S1-Hg2+- S2 structures are combined with padlock-probe again as recycle, realize that the preliminary of signal is put
Greatly.
（3）The base pair complementarity of fluorescence probe and extended chain：The long-chain extended after rolling ring can be with fluorescence probe
Base pair complementarity is carried out, multiple double-strands are formed.Endonuclease IV is added, fluorescence probe can be cut into two parts,
Make fluorophor（FAM）And quencher（Dabcyl）Separation, to generate fluorescence signal.Due to having multiple complementations on ring
The secondary amplification of signal is realized to ensure that more fluorescence probes are combined with extended chain in site.
SEQUENCE LISTING
<110>University Of Ji'nan
<120>A kind of biosensor of detection mercury ion
<130> 20180125
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> S1
<400> 1
agtgacgtgc gctttttttt t 21
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> S2
<400> 2
tttttttttc acctggccgt g 21
<210> 3
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Padlock
<400> 3
gtggcgcacg ttgccttcgc cgttcacact catgccttcg ccgcgcacgg ccag 54
<210> 4
<211> 12
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 4
atgagtgtga ac 12
<210> 5
<211> 9
<212> DNA
<213> Artificial Sequence
<220>
<223> Signal
<400> 5
gccttcgcc 9
Claims (7)
1. a kind of biosensor detecting mercury ion based on neighbouring surface hybridization analysis and rolling circle amplification, which is characterized in that packet
It includes：
a）Hybridization system, including T4 DNA ligases, the few chain DNAs of S1, S2 two and Padlock probes；
b）Amplification system：Including primer, phi29 polymerases and dNTP；
c）Fluorescence system：Endonuclease IV and fluorescence probe；
S1 widow's chain DNA sequence is as shown in SEQ No. 1；S2 widow's chain DNA sequence is as shown in SEQ No. 2；It is described
Padlock probe sequences are as shown in SEQ No. 3；
The primer sequence is as shown in SEQ No. 4；
For the fluorescence probe sequence as shown in SEQ No. 5,5 ' the 5th bases of end are an abasic site, before the site and phase
Fluorophor and corresponding fluorescent quenching group are modified after adjacent base respectively.
2. biosensor according to claim 1, which is characterized in that the fluorophor of the fluorescence probe with it is corresponding glimmering
Optical quenching group is 6- Fluoresceincarboxylic acids and 4- (4 '-dimethylaminoazobenzene base) benzoic acid.
3. a kind of method that biosensor using described in claims 1 or 22 detects mercury ion, which is characterized in that including with
Lower step：
（1）Will, the solution of S1, S2 and padlock probe, T4 DNA ligase solution, 10 × T4 DNA ligase buffer solutions,
Aqua sterilisa is added in EP pipes, adds testing sample solution or serial mercury ion standard solution reaction；
（2）By the solution of Primer, phi29 polymerase and dNTP, 10 × Phi29 DNA polymerase buffer liquid step is added
（1）EP pipes in react；
（3）To step（2）EP pipes in fluorescence probe solution and endonuclease IV solution reactions is added；
（4）By step（3）In reaction solution carry out fluoroscopic examination, according to the fluorescence intensity of mercury ion standard solution make standard song
Line obtains regression equation, calculates the ion concentration of mercury in sample to be tested.
4. according to the method described in claim 3, it is characterized in that, step（1）Middle reaction temperature is 16 DEG C -25 DEG C, reaction
Time is 16 h-24 h.
5. according to the method described in claim 3, it is characterized in that, step（2）With（3）Middle reaction temperature is 37 DEG C, reaction
Time is 2 h.
6. according to the method described in claim 3, it is characterized in that, the step（4）The condition of fluoroscopic examination is：Excitation wave
Long 486nm, launch wavelength 518nm, 450 nm-530 nm of detection range.
7. according to any methods of claim 3-6, which is characterized in that a concentration of 1 μM -10 μM of mercury ion detecting.
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Cited By (6)
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|---|---|---|---|---|
| CN111175268A (en) * | 2020-01-23 | 2020-05-19 | 闽江学院 | Fluorescent sensor for detecting dual signal amplification of mercury ions and preparation method thereof |
| CN112285078A (en) * | 2020-10-14 | 2021-01-29 | 安庆师范大学 | Novel mercury ion on-site detection method based on intelligent DNA hydrogel |
| CN112304913A (en) * | 2020-10-22 | 2021-02-02 | 济南大学 | Hg detection method2+Fluorescent biosensor and preparation method and application thereof |
| CN112345506A (en) * | 2020-11-06 | 2021-02-09 | 济南大学 | Fluorescent biosensor for detecting mercury ions |
| CN113862259A (en) * | 2020-06-30 | 2021-12-31 | 上海健康医学院 | Detection of Hg based on DSN enzyme2+DNA biosensor of |
| CN114235763A (en) * | 2021-12-03 | 2022-03-25 | 济南大学 | Method for detecting mercury ions (Hg)2+) In a biosensor |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111175268A (en) * | 2020-01-23 | 2020-05-19 | 闽江学院 | Fluorescent sensor for detecting dual signal amplification of mercury ions and preparation method thereof |
| CN113862259A (en) * | 2020-06-30 | 2021-12-31 | 上海健康医学院 | Detection of Hg based on DSN enzyme2+DNA biosensor of |
| CN113862259B (en) * | 2020-06-30 | 2024-03-12 | 上海健康医学院 | Detection of Hg based on DSN enzyme 2+ DNA biosensor of (2) |
| CN112285078A (en) * | 2020-10-14 | 2021-01-29 | 安庆师范大学 | Novel mercury ion on-site detection method based on intelligent DNA hydrogel |
| CN112304913A (en) * | 2020-10-22 | 2021-02-02 | 济南大学 | Hg detection method2+Fluorescent biosensor and preparation method and application thereof |
| CN112345506A (en) * | 2020-11-06 | 2021-02-09 | 济南大学 | Fluorescent biosensor for detecting mercury ions |
| CN112345506B (en) * | 2020-11-06 | 2022-08-26 | 济南大学 | Fluorescent biosensor for detecting mercury ions |
| CN114235763A (en) * | 2021-12-03 | 2022-03-25 | 济南大学 | Method for detecting mercury ions (Hg)2+) In a biosensor |
| CN114235763B (en) * | 2021-12-03 | 2023-05-26 | 济南大学 | Detection of mercury ions (Hg) 2+ ) Is a biosensor of (2) |
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