CN107300543B - A method of being based on the AgNCs probe super sensitivity detection mercury ion of T-Hg (II)-T structure - Google Patents
A method of being based on the AgNCs probe super sensitivity detection mercury ion of T-Hg (II)-T structure Download PDFInfo
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- BQPIGGFYSBELGY-UHFFFAOYSA-N mercury(2+) Chemical compound [Hg+2] BQPIGGFYSBELGY-UHFFFAOYSA-N 0.000 title claims abstract description 47
- 239000000523 sample Substances 0.000 title claims abstract description 46
- 238000001514 detection method Methods 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 24
- 230000035945 sensitivity Effects 0.000 title claims abstract description 16
- 102100033072 DNA replication ATP-dependent helicase DNA2 Human genes 0.000 claims abstract description 39
- 101000927313 Homo sapiens DNA replication ATP-dependent helicase DNA2 Proteins 0.000 claims abstract description 39
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 12
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 11
- 238000009396 hybridization Methods 0.000 claims abstract description 10
- 238000001917 fluorescence detection Methods 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 48
- 230000000295 complement effect Effects 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 9
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(I) nitrate Inorganic materials [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 6
- 239000008363 phosphate buffer Substances 0.000 claims description 5
- 230000009870 specific binding Effects 0.000 claims description 5
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 4
- 239000012279 sodium borohydride Substances 0.000 claims description 4
- 101710134784 Agnoprotein Proteins 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 230000002194 synthesizing effect Effects 0.000 claims description 3
- 239000012086 standard solution Substances 0.000 claims description 2
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 abstract description 23
- 229910052709 silver Inorganic materials 0.000 abstract description 21
- 239000004332 silver Substances 0.000 abstract description 21
- 230000008901 benefit Effects 0.000 abstract description 4
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 14
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 12
- 108020004414 DNA Proteins 0.000 description 11
- 229910052753 mercury Inorganic materials 0.000 description 8
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 7
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 7
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 7
- 229940104230 thymidine Drugs 0.000 description 7
- 229910052751 metal Inorganic materials 0.000 description 5
- 239000002184 metal Substances 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 230000006641 stabilisation Effects 0.000 description 4
- 238000011105 stabilization Methods 0.000 description 4
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 3
- ORMNPSYMZOGSSV-UHFFFAOYSA-N mercury(II) nitrate Inorganic materials [Hg+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ORMNPSYMZOGSSV-UHFFFAOYSA-N 0.000 description 3
- VRJVVIKEWDDYOG-UHFFFAOYSA-N mercury;nitric acid Chemical compound [Hg].O[N+]([O-])=O VRJVVIKEWDDYOG-UHFFFAOYSA-N 0.000 description 3
- 150000007523 nucleic acids Chemical group 0.000 description 3
- 229910002651 NO3 Inorganic materials 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 230000003252 repetitive effect Effects 0.000 description 2
- 238000011896 sensitive detection Methods 0.000 description 2
- 229940113082 thymine Drugs 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- 206010019133 Hangover Diseases 0.000 description 1
- 238000004847 absorption spectroscopy Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 229910001431 copper ion Inorganic materials 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 238000004993 emission spectroscopy Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 108010052305 exodeoxyribonuclease III Proteins 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- -1 mercury ions Chemical class 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- RECVMTHOQWMYFX-UHFFFAOYSA-N oxygen(1+) dihydride Chemical compound [OH2+] RECVMTHOQWMYFX-UHFFFAOYSA-N 0.000 description 1
- 238000005424 photoluminescence Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229910001961 silver nitrate Inorganic materials 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 238000000411 transmission spectrum Methods 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
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- 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"
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Abstract
A kind of method of AgNCs probe super sensitivity detection mercury ion based on T-Hg (II)-T structure of the invention, this method is using DNA1 as templated synthesis silver nanoclusters (DNA1-AgNCs), DNA1-AgNCs is combined with DNA2 hybridization, obtain DNA1/DNA2-AgNCs fluorescence probe, the concentration of the Hg (II) in the change in fluorescence detection solution of DNA1/DNA2-AgNCs fluorescence probe is recycled, this method has the advantages that label-free, signal is good, high specificity, high sensitivity, detectable concentration range are wide, detection limit is low.
Description
Technical field
The present invention relates to a kind of Hg2+Detection method, in particular to a kind of DNA that is based on is templated synthesis DNA1/DNA2-
AgNCs fluorescence probe, and using DNA1/DNA2-AgNCs fluorescence probe to Hg2+The method of specificity fluorescent detection, belongs to biology
Field of sensing technologies.
Background technique
Metal mercury ions are widely present in the Nature in the form of inorganic salts and organic coordination compound and metal mercury.
Mercury ion is easy to be accumulated by food chain in vivo, causes brain, liver and central nervous system by permanent wound
Evil.U.S.EPA (EPA) strictly defines concentration≤10nM of Mercury in Drinking Water ion.At present to the micro- of metal mercury ion
Amount detection become one it is too impatient to wait solve the problems, such as, the reliable metal mercury ion detecting method of tradition is mainly based upon atom
Absorption/emission spectrometry.However, such method is time-consuming and expends large number of equipment.In recent years, many team have been devoted to
Metal ion quotes research of the fluorescence metal nanocluster (NCs) as new inducing probes in environment and biosystem, and
And have document report by metal ion and silver nanoclusters occur fluorescent quenching (DNA/AgNCs) (Peng, J., et al.,
Sensitive detection of mercury and copper ions by fluorescent DNA/Ag
nanoclusters in guanine-rich DNA hybridization.Spectrochim Acta A Mol Biomol
Spectrosc, 2015.137:p.1250-7.) or in marking fluorescein to realize in nucleic acid chains the fluorescence of micro mercury ion is examined
Survey (Zhang, H., et al., Hg2+-triggered exonuclease III-assisted dual-cycle targets
recycling amplification for label-free and ultrasensitive colorimetric
detection of Hg2+.Sensors and Actuators B:Chemical,2017.246:p.896-903.).Such side
The maximum defect of method be using fluorescent quenching to mercury ion carry out accuracy in detection it is low, additionally need fluorescein be marked it is glimmering
Light probe, at high cost, synthesis is complicated, detects limit for height, and detection is linear narrow.
Summary of the invention
For Hg in existing detection water sample2+Defect existing for the method for concentration, the purpose of the invention is to provide one
Kind is label-free, signal is good, high specificity, high sensitivity, detectable concentration range are wide and it is low to Hg to detect limit2+Specificity fluorescent
The method of detection.
In order to achieve the above technical purposes, the present invention provides a kind of AgNCs probe based on T-Hg (II)-T structure is super
The method of Sensitive Detection mercury ion, method includes the following steps:
1) using DNA1 as templated synthesis DNA1-AgNCs；
2) DNA1-AgNCs is combined with DNA2 hybridization, obtains DNA1/DNA2-AgNCs fluorescence probe；
3) a series of Hg of the DNA1/DNA2-AgNCs fluorescence probe and various concentrations2+After standard solution reaction, carry out
Fluorescence detection obtains a series of fluorescence signal values, establishes Hg2+The standard curve of solution concentration and fluorescence signal value；
4) by DNA1/DNA2-AgNCs fluorescence probe and Hg to be measured2+After solution reaction, fluorescence detection is carried out, fluorescence is obtained
Signal value, and according to standard curve, calculate Hg to be measured2+The concentration of solution；
The DNA1 includes the segment complementary with DNA2 hybridization, the template segments and and Hg for synthesizing AgNCs2+Specificity knot
The segment of conjunction；
The DNA2 includes the segment complementary with DNA1 hybridization, the segment and and Hg for making AgNCs fluorescence enhancement2+Specificity
In conjunction with segment.
Technical solution of the present invention key is using DNA as templated synthesis AgNCs silver nanoclusters fluorescence probe, then benefit
With AgNCs silver nanoclusters fluorescence probe to Hg2+Specificity fluorescent detection.First with DNA1 as templated synthesis silver nanoclusters
(template segments of synthesizing nano-silver cluster are contained in DNA1) is added DNA2 in system and (wraps in DNA2 after fluorescent stabilization
The segment complementary with DNA1 hybridization, the segment for making silver nanoclusters fluorescence enhancement and continuous thymidine (thymine, T) are contained
Or increase certain repetition AGC sequence after continuous T).After adding DNA2 in system, DNA1 and DNA2 partial complementarity are sent out
Raw to hybridize, part guanine (guanine, G) enhances fluorescence signal close to silver nanoclusters in DNA2.It is added in system again
Hg2+, the Hg of thymidine T and addition in DNA1 and DNA2 chain2+Specific binding forms stable T-Hg2+- T is conducive to
DNA1 and DNA2 chain is straightened, so that the guanine G in DNA2 is remarkably reinforced closer to AgNCs so as to cause fluorescence signal.Cause
This, the trend that the fluorescence of whole system is gradually increased with the increase presentation of ion concentration of mercury.Using the principle, Hg is realized2+
Specificity fluorescent detection, and between 0.00002nM~0.16nM range system fluorescence intensity with concentration increase be in line
Property enhancing.This fluorescence probe is compared with transmission spectra method relatively to the detection of mercury ion, the detection range of linearity of this fluorescence probe
Relatively broader, detection limit is lower.
Preferred scheme, the Serial No. of the DNA1: 5'-TTTTTTCCCCCTAATTCCCAA AGCTCGACGGATT-
3'。
Preferred scheme, the Serial No. of the DNA2: 5'-AATCCGTCGAGCAGGGGAAGGGGAAGGGTTTTTT-
3', or be 5'-AATCCGTCGAGCAGGGGAAGGGGAAGGGTTTTTTCGACGACGACGACGACGAC GACGACGACGA-
3'。
DNA1 and DNA2 of the invention can be bought in Shanghai bioengineering Co., Ltd.The sequence of the two is as follows: DNA1:
5'-TTTTTTCCCCCTAATTCCCAA AGCTCGACGGATT-3'；DNA2:5'-AATCCGTCGAGCAGGGGAAGGGGAAG
GGTTTTTT-3' or 5'-AATCCGTCGAGCAGGGGAAGGGGAAGGGTTTTTTCGACGACGACGACGACGAC GACGACG
ACGA-3'；According to above-mentioned DNA sequence dna it is found that DNA1 and DNA2 are consisted of three parts: in DNA1 12, the end 3' base and
12, the end 5' base carries out complete complementary in DNA2；The middle part CCCCCTAATTCCCAAA segment of DNA1 is as synthesis silver nanoparticle
The template of cluster, the middle section AGG GGA AGGGGAAGGG of DNA2 are the part rich in G base, are mainly used for increasing fluorescence letter
Number, enhance fluorescence signal when guanine G is close to silver nanoclusters；The end 3' is that 6 thymus gland are phonetic in the end 5' and DNA2 in DNA1
Pyridine T.In addition, in the design of DNA2 nucleic acid sequence, experiments prove that increasing certain and DNA1 incomplementarity in the end 3' of DNA2
Base sequence, fluorescence probe can be widened to the detection interval of mercury ion, and the accuracy of enhancing mercury ion detecting.When
Hg is added in DNA1/DNA2-AgNCs fluorescence probe2+Later, the thymidine T and Hg in DNA1 and DNA2 chain2+Specific binding
Form stable rigid structure T-Hg2+3 ends ' of-T, 5 ends ' and DNA2 that make DNA1 are combined to form rectilinear rigid structure.
The AGGGGA repetitive sequence of DNA2 chain middle section is closer to template silver nanoclusters at this time, the fluorescence of whole system with mercury from
The trend gradually increased is presented in the increase of sub- concentration, therefore the fluorescence of probe can enhance with the increase of ion concentration of mercury.It is special
It is not in T-Hg2+When the tail end of-T structure increases certain pulling force (different AGC repetitive sequences), probe can be improved to mercury ion
Detection sensitivity, keep detection interval wider.
Preferred scheme, will be dissolved with the phosphate buffer and AgNO of DNA13After solution mixing, NaBH is added4Solution mixes,
1~3h is reacted under 20~30 DEG C of constant temperature to get DNA1-AgNCs.The method of preferred synthetic DNA 1-AgNCs: first will
DNA1 and silver nitrate solution, phosphate buffer (20mM, pH=7.0) shake at room temperature mixes 15min, and Fresh is then added
Sodium borohydride, rapidly concussion mix, be subsequently placed in 25 DEG C of constant water bath box and react 2h, can be detected its fluorescence signal.Through
Experiments have shown that the method synthesis silver nanoclusters only need 2h, entire DNA1-AgNCs system 2h to can reach stabilization.
More preferably scheme, DNA1, AgNO3And NaBH4Molar ratio be 1:5~7:5~7.More preferably 1:6:6.
Preferred scheme, the DNA1-AgNCs silver nanoclusters are mixed with DNA2 equimolar ratio, react at room temperature to get
DNA1/DNA2-AgNCs fluorescence probe.Each own 12 in the DNA2 and DNA1 nucleic acid sequence used in technical solution of the present invention
It is a continuously to hybridize complementary base sequence.After two DNA chain carry out mixing certain time, 12 bases of two chains
Complementary pairing can be carried out one by one.It was proved that the time needed for two chains carry out complementary pairing is 1h namely DNA1-AgNCs
After the entire solution hybrid reaction 1h of DNA2, system reaches complete and stablizes to get the fluorescence probe of detection mercury ion is arrived
DNA1/DNA2-AgNCs。
Preferred scheme, in step 3), the DNA1/DNA2-AgNCs fluorescence probe and standard Hg2+Solution is at room temperature
React 3~6min.
Preferred scheme, in step 4), the DNA1/DNA2-AgNCs fluorescence probe and Hg to be measured2+Solution is at room temperature
React 3~6min.
Compared with the prior art, technical solution of the present invention bring advantageous effects:
Technical solution of the present invention is realized by constructing special silver nanoclusters fluorescence probe DNA1/DNA2-AgNCs
To Hg2+The fluorescence detection of specificity, with label-free, signal is good, high specificity, high sensitivity, detectable concentration range are wide, detection
Low advantage is limited, is conducive to promote and apply.
Technical solution of the present invention constructs silver nanoclusters fluorescence by two special DNA chain comprising hybridization complementary fragment and visits
Needle DNA1/DNA2-AgNCs recycles Hg2+The characteristics of being specifically bound with thymidine T, Lai Jinhang Hg2+Detection.Hg2+With
In DNA1 in silver nanoclusters fluorescence probe in the end 5' and DNA2 the end 3' thymidine T specific binding, form linear type T-
Hg2+- T rigid structure；Helical form is presented because of the active force between base in the position that base hybridizes in two DNA chain；Two DNA chain
Middle section, due to T-Hg2+The formation of-T structure causes the chain at the end 3' in the end 5' and DNA2 in DNA1 to be straightened, and makes among DNA2 chain
AGGGGA part closer to silver nanoclusters, so that entire fluorescence probe fluorescence signal be made to enhance.DNA1/DNA2-
The fluorescence of AgNCs whole system enhances with the increase of ion concentration of mercury, has the characteristics that signal is strong, specificity is high, and
To Hg2+Response range is wide, the Hg suitable for 0.00002nM~0.16nM concentration range2+Detection, to Hg2+Detection sensitivity is very
Good, detection limit has reached 0.00002nM.In addition, being regulated and controled by the length to 2 chain of complementary DNA, discovery connects in DNA2 sequence
Continuous thymidine (thymine, T) tail end increases the hangover of different bases number, can obviously widen the detection interval of mercury ion, this
Fluorescence probe is suitable for 0.00002nM~1.6nM to the detectable concentration range of mercury ion, well below EPA minimum concentration content
It is required that detection.
Technical solution of the present invention silver nanoclusters fluorescence probe stablizes Nano silver grain by DNA nucleic acid sequence, steady
It is qualitative good, and the size of silver nanoclusters is small, resistance to environmental impact is strong；And the highly selective and specific feature of application DNA
The problem that above-mentioned sensitivity is low and detection limit is low can be effectively improved.
Technical solution of the present invention is using silver nanoclusters fluorescence probe to Hg2+Detection, have quickly, efficiently, accurately
Feature, it is only necessary to which 5min effect can reach stable detection, and the result accuracy of detection is high.
The present invention changes detection Hg by fluorescence signal2+Hg is detected with existing electrochemical method2+Comparing, it is obvious to have
Technical advantage, if property is more stable, operate more easy, the high sensitivity of detection, and detect range it is wide, detection limit
It is low.
Technical solution of the present invention is first with Hg2+It is specifically bound with thymidine T, recycles T-Hg2+- T structure
It is formed, realization regulates and controls guanine G at a distance from silver nanoclusters, to control the fluorescence intensity of fluorescence probe, therefore real
Now object is detected.
Detailed description of the invention
[Fig. 1] is the schematic diagram of the method for the AgNCs probe super sensitivity detection mercury ion based on T-Hg (II)-T structure；
[Fig. 2] is that embodiment 1 detects Hg within the scope of a certain concentration2+The fluorogram of content；
[Fig. 3] is that embodiment 1 detects Hg within the scope of a certain concentration2+The relational graph of concentration and fluorescent value；
[Fig. 4] is that embodiment 1 detects Hg within the scope of a certain concentration2+The standard curve linear relationship chart of content；
[Fig. 5] is that embodiment 2 detects Hg within the scope of a certain concentration2+The relational graph of concentration and fluorescent value；
[Fig. 6] is that embodiment 2 detects Hg within the scope of a certain concentration2+The standard curve linear relationship chart of content.
Specific embodiment
Implement to be intended to further illustrate the content of present invention below, rather than limits the protection scope of the claims in the present invention.
Embodiment 1
The DNA1 Serial No. of use: 5'-TTTTTTCCCCCTAATTCCCAAAGCTCGACGGATT-3'；
The DNA2 Serial No. of use: 5'-AATCCGTCGAGCAGGGGAAG GGGAAGGGTTTTTT-3'；
The preparation method of DNA1-AgNCs, steps are as follows:
The DNA1 (100 μM) of 33 μ L is taken, the phosphate buffer dissolution of DNA1 pH=7.4,10mM are (before being not used
The DNA1 solution for completing centrifugation is placed on 4 DEG C of refrigerator the inside storage).Then by 160 μ L phosphate buffer (20mM, pH=
7.0) it is mixed with 33 above-mentioned μ L DNA1, mixes in solution and 13.2 μ L AgNO are added3Aqueous solution (1.5mM), it is above-mentioned entire mixed
Close solution uses vortex mixer sustained oscillation to mix 15min (1000r/min) at room temperature, then takes 13.2 μ L Fresh
NaBH4(1.5mM) solution is rapidly added into above-mentioned mixed solution, and entire solution is mixed 30s, then sets entire solution
2h is reacted in 25 DEG C of thermostat water baths, measures the fluorescence of the DNA1-AgNCs silver nanoclusters of synthesis after complete reaction.
DNA1/DNA2-AgNCs synthesis step is as follows:
After above-mentioned DNA1-AgNCs is synthesized to fluorescent stabilization, 23 μ L DNA2 are added into DNA1-AgNCs solution by 1:1
(100 μM), to entire solution reaction 1h, the fluorescence of entire DNA1/DNA2-AgNCs reaches stable, can be detected its fluorescence letter
Number.
Hg(NO3)2The preparation steps of solution are as follows:
According to document introduction, mercuric nitrate and its it is not readily dissolved in water, therefore using following when preparing nitric acid mercury solution
Method is prepared: precise 0.0223g Hg (NO3)210 μ L 0.1M HNO are added3Solution is carried out, after solution-stabilized
It is settled to 100mL with ultrapure water, finally obtaining concentration is 0.65mM Hg (NO3)2Solution.
Hg2+Detecting step it is as follows:
After the fluorescence probe fluorescent stabilization of above-mentioned DNA1/DNA2-AgNCs, by synthetic DNA1/DNA2-
AgNCs solution is in charge of in equal volume, then the nitric acid mercury solution of same volume various concentration is taken to be added separately to above-mentioned be in charge of
In DNA1/DNA2-AgNCs solution, entire solution mixes reaction 5min, measures DNA1/DNA2-AgNCs fluorescence signal in solution
Variation.
Hg2+The step of fluorescence standard curve is established is as follows:
Many difference Hg are detected in above-mentioned mercury ion detecting step2+The aqueous solution of concentration, the Hg of each concentration2+It will go out
An existing photoluminescence peak, is repeated 4 times experiment, and the data obtained carry out linear fit with origin, fits corresponding
logcHg2+- Fluorescence intensity canonical plotting.
Detect Hg2+Solution step to be measured is as follows:
Various concentration Hg is prepared with tap water2+Solution to be measured, take with the solution to be measured of nitric acid mercury solution same volume in
In DNA1/DNA2-AgNCs solution, according to the fluorescent value that the mercury ion solution to be measured of known concentration measures, the fluorescent value that will be obtained
It substitutes into known standard curve, calculates in solution to be measured and contain Hg2+Concentration.
As can be seen from Figure 2 fluorescence signal is with Hg2+Concentration increases and increases.
As can be seen from Figure 4 the Hg in the concentration range of 0.00002nM~0.16nM2+With the response signal corresponding to it
There is certain linear relationship.
Embodiment 2
Operating procedure and experiment condition are same as Example 1, and DNA2 sequence number is only substituted for 5'-AATCCGTCGAGCA
GGGGAAGGGGAAGGGTTTTTTCGACGACGACGACGACGACGACGACGACGA-3'。
As can be seen from Figure 5 the Hg in the concentration range of 0.00002nM~1.6nM2+With the response signal corresponding to it
There is certain linear relationship.
Claims (5)
1. the method for AgNCs probe super sensitivity detection mercury ion of the one kind based on T-Hg (II)-T structure, it is characterised in that: including
Following steps:
1) using DNA1 as templated synthesis DNA1-AgNCs；
2) DNA1-AgNCs is combined with DNA2 hybridization, obtains DNA1/DNA2-AgNCs fluorescence probe；
3) a series of Hg of the DNA1/DNA2-AgNCs fluorescence probe and various concentrations2+After standard solution reaction, fluorescence is carried out
Detection, obtains a series of fluorescence signal values, establishes Hg2+The standard curve of solution concentration and fluorescence signal value；
4) by DNA1/DNA2-AgNCs fluorescence probe and Hg to be measured2+After solution reaction, fluorescence detection is carried out, fluorescence signal is obtained
Value, and according to standard curve, calculate Hg to be measured2+The concentration of solution；
The DNA1 includes the segment complementary with DNA2 hybridization, the template segments and and Hg for synthesizing AgNCs2+Specific binding
Segment；
The DNA2 includes the segment complementary with DNA1 hybridization, the segment and and Hg for making AgNCs fluorescence enhancement2+Specific binding
Segment；
The Serial No. of the DNA1: 5'-TTTTTTCCCCCTAATTCCCAA AGCTCGACGGATT-3'；
The Serial No. of the DNA2: 5'-AATCCGTCGAGCAGGGGAAGGGGAAGGGTTTTTT-3' is 5'-AATCCG
TCGAGCAGGGGAAGGGGAAGGGTTTTTTCGACGACGACGACGACGACGACGACGACGA-3。
2. the method for the AgNCs probe super sensitivity detection mercury ion according to claim 1 based on T-Hg (II)-T structure,
It is characterized by: by dissolved with the phosphate buffer and AgNO of DNA13After solution mixing, NaBH is added4Solution mixes, 20~30
1~3h is reacted under DEG C constant temperature to get DNA1-AgNCs.
3. the method for the AgNCs probe super sensitivity detection mercury ion according to claim 2 based on T-Hg (II)-T structure,
It is characterized by: DNA1, AgNO3And NaBH4Molar ratio be 1:5~7:5~7.
4. the method for the AgNCs probe super sensitivity detection mercury ion according to claim 1 based on T-Hg (II)-T structure,
It is characterized by: the DNA1-AgNCs is mixed with DNA2 equimolar ratio, isothermal reaction is to get DNA1/DNA2-AgNCs fluorescence
Probe.
5. according to claim 1, AgNCs probe super sensitivity detection mercury ion described in 3 or 4 based on T-Hg (II)-T structure
Method, it is characterised in that:
In step 3), the DNA1/DNA2-AgNCs fluorescence probe and standard Hg2+Solution reacts 3~6min at room temperature；
In step 4), the DNA1/DNA2-AgNCs fluorescence probe and Hg to be measured2+Solution reacts 3~6min at room temperature.
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