CN102590170A - Method for simultaneously detecting mercury ion and/or silver ion in water solution based on fluorescence resonance energy transfer - Google Patents
Method for simultaneously detecting mercury ion and/or silver ion in water solution based on fluorescence resonance energy transfer Download PDFInfo
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- CN102590170A CN102590170A CN2012100471161A CN201210047116A CN102590170A CN 102590170 A CN102590170 A CN 102590170A CN 2012100471161 A CN2012100471161 A CN 2012100471161A CN 201210047116 A CN201210047116 A CN 201210047116A CN 102590170 A CN102590170 A CN 102590170A
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Abstract
The invention relates to a method for simultaneously detecting a divalent mercury ion and a silver ion in water solution based on fluorescence resonance energy transfer, belonging to the field of heavy metal ion detecting technology. The method comprises the following steps: firstly, preparing a detecting probe, comprising following steps: (1), preparing a cadmium telluride (CdTe) quantum dot which is encapsulated by mercaptopropionic acid (MPA); (2), designing and composing a nucleic acid probe for detection; (3), preparing a fluorescent probe CdTe-DNA of quantum dots and nucleic acid; and secondly, detecting heavy metal ion Ag+ and/or Hg2+. The method provided by the invention can detect two ions simultaneously, the workload is low, the cost is low and the process is not complex.
Description
Technical field
The present invention relates to a kind of method, belong to the detection technique field of heavy metal ion based on dimercurion and silver ion in the FRET detection WS.
Background technology
Heavy metal ion such as mercury ion and/or silver ion are the serious environmental pollutants.Their contaminant water and soil cause serious threat and animals and plants are produced serious poisonous effect human health.Mercury ion exists inrichment and long-term contact with it can cause the lasting and serious damage of central nervous system in environment.Silver ion has been proved to be has strong toxicity to hydrobiont.Therefore, invention is a kind of can be effectively and the method for these two kinds of ions of detection of sensitivity is very important.Traditional detection method such as Cold Atomic Fluorescent Mercury spectroscopic methodology (CV-AFS), cold atomic absorption spectrometry (CV-AAS), inductivity coupled plasma mass spectrometry (ICPMS) etc. has a lot of defectives: workload is big, cost is high and process is complicated.In order to solve the problem that traditional detection method brings, develop new sensor and just be necessary very much.FRET (FRET) is a kind of physical phenomenon that was at first proposed before more than 50 years by Theodor F rster.FRET has multiple application, like developed FRET imaging technique and unimolecule FRET detection technique.In recent years, FRET is applied in the detection of many fluorescent materials, as detecting DNA, lysozyme, lemon yellow and histamine.As everyone knows, dimercurion can specificity bonding thymine alkali bases T and is formed T-Hg
2+-T compound, silver ion can specificity bonding cytimidine base C and are formed C-Ag
+-C compound.Up to now, scientists has developed many FRET sensors based on mercury ion and this special nature of silver ion.But these sensors mainly concentrate on a kind of detection of ion, fail to accomplish to detect simultaneously this two kinds of ions.
Summary of the invention
The purpose of this invention is to provide a kind of method of coming to detect simultaneously dimercurion and silver ion in the WS based on FRET.
Technical scheme of the present invention: a kind of method of coming to detect simultaneously dimercurion and silver ion in the WS based on FRET:
(1) preparation of detector probe
(1) preparation of the cadmium telluride CdTe quantum dot of mercaptopropionic acid MPA parcel
With sodium borohydride NaBH
4, ultrapure water and tellurium Te powder be mixed together in the reaction vessel, makes NaBH
4Concentration be 2-3M, the Te powder content is 0.5-0.6M, again reaction vessel is placed the NaHTe solution for standby of 0 ℃ of ice-water bath reaction 12h with the preparation clarification; Take by weighing a certain amount of inorganic cadmium salt Cd
2+Be dissolved in the water control Cd
2+Concentration is 0.01-0.02M, under violent stirring, drips an amount of MPA, and using NaOH solution to regulate pH is 11.20, logical N
2Add NaHTe solution rapidly behind deoxygenation, the protection 30min, keep Cd
2+: Te
2-: the mol ratio of MPA is 1:0.4-0.5:2.2-2.5; Promptly obtain the precursor solution of CdTe quantum dot after the reaction; Get precursor solution and be added to teflon and do in the stainless steel cauldron of liner, 160 ℃ of reaction 20min down promptly obtain needed CdTe quantum dot;
(2) design of the nucleic acid probe of detection usefulness is with synthetic
The single stranded nucleic acid probe DNA1:5'-NH of amido modified mistake
2-GTACAAGATG-3';
Do not make the single stranded nucleic acid probe DNA2:5'-GAGCTTTTCA GACGCATCTT GTACGACTCG CTCCCCATAC-3' of any modification;
The single stranded nucleic acid probe TAMRA-DNA:5'-TAMRA-TTTTGCTC-3' of fluorescent dye TAMRA modified;
The single stranded nucleic acid probe Cy5-DNA:5'-Cy5-GTATCCCC-3' of indoles cyanine dyes Cy5 modified;
(3) preparation of the fluorescence probe CdTe-DNA of quantum dot and nucleic acid
The coupling matter that at first prepares CdTe and DNA1: 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimides EDC; N-hydroxy-succinamide NHS and CdTe solution are admixed together by proper proportion; Activation 0.5-1 h adds the DNA1 WS then, reacts 4h down at 25 ℃; Carry out ultrafiltration with the DNA1 that thorough removal does not join with the CdTe coupling, promptly prepare CdTe-DNA1; Then, at room temperature carry out hybridization reaction 6h to CdTe-DNA1 and DNA2, ultrafiltration promptly prepares probe CdTe-DNA1-DNA2 of quantum dot and DNA to remove the not DNA2 of hybridization; At last, be added to the WS of Cy5-DNA and TAMRA-DNA respectively among the CdTe-DNA1-DNA2 of above-mentioned preparation, promptly prepare the CdTe-DNA fluorescence probe that detects usefulness;
(2) heavy metal ion Ag
+And Hg
2+Detection
(1) Ag
+Detection
Bonding force between silver ion and 3-(N-morpholine) the propane sulfonic acid MOPS very a little less than, so select the MOPS damping fluid as detection architecture: the CdTe-DNA fluorescence probe of preparation in the 100nM step (), 10mM sodium dihydrogen phosphate/sodium hydrogen phosphate NaH
2PO
4/ Na
2HPO
4, the MOPS of 20mM, the sodium nitrate NaNO of 50mM
3
The WS that will contain 0-50 nM variable concentrations silver ion joins in the above-mentioned detection architecture, and under 25 ℃, keeps 0.5h, makes Ag
+Cy5-DNA that induces and DNA2 hybridization reaction carry out fully, record the fluorescent emission collection of illustrative plates of whole system then;
(2) Hg
2+Detection
Detection method and step (1) are similar, just with Ag in the step (1)
+Solion changes Hg into
2+Solution; Contain 0-50 nM variable concentrations Hg
2+The WS of ion joins in the above-mentioned detection architecture, and under 25 ℃, keeps 0.5h, makes Hg
2+TAMRA-DNA that induces and DNA2 hybridization reaction carry out fully, measure the fluorescent emission collection of illustrative plates of whole system then;
(3) carry out Ag simultaneously
+And Hg
2+Detection
Detection method and step (1) are similar, just with Ag in the step (1)
+Solion changes Ag into
+And Hg
2+Mixed solution; Contain 0-50 nM variable concentrations Ag
+And Hg
2+The WS join in the above-mentioned detection architecture, and under 25 ℃, keep 0.5h, make Hg
2+The TAMRA-DNA that induces and DNA2 hybridization reaction and Ag
+Cy5-DNA that induces and DNA2 hybridization reaction carry out fully, measure the fluorescent emission collection of illustrative plates of whole system then.
Described a kind of method of coming to detect simultaneously dimercurion and silver ion in the WS based on FRET: the preparation of the fluorescence probe CdTe-DNA of quantum dot and nucleic acid: the coupling matter that at first is preparation CdTe and DNA1: 1-ethyl-3-of 320 μ L 0.1mM (3-dimethylamino-propyl)-carbodiimides EDC; The N-hydroxy-succinamide NHS of 200 μ L, 0.0125 mM and 1280 μ L, 1 μ M CdTe solution are admixed together, activation 0.5-1 h; The DNA1 WS that adds 100 μ L, 10 μ M then reacted 4 hours down at 25 ℃; Carry out ultrafiltration 15min with ultra filtration membrane with 5000rpm then, and resuspended with ultrapure water, ultrafiltration once more so circulates 3 times, thoroughly removes not the DNA1 that joins with the CdTe coupling, promptly prepares CdTe-DNA1;
Then, at room temperature carry out hybridization reaction 6h to the CdTe-DNA1 of 900 μ L, 1 μ M and 100 μ L, 20 μ M DNA2, carry out same ultrafiltration step and remove the not DNA2 of hybridization, promptly prepare CdTe-DNA1-DNA2;
At last, be added to the WS of 100 μ L, 20 μ M Cy5-DNA and 100 μ L, 20 μ M TAMRA-DNA respectively among the CdTe-DNA1-DNA2 of above-mentioned preparation, promptly prepare the CdTe-DNA fluorescence probe that detects usefulness.
Beneficial effect of the present invention: the present invention provides a kind of method of coming to detect simultaneously dimercurion and silver ion in the WS based on FRET; The present invention can accomplish to detect simultaneously this two kinds of ions, and workload is little comparatively speaking, cost is not high and process is uncomplicated.
Description of drawings
Fig. 1 the present invention detects the schematic flow sheet of dimercurion and silver ion in the WS simultaneously, wherein, and Ag
+And Hg
2+When existing simultaneously, the energy of quantum dot shifts the dyestuff to correspondence, and FRET takes place.
Fig. 2 the present invention the transmission electron microscope photo of CdTe quantum dot of synthetic mercaptopropionic acid parcel.
Fig. 3 the present invention adopted 1, the CdTe quantum dot, 2, TAMRA, 3, ultraviolet absorpting spectrum (solid line) and the fluorescent emission collection of illustrative plates (dotted line) of Cy5, wherein, excite light wavelength to select 380 nanometers.
Fig. 4 embodiment of the invention 1 detects Ag
+The fluorescent emission collection of illustrative plates, wherein, curve a to f representes the Ag in the solution
+Be respectively 0,5,10,15,20,50nM, excite light wavelength to select 380 nanometers.
Fig. 5 embodiment of the invention 2 detects Hg
2+The fluorescent emission collection of illustrative plates, wherein, curve a to f representes the Hg in the solution
2+Be respectively 0,5,10,15,20,50nM, excite light wavelength to select 380 nanometers.
Fig. 6 embodiment of the invention 3 detects Ag
+And Hg
2+The fluorescent emission collection of illustrative plates, wherein, excite light wavelength to select 380 nanometers.
Embodiment
(1) preparation of the CdTe quantum dot of mercaptopropionic acid MPA parcel
Sodium borohydride NaBH with 0.189g (5mmol)
4Be dissolved in the vial that contains the 2.0mL ultrapure water, add 0.1528g (1.175mmol) tellurium Te powder, on bottle cap, stay an aperture, place 0 ℃ of ice-water bath to react reaction vessel again.Behind the 12h, the Te powder of black disappears, the NaHTe solution for standby that obtains clarifying.Take by weighing 0.57g (2.5mmol) CdCl
22.5H
2O is dissolved in the 150mL water, the MPA of Dropwise 5 00 μ L (5.74mmol) under violent stirring, and using the NaOH solution adjusting pH of 0.1mol/L is 11.20, logical N
2Add the above-mentioned NaHTe solution for preparing rapidly behind deoxygenation, the protection 30min, promptly obtain the precursor solution of CdTe quantum dot after the reaction.Get the 40mL precursor solution and be added to teflon and do in the stainless steel cauldron of liner, 160 ℃ of reactions 20 minutes down promptly obtain needed CdTe quantum dot.
(2) design of the nucleic acid probe of detection usefulness is with synthetic
Select the acceptor of 2 kinds of dyestuffs as energy, these 2 kinds of dyestuffs are modified at the 5' end of corresponding nucleic probe respectively; The terminal modified amino of the 5' of another nucleic acid probe is to join in order to have the quantum dot of carboxyl to carry out the covalency coupling with finishing; In addition, also have one to be the nucleic acid probe of not doing modification, as the bridge that connects energy donor quantum dot and energy acceptor dyestuff.All nucleic acid probes are given birth to worker's bioengineering company limited by Shanghai and are synthesized, and its concrete sequence is following:
The single stranded nucleic acid probe DNA1:5'-NH of amido modified mistake
2-GTACAAGATG-3';
Do not make the single stranded nucleic acid probe DNA2:5'-GAGCTTTTCA GACGCATCTT GTACGACTCG CTCCCCATAC-3' of any modification;
The single stranded nucleic acid probe TAMRA-DNA:5'-TAMRA-TTTTGCTC-3' of fluorescent dye TAMRA modified;
The single stranded nucleic acid probe Cy5-DNA:5'-Cy5-GTATCCCC-3' of indoles cyanine dyes Cy5 modified;
(3) fluorescence probe (CdTe-DNA) of preparation quantum dot and nucleic acid
At first be the coupling matter of preparation CdTe and DNA1: 1-ethyl-3-of 320 μ L 0.1mM (3-dimethylamino-propyl)-carbodiimides (EDC); The N-hydroxy-succinamide (NHS) of 200 μ L, 0.0125 mM is admixed together with 1280 μ L, 1 μ M CdTe solution, activation 0.5-1 h.The DNA1 WS that adds 100 μ L, 10 μ M then reacted 4 hours down at 25 ℃.Carry out ultrafiltration 15min with ultra filtration membrane with 5000rpm then, and resuspended with ultrapure water, ultrafiltration once more so circulates 3 times, thoroughly removes not the DNA1 that joins with the CdTe coupling.So promptly prepare CdTe-DNA1.
Next, be 900 μ L concentration that CdTe-DNA1 and 100 μ L, the 20 μ M DNA2 of 1 μ M at room temperature carry out hybridization reaction 6h.Carry out same ultrafiltration step and remove the not DNA2 of hybridization.So promptly prepare CdTe-DNA1-DNA2.
At last, be added to the WS of 100 μ L, 20 μ M Cy5-DNA and 100 μ L, 20 μ M TAMRA-DNA respectively among the CdTe-DNA1-DNA2 of above-mentioned preparation.So promptly prepare the CdTe-DNA fluorescence probe that detects usefulness.
(4) Ag
+Detection
Because the bonding force between silver ion and 3-(N-morpholine) propane sulfonic acid (MOPS) very a little less than, so select the MOPS damping fluid as detection architecture: the CdTe-DNA fluorescence probe of 100nM step () preparation, 10mM sodium dihydrogen phosphate/sodium hydrogen phosphate NaH
2PO
4/ Na
2HPO
4, the MOPS of 20mM, the sodium nitrate NaNO of 50mM
3
The WS that contains variable concentrations (0 to 50 nM) silver ion joins in the above-mentioned detection architecture, and under 25 ℃, keeps 0.5h, and Cy5-DNA and DNA2 hybridization reaction that silver ion is induced carry out fully.Measure the fluorescent emission collection of illustrative plates of whole system then.
(1) preparation of the CdTe quantum dot of mercaptopropionic acid MPA parcel.
Method such as instance 1 are said.
(2) design of the nucleic acid probe of detection usefulness is with synthetic
Method such as instance 1 are said.
(3) fluorescence probe (CdTe-DNA) of preparation quantum dot and nucleic acid
Method such as instance 1 are said.
(4) Hg
2+Detection
Detection architecture such as instance 1 are said, contain variable concentrations (0 to 50 nM) Hg
2+The WS of ion joins in the above-mentioned detection architecture, and under 25 ℃, keeps 0.5h, makes Hg
2+TAMRA-DNA that induces and DNA2 hybridization reaction carry out fully.Measure the fluorescent emission collection of illustrative plates of whole system then.
(1) preparation of the CdTe quantum dot of mercaptopropionic acid MPA parcel.
Method such as instance 1 are said.
(2) design of the nucleic acid probe of detection usefulness is with synthetic
Method such as instance 1 are said.
(3) fluorescence probe (CdTe-DNA) of preparation quantum dot and nucleic acid
Method such as instance 1 are said.
(4) carry out Ag simultaneously
+And Hg
2+Detection
Detection architecture such as instance 1 are said, contain variable concentrations (0 to 50 nM) Ag
+And Hg
2+The WS join in the above-mentioned detection architecture, and under 25 ℃, keep 0.5h, make Hg
2+The TAMRA-DNA that induces and DNA2 hybridization reaction and Ag
+Cy5-DNA that induces and DNA2 hybridization reaction carry out fully.Measure the fluorescent emission collection of illustrative plates of whole system then.
Claims (2)
1. one kind is come to detect simultaneously the method for dimercurion and silver ion in the WS based on FRET, it is characterized in that:
(1) preparation of detector probe
(1) preparation of the cadmium telluride CdTe quantum dot of mercaptopropionic acid MPA parcel
With sodium borohydride NaBH
4, ultrapure water and tellurium Te powder be mixed together in the reaction vessel, makes NaBH
4Concentration be 2-3M, the Te powder content is 0.5-0.6M, again reaction vessel is placed the NaHTe solution for standby of 0 ℃ of ice-water bath reaction 12h with the preparation clarification; Take by weighing a certain amount of inorganic cadmium salt Cd
2+Be dissolved in the water control Cd
2+Concentration is 0.01-0.02M, under violent stirring, drips an amount of MPA, and using NaOH solution to regulate pH is 11.20, logical N
2Add NaHTe solution rapidly behind deoxygenation, the protection 30min, keep Cd
2+: Te
2-: the mol ratio of MPA is 1:0.4-0.5:2.2-2.5; Promptly obtain the precursor solution of CdTe quantum dot after the reaction; Get precursor solution and be added to teflon and do in the stainless steel cauldron of liner, 160 ℃ of reaction 20min down promptly obtain needed CdTe quantum dot;
(2) design of the nucleic acid probe of detection usefulness is with synthetic
The single stranded nucleic acid probe DNA1:5'-NH of amido modified mistake
2-GTACAAGATG-3';
Do not make the single stranded nucleic acid probe DNA2:5'-GAGCTTTTCA GACGCATCTT GTACGACTCG CTCCCCATAC-3' of any modification;
The single stranded nucleic acid probe TAMRA-DNA:5'-TAMRA-TTTTGCTC-3' of fluorescent dye TAMRA modified;
The single stranded nucleic acid probe Cy5-DNA:5'-Cy5-GTATCCCC-3' of indoles cyanine dyes Cy5 modified;
(3) preparation of the fluorescence probe CdTe-DNA of quantum dot and nucleic acid
The coupling matter that at first prepares CdTe and DNA1: 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimides EDC; N-hydroxy-succinamide NHS and CdTe solution are admixed together by proper proportion; Activation 0.5-1 h adds the DNA1 WS then, reacts 4h down at 25 ℃; Carry out ultrafiltration with the DNA1 that thorough removal does not join with the CdTe coupling, promptly prepare CdTe-DNA1; Then, at room temperature carry out hybridization reaction 6h to CdTe-DNA1 and DNA2, ultrafiltration promptly prepares probe CdTe-DNA1-DNA2 of quantum dot and DNA to remove the not DNA2 of hybridization; At last, be added to the WS of Cy5-DNA and TAMRA-DNA respectively among the CdTe-DNA1-DNA2 of above-mentioned preparation, promptly prepare the CdTe-DNA fluorescence probe that detects usefulness;
(2) heavy metal ion Ag
+And/or Hg
2+Detection
(1) Ag
+Detection
Bonding force between silver ion and 3-(N-morpholine) the propane sulfonic acid MOPS very a little less than, so select the MOPS damping fluid as detection architecture: the CdTe-DNA fluorescence probe of preparation in the 100nM step (), 10mM sodium dihydrogen phosphate/sodium hydrogen phosphate NaH
2PO
4/ Na
2HPO
4, the MOPS of 20mM, the sodium nitrate NaNO of 50mM
3
The WS that will contain 0-50 nM variable concentrations silver ion joins in the above-mentioned detection architecture, and under 25 ℃, keeps 0.5h, makes Ag
+Cy5-DNA that induces and DNA2 hybridization reaction carry out fully, record the fluorescent emission collection of illustrative plates of whole system then;
(2) Hg
2+Detection
Detection method and step (1) are similar, just with Ag in the step (1)
+Solion changes Hg into
2+Solution; Contain 0-50 nM variable concentrations Hg
2+The WS of ion joins in the above-mentioned detection architecture, and under 25 ℃, keeps 0.5h, makes Hg
2+TAMRA-DNA that induces and DNA2 hybridization reaction carry out fully, measure the fluorescent emission collection of illustrative plates of whole system then;
(3) carry out Ag simultaneously
+And Hg
2+Detection
Detection method and step (1) are similar, just with Ag in the step (1)
+Solion changes Ag into
+And Hg
2+Mixed solution; Contain 0-50 nM variable concentrations Ag
+And Hg
2+The WS join in the above-mentioned detection architecture, and under 25 ℃, keep 0.5h, make Hg
2+The TAMRA-DNA that induces and DNA2 hybridization reaction and Ag
+Cy5-DNA that induces and DNA2 hybridization reaction carry out fully, measure the fluorescent emission collection of illustrative plates of whole system then.
2. a kind of method of coming to detect simultaneously dimercurion and silver ion in the WS based on FRET according to claim 1; It is characterized in that: the preparation of the fluorescence probe CdTe-DNA of quantum dot and nucleic acid: the coupling matter that at first is preparation CdTe and DNA1: 1-ethyl-3-of 320 μ L 0.1mM (3-dimethylamino-propyl)-carbodiimides EDC; The N-hydroxy-succinamide NHS of 200 μ L, 0.0125 mM and 1280 μ L, 1 μ M CdTe solution are admixed together, activation 0.5-1 h; The DNA1 WS that adds 100 μ L, 10 μ M then reacted 4 hours down at 25 ℃; Carry out ultrafiltration 15min with ultra filtration membrane with 5000rpm then, and resuspended with ultrapure water, ultrafiltration once more so circulates 3 times, thoroughly removes not the DNA1 that joins with the CdTe coupling, promptly prepares CdTe-DNA1;
Then, at room temperature carry out hybridization reaction 6h to the CdTe-DNA1 of 900 μ L, 1 μ M and 100 μ L, 20 μ M DNA2, carry out same ultrafiltration step and remove the not DNA2 of hybridization, promptly prepare CdTe-DNA1-DNA2;
At last, be added to the WS of 100 μ L, 20 μ M Cy5-DNA and 100 μ L, 20 μ M TAMRA-DNA respectively among the CdTe-DNA1-DNA2 of above-mentioned preparation, promptly prepare the CdTe-DNA fluorescence probe that detects usefulness.
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