CN102200510A - Mercuric ion concentration fluorescence detection method based on T-T mismatched DNA probe - Google Patents
Mercuric ion concentration fluorescence detection method based on T-T mismatched DNA probe Download PDFInfo
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Abstract
The invention relates to the field of mercuric ion concentration detection and analysis and particularly discloses a mercuric ion concentration fluorescence detection method based on a T-T mismatched DNA probe. The method is characterized in that a prober containing T-T mismatched DNA is used as a mercuric ion identification component, a DNA double-strand intercalator SYBRGREENI is used as a fluorescence signal molecule, and then mercuric ion concentration in a sample to be detected is subjected to fluorescence detection on the basis of the difference of fluorescence signal intensity during the single-strand and double-strand binding between the SYBRGREENI and the prober containing the T-T mismatched DNA. The method disclosed by the invention has the advantages of no environmental pollution, good selectivity to mercuric ion, capability of eliminating interference from most of metal ions, low cost, high sensitivity, high detection speed and the like.
Description
Technical field
The present invention relates to the check and analysis field of ion concentration of mercury, relate to a kind of method of dna probe fluorometric assay ion concentration of mercury of the T-T of utilization mispairing particularly.
Background technology
Mercury is a kind of severe toxicity, non-biodegradable heavy metal ion.After the passive plant absorbing of mercury in the environment,, enter inside of human body at last by the inrichment of biology and the transmission of food chain, after entering blood, to combine with plasma proteins or erythrocyte, mainly be distributed to brain and kidney later on, secondly locate for liver, intestines wall, the heart, lung etc.The harm outstanding behaviours of mercury is on its neurotoxicity, and is also influential to respiratory tract, alimentary canal.When mercury content at the human body cylinder accumulation to a certain degree the time, can cause the morbidity of poisoning, severe patient even death.(the Codex Alimentarius Commission of the international food code council, CAC) stipulated in the mineral water with edible salt in the limit standard of mercury be respectively 0.001mg/kg and 0.1mg/kg(CODEX STAN 193-1995, Rev. 2-2006), European Union's limit standard that some fish and products thereof are formulated mercury is 1mg/kg and 0.5mg/kg(COMMISSION REGULATION (EC) No 1881/2006 of 19 December 2006).China has also formulated corresponding food hygienic standard, wherein the minimum 0.01mg/kg (hygienic standard that mercury is limited the quantity of in GB 2762-2005. food) that is that limits the quantity of of mercury in the food.At present, the instrumental method of detection total mercury comprises (Hight, et al. Food Chem. 2005,91,557 such as cold atomic absorption spectrometry, Cold Atomic Fluorescent Mercury spectroscopic methodology and ICP-MS; Roulet, et al. Sci.Total Environ. 2000,26,143; Wuillouda, et al. Spectrochim. Acta Part B 2004,59,755.).But these methods need valuable instrument, and the sample preparation of complicated and time consumption is not suitable for field quick detection.
Because the structure of DNA and performance are very sensitive to the influence of heavy metal, thereby utilize the interaction development high selectivity between DNA and the metallic ion, highly sensitive heavy metal analyzing detecting method that very big value and meaning are arranged.Gao etc. proved the guanine among the DNA can and Cu
2+Specificity is in conjunction with forming stable structure, and (Y.G. Gao, M. Sriam, A.H.J. Wang, Nucleic Acid Res. 21 (1993) 4093 – 4191) also are used for Cu
2+Detection in.Ono group reported first among the DNA base-pair C-C can and Ag
+Specificity is in conjunction with forming stable C-Ag
+-C-structure (Miyake, Y.; Togashi, H.; Tashiro, M.; Yamaguchi, H.; Oda, S.; Kudo, M.; Tanaka, Y.; Kondo, Y.; Sawa, R.; Fujimoto, T.; Machinami, T.; Ono, A. J. Am. Chem. Soc. 2006,128,2172 – 2173.).Dong group is based on C-Ag
+-C-structure has designed detection Ag
+Colorimetric bio sensor (T. Li, L. Shi, E. Wang and S. Dong, Chem. – Eur. J., 2009,15,3347 – 3350.).Utilize biocompatibility and water-soluble all good dna molecular heavy-metal pollution to be detected the concern that has been subjected to domestic and international expert.
Ono group also reported first T-T base-pair energy and the Hg in the dna sequence dna
2+Specificity takes place in conjunction with forming stable T-Hg
2+-T structure.Subsequently, reported both at home and abroad much based on T-Hg
2+The fluorescent optical sensor of-T structure determination mercury ion and colorimetric method (Z. D. Wang, J. H. Lee, Y. Lu, Chem. Commun., 2008,6005 – 6007; C. K. Chiang, C. C. Huang, C. W. Liu, H. T. Chang. Analytical Chemistry, Vol. 80, and No. 10, and May 15,2008; J. S. Lee, M. S. Han, and C. A. Mirkin. Angew. Chem. Int. Ed. 2007,46,4093 – 4096.), these methods all have selectivity and higher sensitivity preferably.But the dna probe that uses in the method all is by making at DNA chain two ends mark fluorescent dye molecule, this labeling process complicated operation, and gained dna probe cost is higher.
The present invention is adopting on the basis of fluorescent dye as signaling molecule that combines with double-stranded specific, overcome the shortcoming in the above-mentioned art methods, this operation of DNA fluorescence labeling comparatively complexity and the high step of cost have been removed in the art methods from, propose a kind of non-marking, utilized the dna probe of T-T mispairing to measure the method for ion concentration of mercury.The inventive method measure ion concentration of mercury have with low cost, simply, advantage and kept specificity good and highly sensitive fast.
Summary of the invention
The present invention proposes a kind of method of dna probe fluorometric assay ion concentration of mercury of the T-T of utilization mispairing, it is characterized in that, described method is with the dna probe that contains the T-T mispairing recognition component as mercury ion, with dna double chain intercalator SYBR GREEN I is the fluorescence signal molecule, the difference of fluorescence signal intensity is carried out fluoroscopic examination to the ion concentration of mercury in the testing sample when utilizing described SYBR GREEN I to combine with the dna probe list two strands of the described T-T of containing mispairing;
Wherein, shown in the following chemical formula of structural formula (I) of described SYBR GREEN I:
(I)
Wherein, the sequence of the dna probe of the described T-T of containing mispairing is:
Dna probe 1:5 '-CTCTTTCTC-3 ';
Dna probe 2:5 '-GTGTTTGTG-3 '.
The present invention utilizes in the method for dna probe fluorometric assay ion concentration of mercury of T-T mispairing, adds HEPES damping fluid (4-hydroxyethyl piperazine ethanesulfonic acid damping fluid) in the described dna probe that contains the T-T mispairing, and uses NaNO
3Solution is regulated ionic strength, makes dna probe solution; The gained probe solution is adjusted to pH neutrality with NaOH solution.
The present invention utilizes in the method for dna probe fluorometric assay ion concentration of mercury of T-T mispairing, and described testing sample filters before detection, and regulates acidity to neutral with NaOH solution.
The present invention utilizes in the method for dna probe fluorometric assay ion concentration of mercury of T-T mispairing, and described fluoroscopic examination is 479nm in excitation wavelength, and emission wavelength 524nm carries out at the place.
The present invention utilizes in the method for dna probe fluorometric assay ion concentration of mercury of T-T mispairing, and detecting of described ion concentration of mercury is limited to 3n mol/L.
The present invention utilizes that employed ssDNA probe 1,2 contains 5 thymine-thymine(T-T, thymine-thymine in the method for dna probe fluorometric assay ion concentration of mercury of T-T mispairing) mismatch.When mercury ion and the coexistence of described probe solution, the T-T mispairing and the mercury ion of dna probe 1,2 form stable thymine-Hg
2+-thymine complex, two dna probes are combined into double-stranded DNA simultaneously, form and structure like the natural dna double Corkscrews.Employed fluorescence signal molecule SYBR GREEN I only sends very low fluorescence signal when combining with ssDNA probe, and after single-stranded probe DNA was combined into two strands, SYBR GREEN I embedded double-stranded, discharges strong fluorescence signal.The concentration of the mercury ion in fluorescence intensity and the solution is relevant, by setting up the typical curve of fluorescence signal molecule intensity and ion concentration of mercury linear relationship, and detects the fluorescence signal intensity of testing sample, can calculate the ion concentration of mercury in the testing sample.
" dna probe that contains the T-T mispairing " is meant between two ssDNA probe sequences because of existing the T-T mispairing can't form the situation of dna double chain in the inventive method.In dna probe, the T-T mispairing can occur in the one or more arbitrarily site/sections in the probe sequence.Under the situation that mercury ion exists, thereby T base and mercury ion generation coordination reaction form stable T-Hg in the dna probe
2+-T complex, thus the dna double chain obtained, be similar to the dna double chain of " zip mode ".
The present invention utilizes the dna probe that designs in the method for dna probe fluorometric assay ion concentration of mercury of T-T mispairing, article two, a pair of complete complementary base is contained at the two ends of short chain DNA respectively, 5 T-T base mismatch are contained in the centre, when mercury ion occurs, guarantee the formation of dna double chain.Simultaneously, specially designed dna probe length is shorter in the inventive method, is 9bp only, and it is low also to have cost when stating purpose in realization, the advantage that background is little.
Testing sample is carried out in the mensuration of ion concentration of mercury in the inventive method, the testing sample solution that will contain mercury ion is added drop-wise in the HEPES damping fluid of dna probe of above-mentioned preparation, shakes up; Add a certain amount of SYBR GREEN I then, in the interscan of 510～650nm wavelength coverage, the fluorescence intensity at record 524nm place is calculated ion concentration of mercury in the solution to be measured according to this fluorescence intensity with fluorospectrophotometer.
The present invention utilizes the method for the dna probe fluorometric assay ion concentration of mercury of T-T mispairing, the mensuration of ion concentration of mercury is limited to 3n mol/L, be 0.0006mg/kg, can satisfy the requirement of limiting the quantity of international and domestic relevant criterion fully, and have the following advantages: the inventive method adopts eco-friendly DNA as detector probe, specificity is good, and can not cause second environmental pollution; Adopt the fluorometric assay ion concentration of mercury, highly sensitive, selectivity good; Simultaneously, because the dna probe in the detection method of the present invention has been removed in the art methods this operation of fluorescence labeling comparatively complexity and the high step of cost from, SYBR GREEN I is inserted in the dna double chain ditch automatically by the hydrogen bond action with base in the detection method of the present invention, so have advantage easy and simple to handle, with low cost.
Description of drawings
Fig. 1 is the principle of work synoptic diagram of the inventive method.
Fig. 2 adds 100 nM Hg in the inventive method
2+The comparison synoptic diagram of the fluorescence spectrum figure that front and back solution records.
Fig. 3 adds the fluorescence spectrum figure that mercury ion standard solution (from top to bottom, concentration is respectively 5n mol/L, 15n mol/L, 20n mol/L, 40n mol/L, 60n mol/L, 80n mol/L, 100n mol/L) back obtains in dna probe solution in the inventive method.
Fig. 4 is the canonical plotting of 524nm place fluorescence intensity and ion concentration of mercury in the inventive method.
Embodiment
In conjunction with following specific embodiments and the drawings, the present invention is described in further detail, and protection content of the present invention is not limited to following examples.Under the spirit and scope that do not deviate from inventive concept, variation and advantage that those skilled in the art can expect all are included among the present invention, and are protection domain with the appended claims.
As shown in Figure 1, at 10mmol/LHEPES(pH7.4,100mM NaNO
3) in the buffer solution, when not having mercury ion to exist, because of the existence of 5 the T-T mispairing form with strand exists, SYBR GREEN I sends very low fluorescence signal when combining with single stranded DNA between two dna probes; When having mercury ion in the solution, T base and mercury ion generation coordination reaction form stable T-Hg among the DNA
2+-T complex makes two short chain dna probes form the structure that is as good as with natural dna double spiral simultaneously, in the SYBR GREEN I intercalation of DNA two strands of Jia Ruing, discharges strong fluorescence signal then.The fluorescence intensity of SYBR GREEN I and the concentration of the mercury ion in the solution are linear dependence, and the fluorescence signal intensity that can record by the typical curve and the testing sample of foundation calculates the ion concentration of mercury in the testing sample thus.
Embodiment 1:
The present invention measures the method for mercury ion to contain the dna probe of T-T mispairing, and selectivity is good, sensitive, easy, the ion concentration of mercury in the rapid fluorescence working sample.Utilization of the present invention contains the method for ion concentration of mercury in the dna probe working sample of T-T mispairing, with the dna probe that contains the T-T mispairing recognition component as mercury ion, with fluorescent dye SYBR GREEN I is the fluorescence signal molecule, the difference of fluorescence signal intensity detects ion concentration of mercury in the testing sample when utilizing described fluorescent dye to combine with the dna probe list two strands of the described T-T of containing mispairing.
The present invention utilizes the method for the dna probe fluorometric assay ion concentration of mercury of T-T mispairing, specifically may further comprise the steps:
(1) foundation of typical curve: in the fragrant doffer's pipe of the dust of 7 2.0mL scales, (ultimate density all is 6.6 * 10 to add two short chain dna probes respectively
-8Mol/L).It is mixed with 1.5 mL 10mM HEPES damping fluids (4-hydroxyethyl piperazine ethanesulfonic acid damping fluid), use 100mM NaNO
3Solution is regulated ionic strength, and regulates the HEPES damping fluid to pH7.4 with 0.1mol/L NaOH solution, makes probe solution.Prepare a series of mercury ion standard solution (concentration is 5nmol/L, 15nmol/L, 20nmol/L, 40nmol/L, 60nmol/L, 80nmol/L, 100nmol/L), add probe solution and cultivate 2min down at 20～25 ℃;
Wherein the sequence of dna probe is as follows:
Dna probe 1:5 '-CTCTTTCTC-3 ',
Dna probe 2:5 '-GTGTTTGTG-3 ';
Dna probe is synthetic by Shanghai biotechnology company limited, and uses the HPLC purifying.
(2) do not add mercury ion with step (1) method and do blank solution;
(3) get an amount of above-mentioned solution respectively, place quartz colorimetric utensil, in above-mentioned solution, add 5 μ L SYBR GREEN I then, use fluorospectrophotometer behind the 3min, slit 5nm, excitation wavelength 479nm in the interscan of 510～650nm wavelength coverage, writes down the fluorescence intensity (I at 524nm place respectively
524nm), and record blank value (I
b).Calculate Δ I
524nm(Δ I
524nm=I
524nm-I
b);
Wherein, SYBR GREEN I(10000 *) available from handsome company (invitrogen inc).At first use dimethyl sulfoxide (DMSO) (DMSO) with 10000 * SYBR GREEN I be diluted to 250 *, then water further it is diluted to 100 * SYBR GREEN I(experiment with).The amount of substance concentration about 2.03 * 10 of list of references [H. Zipper, H. Brunner, J. Bernhagen and F. Vitzthum, Nucleic Acids Research, 2004,32, e103.] 100 * SYBR GREEN I
-6Mol/L.Lucifuge, 4
0C preserves.
(4) with the variable concentrations mercury ion to Δ I
524nmMapping, the drawing standard curve;
(5) pre-service of testing sample:, filter the back and use 1mol/L NaOH to regulate acidity to neutral for fluid sample; For solid sample, use nitric acid-hydrogen peroxide (7mL+1.5mL) micro-wave digestion, catch up with acid and filtration back to use 1mol/LNaOH to regulate acidity to neutrality.Get a certain amount of gained sample solution and replace the mercury ion standard solution, Δ I is asked in (1) set by step～(3) operation
Sample
(6) the Δ I that records according to testing sample
Sample, look into typical curve, try to achieve the mercury content of testing sample.
The fluoroscopic examination of testing sample is at 10mM HEPES(pH7.4,100mM NaNO
3) damping fluid carries out the fluoroscopic examination of mercury ion.
The mensuration of ion concentration of mercury is limited to 3n mol/L in the inventive method, and promptly 0.0006mg/kg can satisfy the requirement of limiting the quantity of international and domestic relevant criterion fully.
Embodiment 2: soluble mercury ion determination in the toy sample
Normative reference: EN71-3:1994+A1:2001
Situation when present embodiment is depicted as testing sample and is toy is all tested all the touched parts on the toy sample.
One, sample preparation
Toy for types such as weaving face fabric, plastics or rubber is cut into the fritter of 5mm * 5mm as testing sample with scissors or pliers with material.
For toy, will scrape with clean blade and to get part as testing sample with paint, printing ink or similar coatings.
For the toy that is liquid paint or printing ink, get part and be applied on the clean glass plate after the air dry at room temperature, scrape as testing sample.Can not be at room temperature dry as testing sample, then press serviceability temperature in drying in oven, scrape the acquisition testing sample.
All powdery testing samples must use the aperture to sieve as the stainless steel metal of 0.5mm and sieve.
For the toy that contains grease, oils, wax or similar material, sample is wrapped in the toughened filter paper, use the normal heptane will contained grease by dissolution extraction and after analogous components removes, the acquisition testing sample.
Two, the pre-service before the acid extractants of testing sample and the sample detection
With load weighted testing sample be equivalent to 50 times of testing sample quality, temperature is 37 ± 2 ℃, concentration is the combined of 0.07 mol/L.Potpourri is shaken 1min, and check its acidity.If the pH value is greater than 1.5, shake potpourri, and meanwhile dropwise add 2 mol/L hydrochloric acid up to the pH value between 1.0 ~ 1.5.Make the potpourri lucifuge, stir 1h down, under 37 ± 2 ℃, leave standstill 1h again at 37 ± 2 ℃.
Use filter membrane (aperture is 0.45 μ m) to filter, gained filtrate neutralizes with 1 mol/L NaOH, measures the fluorescence signal intensity of testing sample in the HEPES damping fluid.
Three, measure
At the 10mmol/L HEPES(pH7.4 that contains two DNA short chain probes, 100mmol/L NaNO
3) add the testing sample of 20 μ L, reaction 2min in the buffer solution; Add 5 μ L, 100 * SYBR GREEN I again, detect in the scanning of 510-650nm scope with fluorospectrophotometer behind the 3min.After adding testing sample, solution is 104nm in the fluorescence intensity at 524nm place, and the content that the typical curve from Fig. 4 can draw mercury ion in the toy sample to be measured is 40n mol/L.
Embodiment 3: the mensuration of mercury ion in the waste water sample
One, the pre-service before the sample detection
Wastewater sample uses quick filter paper filtering, checks gained filtrate acidity, greater than 7, then uses 1 molL as the pH value
-1Fluorometric assay is carried out in the NaOH neutralization in the HEPES damping fluid.
Two, measure
At the 10mmol/L HEPES(pH7.4 that contains two DNA short chain probes, 100mmol/L NaNO
3) add the testing sample of 20 μ L, reaction 2min in the buffer solution; Add 5 μ L, 100 * SYBR GREEN I again, detect in the scanning of 510-650nm scope with fluorospectrophotometer behind the 3min.After adding testing sample, solution is 204 in the fluorescence intensity at 524nm place, and the content that the typical curve from Fig. 4 can draw mercury ion in the wastewater sample to be measured is 80 n mol/L.
Embodiment 4: total mercury content is measured in the rice sample
One, the pre-service before the sample detection
Rice sample is pulverized, taken by weighing 0.5g, be accurate to 0.1mg to counteracting tank, add 7mLHNO
3And 1.5mLH
2O
2With the counteracting tank sealing, place microwave digestion system to clear up.Heating schedule (reference instrument, Anton Paar Microwave 3000) as shown in table 1 below.
Table 1. micro-wave digestion heating schedule
The gained digestion solution catches up with acid to the solution 1mL in low-grade fever on the electric hot plate, adds ultrapure water then, is settled to about 50mL.Measure solution acidity, greater than 7, then use 1 mol/LNaOH neutralization, in the HEPES damping fluid, carry out fluorometric assay as the pH value.
Two, measure
At the 10mmol/L HEPES(pH7.4 that contains two DNA short chain probes, 100mM NaNO
3) add the testing sample of 20 μ L, reaction 2min in the buffer solution; Add 5 μ L, 100 * SYBR GREEN I again, detect in the scanning of 510-650nm scope with fluorospectrophotometer behind the 3min.After adding testing sample, solution is 30 in the fluorescence intensity at 524nm place, and the content that the typical curve from Fig. 4 can draw mercury ion in the rice sample to be measured is 3.2n mol/L.
Claims (5)
1. method of utilizing the dna probe fluorometric assay ion concentration of mercury of T-T mispairing, it is characterized in that, described method is with the dna probe that contains the T-T mispairing recognition component as mercury ion, with dna double chain intercalator SYBR GREEN I is the fluorescence signal molecule, the difference of fluorescence signal intensity is carried out fluoroscopic examination to the ion concentration of mercury in the testing sample when utilizing described SYBR GREEN I to combine with the dna probe list two strands of the described T-T of containing mispairing;
Wherein, shown in the following chemical formula of structural formula (I) of described SYBR GREEN I:
(I)
Wherein, the sequence of the dna probe of the described T-T of containing mispairing is:
Dna probe 1:5 '-CTCTTTCTC-3 ';
Dna probe 2:5 '-GTGTTTGTG-3 '.
2. the method for claim 1 is characterized in that, adds the HEPES damping fluid in the described dna probe that contains the T-T mispairing, and uses NaNO
3Solution is regulated ionic strength, makes dna probe solution; The gained probe solution is adjusted to pH neutrality with NaOH solution.
3. the method for claim 1 is characterized in that, described testing sample filters before detection, and regulates acidity to neutral with NaOH solution.
4. the method for claim 1 is characterized in that, described fluoroscopic examination is 479nm in excitation wavelength, and emission wavelength 524nm carries out at the place.
5. the method for claim 1 is characterized in that, detecting of described ion concentration of mercury is limited to 3n mol/L.
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