CN102183496B - Fluorescence detection method of potassium ion - Google Patents
Fluorescence detection method of potassium ion Download PDFInfo
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- CN102183496B CN102183496B CN201110034763A CN201110034763A CN102183496B CN 102183496 B CN102183496 B CN 102183496B CN 201110034763 A CN201110034763 A CN 201110034763A CN 201110034763 A CN201110034763 A CN 201110034763A CN 102183496 B CN102183496 B CN 102183496B
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- potassium ion
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Abstract
The invention discloses a fluorescence detection method of a potassium ion. The specific DNA (5'-GGGTTAGGGTTAGGG TTAGGG-3') of the potassium ion is in an irregular curling state when no potassium ion exists, fluorescent dye thiazole orange and a G-rich sequence are acted to generate very strong fluorescence, after the potassium ion is added, the specific DNA of the potassium ion forms a G tetrahedral structure, and the fluorescence is decreased. The fluorescence detection method disclosed by the invention not only has high sensitivity and better specificity, but also is simple and rapid, and the whole process can be completed in five minutes.
Description
Technical field
The present invention relates to the detection range of potassium ion, particularly a kind of fluorescence detection method of potassium ion.
Background technology
Potassium is indispensable macroelement in the human body, and its effect mainly is a normal function of keeping nerve, muscle.K
+Can participate in such as bioprocess such as nerve impulse transmission and cellular activity adjustings; The content of potassium ion is human physiological activity's important indicator; In blood and urine, exist level to clinical diagnosis particularly important [KOFUJI P, NEWMAN E A.Potassium buffering in the central nervous system [J] .Neuroscience, 2004; 129:1045-1056.], so the mensuration of potassium content has great importance.At present, the method that potassium ion detects is a lot, like [Ji Guoliang such as flame photometry, ISE method, atomic absorption spectrography (AAS), potentiometric titrations; Kong Xiaoling. the application [J] in the mensuration of potassium electrode quick-acting potassium in soil. chemical sensor, 1997,17 (1): 53-58. Liu Dong; Open the lady in the moon, Yang Shikui. the method research [J] of Trace Sodium and Potassium ion in the hard calcium acetate of aas determination. Southwest Nationalities College's journal: natural science edition, 2002; 28 (2): 234-235. leaf milk justice. the rapid assay methods of potassium ion [J] in the polyether glycol. the Fujian chemical industry, 2005, (5): 51-54. Dong Li is beautiful. the potassium content [J] in the automatic potentiometric titration fast measuring sylvite. assay office; 2004,23 (1): 83-84.], but the most complicated operation of these methods; Ion interference is big, and influence factor is more, and accuracy is good inadequately.
Aptamer (Aptamer) is one type and through what SELEX technology in-vitro screening went out target molecule (Target) is had the one type of DNA or the RNA molecule of high affinity, has affinity height (K
dValue is at nM-pM), good stability, target molecule scope be wide, be easy to advantages such as synthetic and modification, can satisfy in the testing process requirement to aspects such as sensitivity, specificity, operability, costs.The detection method based on aptamer that developed recently gets up has shown good prospects for application in fields such as biology, environment, safety.At present; Detect existing bibliographical information [the UEYAMA H of method of potassium ion based on the aptamer conformation change; TAKAGI M, TAKENAKA S.A novel potassium sensing in aqueous media with a synthetic oligonucleotide derivative.Fluorescence resonance energy transfer associated with guanine quartet-potassium ion complex formation [J] .J Am Chem Soc, 2002; 124 (48): 14286-14287.HO H A; LECLERC M.Optical sensors based on hybrid aptamer/conjugated polymer complexes [J] .J Am Chem Soc, 2004,126 (5): 1384-1387.]; But still exist the not high shortcoming of sensitivity and selectivity; The need that have carry out the dna double mark, and it is high to detect cost, has limited its practical application.[CHOI M S such as nearest Choi; YOON M; BAEG J O; KIM J.Label-free dual assay of DNA sequences and potassium ions using an aptamer probe and a molecular light switch complex [J] .Chem Commun; 2009:7419-7421.] utilize ruthenium complex and aptamer to realize the double check of dna sequence dna and potassium ion, but method needs under 70 ℃ of conditions, to react and need in ice-water bath, cool off complex operation.
Summary of the invention
The objective of the invention is and to address the deficiencies of the prior art, a kind of high sensitivity and specificity are provided, potassium ion fluorescence detection method simple to operate.
For realizing above-mentioned purpose, technical scheme of the present invention is following:
(1) in cuvette, add buffer solution, with potassium ion specific DNA, thiazole orange and metal cation hybrid reaction to be measured,
(2) measure fluorescence intensity.
The used buffer solution of the present invention is 10-50mM Tris-HCl (pH 7.0-7.5), and (pH 7. to be preferably 50mM Tris-HCl.4)。
Said potassium ion specific DNA sequences is 5 '-GGGTTAGGGTTAGGGTTAGGG-3 ' among the present invention.When having potassium ion in the system, can form the G tetrahedral structure.Its concentration is 150nM.
Said metal cation to be measured is a potassium ion among the present invention, lithium ion, calcium ion, magnesium ion, ammonium radical ion, sodion.
Said metal cation concentration to be measured is 0-10mM among the present invention.
The said thiazole orange concentration of the present invention is 100nM.
Said potassium ion specific DNA among the present invention, thiazole orange and metal cation to be measured were 25 ℃ of-30 ℃ of hybrid reactions 4 minutes.
Positive progressive effect of the present invention is:
1, the present invention only needed for two steps can accomplish, and had simplified operation steps greatly.The general work person only need can accomplish through simple exercise, need not the professional staff and operates.
2, the present invention can accomplish detection within 5 minutes.Practiced thrift detection time greatly.
3, the present invention need not DNA is carried out mark, has practiced thrift the detection cost.
4, the present invention has the sensitivity of height, and detectability can reach 0.22 μ M.
Description of drawings
Fig. 1 is the fluorescence spectrum of the various metal cations measured according to the method for the invention.[thiazole orange]=100nM wherein, [potassium ion specific DNA]=150nM, [potassium ion]=[lithium ion]=[calcium ion]=[magnesium ion]=[ammonium radical ion]=[sodion]=1mM.
Fig. 2 be the different potassium concentrations measured according to the method for the invention fluorescence spectrum (Fig. 2 a) with the linear dynamics scope (Fig. 2 b, c).[thiazole orange]=100nM wherein, [potassium ion specific DNA]=150nM.Potassium concentration is followed successively by from top to bottom among Fig. 2 a: 0,1,10,30,50,100,300,500, and 700,900 μ M, 2,5,10mM.Fig. 2 b and c are two linear dynamics scopes of potassium ion.
Embodiment
According to accompanying drawing, provide the preferred embodiment of the invention below, and give detailed description, enable to understand better function of the present invention, characteristics.
Experimental apparatus
Used instrument is fluorospectrophotometer (LS-55; U.S. Perkins Elmer Instr Ltd.), the Instrument measuring condition is: the pulsed xenon lamp excites, and excitation wavelength is 488nm; The sweep limit 500-650nm of fluorescence spectrum; Excite and launch slit width to be 5nm, measure sample volume 2mL with width 10mm quartz colorimetric utensil; Room temperature.
Following as test in the embodiment of the invention with material potassium ion specific DNA sequences, synthesize and through the HPLC purifying by Takara company.
5’-GGGTTAGGGTTAGGGTTAGGG-3’
Analytically pure ammonium chloride, magnesium chloride, lime chloride, sodium chloride, potassium chloride, lithium chloride is available from traditional Chinese medicines chemical reagent company limited.Thiazole orange (Thiazole orange, TO), structural formula is following, available from Sigma-Aldrich company, is diluted to desired concn with dimethyl sulfoxide (DMSO) during use; All solution are all with three distilled water preparations.
Embodiment 1
In cuvette, add 2mL buffer (50mM Tris-HCl, pH 7.40), add 5 μ L 4 * 10
-5The M thiazole orange, 5 μ L 6 * 10
-5M potassium ion specific DNA and 5 μ L 0.4M potassium chloride mix 25 ℃ of reactions 4 minutes, carry out fluorometric investigation.This moment, each material ultimate density was: [thiazole orange]=100nM, [potassium ion specific DNA]=150nM, [potassium ion]=1mM.
Fluorescence spectrum figure before and after potassium ion adds is as shown in Figure 1, and before potassium ion added, the fluorescence intensity at maximum emission wavelength place was 353.06, and the fluorescence intensity that potassium ion adds maximum emission wavelength place, back is 151.42, so the present invention can the detection by quantitative potassium ion.
Embodiment 2
Change potassium ion into other metal cation such as calcium ion, magnesium ion, lithium ion, ammonium ion, sodion repeats embodiment 1 step, and the result is as shown in Figure 1.Calcium ion, magnesium ion, lithium ion, ammonium ion, sodion also can make fluorescence intensity reduce, but compare with potassium ion, and the reduction amplitude is very little.Explain that the present invention receives the interference of other metal cation less, has reasonable selectivity.
Embodiment 3
The potassium ion that potassium concentration is changed into variable concentrations repeats embodiment 1 step, and the result is as shown in Figure 2.The potassium ion that the present invention can the detection by quantitative variable concentrations is described.As [thiazole orange]=100nM, during [potassium ion specific DNA]=150nM, the concentration of potassium ion is proportional with fluorescence intensity in 1～60 μ M and 400～900 μ M scopes.
Sequence table
< 110>Ludong University
< 120>a kind of fluorescence detection method of potassium ion
<140>201110034763.4
<141>2011-01-26
<160>1
<170>PatentIn?version?3.3
<210>1
<211>21
<212>DNA
< 213>artificial sequence
<400>1
gggttagggt?tagggttagg?g 21
Claims (10)
1. the fluorescence detection method of a potassium ion, it comprises the following steps:
(1) in cuvette, add buffer solution, with potassium ion specific DNA, thiazole orange and metal cation hybrid reaction to be measured,
(2) measure fluorescence intensity.
2. fluorescence detection method as claimed in claim 1 is characterized in that buffer solution is 10-50mM pH 7.0-7.5 Tris-HCl.
3. according to claim 1 or claim 2 fluorescence detection method is characterized in that buffer solution is preferably 50mM pH 7.4Tris-HCl.
4. fluorescence detection method as claimed in claim 1 is characterized in that the potassium ion specific DNA sequences is 5 '-GGGTTAGGGTTAGGGTTAGGG-3 '.
5. like claim 1 or 4 described fluorescence detection methods, it is characterized in that potassium ion specific DNA concentration is 150nM.
6. fluorescence detection method as claimed in claim 1 is characterized in that metal cation to be measured is a potassium ion, lithium ion, calcium ion, magnesium ion, ammonium radical ion, sodion.
7. like claim 1 or 6 described fluorescence detection methods, it is characterized in that metal cation concentration to be measured is 0-10mM.
8. fluorescence detection method as claimed in claim 1 is characterized in that thiazole orange concentration is 100nM.
9. fluorescence detection method as claimed in claim 1 is characterized in that the potassium ion specific DNA, and thiazole orange and metal cation to be measured are 25 ℃ of-30 ℃ of hybrid reactions.
10. fluorescence detection method as claimed in claim 9 is characterized in that the potassium ion specific DNA, and thiazole orange and metal cation to be measured were 25 ℃ of-30 ℃ of hybrid reactions 4 minutes.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1192458B1 (en) * | 1999-07-07 | 2003-04-16 | Fritz Andreae | Optical sensor for potassium ions |
| CN101464299A (en) * | 2007-12-19 | 2009-06-24 | 苏州艾杰生物科技有限公司 | Kalium ion diagnosis/measuring reagent kit and kalium ion concentration determination method |
-
2011
- 2011-01-26 CN CN201110034763A patent/CN102183496B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1192458B1 (en) * | 1999-07-07 | 2003-04-16 | Fritz Andreae | Optical sensor for potassium ions |
| CN101464299A (en) * | 2007-12-19 | 2009-06-24 | 苏州艾杰生物科技有限公司 | Kalium ion diagnosis/measuring reagent kit and kalium ion concentration determination method |
Non-Patent Citations (2)
| Title |
|---|
| Min Sun Choi et al..Label-free dual assay of DNA sequences and potassium ions using an aptamer probe and a molecular light switch complex.《Chem.Comm.》.2009, * |
| 高淑丽等.基于荧光探针噻唑橙与核酸适体构象变化的钾离子检测.《分析测试学报》.2011,第30卷(第2期), * |
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