CN104076004A - Method for detecting mercury ionic concentration in sample - Google Patents
Method for detecting mercury ionic concentration in sample Download PDFInfo
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- CN104076004A CN104076004A CN201410339731.9A CN201410339731A CN104076004A CN 104076004 A CN104076004 A CN 104076004A CN 201410339731 A CN201410339731 A CN 201410339731A CN 104076004 A CN104076004 A CN 104076004A
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Abstract
The invention discloses a method for detecting mercury ionic concentration in a sample. The method for detecting mercury ionic concentration in the sample comprises the following steps: (1) DNA (deoxyribonucleic acid) probe molecules capable of being combed with mercury ionic specificities are designed and synthesized; (2) the DNA probe molecules and cationic polymer are added into a buffer solution and a sample to be detected and a gold nano solution are added for a T-Hg <2+>-T hybridization reaction; (3) gold nanometer particles are congregated and the system color changes; relative absorbency value is measured, so that the mercury ionic concentration in the sample to be detected is determined. The method for detecting the mercury ionic concentration in the sample provided by the invention can simply, quickly and accurately detect the mercury ionic concentration in the sample.
Description
Technical field
The invention belongs to analytical chemistry field, relate to a kind of method that detects ion concentration of mercury in sample.
Background technology
Nucleic acid gamete is DNA or the RNA molecule of a strand, is optionally tied to the various objects with high-affinity as little molecule, protein and medicine etc.Compare with traditional molecular recognition system, oligonucleotide aptamer is owing to easily synthesizing and having very large advantage.Oligonucleotide aptamer is easily labeled, and can steady in a long-term store, and has good stability,, applicability, and high sensitivity characteristic widely.Therefore, their excellent attribute makes it in the application aspect medical diagnosis, environmental monitoring, bioanalysis, have very large potentiality.In addition nucleic acid gamete can be with cationic polymer because electrostatic attraction attracts mutual winding.
Mercury is a kind of global environmental contaminants of severe toxicity.The long-time deposition of mercury ion can cause the nonreversibility of soil and water environment to pollute, water-soluble mercury ion be wherein the most common be also the most reliable mercury contaminants.Inorganic mercury can damage mammiferous heart, kidney, and stomach and small intestine system, apparent to the mankind's harm.Therefore, following for the exploitation of technology that detects water-soluble mercury ion.At present the detection of mercury ion is mainly contained to spectroscopic methodology and the large class of galvanochemistry two, but these methods need a large amount of pre-service toward contact, increased a lot of costs, and operating personnel have been had to very high technical requirement, so can not meet the demand day by day increasing that society is detected actual sample.Have scholar found divalence mercury metal ion can with nucleic acid in two thymine alkali bases (T) form highly stable T-Hg
2+-T complex structure, based on this great discovery, a lot of scholars utilize the specific recognition effect of oligonucleotide to mercury ion, a series of biology sensors to mercury ion detecting have been built respectively, it is good that this class sensor has selectivity, the advantage that specificity is good, becomes the focus that nearest people study.
As the nano gold mark technology (nanogold labelling techique) of one of modern four large labelling techniques, it is in fact the coated process that the macromolecules such as protein are adsorbed to nanogold particle surface.Adsorption mechanism may be nanogold particle surface negative charge, form strong bonded with the positive charge group of protein because of Electrostatic Absorption, and can not make biomolecule sex change after absorption, because gold grain has the characteristic of high electron density, at gold mark protein combination place, visible pitchy particle under the microscope, when these labels are assembled in a large number at corresponding part place, naked eyes red color visible or pink spot, thereby for qualitative or semiquantitative tachysynthesis detection method.Utilizing the nano-Au solution that gold chloride restores is claret, after adding therein a small amount of electrolyte, induction gold nano grain is reunited, can make solution become blueness from ruby, and finally aggegation is colourless, therefore can cause the variation of solution colour while adding therein suitable salt solusion or cationic polymer.
But there is not the desirable technical scheme with ion concentration of mercury in nano gold mark technology for detection sample at present.
Summary of the invention
The object of this invention is to provide a kind of method that detects ion concentration of mercury in sample, in order to realize simple, quick, the highly sensitive detection of mercury ion in water.
The present invention is achieved through the following technical solutions:
A method that detects ion concentration of mercury in sample, is characterized in that, comprising:
(1) design with synthetic can with the DNA probe molecule that is rich in T base of mercury ion specific binding;
(2) DNA probe molecule and cationic polymer are joined in damping fluid, and add wherein testing sample and gold nano solution, carry out T-Hg
2+-T hybridization reaction;
(3) golden nanometer particle is assembled, and system color changes, and measures relative absorbance, determines the ion concentration of mercury in described testing sample.
Wherein, the described DNA probe molecule that is rich in T base has the nucleotide sequence as shown in SEQ ID NO:1 to DNA probe molecule, and its concentration with respect to damping fluid is 10nM.
Wherein, in conjunction with the synthetic method in document, the concentration of described golden nanometer particle is 4.9nM, and diameter is 15nm, and it has good absorbance at visible ray 523nm place.
Wherein, the described cationic polymer of step (2) is diallyl dimethyl ammoniumchloride (PDDA), and PDDA has higher detection limit, and can with the good combination of DNA.Described damping fluid is Tris-HCl solution.
Wherein, described diallyl dimethyl ammoniumchloride (PDDA) is 10nM with respect to the concentration of damping fluid, to obtain testing result more accurately.
The method of the invention is limited to and is not less than 0.02ng/mL for the detection of mercury ion.Preferably in described testing sample, ion concentration of mercury is 0.05-10 ng/mL.
Further, based on following linear equation, determine the concentration of mercury ion in described sample:
Y=0.177+0.064x, y is the relative absorbance under different ion concentration of mercury, the concentration that x is corresponding mercury ion.Thus, can further improve efficiency and the sensitivity that utilizes the inventive method to carry out ion concentration of mercury detection.
According to technical solutions according to the invention, change color based on system, determine that the ion concentration of mercury in described sample is by the color of described system and typical curve have been compared, wherein, the standard model that described typical curve is respectively 0ng/mL, 0.05ng/mL, 0.1ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL based on known ion concentration of mercury carries out parallel laboratory test and sets up.Thereby further improve and utilize the inventive method to carry out efficiency and the sensitivity of ion concentration of mercury detection.
Additional aspect of the present invention and advantage in the following description part provide, and part will become obviously from the following description, or recognize by practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage accompanying drawing below combination obviously and is easily understood becoming the description of embodiment, wherein:
Fig. 1 utilizes different ion concentration of mercury standard models to detect resulting ultraviolet absorption curve, and wherein ion concentration of mercury is got respectively 0ng/mL, 0.05ng/mL, 0.1ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL;
Fig. 2 is the mercury ion canonical plotting that one embodiment of the invention is set up;
Fig. 3 has shown specificity analyses figure according to an embodiment of the invention.
Embodiment
For making to test sensed condition optimization, the present invention has made following shaker test to the optium concentration of PDDA and TBA:
Get 7 little centrifuge tubes, all add 500uL Tris-HCl (20 mM, pH 7.41) damping fluid, in each test tube, add diallyl dimethyl ammoniumchloride (PDDA), make its ultimate density be respectively 1,3,5,8,10,30 and 50 nM, then add wherein the gold nano solution of 500uL.The demonstration of visible ray collection of illustrative plates, when the concentration of PDDA is 10nM, visible absorption value is minimum, and golden nanometer particle reunion degree is maximum, and the optimal concentration of known PDDA is 10nM.Get again 7 centrifuge tubes, all add 500uL Tris-HCl (20 mM, pH 7.41) damping fluid and the final concentration PDDA that is 10nM, TBA is joined in each centrifuge tube, make its ultimate density be respectively 1,3,5,8,10,30 and 50 nM, mixing and at room temperature stablize 20min, then to the gold nano solution that adds 500uL in each centrifuge tube, is 10nM by the optimal concentration of the known TBA of visible absorption collection of illustrative plates.Therefore below in experiment, the concentration of PDDA and TBA all selects 10nM to survey the mercury ion in solution as optimum reaction condition.
Embodiment 1 design and synthetic corresponding oligonucleotide fragment
By consulting pertinent literature, designing one section can to mercury ion specific recognition and containing the DNA fragmentation of T base, sequence be prepared by DNA synthesizer.
DNA probe molecule (TBA) sequence:
TTCTTTCTTCCCCTTGTTTGTT（SEQ?ID?NO：1）
Synthesizing of embodiment 2 gold nano grains;
According to bibliographical information, the sodium citrate of take reduction gold chloride obtains the golden nanometer particle that diameter is 15nm, and the concentration of the gold nano solution of acquisition is 4.9nM, is cooled to room temperature, at 4 ℃, preserves.
The foundation of embodiment 3 mercury ion typical curves;
Get 7 centrifuge tubes, TBA (10nM) and PDDA (10nM) are joined in the Tris-HCl damping fluid of 500uL, in centrifuge tube, add mercury ion, make its final concentration be respectively 0,0.05,0.1,0.5,1,5,10,50 and 100 ng/mL, mix and also at room temperature stablize 20min, then to the gold nano solution that adds 500uL in each centrifuge tube, at wavelength, be that visible absorption value is measured at 558 places, according to visible absorption value under the different ion concentration of mercury of measuring, draw the typical curve of ion concentration of mercury.As shown in Figure 1, along with adding of mercury ion, gold nano grain concentration class increases, thereby causes absorbance to reduce, and when ion concentration of mercury is zero, the absorbance of gold nano solution is 0.91; For making experiment shows go out good effect, linear obvious, the absorbance in Fig. 2 is that blank solution light absorption value (0.91) deducts and adds the absorbance obtaining after mercury ion solution.The range of linearity 0.05-10ng/mL of this sensor, detects and is limited to 0.02ng/mL.The linear equation of typical curve is y=0.177+0.064x, and y is the visible absorption value obtaining after proofreading and correct under different ion concentration of mercury, the concentration that x is corresponding mercury ion, linear dependence >0.98(is shown in Fig. 1 and Fig. 2)
The specific assay of embodiment 4 mercury ions;
Get 5 little centrifuge tubes, TBA (10nM) and PDDA (10nM) are joined in the Tris-HCl damping fluid of 500uL, in each centrifuge tube, add respectively Hg
2+, Cu
2+, Cd
2+, Pb
2+, Ni
2+, Fe
2+, making to add the ultimate density of metallic ion is 10ng/mL, mix and at room temperature react 20 minutes, and, to the gold nano solution that adds 500uL in each centrifuge tube, at wavelength, be then that visible absorption values are measured at 558 places.By experimental result, learn that this mercury ion detecting sensor based on colorimetric detection technology has very high specificity (referring to Fig. 3).
The mensuration of the actual mercurous particle testing sample of embodiment 5;
To the mercury ion that adds respectively 0.1 ng/mL, 0.5ng/mL, 1 ng/mL and 5 ng/mL in tap water sample as testing sample, according to the method design described in embodiment 1 with synthetic can with the DNA probe molecule that is rich in T base of mercury ion specific binding, according to the method described in embodiment 2, prepare gold nano grain solution; Get 4 centrifuge tubes, therein TBA (10nM) and PDDA (10nM) are joined in the Tris-HCl damping fluid of 500uL, in centrifuge tube, add respectively above-mentioned 4 testing samples, mix and also at room temperature stablize 20min, then to the gold nano solution that adds 500uL in each centrifuge tube, at wavelength, be that visible absorption value is measured at 558 places, typical curve (the y=0.177+0.064x that substitution embodiment 3 sets up, y is the relative absorbance under different ion concentration of mercury, x is the concentration of corresponding mercury ion), calculate ion concentration of mercury, the results are shown in Table 1:
Table 1: the mensuration of mercury ion water sample
Above-mentioned test findings shows: adopt the interpolation recovery of the mercury ion in the mercury ion detecting new method mensuration actual sample based on colorimetric technology between 98.0%-108.0%, standard deviation is less than 5.67%, can meet the detection demand to mercury ion in actual life completely.
More than show and described ultimate principle of the present invention and principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that in above-described embodiment and instructions, describes just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.
Claims (10)
1. a method that detects ion concentration of mercury in sample, is characterized in that, comprising:
(1) design with synthetic can with the DNA probe molecule that is rich in T base of mercury ion specific binding;
(2) DNA probe molecule and cationic polymer are joined in damping fluid, and add wherein testing sample and gold nano solution, carry out T-Hg
2+-T hybridization reaction;
(3) golden nanometer particle is assembled, and system color changes, and measures relative absorbance, determines the ion concentration of mercury in described testing sample.
2. method according to claim 1, is characterized in that: the described DNA probe molecule that is rich in T base has the nucleotide sequence as shown in SEQ ID NO:1.
3. method according to claim 1, is characterized in that: the described DNA probe molecule that is rich in T base is 10nM with respect to the concentration of damping fluid.
4. method according to claim 1, is characterized in that: the concentration of described golden nanometer particle is 4.9nM, and diameter is 15nm.
5. method according to claim 1, is characterized in that: the described cationic polymer of step (2) is diallyl dimethyl ammoniumchloride, and damping fluid is Tris-HCl solution.
6. method according to claim 5, is characterized in that: described diallyl dimethyl ammoniumchloride is 10nM with respect to the concentration of damping fluid.
7. method according to claim 1, is characterized in that: described method is limited to and is not less than 0.02ng/mL for the detection of mercury ion.
8. according to the method described in claim 1 or 7, it is characterized in that: in described testing sample, ion concentration of mercury is 0.05-10 ng/mL.
9. method according to claim 1, is characterized in that: based on following linear equation, determine the concentration of mercury ion in described sample:
Y=0.177+0.064x, y is the relative absorbance under different ion concentration of mercury, the concentration that x is corresponding mercury ion.
10. according to the method described in claim 1 or 9, it is characterized in that: described relative absorbance measures at wavelength 558 places.
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Cited By (12)
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| CN104946770A (en) * | 2015-07-02 | 2015-09-30 | 常熟理工学院 | Novel exonuclease-III-based mercury ion detection method |
| CN105652012A (en) * | 2014-11-28 | 2016-06-08 | 惠州市银嘉环保科技有限公司 | Nanogold-DNA composite detection test paper for mercury ions, preparation method and use thereof |
| CN106442345A (en) * | 2016-09-08 | 2017-02-22 | 常熟理工学院 | Method of detecting lead ion based on CD spectrum detection technology |
| CN107462579A (en) * | 2017-07-03 | 2017-12-12 | 北京欧凯纳斯科技有限公司 | A kind of kit and detection method for detecting mercury ion |
| CN107589085A (en) * | 2017-08-16 | 2018-01-16 | 广西师范大学 | It is a kind of to regulate and control nano silicon active absorption spectroscopic assay Hg with aptamers2+Method |
| CN107727596A (en) * | 2017-09-30 | 2018-02-23 | 中国石油大学(华东) | A kind of method of the micro mercury ion of quick detection |
| CN108526484A (en) * | 2018-04-25 | 2018-09-14 | 安徽师范大学 | Sulfydryl DNA modification gold nanorods and preparation method thereof, the detection method of metal mercury ions and application |
| CN109212123A (en) * | 2018-09-10 | 2019-01-15 | 陕西师范大学 | A method of mercury ion is detected based on air pressure change immediately |
| WO2020000598A1 (en) * | 2018-06-28 | 2020-01-02 | 中国农业大学 | Mercury ion detection product and method and smart phone imaging analysis system |
| CN114034694A (en) * | 2021-11-11 | 2022-02-11 | 中国热带农业科学院南亚热带作物研究所 | Colorimetric sensor for visually detecting divalent mercury ions and preparation method and application thereof |
| CN114441521A (en) * | 2022-01-27 | 2022-05-06 | 宜宾五粮液股份有限公司 | Preparation method of white spirit ethyl carbamate quick-detection test paper |
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Cited By (15)
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| CN105652012A (en) * | 2014-11-28 | 2016-06-08 | 惠州市银嘉环保科技有限公司 | Nanogold-DNA composite detection test paper for mercury ions, preparation method and use thereof |
| CN104730236A (en) * | 2015-04-16 | 2015-06-24 | 三诺生物传感股份有限公司 | Protein fixing reagent and application thereof |
| CN104946770A (en) * | 2015-07-02 | 2015-09-30 | 常熟理工学院 | Novel exonuclease-III-based mercury ion detection method |
| CN106442345B (en) * | 2016-09-08 | 2019-03-15 | 常熟理工学院 | A kind of lead ion detection method based on CD spectrum detection technique |
| CN106442345A (en) * | 2016-09-08 | 2017-02-22 | 常熟理工学院 | Method of detecting lead ion based on CD spectrum detection technology |
| CN107462579A (en) * | 2017-07-03 | 2017-12-12 | 北京欧凯纳斯科技有限公司 | A kind of kit and detection method for detecting mercury ion |
| CN107589085A (en) * | 2017-08-16 | 2018-01-16 | 广西师范大学 | It is a kind of to regulate and control nano silicon active absorption spectroscopic assay Hg with aptamers2+Method |
| CN107589085B (en) * | 2017-08-16 | 2020-03-31 | 广西师范大学 | Method for measuring Hg by using aptamer to regulate and control nano-silica activity-absorption spectrum2+Method (2) |
| CN107727596A (en) * | 2017-09-30 | 2018-02-23 | 中国石油大学(华东) | A kind of method of the micro mercury ion of quick detection |
| CN107727596B (en) * | 2017-09-30 | 2019-11-08 | 中国石油大学(华东) | A method of quickly detecting micro mercury ion |
| CN108526484A (en) * | 2018-04-25 | 2018-09-14 | 安徽师范大学 | Sulfydryl DNA modification gold nanorods and preparation method thereof, the detection method of metal mercury ions and application |
| WO2020000598A1 (en) * | 2018-06-28 | 2020-01-02 | 中国农业大学 | Mercury ion detection product and method and smart phone imaging analysis system |
| CN109212123A (en) * | 2018-09-10 | 2019-01-15 | 陕西师范大学 | A method of mercury ion is detected based on air pressure change immediately |
| CN114034694A (en) * | 2021-11-11 | 2022-02-11 | 中国热带农业科学院南亚热带作物研究所 | Colorimetric sensor for visually detecting divalent mercury ions and preparation method and application thereof |
| CN114441521A (en) * | 2022-01-27 | 2022-05-06 | 宜宾五粮液股份有限公司 | Preparation method of white spirit ethyl carbamate quick-detection test paper |
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