CN109655505A - A kind of method of electrochemistry amplification detection mercury ion - Google Patents
A kind of method of electrochemistry amplification detection mercury ion Download PDFInfo
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- CN109655505A CN109655505A CN201910121496.0A CN201910121496A CN109655505A CN 109655505 A CN109655505 A CN 109655505A CN 201910121496 A CN201910121496 A CN 201910121496A CN 109655505 A CN109655505 A CN 109655505A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/333—Ion-selective electrodes or membranes
Abstract
A kind of method that the present invention discloses electrochemistry amplification detection mercury ion includes the following steps: a, glass-carbon electrode is placed in aqueous solution of chloraurate, deposits one layer of gold nano layer in glassy carbon electrode surface using electrochemical deposition method；B, the obtained electrode of step a is placed in mercapto-modified aptamer and mercapto-modified ankyrin nucleic acid mixed liquor, obtains the glass-carbon electrode of gold modification, taken out after placing a period of time and then electrode is placed in sulfydryls hexanol aqueous solution, then taken out after placing a period of time；C, the glass-carbon electrode by the gold modification that step b is obtained is placed in the sample solution containing mercury ion, is taken out after placing a period of time；D, the electrode after taking out step c is placed in the mixing buffer solution of deoxynucleotide, terminal enzyme (DNA) and signal nucleic acid, is taken out after placing a period of time；E, the electric current after being taken out using square wave voltammetry detecting step d on electrode, the content of mercury ion in sample solution is calculated thus according to standard curve.
Description
Technical field
The invention belongs to electrochemistry and biochemical analysis detection fields, and in particular to a kind of electrochemistry amplification detection mercury
The method of ion.
Background technique
With the continuous improvement of living standards and the continuous enhancing of environmental consciousness, people are also increasingly heavier to metallic pollution
Depending on the especially significant metallic pollution of bio-toxicity.Metal ion is due to unprocessed or do not handle as required and be just discharged into river
In, they cannot be biodegradable, and can be rich in animals and plants under the biological magnification of food chain or soil cultivation
Collection, finally enters human body.Metal ion is accumulated in the organ-tissues such as liver, the kidney of human body, cause each organ-tissue damage and
Slow poisoning.Therefore, it protects the mankind from the injury of metal residual in metallic pollution and food, finds simple and convenient efficient
The method of detection metal residual is of great significance.
Common metal analysis method include it is ultraviolet can spectrophotometry, atomic absorption method, atomic fluorescence method, inductive coupling
The methods of plasma method, Inductively coupled plasma-mass spectrometry.However these methods take time and effort, need expensive instrument and
Skilled operator, most importantly, the not strong and sensitivity of selectivity be not high.
In consideration of it, overcoming defect present in the above-mentioned prior art, a kind of simple, selective strong, high sensitivity is provided
Metal ion inspection, be this field urgent problem to be solved.
Summary of the invention
The present invention provides a kind of method of electrochemistry amplification detection mercury ion, efficiently solves existing metal analysis side
The problem that method testing cost is high, selectivity is not strong, sensitivity is not high.
A kind of method of electrochemistry amplification detection mercury ion, includes the following steps:
A, glass-carbon electrode is placed in aqueous solution of chloraurate, deposits one layer in glassy carbon electrode surface using electrochemical deposition method
Gold nano layer；
B, the obtained electrode of step a is placed in mercapto-modified aptamer and mercapto-modified ankyrin nucleic acid mixed liquor,
The glass-carbon electrode of gold modification is obtained, takes out, then electrode is placed in sulfydryls hexanol aqueous solution, then place after placing a period of time
It is taken out after a period of time；
C, the glass-carbon electrode by the gold modification that step b is obtained is placed in the sample solution containing mercury ion, when placing one section
Between after take out；
D, the mixing buffering that the electrode after taking out step c is placed in deoxynucleotide, terminal enzyme (DNA) and signal nucleic acid is molten
In liquid, taken out after placing a period of time；
E, the electric current after being taken out using square wave voltammetry detecting step d on electrode, calculates sample thus according to standard curve
The content of mercury ion in product solution.
In the preferred embodiment of the invention, in step a, the concentration of chlorauric acid solution is 1.0wt%, dosage 2mL, electricity
The current potential of chemical deposition is -0.2V, sedimentation time 60s.
In the preferred embodiment of the invention, mercapto-modified aptamer concentration is 0.2 μM in step b；Sulfydryl modification
Ankyrin nucleic acid concentration be 0.5~5.0 μM, the concentration of sulfydryls hexanol aqueous solution is 1mM.
In the preferred embodiment of the invention, in step b, electrode is placed in mercapto-modified aptamer and mercapto-modified
Time in ankyrin nucleic acid mixed liquor is 1h, and temperature is 20~30 DEG C, and the time that electrode is placed in sulfydryls hexanol aqueous solution is 1h.
In the preferred embodiment of the invention, in step d, semiochemicals used in signal nucleic acid are methylene blue or two
Luxuriant iron, the concentration of deoxynucleotide, terminal enzyme (DNA) and signal nucleic acid are respectively 50 μM, 2U/ μ L and 1 μM.
In the preferred embodiment of the invention, the time that electrode is placed in mixing buffer solution in step d is 0.5~2h,
Temperature is 37 DEG C.
In the present invention, used glass-carbon electrode diameter 3mm is bought in Tianjin Aidahengsheng Technology Development Co., Ltd.
In the present invention, mercapto-modified aptamer refers to 5 ' ends of nucleic acid by mercapto-modified nucleic acid.
In the present invention, mercapto-modified ankyrin nucleic acid refers to 3 ' ends of nucleic acid by mercapto-modified nucleic acid.
In the present invention, signal nucleic acid refers to that 5 ' ends of nucleic acid are modified by methylene blue, and 3 ' ends are by amido modified nucleic acid.
In the present invention, the purpose that sulfydryls hexanol is added is enclosed-electrode surface, is reduced unrelated non-specific with detection mercury ion
The interference of property signal.
The testing principle of the course of work and mercury ion of the invention is as follows:
As shown in Figure 1, glass-carbon electrode is placed in chlorauric acid solution, one is deposited in electrode surface by electrochemical deposition method
Layer gold nano layer obtains the glass-carbon electrode of electrode surface deposition gold nano layer；Then the glass-carbon electrode for depositing gold nano layer is set
In mercapto-modified aptamer and mercapto-modified ankyrin nucleic acid mixed solution, mercapto-modified aptamer and sulfydryl modification
Ankyrin nucleic acid and the gold of electrode surface electrode surface is fixed on by sulphur gold key, obtain the glass-carbon electrode of gold modification；Gold is modified
Glass-carbon electrode be sequentially placed into the sample solution containing mercury ion and be made of deoxynucleotide, terminal enzyme (DNA) and signal nucleic acid
Mixing buffer solution in, if in sample solution not with the matched mercury ion of mercapto-modified aptamer, sulfydryl modification
Aptamer 3 ' hold under the action of terminal enzyme (DNA) with mixing buffer solution in deoxynucleotide in conjunction with, it is mercapto-modified
The end of aptamer 3 ' is extended, and the signal nucleic acid comprising methylene blue or ferrocene is in the prolongation and mercapto-modified anchor core
Electrode surface, methylene blue or ferrocene are fixed under the synergistic effect of acid under external voltage effect, methylene blue or two cyclopentadienyls
Electronics in iron moves, to generate electric signal, improves the electrochemical signals of electrode surface；If had in sample solution
With the matched mercury ion of mercapto-modified aptamer, mercapto-modified aptamer is in conjunction with mercury ion thus by sulfydryl modification
The end of aptamer 3 ' it is buried in electrode surface, terminal enzyme (DNA) can not hold mercapto-modified aptamer 3 ' and identify,
The combination at mercapto-modified aptamer 3 ' end and deoxynucleotide is hindered, is caused only on a small quantity comprising methylene blue or two cyclopentadienyls
The signal cDNA chip of iron is in electrode surface, to reduce the electrochemical signals of electrode surface.
In the present invention, by adding mercapto-modified ankyrin nucleic acid, the letter comprising methylene blue or ferrocene is substantially increased
Number nucleic acid does not increase mercapto-modified ankyrin nucleic acid in the fixed amount of electrode surface, and most of mercapto-modified aptamer 3 ' is held
Prolongation far from electrode surface so that the signal cDNA chip comprising methylene blue or ferrocene is big in the amount of electrode surface
It is big to reduce, cause the electrochemical signals of electrode surface low, after adding mercapto-modified ankyrin nucleic acid, mercapto-modified aptamer 3 '
The prolongation at end under the synergistic effect of mercapto-modified ankyrin nucleic acid with multiple signal cores comprising methylene blue or ferrocene
Acid is combined in electrode surface, substantially increases methylene blue or ferrocene in the fixed amount of electrode surface, the electrification of electrode surface
It learns signal to greatly improve, and then improves the sensitivity of detection method.
In the preferred embodiment of the invention, pass through the mercapto-modified nucleic acid for selecting to match with mercury ion to be detected
Aptamer substantially increases the selectivity of mercury ion to be detected.
In the preferred embodiment of the invention, match when existing in mercury ion to be detected with mercapto-modified aptamer
Ion when, mercury ion is combined with mercapto-modified aptamer, and by mercapto-modified aptamer 3 ' hold it is buried in electricity
Pole surface hinders the identification that terminal enzyme (DNA) holds mercapto-modified aptamer 3 ', and then influences signal nucleic acid and sulfydryl
The combination of the aptamer of modification causes no methylene blue or ferrocene to be incorporated in electrode surface, no electrochemical signals detection.
Standard working curve in the present invention is the electric current that known ion concentration of mercury is measured with detection method of the invention, is drawn
Standard working curve processed is measured produced by unknown ion concentration of mercury when measuring unknown ion concentration of mercury using the method for the present invention
Electric current, then compared with the electric current in standard working curve, and then calculate the content of unknown ion concentration of mercury.
In the present invention, terminal enzyme (DNA) and its buffer solution are commercial prod, and purchase is public in thermo Scientific
Department.
In the present invention, all nucleic acid and deoxynucleotide are bought from Sangon Biotech (Shanghai) Co., Ltd.) limited liability company,
In mercapto-modified aptamer particular sequence are as follows: 5 '-SH-TTCTCTCTTCGACGTTGTGTGTT-3 ', SH represent 5 ' end
By sulfydryl modification；Ankyrin nucleic acid particular sequence is 5 '-CACAGCTCT-SH-3 ', and SH represents 3 ' ends by sulfydryl modification；Signal nucleic acid
Particular sequence is 5 '-MB-GAGCTGTGGTTTTTTTTT-NH2- 3 ', MB represent 5 ' ends and are modified by methylene blue, NH2Represent 3 ' ends
It is amido modified；Used deoxynucleotide is deoxy adenosine triphosphate disodium.
The invention has the advantages that:
(1) present invention firstly provides be used to detect mercury ion in conjunction with terminal enzyme (DNA) for mercapto-modified aptamer
Content, the participation of terminal enzyme (DNA) improve the sensitivity of mercury ion assay.
(2) present invention passes through addition on the basis of proposing mercapto-modified aptamer in conjunction with terminal enzyme (DNA)
Mercapto-modified ankyrin nucleic acid, substantially increases the signal nucleic acid comprising methylene blue or ferrocene in the fixed amount of electrode surface,
Mercapto-modified ankyrin nucleic acid is not increased, the prolongation that most of mercapto-modified aptamer 3 ' is held makes far from electrode surface
It obtains the signal cDNA chip comprising methylene blue or ferrocene to substantially reduce in the fixed amount of electrode surface, leads to electrode surface
Electrochemical signals are low, and after adding mercapto-modified ankyrin nucleic acid, the prolongation that mercapto-modified aptamer 3 ' is held is repaired in sulfydryl
It is incorporated in electrode surface with multiple signal nucleic acid comprising methylene blue or ferrocene under the synergistic effect of the ankyrin nucleic acid of decorations, significantly
Methylene blue or ferrocene are improved in the fixed amount of electrode surface, the electrochemical signals of electrode surface greatly improve and amplify,
And then greatly improve the sensitivity of detection method.
(3) present invention is greatly improved by the mercapto-modified aptamer for selecting to match with mercury ion to be detected
The selectivity of mercury ion to be detected.
(4) method of present invention detection mercury ion is easy to operate, without expensive instrument, selective strong, high sensitivity.
Detailed description of the invention
Fig. 1 is detection schematic diagram of the invention；
Fig. 2 is to detect the curent change figure of mercury ion and other metal ions in embodiment 1 using the method for the present invention；
Fig. 3 is the current graph that various concentration mercury ion in embodiment 1 is detected using the method for the present invention；
Fig. 4 is that sensitivity, (electric current becomes using whether there is or not mercapto-modified ankyrin nucleic acids in the method for the present invention detection embodiment 2
Change) influence diagram；
Appended drawing reference meaning is as follows in figure:
1- aptamer；2- ankyrin nucleic acid；3- mercury ion；4- deoxynucleotide；5- signal nucleic acid；6- methylene blue or two cyclopentadienyls
Iron.
Specific embodiment
Below in conjunction with specific example, the present invention will be described in detail, but is not intended to limit the present invention.
Embodiment 1:
A kind of method of electrochemistry amplification detection mercury ion, includes the following steps:
A, the cleaned glass-carbon electrode that will polish is placed in 1.0wt% chlorauric acid solution, using electrochemical deposition method in glass
Carbon electrodes deposit one layer of gold nano layer, and controlling potential is -0.2V, sedimentation time 60s, cleaning electrode；
B, electrode made from step a is placed in mercapto-modified mercury ion aptamer that 10 μ L contain 0.2 μM and 3 μM
In mercapto-modified ankyrin nucleic acid mixed liquor, 25 DEG C of placement 1h, cleaning electrode；Then the sulfydryls hexanol for electrode being placed in 1mM is water-soluble
0.5h in liquid, cleaning electrode obtain the glass-carbon electrode of gold modification；
C, the glass-carbon electrode for the gold modification that step b is obtained is placed in 10 μ L to contain in the solution of various concentration mercury ion, 25
It is placed 1 hour at DEG C, cleaning electrode；
D, the electrode after taking out step c is placed in containing 50 μM of deoxy adenosine triphosphates, 2U/ μ L terminal enzyme (DNA) and 1 μM
In 10 μ L mixing buffer solutions of signal nucleic acid, 45min is placed, temperature is 37 DEG C, cleaning electrode；
E, the electric current after being taken out using square wave voltammetry detecting step d on electrode, calculates sample thus according to standard curve
The content of mercury ion in product solution, setting current potential are -0.35 to -0.15V.
Mercapto-modified mercury ion aptamer refers to the aptamer to match with mercury ion in the present embodiment, can be used
In the measurement of ion concentration of mercury, particular sequence 5 '-SH-TTCTCTCTTCGACGTTGTGTGTT-3 ', SH represent 5 ' end quilts
Sulfydryl modification.
Other metal ion curent changes are also detected using method identical with detection mercury ion in the present embodiment, it is specific to tie
Fruit is as shown in Figure 2.Used ion concentration of mercury is 0.2 μM, other ion concentrations are 2 μM.From figure 2 it can be seen that mercury from
The curent change of son is obvious, this is because mercapto-modified mercury ion aptamer causes mercapto-modified in conjunction with mercury ion
The end of aptamer 3 ' is buried in electrode surface, and terminal enzyme (DNA) can not hold mercapto-modified aptamer 3 ' and identify, hinder
The combination for having hindered mercapto-modified aptamer 3 ' end and deoxynucleotide, leads to the signal core comprising methylene blue or ferrocene
Acid cannot be fixed on electrode surface, to reduce the electrochemical signals of electrode surface, bigger variation occurs in electric current, from figure
It can be seen that the mercapto-modified aptamer in the present embodiment has very strong selectivity and sensibility to mercury ion in 2.
The electrochemistry spectrogram obtained using the mercury ion detecting of method of the invention to various concentration is as shown in figure 3, from figure
In as can be seen that with ion concentration of mercury increase, the electric current detected is gradually reduced, this is because mercapto-modified nucleic acid is suitable
Body is buried in electrode surface by mercapto-modified aptamer 3 ' end in conjunction with mercury ion, and terminal enzyme (DNA) can not be to sulfydryl modification
The end of aptamer 3 ' identified, hinder the combination at mercapto-modified aptamer 3 ' end and deoxynucleotide, cause to wrap
Signal nucleic acid containing methylene blue or ferrocene cannot be fixed on electrode surface, to reduce the electrochemistry letter of electrode surface
Number, i.e., current signal weakens.The detection range of mercury ion is 2pM to 20nM in the present embodiment.
Embodiment 2:
Influence whether there is or not ankyrin nucleic acid to method sensitivity, the specific steps are as follows:
A, identical with step a, b, c in embodiment 1, difference is not increase mercapto-modified ankyrin nucleic acid.
B, the obtained electrode of step A is placed in containing 50 μM of deoxy adenosine triphosphates, 2U/ μ L terminal enzyme (DNA) and 1 μM of letter
In 10 μ L mixing buffer solutions of number nucleic acid, 45min is placed, temperature is 37 DEG C, cleaning electrode；
C, identical with the step e in embodiment 1.
The result of the present embodiment has mercapto-modified anchor as shown in figure 4, under the conditions of existing for the mercury ion of same concentrations
The current signal detected in the presence of nucleic acid is much larger than without the current signal detected in the presence of mercapto-modified ankyrin nucleic acid, this is
After adding mercapto-modified ankyrin nucleic acid, the prolongation that mercapto-modified aptamer 3 ' is held is in mercapto-modified ankyrin nucleic acid
Synergistic effect under with multiple signal nucleic acid comprising methylene blue or ferrocene be incorporated in electrode surface, substantially increase methylene
Base is blue or ferrocene is in the fixed amount of electrode surface, and the electrochemical signals of electrode surface are amplified, and detection spirit is greatly improved
Quick property and then and solution in mercury ion detection limit.
Embodiment 3:
Utilize the content of mercury ion in method detection tobacco sample of the invention, the specific steps are as follows:
1, the extraction step of tobacco sample is as follows:
A, the artificial body fluid of 20mL is added in the cambridge filter for having trapped Smoke Particulate using smoking machine at 37 DEG C
Ultrasonic extraction 40min；
B, the solution after step a ultrasound is filtered into obtain extracting solution, then manually body fluid washs cambridge filter, it is repeated 4 times,
Combined extract after ultrasound filtration；
C, the nitric acid that 5mL concentration is 5wt% is added in the extracting solution obtained by step b, heating evaporation to 5mL or so is simultaneously accurate
It is settled to 5mL.
2, in tobacco sample mercury ion content detection, include the following steps:
A, the cleaned glass-carbon electrode that will polish is placed in 1wt% chlorauric acid solution, using electrochemical deposition method in glass carbon
Electrode surface deposits one layer of gold nano layer, and controlling potential is -0.2V, sedimentation time 60s, cleaning electrode；
B, electrode made from step a is placed in the mercapto-modified aptamer of mercury ion that 10 μ L contain 0.2 μM and 3 μM
In mercapto-modified ankyrin nucleic acid mixed liquor, 1h, cleaning electrode are placed at 25 DEG C；Then electrode is placed in the sulfydryls hexanol water of 1mM
0.5h in solution, cleaning electrode obtain the glass-carbon electrode of gold modification；
C, the glass-carbon electrode for the gold modification that step b is obtained is placed in 10 μ L to contain in the solution of various concentration mercury ion, 25
It is placed 1 hour at DEG C, cleaning electrode；
D, the obtained electrode of step c is placed in containing 50 μM of deoxy adenosine triphosphates, 2U/ μ L terminal enzyme (DNA) and 1 μM of letter
In 10 μ L mixing buffer solutions of number nucleic acid, 45min is placed, temperature is 37 DEG C, cleaning electrode；
E, the curent change after being taken out using square wave voltammetry detecting step d on electrode is calculated thus according to standard curve
Out in sample solution mercury ion content, setting current potential be -0.35 to -0.15V.
Artificial body fluid in the present embodiment is to include Na+、Cl-、HCO3 2-, glucose, K+、Ca2+、Mg2+、SO4 2-、HPO4 2-、
The inorganic ions such as amino acid, nucleotide, protein, polysaccharide and organic matter, concentration is suitable with practical body fluid, and pH value is in 7.3-7.5
Between.
The content of mercury ion is to be not detected in tobacco sample measured by the present embodiment.
Claims (5)
1. a kind of method of electrochemistry amplification detection mercury ion, which comprises the steps of:
A, glass-carbon electrode is placed in aqueous solution of chloraurate, deposits one layer of Jenner in glassy carbon electrode surface using electrochemical deposition method
Rice layer；
B, the obtained electrode of step a is placed in mercapto-modified aptamer and mercapto-modified ankyrin nucleic acid mixed liquor, is obtained
The glass-carbon electrode of gold modification, takes out after placing a period of time, then electrode is placed in sulfydryls hexanol aqueous solution, then place one section
It is taken out after time；
C, the glass-carbon electrode by the gold modification that step b is obtained is placed in the sample solution containing mercury ion, after placing a period of time
It takes out；
D, the electrode after taking out step c is placed in the mixing buffer solution of deoxynucleotide, terminal enzyme (DNA) and signal nucleic acid,
It is taken out after placing a period of time；
E, the electric current after being taken out using square wave voltammetry detecting step d on electrode, it is molten to calculate sample thus according to standard curve
The content of mercury ion in liquid.
2. the method for electrochemistry amplification detection mercury ion according to claim 1, which is characterized in that in step b, sulfydryl is repaired
The particular sequence of the aptamer of decorations is 5 '-SH-TTCTCTCTTCGACGTTGTGTGTT-3 ', and SH represents 5 ' ends and repaired by sulfydryl
Decorations.
3. the method for electrochemistry amplification detection mercury ion according to claim 1, which is characterized in that sulfydryl is repaired in step b
The ankyrin nucleic acid particular sequence of decorations is 5 '-CACAGCTCT-SH-3 ', and SH represents 3 ' ends by sulfydryl modification.
4. the method for electrochemistry amplification detection mercury ion according to claim 1, which is characterized in that in step d, deoxidation core
Thuja acid is deoxy adenosine triphosphate disodium.
5. the method for electrochemistry amplification detection mercury ion according to claim 1, which is characterized in that in step d, signal core
The particular sequence of acid is 5 '-MB-GAGCTGTGGTTTTTTTTT-NH2- 3 ', MB represent 5 ' ends and are modified by methylene blue, NH2It represents
3 ' ends are amido modified.
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