CN104059964A - Method For Detecting Mercury Ions In Solution, And Kit - Google Patents
Method For Detecting Mercury Ions In Solution, And Kit Download PDFInfo
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- CN104059964A CN104059964A CN201410100363.2A CN201410100363A CN104059964A CN 104059964 A CN104059964 A CN 104059964A CN 201410100363 A CN201410100363 A CN 201410100363A CN 104059964 A CN104059964 A CN 104059964A
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Abstract
The invention provides a method for detecting mercury ions in a solution, and a kit. The method comprises: 1) mixing the solution with single-stranded DNA containing thymine to form a mixture so that the single-stranded DNA containing the thymine forms double-stranded DNA through T-Hg2+-T base pairs wherein at least one deoxyribonucleotide of the single-stranded DNA containing the thymine is connected with a signal generating molecule; 2) contacting the double-stranded DNA obtained from the step 1) with exonuclease specifically different from the double-stranded DNA so that the exonuclease cuts the double-stranded DNA to release the deoxyribonucleotides connected with the signal generating molecules; and 3) detecting the signal generating molecules connected with the deoxyribonucleotides released in the detection step 2). The method has high specificity and sensitivity, does not need fixed DNA, is convenient to operate and can realize rapid detection.
Description
Technical field
The present invention relates to biological chemistry and electrochemical field, be specifically related to detect method and the test kit of the mercury ion in solution.
Background technology
Between metal ion and nucleic acid base, there is specific binding interactions.For example,, such as Ag
+, Hg
2+, Cu
2+, Ni
2+can be combined with specific natural or synthesizing ribonucleotide by coordination power Deng metal ion, form the base pair of metal ion mediation, wherein Watson-Crick(W-C) hydrogen bond in type base pair replaced by metal-base key.The Novel photo mutual effect of this nucleic acid and metal ion has huge prospect in multiple application, comprises the biosensor of preparation based on DNA, the logical gate (logic gate) of design molecular level and adjusting DNA function.
Mercury ion (Hg
2+) be a kind of biological accumulation and there are highly toxic environmental pollutant, it can cause serious human health problems, for example the destruction of DNA damage, brain injury, organ failure and immunity system balance.Due to the Hg in environment and food
2+the concern of polluting increases day by day, and needing to develop can be to the Hg in aqueous medium
2+carry out the method for sensitive Site Detection, in the prevention for environment protection and disease.Recently, the Hg of Ono and Togashi report
2+and the Coordination interaction between two thymus pyrimidines (T) has inspired the Hg based on DNA
2+the novel method of biosensor (refers to document " Ono, A.; Togashi, H.Angew.Chem., Int.Ed.2004,43,4300-4302 ", " Tanaka, Y.; Oda, S.; Yamaguchi, H.; Kondo, Y.; Kojima, C.; Ono, A.J.Am.Chem.Soc.2007,129,244 245 " and " Miyake, Y.; Togashi, H.; Tashiro, M.; Yamaguchi, H.; Oda, S.; Kudo, M.; Tanaka, Y.; Kondo, Y.; Sawa, R.; Fujimoto, T.; Machina-mi, T.; Ono, A.J.Am.Chem.Soc.2006,128,2,172 2173 ").Specifically, the T-T mispairing in DNA double chain (T-T mismatch) can attract the Hg in the aqueous solution
2+thereby, form stable with Hg
2+the DNA double chain of the T-T DNA base pairing of mediation.T-Hg
2+the binding constant of-T base pair is more much higher than natural A-T base pair.The more important thing is this T-Hg
2+-T interacts and has high degree of specificity, and only has Hg
2+can make T-T base pair stable.This discovery causes having developed much fluorescence, colourity and electrochemical sensor, and they have good selectivity for the interference of other metal ions.But the major part in these sensors nearly all can not realize the detection within the scope of lower nmole, this has limited the Hg of this technology in tap water
2+application in detection, because the Hg specifying according to USEPA (EPA)
2+toxic level, this testing requirement detects lower limit will reach 10nM.
Recently, based on the similarity having between the DNA double chain (M-dsDNA) of base pair and the secondary structure of natural double-stranded DNA (dsDNA) of metal ion mediation, several research groups have reported that a lot of function and applications of normal dsDNA are also applicable to M-dsDNA, and have reported to have more highly sensitive Hg
2+sensor.For example, the people such as Liu reported can be by by Hg
2+the catalytic activity that the allosteric interaction causing with the coordination of T-T mispairing is opened DNA enzyme (DNAzyme), and developed thus a kind of lower sensitivity limit and reach the Hg of 2.4nM
2+detection platform (refers to document " Liu, J.W.; Lu, Y.Angew.Chem., Int.Ed.2007,119,7,731 7734 ").But this sensor based on DNA enzyme is easily subject to the interference of other metal ions.The people such as Li have reported Hg
2+can open indentation endonuclease (nicking endonuclease) nicking activity of the DNA double chain with T-T mispairing (is referred to document " Li, F.; Feng, Y.; Liu, S.F.; Tang, B.Chem.Commun.2011,47,6,347 6349 ").People and the Park etc. such as Urata have reported that archaeal dna polymerase can identify the metal-complexing T-Hg of primer 3 ' end per capita
2+-T base pair also makes primer extend and (refer to document " Urata, H. along template DNA; Yamaguchi, E.; Funai, T.; Matsumura, Y.; Wada, S.Angew.Chem., Int.Ed.2010,49,6,516 6519 " and " Park, K.S.; Jung, C.; Park, H.G.Angew.Chem., Int.Ed.2010,49,9,757 9760 ").After this, the people such as Zhu has reported that one exempts from mark Hg
2+sensor, the low-detection lower limit that has reached 40pM by the auxiliary amplified fluorescence of polysaccharase (refers to document " Zhu, X.; Zhou, X.M.; Xing, D.Biosens.Bioelectron.2011,26,2,666 2669 ").But this method needs operation and the complicated fluorescent instrument of multiple steps, is not therefore suitable for Site Detection.
Summary of the invention
For solving existing problem in above-mentioned prior art, the invention provides a kind of method that detects the mercury ion in solution, a kind of test kit that detects the mercury ion in solution is also provided.
Particularly, the invention provides:
(1) detect the method for the mercury ion in solution, the method comprises:
1) described solution and the single stranded DNA that contains thymus pyrimidine (T) are mixed to form to mixture, the single stranded DNA that contains thymus pyrimidine described in making passes through T-Hg
2+-T base pair forms double-stranded DNA, is connected with signal and produces molecule at least one deoxyribonucleotide of the wherein said single stranded DNA that contains thymus pyrimidine;
2) double-stranded DNA of step 1) gained is contacted with the exonuclease that is specific to double-stranded DNA, make described exonuclease cut described double-stranded DNA, discharge the deoxyribonucleotide that is connected with described signal generation molecule; And
3) detecting step 2) in the signal that connects of the described deoxyribonucleotide that discharges produce molecule.
(2) according to the method (1) described, the ion concentration of mercury of wherein said solution is 0.2nM to 200 μ M.
(3) according to the method (1) or (2) described, wherein said thymus pyrimidine accounts for the 50%-80% of the total alkali radix of described single stranded DNA.
(4) according to the method described in any one in (1)-(3), the wherein said single stranded DNA that contains thymus pyrimidine has the length of 20-40 Nucleotide.
(5) according to the method described in any one in (1)-(4), wherein in the mixture described in step 1), described in contain thymus pyrimidine the concentration of single stranded DNA be 1-5 μ M.
(6) according to the method described in any one in (1)-(5), wherein, in the described single stranded DNA that contains thymus pyrimidine, described in be connected with deoxyribonucleotide that signal produces molecule and account for the 3.8%-30.8% of the total nucleotide number of described single stranded DNA.
(7) according to the method described in any one in (1)-(6), it is electric active molecule that wherein said signal produces molecule.
(8) according to the method (7) described, wherein said electric active molecule is selected from methylene blue and ferrocene.
(9) according to the method described in any one in (1)-(8), wherein in step 2) in, described double-stranded DNA reacts 10-30 minute with described exonuclease at 30-50 DEG C.
(10) according to the method described in any one in (1)-(9), wherein said exonuclease is selected from Escherichia coli nucleic acid exonucleaseⅢ, lambda particles phage exonuclease and T7 phage gene 6 exonucleases.
(11) according to the method described in any one in (1)-(10), it is methylene blue that wherein said signal produces molecule, 3 ' end part of the single stranded DNA that contains thymus pyrimidine described in this signal generation molecule is connected to; Described exonuclease is Escherichia coli nucleic acid exonucleaseⅢ.
(12), according to the method described in any one in (1)-(11), wherein in step 3), utilize to be positioned at the signal that described deoxyribonucleotide that the electronegative indium oxide tin glass electrode detection on electrode chip discharges connects and to produce molecule.
(13) according to the method described in any one in (1)-(12), wherein step 2) described in exonuclease cutting that described double-stranded DNA is carried out also comprise: cut the T-Hg in this double-stranded DNA
2+-T base pair, makes the Hg in this base pair
2+dissociate out, thereby be combined with other single stranded DNAs that contain thymus pyrimidine.
(14) detect the test kit of the mercury ion in solution, comprising:
The single stranded DNA that contains thymus pyrimidine, this single stranded DNA that contains thymus pyrimidine comprises at least one and is connected with signal and produces the deoxyribonucleotide of molecule;
Be specific to the exonuclease of double-stranded DNA; And
Detection reagent, the signal that this detection reagent can be connected with the described deoxyribonucleotide of free state produces molecularity and produces signal.
(15) according to the test kit (14) described, wherein said thymus pyrimidine accounts for the 50%-80% of the total alkali radix of described single stranded DNA.
(16) according to the test kit (14) or (15) described, the wherein said single stranded DNA that contains thymus pyrimidine has the length of 20-40 Nucleotide.
(17), according to the test kit described in any one in (14)-(16), wherein, in the described single stranded DNA that contains thymus pyrimidine, the deoxyribonucleotide that is connected with described signal generation molecule accounts for the 3.8%-30.8% of the total nucleotide number of described single stranded DNA.
(18) according to the test kit described in any one in (14)-(17), it is electric active molecule that wherein said signal produces molecule.
(19) according to the test kit (18) described, wherein said electric active molecule is selected from methylene blue and ferrocene.
(20) according to the test kit described in any one in (14)-(19), wherein said exonuclease is selected from Escherichia coli nucleic acid exonucleaseⅢ, lambda particles phage exonuclease and T7 phage gene 6 exonucleases.
(21) according to the test kit described in any one in (14)-(20), it is methylene blue that wherein said signal produces molecule, 3 ' end part of the single stranded DNA that contains thymus pyrimidine described in this signal generation molecule is connected to; Described exonuclease is Escherichia coli nucleic acid exonucleaseⅢ.
(22) according to the test kit described in any one in (14)-(21), wherein said detection reagent is the form of electronegative indium oxide tin glass electrode, and this electronegative indium oxide tin glass electrode is positioned on electrode chip.
The present invention, by the configuration dependency exonuclease activity being caused by metal ion, has developed a kind of electrochemistry Hg
2+sensor platform, it compared with prior art has the following advantages and positively effect:
1. the present invention is had signal to produce the single stranded DNA that is rich in thymus pyrimidine of molecule and is specific to the exonuclease of double-stranded DNA by applying marking, makes it possible to high degree of specificity ground quantitative and qualitative analysis and detects the Hg in solution
2+;
2. described in, exonuclease, by cutting double-stranded DNA, can make Hg
2+from T-Hg
2+in-T base pair, discharge, it is got back in solution phase, be then combined with other single stranded DNAs that are rich in T, form thus Hg
2+circulative metabolism, thereby the electrical signal that amplification detection system produces, improved detection sensitivity thus greatly;
Method of the present invention without fixed dna, easy and simple to handle, can realize rapid detection.
Brief description of the drawings
Figure 1A is for illustrating the schematic diagram of the Degradation of Escherichia coli nucleic acid exonucleaseⅢ (Exo III) to the Ref-oligon1 in Ref-oligo1/Ref-oligo2 two strands; Figure 1B illustrates Hg
2+the PAGE analytical results of the active retarding effect to Exo III; Wherein, a:2 μ MRef-oligo1+2 μ M Ref-oligo2; B:2 μ M Ref-oligo2; C:2 μ M Ref-oligo1+2 μ M Ref-oligo2+Exo III; D:2 μ M Ref-oligo1+2 μ M Ref-oligo2+Exo III+10 μ M Hg
2+; E:2 μ M Ref-oligo1+2 μ M Ref-oligo2+ExoIII+100 μ M Hg
2+; F:2 μ M Ref-oligo1+2 μ M Ref-oligo2+Exo III+200 μ M Hg
2+; G:2 μ M Ref-oligo1+2 μ M Ref-oligo2+Exo III+500 μ M Hg
2+.
Fig. 2 A illustrates the T-Hg that contains different numbers
2+the sequence of the DNA double chain of-T base pair; Fig. 2 B illustrates the PAGE analytical results of the degraded product of the DNA double chain shown in A; Wherein, a:2 μ M Ref-oligo1+2 μ M Ref-oligo2; B:2 μ M Ref-oligo2; C:2 μ M Ref-oligo1+2 μ M Ref-oligo2+Exo III+20 μ M Hg
2+; D:2 μ MRef-oligo3+2 μ M Ref-oligo4+Exo III+20 μ M Hg
2+; E:2 μ MRef-oligo5+2 μ M Ref-oligo6+Exo III+20 μ M Hg
2+; F:2 μ MRef-oligo7+2 μ M Ref-oligo8+Exo III+20 μ M Hg
2+; G:2 μ MRef-oligo9+2 μ M Ref-oligo10+Exo III+20 μ M Hg
2+.
Fig. 3 is the schematic diagram of exempting from fixing electrochemical detection method and detect the degraded of designed e-T-rich probe illustrating by one embodiment of the invention, and the degraded of wherein said e-T-rich probe is by Hg
2+trigger, and by Exo III catalysis.
Fig. 4 illustrates that shown differential responses mixture is at Differential Pulse Voltammetry (DPV) scanning result of 37 DEG C (A) or 45 DEG C (B) lower incubation respectively after 20 minutes; Probe in figure represents e-T-rich probe.
Fig. 5 A is illustrated in and has respectively 20 μ M or 0.1 μ M Hg
2+situation under, the graphic representation of DPV peak current to incubative time; Fig. 5 B illustrates the PAGE analytical results of the reaction mixture in A; Wherein, a:2 μ M e-T-rich probe; B:2 μ M e-T-rich probe+ExoIII; C:2 μ M e-T-rich probe+Exo III+0.1 μ M Hg
2+; D:2 μ M e-T-rich probe+Exo III+20 μ M Hg
2+.
Fig. 6 illustrates the sensitivity of the inventive method; Wherein, A illustrates and contains 2 μ M e-T-rich probes, the 20 Exo III of unit and different concns Hg
2+reaction mixture incubation DPV scanning result after 20 minutes at 45 DEG C; B illustrates that DPV peak current is to Hg
2+the graphic representation of concentration, small icons wherein goes out DPV peak current to lower Hg
2+the graphic representation of concentration; Mean value and standard deviation are obtained by three independent experiments.
Fig. 7 illustrates the DPV scanning result of differential responses mixture, and this result shows the selection specificity of the inventive method, and wherein all competition metal ions are tested under 1 μ M, 5 μ M and 10 μ M; Also show the Hg to 10nM, 50nM and 500nM
2+the reaction signal of ion, with as a comparison.
Embodiment
The invention provides a kind of method that detects the mercury ion in solution, the method comprises:
1) described solution and the single stranded DNA that contains thymus pyrimidine (T) are mixed to form to mixture, the single stranded DNA that contains thymus pyrimidine described in making passes through T-Hg
2+-T base pair forms double-stranded DNA, is connected with signal and produces molecule at least one deoxyribonucleotide of the wherein said single stranded DNA that contains thymus pyrimidine;
2) double-stranded DNA of step 1) gained is contacted with the exonuclease that is specific to double-stranded DNA, make described exonuclease cut described double-stranded DNA, discharge the deoxyribonucleotide that is connected with described signal generation molecule; And
3) detecting step 2) in the signal that connects of the described deoxyribonucleotide that discharges produce molecule.
In this article, term " signal generation molecule " thus refer to that can instruction sheet chain DNA forming double-stranded DNA be specific to the exonuclease cutting of double-stranded DNA and the material that can be detected.Especially, described signal produces molecule can not produce signal in the time being connected on DNA, only has in the time that DNA is cut into free deoxyribonucleotide, and the signal generation molecule being connected on this free deoxyribonucleotide can produce signal.
In the present invention, it can be electric active molecule that signal produces molecule, for example, is easily marked at the electric active molecule on deoxyribonucleotide such as methylene blue and ferrocene etc.Can utilize can interactional detection reagent occur with this electric active molecule this electric active molecule being connected on free deoxyribonucleotide is detected, described detection reagent is preferably electronegative and be positioned on electrode chip.Interaction between described electric active molecule and described detection reagent can be for example redox reaction, produces thus electronics transmission, thereby electrochemical signals is provided.
In this case, when there is no Hg
2+time, the single stranded DNA that contains thymus pyrimidine is the single-stranded structure of random coil, can not be specific to the exonuclease degraded of double-stranded DNA.Because the negative electricity skeleton of DNA makes to have electrostatic repulsion between itself and electronegative detection reagent, be connected to signal on DNA and produce molecule and cannot contact with detection reagent, therefore almost do not observe electrochemical signals.When adding Hg
2+time, the single stranded DNA that contains thymus pyrimidine can form T-Hg
2+-T base pair, thereby formation double-stranded DNA, it can be specific to the exonuclease identification of double-stranded DNA, thereby mononucleotide is cut one by one, when be marked with signal produce the mononucleotide of molecule cut come after, due to the less negative charge of this mononucleotide band and volume less, it is more easily diffused into described electronegative detection reagent surface, can interact with described detection reagent and produce electrochemical signals thereby make signal produce molecule.
In a specific embodiment, described detection reagent is electronegative indium oxide tin glass electrode, and this electronegative indium oxide tin glass electrode is positioned on electrode chip; Described electrode chip is mainly made up of electrode, an accurate reference electrode of platinum and four circular ITO electrodes as working electrode a platinum, and successively through Alconox solution clean, Virahol cleans, water cleans and form electronegative working electrode surface.
In the present invention, in the process of the described exonuclease degraded double-stranded DNA that is specific to double-stranded DNA, can make Hg
2+from T-Hg
2+in-T structure, discharge the Hg discharging
2+form double-stranded DNA thereby can be combined with other the single stranded DNA that contains thymus pyrimidine again, carry out thus the degradation process of a new round.By this way, Hg
2+can be at solution phase and T-Hg
2+in-T, recycle, thus the degradation process of amplifying double-stranded DNA as enzyme catalysis initiator, thus produce the electrochemical signals amplifying.
Preferably, in the method for the invention, for example described in, contain the single stranded DNA of thymus pyrimidine, for thering are 20-40 (20,21,22,23,24,25 ... 36,37,38,39,40) oligonucleotide of deoxyribonucleotide.Described thymus pyrimidine preferably accounts for the 50%-80%(for example 50%, 55%, 60%, 65%, 70%, 75%, 80% of the total alkali radix of described single stranded DNA).
Preferably, in described step 1) of the present invention, in solution to be measured, the concentration of mercury ion is preferably in the scope of 0.2nM to 200 μ M.In the mixture described in step 1), described in contain thymus pyrimidine the concentration of single stranded DNA be preferably 1-5 μ M.
Also preferably, in described step 1), make described mercury ion with described in contain thymus pyrimidine single stranded DNA at 20-45 DEG C, react at least 1-30 minute, preferably at 37-45 DEG C, react at least 1-30 minute, more preferably at 45 DEG C, react 30 minutes.
Preferably, the exonuclease that is specific to double-stranded DNA described in is selected from Escherichia coli nucleic acid exonucleaseⅢ (Exo III), lambda particles phage exonuclease and T7 phage gene 6 exonucleases, wherein more preferably Escherichia coli nucleic acid exonucleaseⅢ.
Preferably, in the described single stranded DNA that contains thymus pyrimidine, described in be connected with signal and produce the deoxyribonucleotide of molecule and account for the 3.8%-30.8% of the total nucleotide number of described single stranded DNA.
In the time that use starts the exonuclease of cutting from 3 ' end of double-stranded DNA, 3 ' end part of the single stranded DNA that contains thymus pyrimidine described in described signal generation molecule is preferably connected to, more preferably described in being connected to, contain in the T base of 3 ' end part of the single stranded DNA of thymus pyrimidine, be more preferably connected in the T base of 3 ' end; In the time that use starts the exonuclease of cutting from 5 ' end of double-stranded DNA, 5 ' end part of the single stranded DNA that contains thymus pyrimidine described in described signal generation molecule is preferably connected to, more preferably described in being connected to, contain in the T base of 5 ' end part of the single stranded DNA of thymus pyrimidine, be more preferably connected in the T base of 5 ' end.
In this article, term " 3 ' end part " refers to the 3 ' terminal nucleotide of DNA or the Nucleotide near 3 ' end; Term " 5 ' end part " refers to the 5 ' terminal nucleotide of DNA or the Nucleotide near 5 ' end; Term " 3 ' end " refers to first Nucleotide starting from the 3 ' end of DNA; Term " 5 ' end " refers to first Nucleotide starting from the 5 ' end of DNA.
It is known in the art to the method on the deoxyribonucleotide in DNA that signal is produced to molecule marker.For example, in the time that signal generation molecule is methylene blue, can pass through document " Xiao, Y.; Qu, X.; Plaxco, K.W.; Heeger, A.J.J.Am.Chem.Soc., 2007,129 (39), pp11896 – 11897 " described in method carry out mark.
Preferably, in described step 2) in, make formed double-stranded DNA with described in be specific to double-stranded DNA exonuclease at 30-45 DEG C, react 1-30 minute, preferably at 37-45 DEG C, react 1-30 minute, more preferably at 45 DEG C, react 30 minutes.In the present invention, the endonuclease reaction that is specific to the exonuclease of double-stranded DNA also can carry out at 4 DEG C, and this enzyme is higher to the specificity of double-stranded DNA like this, but can greatly extend the reaction times (for example needing to react 24 hours).
The method of the electrochemical signals that in the present invention, detection produces is known in the art.For example, can utilize the Autolab PGSTAT30 potentiostat/galvanostat (Eco Chemie, Holland) of being controlled by General Purpose Electrochemical System (GPES) (general electrochemical system) software (Eco Chemie) to carry out electrochemical measurement.
In the method for the invention, can after the reaction of described step 1) completes, in system, add described step 2 again) exonuclease used, also all reactants (, mercury ion, be marked with signal produce the single stranded DNA that contains thymus pyrimidine of molecule and be specific to the exonuclease of double-stranded DNA) can be mixed simultaneously.
In the method for the invention, can be in described step 1) and 2) reaction complete after, resampling carries out separately the detection described in described step 3).Also can in same system, carry out described step 1), 2) and 3).
The present invention also provides a kind of test kit that detects the mercury ion in solution, comprising:
The single stranded DNA that contains thymus pyrimidine, this single stranded DNA that contains thymus pyrimidine comprises at least one and is connected with signal and produces the deoxyribonucleotide of molecule;
Be specific to the exonuclease of double-stranded DNA; And
Detection reagent, the signal that this detection reagent can be connected with the described deoxyribonucleotide of free state produces molecularity and produces signal.
Preferably, the single stranded DNA that contains thymus pyrimidine described in is the oligonucleotide with 20-40 deoxyribonucleotide.Described thymus pyrimidine preferably accounts for the 50%-80% of the total alkali radix of described single stranded DNA.
Also preferably, in the described single stranded DNA that contains thymus pyrimidine, the deoxyribonucleotide that is connected with described signal generation molecule accounts for the 3.8%-30.8% of the total nucleotide number of described single stranded DNA.
Preferably, it is electric active molecule that described signal produces molecule, for example, is easily marked at the electric active molecule on deoxyribonucleotide such as methylene blue and ferrocene etc.
Described exonuclease is preferably selected from Escherichia coli nucleic acid exonucleaseⅢ, lambda particles phage exonuclease and T7 phage gene 6 exonucleases, wherein more preferably Escherichia coli nucleic acid exonucleaseⅢ.
In a preferred embodiment, it is methylene blue that described signal produces molecule, 3 ' end part of the single stranded DNA that contains thymus pyrimidine described in this signal generation molecule is connected to; Described exonuclease is Escherichia coli nucleic acid exonucleaseⅢ.More preferably, in the T base of 3 ' end part of the single stranded DNA that contains thymus pyrimidine described in described signal generation molecule is connected to, be more preferably connected in the T base of 3 ' end.
Described detection reagent is preferably and can interactional material occurs with described electric active molecule, and it is preferably electronegative and be positioned on electrode chip.Interaction between described electric active molecule and described detection reagent can be for example redox reaction, produces thus electronics transmission, thereby electrochemical signals is provided.
In a specific embodiment, described detection reagent is electronegative indium oxide tin glass electrode, and this electronegative indium oxide tin glass electrode is positioned on electrode chip; Described electrode chip is mainly made up of electrode, an accurate reference electrode of platinum and four circular ITO electrodes as working electrode a platinum, and successively through Alconox solution clean, Virahol cleans, water cleans and form electronegative working electrode surface.
Example
Reagent
Exo III is purchased from New England Biolabs company (USA), without being further purified.
10 × NEBuffer1 (1 × NEBuffer1:10mM Bis-Tris-propane-HCl, 10mM MgCl2,1mM dithiothreitol (DTT), pH7.0,25 DEG C) is purchased from New EnglandBiolabs, and Inc. (USA), without being further purified.
Methylene blue is modified rich T oligonucleotide (e-T-rich probe) purchased from BiosearchTechnologies company (USA), sequence is as follows: 5 '-CATTCTTTCTTCCCCTTGTTTGTTT-(methylene blue)-G-3 ', its Methylene Blue is modified on italic T.
Following reference oligonucleotide is purchased from Invitrogen (USA), and with standard desalting and purifying: reference oligonucleotide 1 (Ref-oligo1:5 '-GTAGCGGTCTTGCGG-3 '), reference oligonucleotide 2 (Ref-oligo2:5 '-CCGCAAGACCGCTACAAAAA-3 '), reference oligonucleotide 3 (Ref-oligo3:5 '-GTAGCGGTCTTGCGG-3 '), reference oligonucleotide 4 (Ref-oligo4:5 '-CCGCTAGACCGCTACAAAAA-3 '), reference oligonucleotide 5 (Ref-oligo5:5 '-GTAGCGGTCTTGCGG-3 '), reference oligonucleotide 6 (Ref-oligo6:5 '-CCGCTAGTCCGCTACAAAAA-3 '), reference oligonucleotide 7 (Ref-oligo7:5 '-GTAGCGGTCTTGCGG-3 '), reference oligonucleotide 8 (Ref-oligo8:5 '-CCGCTTGACCGCTACAAAAA-3 '), reference oligonucleotide 9 (Ref-oligo9:5 '-GTAGCGGCTTTGCGG-3 ') and reference oligonucleotide 10 (Ref-oligo10:5 '-CCGCTTTGCCGCTACAAAAA-3 ').
10 × TBE (Tris-borate-EDTA) damping fluid is purchased from Fermentas (Canada).
GelRed
tMgel-colored dose of nucleic acid (10,000 ×, soluble in water) purchased from Biotium (USA).
Acrylamide/bisacrylamide (29:1) 30% gel mother liquor is purchased from Bio-Rad (USA).
Ammonium persulphate (APS) is purchased from USB company (USA).
N, N, N ', N '-Tetramethyl Ethylene Diamine (TEMED), metal-salt (Hg (NO
3)
2, Pb (NO
3)
2, Ni (NO
3)
2, Cd (NO
3)
2, FeCl
2, FeCl
3, ZnCl
2, CuCl
2and MnCl
2) purchased from Sigma-Aldrich (St.Louis, MO, USA).
All aqueous solutions all with Milli-Q SILVER REAGENT water system (Millipore, USA) deionized water (resistivity >18.2M Ω/
cm) preparation.
In following test example and embodiment, adopt following method to carry out processing and the electrochemical measurement of ITO electrode chip.
The glass-chip with ITO coating for electrochemical measurement is prepared institute (NFF) preparation at the nanoelectronic of Hong Kong University of Science and Thchnology.Its design and this research group previously report for exempting from, the microchip of fixing electrochemical DNA detection is identical (refers to document " Xuan, F.; Luo, X.; Hsing, I.M.Anal.Chem.2012,84,5216-5220 "), this chip is mainly made up of electrode, an accurate reference electrode of platinum and four circular ITO electrodes as working electrode a platinum.The useful area of each ITO working electrode is 7.85 × 10
-3cm
2.The accurate reference electrode of platinum with respect to the electromotive force of silver/silver chloride reference electrode is+0.36V in 1 × NEBuffer1.Before each electrochemical measurement, the detection chip using is successively at Alconox solution (8 grams of Alconox(Sigma-Aldrich)/premium on currency) in ultrasonic cleaning 15 minutes, ultrasonic cleaning 15 minutes in Virahol, 15 minutes twice of ultrasonic cleaning in water.Through these wash procedures, form electronegative working electrode surface.Adopt the Autolab PGSTAT30 potentiostat/galvanostat (Eco Chemie, Holland) of being controlled by GeneralPurpose Electrochemical System (GPES) (general electrochemical system) software (EcoChemie) to carry out electrochemical measurement.At C1000
tMincubation sample in thermal cycler (Bio-Rad, USA).In the time of each measurement, every kind of testing sample solution of 1.5 microlitres is added on chip, to cover platinum to electrode, the accurate reference electrode of platinum and an ITO working electrode, utilize differential pulse voltammetry (DPV) to scan, measure electric current.
If no special instructions, in following test example and embodiment, utilize polyacrylamide gel electrophoresis to detect the concrete steps of analyzing as follows:
Acrylamide/bisacrylamide of 5mL30% (29:1) gelating soln, 1mL tbe buffer liquid (10 ×), 100 μ L APS, 10 μ L TEMED and 3.89mL deionized water are mixed, to prepare hydrogel.The final gel per-cent of this mixture is 15%.Make at room temperature polymerization of gel 1 hour, be immersed in afterwards in 1 × tbe buffer liquid (pH=8.3) in order to using.The each test sample of 5 μ L is mixed with 1 μ L6 × sample-loading buffer (loading buffer), then carry out 15% native polyacrylamide gel electrophoresis (PAGE).This PAGE carries out about 40 minutes in 1 × tbe buffer liquid, under room temperature, 150V constant voltage.With the Gel-Red solution-dyed of dilution, use afterwards Gel Doc
tMxR register system (Bio-Rad, USA) scanning gel.
Test example 1:Hg
2+to the inhibiting research of exonuclease (Exo III)
2 μ M Ref-oligo1 and 2 μ M Ref-oligo2 are hybridized 20 minutes under room temperature in damping fluid 1 × NEBuffer1.Add subsequently the Hg of different concns
2+(0-500 μ M), at room temperature places the solution mixing 20 minutes.Then, the ExoIII that adds 20 units to cumulative volume be 50 μ L, and place 10 minutes in 37 DEG C.Finally, these mixed solutions are heated to 80 DEG C and keep 10 minutes, so that Exo III loses activity.Make all sample solutions be cooled to lentamente room temperature (about 20 minutes), in order to carrying out polyacrylamide gel electrophoresis (PAGE) analysis.
Exo III has 3 ' → 5 ' 5 prime excision enzyme activity that acts on specifically double-stranded DNA (dsDNA), starts degraded from one end, obtains complementary single stranded DNA (ssDNA) and 5 '-P mononucleotide.This enzyme has high degree of specificity to duplex structure, can and have the DNA site of otch to start degraded from flat end, 3 ' recessed end, but in the time that 3 ' end contains 4 more than base protruding terminuses, its degrading activity is restricted and (refers to document " Zuo, X.; Xia, F.; Xiao, Y.; Plaxco, K.W.J.Am.Chem.Soc.2010,132,1816-1818 ").The activity of Exo III is affected by some positively charged ions.For example, Mg
2+and Co
2+support its activity as necessary metal cofactor, and Na
+, Pb
2+, Fe
2+and Cd
2+suppress its activity.Recently, Yuan group has reported Hg
2+may there is restraining effect to Exo III activity.At the Hg based on Exo III
2+in test experience, in order to eliminate Hg
2+the possible restraining effect to Exo III activity own, first contriver has studied at different Hg
2+under concentration, the situation that dsDNA is degraded by Exo III.As shown in Figure 1A, Ref-oligo1 sequence is designed to the 5 ' part of Ref-oligo2 complementary, forms flat end.Therefore, Exo III starts the 3 ' end from Ref-oligo1 to its degraded, and cutting obtains 5 '-P mononucleotide, until degrade to the end of Ref-oligo1, or the remaining fragment of Ref-oligo1 separates from Ref-oligo2.Because 3 ' of Ref-oligo2 holds the protruding terminus with 5nt, therefore can not be degraded.As shown in Figure 1B, swimming lane a and swimming lane b corresponding Ref-oligo1/Ref-oligo2 two strands and Ref-oligo2 respectively.The band of swimming lane c and swimming lane b, at same position, illustrates when there is no Hg
2+time, Ref-oligo1 is degradable by Exo III.Work as Hg
2+when concentration is 10 μ M or 100 μ M, the band of swimming lane d and swimming lane e and the band of swimming lane b, also at same position, illustrate that Ref-oligo1 is also degradable by Exo III.Hg is worked as in this explanation
2+concentration is during lower than 100 μ M, and its activity on Exo III does not almost affect.Work as Hg
2+when concentration is 200 μ M, in swimming lane f, there is the band that represents Ref-oligo1/Ref-oligo2 two strands, because the Ref-oligo1 in Ref-oligo1/Ref-oligo2 two strands is not completely degraded, therefore the Hg of this concentration is described
2+suppress the activity of Exo III.Work as Hg
2+when concentration increases to 500 μ M, the activity of Exo III is suppressed completely, does not therefore observe the band that represents Ref-oligo2.These the results shows, under this experiment condition, only have the Hg of working as
2+while reaching quite high concentration, just the activity of Exo III is had to restraining effect.
Test example 2:Exo III is to having T-Hg
2+the enzyme of the DNA double chain of-T base pair is cut active research
First, in damping fluid 1 × NEBuffer1, mix respectively 2 μ M Ref-oligo1 and 2 μ M Ref-oligo2,2 μ M Ref-oligo3 and 2 μ M Ref-oligo4,2 μ M Ref-oligo5 and 2 μ M Ref-oligo6,2 μ M Ref-oligo7 and 2 μ M Ref-oligo8,2 μ MRef-oligo9 and 2 μ M Ref-oligo10, and under room temperature, hybridize 20 minutes.Afterwards, by 20 μ M Hg
2+be added in above five kinds of mixed solutions, and room temperature is placed 20 minutes.Then, by the 20 Exo III of unit join in above system to cumulative volume be 50 μ L, and at 37 DEG C, place 10 minutes.Finally, these mixed solutions being heated to 80 DEG C keeps 10 minutes.Make all sample solutions slowly cool to room temperature (about 20 minutes), analyze in order to polyacrylamide gel electrophoresis (PAGE).
Above-mentioned to the inhibiting research of Exo III activity in, Hg
2+be not introduced in DNA double chain.Another kind of possible approach is, Hg
2+likely by form T-Hg in double-stranded DNA
2+-T base pair, thereby the activity of inhibition Exo III.Do not have article to report T-Hg before
2+whether-T base pair can suppress Exo III degradation of dna.At T-Hg
2+in-T base pair, Hg
2+can be directly and the N3 position coordination of thymus pyrimidine, substituted imido proton, thereby with the thymine residue formation pair of keys of two opposed orientation.Hg
2+van der Waals radius be about
, the distance in DNA double chain between W-C type base pair is about
, therefore, Hg
2+be inserted into rigidity and Hg that T-T base mismatch centering can have influence on DNA double spirane structure hardly
2+combination with Exo III.On the other hand, because dsDNA is base of a base cut next process continuously by Exo III degraded, wherein the obstruction of any relevant step (dissociating of the Nucleotide for example, cutting away) all can cause the reduction of Exo III activity.At T-Hg
2+in-T base pair, thymus pyrimidine is by covalent linkage N-Hg and Hg
2+in conjunction with.Hydrogen bond action in strength ratio A-T and the G-C of this covalent linkage (refers to document " Miyake, Y. more by force; Togashi, H.; Tashiro, M.; Yamaguchi, H.; Oda, S.; Kudo, M.; Tanaka, Y.; Kondo, Y.; Sawa, R.; Fujimoto, T.; Machina-mi, T.; Ono, A.J.Am.Chem.Soc.2006,128,2,172 2173 ").Therefore the T base, being cut down by Exo III is from T-Hg
2+dissociating probably at Exo III to thering is T-Hg on-T base pair
2+in the degraded of the dsDNA of-T base pair, play a role.Ono group is to having T-Hg
2+the thermal induction of the DNA double chain of-T base pair is dissociated and is studied in conjunction with situation, and proposes, and implements in the process of degraded at Exo III, and the T base cutting down may be from T-Hg
2+in-T base pair, disintegrate down.
As shown in Figure 2 A, in order to study T-Hg
2+the impact of-T base pair on Exo III activity, changed Ref-oligo1 and Ref-oligo2 sequence to introduce the T-T mispairing of different numbers, thus built five kinds different from T-Hg
2+the double-stranded DNA of-T.These DNA double chains all with excessive Hg
2+hatch, to ensure each T-T base mismatch pair and Hg
2+effect completely.In addition, Hg
2+concentration be 20 μ M, well below 100 μ M, thereby eliminated Hg
2+restraining effect (referring to Fig. 1) to Exo III.As shown in Figure 2 B, analyze the Degradation of Exo III to these double-stranded DNAs with PAGE.Consistent with experimental result before, when there being 20 μ MHg
2+time, Exo III has activity, Ref-oligo1/Ref-oligo2 two strands (there is no T-T mispairing) be completely degraded into Ref-oligo2(swimming lane c).When there being the T-Hg of one or two separation in two strands
2+when-T base pair, the activity of Exo III does not have suppressed, and two strands is still degraded to ssDNA, as shown in swimming lane d and swimming lane e.When there being two or three continuous T-Hg in two strands
2+when-T base pair, because the double-stranded DNA not being degraded in swimming lane f and swimming lane g, the therefore known Exo III double-stranded DNA of still can effectively degrading.These experiment showed, as normal double-stranded DNA, and Exo III is to containing T-Hg
2+the double-stranded DNA of-T base pair has Degradation.Also illustrate that N-Hg key can not hinder the Degradation of Exo III, also can not hinder T base from T-Hg simultaneously
2+on-T base pair, dissociate.
Ironically, in swimming lane f and swimming lane g, there are two shorter bands (as arrow in Fig. 2 B marks), this shows after Ref-oligo7 is degraded, the complete Ref-oligo8 of some is released, and after Ref-oligo9 is degraded, all complete Ref-oligo10 are released, and they are further degraded to shorter oligonucleotide.This may be (being represented by bold-faced letter in Fig. 2 A) being caused by the inside base pair of Ref-oligo8 and Ref-oligo10.The solvent temperature of certainly drawing (self-primed) sequence Ref-oligo10 self with 4 complementary bases is 40 DEG C.After Ref-oligo9 is degraded, in the time that temperature of reaction is 37 DEG C, the Ref-oligo10 being released has from drawing to form the 3 ' end that is less than 4 outstanding bases, and then is degraded by Exo III.Apparently, the solvent temperature of certainly drawing sequence Ref-oligo8 of 3 complementary bases of self tool is about 25 DEG C, and this is more much lower than temperature of reaction (37 DEG C).In theory, the Ref-oligo8 discharging should be unable to certainly draw to form to have and be less than 3 ' of 4 outstanding bases and hold and then be degraded.But as shown in swimming lane f in Fig. 2 B, the Ref-oligo8 that contriver finds that there is almost half is degraded.Contriver thinks, this significant experimental result may be its corresponding coefficient result of thermodynamic(al)equilibrium from drawing sequence of Exo III enzyme kinetics and Ref-oligo8.From thermodynamics aspect, in the time that temperature of reaction is 37 DEG C, although high temperature (37 DEG C) be conducive to form strand Ref-oligo8 instead of solvent temperature (25 DEG C) lower certainly draw Ref-oligo8 structure, but due to the existence of inner base pair, still have and a small amount of certainly draw Ref-oligo8 structure and strand Ref-oligo8 exists simultaneously.But, owing to there being Exo III, forming once certainly draw Ref-oligo8 structure, 3 ' end has the duplex structure that is less than 4 outstanding bases will induce ExoIII activity, thereby causes further being degraded by Exo III from drawing Ref-oligo8 structure.The enzyme process of cutting has been broken thermodynamic(al)equilibrium, and this process is moved to the direction of certainly drawing the formation of Ref-oligo8 structure, is further degraded thereby certainly draw Ref-oligo8 structure.This may be able to explain that the Exo III reporting in some documents has the active reason of degraded ssDNA.
Test example 3:Hg
2+the Degradation of the Exo III causing to DNA
Upper one experimental results show that containing T-Hg
2+the DNA double chain of-T base pair is the same with normal double-stranded DNA, can effectively be degraded by Exo III, and therefore contriver has designed a ssDNA who is rich in T, and it is at Hg
2+initiation under can be degraded by Exo III.As shown in Figure 3, this degradation process detects by the Electrochemical Detection platform without fixing DNA probe of the present invention.Its principle is to utilize oligonucleotide and mononucleotide in the time that electronegative ITO electrode surface spreads, to have different velocity of diffusion.The DNA sequence dna (e-T-rich probe) of designed this rich T comprises 26 nucleosides and the methylene blue group (MB) of institute's mark on the T that closes on 3 ' end.When there is no Hg
2+time, e-T-rich probe is the single-stranded structure of random coil, can not be degraded by Exo III.Due to the electrostatic repulsion between its negative electricity skeleton and negative electricity ITO electrode surface, its cannot with electrode contact, so almost do not observe electrochemical signals.When there being Hg
2+time, Hg
2+interact with T base, can form T-Hg
2+-T structure, thus make the e-T-rich probe of line style be folded into the hairpin structure with 3 ' flat end containing 4nt ring (CCCC) and 11bp stem.This structure can be identified by Exo III, thereby mononucleotide is degraded one by one from 3 ' end, when be marked with the T of MB cut come after, due to its compared with e-T-rich probe with less negative charge and there is less volume, it is more easily diffused into electronegative electrode surface, thereby provides the electrical signal of amplification.The electrical signal of this enhancing has proved Hg
2+cause the activity of Exo III.In addition, contriver infers, Hg in degradation process
2+can be from T-Hg
2+in-T structure, discharge, make another new e-T-rich probe not being degraded be folded into two strands, thereby carry out the degradation process (being below verified in test example 4) of a new round.By this way, Hg
2+can in solution phase and e-T-rich probe, recycle, and amplify the degradation process of a large amount of e-T-rich probes as catalysis initiator.
Differential responses mixture is as shown in Figure 4 distinguished down to incubation 20 minutes at 37 DEG C (Fig. 4 A) or 45 DEG C (Fig. 4 B), carry out afterwards Differential Pulse Voltammetry (DPV) scanning.As shown in Figure 4 A, when adding Hg in the system that is containing e-T-rich probe and Exo III
2+time, to observe obvious electrical signal and strengthen, this has illustrated Hg
2+cause the degrading activity of Exo III.But it should be noted that when there is no Hg
2+but while having Exo III, and there is no Hg
2+signal (very weak background signal only being detected) during with Exo III is compared, and occurs slightly strong signal, and this has implied even without Hg
2+initiation, also have a small amount of e-T-rich probe to be degraded, may be because Exo III has remaining exonuclease activity to not folding e-T-rich probe.Although some study group has reported Exo III and had better specificity 37 DEG C of 4 DEG C of ratios, reduce the temperature Extending culture time (placing 24 hours for 4 DEG C in some instances) greatly.From this paper experimental result above, e-T-rich probe is likely owing to there being base pairing in molecule in e-T-rich probe by the non-specific degraded of Exo III.Therefore, can reduce base pairing in molecule by rising temperature of reaction, thereby be reduced in without Hg
2+time the electrical signal being caused by non-specific degraded.Fig. 4 B has shown the result 45 DEG C of reactions.Compared with the result of 37 DEG C, without Hg
2+time signal significantly reduce, this situation that shows that e-T-rich probe is degraded greatly reduces.Meanwhile, there is Hg
2+time signal slightly strengthen, this may be because Exo III has better enzymic activity 45 DEG C time.With this understanding, just obtained better signal to noise ratio without extending the reaction times.These results have further confirmed the supposition before contriver, and in molecule, base pairing causes ssDNA by the non-specific degraded of Exo III.
In order to confirm Hg
2+can circulate between solution phase and the e-T-rich probe mechanism of release-bonding, contriver has carried out time-histories experiment.At 45 DEG C, by the 50 μ L solution that contain 2 μ M e-T-rich probes and the 20 Exo III of unit respectively with the Hg of 20 μ M or 0.1 μ M
2+mix for some time, observe the difference along with mixing time, the variation of electrochemical signals.As shown in Figure 5A, work as Hg
2+be that 20 μ M(are greatly excessive) time, electrochemical signals is rapidly state of saturation, and this shows when adding Hg
2+time, e-T-rich probe is all hairpin structure and is degraded rapidly.Work as Hg
2+be that 0.1 μ M(is inadequate) time, electrochemical signals increases continuously with speed relatively slowly, finally arrives identical platform.Because the concentration of e-T-rich probe is Hg
2+20 times of concentration, and last e-T-rich probe can be completely degraded, and therefore Hg is described
2+really release-bonding can circulate between solution phase and e-T-rich probe.The PAGE of reaction product analyzes referring to Fig. 5 B.Consistent with expection, even Hg
2+concentration is only 0.1 μ M, and all e-T-rich probes are also all degraded into shorter oligonucleotide by Exo III.
Test example 4: the detectability of the inventive method
Experimental result explanation above, the activity of Exo III " being initiated " can be used for Hg
2+signal amplification detection, and there is hypersensitivity.Contriver has attempted the Hg of different concns
2+to test method of the present invention.0.1 μ M Hg before
2+time-histories experiment show, at first 20 minutes that hatch, signal was coarse linear growth, within 30 minutes, reached plateau hatching later.Therefore,, before carrying out Electrochemical Detection, will contain 2 μ M e-T-rich probes, the 20 Exo III of unit and different concns Hg
2+reaction mixture at 45 DEG C, hatch 20 minutes respectively.As shown in Figure 6A, in the situation that eliminating without any background, be low to moderate the Hg of 0.5nM
2+can realize easily detectable signal and increase, the detectable concentration of this method well below EPA standard (for tap water, 10nM Hg
2+).Fig. 6 B has described peak point current and Hg
2+the funtcional relationship of concentration, its S-shaped curve.When there being two or three Hg
2+during with e-T-rich probe chelating, although there are 7 pairs of T-T base mismatch pair on each e-T-rich probe, e-T-rich probe still can fold.As shown in the illustration in Fig. 6 B, within the scope of low concentration, peak current and Hg
2+concentration linear dependence.Its calibration equation is: peak value (nA)=1.38C+1.26, wherein C is Hg
2+concentration, relation conefficient is 0.97426.The lowest signal value that can detect be as seen from the figure background signal and three times of standard deviations and, determine that according to described calibration equation lowest detection is limited to 0.2nM thus.This is the electrochemistry Hg reporting at present
2+the lowest detectable limit of sensor, the even Hg based on fluorescence than most
2+the detectability of detector is also low.
Test example 5:Hg
2+specificity
Subsequently, we have detected this method to Hg
2+selectivity (specificity).By the Hg of 2 μ Me-T-rich probes and different concns
2+, Pb
2+, Mn
2+, Ni
2+, Cu
2+, Fe
2+, Fe
3+or Cd
2+at room temperature mix, place 20 minutes.Add subsequently 20 Exo III to the 50 μ L of unit, place 20 minutes for 37 DEG C or 45 DEG C.Then be heated to 80 DEG C and keep 10 minutes, so that Exo III loses activity.Finally slowly cool to room temperature (about 20 minutes), prepare against PAGE analysis and on ITO electrode chip, carry out Electrochemical Detection.
For example, with ubiquitous many kinds of metal ions (Pb in actual sample
2+, Mn
2+, Zn
2+, Ni
2+, Cu
2+, Fe
2+, Fe
3+and Cd
2+) alternative Hg
2+detect.As shown in Figure 7, under identical test condition (Exo III temperature of reaction is 45 DEG C), every kind of alternative metals (1 μ M, 5 μ M and 10 μ M) under three kinds of relatively high concentration is tested.Find can provide than 10nM Hg without any a kind of detected alternative metals
2+the high peak point current of half of signal.The selectivity of this excellence is owing to specific T-Hg
2+the preference activity of-T base pair and ExoIII.
Embodiment 1
In 1 × NEBuffer1 damping fluid, by e-T-rich single stranded DNA respectively with three part of 10 μ L, Hg
2+the solution example to be measured (being called respectively sample 1, sample 2 and sample 3) that concentration is different at room temperature mixes, and makes the concentration of e-T-rich reach 2 μ M.Under room temperature, place this mixture 20 minutes.Add subsequently 20 Exo III to the 50 μ L of unit, place 20 minutes for 45 DEG C.Then be heated to 80 DEG C and keep 10 minutes, so that Exo III loses activity.Finally slowly cool to room temperature (about 20 minutes), prepare against PAGE analysis and on ITO electrode chip, carry out Electrochemical Detection.The results listed in the following table for this embodiment:
| Specimen coding | Size of current | The Hg detecting 2+Concentration |
| Sample 1 | 4.23 | 10nM |
| Sample 2 | 6.15 | 50nM |
| Sample 3 | 11.06 | 500nM |
Embodiment 2
To contain 2 μ M e-T-rich probes, the 20 Exo III of unit and 10 μ L containing different concns Hg
2+the reaction mixture of testing sample in 1 × NEBuffer1 damping fluid, at 45 DEG C, hatch 20 minutes respectively.Then be heated to 80 DEG C and keep 10 minutes, so that Exo III loses activity.Finally slowly cool to room temperature (about 20 minutes), prepare against PAGE analysis and on ITO electrode chip, carry out Electrochemical Detection.The results listed in the following table for this embodiment:
| Specimen coding | Size of current | The Hg detecting 2+Concentration |
| Sample 1 | 1.03 | 0nM |
| Sample 2 | 1.96 | 0.5nM |
| Sample 3 | 2.61 | 1nM |
| Sample 4 | 3.33 | 5nM |
| Sample 5 | 4.18 | 10nM |
| Sample 6 | 4.85 | 20nM |
| Sample 7 | 5.77 | 40nM |
| Sample 8 | 6.66 | 80nM |
Conclusion:
Each test example and embodiment prove above, detection Hg solution of the present invention
2+method can successfully realize quantitative and qualitative analysis detect, there is excellent selectivity (specificity) and sensitivity, and easy and simple to handle, quick, without fixed dna.
Claims (22)
1. a method that detects the mercury ion in solution, the method comprises:
1) described solution and the single stranded DNA that contains thymus pyrimidine (T) are mixed to form to mixture, the single stranded DNA that contains thymus pyrimidine described in making passes through T-Hg
2+-T base pair forms double-stranded DNA, is connected with signal and produces molecule at least one deoxyribonucleotide of the wherein said single stranded DNA that contains thymus pyrimidine;
2) double-stranded DNA of step 1) gained is contacted with the exonuclease that is specific to double-stranded DNA, make described exonuclease cut described double-stranded DNA, discharge the deoxyribonucleotide that is connected with described signal generation molecule; And
3) detecting step 2) in the signal that connects of the described deoxyribonucleotide that discharges produce molecule.
2. method according to claim 1, the ion concentration of mercury of wherein said solution is 0.2nM to 200 μ M.
3. method according to claim 1, wherein said thymus pyrimidine accounts for the 50%-80% of the total alkali radix of described single stranded DNA.
4. method according to claim 3, the wherein said single stranded DNA that contains thymus pyrimidine has the length of 20-40 Nucleotide.
5. method according to claim 1, wherein in the mixture described in step 1), described in contain thymus pyrimidine the concentration of single stranded DNA be 1-5 μ M.
6. method according to claim 1, wherein, in the described single stranded DNA that contains thymus pyrimidine, described in be connected with signal and produce the deoxyribonucleotide of molecule and account for the 3.8%-30.8% of the total nucleotide number of described single stranded DNA.
7. method according to claim 1, it is electric active molecule that wherein said signal produces molecule.
8. method according to claim 7, wherein said electric active molecule is selected from methylene blue and ferrocene.
9. method according to claim 1, wherein in step 2) in, described double-stranded DNA reacts 10-30 minute with described exonuclease at 30-50 DEG C.
10. method according to claim 1, wherein said exonuclease is selected from Escherichia coli nucleic acid exonucleaseⅢ, lambda particles phage exonuclease and T7 phage gene 6 exonucleases.
11. methods according to claim 1, it is methylene blue that wherein said signal produces molecule, 3 ' end part of the single stranded DNA that contains thymus pyrimidine described in this signal generation molecule is connected to; Described exonuclease is Escherichia coli nucleic acid exonucleaseⅢ.
12. methods according to claim 1, wherein in step 3), utilize to be positioned at the signal that described deoxyribonucleotide that the electronegative indium oxide tin glass electrode detection on electrode chip discharges connects and to produce molecule.
13. according to the method described in any one in claim 1-12, wherein step 2) described in exonuclease cutting that described double-stranded DNA is carried out also comprise: cut the T-Hg in this double-stranded DNA
2+-T base pair, makes the Hg in this base pair
2+dissociate out, thereby be combined with other single stranded DNAs that contain thymus pyrimidine.
14. 1 kinds are detected the test kit of the mercury ion in solution, comprising:
The single stranded DNA that contains thymus pyrimidine, this single stranded DNA that contains thymus pyrimidine comprises at least one and is connected with signal and produces the deoxyribonucleotide of molecule;
Be specific to the exonuclease of double-stranded DNA; And
Detection reagent, the signal that this detection reagent can be connected with the described deoxyribonucleotide of free state produces molecularity and produces signal.
15. test kits according to claim 14, wherein said thymus pyrimidine accounts for the 50%-80% of the total alkali radix of described single stranded DNA.
16. test kits according to claim 14, the wherein said single stranded DNA that contains thymus pyrimidine has the length of 20-40 Nucleotide.
17. test kits according to claim 14, wherein, in the described single stranded DNA that contains thymus pyrimidine, the deoxyribonucleotide that is connected with described signal generation molecule accounts for the 3.8%-30.8% of the total nucleotide number of described single stranded DNA.
18. test kits according to claim 14, it is electric active molecule that wherein said signal produces molecule.
19. test kits according to claim 18, wherein said electric active molecule is selected from methylene blue and ferrocene.
20. test kits according to claim 14, wherein said exonuclease is selected from Escherichia coli nucleic acid exonucleaseⅢ, lambda particles phage exonuclease and T7 phage gene 6 exonucleases.
21. test kits according to claim 14, it is methylene blue that wherein said signal produces molecule, 3 ' end part of the single stranded DNA that contains thymus pyrimidine described in this signal generation molecule is connected to; Described exonuclease is Escherichia coli nucleic acid exonucleaseⅢ.
22. according to the test kit described in any one in claim 14-21, and wherein said detection reagent is the form of electronegative indium oxide tin glass electrode, and this electronegative indium oxide tin glass electrode is positioned on electrode chip.
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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 |
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| CN107228892A (en) * | 2017-05-19 | 2017-10-03 | 福州大学 | Electrochemistry mercury ion sensor of temperature-controllable and preparation method thereof |
| CN107228892B (en) * | 2017-05-19 | 2019-08-09 | 福州大学 | Electrochemistry mercury ion sensor of temperature-controllable and preparation method thereof |
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