CN106501343B - A method of heavy metal ion is detected based on polymerase isothermal amplification technique - Google Patents
A method of heavy metal ion is detected based on polymerase isothermal amplification technique Download PDFInfo
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Abstract
The method based on magnetic core-core/shell nanoparticles and polymerase isothermal amplification signal amplification technique detection heavy metal nickel ion that the present invention relates to a kind of, belongs to analytical chemistry or environmental monitoring technology field.Using self-assembling technique, DNA chain S1 is fixed to Fe by Au-S key3O4@Au core-shell structure copolymer magnetic nano-particle surface.Nickel ion (Ni2+) in the presence of, nucleic acid chains S1 is in enzyme site by Ni2+Cutting, the number of base missing at the 5 ' ends of nucleic acid chains S1 cause its 3 ' end nucleic acid chains to become single-stranded, hybridization then occur with the single-stranded S2 of the DNA of another complementary series and is combined.After polymerase and substrate dNTP is added, promote S1 to open under the induction progradation of S2 and isothermal duplication iodine occurs, methylene blue MB is finally embedded into double-stranded DNA gap, the compound of formation realizes the amplification of electrochemical response signal in golden magnetoelectricity pole surface by magnetite gathering.It is realized according to the enhancing of electrochemical signals to Ni in solution2+Measurement, which has the characteristics that highly sensitive, highly selective, simple, quick.
Description
Technical field
The invention belongs to analytical chemistry or environmental monitoring technology fields, and in particular to one kind is based on magnetic nano-particle and gathers
The electrochemical method of synthase isothermal amplification signal amplification technique detection heavy metal nickel ion.
Background technique
Nickel be maintain body metabolism process necessary to microelement, biological action is extremely wide, but nickel and
Its compound is common environmental pollution factors again.Nickel is widely used in the row such as plating, automobile, electrolysis, pesticide, medicine, paint
Industry.Nickel can be sucked by atmosphere and the number of ways such as diet enter human body, will cause when internal savings is excessive nasal cavity, lung and
A series of diseases such as heart, or even induce cancer.Therefore, nickel is one of the heavy metal for needing to monitor in the media such as environment, food.
Currently, the common measuring method of nickel has spectrophotometry, atomic spectroscopy, inductively coupled plasma mass spectrometry and fluorescence method
Deng.These methods not only need the instrument of high precision, require height to operating technology, and detection cycle is long, testing cost is high.Cause
This, needs to develop new method and new technology simple, quick, and that can realize trace detection nickel.
Nucleic acid isothermal amplification technology is a kind of completely new Progress of Nucleic Acid Amplification Technologies that developed recently gets up, reaction process
Always it maintains at a constant temperature, to reach quick by adding enzyme and the respective specific primer (or being not added) of different activities
The purpose of nucleic acid amplification.Compared with Standard PCR technology, thermal denaturation, temperature cycles, electrophoresis and ultraviolet visualization of template etc. are not needed
Process.Requirement of the nucleic acid isothermal amplification technology to instrument greatly simplifies, and the reaction time greatly shortens, and has simple, quick, special
The strong feature of property, has been widely used in all kinds of nucleic acid, virus and the detection of microorganism.1994, pass through in-vitro screening skill
Art obtains the discovery of deoxyribozyme (DNAzyme) and its catalytic performance, promotes the fast development of nucleic acid isothermal amplification technology.It is de-
Oxygen ribozyme is the single stranded DNA for having high-efficiency catalytic activity and structure recognition ability using one kind that directed evolution technology obtains
Molecule.Similar to RNA, deoxyribozyme can form complicated and diversified foldable structure and show catalysis, can be with catalytic dna
Or the multiclass reaction such as RNA cutting, DNA connection, DNA hydrolysis.Compared with traditional protease, deoxyribozyme has stability height, phase
It is small to molecular mass, chemically reactive synthesis, modification and duplication, by the advantages such as the such environmental effects such as acidity are small.Deoxyribozyme is very
It is easy construct by recognition mode or the biosensor of signal representation mode of nucleic acid.In addition, deoxyribozyme have pair
DNA is attached the catalytic capability with fracture, can be applied to gene regulation and treatment.
Magnetic nano-particle is a kind of novel nano-material developed in recent years, since magnetic nano-particle has spy
Different magnetic conductance tropism, superparamagnetism, easy cleaning, easily separated and surface can connect the characteristics such as biochemical activity functional group, make it
Application in fields such as foranalysis of nucleic acids, clinical diagnosis, targeted drug, cell separation and enzyme immobilizations has obtained extensive development.
These characteristics of magnetic nano-particle can improve the sensitivity of biosensor detection significantly, shorten the biochemical reaction time,
Detection flux is improved, opens wide prospect for the application of field of biosensors.
The present invention is based on magnetic nano-particles and polymerase isothermal amplification as signal amplification technique, utilizes Ni2+It is right
The RNA locus specificity of DNA enzymatic is cut and electrochemical method, realizes to heavy metal Ni2+Ultra trace detection.In order to increase analysis
Rate and stability, magnetic nanometer surface occur the long chain DNA compound that polymerase isothermal amplification is formed and use golden magnetic
Electrode adsorption is fixed, and this method has many advantages, such as simple, quick, high sensitivity.
Summary of the invention
An object of the present invention is to design not homotactic DNA, constructs a kind of quick, sensitive electrochemical sensor.
It is a kind of based on magnetic nano-particle and polymerase isothermal amplification amplification skill the second object of the present invention is to provide
The preparation method of art combination building electrochemical sensor.
The third object of the present invention is that the electrochemical sensor of building is used for highly sensitive, the quick inspection of heavy metal ion
It surveys.
The specific technical solution of the present invention is as follows:
1. one kind is based on magnetic nano-particle and polymerase isothermal amplification amplifying technique combination building heavy metal ion
Electrochemical sensor
Fe is prepared first3O4DNA nucleic acid chains S1 is fixed to Fe by Au-S key by@Au core-shell structure copolymer magnetic nano-particle3O4@
The surface Au.Ni2+In the presence of, nucleic acid chains S1 is in the site rA by Ni2+Two sections are cut into, its 3 ' end nucleic acid chains is caused to become single-stranded, S1
The single-stranded S2 single-stranded with the DNA of another complementary series in 3 ' ends occur to hybridize and combined.After polymerase and substrate dNTP is added,
Promote S1 to open under the induction progradation of S2 and isothermal duplication iodine occurs, is finally embedded into methylene blue (MB) double
Chain DNA gap.The long chain DNA compound formed realizes putting for electrochemical response signal in golden magnetoelectricity pole surface by magnetite gathering
Greatly.It is realized according to the enhancing of electrochemical signals to Ni in standard solution or sample solution2+The quantitative determination of concentration.
2. one kind is based on magnetic nano-particle and polymerase isothermal amplification amplifying technique combination building heavy metal ion
The preparation method of electrochemical sensor
(1) Fe3O4The preparation of@Au core-shell structure copolymer magnetic nano-particle
Fe is prepared using coprecipitation first3O4, by FeCl2、FeCl3It is dissolved in ultrapure water with dense HCl, ultrasonic deoxidation.It will
Above-mentioned mixed solution is added drop-wise in NaOH solution, the lower logical N of 80 DEG C of stirrings2Gas shielded obtains Fe3O4Magnetic nano-particle.?
Fe3O43- aminopropyl triethoxysilane is added in/ethanol solution, to Fe3O4Particle surface carries out amination.In above-mentioned solution
Middle addition HAuCl4Aqueous solution obtains Fe using sodium citrate as reducing agent3O4@Au core-shell structure copolymer magnetic nano-particle.
(2) double-stranded DNA flag F e3O4The preparation of@Au particle
The Fe that step (1) is obtained3O4@Au magnetic nano-particle reacts 12 h with DNA nucleic acid chains S1 at 37 DEG C, hatching,
Magneto separate obtains the Fe of S1 label3O4@Au.By Ni2+With the Fe of S1 label3O4@Au hatches 1 h at 37 DEG C, and nucleic acid chains S1 exists
DNA enzymatic site is by Ni2+Two sections are cut into, the number of base missing at the 5 ' ends of nucleic acid chains S1 causes its 3 ' end nucleic acid chains to become single
Chain, later in 95 DEG C of 5 min of heating, causing S2 can hold complementary single strand to hybridize 3 H-shapeds into double-stranded DNA at 37 DEG C with S1 3 ', obtain
The Fe of double-stranded DNA label3O4@Au magnetic nano-particle.
(3) polymerase isothermal amplification
In the Fe for the double-stranded DNA label that step (2) obtains3O4Polymerase and substrate are added in@Au Tris-HCl buffer
DNTP, 2 h of hybridization reaction at 37 DEG C, obtains long chain DNA compound.Finally, DNA compound and methylene blue (MB) are anti-at 37 DEG C
3 h are answered, MB is embedded into Fe3O4In the double-stranded DNA on the surface@Au.
(4) Ni2+The preparation of electrochemical sensor
Working electrode (golden carbon electrode) is impregnated into 1 h, ultrapure water cleaning in piranha solution.Then respectively with 0.3
The Al of mm and 0.05 mm2O3Polishing powder polishing, is successively cleaned by ultrasonic in ultrapure water, dehydrated alcohol, ultrapure water.Finally 0.5
M H2SO4In solution, the circle of cyclic voltammetry scan 30, activated electrode are carried out between -0.2 ~ 1.6 V with the speed of sweeping of 50 mV/s.N2
Air-blowing is done spare.The golden carbon electrode table that the long chain DNA compound that step (3) obtains passes through magnetite gathering effect modification to activation
Face constructs heavy metal Ni2+Electrochemical sensor.
3. Ni2+The detection method of ion
(1) it is tested using electrochemical workstation with three-electrode system, saturated calomel electrode is reference electrode, platinum filament electricity
Extremely to electrode, the golden magnetoelectricity extremely working electrode of modification (includes 20 mM in the PBS buffer solution of 10 mM pH 7.4
NaCl and 0.1 M NaClO4) in tested；
(2) heavy metal ion is detected with Differential Pulse Voltammetry (DPV), setting scanning potential range is 0 ~ 1.2
V, sweep speed are 0.1 V/S, record the size of peak current, and save data；
(3) according to the size and Ni of gained DPV peak current2+The relationship of ion concentration draws working curve；
(4) standard addition method is used, the Ni of various concentration is added in water sample solution2+Ion standard solution, according to work
Ni of the method for drafting of curve to various concentration2+Ion is detected.
Compared with prior art, the present invention has the following advantages that and beneficial achievement:
(1) present invention utilizes magnetic nano-particle and the combination building of polymerase isothermal amplification dual signal amplifying technique
Heavy metal ion electrochemical sensor.
(2) of the invention by Fe3O4@Au core-shell structure copolymer magnetic nano-particle is introduced into the building of electrochemical sensor, using gold
Carbon electrode, to increase the stability of modified electrode.
(3) present invention utilizes Ni2+To DNA enzymatic specificity dissection, the DNA of particular sequence is designed, to enhance electricity
Extremely to the selectivity of heavy metal ion, the specificity of sensor is increased.
(4) electrochemical sensor prepared by the present invention is used for heavy metal Ni2+The detection of ion, selectivity with higher and
Anti-interference ability, the range of linearity is wide, and detection limit is low, can be used for ultra trace Ni in actual water sample2+The measurement of ion.
Detailed description of the invention
Fig. 1 is using magnetic nano-particle of the invention and polymerase isothermal amplification amplifying technique detection heavy metal
Ni2+Schematic diagram；
Fig. 2 is using magnetic nano-particle of the invention and polymerase isothermal amplification technique detection various concentration Ni2+DPV
Curve graph；
Fig. 3 is using magnetic nano-particle of the invention and polymerase isothermal amplification technique detection Ni2+Canonical plotting,
Wherein abscissa is Ni2+Concentration, unit is mM or M, and ordinate is DPV peak current, and unit is mA.
Specific embodiment
The present invention is further illustrated combined with specific embodiments below, but does not constitute the further limitation to invention.
Embodiment 1 is a kind of based on magnetic nano-particle and a polymerase isothermal amplification amplifying technique combination detection huge sum of money
Belong to Ni2+Method
The present invention is based on magnetic nano-particles and polymerase isothermal amplification amplifying technique combination detection heavy metal Ni2+'s
Method And Principle is as shown in Figure 1.Nucleic acid chains S1 is fixed to Fe by Au-S key3O4@Au magnetic nano-particle surface.Ni2+In the presence of,
Nucleic acid chains S1 is in DNA enzymatic site by Ni2+Cutting, the number of base missing at the 5 ' ends of nucleic acid chains S1 cause its 3 ' end nucleic acid chains to become
It is single-stranded, and then hybridization occurs with the single-stranded S2 of the DNA of another complementary series and is combined.After polymerase and substrate dNTP is added,
Promote S1 to open under the induction progradation of S2 and isothermal duplication iodine occurs.MB is finally embedded into double-stranded DNA gap,
The long chain DNA compound formed realizes the amplification of electrochemical response signal by magnetite gathering in golden magnetoelectricity pole surface.According to electricity
The enhancing of chemical signal is realized to Ni in standard solution or sample solution2+The quantitative determination of concentration.
The design of 2 oligonucleotide sequence of embodiment
Oligonucleotide sequence designed by the present invention is limited by Chinese Shanghai Sangon raw work bioengineering Shanghai (share)
Company's synthesis, and purified and examined by HPLC, freeze-drying.The oligonucleotide sequence that the present invention designs is as follows:
S1:5 '-ACT CAC TAT ︱ RA ︱ GGA AGA GAT GGA CGT GAG TCG ACT AGA CAC GTC CAT
CTC TGC AGT CGG GTA GTT AAA CCG ACC TTC AGA CAT AGT GAG TAG CA -3'-SH；
S2:5 '-CTA CTC ACT ATG TCT -3 '；
Oligonucleotides is dissolved in ultrapure aqua sterilisa, -18 DEG C save backup.
3 Fe of embodiment3O4The preparation of@Au core-shell structure copolymer magnetic nano-particle
Take 1 g FeCl2×4H2O、2.6 g FeCl3×6H2O and the dense HCl of 0.425 mL are dissolved in 100 mL water, ultrasound
Deoxidation.Above-mentioned mixed liquor is added drop-wise in 125 mL, 0.75 M NaOH solution, N2Lower 80 DEG C of stirrings are protected, Fe is obtained3O4It is magnetic
Nanoparticle.Prepare the Fe of 5 g/L3O40.4 mL 3- aminopropyl-triethoxy silicon is added dropwise in 25 mL of/ethanol solution, ultrasonic disperse
Alkane stirs 7 h at room temperature, is diluted to 1 g/L.It is separately added into 14 mL, 0.6 M HAuCl4×4H20.2 M lemon of O and 0.3 mL
Lemon acid sodium solution, is ultrasonically treated at room temperature, and solution colour becomes black from pale yellow, obtains Fe3O4@Au core-shell structure copolymer magnetic nano particle
Son.
4 oligonucleotides S1 and S2 flag F e of embodiment3O4@Au core-shell structure copolymer magnetic nano-particle
Take 1 mg Fe3O4@Au particle is scattered in the S1 solution of 1 mL, 0.6 mM, and 37 DEG C of waters bath with thermostatic control mix 12 h,
PBS buffer solution cleaning, is made the Fe of S1 label3O4@Au particle.The Fe for taking 60 mL S1 to mark3O4@Au and 60 mL various concentrations
Ni2+Solution hatches 1 h, Tris-HCl buffer solution for cleaning, 95 DEG C of 5 min of heating in 37 DEG C of waters bath with thermostatic control.60 mL 1 are taken again
The Fe of S1 and S2 double-stranded DNA label is made in mM S2 and above-mentioned 3 h of solution hybridization3O4@Au core-shell structure copolymer magnetic nano-particle.
5 polymerase isothermal amplification of embodiment
Take double-stranded DNA flag F e made from 60 mL embodiments 43O4@Au core-shell structure copolymer magnetic nano-particle, with 20 mM pH
7.4 Tris-HCl(contain 60 mM KCl and 10 mM MgCl) above-mentioned solution is diluted to 600 mL, then with 200 mM dNTP
With 25 U/mL polymerases at 37 DEG C 2 h of hybridization reaction, Tris-HCl buffer solution for cleaning, the modification of long chain DNA compound is made
Fe3O4@Au particle.Finally hybridize 3 h, Tris-HCl buffer solution for cleaning at 37 DEG C with 20 mM MB again.
The preparation of 6 electrochemical sensor of embodiment
Golden carbon electrode is impregnated into 1 h, ultrapure water cleaning in piranha solution.Then respectively with 0.3 mm and 0.05 mm
Al2O3Polishing powder polishing, is successively cleaned by ultrasonic in ultrapure water, dehydrated alcohol.In 0.5 M H2SO4In solution, with 50 mV/
The speed of sweeping of s carries out the circle of cyclic voltammetry scan 30, activated electrode between -0.2 ~ 1.6 V.N2Air-blowing is done spare.By what is handled well
Golden carbon electrode immerses in solution obtained by embodiment 5, is fixed in golden magnetoelectricity pole surface using magnetite gathering effect, is made
Ni2+Electrochemical sensor.
The detection of 7 heavy metal ion of embodiment
It is tested using electrochemical workstation with three-electrode system, saturated calomel electrode is reference electrode, platinum electrode
For to electrode, golden magnetoelectricity extremely working electrode.Using Differential Pulse Voltammetry heavy metal Ni2+Concentration, with 10 mM pH
7.4 PBS (contain 20 mM NaCl and 0.1 M NaClO4) it is buffer, potential range is 0 ~ 1.2V, and current potential amplification is 4
MV, pulse period are 0.5 s.Differentiated pulse curve peak current and different Ni2+The relation curve of concentration is as shown in Fig. 2, the peak DPV is electric
Stream and Ni2+Good linear relationship (Fig. 3), related coefficient is presented in concentration within the scope of the mM of 100 aM ~ 1RIt is 0.9962, line
Property equation isi pc (mA) = 2.01logc(M)+37.56, detection is limited to 10 aM.Sensor proposed by the present invention compares
Other sensors have the broader range of linearity and lower detection limit, using magnetic nano-particle and polymerase isothermal duplication
Reaction amplifying technique can realize Ni2+Ultra trace detection.
The measurement of 8 sample of embodiment
Testing sample solution is replaced into Ni2+Ion standard solution, using standard addition method, according to standard curve method to sample
Ni in product solution2+It is measured.Measurement result shows the Ni of testing result and addition2+Concentration of standard solution is consistent, has
The satisfied rate of recovery and relative standard deviation, electrochemical sensor prepared by the present invention can be used for Ni in actual sample2+Measurement.
The embodiment of the present invention is illustrated above in conjunction with attached drawing, but the present invention is not limited to the above embodiments, it can be with
The purpose of innovation and creation according to the present invention makes a variety of variations, under the Spirit Essence and principle of all technical solutions according to the present invention
The changes, modifications, substitutions, combinations, simplifications done should be equivalent substitute mode, as long as meeting goal of the invention of the invention, only
Otherwise away from the present invention is based on magnetic nano-particles and polymerase isothermal amplification amplifying technique combination building heavy metal ion
The technical principle and invention structure of the preparation method of electrochemical sensor, ultra trace heavy metal ion electrochemical determination method and application
Think, belongs to protection scope of the present invention.
Sequence table
Sangon Biotech (Shanghai) Co., Ltd.
DNA artificial sequence
S1:5'-ACT CAC TAT ︱ RA ︱ GGA AGA GAT GGA CGT GAG TCG ACT AGA CAC GTC CAT
CTC TGC AGT CGG GTA GTT AAA CCG ACC TTC AGA CAT AGT GAG TAG CA -3'-SH；
S2:5'-CTA CTC ACT ATG TCT-3'；
Claims (6)
1. a kind of electrochemical method based on polymerase isothermal amplification signal amplification technique detection metal ion, it is characterized in that
By containing DNA enzymatic and can part from hybridization nucleic acid chains S1 by Au-S key be fixed to gold package magnetic nano-particle surface；Ni2+
In the presence of, the DNA enzymatic site of nucleic acid chains S1 is by Ni2+Cutting, nucleic acid chains S1 5 ' end excalations cause 3 ' end nucleic acid chains at
To be single-stranded, the single-stranded S2 of complementary series DNA hybridizes with S1 single stranded portion；After polymerase and substrate dNTP is added, in the induction of S2
Promote S1 to open under progradation and isothermal duplication iodine occur, methylene blue MB is finally embedded into double-stranded DNA gap,
The compound of formation realizes the amplification of electrochemical response signal in golden magnetoelectricity pole surface by magnetite gathering, according to electrochemical signals
Enhancing realize solution in Ni2+The measurement of concentration, comprising the following steps:
(1) design of DNA sequence dna
DNA sequence dna designed by the present invention is as follows:
S1:5 '-ACT CAC TAT ︱ RA ︱ GGA AGA GAT GGA CGT GAG TCG ACT AGA CAC GTC CAT CTC
TGC AGT CGG GTA GTT AAA CCG ACC TTC AGA CAT AGT GAG TAG CA -3'-SH；
S2:5 '-CTA CTC ACT ATG TCT-3 '；
(2) Fe3O4The preparation of@Au core-shell structure copolymer magnetic nano-particle
Fe is prepared using coprecipitation under alkaline condition first3O4Magnetic nano-particle passes through 3- aminopropyl triethoxysilane
Amination is carried out to its surface, utilizes reduction of sodium citrate HAuCl later4·4H2O aqueous solution, in Fe3O4Particle surface is formed
Golden shell obtains Fe3O4@Au core-shell structure copolymer magnetic nano-particle；
(3) double-stranded DNA flag F e3O4The preparation of@Au particle
By Fe3O4@Au particle reacts 12 h with nucleic acid chains S1 at 37 DEG C, hatching, and Magneto separate obtains the Fe of S1 label3O4@Au,
By Ni2+With the Fe of S1 label3O4@Au hatches 1 h at 37 DEG C, and the DNA enzymatic site of nucleic acid chains S1 is by Ni2+Cutting, the 5 ' ends of S1
Excalation cause 3 ' end nucleic acid chains to become single-stranded, in 95 DEG C of 5 min of heating, nucleic acid chains S2 is with S1 single stranded portion in 37 later
DEG C 3 H-shapeds of hybridization obtain the Fe of double-stranded DNA label at double-stranded DNA3O4@Au magnetic nano-particle；
(4) polymerase isothermal amplification
The Fe of double-stranded DNA label3O4In@Au solution, polymerase is added at 37 DEG C and substrate dNTP reacts 2 h, in the induction of S2
Promote S1 to open under progradation and isothermal duplication iodine occurs, finally reacts 3 h at 37 DEG C with methylene blue MB, make MB
It is embedded into double-stranded DNA gap, the compound of formation realizes electrochemical response signal by magnetite gathering in golden magnetoelectricity pole surface
Amplification；
(5) analysis detection
The detection of electrochemical response signal is carried out using electrochemical workstation, it is molten to standard according to the size of electrochemical response signal
Ni in liquid or sample solution2+Concentration is quantitative determined.
2. according to claim 1 a kind of based on polymerase isothermal amplification signal amplification technique detection metal ion
Electrochemical method, which is characterized in that the nanoparticle is Fe3O4@Au core-shell structure copolymer magnetic nano-particle.
3. according to claim 1 a kind of based on polymerase isothermal amplification signal amplification technique detection metal ion
Electrochemical method, which is characterized in that electrochemical signals amplifying technique is based on Fe3O4@Au core-shell structure copolymer magnetic nano-particle and polymerization
Enzyme isothermal amplification dual amplification technology.
4. according to claim 1 a kind of based on polymerase isothermal amplification signal amplification technique detection metal ion
Electrochemical method, which is characterized in that the electrode is golden carbon electrode.
5. according to claim 1 a kind of based on polymerase isothermal amplification signal amplification technique detection metal ion
Electrochemical method, which is characterized in that the electrochemical method is Differential Pulse Voltammetry.
6. according to claim 1 a kind of based on polymerase isothermal amplification signal amplification technique detection metal ion
Electrochemical method, which is characterized in that the isothermal amplification is to use electroactive material methylene blue (MB) as indicator.
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