CN110044993A - A kind of mercury ion fax sense analyzing detecting method based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition - Google Patents

A kind of mercury ion fax sense analyzing detecting method based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition Download PDF

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CN110044993A
CN110044993A CN201910192948.4A CN201910192948A CN110044993A CN 110044993 A CN110044993 A CN 110044993A CN 201910192948 A CN201910192948 A CN 201910192948A CN 110044993 A CN110044993 A CN 110044993A
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electrode
single stranded
stranded dna
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谢顺碧
唐英
滕柳梅
张进
彭琴
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Chongqing University of Arts and Sciences
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    • 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
    • G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
    • 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
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
    • 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
    • G01N27/416—Systems
    • G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage

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Abstract

The invention discloses a kind of mercury ion fax sense analyzing detecting methods based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition, comprising the following steps: cleans glass-carbon electrode polishing grinding；The electrode of decorated by nano-gold is made in glassy carbon electrode surface electro-deposition nanogold；Amidized beta-cyclodextrin modified is made to the electrode of beta-cyclodextrin modified on nanogold particle, by Hg2+It is added in the mixed solution of DNA A1 and DNA A2 and forms double-strand solution；The DNA A3 of ferrocene label is added in above-mentioned double-strand solution and is incubated for, T-type DNA structure is formed；Exonuclease III is added in T-type DNA structure and forms mixed solution；Mixed solution is added drop-wise to the electrode surface of beta-cyclodextrin modified；Modified electrode is immersed in test bottom liquid and detects its chemical property, realizes Hg according to electrochemical signals2+Quantitative determination；Test the linear and selective of electrochemica biological sensor.

Description

A kind of mercury ion electricity based on III auxiliary mark object of excision enzyme circulation with Host-guest Recognition Sensing analysis detection method
Technical field
The present invention relates to technical field of biological, it particularly relates to which a kind of followed based on III auxiliary mark object of excision enzyme The mercury ion fax sense analyzing detecting method of ring and Host-guest Recognition.
Background technique
Metal not merely depends on the type of metal, physicochemical property to the pros and cons of human body or environment, and metal is present in certainly Concentration and its chemical form in right boundary more play decisive role, even if beneficial metallic element content is above standard Value can also cause body to be poisoned, or even dead.Heavy metal ion is once absorbed by the body, and will produce with intracorporal protein and enzyme Raw huge interaction.Micro heavy metal ion will not cause harm to the human body, still, if heavy metal ion is dense in vivo Height is spent, then can change other essential metal ions in the three-dimensional structure of enzyme, the charge in activated centre or substituted enzyme, make enzyme Inactivation, can also largely assemble in the intracorporal certain organs of people, if it exceeds the range that human body is able to bear, it will be to human body Generate very big toxic action.Wherein mercury ion is exactly to one of maximum heavy metal ion of human health risk, is common people Known Japanese minamata disease is to lead to patient's nervous centralis by the fish of Methylmercury pollution Riverfish and shellfish due to edible for patients Systemic disease, methyl mercury are a kind of organic mercuries, and physical aspect is the powder of white, it is easy to be absorbed by stomach, pass through blood Liquid recycles the liver stomach function regulating for being first into human body, is then transferred in the brain or fetus body of human body, and accumulate in this always, Influence and damage very serious are brought to human health.
The technology of detection heavy metal mainly has spectroscopic methodology such as Atomic absorption, emission spectrum, spectrophotometry, chromatography at present Method, mass spectrography and electrochemical process are if any cyclic voltammetry, polarography, potentiometry, ampere, conductimetry etc..Pass through electrochemistry Anodic stripping voltammetry is not necessarily to high pressure gas, flame, atomic lamp, experiment is simple, low in cost etc. excellent though having high sensitivity Point, but anodic stripping voltammetry operation needs personnel's operation of profession, but mercury brings new heavy metal to environment as electrode Pollution.The testing cost of spectrophotometry etc., this method is low, but sensitivity is not high, and the detection for not being able to satisfy national standard is wanted It asks；And the low, high sensitivity of the detections such as atomic absorption spectrography (AAS), inductively coupled plasma body (ICP) method limit, selectivity are good, but valence Lattice are expensive, need technical professional, and detection process is relatively laborious, time-consuming etc..In real time, monitor on-line environment in heavy metal from Son pollution, sensitivity, stability, simplification and the high efficiency for improving detection method are very important.
In recent years, electrochemical sensor is because it is easy, quick, sensitive, at low cost, be easy to be miniaturized, online inspection can be achieved The features such as survey, has attracted the extensive concern of researcher.Wherein, electrochemica biological sensor turns due to combining electrochemistry well The high sensitivity of changing device and bio-identification system it is highly selective, have biggish application can in Analysis of Heavy Metal Ions field Row and bright prospects.When building is used for the electrochemica biological sensor of detection of heavy metal ion, in order to guarantee the spy of sensor The selection of the opposite sex, recognition component is most important.Currently, by the phyletic evolution technology (SELEX) of index concentration ligand from people The aptamer for capableing of the certain heavy metal ion of specific recognition has been filtered out in the random single chain oligonucleotide library of work building (aptamer) and the deoxyribozyme with catalytic activity (DNAzyme), the detection for heavy metal ion.2006, Miyake et al. reports Hg2+It can be a kind of also more stable than base pairing in conjunction with being formed with thymidine T base specific Structure T-Hg2+- T compound, this discovery, which has been established, detects Hg using aptamer probe2+Basis.This patent utilizes T-Hg2+-T Coordinative Chemistry, using DNA as pickup probe, introduce EXOIII auxiliary object recycle amplifying technique, in conjunction with ferrocene and β- The Host-guest Recognition of cyclodextrin acts on, and building electrochemica biological sensor realizes the highly sensitive detection to mercury ion.
Summary of the invention
For the problems in the relevant technologies, the present invention proposes a kind of based on III auxiliary mark object of excision enzyme circulation and Subjective and Objective The mercury ion fax sense analyzing detecting method of identification, to overcome above-mentioned technical problem present in existing the relevant technologies.
For this purpose, the specific technical solution that the present invention uses is as follows:
A kind of mercury ion fax sense analyzing detecting method based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition.
Method includes the following steps:
Preprepared glass-carbon electrode is subjected to polishing grinding, it is clear that the glass-carbon electrode after polishing is carried out ultrasound It washes, juxtaposition is dried spare at room temperature；
The glass-carbon electrode after drying is immersed in HAuCl4In solution and the deposition that is powered, nanogold electrode is made；
The nanogold electrode is immersed in preprepared amidized beta-cyclodextrin, beta-cyclodextrin modified is made Electrode, be placed in room temperature storage it is spare；
By the Hg of preprepared various concentration2+It is added in the mixed solution of single stranded DNA A1 and single stranded DNA A2, Being formed has T-Hg2+The partial complementarity of-T structure matches DNA double-strand solution；
The single stranded DNA A3 for first passing through ferrocene label in advance is added in the DNA double chain solution and is incubated for, T-type structure is formed DNA solution；
Preprepared exonuclease III is added in the T-type structure DNA solution and is incubated for, is formed rich in described The mixed solution of ferrocene；
The mixed solution is added drop-wise to the electrode surface of the beta-cyclodextrin modified, forms the master for being enriched with the ferrocene Modified electrode is made in guest complex；
The modified electrode is immersed in preprepared PBS test bottom liquid and connects electrochemical workstation, is used Cyclic voltammetry CV carries out electrochemical Characterization measurement to the modified electrode, and electrochemica biological sensor is made；
The electrochemica biological sensor is immersed in PBS detection bottom liquid and connects electrochemical workstation, using difference Pulse voltammetry DPV detects the chemical property of the electrochemica biological sensor and measures Hg in the PBS detection bottom liquid2+'s Concentration；
The electrochemica biological sensor is tested to Hg using differential pulse voltammetry DPV2+Specific selectivity.
Further, by preprepared glass-carbon electrode carry out polishing grinding, by the glass-carbon electrode after polishing into Row ultrasonic cleaning the step of include:
Successively use preprepared 0.3 μM and 0.05 μM of aluminum oxide powder on chamois leather the glass-carbon electrode Polishing grinding, also, need to fix when polishing and be ground clockwise or counterclockwise；
The glass-carbon electrode after polishing is successively subjected to ultrasonic cleaning number in preprepared ethyl alcohol and ultrapure water, Scavenging period is 3-5 minutes.
Further, the HAuCl4The mass fraction of solution is 1%, and the current potential of the deposition that is powered is -0.2V, described The time for the deposition that is powered is 30 seconds.
Further, the concentration of the amidized beta-cyclodextrin is 1mM, and the modified nano gold electrode will be made and be placed in The room temperature resting period is 12 hours.
Further, the sequence of the single stranded DNA A1 is 5'-TCTTCTTCTTAACACACGG-3', the single stranded DNA The sequence of A2 is 5'-GCTGAGGGCGTTGTTGTTGT-3', and the sequence of the single stranded DNA A3 is 5'- CCGTGTGTGGCGCCCTCAGC-Fc-3', the single stranded DNA A1 and the single stranded DNA A2 and the single stranded DNA A3's Volume is that the concentration of 10 μ L, the single stranded DNA A1 and the single stranded DNA A2 and single stranded DNA A3 is 2 μM, institute Stating incubation temperature is 37 DEG C, and the incubation time is 1 hour.
Further, it is 0.5U/ that the volume of the exonuclease III, which is the concentration of 10 μ L, the exonuclease III, μL。
Further, the modified electrode is immersed in preprepared PBS test bottom liquid and connects electrochemistry work It stands, electrochemical Characterization measurement is carried out to the modified electrode using cyclic voltammetry CV, electrochemica biological sensor is made Step includes:
Preprepared 2mL is contained to the 5mmol/L [Fe (CN) of 0.2mol/LKCl6]3-/4-The PBS test bottom Liquid energization is scanned characterization to the variation of modified electrode modification median surface electrochemical properties；
Wherein, scanning current potential is -0.6V-0.2V, potential scanning speed 100mV/s, the pH=of the PBS test bottom liquid 7.0。
Further, the electrochemica biological sensor is immersed in PBS detection bottom liquid and rather connects electrochemical workstation, The chemical property of the electrochemica biological sensor is detected using differential pulse voltammetry DPV and measures PBS detection bottom Hg in liquid2+Concentration the step of include:
The test bottom liquid of PBS described in preprepared 0.1mol/L is powered, electricity is carried out to the electrochemica biological sensor The test of the response performance of pole；
By the Hg of the electrochemica biological sensor detection various concentration after test2+And the DPV for recording its electrochemistry is rung It answers；
Wherein, the amplitude of the differential pulse voltammetry DPV is 0.05V, sampling width 0.0167s, and pulse width is 0.1s, scanning current potential are 0.2-0.7V.
Further, the electrochemica biological sensor is tested to Hg using differential pulse voltammetry DPV2+Special selection The step of property includes:
Using differential pulse voltammetry DPV respectively to the Mn of pre-prepd 100nmol/L2+, 100nmol/L Cd2+、 The Pb of 100nmol/L2+, 100nmol/L Cu2+And the Hg of 2nmol/L2+Detection pair is carried out in same experiment condition Than.
Further, the experimental system of the cyclic voltammetry CV and differential pulse voltammetry DPV is three electrodes System, working electrode are glass-carbon electrode, are platinum filament to electrode, reference electrode is saturated calomel electrode.
The invention has the benefit that
(1), the structure of the design and characterization and highly sensitive heavy metal ion electrochemica biological sensor of signal amplification strategy It builds, response mechanism and its application study；
(2), the immobilized rate and rush of beta-cyclodextrin are improved using the big specific surface area of nanogold and good electric action Into electron transmission to achieve the purpose that improve sensitivity；
(3), combine T-Hg2+- T structure and exonuclease Ш realize recycling for object to the shear action of DNA To amplify response signal；
(4), it is acted on by the Host-guest Recognition of ferrocene Fc and beta-cyclodextrin and realizes object quantitative detection；
(5), using the power of electrochemical techniques inspecting electrode assembling process and signal amplification mechanism and nucleic acid interaction Learn feature；
(6), constructed electrochemica biological sensor has the characteristics that highly sensitive, biological recognition system is selectively high, And it also has many advantages, such as that simple structure, low in cost, fast response time, stability are high.
Detailed description of the invention
It in order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, below will be to institute in embodiment Attached drawing to be used is needed to be briefly described, it should be apparent that, the accompanying drawings in the following description is only some implementations of the invention Example, for those of ordinary skill in the art, without creative efforts, can also obtain according to these attached drawings Obtain other attached drawings.
Fig. 1 is a kind of mercury based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition according to an embodiment of the present invention The flow chart of ion-conductance sensing analysis detection method；
Fig. 2 is a kind of mercury based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition according to an embodiment of the present invention The building schematic diagram of the electrochemica biological sensor of ion-conductance sensing analysis detection method；
Fig. 3 is a kind of mercury based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition according to an embodiment of the present invention The electrode of the ion-conductance sensing analysis detection method CV response diagram that gradually modification characterizes；
Fig. 4 is a kind of mercury based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition according to an embodiment of the present invention The optimization figure of the incubation time of ion-conductance sensing analysis detection method；
Fig. 5 is a kind of mercury based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition according to an embodiment of the present invention The various concentration Hg of ion-conductance sensing analysis detection method2+The DPV response diagram of detection；
Fig. 6 is a kind of mercury based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition according to an embodiment of the present invention Ion-conductance sensing analysis detection method to Hg2+The canonical plotting of detection；
Fig. 7 is a kind of mercury based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition according to an embodiment of the present invention The selective analysis chart of the sensor of ion-conductance sensing analysis detection method.
Specific embodiment
To further illustrate that each embodiment, the present invention are provided with attached drawing, these attached drawings are that the invention discloses one of content Point, mainly to illustrate embodiment, and the associated description of specification can be cooperated to explain the operation principles of embodiment, cooperation ginseng Examine these contents, those of ordinary skill in the art will be understood that other possible embodiments and advantages of the present invention, in figure Component be not necessarily to scale, and similar component symbol is conventionally used to indicate similar component.
According to an embodiment of the invention, providing a kind of based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition Mercury ion fax sense analyzing detecting method.
Now in conjunction with the drawings and specific embodiments, the present invention is further described, as shown in Figure 1, according to embodiments of the present invention Based on III auxiliary mark object of excision enzyme circulation and the mercury ion fax sense analyzing detecting method of Host-guest Recognition.
Method includes the following steps:
Step S101, by preprepared glass-carbon electrode carry out polishing grinding, by the glass-carbon electrode after polishing into Row ultrasonic cleaning, juxtaposition is dried spare at room temperature；
Step S102, the glass-carbon electrode after drying are immersed in HAuCl4In solution and the deposition that is powered, nanometer is made Gold electrode；
The nanogold electrode is immersed in preprepared amidized β-cyclodextrin by step S103, and β-is made It is spare to be placed in room temperature storage for cyclodextrin modified electrode；
Step S104, by the Hg of preprepared various concentration2+It is added to the mixed of single stranded DNA A1 and single stranded DNA A2 It closes in solution, being formed has T-Hg2+The partial complementarity of-T structure matches DNA double chain solution；
The single stranded DNA A3 for first passing through ferrocene label in advance is added in the DNA double-strand solution and is incubated for by step S105, Form T-type structure DNA solution；
Preprepared exonuclease III is added in the T-type structure DNA solution and is incubated for by step S106, shape At the mixed solution for being rich in the ferrocene；
The mixed solution is added drop-wise to the electrode surface of the beta-cyclodextrin modified by step S107, is formed described in enrichment Modified electrode is made in the host-guest complex of ferrocene；In specific application, being sheared the ferrocene Fc to get off can be concentrated In the modified nano gold electrode surface, a detectable electrochemical signals are thus obtained；
The modified electrode is immersed in preprepared PBS test bottom liquid and connects electrochemistry work by step S108 It stands, electrochemical Characterization measurement is carried out to the modified electrode using cyclic voltammetry CV, electrochemica biological sensor is made；Institute Building and the principle for stating electrochemica biological sensor are as shown in Figure 2；
The electrochemica biological sensor is immersed in PBS detection bottom liquid and is connected electrochemical workstation by step S109, The chemical property of the electrochemica biological sensor is detected using differential pulse voltammetry DPV and measures PBS detection bottom Hg in liquid2+Concentration；
Step S110 tests the electrochemica biological sensor to Hg using differential pulse voltammetry DPV2+Special selection Property.
In one embodiment, preprepared glass-carbon electrode is subjected to polishing grinding, by the glass carbon after polishing The step of electrode is cleaned by ultrasonic include:
Successively use preprepared 0.3 μM and 0.05 μM of aluminum oxide powder on chamois leather the glass-carbon electrode Polishing grinding, also, need to fix when polishing and be ground clockwise or counterclockwise；
The glass-carbon electrode after polishing is successively subjected to ultrasonic cleaning number in preprepared ethyl alcohol and ultrapure water, Scavenging period is 3-5 minutes.
In one embodiment, the HAuCl4The mass fraction of solution be 1%, it is described be powered deposition current potential be- The time of 0.2V, the deposition that is powered are 30 seconds；To deposit one layer of nanogold particle (AuNPs) in glassy carbon electrode surface, into And nanogold electrode is made.
In one embodiment, the concentration of the amidized beta-cyclodextrin is 1mM, and the modified nano gold electricity will be made Pole be placed in the room temperature resting period be 12 hours；To which by Au-N key, amidized beta-cyclodextrin is assembled in nanogold particle (AuNPs) on, and then modified nano gold electrode is made.
In one embodiment, the sequence of the single stranded DNA A1 is 5'-TCTTCTTCTTAACACACGG-3', described The sequence of single stranded DNA A2 is 5'-GCTGAGGGCGTTGTTGTTGT-3', and the sequence of the single stranded DNA A3 is 5'- The body of CCGTGTGTGGCGCCCTCAGC-Fc-3', the single stranded DNA A1 and the single stranded DNA A2 and the single stranded DNA A3 Product is that the concentration of 10 μ L, the single stranded DNA A1 and the single stranded DNA A2 and single stranded DNA A3 is 2 μM, described to incubate Educating temperature is 37 DEG C, and the incubation time is 1 hour.
In addition, in specific application, by the Hg of preprepared various concentration2+Be added to single stranded DNA A1 with it is single-stranded In the mixed solution of DNA A2, being formed has T-Hg2+The partial complementarity of-T structure matches DNA double chain solution；Two will be first passed through in advance The step of being incubated for, forming T-type structure DNA solution is added in the DNA double chain solution in the single stranded DNA A3 of luxuriant iron label specifically: By the Hg of various concentration2+It is added in the mixed solution of each 2 μM of 10 μ L of single stranded DNA A1 and single stranded DNA A2, in Hg2+It deposits Under the conditions, single stranded DNA A1 and single stranded DNA A2 forms T-Hg2+- T structure, to form the DNA double of part complementary pairing Chain.
In 37 after the single stranded DNA A3 for electroactive substance ferrocene (Fc) label that 2 μM of 10 μ L is further added It is incubated for 1 hour at DEG C, single stranded DNA A3 is complementary with the exposed cohesive end of single stranded DNA A1 and single stranded DNA A2 at this time, in turn Form the DNA structure of T-type.
In one embodiment, the volume of the exonuclease III is 10 μ L, the concentration of the exonuclease III For 0.5U/ μ L.In addition, in specific application, the exonuclease III of 10 μ L 0.5U/ μ L is added in above-mentioned mixed liquor (EXO III) is incubated for 1 hour at 37 DEG C, and exonuclease III is sheared from the flush end of complementary DNA double-strand from the end 3'-5' at this time Single stranded DNA A3 forms the part of DNA double chain, and ferrocene Fc of the label on single stranded DNA A3 is shear off, and is discharged into In solution；Single stranded DNA A1, single stranded DNA A2 and Hg simultaneously2+The DNA double chain structure of formation participates in next complementary and shearing Circulation, can be obtained by the ferrocene Fc largely shear off by multiple circulation shear.
In one embodiment, the modified electrode is immersed in preprepared PBS test bottom liquid and connects electricity Chem workstation carries out electrochemical Characterization measurement to the modified electrode using cyclic voltammetry CV, electrochemica biological is made and passes The step of sensor includes:
Preprepared 2mL is contained to the 5mmol/L [Fe (CN) of 0.2mol/LKCl6]3-/4-The PBS test bottom Liquid energization is scanned characterization to the variation of modified electrode modification median surface electrochemical properties；
Wherein, scanning current potential is -0.6V-0.2V, potential scanning speed 100mV/s, the pH of the PBS test bottom liquid =7.0.
In addition, electrochemical Characterization measurement is carried out in specific application to the modified electrode using cyclic voltammetry CV, 5mmol/L[Fe(CN)6]3-/4-Test bottom liquid in record different modifying electrode CV response, as shown in figure 3, in figure I As can be seen that curve b (GCE) corresponding to bare electrode shows a pair of [Fe (CN) that will definitely be inverse6]3-/4-Redox Peak；After glass-carbon electrode deposits one layer of nanogold, since nanogold can promote electron-transport, CV response current value is Increase (curve a, GCE/Au), after electrode surface is incubated for beta-cyclodextrin, observed the response current value (curve further decreased C, GCE/Au/ β-CD) illustrate that β-cyclodextrin has successfully been assembled in electrode surface；The corresponding CV response of electrode different modifying stage Variation, illustrate that modified electrode is successfully prepared.
In one embodiment, the electrochemica biological sensor is immersed in PBS detection bottom liquid and rather connects electrochemistry Work station detects the chemical property of the electrochemica biological sensor using differential pulse voltammetry DPV and measures the PBS Detect Hg in the liquid of bottom2+Concentration the step of include:
The test bottom liquid of PBS described in preprepared 0.1mol/L is powered, electricity is carried out to the electrochemica biological sensor The test of the response performance of pole；
By the Hg of the electrochemica biological sensor detection various concentration after test2+And the DPV for recording its electrochemistry is rung It answers；
Wherein, the amplitude of the differential pulse voltammetry DPV is 0.05V, sampling width 0.0167s, and pulse width is 0.1s, scanning current potential are 0.2-0.7V.
In addition, being powered the test bottom liquid of PBS described in preprepared 0.1mol/L to the electrochemica biological sensor Carry out electrode response performance test in specific application, in order to increase the sensitivity and selection of electrochemica biological sensor Property, we optimize the incubation time of exonuclease III (EXO III), finally still use Impulsive Difference voltammetry DPV detects to study the chemical property of electrochemica biological sensor, as shown in figure 4, prolonging with EXO III incubation time Long, we can significantly observe that current-responsive gradually increases and current value tends to be steady when 50min, therefore select The incubation time of EXO III is optimum performance of the 50min to ensure sensor.
By the Hg of the electrochemica biological sensor detection various concentration after test2+And the DPV for recording its electrochemistry is rung It should in specific application, in order to assess the sensitivity of constructed electrochemica biological sensor and the potential of quantitative detection, most Under excellent experiment condition, we detect the Hg of various concentration with the electrochemica biological sensor of building2+And record the DPV of its electrochemistry Response, that electrochemica biological sensor detection is 0.002 Hg for arriving 100nmol/L various concentration as can be seen from Figure 52+Peak Current value, and reduction peak current value is with Hg2+The increase of concentration and gradually increase, it means that under the action of EXO III, be sheared The electric active molecule ferrocene Fc to get off, the beta-cyclodextrin for being modified at electrode surface successfully capture；Hg2+The standard of detection is bent Line as shown in fig. 6, current-responsive value 0.002 to 100nmol/L concentration range in and Hg2+Logarithm between present Good linear relationship；Its linear equation is I=-0.0608 lg c-2.1413, c Hg2+Concentration, relative coefficient is R=0.9971；According to this, the experimental results showed that, the constructed electrochemica biological sensor of this experiment can be used in Hg2+Gao Ling The detection of quick property.
In one embodiment, the electrochemica biological sensor is tested to Hg2+'s using differential pulse voltammetry DPV The step of specific selectivity includes:
Using differential pulse voltammetry DPV respectively to the Mn of pre-prepd 100nmol/L2+, 100nmol/L Cd2+、 The Pb of 100nmol/L2+, 100nmol/L Cu2+And the Hg of 2nmol/L2+Detection pair is carried out in same experiment condition Than.
In addition, in specific application, in order to test the electrochemica biological sensor of this experimental construction to Hg2+Special selection Property, we use the Mn of 100nmol/L respectively2+, 100nmol/L Cd2+, 100nmol/L Pb2+And the Cu of 100nmol/L2+Four Kind of ion in same experiment condition with the Hg of 2nmol/L2+Testing result compare；As shown in fig. 7, a is sky White value, b are the Mn of 100nmol/L2+, c is the Cu of 100nmol/L2+, d is the Pb of 100nmol/L2+, e is 100nmol/L's Cd2+, f is the Hg of 2nmol/L2+, the electric signal of this four kinds of ions is all lower and suitable with blank value, only object Hg2+It generates Obviously electrochemical signals, this illustrates the electrochemica biological sensor detection method of this experimental construction to Hg2+Detection Specificity with higher.
In one embodiment, the experimental system of the cyclic voltammetry CV and the differential pulse voltammetry DPV are Three-electrode system, working electrode are glass-carbon electrode (mm of GCE, Φ=4), are platinum filament to electrode, and reference electrode is saturation calomel electricity Pole (SCE).
In conclusion above-mentioned technical proposal of the invention, it is related to the design and characterization and highly sensitive of signal amplification strategy Building, response mechanism and its application study of heavy metal ion electrochemica biological sensor；Utilize the big specific surface area of nanogold And good electric action come improve beta-cyclodextrin immobilized rate and promote electron transmission with achieve the purpose that improve sensitivity；Connection Close T-Hg2+- T structure and exonuclease Ш realize that recycling for object is believed with amplification response to the shear action of DNA Number；It is acted on by the Host-guest Recognition of ferrocene Fc and beta-cyclodextrin and realizes object quantitative detection；It is examined using electrochemical techniques Examine the dynamic characteristic of electrode assembling process and signal amplification mechanism and nucleic acid interaction；And then constructed electrochemica biological Sensor has the characteristics that highly sensitive, biological recognition system is selectively high, and its also have simple structure, it is low in cost, The advantages that fast response time, high stability.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.

Claims (10)

1. a kind of mercury ion fax sense analyzing detecting method based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition, It is characterized in that, comprising the following steps:
Preprepared glass-carbon electrode is subjected to polishing grinding, the glass-carbon electrode after polishing is cleaned by ultrasonic, and Be placed in dry at room temperature it is spare；
The glass-carbon electrode after drying is immersed in HAuCl4In solution and the deposition that is powered, nanogold electrode is made；
The nanogold electrode is immersed in preprepared amidized beta-cyclodextrin, the electricity of beta-cyclodextrin modified is made It is spare to be placed in room temperature storage for pole；
By the Hg of preprepared various concentration2+It is added in the mixed solution of single stranded DNA A1 and single stranded DNA A2, is formed With T-Hg2+The partial complementarity of-T structure matches DNA double chain solution；
The single stranded DNA A3 for first passing through ferrocene label in advance is added in the DNA double chain solution and is incubated for, T-type structure DNA is formed Solution；
Preprepared exonuclease III is added in the T-type structure DNA solution and is incubated for, is formed rich in two cyclopentadienyl The mixed solution of iron；
The mixed solution is added drop-wise to the electrode surface of the beta-cyclodextrin modified, forms the Subjective and Objective for being enriched with the ferrocene Modified electrode is made in compound；
The modified electrode is immersed in preprepared PBS test bottom liquid and connects electrochemical workstation, using circulation Voltammetry CV carries out electrochemical Characterization measurement to the modified electrode, and electrochemica biological sensor is made；
The electrochemica biological sensor is immersed in PBS detection bottom liquid and connects electrochemical workstation, using differential pulse Voltammetry DPV detects the chemical property of the electrochemica biological sensor and measures Hg in the PBS detection bottom liquid2+It is dense Degree；
The electrochemica biological sensor is tested to Hg using differential pulse voltammetry DPV2+Specific selectivity.
2. the mercury ion fax sense according to claim 1 based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition Analyzing detecting method, which is characterized in that preprepared glass-carbon electrode is subjected to polishing grinding, by the glass carbon after polishing The step of electrode is cleaned by ultrasonic include:
The glass-carbon electrode is successively polished on chamois leather with the aluminum oxide powder of preprepared 0.3 μM and 0.05 μM Polishing, also, need to fix when polishing and be ground clockwise or counterclockwise；
The glass-carbon electrode after polishing is successively subjected to ultrasonic cleaning number in preprepared ethyl alcohol and ultrapure water, is cleaned Time is 3-5 minutes.
3. the mercury ion fax sense according to claim 1 based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition Analyzing detecting method, which is characterized in that the HAuCl4The mass fraction of solution be 1%, it is described be powered deposition current potential be- The time of 0.2V, the deposition that is powered are 30 seconds.
4. the mercury ion fax sense according to claim 1 based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition Analyzing detecting method, which is characterized in that the concentration of the amidized beta-cyclodextrin is 1mM, and the modified nano gold will be made Electrode be placed in the room temperature resting period be 12 hours.
5. the mercury ion fax sense according to claim 1 based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition Analyzing detecting method, which is characterized in that the sequence of the single stranded DNA A1 is 5'-TCTTCTTCTTAACACACGG-3', described The sequence of single stranded DNA A2 is 5'-GCTGAGGGCGTTGTTGTTGT-3', and the sequence of the single stranded DNA A3 is 5'- The body of CCGTGTGTGGCGCCCTCAGC-Fc-3', the single stranded DNA A1 and the single stranded DNA A2 and the single stranded DNA A3 Product is that the concentration of 10 μ L, the single stranded DNA A1 and the single stranded DNA A2 and single stranded DNA A3 is 2 μM, described to incubate Educating temperature is 37 DEG C, and the incubation time is 1 hour.
6. the mercury ion fax sense according to claim 1 based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition Analyzing detecting method, which is characterized in that the volume of the exonuclease III is 10 μ L, the concentration of the exonuclease III For 0.5U/ μ L.
7. the mercury ion fax sense according to claim 1 based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition Analyzing detecting method, which is characterized in that the modified electrode is immersed in preprepared PBS test bottom liquid and connects electricity Chem workstation carries out electrochemical Characterization measurement to the modified electrode using cyclic voltammetry CV, electrochemica biological is made and passes The step of sensor includes:
Preprepared 2mL is contained to the 5mmol/L [Fe (CN) of 0.2mol/L KCl6]3-/4-PBS test bottom liquid it is logical Electricity is scanned characterization to the variation of modified electrode modification median surface electrochemical properties；
Wherein, scanning current potential is -0.6V-0.2V, potential scanning speed 100mV/s, the pH=7.0 of the PBS test bottom liquid.
8. the mercury ion fax sense according to claim 1 based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition Analyzing detecting method, which is characterized in that the electrochemica biological sensor is immersed in PBS detection bottom liquid and connects electrochemistry work It stands, the chemical property of the electrochemica biological sensor is detected using differential pulse voltammetry DPV and measures the PBS and is examined Survey Hg in the liquid of bottom2+Concentration the step of include:
The test bottom liquid of PBS described in preprepared 0.1mol/L is powered, electrode is carried out to the electrochemica biological sensor The test of response performance；
By the Hg of the electrochemica biological sensor detection various concentration after test2+And record the DPV response of its electrochemistry；
Wherein, the amplitude of the differential pulse voltammetry DPV be 0.05V, sampling width 0.0167s, pulse width 0.1s, Scanning current potential is 0.2-0.7V.
9. the mercury ion fax sense according to claim 1 based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition Analyzing detecting method, which is characterized in that the electrochemica biological sensor is tested to Hg using differential pulse voltammetry DPV2+'s The step of specific selectivity includes:
Using differential pulse voltammetry DPV respectively to the Mn of pre-prepd 100nmol/L2+, 100nmol/L Cd2+、 The Pb of 100nmol/L2+, 100nmol/L Cu2+And the Hg of 2nmol/L2+Detection pair is carried out in same experiment condition Than.
10. the mercury ion fax according to claim 1 based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition Feel analyzing detecting method, which is characterized in that the experimental system of the cyclic voltammetry CV and the differential pulse voltammetry DPV are equal For three-electrode system, working electrode is glass-carbon electrode, is platinum filament to electrode, reference electrode is saturated calomel electrode.
CN201910192948.4A 2019-03-14 2019-03-14 A kind of mercury ion fax sense analyzing detecting method based on III auxiliary mark object of excision enzyme circulation and Host-guest Recognition Withdrawn CN110044993A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111398396A (en) * 2020-05-22 2020-07-10 河南工业大学 Heavy metal Hg2+And Pb2+Preparation method of electrochemical sensor capable of detecting simultaneously

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111398396A (en) * 2020-05-22 2020-07-10 河南工业大学 Heavy metal Hg2+And Pb2+Preparation method of electrochemical sensor capable of detecting simultaneously

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