CN110068602A - A kind of detection method hindering sensitive membrane time-measuring electric potential based on polymer - Google Patents
A kind of detection method hindering sensitive membrane time-measuring electric potential based on polymer Download PDFInfo
- Publication number
- CN110068602A CN110068602A CN201910374232.6A CN201910374232A CN110068602A CN 110068602 A CN110068602 A CN 110068602A CN 201910374232 A CN201910374232 A CN 201910374232A CN 110068602 A CN110068602 A CN 110068602A
- Authority
- CN
- China
- Prior art keywords
- polymer
- sensitive membrane
- electrode
- electric potential
- ion
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/333—Ion-selective electrodes or membranes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Electrochemistry (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Hematology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to the sensitive membrane potentiometric analysis technology based on obstruction, specifically a kind of detection method that sensitive membrane time-measuring electric potential is hindered based on polymer.Using polymer film ion selective electrode as working electrode, using the aptamer, DNA or antigen/antibody of target to be measured as identification molecule, by the load on probe base or marker enzyme or its analog molecule as identification probe, quantitative/qualitative detect is carried out to object in liquid to be detected using the time-measuring electric potential technology of pulse constant current controlling；The method of the present invention has certain versatility, by changing the aptamer sequence of identification molecule or can be realized the detection to different target object using DNA, antigen/antibody.The sensitive membrane containing ion-exchanger can also be utilized in the detection process, and the open circuit potential by measuring electrode realizes the detection of object.
Description
Technical field
It is specifically a kind of quick based on polymer obstruction the present invention relates to the sensitive membrane potentiometric analysis technology based on obstruction
Feel the detection method of film time-measuring electric potential.
Background technique
Copper diethlydithiocarbamate based on polymer film ion selective electrode is that one of electrochemical sensor field is important
Branch.By the change of the ionic flux of polymer sensitive membrane phase, have to the ion conduction processes of research liquid/liquid interface important
Effect.The Copper diethlydithiocarbamate based on stopping effect has excited the great interest of experts and scholars in recent years.However, existing
Potentiometric sensor sensitivity based on stopping effect is not high, and this is mainly due to be difficult to obtain effectively to hinder sensitive membrane interface
The method hindered.In addition, the ionic flux by polymer sensitive membrane phase is smaller, and potential response is low under the conditions of traditional electrode, thus
Sensitivity is to be improved.In order to improve the sensitivity of potentiometric sensor, develops and ion selective electrode sensitive membrane has mutually been applied
The chronopotentiometry technology of pulse constant current, the technology can rapidly and accurately control the ion by polymer sensitive membrane phase
Flux.In the art, ionic flux is the most obvious in the stopping effect of this super special response region, under electric potential signal is obvious
Drop, sensitivity effectively improve.It is therefore possible to use obstruction of the highly sensitive time-measuring electric potential technology to polymer sensitive membrane surface
Effect is effectively monitored.
Summary of the invention
It is an object of the invention to overcome Copper diethlydithiocarbamate under the conditions of traditional electrode, ionic flux is small, and sensitivity is low
Deficiency, provide it is a kind of based on polymer hinder sensitive membrane time-measuring electric potential detection method.
To achieve the above object, the invention adopts a technical scheme as:
A kind of detection method hindering sensitive membrane time-measuring electric potential based on polymer, it is characterised in that: with polymer film ion
Electrodes selective is working electrode, using the aptamer, DNA or antigen/antibody of target to be measured as identification molecule, by
Load or marker enzyme or its analog molecule are as identification probe on probe base, using the time-measuring electric potential of pulse constant current controlling
Technology carries out quantitative/qualitatively detection to object in liquid to be detected；
Object in the solution to be measured is immunoreacted mode or substitution by sandwich in conjunction with identification molecule
Model function, and then change the amount of identification probe, it is made by load on probe or the enzyme marked or its analog catalysis substrate
Polymer is generated in polymer sensitive membrane substrate, polymer hinders the ionic flux of indication ion, reduces electric potential signal, realizes
/ qualitatively detection quantitative to object to be detected.
The probe is to load or mark biologically active enzyme molecule or class enzyme molecule on probe base；
Probe is combined with magnetic bead by DNA hybridization or antigen and antibody specific identification, and the immune knot of sandwich is formed
Structure；Wherein, the DNA fragmentation for capableing of base pair complementarity with identification molecule both ends is marked respectively on probe and magnetic bead, or respectively
The antibody that label can be identified with antigentic specificity on probe and magnetic bead.
The probe base is graphene, single-walled carbon nanotube, double-walled carbon nano-tube, multi-walled carbon nanotube, carbon nanotube
Array, nano wire, fullerene, quantum dot or MOF material；
The enzyme molecule be horseradish peroxidase, alkaline phosphatase lipase, soybean peroxidase, butyrylcholine esterase,
Urinate enzyme, beta galactosidase, laccase, bilirubin oxidase, polyphenol oxidase, tyrosinase, G- tetrad functional enzyme or manganese porphin
Quinoline-DNA chelate；Class enzyme molecule are as follows: graphene oxide, ferroso-ferric oxide, carbon nanotube, gold nano, molybdenum disulfide, MOFs,
Metal oxide nano-material or ferroheme.
The polymer for hindering indication ion flux is that substrate passes through the enzyme or its analog of load or label on probe
The polymer for the obstruction indication ion flux that Journal of Molecular Catalysis is formed in polymer film ion selective electrode sensitive membrane substrate, jail
Admittedly attachment 10-120 minutes；The substrate is dopamine, catechol, p-tert-butyl catechol, aniline, adrenaline, demethyl
Adrenaline or tannic acid.
The target to be measured is antibiotic, bacterium, albumen, cell, virus, heavy metal ion, amino acid, pesticide, algae
Toxin, small organic molecule or DNA；It is described identification molecule be specifically bound with target to be measured sequence, DNA or antigen/
Antibody.
The indication ion is cation, anion or polyion；Such as: calcium ion, magnesium ion, potassium ion, silver ion,
Lead ion, cadmium ion, copper ion, sulfate ion, carbanion, phosphate anion, nucleoprotamine or heparin etc.；It is described poly-
The polymer sensitive membrane of compound film ion selective electrode includes the carrier of indication ion.
The chronopotentiometry is using polymer film ion selective electrode as working electrode, using working electrode, right
The indication ion that the three-electrode system of electrode and reference electrode hinders polymer detects, and obtains indication ion potential response
The qualitative detection to target to be measured can be realized in value；Further according to above-mentioned generation indication ion polymer obstruction before and after electricity
The difference of position variation, can be realized the quantitative detection to target to be measured.
The constant pulse cathode current size applied to working electrode is 0.1-10 μ A, the pulse electricity applied
The stream time is 0.1-10s；
After the application pulse current, by further applying open circuit potential 10-600s to electrode, electrode renaturation is realized more
Newly.
The polymer sensitive membrane is that the film component mainly consists of three parts: for according to parts by weight, 20%-80%
Film matrix, 20%-80% plasticizer, surplus are ion-exchanger；Film matrix be polyvinyl chloride, poly- butyl propyleneglycol acid esters, poly- third
Olefin(e) acid butyl ester, polyetherimide are pressed, rubber or sol-gel film；Plasticizer be ortho-nitrophenyl octyl ether, two -2- ethylhexyl last of the ten Heavenly stems esters,
Dibutyl sebacate or dioctyl sebacate；Ion-exchanger is anionite or cation-exchanger；Wherein, anion
Exchanger is tridodecylmethylammonium ammonium chloride, tridodecylmethylammonium chlorination ammonium derivative, tetratriacontane methyl ammonium
Or tetratriacontane methyl ammonium derivative；Cation-exchanger is four (4- chlorphenyl) potassium borates, four (p-methylphenyl) boron
Sour sodium, four (3,5- bis- (trifluoromethyl) phenyl) Boratex, dinonylnaphthalene sulfonic acid, dinonylnaphthalene sulfonic acid salt or borates or above-mentioned
Each compound derivatives.The conducting shell is obtained by existing usual manner, such as PEDOT/PSS.
Further,
To load HRP enzyme molecule catalysis dopamine on multi-walled carbon nanotube in Calcium signaling sensitivity film surface
For polymerization:
On functionalized multi-wall carbonnanotubes matrix load HRP and Linker DNA molecular, formed have catalytic action and
The identification probe of bonding action.With the aptamer of object to be detected be identification molecule, by DNA hybridization will identify probe and
Aptamer on magnetic bead combines.Identification molecule and probe are fixed on magnetic bead.In the presence of not having object, probe is bonded in
Magnetic bead surfaces, by dopamine and H2O2It is added in alkaline magnetic bead solution, takes appropriate drop on polymer sensitive membrane surface with pipettor
React certain time.HRP molecule on probe can effectively be catalyzed dopamine in polymer sensitive membrane surface aggregate, form one layer
Fine and close poly-dopamine film hinders the ionic flux for passing through sensitive membrane phase under the conditions of time-measuring electric potential, reduces electric potential signal.When having
In the presence of object, object and identification molecule-aptamer are acted on, and probe is released from magnetic bead, by dopamine and H2O2
It is added in alkaline magnetic bead solution, takes appropriate drop to react certain time on polymer sensitive membrane surface with pipettor.Dopamine exists
The auto polymerization of polymer sensitive membrane surface hinders the effect of calcium ion flux unobvious, and electric potential signal decline is little.Therefore, pass through
The detection to object is realized in electric potential signal variation.
Further: using polymer film Calcium signaling as working electrode, using working electrode, to electrode and
The three-electrode system of reference electrode hinders time-measuring electric potential response to detect poly-dopamine.By O2Oxidation dopamine auto polymerization is repaired
The Sensitive membrane electrode of decorations is inserted into 10 with to electrode and reference electrode-5M and 10-4M CaCl2In solution, the amine-modified calcium of DOPA is obtained
Ion sensitive membrane is 10-5—10-4M Ca2+Time-measuring electric potential respond background value.Then HRP is catalyzed H2O2Aoxidize dopamine polymerization
The Sensitive membrane electrode of modification is inserted into 10 with to electrode and reference electrode-5M and 10-4M CaCl2In solution, 10 are obtained-5—10-4M
Ca2+Time-measuring electric potential response.It is finally to quantify current potential by resulting time-measuring electric potential response and potential response background value difference
Value, potential difference are used for the quantitative detection of target to be measured.
The working electrode is the ion selective electrode of mucoadhesive polymers film；Wherein, polymer film is handed over without containing ion
Change agent.By polyvinyl chloride, ortho-nitrophenyl octyl ether, inertia lipophilic salt four (dodecyl)-four (4- chlorphenyl) ammonium borate (ETH
500) it is dissolved in tetrahydrofuran with Calcium ionophore II (ETH 129), it is sensitive that calcium ion polymer can be obtained after mixing evenly
Coating solution.
The constant pulse cathode current size applied to working electrode is 0.1-10 μ A, the pulse electricity applied
The stream time is 0.1-10s；After applying pulse current every time, by further applying open circuit potential 10-600s to electrode, make to be extracted
The indication ion for getting film phase is released back into solution, realizes that electrode updates.
The current potential output signal can also be opening for the polymer film ion selective electrode containing ion-exchanger
Road potential response.
The present invention has the advantages that
The method of the present invention has certain versatility, can be realized by changing the aptamer sequence of identification molecule to different mesh
Mark the detection of object.The sensitive membrane containing ion-exchanger can also be utilized in the detection process, by the open circuit electricity for measuring electrode
Realize the detection of object in position.For using terramycin as object:
1. secured by being generated in sensitive film surface present invention introduces the new technology that polymer hinders sensitive membrane time-measuring electric potential
The polymer of attachment hinders to pass through the ionic flux of sensitive membrane phase, reduces time-measuring electric potential response, determines to realize terramycin
Amount detection.
2. modifying HRP enzyme molecule and Linker DNA binding molecule, Linker DNA and terramycin on multi-walled carbon nanotube
Aptamer connection mutually generates polymer in sensitive membrane using enzymatic reaction, hinders ionic flux, reduce by screening substrate
Time-measuring electric potential response, designs electric potential type aptamer biosensor.
3. multi-walled carbon nanotube specific surface area with higher can load a large amount of HRP and Linker DNA and carry out signal product
Tired and amplification, improves the sensitivity of sensor.
4. the present invention is bonded HRP as enzyme molecule on multi-walled carbon nanotubes, HRP is not only catalyzed dopamine polymerization, and
And anchor point is deposited as poly-dopamine, fine and close poly-dopamine film is formed in sensitive film surface, effectively obstruction ionic flux, mention
The sensitivity of high sensor.
5. this super special response range of chronoptentiometry is most sensitive to surface stopping effect.Select this super spy's response
Quantitative response range as terramycin substantially increases the detection range and sensitivity of sensor.
6. detection method high sensitivity.Using the method for the present invention by various reagents dosage optimization, mould for soil
The detection detection range of element is 0.1-1000nM, and detection is limited to 0.028nM.
7. detection method is versatile.By simply changing corresponding aptamers, the electricity based on time-measuring electric potential response
Bit-type aptamer sensor can be generalized in bioanalysis and environmental monitoring and detect to the sensitivity of other analytes.
8. the present invention combines galvanostatic technique and constant potential technology, permanent by applying cathode pulse to electrode sensitive film
Electric current makes the Ca in solution to be measured2+It is extracted efficiently into film phase, generates time-measuring electric potential response, the detection for terramycin.Pass through
Further apply open circuit potential to electrode, makes the Ca for being extracted to film phase2+It is released back into solution, realizes that electrode updates.Electrode can
With reversible, continuous detection, workload is reduced, can be improved the stability of analysis, reduces experimental error.
9. inert solid contact ion selective electrode of the present invention building containing Lipophilic salts, eliminates ion exchange effect
The interference of electrode stability, sensitivity is coped with, can realize same electrode point by conditions such as optimization sense of current, sizes
The other chronopotentiometry to positive and negative charge indication ion.Further expand its application space.
Detailed description of the invention
Fig. 1 be detection device of the present invention schematic diagram (wherein 1 be glass-carbon electrode, and 2 be PEDOT/PSS conducting shell, 3 be from
The polymer sensitive membrane of sub- electrodes selective, 4 be pvc pipe, and 5 be auxiliary electrode, and 6 be reference electrode).
Fig. 2 is time-measuring electric potential of the solid contact polymer film ion selective electrode of the present invention under poly-dopamine obstruction
Detect the schematic illustration of calcium ion.(R+R-For the Lipophilic salts of dissociation, can mutually be migrated in sensitive membrane.)
Fig. 3 is that the present invention is based on the detection schematic diagrams of the terramycin of stopping effect.
Fig. 4 is that the present invention is based on the calcium ion potential response figures (A) of stopping effect.
Fig. 5 is that the present invention is based on the time-measuring electric potential response diagram (A) of the terramycin of stopping effect and linear response figures (B).
Specific embodiment
Explanation that the present invention will be further explained with reference to the accompanying drawings and examples.
The present invention with the aptamer of object to be detected be identification molecule, by probe fixed substrate load or
Mark biologically active enzyme molecule building that there is the identification probe of catalytic action on probe；Target to be measured and aptamer
After being immunoreacted mode or substitute mode effect by sandwich, it can change with catalytic action identification probe
Amount.Using the chronopotentiometry technology of pulse constant current controlling, produced in polymer sensitive membrane substrate by substrate for enzymatic activity
Raw polymer hinders indication ion, reduces electric potential signal, realizes the detection to object.
Embodiment 1
HRP is not only catalyzed dopamine polymerization, but also deposits anchor point as poly-dopamine, forms densification in sensitive film surface
Poly-dopamine film effectively hinders to mention by the ionic flux of sensitive membrane phase to reduce electric potential signal under the conditions of time-measuring electric potential
The sensitivity of high potential type sensor.
(1) 60mg polyvinyl chloride, 120mg adjacent nitro the preparation of polymer film Calcium signaling sensitive membrane: are weighed
Benzene octyl ether, 20mg inertia lipophilic salt four (dodecyl)-four (4- chlorphenyl) ammonium borate (ETH 500) and 1.84mg calcium ion carry
Body II (ETH 129) is dissolved in 2mL tetrahydrofuran, and calcium ion polymer sensitive membrane solution can be obtained after mixing evenly.Current potential
Conducting shell is poly- (3,4- ethene dioxythiophene)-polystyrolsulfon acid；It is using electro-deposition that poly- (3,4- ethene dioxythiophene)-is poly-
Styrene sulfonic acid solution (PEDOT-PSS) deposits on glass-carbon electrode, dries at room temperature, then puts on ready pvc pipe,
80 microlitres of calcium ion polymer sensitive membrane solution prepared are added dropwise on potential conductance layer, after room temperature is dried, in 0.01M
12h is activated in NaCl solution, i.e. acquisition polymer sensitive membrane Calcium signaling, as working electrode.
(2) ion selective electrode, the reference of calcium ion sensitive membrane the potentiometric detection of Calcium signaling: will be added dropwise
Electrode (silver/silver chloride electrode) and auxiliary electrode (platinum electrode) are connected with electrochemical workstation by conducting wire as inspection respectively
It surveys in device (referring to Fig. 1) insertion 0.01M NaCl solution, surveys open circuit potential under conditions of zero current first, be confirmed as work
The baseline current potential of electrode；Then according to program is detected associated with designed time-measuring electric potential and chrono-amperometric, apply 2s, 2 μ A
Cathode constant current makes sodium ion in liquor be extracted into polymer sensitive membrane phase, meanwhile, the 500 (R of inertia lipophilic salt ETH of dissociation+
R-) mutually migrated in sensitive membrane, utilize the R in the Lipophilic salts of current control dissociation+It is migrated to electrode basement direction, R-To solution
It mutually migrates, the sodium ion complexing of sensitive membrane phase, therefore generates time-measuring electric potential response (referring to fig. 2).Finally set according to detection program
Meter applies the baseline current potential that 100s open circuit potential makes working electrode since be sent back to；The potential change obtained when applying constant current
Signal is the background value of 0.01M NaCl solution.Under the premise of keeping original device motionless, by into 0.01M NaCl solution
The CaCl of high concentration is added dropwise2Solution changes calcium ion concentration in concentration gradient.The every variation an order of magnitude of calcium ion concentration,
Apply 100s open circuit potential again after applying the cathode constant current of 2 μ A of 2s.Thus the potential value of different calcium ion concentrations is obtained, point
Potential value and calcium ion activity when applying constant current 2s is not taken to map, 10-5M and 10-4M CaCl2Obtained in range it is super this
Spy's response (referring to fig. 4, black curve).
(3) dopamine is catalyzed in the potentiometric detection of polymer sensitive membrane surface aggregate without HRP: firstly, taking 100 μ L dopamines
Alkalescent PBS solution drop is reacted 60 minutes on polymer sensitive membrane surface, makes dopamine in calcium ion sensitivity film surface auto polymerization.
By Calcium signaling, reference electrode (silver/silver chloride electrode) and auxiliary electrode (the platinum filament electricity of poly-dopamine modification
Pole) it is connected by conducting wire with electrochemical workstation be inserted into 0.01M NaCl solution as detection device (referring to Fig. 1) respectively, it is first
Open circuit potential first is surveyed under conditions of zero current, is confirmed as the baseline current potential of working electrode；Then according to designed timing electricity
Program is detected associated with position and chrono-amperometric, applies the cathode constant current of 2s, 2 μ A, sodium ion in liquor is made to be extracted into polymer
Sensitive membrane phase, meanwhile, the 500 (R of inertia lipophilic salt ETH of dissociation+R-) mutually migrated in sensitive membrane, the parent dissociated using current control
R in lipid salt+It is migrated to electrode basement direction, R-To molten migration of liquid, the sodium ion of sensitive membrane phase is complexed, therefore generates meter
When potential response (referring to fig. 2).Finally applying 100s open circuit potential according to detection programming is sent back to out working electrode certainly
The baseline current potential of beginning；The potential change signal obtained when applying constant current is the background value of 0.01M NaCl solution.Keep former
Have device it is motionless under the premise of, pass through into 0.01M NaCl solution be added dropwise high concentration CaCl2Solution makes calcium ion concentration in dense
Spend change of gradient.The every variation an order of magnitude of calcium ion concentration applies 100s open circuit after applying the cathode constant current of 2 μ A of 2s again
Current potential.Thus the potential value of different calcium ion concentrations is obtained, takes potential value and calcium ion activity when applying constant current 2s respectively
Mapping, due to the stopping effect of poly-dopamine, 10-5M and 10-4M CaCl2(referring to fig. 4, this super spy's response in range reduces
Red curve).
(4) there is HRP catalysis dopamine in the potentiometric detection of polymer sensitive membrane surface aggregate: firstly, 100 μ L is taken to contain
The dopamine alkalescent PBS solution drop of HRP is reacted 60 minutes on polymer sensitive membrane surface, makes dopamine in calcium ion sensitive membrane
Surface rapid polymerization.By the Calcium signaling of poly-dopamine modification, reference electrode (silver/silver chloride electrode) and auxiliary
Electrode (platinum electrode) is connected by conducting wire with electrochemical workstation respectively is inserted into 0.01M as detection device (referring to Fig. 1)
In NaCl solution, open circuit potential is surveyed under conditions of zero current first, is confirmed as the baseline current potential of working electrode；Then basis is set
Program is detected associated with the time-measuring electric potential and chrono-amperometric counted, and is applied the cathode constant current of 2s, 2 μ A, is made sodium ion in liquor
It is extracted into polymer sensitive membrane phase, meanwhile, the 500 (R of inertia lipophilic salt ETH of dissociation+R-) mutually migrated in sensitive membrane, utilize electric current
The R+ controlled in the Lipophilic salts of dissociation is migrated to electrode basement direction, R-To molten migration of liquid, the sodium ion network of sensitive membrane phase
It closes, therefore generates time-measuring electric potential response (referring to fig. 2).Finally applying 100s open circuit potential according to detection programming keeps work electric
Baseline current potential of the pole since be sent back to；The potential change signal obtained when applying constant current is the back of 0.01M NaCl solution
Scape value.Under the premise of keeping original device motionless, by the CaCl that high concentration is added dropwise into 0.01M NaCl solution2Solution makes calcium
Ion concentration changes in concentration gradient.The every variation an order of magnitude of calcium ion concentration is applied again after applying the cathode constant current of 2s2 μ A
Add 100s open circuit potential.Thus obtain the potential value of different calcium ion concentrations, take respectively apply constant current 2s when potential value with
The mapping of calcium ion activity, since HRP is catalyzed dopamine rapid polymerization, stopping effect is remarkably reinforced, and 10-5M and 10-4M CaCl2Model
This super spy's response in enclosing is substantially reduced (referring to fig. 4, blue curve).
From the foregoing, it can be seen that the presence of HRP can significantly improve dopamine in the polymerization speed of calcium ion sensitivity film surface, hinder
Effect significantly increases.In time-measuring electric potential technology, poly-dopamine hinders calcium ion flux the brightest in this super special response region
Aobvious, the sensitivity of Copper diethlydithiocarbamate can be improved in declining to a great extent for this super special response region current potential.
The detection of 2 object terramycin of embodiment
On functionalized multi-wall carbonnanotubes matrix load HRP and Linker DNA molecular, formed have catalytic action and
The identification probe of bonding action is identification molecule with the aptamer of object to be detected.By DNA hybridization by probe and magnetic bead
On aptamer combine, identification molecule and probe be fixed on magnetic bead.In the presence of not having object, probe is bonded in magnetic bead
Surface, by dopamine and H2O2It is added in alkaline magnetic bead solution, takes appropriate drop to react on polymer sensitive membrane surface with pipettor
Certain time.HRP molecule on probe can effectively be catalyzed dopamine in polymer sensitive membrane surface aggregate, form one layer of densification
Poly-dopamine film, hinder to reduce electric potential signal by the ionic flux of sensitive membrane phase under the conditions of time-measuring electric potential.When there is target
In the presence of object, object and identification molecule-aptamer are acted on, and probe is released from magnetic bead, by dopamine and H2O2It is added
Into alkaline magnetic bead solution, appropriate drop is taken to react certain time on polymer sensitive membrane surface with pipettor.Dopamine is polymerizeing
Object sensitivity film surface auto polymerization hinders the effect of calcium ion flux unobvious, and electric potential signal decline is little.Therefore, pass through current potential
Signal intensity realizes the detection to object.
(1) preparation of multi-walled carbon nanotube probe: 1mg functionalized multi-wall carbonnanotubes are added and contain 110.0mg 1-
The 2- of (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) and 13.0mg N- hydroxysuccinimide (NHS)
It is stirred 2 hours in (N- morpholine) ethanesulfonic acid (MES, 0.05M pH=6.0) buffer solution, activates the carboxylic on multi-walled carbon nanotube
Then multi-walled carbon nanotube is centrifuged 15min at 12,000rpm by base, discard upper solution, and ultrapure water is added, is centrifuged repeatedly
Multi-walled carbon nanotube, is finally dispersed in PBS (0.01M pH=7.4) buffer solution by separation 3 times.It is added into buffer solution
Linker DNA and HRP, respective concentration are 1 μM and 0.5mg mL-1, magnetic agitation, reacts 8 hours at room temperature.After having reacted,
Three's mixed solution is centrifuged 15min at 12,000rpm, discards upper solution, PBS buffer solution is added, is centrifuged repeatedly point
From 3 times, finally by Linker DNA and HRP modify multi-walled carbon nanotube probe be dispersed in PBS buffer solution be used for it is next
Step uses (referring to Fig. 3).
(2) preparation of functionalization magnetic bead: the magnetic bead of 100 μ L marked by streptavidin is taken to add through PBS elution 3 times
Entering 100 μ L concentration is 5 × 10-5Immobilization probe (capture DNA) solution of M shakes 30 minutes, elution at room temperature
3 times magnetic beads for obtaining fixing capture DNA.Then the Beads enrichment for taking the fixed capture DNA of 10 μ L, is added 1 μ L, 100 μ
The OTC-aptamer of M and 100 μ L multi-walled carbon nanotube probes shake 1 hour at room temperature, through elution 3 times, are visited
The functionalization magnetic bead of needle modification (referring to Fig. 3).Three DNA chain base sequences are followed successively by
Capture DNA:5 '-biotin-TTTTTT ACACAACAC-3 '
OTC-aptamer:
5’-ACGTTGACGCTGGTGCCCGGTTGTGGTGCGAGTGTTGTGT-3’
Linker DNA:5’-CGTCAACGTTTTTTTTTT-NH2-3’
(3) 60mg polyvinyl chloride, 120mg adjacent nitro the preparation of polymer film Calcium signaling sensitive membrane: are weighed
Benzene octyl ether, four (dodecyl) _ tetra- (4- chlorphenyl) ammonium borate (ETH 500) of 20mg inertia lipophilic salt and 1.84mg calcium ion carry
Body II (ETH 129) is dissolved in 2mL tetrahydrofuran, and calcium ion polymer sensitive membrane solution can be obtained after mixing evenly.Current potential
Conducting shell is poly- (3,4- ethene dioxythiophene)-polystyrolsulfon acid；It is using electro-deposition that poly- (3,4- ethene dioxythiophene)-is poly-
Styrene sulfonic acid solution (PEDOT-PSS) deposits on glass-carbon electrode, dries at room temperature, then puts on ready pvc pipe,
80 microlitres of calcium ion polymer sensitive membrane solution prepared are added dropwise on potential conductance layer, after room temperature is dried, in 0.01M
12h is activated in NaCl solution, i.e. acquisition polymer sensitive membrane Calcium signaling, as working electrode.
(4) Calcium signaling is 10 in the presence of having object-5M—10-4M CaCl2Range inner potential variation: first
First, into the functionalization magnetic bead of probe modification be added various concentration terramycin solution, at room temperature shake 1 hour, terramycin with
OTC-aptamer on magnetic bead is combined, and aptamer-terramycin compound is constituted, thus by multi-walled carbon nanotube probe from magnetic bead
It releases, after magnetic bead is eluted 3 times, is dispersed in alkalescent PBS (0.01M pH=8.0) buffer solution again.By DOPA
Amine and H2O2It is added in alkalescent PBS solution, is dissolved to dopamine, after mixing, take 100 μ L drops quick in polymer with pipettor
Feel film surface and reacts certain time.During the reaction, the HRP molecule for remaining in the probe load on magnetic bead can be different degrees of
Ground is catalyzed dopamine in sensitive membrane surface aggregate, thus under the conditions of hindering time-measuring electric potential to some extent by sensitive membrane phase from
Sub- flux (referring to Fig. 3).By poly-dopamine modification Calcium signaling, reference electrode (silver/silver chloride electrode) and
Auxiliary electrode (platinum electrode) is connected by conducting wire with electrochemical workstation respectively to be inserted into as detection device (referring to Fig. 1)
In 0.01M NaCl solution, open circuit potential is surveyed under conditions of zero current first, is confirmed as the baseline current potential of working electrode；Then
According to program is detected associated with designed time-measuring electric potential and chrono-amperometric, applies the cathode constant current of 2 μ A of 2s, make in solution
On calcium ion high-efficiency accumulation to the polymer sensitive membrane of working electrode, meanwhile, the inertia lipophilic salt ETH500 (R of dissociation+R-) quick
Sense film mutually migrates, and utilizes the R in the Lipophilic salts of current control dissociation+It is migrated to electrode basement direction, R-To molten migration of liquid,
It is complexed with the calcium ion gathered in working electrode sensitive membrane, generates time-measuring electric potential response (referring to fig. 2).Finally according to detection journey
Sequence design applies the baseline current potential that 100s open circuit potential makes working electrode since be sent back to；The current potential obtained when applying constant current
Variable signal is the background value of 0.01M NaCl solution.Under the premise of keeping original device motionless, by molten to 0.01M NaCl
The CaCl of high concentration is added dropwise in liquid2Solution, making calcium ion concentration is respectively 10-5M and 10-4M.Calcium ion concentration is 10-5M and 10- 4When M, apply 100s open circuit potential again after applying the cathode constant current of 2 μ A of 2s.Take calcium ion selection when applying constant current 2s
Property electrode is 10-5M—10-4M CaCl2Potential difference and terramycin concentration map, due to poly-dopamine hinder calcium ion flux
Effect, 10-5M—10-4M CaCl2Potential difference is increased with the raising of terramycin concentration (referring to Fig. 5).
(5) without Calcium signaling in the presence of object 10-5M—10-4M CaCl2The variation of range inner potential: it visits
It is added, equally shakes 1 hour at room temperature, no aptamer-terramycin compound structure without terramycin in the functionalization magnetic bead of needle modification
At, on magnetic bead still contain a large amount of multi-walled carbon nanotube probe, by magnetic bead elute 3 times after, be dispersed in alkalescent PBS again
In (0.01M pH=8.0) buffer solution.By dopamine and H2O2It is added in alkalescent PBS solution, is dissolved to dopamine, mixed
After even, 100 μ L drops are taken to react certain time on polymer sensitive membrane surface with pipettor.During the reaction, remain in magnetic bead
On a large amount of HRP molecules of probe load can be catalyzed dopamine in polymer sensitive membrane surface rapid polymerization, it is quick in polymer
Feel film surface and form one layer of fine and close poly-dopamine film (referring to Fig. 3).By poly-dopamine modification Calcium signaling,
Reference electrode (silver/silver chloride electrode) and auxiliary electrode (platinum electrode) pass through conducting wire and electrochemical workstation phase continuous cropping respectively
It is inserted into 0.01M NaCl solution for detection device (referring to Fig. 1), surveys open circuit potential under conditions of zero current first, be confirmed as
The baseline current potential of working electrode；Then according to program is detected associated with designed time-measuring electric potential and chrono-amperometric, apply 2 μ of 2s
The cathode constant current of A, makes in solution on calcium ion high-efficiency accumulation to the polymer sensitive membrane of working electrode, meanwhile, dissociation it is lazy
500 (R of property lipophilic salt ETH+R-) mutually migrated in sensitive membrane, utilize the R in the Lipophilic salts of current control dissociation+To electrode basement
Direction migration, R-It to molten migration of liquid, is complexed with the calcium ion gathered in working electrode sensitive membrane, generates time-measuring electric potential response
(referring to fig. 2).Finally applying 100s open circuit potential according to detection programming keeps working electrode electric since the baseline being sent back to
Position；The potential change signal obtained when applying constant current is the background value of 0.01M NaCl solution.Keep original device motionless
Under the premise of, by the CaCl that high concentration is added dropwise into 0.01M NaCl solution2Solution, making calcium ion concentration is respectively 10-5M and
10-4M.Calcium ion concentration is 10-5M and 10-4When M, apply 100s open circuit potential again after applying the cathode constant current of 2 μ A of 2s.By
This obtains 10-5M and 10-4M CaCl2Potential value, stopping effect in the presence of object is the most obvious due to not having, apply
10 when constant current 2s-5M—10-4M CaCl2Potential difference minimum (referring to Fig. 5).
From the foregoing, it can be seen that the presence of object terramycin can reduce dopamine in the polymerization speed of calcium ion sensitivity film surface
Degree, and with the raising of target concentration, stopping effect gradually weakens.
Embodiment 3: the detection of object DNA
On functionalized multi-wall carbonnanotubes matrix load HRP and Linker DNA molecular, formed have catalytic action and
The identification probe of bonding action, using Escherichia coli (E.coli-DNA) as object.Object can pass through DNA hybridization and probe
It is combined with the nucleotide on magnetic bead, forms magnetic bead-object-probe complex.By dopamine and H2O2It is molten to be added to alkaline magnetic bead
In liquid, appropriate drop is taken to react certain time on polymer sensitive membrane surface with pipettor, the HRP molecule on probe can be urged effectively
Change dopamine in polymer sensitive membrane surface aggregate, form one layer of fine and close poly-dopamine film, under the conditions of hindering time-measuring electric potential
By the ionic flux of sensitive membrane phase, electric potential signal is reduced.In the presence of not having object, probe and magnetic bead are two separated
Point, after Magnetic Isolation, by dopamine and H2O2It is added in alkaline magnetic bead solution, takes appropriate drop in polymer sensitivity with pipettor
Film surface reacts certain time, and dopamine hinders the effect of calcium ion flux unobvious in the auto polymerization of polymer sensitive membrane surface,
Electric potential signal decline is little.Therefore, the detection to Escherichia coli is realized by electric potential signal variation.
(1) preparation of multi-walled carbon nanotube probe: with (1) in embodiment 2
(2) preparation of functionalization magnetic bead: the magnetic bead of 100 μ L marked by streptavidin is taken to add through PBS elution 3 times
Entering 100 μ L concentration is 5 × 10-5Immobilization probe (capture DNA) solution of M shakes 30 minutes, elution at room temperature
3 times magnetic beads for obtaining fixing capture DNA.
Three DNA chain base sequences are followed successively by
Capture DNA:5 '-biotin-TTTTTT ACACAACAC-3 '
E.coli-DNA (object):
5’-TATCAGGCATGGCTCTTGATAACGAACTGTTCGCGGTGTTGTGT-3’
Linker DNA:5’-ATAGTCCGTTTTTTTTTT-NH2-3’
(3) preparation of polymer film Calcium signaling sensitive membrane: with (3) in embodiment 2
(4) Calcium signaling is 10 in the presence of having object-5M—10-4M CaCl2Range inner potential variation: first
First, the Escherichia coli of various concentration are added into probe and magnetic bead mixed solution, shake 1 hour at room temperature, Escherichia coli pass through
It is compound to form magnetic bead-object-probe for the combination of Capture DNA on Linker DNA and magnetic bead in DNA hybridization and probe
Object.After magnetic bead is eluted 3 times, it is dispersed in alkalescent PBS (0.01M pH=8.0) buffer solution again.By dopamine and H2O2
It is added in alkalescent PBS solution, is dissolved to dopamine, after mixing, take 100 μ L drops on polymer sensitive membrane surface with pipettor
React certain time.During the reaction, the HRP molecule of the probe load on magnetic bead can be catalyzed to some extent dopamine and exist
Sensitive membrane surface aggregate, to pass through the ionic flux of sensitive membrane phase under the conditions of hindering time-measuring electric potential to some extent.It will be poly- more
Bar amine-modified Calcium signaling, reference electrode (silver/silver chloride electrode) and auxiliary electrode (platinum electrode) are respectively
It is connected by conducting wire with electrochemical workstation and is inserted into 0.01M NaCl solution as detection device (referring to Fig. 1), first zero
Open circuit potential is surveyed under conditions of electric current, is confirmed as the baseline current potential of working electrode；Then according to designed time-measuring electric potential and meter
When electric current associated with detect program, apply the cathode constant current of 2 μ A of 2s, make in solution calcium ion high-efficiency accumulation to working electrode
Polymer sensitive membrane on, meanwhile, the 500 (R of inertia lipophilic salt ETH of dissociation+R-) mutually migrated in sensitive membrane, utilize current control
R in the Lipophilic salts of dissociation+It is migrated to electrode basement direction, R-To molten migration of liquid, and gather in working electrode sensitive membrane
Calcium ion complexing, generate time-measuring electric potential response (referring to fig. 2).Finally applying 100s open circuit potential according to detection programming makes
Baseline current potential of the working electrode since be sent back to；The potential change signal obtained when applying constant current is 0.01M NaCl molten
The background value of liquid.Under the premise of keeping original device motionless, by the CaCl that high concentration is added dropwise into 0.01M NaCl solution2It is molten
Liquid, making calcium ion concentration is respectively 10-5M and 10-4M.Calcium ion concentration is 10-5M and 10-4When M, the cathode for applying 2 μ A of 2s is permanent
Apply 100s open circuit potential after electric current again.Take Calcium signaling when applying constant current 2s 10-5M—10-4M CaCl2
Potential difference and e. coli concentration map, due to poly-dopamine hinder calcium ion flux effect, 10-5M—10-4M
CaCl2Potential difference is reduced with the raising of e. coli concentration.
(5) without Calcium signaling in the presence of object 10-5M—10-4M CaCl2The variation of range inner potential: it visits
Escherichia coli are added without in needle and magnetic bead mixed solution, are shaken 1 hour at room temperature, no magnetic bead-object-probe complex shape
At.After Magnetic Isolation, magnetic bead is eluted 3 times, is dispersed in alkalescent PBS (0.01M pH=8.0) buffer solution again.It will be more
Bar amine and H2O2It is added in alkalescent PBS solution, is dissolved to dopamine, after mixing, take 100 μ L drops in polymer with pipettor
Sensitive film surface reacts certain time.During the reaction, due to not having probe combination on magnetic bead, dopamine is in polymer sensitivity
Film surface auto polymerization.By the Calcium signaling of poly-dopamine modification, reference electrode (silver/silver chloride electrode) and auxiliary
Electrode (platinum electrode) is connected by conducting wire with electrochemical workstation respectively is inserted into 0.01M as detection device (referring to Fig. 1)
In NaCl solution, open circuit potential is surveyed under conditions of zero current first, is confirmed as the baseline current potential of working electrode；Then basis is set
Program is detected associated with the time-measuring electric potential and chrono-amperometric counted, and is applied the cathode constant current of 2 μ A of 2s, is made calcium ion in solution
On high-efficiency accumulation to the polymer sensitive membrane of working electrode, meanwhile, the 500 (R of inertia lipophilic salt ETH of dissociation+R-) in sensitive membrane
It mutually migrates, utilizes the R in the Lipophilic salts of current control dissociation+It is migrated to electrode basement direction, R-It is and poly- to molten migration of liquid
Collect the calcium ion complexing in working electrode sensitive membrane, generates time-measuring electric potential response (referring to fig. 2).Finally set according to detection program
Meter applies the baseline current potential that 100s open circuit potential makes working electrode since be sent back to；The potential change obtained when applying constant current
Signal is the background value of 0.01M NaCl solution.Under the premise of keeping original device motionless, by into 0.01M NaCl solution
The CaCl of high concentration is added dropwise2Solution, making calcium ion concentration is respectively 10-5M and 10-4M.Calcium ion concentration is 10-5M and 10-4When M,
Apply 100s open circuit potential again after applying the cathode constant current of 2 μ A of 2s.Thus 10 are obtained-5M and 10-4M CaCl2Potential value,
Stopping effect in the presence of object is smaller due to not having, apply 10 when constant current 2s-5M—10-4M CaCl2Potential difference
Value is maximum.From the foregoing, it can be seen that dopamine can be improved in the polymerization speed of calcium ion sensitivity film surface in the presence of object Escherichia coli
Degree, and with the raising of target concentration, stopping effect gradually increases.
Embodiment 4: the detection of object antigen
HRP and antibody molecule (anti-mouse immunoglobulin A b is loaded on functionalized multi-wall carbonnanotubes matrix2), it is formed
Identification probe with catalytic action and antigen-specific sexual reaction.Antibody molecule is modified on magnetic bead, and (ball is immunized in anti-mouse
Albumin A b1), form the magnetic bead with antigen-specific sexual reaction.Using antigen (mouse immune globulin Ag) as object,
Object can form magnetic bead-target by antigen-antibody specificity identification in conjunction with the antibody specificity on probe and magnetic bead
Object-probe complex.After Magnetic Isolation, by dopamine and H2O2It is added in alkaline magnetic bead solution, takes appropriate drop to exist with pipettor
React certain time on polymer sensitive membrane surface, and the HRP molecule on probe can effectively be catalyzed dopamine in polymer sensitive membrane
Surface aggregate forms one layer of fine and close poly-dopamine film, passes through the ionic flux of sensitive membrane phase under the conditions of obstruction time-measuring electric potential,
Reduce electric potential signal.In the presence of not having object, probe and magnetic bead are isolated two parts, after Magnetic Isolation, by dopamine
And H2O2It is added in alkaline magnetic bead solution, takes appropriate drop to react certain time, DOPA on polymer sensitive membrane surface with pipettor
Amine hinders the effect of calcium ion flux unobvious in the auto polymerization of polymer sensitive membrane surface, and electric potential signal decline is little.Therefore,
The detection to object is realized by electric potential signal variation.
(1) preparation of multi-walled carbon nanotube probe: 1mg functionalized multi-wall carbonnanotubes are added and contain 110.0mg 1-
The 2- of (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) and 13.0mg N- hydroxysuccinimide (NHS)
It is stirred 2 hours in (N- morpholine) ethanesulfonic acid (MES, 0.05M pH=6.0) buffer solution, activates the carboxylic on multi-walled carbon nanotube
Then multi-walled carbon nanotube is centrifuged 15min at 12,000rpm by base, discard upper solution, and ultrapure water is added, is centrifuged repeatedly
Multi-walled carbon nanotube, is finally dispersed in PBS (0.01M pH=7.4) buffer solution by separation 3 times.By being crosslinked with amino,
By anti-mouse immunoglobulin A b2It is covalently fixed on multi-walled carbon nanotubes with HRP.After having reacted, three's mixed solution is existed
It is centrifuged 15min under 12,000rpm, discards upper solution, PBS buffer solution is added, separation 3 times is centrifuged repeatedly, finally by Ab2With
The multi-walled carbon nanotube probe of HRP modification is dispersed in PBS buffer solution for using (referring to Fig. 3) in next step.
(2) preparation of functionalization magnetic bead: the magnetic bead of 100 μ L marked by streptavidin is taken to add through PBS elution 3 times
Enter the anti-mouse immunoglobulin A b of 100 μ L biotin labelings1Solution shakes 30 minutes at room temperature, obtains for elution 3 times
Ab1The magnetic bead of modification.
(3) preparation of polymer film Calcium signaling sensitive membrane: with (3) in embodiment 2
(4) Calcium signaling is 10 in the presence of having object-5M—10-4M CaCl2Range inner potential variation: first
First, the object antigenic solution of various concentration is added into probe and magnetic bead mixed solution, shakes 1 hour at room temperature, object
It is compound can to form magnetic bead-object-probe by antigen-antibody specificity identification in conjunction with the antibody on probe and magnetic bead
Object.After magnetic bead is eluted 3 times, it is dispersed in alkalescent PBS (0.01M pH=8.0) buffer solution again.By dopamine and H2O2
It is added in alkalescent PBS solution, is dissolved to dopamine, after mixing, take 100 μ L drops on polymer sensitive membrane surface with pipettor
React certain time.During the reaction, the HRP molecule of the probe load on magnetic bead can be catalyzed to some extent dopamine and exist
Sensitive membrane surface aggregate, to pass through the ionic flux of sensitive membrane phase under the conditions of hindering time-measuring electric potential to some extent.It will be poly- more
Bar amine-modified Calcium signaling, reference electrode (silver/silver chloride electrode) and auxiliary electrode (platinum electrode) are respectively
It is connected by conducting wire with electrochemical workstation and is inserted into 0.01M NaCl solution as detection device (referring to Fig. 1), first zero
Open circuit potential is surveyed under conditions of electric current, is confirmed as the baseline current potential of working electrode；Then according to designed time-measuring electric potential and meter
When electric current associated with detect program, apply the cathode constant current of 2 μ A of 2s, make in solution calcium ion high-efficiency accumulation to working electrode
Polymer sensitive membrane on, meanwhile, the 500 (R of inertia lipophilic salt ETH of dissociation+R-) mutually migrated in sensitive membrane, utilize current control
R in the Lipophilic salts of dissociation+It is migrated to electrode basement direction, R-To molten migration of liquid, and gather in working electrode sensitive membrane
Calcium ion complexing, generate time-measuring electric potential response (referring to fig. 2).Finally applying 100s open circuit potential according to detection programming makes
Baseline current potential of the working electrode since be sent back to；The potential change signal obtained when applying constant current is 0.01M NaCl molten
The background value of liquid.Under the premise of keeping original device motionless, by the CaCl that high concentration is added dropwise into 0.01M NaCl solution2It is molten
Liquid, making calcium ion concentration is respectively 10-5M and 10-4M.Calcium ion concentration is 10-5M and 10-4When M, the cathode for applying 2 μ A of 2s is permanent
Apply 100s open circuit potential after electric current again.Take Calcium signaling when applying constant current 2s 10-5M—10-4M CaCl2
Potential difference and antigen concentration map, due to poly-dopamine hinder calcium ion flux effect, 10-5M—10-4M CaCl2Electricity
Potential difference value is reduced with the raising of antigen concentration.
(5) without Calcium signaling in the presence of object 10-5M—10-4M CaCl2The variation of range inner potential: it visits
It is added without object antigen in needle and magnetic bead mixed solution, is shaken 1 hour at room temperature, no magnetic bead-object-probe complex shape
At.After Magnetic Isolation, magnetic bead is eluted 3 times, is dispersed in alkalescent PBS (0.01M pH=8.0) buffer solution again.It will be more
Bar amine and H2O2It is added in alkalescent PBS solution, is dissolved to dopamine, after mixing, take 100 μ L drops in polymer with pipettor
Sensitive film surface reacts certain time.During the reaction, due to not having probe combination on magnetic bead, dopamine is in polymer sensitivity
Film surface auto polymerization.By the Calcium signaling of poly-dopamine modification, reference electrode (silver/silver chloride electrode) and auxiliary
Electrode (platinum electrode) is connected by conducting wire with electrochemical workstation respectively is inserted into 0.01M as detection device (referring to Fig. 1)
In NaCl solution, open circuit potential is surveyed under conditions of zero current first, is confirmed as the baseline current potential of working electrode；Then basis is set
Program is detected associated with the time-measuring electric potential and chrono-amperometric counted, and is applied the cathode constant current of 2 μ A of 2s, is made calcium ion in solution
On high-efficiency accumulation to the polymer sensitive membrane of working electrode, meanwhile, the 500 (R of inertia lipophilic salt ETH of dissociation+R-) in sensitive membrane
It mutually migrates, utilizes the R in the Lipophilic salts of current control dissociation+It is migrated to electrode basement direction, R-It is and poly- to molten migration of liquid
Collect the calcium ion complexing in working electrode sensitive membrane, generates time-measuring electric potential response (referring to fig. 2).Finally set according to detection program
Meter applies the baseline current potential that 100s open circuit potential makes working electrode since be sent back to；The potential change obtained when applying constant current
Signal is the background value of 0.01M NaCl solution.Under the premise of keeping original device motionless, by into 0.01M NaCl solution
The CaCl of high concentration is added dropwise2Solution, making calcium ion concentration is respectively 10-5M and 10-4M.Calcium ion concentration is 10-5M and 10-4When M,
Apply 100s open circuit potential again after applying the cathode constant current of 2 μ A of 2s.Thus 10 are obtained-5M and 10-4M CaCl2Potential value,
Stopping effect in the presence of object is smaller due to not having, apply 10 when constant current 2s-5M—10-4M CaCl2Potential difference
Value is maximum.
From the foregoing, it can be seen that dopamine can be improved in the polymerization speed of calcium ion sensitivity film surface in the presence of object antigen,
And with the raising of target concentration, stopping effect is gradually increased.
Claims (8)
1. a kind of detection method for hindering sensitive membrane time-measuring electric potential based on polymer, it is characterised in that: with the choosing of polymer film ion
Selecting property electrode is working electrode, using the aptamer, DNA or antigen/antibody of target to be measured as identification molecule, by visiting
Load or marker enzyme or its analog molecule are as identification probe on needle matrix, using the time-measuring electric potential skill of pulse constant current controlling
Art carries out quantitative/qualitatively detection to object in liquid to be detected；
Object in the solution to be measured is immunoreacted mode or substitute mode by sandwich in conjunction with identification molecule
Effect, and then change the amount of identification probe, make it poly- by load on probe or the enzyme marked or its analog catalysis substrate
It closes and generates polymer in object sensitive membrane substrate, polymer hinders the ionic flux of indication ion, reduces electric potential signal, and realization is treated
Detect quantitative/qualitative detection of object.
2. the detection method according to claim 1 for hindering sensitive membrane time-measuring electric potential based on polymer, it is characterised in that: described
Probe base be graphene, single-walled carbon nanotube, double-walled carbon nano-tube, multi-walled carbon nanotube, carbon nano pipe array, nano wire,
Fullerene, quantum dot or MOF material；
The enzyme molecule be horseradish peroxidase, alkaline phosphatase lipase, soybean peroxidase, butyrylcholine esterase, urine enzyme,
Beta galactosidase, laccase, bilirubin oxidase, polyphenol oxidase, tyrosinase, G- tetrad functional enzyme or manganoporphyrin-DNA
Chelate；Class enzyme molecule are as follows: graphene oxide, ferroso-ferric oxide, carbon nanotube, gold nano, molybdenum disulfide, MOFs, metal oxygen
Compound nano material or ferroheme.
3. the detection method according to claim 1 for hindering sensitive membrane time-measuring electric potential based on polymer, it is characterised in that: described
Working electrode bottom has polymer sensitive membrane, and polymer sensitive membrane surface forms the obstruction polymerization for hindering indication ion flux
Object；It wherein, is conducting shell between the polymer sensitive membrane and working electrode bottom.
4. by the detection method for hindering sensitive membrane time-measuring electric potential described in claim 1 or 3 based on polymer, it is characterised in that:
The polymer for hindering indication ion flux is that substrate passes through the enzyme or its analog Journal of Molecular Catalysis of load or label on probe
The polymer of the obstruction indication ion flux formed in polymer film ion selective electrode sensitive membrane substrate, firm attachment
10-120 minutes；The substrate is dopamine, catechol, p-tert-butyl catechol, aniline, adrenaline, demethyl adrenal gland
Element or tannic acid.
5. the detection method according to claim 1 for hindering sensitive membrane time-measuring electric potential based on polymer, it is characterised in that: described
Target to be measured is antibiotic, bacterium, albumen, cell, virus, heavy metal ion, amino acid, pesticide, algae toxin, small point organic
Son or DNA；The identification molecule is sequence, DNA or the antigen/antibody specifically bound with target to be measured.
6. the detection method according to claim 1 for hindering sensitive membrane time-measuring electric potential based on polymer, it is characterised in that: described
Indication ion is cation, anion or polyion；The polymer sensitive membrane of the polymer film ion selective electrode includes
The carrier of indication ion.
7. the detection method according to claim 1 for hindering sensitive membrane time-measuring electric potential based on polymer, it is characterised in that: described
Chronopotentiometry is using polymer film ion selective electrode as working electrode, using working electrode, to electrode and reference electricity
The indication ion that the three-electrode system of pole hinders polymer detects, and show that indication ion potential response value can be realized pair
The qualitative detection of target to be measured；Further according to above-mentioned generation indication ion polymer obstruction before and after potential change difference
Value, can be realized the quantitative detection to target to be measured.
8. the detection method according to claim 1 for hindering sensitive membrane time-measuring electric potential based on polymer, it is characterised in that:
The constant pulse cathode current size applied to working electrode is 0.1-10 μ A, when the pulse current applied
Between be 0.1-10s；
After the application pulse current, by further applying open circuit potential 10-600s to electrode, realize that electrode renaturation updates.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910374232.6A CN110068602B (en) | 2019-05-07 | 2019-05-07 | Detection method based on polymer blocking sensitive membrane time-counting potential |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910374232.6A CN110068602B (en) | 2019-05-07 | 2019-05-07 | Detection method based on polymer blocking sensitive membrane time-counting potential |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110068602A true CN110068602A (en) | 2019-07-30 |
| CN110068602B CN110068602B (en) | 2021-04-16 |
Family
ID=67370020
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910374232.6A Active CN110068602B (en) | 2019-05-07 | 2019-05-07 | Detection method based on polymer blocking sensitive membrane time-counting potential |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110068602B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110487870A (en) * | 2019-08-23 | 2019-11-22 | 中国科学院烟台海岸带研究所 | It is a kind of detection polypeptide and with polypeptide be identification Molecular Detection bacterium method and device |
| CN111253758A (en) * | 2020-01-17 | 2020-06-09 | 首都师范大学 | Detection reagent for detecting adenosine triphosphate, preparation method thereof, method and device for detecting adenosine triphosphate by using detection reagent |
| CN111505287A (en) * | 2020-05-07 | 2020-08-07 | 军事科学院军事医学研究院军事兽医研究所 | Biosensor for detecting food-borne pathogenic bacteria by taking acetaldehyde dehydrogenase as signal transduction system and preparation method and application thereof |
| CN112485313A (en) * | 2020-11-16 | 2021-03-12 | 中国科学院苏州生物医学工程技术研究所 | Electrochemical sensor for detecting virus and preparation method thereof |
| CN114544728A (en) * | 2020-11-25 | 2022-05-27 | 中国科学院烟台海岸带研究所 | Thin-layer polymer film ion selective electrode based on pulse constant current control and detection method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102235995A (en) * | 2010-05-07 | 2011-11-09 | 中国科学院烟台海岸带研究所 | Polymer liquid film potential sensor for detecting enzymatic activity and detection method thereof |
| CN102980929A (en) * | 2012-12-06 | 2013-03-20 | 江南大学 | Nano photoelectronchemical sensor for detecting dopamine with high sensitivity |
| CN108020589A (en) * | 2016-10-28 | 2018-05-11 | 中国科学院烟台海岸带研究所 | A kind of detection method of Calcium in Seawater ion |
| CN108226240A (en) * | 2017-12-11 | 2018-06-29 | 江苏奔拓电气科技有限公司 | A kind of preparation method for the marking sensor for detecting terramycin |
-
2019
- 2019-05-07 CN CN201910374232.6A patent/CN110068602B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102235995A (en) * | 2010-05-07 | 2011-11-09 | 中国科学院烟台海岸带研究所 | Polymer liquid film potential sensor for detecting enzymatic activity and detection method thereof |
| CN102980929A (en) * | 2012-12-06 | 2013-03-20 | 江南大学 | Nano photoelectronchemical sensor for detecting dopamine with high sensitivity |
| CN108020589A (en) * | 2016-10-28 | 2018-05-11 | 中国科学院烟台海岸带研究所 | A kind of detection method of Calcium in Seawater ion |
| CN108226240A (en) * | 2017-12-11 | 2018-06-29 | 江苏奔拓电气科技有限公司 | A kind of preparation method for the marking sensor for detecting terramycin |
Non-Patent Citations (2)
| Title |
|---|
| JIAWANG DING ET AL.: "Polymeric Membrane Ion-Selective Electrode for Butyrylcholinesterase Based on Controlled Release of Substrate", 《ELECTROANALYSIS》 * |
| SHUWEN LIU ET AL.: "Dual-Analyte Chronopotentiometric Aptasensing Platform Based on a G‑Quadruplex/Hemin DNAzyme and Logic Gate Operations", 《ANAL. CHEM.》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110487870A (en) * | 2019-08-23 | 2019-11-22 | 中国科学院烟台海岸带研究所 | It is a kind of detection polypeptide and with polypeptide be identification Molecular Detection bacterium method and device |
| CN110487870B (en) * | 2019-08-23 | 2022-01-11 | 中国科学院烟台海岸带研究所 | Method and device for detecting polypeptide and detecting bacteria by taking polypeptide as recognition molecule |
| CN111253758A (en) * | 2020-01-17 | 2020-06-09 | 首都师范大学 | Detection reagent for detecting adenosine triphosphate, preparation method thereof, method and device for detecting adenosine triphosphate by using detection reagent |
| CN111505287A (en) * | 2020-05-07 | 2020-08-07 | 军事科学院军事医学研究院军事兽医研究所 | Biosensor for detecting food-borne pathogenic bacteria by taking acetaldehyde dehydrogenase as signal transduction system and preparation method and application thereof |
| CN112485313A (en) * | 2020-11-16 | 2021-03-12 | 中国科学院苏州生物医学工程技术研究所 | Electrochemical sensor for detecting virus and preparation method thereof |
| CN114544728A (en) * | 2020-11-25 | 2022-05-27 | 中国科学院烟台海岸带研究所 | Thin-layer polymer film ion selective electrode based on pulse constant current control and detection method |
| CN114544728B (en) * | 2020-11-25 | 2023-10-27 | 中国科学院烟台海岸带研究所 | Thin-layer polymer film ion selective electrode based on pulse constant current control and detection method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110068602B (en) | 2021-04-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110068602A (en) | A kind of detection method hindering sensitive membrane time-measuring electric potential based on polymer | |
| EP1108213B1 (en) | Method of electrochemical analysis of an analyte | |
| Jampasa et al. | Electrochemical detection of c-reactive protein based on anthraquinone-labeled antibody using a screen-printed graphene electrode | |
| Jalalvand | Fabrication of a novel and ultrasensitive label-free electrochemical aptasensor for detection of biomarker prostate specific antigen | |
| Rishpon et al. | An amperometric enzyme-channeling immunosensor | |
| US8198039B2 (en) | Biosensors and related methods | |
| JP3329449B2 (en) | Conductivity membrane strip biosensor | |
| Kong et al. | Simultaneous electrochemical immunoassay using CdS/DNA and PbS/DNA nanochains as labels | |
| Akter et al. | A highly sensitive quartz crystal microbalance immunosensor based on magnetic bead-supported bienzymes catalyzed mass enhancement strategy | |
| US20020037530A1 (en) | Detection of binding reactions using labels detected by mediated catalytic electrochemistry | |
| Xia et al. | Electrochemical immunosensors with protease as the signal label for the generation of peptide-Cu (II) complexes as the electrocatalysts toward water oxidation | |
| Han et al. | Electrochemical immunoassay for thyroxine detection using cascade catalysis as signal amplified enhancer and multi-functionalized magnetic graphene sphere as signal tag | |
| CN107389949A (en) | A kind of electrochemical immunosensor preparation method for PCSK9 Protein Detections | |
| KR102216258B1 (en) | Sensor of detecting norovirus and sensing method using the sensor | |
| Díaz-González et al. | Development of an immunosensor for the determination of rabbit IgG using streptavidin modified screen-printed carbon electrodes | |
| Xu et al. | Fractal gold modified electrode for ultrasensitive thrombin detection | |
| Wang et al. | Electrochemiluminescence immunosensor for α-fetoprotein using Ru (bpy) 32+-encapsulated liposome as labels | |
| Kakhki | Application of nanoparticles in the potentiometric ion selective electrodes | |
| Yin et al. | Ultrasensitive electrochemical immunoassay based on cadmium ion-functionalized PSA@ PAA nanospheres | |
| Lv et al. | A novel immunosensor for carcinoembryonic antigen based on poly (diallyldimethylammonium chloride) protected prussian blue nanoparticles and double-layer nanometer-sized gold particles | |
| Liang et al. | A novel, label-free immunosensor for the detection of α-fetoprotein using functionalised gold nanoparticles | |
| Liu et al. | A renewable electrochemical magnetic immunosensor based on gold nanoparticle labels | |
| Xiang et al. | Multi-enzyme layer-by-layer assembly for dual amplified ultrasensitive electronic detection of cancer biomarkers | |
| CN101923092A (en) | Method for preparing carcinoembryonic antigen working electrode for screen printing electrode | |
| Liang et al. | A novel label-free voltammetric immunosensor for the detection of α-fetoprotein using functional titanium dioxide nanoparticles |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |