CN104034782B - The potentiometric sensor of a kind of detection dichloro three (1,10-phenanthrolene) ruthenium (II) and detection method thereof and application - Google Patents
The potentiometric sensor of a kind of detection dichloro three (1,10-phenanthrolene) ruthenium (II) and detection method thereof and application Download PDFInfo
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- CN104034782B CN104034782B CN201410242054.9A CN201410242054A CN104034782B CN 104034782 B CN104034782 B CN 104034782B CN 201410242054 A CN201410242054 A CN 201410242054A CN 104034782 B CN104034782 B CN 104034782B
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Abstract
The present invention relates to sensor, the potentiometric sensor of specifically a kind of detection dichloro three (1,10 phenanthrolene) ruthenium (II) and detection method thereof and application.Potentiometric sensor includes working electrode, reference electrode and potential measurement instrument, potential measurement instrument connects working electrode and outer reference electrode respectively by wire, working electrode and outer reference electrode insert in the detection cell filling detection liquid, it is stained with polymer sensitive membrane bottom working electrode, detection cell is provided with the power set with disturbance solution effects；The potentiometric sensor being used for detecting dichloro three (1,10 phenanthrolene) ruthenium (II) that the present invention provides is highly sensitive, selectivity is high, and this potentiometric sensor can be used in bio-sensing based on DNA simultaneously；Bio-sensing based on DNA can be by selecting different aptamers, it is capable of the detection of different target molecule, can combine with circulation isothermal strand displacement polyreaction and hybridization chain reaction and nano material amplifying technique during detection, improve further sensitivity, the selectivity detected.
Description
Technical field
The present invention relates to sensor, specifically a kind of detection dichloro three (1,10-neighbour's diaza
Luxuriant and rich with fragrance) ruthenium (II) (Dichlorotris (1,10-phenanthroline) ruthenium (II))
Potentiometric sensor and detection method and application.
Background technology
Dichloro three (1,10-phenanthrolene) ruthenium (II) is a kind of efficient chemical illuminating reagent,
Extensively apply the analytical technology of chemically based luminescence.But above-mentioned chemiluminescence is easily subject to
The color of sample and the impact of turbidity, thus limit it and extensively apply.Sensitive based on polymer
The potential monitoring technique of film ion-selective electrode is highly sensitive, selectivity good, fast response time,
And detection process do not disturbed by color and the turbidity of sample.Thus develop high-sensitive dichloro three
(1,10-phenanthrolene) ruthenium (II) polymer sensitive membrane ion-selective electrode has important
Using value.
Polymer film ion selective electrode have easy and simple to handle, response quickly, equipment inexpensive etc.
Advantage, is particularly suitable for the detection of Site Detection and gross sample, be widely used to whole blood,
Serum, urinate, organize, the direct mensuration of various electrolyte ions in intracellular fluid and diluent thereof.
But its application focuses mostly on and detects at electrolyte ion.Aptamer (Aptamer) refers to utilize
The part evolution technology of index concentration filters out from specific oligonucleotide library to be had target
The oligonucleotide (DNA or RNA) that specificity interacts, as a kind of novel molecular recognition
Element, it is possible to combine various target molecule efficiently, specifically, has and is easily-synthesized, easily stores and steady
The qualitative advantage such as good；But aptamer itself does not have the function of display signal.It is currently based on
The detection method of aptamer typically requires mark fluorescent, nano material, thus can produce detection
Signal, this kind of detection method on the one hand testing cost is high, complex operation；The most labeled
Journey also have impact on the binding ability of aptamer.
Summary of the invention
Present invention aim at providing a kind of detection dichloro three (1,10-phenanthrolene) ruthenium (II)
The current potential of (Dichlorotris (1,10-phenanthroline) ruthenium (II)) passes
Sensor and detection method thereof and application.
For achieving the above object, the technical solution used in the present invention is:
A kind of potentiometric sensor of detection dichloro three (1,10-phenanthrolene) ruthenium (II), including
Working electrode, reference electrode and potential measurement instrument, it is characterised in that: potential measurement instrument is by leading
Line connects working electrode and outer reference electrode, working electrode and the insertion of outer reference electrode respectively and fills
In the detection cell of detection liquid, it is provided with polymer sensitive membrane bottom working electrode, detection cell is provided with
There are the power set of disturbance solution effects；
Described polymer sensitive membrane is pressed by polymeric matrix material, plasticizer and cationite
The ratio of weight and number mixing of 20-40:40-80:0.1-10:0.1-10, is then dissolved into four
In hydrogen tetrahydrofuran solution, at room temperature place 12-24h after being mixed, i.e. obtain resilient polymerization
Thing sensitive membrane；
Described polymeric matrix material is polrvinyl chloride, carboxylated polrvinyl chloride, poly-butyl propyleneglycol
Acid esters, butyl polyacrylate, Polyetherimide, rubber, sol-gel film, methacrylic acid
Methyl ester-certain herbaceous plants with big flowers methacrylic acid methyl terpolymer or n-butyl acrylate-hydroxyethyl methacrylate second
Ester copolymer；Plasticizer is o-Nitrobenzol octyl ether, two-2-ethylhexyl esters in the last of the ten Heavenly stems, decanedioic acid two
Butyl ester or di-n-octyl sebacate；Cationite is four (3,5-bis-(trifluoromethyl) phenyl) boron
Acid sodium, dinonylnaphthalene sulfonic acid or dinonylnaphthalene sulfonic acid salt or borate.
Conducting shell material it is provided with between described working electrode and polymer sensitive membrane.
Described working electrode is fluid electrode or solid electrode；Wherein, liquid working electrode is interior
Fill internal-filling liquid and be inserted with the electrode of internal reference electrode；Sensitivity when working electrode is fluid electrode
Film is dissolved in tetrahydrofuran solution, at room temperature places 12-24h, then stick after being mixed
Bottom electrode holder；When working electrode is solid electrode, it is (pre-that sensitive membrane is dissolved in dichloromethane
First lead to nitrogen deoxygenation) in, membrane material dropping is coated in solid electrode lower surface.
Described solid working electrode be carbon electrode, gold electrode, copper electrode, silver electrode, platinum electrode,
Indium-tin oxide electrode, zinc oxide electrode, silicon electrode, carbon fiber electrode, silicon carbide electrode, many
Hole gold electrode, carbon nano tube modified electrode, gold nano modified electrode, platinum Nanoparticle Modified Electrode,
Graphene modified electrode, C60 modified electrode, porous carbon modified electrode, carbon screen printed electrodes,
Gold screen printing electrode, paper chip electrode or ceramic chip electrode；
Described conducting shell material is 3-alkylthrophene, polypyrrole, polythiophene, polyaniline, poly-
Acetylene, poly-n-octyl thiophene, six polythiophenes, Polyglycolic acid fibre, CNT, graphite
Alkene, platinum nanometer, gold nano, molybdenum bisuphide, mesoporous carbon, porous gold or tin indium oxide nanometer.
A kind of method of detection dichloro three (1,10-phenanthrolene) ruthenium (II), by described current potential
The working electrode of sensor and outer reference electrode insert containing dichloro three (1,10-neighbour's phenodiazine to be measured
Miscellaneous phenanthrene) ruthenium (II) detection liquid in, measure polymer on working electrode by potential measurement instrument quick
Potential change on sense film, records to be measured two according to potential change signal and standard working curve
Chlorine three (1,10-phenanthrolene) ruthenium (II) concentration.
Answering of the potentiometric sensor of a kind of detection dichloro three (1,10-phenanthrolene) ruthenium (II)
With, described potentiometric sensor can be as biosensor based on DNA or aptamer, to not
With the detection that target molecule is quantitative.
Further, using aptamer as identifying molecule, dichloro three (1,10-neighbour's diaza
Luxuriant and rich with fragrance) ruthenium (II) conduct embedding molecule, aptamer and complementary DNA form double-stranded DNA, dichloro
Three (1,10-phenanthrolene) ruthenium (II) embeds double-stranded DNA, by described potentiometric sensor
The indication ion dichloro three (1,10-that potentiometer causes after measuring target molecule and aptamer effect
Phenanthrolene) potential change that causes of ruthenium (II) concentration change, according to potential change signal with
And standard working curve records target molecule testing concentration.
Moreover, dichloro three (1,10-phenanthrolene) ruthenium (II) concentration is added
10-1-10-9Mole, and 0.1-10 hour interface interaction time of double-stranded DNA, measure in solution
The potential response that residue dichloro three (1,10-phenanthrolene) ruthenium (II) produces；Then measure target
After molecule and aptamer effect, add dichloro three (1,10-phenanthrolene) ruthenium (II) concentration
10-1-10-9Mole, with 0.1-10 hour interface interaction time of double-stranded DNA, residue in solution
The potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces, the current potential of both differences
Signal records the concentration of target molecule according to potential change signal by standard working curve.
Or, add dichloro three (1,10-phenanthrolene) ruthenium (II) concentration 10-1-10-9Mole,
With the 0.1-10 hour interface interaction time of double-stranded DNA, then by adjusting the temperature to 95 degree,
Interface double-stranded DNA unwinds, and discharges dichloro three (1,10-phenanthrolene) ruthenium (II) to buffering
In solution, measure the electricity that in buffer solution, dichloro three (1,10-phenanthrolene) ruthenium (II) produces
Position response；After measuring target molecule and aptamer effect, add dichloro three (1,10-neighbour's diaza
Luxuriant and rich with fragrance) ruthenium (II) concentration 10-1-10-9Mole, little with double-stranded DNA interface interaction time 0.1-10
Time, then by adjusting the temperature to 95 degree, interface double-stranded DNA unwinds, and discharges dichloro three
(1,10-phenanthrolene) ruthenium (II), in buffer solution, measures buffer solution double center chain DNA
The potential response that the dichloro three (1,10-phenanthrolene) ruthenium (II) of release produces, both differences
Recorded the concentration of target molecule by standard working curve according to potential change signal.
Described aptamer is incorporated in liquid to be detected, or fixing with substrate after be incorporated in detection liquid
In.
Described fixed nucleic acid is fit and the substrate of complementary DNA is ferroferric oxide magnetic nano
At the bottom of grain, magnetic graphene substrate, carbon substrate, gold substrate, cuprio, at the bottom of SERS substrate, platino,
At the bottom of tin indium oxide substrate, Zinc oxide-base, carbon fiber substrates, silicon carbide substrate, porous gold substrate,
Carbon nano tube modified substrate, gold nano modify substrate, the nano-modified substrate of platinum, graphene modified
Substrate, C60 modify substrate, porous carbon modify the surfaces such as substrate, carbon screen printed electrodes substrate,
Gold screen printing electrode substrate, paper chip electrode basement or ceramic chip electrode basement.
It is aptamer and wall scroll complementary strand that described aptamer and complementary DNA form double-stranded DNA
Form double-stranded DNA；Or aptamer is by circulation isothermal strand displacement polyreaction and hybridization chain type
Reaction (Hybridization chain reaction, HCR) forms long double-stranded DNA.
Described aptamer includes: with heavy metal ion, organic dyestuff, aminoacid, You Ji little
Molecule, nucleic acid, RNA, polysaccharide, enzyme, somatomedin, antibody, antibacterial, cell, virus
All kinds of aptamers for part.Wherein, aptamer can also be with containing specific base sequence
DNA replaced.
Advantage for present invention:
What the present invention provided is used for detecting dichloro three (1,10-phenanthrolene) ruthenium (II)
The current potential of (Dichlorotris (1,10-phenanthroline) ruthenium (II)) passes
Sensor is highly sensitive, selectivity is high, and this potentiometric sensor can be used in life based on DNA simultaneously
Thing senses；Bio-sensing based on DNA can be by selecting different aptamers, it is possible to realizes
The detection of different target molecules, can be with circulation isothermal strand displacement polyreaction and hybridization chain during detection
Formula reaction and nano material amplifying technique combine, and improve the sensitivity of detection, selection further
Property.
Separately, dichloro three (1,10-phenanthrolene) ruthenium (II) is a kind of little molecule, it is possible to select
The embedding double-stranded DNA of property.Aptamer is that one can identify that particular target is divided efficiently, specifically
The DNA fragmentation of son.By introducing the DNA strand complementary with aptamer, form DNA double chain,
Now dichloro three (1,10-phenanthrolene) ruthenium (II) can optionally embed double-stranded DNA.
Before and after target molecule and aptamer effect, change in solution or aptamer functional substrate
The concentration of surface dichloro three (1,10-phenanthrolene) ruthenium (II), thus produce electrode potential
Change, realizes the detection of target molecule accordingly.By selecting different aptamers, the method has
There is certain versatility, it is possible to realize the detection of different target molecule.By introducing circulation isothermal chain
Metathesis polymerizable reaction and hybridization chain reaction (Hybridization chain reaction,
HCR) long double-stranded DNA is formed, or a large amount of DNA of nanomaterial loadings, it is achieved potentiometric detection
The amplification of signal.
Accompanying drawing explanation
The sensor schematic diagram that Fig. 1 provides for the embodiment of the present invention.
The sensor schematic diagram that Fig. 2 provides for the embodiment of the present invention.
Liquid membrane electrode detection dichloro three (1,10-neighbour's diaza that Fig. 3 provides for the embodiment of the present invention
Luxuriant and rich with fragrance) ruthenium (II) potential response figure in real time.
(1,10-is adjacent for the solid contact electrode detection dichloro three that Fig. 4 provides for the embodiment of the present invention
Phenanthroline) ruthenium (II) potential response figure in real time.
The magnetic Nano surface nucleic acid that Fig. 5 provides for the embodiment of the present invention is fit miscellaneous with complementary DNA
Chain reaction is handed over to form long double-stranded DNA figure.
Detailed description of the invention
Embodiment 1
Potentiometric sensor, including working electrode 1, outer reference electrode 2, internal reference electrode 3 and
Potential measurement instrument 4, potential measurement instrument 4 connects internal reference electrode 3 and outer ginseng respectively by wire
Ratio electrode 2, internal reference electrode 3 insertion fills internal-filling liquid 8, and (internal-filling liquid is 10-3Mole dichloro
Three (1,10-phenanthrolene) ruthenium (II)) working electrode 1 in, working electrode 1 and outer ginseng
Inserting than electrode 2 in the detection cell 5 filling detection liquid 6, working electrode 1 is for being fixed with polymerization
The electrode holder of thing sensitive membrane 7, is provided with the magnetic stirrer with stirring action in detection cell 5
9, it is configured with magnetic stirring apparatus at the bottom of detection cell to rotate magnetic stir bar.It is fixed with polymer quick
The electrode holder of sense film 7 is polyvinyl chloride pipe.
Detection method:
A. gathering with the doping of cationite four (3,5-bis-(trifluoromethyl) phenyl) sodium borate
Compound film is working electrode sensitive membrane, and silver-silver chloride electrode is inside and outside reference electrode, interior reference
Electrode is connected with the working electrode joint of electrochemical workstation CHI760D, outer reference electrode with
Reference electrode joint connects (seeing Fig. 1), selects " OCP-time " technical measurement electricity
Place value.
The preparation of working electrode sensitive membrane: according to mass percent 1%:33%:66% by four (3,5-
Two (trifluoromethyl) phenyl) sodium borate, polrvinyl chloride, o-Nitrobenzol octyl ether mixing, gained mix
Compound 345mg altogether, is dissolved in 3.5mL tetrahydrofuran solution, flat under room temperature
Placing in glass ring (internal diameter is 3.6cm), after about 8h, oxolane evaporates, and to obtain final product
To uniform polymer sensitive membrane.
It is fixed with the preparation of the electrode holder working electrode of polymer sensitive membrane: gathered by above-mentioned gained
The polymer sensitive membrane of cut-off footpath about 0.7cm beaten by compound sensitive membrane card punch, and passes through tetrahydrochysene
Furan is fixed on beating the film taken bottom polyvinyl chloride pipe.Electrode uses front 10-3M dichloro three
(1,10-phenanthrolene) ruthenium (II) activates about 8h.
B., under stirring condition, electrode is measured 10-3-10-9M standard solution (10-3-10-9M
Standard solution is 10-3-10-9Dichloro three (1,10-phenanthrolene) ruthenium (II) of M) in electricity
Position response (seeing Fig. 3), according to potential response, makes potential changing value to dichloro three (1,10-
Phenanthrolene) standard working curve of ruthenium (II) concentration.
C. electrode is inserted the to be measured of unknown concentration dichloro three (1,10-phenanthrolene) ruthenium (II)
In sample liquid, the response of recording electrode current potential, then try to achieve dichloro three according to standard curve
The concentration of (1,10-phenanthrolene) ruthenium (II).
Embodiment 2
Potentiometric sensor, including working electrode 1, reference electrode 2 and potential measurement instrument 3, electricity
Position-finding instrument 3 connects working electrode 1 and outer reference electrode 2, working electrode respectively by wire
1 and outer reference electrode 2 insert fill detection liquid detection cell 4 in, working electrode 1 is fixing
There is the solid electrode of polymer sensitive membrane 5, detection cell 4 is provided with the magnetic with stirring action
Stirrer 6, is configured with magnetic stirring apparatus to rotate magnetic stir bar (seeing Fig. 2) at the bottom of detection cell.
Detection method:
A. gathering with the doping of cationite four (3,5-bis-(trifluoromethyl) phenyl) sodium borate
Compound film is working electrode sensitive membrane, and silver-silver chloride electrode is outer reference electrode, selects " to open
Road current potential-time " technical measurement potential value.
By 50mg (wherein four (3,5-bis-(trifluoromethyl) phenyl) sodium borate 0.5mg, four (ten
Dialkyl group)-four (4-chlorphenyl) ammonium borate (ETH500) 5mg, polymeric film methyl
Acrylic acid methyl ester .-certain herbaceous plants with big flowers methacrylic acid methyl ester 44.5mg) membrane component is dissolved in 0.5mL dichloromethane
In alkane (leading to nitrogen deoxygenation in advance), standby after leading to nitrogen deoxygenation again after stirring two hours.Take 100
The above-mentioned membrane material of microlitre drips on the solid carbon electrode of polishing, and this electrode is placed in perseverance
More than volatile dry 12h in temperature drying baker.
Dried electrode can be soaked in 10-4moL/L Ru2+Measure after solution activates 48h.
Ag-AgCl is used to make reference electrode, during mensuration during determination of electrode: solid electrode | polymer is sensitive
Film | | sample solution | 3M KCl solution | Ag-AgCl.
B., under stirring condition, electrode is measured 10-3-10-10Potential response in M standard solution,
According to potential response (seeing Fig. 4), make potential changing value to dichloro three (1,10-neighbour's phenodiazine
Miscellaneous phenanthrene) standard working curve of ruthenium (II) concentration.
C. electrode is inserted treating of unknown concentration dichloro three (1,10-phenanthrolene) ruthenium (II)
Test sample product, the response of recording electrode current potential, then try to achieve dichloro three (1,10-according to standard curve
Phenanthrolene) concentration of ruthenium (II).
Embodiment 3
It is with 2 differences with embodiment 1: the present embodiment develops electricity based on above-described embodiment
Position sensing, and be to refer to aptamer identification and dichloro three (1,10-phenanthrolene) ruthenium (II)
Show ion, use the dichloro three (1,10-phenanthrolene) ruthenium (II) of development as detector
Potentiometric sensors method.The present embodiment is as a example by pesticide buttocks worm amidine detects, and detection method is as follows:
Potentiometric sensors: include working electrode 1, reference electrode 2 and potential measurement instrument 3, current potential
Analyzer 3 connects working electrode 1 and outer reference electrode 2, working electrode 1 respectively by wire
Inserting in the detection cell 4 filling detection liquid with outer reference electrode 2, working electrode 1 is for being fixed with
The solid electrode of polymer sensitive membrane 5, is provided with in detection cell 4 and has the magnetic of stirring action and stir
Mix son 6, be configured with magnetic stirring apparatus at the bottom of detection cell to rotate magnetic stir bar (seeing Fig. 2).
Polymerization with the doping of cationite four (3,5-bis-(trifluoromethyl) phenyl) sodium borate
Thing film is working electrode sensitive membrane, and silver-silver chloride electrode is outer reference electrode, selects " open circuit
Current potential-time " technical measurement potential value.
10 μ L25mmol/LPOT-CH2Cl2 solution are dripped in gold electrode surfaces, is completely dried
After again by 50mg (wherein four (3,5-bis-(trifluoromethyl) phenyl) sodium borate 0.5mg, four (ten
Dialkyl group)-four (4-chlorphenyl) ammonium borate (ETH500) 5mg, polymeric film methyl
Acrylic acid methyl ester .-certain herbaceous plants with big flowers methacrylic acid methyl ester 44.5mg) membrane component is dissolved in 0.5mL dichloromethane
In alkane (leading to nitrogen deoxygenation in advance), standby after leading to nitrogen deoxygenation again after stirring two hours.Take 100
The above-mentioned membrane material of microlitre drips on the solid gold electrode of polishing, and this electrode is placed in perseverance
More than volatile dry 12h in temperature drying baker.Dried electrode can be soaked in 10-4moL/L
Ru2+Measure after solution activates 48h.
Detection:
The preparation at (a) aptamer functionalization double-stranded DNA interface: working electrode is placed in 10-6
In aptamer (as a example by the buttocks worm amidine) solution that M5' end is mercapto-functionalized, aptamer exists
Electrode surface reacts 24 hours, further gold electrode is placed in 10-6In the sulfydryl hexanol of M instead
Answer 12h, above-mentioned gold electrode is placed in 10-6M and the DNA of buttocks worm amidine aptamer partial complementarity
Effect 2h, obtains aptamer functionalization double-stranded DNA interface.
(b) Criterion working curve: step a gained double-stranded DNA interface is placed in 1mL10-5
In mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour, interface with
Solution separating, is placed in solution after separation by working electrode, measures and remains dichloro three in solution
The potential response that (1,10-phenanthrolene) ruthenium (II) produces, as blank.
Step a gained double-stranded DNA interface and 10-3-10-10Mole Acetamiprid standard solution effect
After 1h, interface and solution separating, double-stranded DNA interface reacted with above-mentioned standard solution is put
In 1mL10-5In mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, effect 1 is little
Time, interface and solution separating, working electrode is placed in solution after separation, measures in solution and remain
The potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces, as standard solution
Potential response.
According to standard solution remains the electricity that dichloro three (1,10-phenanthrolene) ruthenium (II) produces
Position is in response to the potential change signal Criterion working curve of blank difference.
The mensuration of the testing sample of (c) unknown concentration Acetamiprid:
Step a gained double-stranded DNA interface is placed in 1mL10-5(1,10-neighbour two for mole dichloro three
Aza-phenanthrenes) in ruthenium (II) solution, act on 1 hour, interface and solution separating, by working electrode
It is placed in solution after separation, measures and solution remains dichloro three (1,10-phenanthrolene) ruthenium (II)
The potential response produced, as blank.
Step a gained double-stranded DNA interface and the testing sample effect 1h of unknown concentration Acetamiprid
After, interface and solution separating, double-stranded DNA interface reacted with above-mentioned testing sample is placed in 1
mL10-5In mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour,
Interface and solution separating, be placed in solution after separation by working electrode, measures and remains dichloro in solution
The potential response that three (1,10-phenanthrolene) ruthenium (II) produce, as the current potential of testing sample
Response.
Testing sample solution remains the electricity that dichloro three (1,10-phenanthrolene) ruthenium (II) produces
Position in response to blank difference, difference again by standard working curve, finally give record to be measured
The concentration of Acetamiprid in sample.
Or,
Step a gained double-stranded DNA interface is placed in 1mL10-5(1,10-neighbour two for mole dichloro three
Aza-phenanthrenes) in ruthenium (II) solution, act on 1 hour, interface and solution separating, the most again by boundary
Face is placed in pH7.410-3Mole of phosphoric acid salt buffer solution is heated to 95 degree, interface combines
Dichloro three (1,10-phenanthrolene) ruthenium (II) be discharged in buffer solution, then by work electricity
Pole is placed in buffer solution, measures dichloro three (1,10-phenanthrolene) ruthenium (II) in buffer solution
The potential response produced, as blank.
Step a gained double-stranded DNA interface and the testing sample effect 1h of unknown concentration Acetamiprid
After, interface and solution separating, double-stranded DNA interface reacted with above-mentioned testing sample is placed in 1
mL10-5In mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour,
Interface and solution separating, be placed in pH7.410 by interface the most again-3Mole of phosphoric acid salt buffer is molten
Liquid is heated to 95 degree, dichloro three (1, the 10-phenanthrolene) ruthenium (II) that interface combines
It is discharged in buffer solution, then working electrode is placed in buffer solution, measure in buffer solution
The potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces, in testing sample solution
It is discharged into the potential response that the dichloro three (1,10-phenanthrolene) ruthenium (II) of buffer solution produces
In blank difference, difference by standard working curve, finally gives and records in testing sample again
The concentration of Acetamiprid.
Aptamer sequence 5-(SH)-(CH of Acetamiprid2)6-TGTAA TTTGTCTGCAG
CGGTTCTTGATCGCTGA-CACCATATTATGAA GA-3. and buttocks worm amidine aptamer part
Complementary DNA sequence is: 3-AAGAACTAGC GACTGTGGR ATAATAC TTCT-5
Embodiment amplifying nucleic acid is fit can also with heavy metal ion such as potassium, hydrargyrum, lead, copper, zinc,
Uranium；Organic dyestuff such as Sulforhodamine B, reactive dark green KE-4BD；Aminoacid such as arginine,
TYR amine；Organic molecule such as cocaine, cholic acid, aspartame (containing phenylalanine),
(R)-Thalidomide, 17 beta estradiols, bisphenol-A, buttocks worm amidine；Nucleic acid such as adenosine triphosphate,
Adenylic acid, adenosine diphosphate (ADP)；RNA such as TAR-RNA；Polysaccharide such as cellobiose, saliva lactose；
Enzyme such as thrombin, protein kinase, centriole cell elastase, HIV1-RT；
Toxin such as Ricin；Somatomedin such as basic fibroblast growth factor, platelet derived
Growth factor B chain；Antibody such as human normal immunoglobulin IgE；Antibacterial and cell such as spore, pulmonary carcinoma
Cell, pathogenic bacterium are that all kinds of aptamers of part are replaced.
Embodiment 4
Use the working electrode of embodiment 3, tie mutually with DNA hybridization chain reaction with aptamer
Close, it is achieved potentiometric detection signal amplifies.The present embodiment by bisphenol-A detect as a example by,
Detection method is as follows:
A. the magnetic Nano that 50ul isosulfocyanate radical is modified is placed in 5 × 10-6M5' Amino End Group
In aptamer (as a example by the bisphenol-A) solution of functionalization, 37 degree are reacted 1 hour, magnetic
Property separate, more above-mentioned aptamer modified magnetic Nano is placed in 5% bovine serum albumin
Reacting 1 hour in solution, Magnetic Isolation obtains aptamer functional magnetic nano-particle.Will
Aptamer functional magnetic nano-particle reacts 2 hours with auxiliary DNA solution, receives in magnetic
There is hybridization chain reaction, Magnetic Isolation in rice surface, magnetic Nano is placed in DNA hybridization buffer
Solution (10mM Tris-HCl+1mM EDTA+500mM NaCl+1mM MgCl2,
PH7.4), in, aptamer functionalization double-stranded DNA interface is constructed.
Wherein, after auxiliary DAN solution reacts 2 hours for auxiliary DNA2 and DNA3, add auxiliary
DNA1 is helped to obtain.
(b) Criterion working curve: 30 μ L step a gained double-stranded DNA interfacial separation
After, it is placed in 1mL10-5In mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, make
With 1 hour, interface and solution separating, working electrode is placed in solution after separation, measures solution
The potential response that middle residue dichloro three (1,10-phenanthrolene) ruthenium (II) produces, as blank.
Step a gained double-stranded DNA interface and 10-3-10-12M bisphenol-A standard solution effect 1h
After, interface and solution separating, double-stranded DNA interface reacted with above-mentioned standard solution is placed in 1
mL10-5In mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour,
Interface and solution separating, be placed in solution after separation by working electrode, measures and remains dichloro in solution
The potential response that three (1,10-phenanthrolene) ruthenium (II) produce, as the current potential of standard solution
Response.
According to standard solution remains the electricity that dichloro three (1,10-phenanthrolene) ruthenium (II) produces
Position is in response to the potential change signal Criterion working curve of blank difference.
The mensuration of the testing sample of (c) unknown concentration bisphenol-A:
After 30 μ L step a gained double-stranded DNA INTERFACE MAGNETISM separate, it is placed in 1mL10-5Rub
In your dichloro three (1,10-phenanthrolene) ruthenium (II) solution, acting on 1 hour, interface is with molten
Liquid separates, and working electrode is placed in solution after separation, measures and remains dichloro three (1,10-in solution
Phenanthrolene) potential response that produces of ruthenium (II), as blank.
Step a gained double-stranded DNA interface and the testing sample effect 1h of unknown concentration bisphenol-A
After, interface and solution separating, double-stranded DNA interface reacted with above-mentioned testing sample is placed in 1
mL10-5In mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour,
Interface and solution separating, be placed in solution after separation by working electrode, measures and remains dichloro in solution
The potential response that three (1,10-phenanthrolene) ruthenium (II) produce, as the current potential of testing sample
Response
Testing sample solution remains the electricity that dichloro three (1,10-phenanthrolene) ruthenium (II) produces
Position in response to blank difference, difference again by standard working curve, finally give record to be measured
The concentration of bisphenol-A in sample.
In described step a, HCR reaction is following (as shown in Figure 5):
(1) isosulfocyanate radical end magnetic particle solution is purchased from Xi'an gold magnetic nano biological science and technology
Co., Ltd (xMagTM).Experiment carries out aptamer mark according to kit standard program
Note.Take 2 1.5mL centrifuge tubes and be once labeled as 1,2, be then separately added into 30 μ L
The xMag that labelling is goodTMIsosulfocyanate radical end magnetic particle solution, Magnetic Isolation removes supernatant.
(2) centrifuge tube 1 adds 30 microlitre DNA hybridization buffer solution (10mM Tris-HCl
+1mM EDTA+500mM NaCl+1mM MgCl2, pH7.4), in centrifuge tube 2
Adding 30 microlitre DNA1 liquid, react 1h in 37 DEG C of water-baths, then Magnetic Isolation takes supernatant.
(3) for the centrifuge tube in step 2,40 microlitre DNA hybridization buffer solution (10 in 1, are added
mM Tris-HCl+1mM EDTA+500mM NaCl+1mM MgCl2, pH7.4),
40 microlitre DNA2 and DNA3 hybridization solution (hybridization time 2h before adding) is added in 2,
Reacting 2h in 37 DEG C of water-baths, then Magnetic Isolation removes supernatant.Wherein bisphenol-A aptamer sequence
As follows: 5'-GGG CCG TTC GAA CAC GAG CAT GCC GGT GGG TGG TCA GGT
GGG ATA GCG TTC CGC GTA TGG CCC AGC GCA TCA CGG GTT CGC ACC AGG
ACA GTA CTC AGG TCA TCC TAG-3', the sequence of auxiliary DNA1: 3'-GCG TGG TCC
TGT CAT GAG TCC AGT AGG ATC ACT AAA AGG GTC TGA GGG-5'；Auxiliary
The sequence of DNA2: 5'-TGA TTT TCC CAG ACT CCC CGT GGA CCC CCT CAT-3'；
The sequence of auxiliary DNA3: 3'GCA CCT GGG GGA GTA ACT AA A AGG GTC TGA
GGG-5'。
Embodiment amplifying nucleic acid is fit can also with heavy metal ion such as potassium, hydrargyrum, lead, copper, zinc,
Uranium；Organic dyestuff such as Sulforhodamine B, reactive dark green KE-4BD；Aminoacid such as arginine,
TYR amine；Organic molecule such as cocaine, cholic acid, aspartame (containing phenylalanine),
(R)-Thalidomide, 17 beta estradiols, bisphenol-A, buttocks worm amidine；Nucleic acid such as adenosine triphosphate,
Adenylic acid, adenosine diphosphate (ADP)；RNA such as TAR-RNA；Polysaccharide such as cellobiose, saliva lactose；
Enzyme such as thrombin, protein kinase, centriole cell elastase, HIV1-RT；
Toxin such as Ricin；Somatomedin such as basic fibroblast growth factor, platelet derived
Growth factor B chain；Antibody such as human normal immunoglobulin IgE；Antibacterial and cell such as spore, pulmonary carcinoma
Cell, pathogenic bacterium are that all kinds of aptamers of part are replaced.
Embodiment amplifying nucleic acid is fit can also be fixed on CNT, gold nano, ferroso-ferric oxide
Magnetic nanoparticle, graphene-supported magnetic nanoparticle, graphenic surface, gold electrode surfaces
Or carbon electrodes.
Embodiment 5
Use the working electrode of embodiment 3, as a example by nuclease and radical damage DNA detection,
Detection method is as follows:
A. the preparation at aptamer functionalization double-stranded DNA interface: working electrode is placed in 10-6M
In the random sequence DNA solution that 5' end is mercapto-functionalized, it is little that DNA reacts 24 at electrode surface
Time, further gold electrode is placed in 10-6The sulfydryl hexanol of M reacts 12h, by above-mentioned gold electricity
Pole is placed in 10-6The DNA of the above-mentioned random sequence DNA partial complementarity that M with 5' end is mercapto-functionalized
Effect 2h, obtains DNA functionalization double-stranded DNA interface.
B. Criterion working curve: step a gained double-stranded DNA interface is placed in 1mL10-5
In mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour, interface with
Solution separating, is placed in solution after separation by working electrode, measures and remains dichloro three in solution
The potential response that (1,10-phenanthrolene) ruthenium (II) produces, as blank.
Step a gained double-stranded DNA interface respectively at 0.1U/mL, 1U/mL, 5U/mL,
The nuclease effect 10min of 10U/mL, 30U/mL, 50U/mL, is then separately added into 1
MM ethylenediaminetetraacetic acid (EDTA) stops enzyme reaction, and interface and solution separating are different from above-mentioned
Double-stranded DNA interface after the nuclease solution reaction of concentration is placed in 1mL10-5Mole dichloro three
In (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour, interface and solution separating,
Working electrode is placed in solution after separation, measures and solution remains dichloro three (1,10-neighbour's diaza
Luxuriant and rich with fragrance) potential response that produces of ruthenium (II), as the potential response of standard solution.
According to standard solution remains the electricity that dichloro three (1,10-phenanthrolene) ruthenium (II) produces
Position is in response to the potential change signal Criterion working curve of blank difference.
The mensuration of the testing sample of (c) unknown concentration sample enzyme: step a gained double-stranded DNA circle
Face is placed in 1mL10-5In mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, effect
1 hour, interface and solution separating, working electrode is placed in solution after separation, measures in solution
The potential response that residue dichloro three (1,10-phenanthrolene) ruthenium (II) produces, as blank.
Behind step a gained double-stranded DNA interface and sample enzymatic solution effect 10min to be measured, add
Entering 1mM ethylenediaminetetraacetic acid (EDTA) to stop enzyme reaction, interface and solution separating, with upper
State testing sample reacted double-stranded DNA interface and be placed in 1mL10-5Mole dichloro three (1,10-
Phenanthrolene) in ruthenium (II) solution, act on 1 hour, interface and solution separating, by work
Electrode is placed in solution after separation, measures and remains dichloro three (1,10-phenanthrolene) ruthenium in solution
(II) potential response produced, as the potential response of testing sample.
Testing sample solution remains the electricity that dichloro three (1,10-phenanthrolene) ruthenium (II) produces
Position in response to blank difference, difference again by standard working curve, finally give record to be measured
Sample enzyme activity in sample.
In step a, the sequence of aptamer/DNA can be according to the selectively acting site of enzyme
It is designed.As the base sequence of restriction enzyme A nal contains 5'-TTA ... TAA3',
Clipped is random sequence, containing 3'-AAT in sequence complementary therewith ... ATT5'.Its
Its restricted enzyme is designed according to mentioned above principle.
In step b, determinand can also be used for the material interacted with aptamer/DNA,
Including deoxyribonuclease, restricted enzyme, exonuclease, ligase, nuclease
The mixture of S1, nuclease and its inhibitor, hydroxyl radical free radical, or antioxidant and hydroxyl
Free radical mixed solution.
Embodiment 6
Use the working electrode of embodiment 3, as a example by adenosine triphosphate detects, use Graphene
As the embedding carrier of dichloro three (1,10-phenanthrolene) ruthenium (II), detection method is as follows:
The preparation at (a) aptamer functionalization DNA interface: working electrode is placed in 10-6M5'
In the aptamer (as a example by adenosine triphosphate atp) of Amino End Group or carboxyl-functional, nucleic acid
Fit electrode surface reaction 24 hours, further gold electrode is placed in 10-6The sulfydryl of M is own
Alcohol reacts 12h,
(b) Criterion working curve: step a gained DNA interface is placed in 1mg/mL graphite
2h in alkene solution, is placed in 1mL10 the most again-5Mole dichloro three (1,10-phenanthrolene)
In ruthenium (II) solution, act on 1 hour, interface and solution separating, working electrode is placed in separation
Rear solution, measures and remains the electricity that dichloro three (1,10-phenanthrolene) ruthenium (II) produces in solution
Position response, as blank.
Step a gained DNA interface and 10-2-10-9M adenosine triphosphate standard solution effect 1h
After, it is placed in 2h in 1mg/mL graphene solution, interface and solution separating the most again, with above-mentioned
Standard solution reacted DNA interface is placed in 1mL10-5Mole dichloro three (1,10-neighbour's phenodiazine
Miscellaneous phenanthrene) in ruthenium (II) solution, act on 1 hour, interface and solution separating, working electrode is put
Solution after separating, dichloro three (1,10-phenanthrolene) ruthenium (II) interacts with Graphene,
Measure and solution remain the potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces,
Potential response as standard solution.
According to standard solution remains the electricity that dichloro three (1,10-phenanthrolene) ruthenium (II) produces
Position is in response to the potential change signal Criterion working curve of blank difference.
The mensuration of the testing sample of (c) unknown concentration adenosine triphosphate:
Step a gained DNA interface is placed in 2h in 1mg/mL graphene solution, is placed in the most again
1mL10-5In mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour,
Interface and solution separating, be placed in solution after separation by working electrode, measures and remains dichloro in solution
The potential response that three (1,10-phenanthrolene) ruthenium (II) produce, as blank.
Step a gained DNA interface and the testing sample solution effect of unknown concentration adenosine triphosphate
After 1h, and being placed on 2h in 1mg/mL graphene solution, interface and solution separating, with above-mentioned
Testing sample reacted DNA interface is placed in 1mL10-5Mole dichloro three (1,10-neighbour's phenodiazine
Miscellaneous phenanthrene) in ruthenium (II) solution, act on 1 hour, interface and solution separating, working electrode is put
Solution after separating, dichloro three (1,10-phenanthrolene) ruthenium (II) interacts with Graphene,
Measure and solution remain the potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces,
Potential response as testing sample.
Testing sample solution remains the electricity that dichloro three (1,10-phenanthrolene) ruthenium (II) produces
Position in response to blank difference, difference again by standard working curve, finally give record to be measured
Adenosine triphosphate concentration in sample.
Embodiment amplifying nucleic acid is fit can also with heavy metal ion such as potassium, hydrargyrum, lead, copper, zinc,
Uranium；Organic dyestuff such as Sulforhodamine B, reactive dark green KE-4BD；Aminoacid such as arginine,
TYR amine；Organic molecule such as cocaine, cholic acid, aspartame (containing phenylalanine),
(R)-Thalidomide, 17 beta estradiols, bisphenol-A, buttocks worm amidine；Nucleic acid such as adenosine triphosphate,
Adenylic acid, adenosine diphosphate (ADP)；RNA such as TAR-RNA；Polysaccharide such as cellobiose, saliva lactose；
Enzyme such as thrombin, protein kinase, centriole cell elastase, HIV1-RT；
Toxin such as Ricin；Somatomedin such as basic fibroblast growth factor, platelet derived
Growth factor B chain；Antibody such as human normal immunoglobulin IgE；Antibacterial and cell such as spore, pulmonary carcinoma
Cell, pathogenic bacterium are that all kinds of aptamers of part are replaced.
Meanwhile, Graphene carrier can also use CNT, molybdenum bisuphide or tungsten disulfide to enter
Row is replaced.
Embodiment 7
Use the working electrode of embodiment 4, for the detection of DNA, particularly as follows:
Detection method is as follows:
A. the magnetic Nano that 50ul isosulfocyanate radical is modified is placed in DNA and identifies probe (this spy
Pin end section and DNA base complementary to be measured) in solution, 37 degree reactions 1 hour,
Magnetic Isolation, more above-mentioned aptamer modified magnetic Nano is placed in 5% bovine serum albumin
Solution in react 1 hour, Magnetic Isolation obtains DNA functional magnetic nano-particle.Will
DNA functional magnetic nano-particle reacts 2 hours, at magnetic Nano table with auxiliary DNA solution
There is hybridization chain reaction, Magnetic Isolation in face, magnetic Nano is placed in DNA hybridization buffer solution
(10mM Tris-HCl+1mM EDTA+500mM NaCl+1mM MgCl2,pH7.4)
In, construct aptamer functionalization double-stranded DNA interface.
Wherein, after auxiliary DAN solution reacts 2 hours for auxiliary DNA2 and DNA3, add auxiliary
DNA1 is helped to obtain.
(b) Criterion working curve:
30 μ L step a gained double-stranded DNA interfaces, Magnetic Isolation is placed on 1mL10-5Mole
In dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour, interface and solution
Separate, working electrode is placed in solution after separation, measure and solution remains dichloro three (1,10-
Phenanthrolene) potential response that produces of ruthenium (II), as blank.
Step a gained double-stranded DNA interface and 10-6-10-18After mole DNA effect 1h to be measured,
Interface and solution separating, double-stranded DNA interface reacted with above-mentioned standard solution is placed in 1mL
10-5In mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour, interface
With solution separating, working electrode is placed in solution after separation, measures and solution remains dichloro three
The potential response that (1,10-phenanthrolene) ruthenium (II) produces, the current potential as standard solution rings
Should.
According to standard solution remains the electricity that dichloro three (1,10-phenanthrolene) ruthenium (II) produces
Position is in response to the potential change signal Criterion working curve of blank difference.
The mensuration of the testing sample of (c) unknown concentration DNA:
After 30 μ L step a gained double-stranded DNA INTERFACE MAGNETISM separate, it is placed in 1mL10-5Rub
In your dichloro three (1,10-phenanthrolene) ruthenium (II) solution, acting on 1 hour, interface is with molten
Liquid separates, and working electrode is placed in solution after separation, measures and remains dichloro three (1,10-in solution
Phenanthrolene) potential response that produces of ruthenium (II), as blank.
Step a gained double-stranded DNA INTERFACE MAGNETISM separate after with testing sample effect 1h after, boundary
Face and solution separating, double-stranded DNA interface reacted with above-mentioned testing sample is placed in 1mL10-5
In mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour, interface with
Solution separating, is placed in solution after separation by working electrode, measures and remains dichloro three in solution
The potential response that (1,10-phenanthrolene) ruthenium (II) produces, the current potential as testing sample rings
Should
Testing sample solution remains the electricity that dichloro three (1,10-phenanthrolene) ruthenium (II) produces
Position in response to blank difference, difference again by standard working curve, finally give record to be measured
The concentration of DNA in sample.
In described step a, HCR reaction is following (as shown in Figure 5):
(1) isosulfocyanate radical end magnetic particle solution is purchased from Xi'an gold magnetic nano biological science and technology
Co., Ltd (xMagTM).Experiment carries out aptamer mark according to kit standard program
Note.Take 2 1.5mL centrifuge tubes and be once labeled as 1,2, be then separately added into 30 μ L
The xMag that labelling is goodTMIsosulfocyanate radical end magnetic particle solution, Magnetic Isolation removes supernatant.
(2) centrifuge tube 1 adds 30 microlitre DNA hybridization buffer solution (10mM Tris-HCl
+1mM EDTA+500mM NaCl+1mM MgCl2, pH7.4), in centrifuge tube 2
Adding 30 microlitre DNA1 liquid, react 1h in 37 DEG C of water-baths, then Magnetic Isolation takes supernatant.
(3) for the centrifuge tube in step 2,40 microlitre DNA hybridization buffer solution (10 in 1, are added
mM Tris-HCl+1mM EDTA+500mM NaCl+1mM MgCl2, pH7.4),
40 microlitre DNA2 and DNA3 hybridization solution (hybridization time 2h before adding) is added in 2,
Reacting 1h in 37 DEG C of water-baths, then Magnetic Isolation removes supernatant.Wherein bisphenol-A aptamer sequence
As follows: 5'-GGG CCG TTC GAA CAC GAG CAT GCC GGT GGG TGG TCA GGT
GGG ATA GCG TTC CGC GTA TGG CCC AGC GCA TCA CGG GTT CGC ACC AGG
ACA GTA CTC AGG TCA TCC TAG-3', the sequence of auxiliary DNA1: 3'-GCG TGG TCC
TGT CAT GAG TCC AGT AGG ATC ACT AAA AGG GTC TGA GGG-5'；Auxiliary
The sequence of DNA2: 5'-TGA TTT TCC CAG ACT CCC CGT GGA CCC CCT CAT-3'；
The sequence of auxiliary DNA3: 3'GCA CCT GGG GGA GTA ACT AA A AGG GTC TGA
GGG-5'。
In the present embodiment step a, the sequence of DNA can be designed according to DNA sequence to be measured.
Embodiment 8
Detection method is as follows:
Potentiometric sensors: include working electrode 1, reference electrode 2 and potential measurement instrument 3, current potential
Analyzer 3 connects working electrode 1 and outer reference electrode 2, working electrode 1 respectively by wire
Inserting in the detection cell 4 filling detection liquid with outer reference electrode 2, working electrode 1 is for being fixed with
The solid electrode of polymer sensitive membrane 5, is provided with in detection cell 4 and has the magnetic of stirring action and stir
Mix son 6, be configured with magnetic stirring apparatus at the bottom of detection cell to rotate magnetic stir bar (seeing Fig. 2).
Quick for working electrode with the polymeric film of cationite tetramethylphenyl sodium borate doping
Sense film, silver-silver chloride electrode is outer reference electrode, selects " OCP-time " technology
Measure potential value.
By 10 μ L25mmol/LPOT-CH2Cl2Solution drips in copper electrode surface, is completely dried
After again by 50mg (wherein 0.5mg tetramethylphenyl sodium borate, four (dodecyl)-four (4-
Chlorphenyl) ammonium borate (ETH500) 5mg, polymeric film methyl methacrylate-certain herbaceous plants with big flowers base
Methyl methacrylate 44.5mg) membrane component is dissolved in 0.5mL dichloromethane and (leads to nitrogen in advance
Deoxygenation) in, standby after leading to nitrogen deoxygenation again after stirring two hours.Take 100 microlitre above-mentioned film material
Gob is added on the solid copper electrode of polishing, and is placed in thermostatic drying chamber by this electrode and waves
Dry dry more than 12h.Dried electrode can be soaked in 10-3moL/L Ru2+Solution activates
24h, 10-9moL/L Ru2+It is used for measuring after solution activates 24h.
The present embodiment is for the detection of lgG, and detection process is as follows:
By 1mL xMagTMIt is little that isosulfocyanate radical end magnetic particle Magnetic Isolation is placed on 1mL goat-anti
In Mus lgG antibody-solutions, 37 degree are reacted 0.5 hour, use cleanout fluid to clean, and magnetic is divided
From, more above-mentioned aptamer modified magnetic Nano is placed in the solution of 5% bovine serum albumin
Middle reaction 1 hour, Magnetic Isolation obtains the magnetic nanoparticle of antibody labeling.
(b) Criterion working curve:
The magnetic nanoparticle of 20 μ L antibody labelings, anti-with 20 μ L immunity after Magnetic Isolation
(500mL contains 4g NaCl, 0.1g KCl, 0.72g Na to answer buffer2HPO4,0.12g
KH2PO4), immunoreation one hour, the goat-anti that Magnetic Isolation adds 20 μ L DNA markers is little
Mus lgG antibody, immunoreation one hour.Add after Magnetic Isolation and marker DNA complementation
Reacting one hour in DNA, Magnetic Isolation is placed on 1mL10-6(1,10-neighbour two for mole dichloro three
Aza-phenanthrenes) in ruthenium (II) solution, act on 1 hour, interface and solution separating, by working electrode
It is placed in solution after separation, measures and solution remains dichloro three (1,10-phenanthrolene) ruthenium (II)
The potential response produced, as blank.
The magnetic nanoparticle of 20 μ L antibody labelings, with 20 μ L10 after Magnetic Isolation-6-106
(500mL contains 4g NaCl, 0.1g KCl, 0.72g to ng/mL lgG standard solution
Na2HPO4,0.12g KH2PO4), immunoreation one hour, Magnetic Isolation adds 20 μ L DNA
The sheep anti-Mouse lgG antibody of labelling, immunoreation one hour.Add and labelling after Magnetic Isolation
Reacting one hour in the DNA that DNA is complementary, Magnetic Isolation is placed on 1mL10-5Mole dichloro three
In (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour, interface and solution separating,
Working electrode is placed in solution after separation, measures and solution remains dichloro three (1,10-neighbour's diaza
Luxuriant and rich with fragrance) potential response that produces of ruthenium (II), as the potential response of standard solution.
According to standard solution remains the electricity that dichloro three (1,10-phenanthrolene) ruthenium (II) produces
Position is in response to the potential change signal Criterion working curve of blank difference.
The mensuration of the testing sample of (c) unknown concentration lgG:
The magnetic nanoparticle of 20 μ L antibody labelings, anti-with 20 μ L immunity after Magnetic Isolation
(500mL contains 4g NaCl, 0.1g KCl, 0.72g Na to answer buffer2HPO4,0.12g
KH2PO4), immunoreation one hour, the goat-anti that Magnetic Isolation adds 20 μ L DNA markers is little
Mus lgG antibody, immunoreation one hour.Add after Magnetic Isolation and marker DNA complementation
Reacting one hour in DNA, Magnetic Isolation is placed on 1mL10-6(1,10-neighbour two for mole dichloro three
Aza-phenanthrenes) in ruthenium (II) solution, act on 1 hour, interface and solution separating, by working electrode
It is placed in solution after separation, measures and solution remains dichloro three (1,10-phenanthrolene) ruthenium (II)
The potential response produced, as blank.
The magnetic nanoparticle of 20 μ L antibody labelings, with 20 μ L the unknown samples after Magnetic Isolation
Product solution, immunoreation one hour, the goat-anti that Magnetic Isolation adds 20 μ L DNA markers is little
Mus lgG antibody, immunoreation one hour.Add after Magnetic Isolation and marker DNA complementation
Reacting one hour in DNA, Magnetic Isolation is placed on 1mL10-5(1,10-neighbour two for mole dichloro three
Aza-phenanthrenes) in ruthenium (II) solution, act on 1 hour, interface and solution separating, by working electrode
It is placed in solution after separation, measures and solution remains dichloro three (1,10-phenanthrolene) ruthenium (II)
The potential response produced, as the potential response of testing sample.
Testing sample solution remains the electricity that dichloro three (1,10-phenanthrolene) ruthenium (II) produces
Position in response to blank difference, difference again by standard working curve, finally give record to be measured
The concentration of lgG in sample.
The present embodiment can be sodium tetraphenylborate such as cationite
(tetraphenylborate), tetramethylphenyl sodium borate (tetra (p-tolyl) borate),
Four ptolylboronic acid sodium, four (the fluorine-based phenyl of 4-) tetrakis sodium borate
(4-fluorophenyl), double (1,1,1,3,3,3-perfluoro-2 methoxyl group-2 third of 43,5-
Base phenyl) sodium borate
(tetrakis-[3,5-bis(1,1,1,3,3,3-hexafluoro-2-methoxy-2-prop
yl)phenyl]-borate)。
Antibody in the present embodiment can according to the difference of test substance, select corresponding antibody and
DNA marker resists.
Claims (9)
1. the potentiometric sensor of detection dichloro three (1,10-phenanthrolene) ruthenium (II), bag
Include working electrode, reference electrode and potential measurement instrument, it is characterised in that: potential measurement instrument passes through
Wire connects working electrode and outer reference electrode, working electrode and outer reference electrode respectively and inserts Sheng
Have in the detection cell of detection liquid, be provided with polymer sensitive membrane bottom working electrode, detection cell sets
There are the power set with disturbance solution effects；
Described polymer sensitive membrane is pressed by polymeric matrix material, plasticizer and cationite
20-40:40-80:0.1-10 ratio of weight and number mixing, i.e. obtain resilient polymer
Sensitive membrane；
Described polymeric matrix material is polrvinyl chloride, carboxylated polrvinyl chloride, poly-butyl propyleneglycol
Acid esters, butyl polyacrylate, Polyetherimide, rubber, sol-gel film, methacrylic acid
Methyl ester-certain herbaceous plants with big flowers methacrylic acid methyl terpolymer or n-butyl acrylate-hydroxyethyl methacrylate second
Ester copolymer；Plasticizer is o-Nitrobenzol octyl ether, two-2-ethylhexyl esters in the last of the ten Heavenly stems, decanedioic acid two
Butyl ester or di-n-octyl sebacate；Cationite is four (3,5-bis-(trifluoromethyl) phenyl) boron
Acid sodium, dinonylnaphthalene sulfonic acid or dinonylnaphthalene sulfonic acid salt or borate；
Conducting shell material it is provided with between described working electrode and polymer sensitive membrane；
Described conducting shell material is 3-alkylthrophene, polypyrrole, polythiophene, polyaniline, gathers
Acetylene, poly-n-octyl thiophene, six polythiophenes, Polyglycolic acid fibre, CNT, graphite
Alkene, platinum nanometer, gold nano, molybdenum bisuphide, mesoporous carbon, porous gold or tin indium oxide nanometer.
2. detection dichloro three (1,10-phenanthrolene) ruthenium (II) as described in claim 1
Potentiometric sensor, it is characterised in that: described working electrode is fluid electrode or solid electrode；Its
In, liquid working electrode fills internal-filling liquid and is inserted with the electrode of internal reference electrode in being.
3. detection dichloro three (1,10-phenanthrolene) ruthenium (II) as described in claim 2
Potentiometric sensor, it is characterised in that: described solid electrode be carbon electrode, gold electrode, copper electrode,
Silver electrode, platinum electrode, indium-tin oxide electrode, zinc oxide electrode, silicon electrode, carbon fiber electrode,
Silicon carbide electrode, porous gold electrode, carbon nano tube modified electrode, gold nano modified electrode, platinum
Nanoparticle Modified Electrode, graphene modified electrode, C60 modified electrode, porous carbon modified electrode,
Carbon screen printed electrodes, gold screen printing electrode, paper chip electrode or ceramic chip electrode.
4. the method for detection dichloro three (1,10-phenanthrolene) ruthenium (II), its feature exists
In:
Working electrode and the outer reference electrode of potentiometric sensor described in claim 1 are inserted and contain
Have in the detection liquid of dichloro three (1,10-phenanthrolene) ruthenium (II) to be measured, pass through potential measurement
Instrument measures the potential change on working electrode on polymer sensitive membrane, according to potential change signal with
And standard working curve records dichloro three (1,10-phenanthrolene) ruthenium (II) concentration to be measured.
5. one kind detection dichloro three (1,10-phenanthrolene) ruthenium (II) potentiometric sensor should
With, it is characterised in that: potentiometric sensor described in claim 1 can be as based on DNA or core
The biosensor that acid is fit, the detection quantitative to different target molecules.
6. detection dichloro three (1,10-phenanthrolene) ruthenium (II) as described in claim 5
The application of potentiometric sensor, it is characterised in that: using aptamer as identifying molecule, dichloro three
(1,10-phenanthrolene) ruthenium (II) is formed as embedding molecule, aptamer and complementary DNA
Double-stranded DNA, dichloro three (1,10-phenanthrolene) ruthenium (II) embeds double-stranded DNA, by described
The instruction that potentiometer in potentiometric sensor causes after measuring target molecule and aptamer effect from
The potential change that sub-dichloro three (1,10-phenanthrolene) ruthenium (II) concentration change causes, according to
Potential change signal and standard working curve record target molecule testing concentration.
7. detection dichloro three (1,10-phenanthrolene) ruthenium (II) as described in claim 6
The application of potentiometric sensor, it is characterised in that: described aptamer adds in liquid to be detected,
Or add in detection liquid after fixing with substrate.
8. detection dichloro three (1,10-phenanthrolene) ruthenium (II) as described in claim 7
The application of potentiometric sensor, it is characterised in that: fixed nucleic acid is fit and complementary DNA
Substrate be ferroferric oxide magnetic nanoparticle, magnetic graphene substrate, carbon substrate, gold substrate,
At the bottom of cuprio, at the bottom of SERS substrate, platino, tin indium oxide substrate, at the bottom of Zinc oxide-base, carbon fiber substrates,
Silicon carbide substrate, porous gold substrate, carbon nano tube modified substrate, gold nano modify substrate, platinum
Nano-modified substrate, graphene modified substrate, C60 modify substrate, porous carbon modification substrate
, carbon screen printed electrodes substrate, gold screen printing electrode substrate, paper chip electrode basement
Or ceramic chip electrode basement.
9. detection dichloro three (1,10-phenanthrolene) ruthenium (II) as described in claim 7
The application of potentiometric sensor, it is characterised in that: described aptamer and complementary DNA are formed double
Chain DNA is that aptamer forms double-stranded DNA with wall scroll complementary strand；Or aptamer passes through
Circulation isothermal strand displacement polyreaction and hybridization chain reaction (Hybridization chain
Reaction, HCR) form long double-stranded DNA.
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| CN105738452B (en) * | 2016-02-04 | 2019-12-10 | 中国科学院烟台海岸带研究所 | Method and device for detecting ammonia nitrogen by using all-solid-state ammonia nitrogen sensor |
| CN105606677B (en) * | 2016-03-09 | 2018-02-23 | 山东理工大学 | A kind of preparation method and application of the molybdenum sulfide multi-wall carbon nano-tube tube sensor of silver nano flower-like hydridization |
| CN107402249B (en) * | 2016-05-21 | 2019-07-09 | 云南民族大学 | A method of metal ion is detected based on hybridization chain reaction signal amplification technique |
| CN109317201A (en) * | 2018-08-23 | 2019-02-12 | 南京理工大学 | Cyclodextrin modified organic aryne nanobelt functionalization nitrogen-doped graphene electrochemical sensing material and preparation method thereof |
| CN109374710B (en) * | 2018-10-30 | 2020-08-04 | 浙江大学 | Conductive tungsten disulfide nanosheet-based modified ion selective electrode and preparation method thereof |
| KR102153732B1 (en) * | 2019-04-19 | 2020-09-08 | 울산과학기술원 | Measuring apparatus for unimolecular, manufacturing method for the same and measuring method for unimolecular |
| CN116989652B (en) * | 2023-09-25 | 2023-12-08 | 科瑞工业自动化系统（苏州）有限公司 | Displacement sensor capable of being used as reference electrode and using method thereof |
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