CN108490054A - Photoelectrochemical DNA sensor for detecting DNA sequence and preparation method and detection method thereof - Google Patents
Photoelectrochemical DNA sensor for detecting DNA sequence and preparation method and detection method thereof Download PDFInfo
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- 108091028043 Nucleic acid sequence Proteins 0.000 title claims abstract description 63
- 108020004414 DNA Proteins 0.000 title claims abstract description 47
- 238000001514 detection method Methods 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 26
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims abstract description 73
- 239000004408 titanium dioxide Substances 0.000 claims abstract description 27
- 239000010931 gold Substances 0.000 claims abstract description 19
- 239000002131 composite material Substances 0.000 claims abstract description 18
- 229910001258 titanium gold Inorganic materials 0.000 claims abstract description 16
- 230000003321 amplification Effects 0.000 claims abstract description 15
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 15
- 239000011521 glass Substances 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 12
- 239000002086 nanomaterial Substances 0.000 claims abstract description 9
- 230000005518 electrochemistry Effects 0.000 claims description 55
- 230000003287 optical effect Effects 0.000 claims description 54
- 239000000243 solution Substances 0.000 claims description 43
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 17
- 239000002585 base Substances 0.000 claims description 14
- 238000012360 testing method Methods 0.000 claims description 14
- UHYPYGJEEGLRJD-UHFFFAOYSA-N cadmium(2+);selenium(2-) Chemical class [Se-2].[Cd+2] UHYPYGJEEGLRJD-UHFFFAOYSA-N 0.000 claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 239000000463 material Substances 0.000 claims description 11
- CWERGRDVMFNCDR-UHFFFAOYSA-N thioglycolic acid Chemical compound OC(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-N 0.000 claims description 10
- -1 carbodiimide hydrochlorides Chemical class 0.000 claims description 9
- 235000011121 sodium hydroxide Nutrition 0.000 claims description 9
- 239000007984 Tris EDTA buffer Substances 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 239000002105 nanoparticle Substances 0.000 claims description 8
- 239000008055 phosphate buffer solution Substances 0.000 claims description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- SJUCACGNNJFHLB-UHFFFAOYSA-N O=C1N[ClH](=O)NC2=C1NC(=O)N2 Chemical compound O=C1N[ClH](=O)NC2=C1NC(=O)N2 SJUCACGNNJFHLB-UHFFFAOYSA-N 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 239000003643 water by type Substances 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 5
- 235000013339 cereals Nutrition 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 4
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 3
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 3
- NLZOGIZKBBJWPB-UHFFFAOYSA-N [Na].[SeH2] Chemical compound [Na].[SeH2] NLZOGIZKBBJWPB-UHFFFAOYSA-N 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 230000003139 buffering effect Effects 0.000 claims description 3
- 239000007795 chemical reaction product Substances 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 235000019441 ethanol Nutrition 0.000 claims description 3
- 229910052737 gold Inorganic materials 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 229910052698 phosphorus Inorganic materials 0.000 claims description 3
- 239000011574 phosphorus Substances 0.000 claims description 3
- KOUKXHPPRFNWPP-UHFFFAOYSA-N pyrazine-2,5-dicarboxylic acid;hydrate Chemical compound O.OC(=O)C1=CN=C(C(O)=O)C=N1 KOUKXHPPRFNWPP-UHFFFAOYSA-N 0.000 claims description 3
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 3
- 239000012498 ultrapure water Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 240000007594 Oryza sativa Species 0.000 claims description 2
- 235000007164 Oryza sativa Nutrition 0.000 claims description 2
- XEIPQVVAVOUIOP-UHFFFAOYSA-N [Au]=S Chemical compound [Au]=S XEIPQVVAVOUIOP-UHFFFAOYSA-N 0.000 claims description 2
- 239000003513 alkali Substances 0.000 claims description 2
- 238000001354 calcination Methods 0.000 claims description 2
- 230000005622 photoelectricity Effects 0.000 claims description 2
- 235000009566 rice Nutrition 0.000 claims description 2
- 229910052724 xenon Inorganic materials 0.000 claims description 2
- FHNFHKCVQCLJFQ-UHFFFAOYSA-N xenon atom Chemical compound [Xe] FHNFHKCVQCLJFQ-UHFFFAOYSA-N 0.000 claims description 2
- 229920002892 amber Polymers 0.000 claims 2
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims 1
- 238000005660 chlorination reaction Methods 0.000 claims 1
- 229920006395 saturated elastomer Polymers 0.000 claims 1
- 229910052709 silver Inorganic materials 0.000 claims 1
- 239000004332 silver Substances 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 239000000523 sample Substances 0.000 description 26
- 239000002096 quantum dot Substances 0.000 description 4
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical compound O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 239000000090 biomarker Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 229910052793 cadmium Inorganic materials 0.000 description 2
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000000460 chlorine Substances 0.000 description 2
- 229910052801 chlorine Inorganic materials 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000005611 electricity Effects 0.000 description 2
- 239000007772 electrode material Substances 0.000 description 2
- 229960002317 succinimide Drugs 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229910021607 Silver chloride Inorganic materials 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001378 electrochemiluminescence detection Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 1
- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
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- 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
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- 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
-
- 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
- G01N27/36—Glass electrodes
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- Spectroscopy & Molecular Physics (AREA)
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Abstract
The invention discloses a photoelectrochemical DNA sensor for detecting a DNA sequence, a preparation method thereof and a detection method thereof, wherein the photoelectrochemical DNA sensor is characterized in that a titanium dioxide/gold composite nano material is covered on the surface of an FTO conductive glass electrode, a basic base sequence is fixed on the surface of the titanium dioxide/gold composite nano material through a gold-sulfur bond, and the basic base sequence is connected with a signal amplification factor. The invention realizes the detection of the target DNA sequence by the photoelectrochemistry DNA sensor, and has the advantages of simple method, high sensitivity and easy operation.
Description
Technical field
The present invention relates to a kind of optical electro-chemistry DNA sensor, specifically a kind of optical electro-chemistry of detection DNA sequence dna
DNA sensor and preparation method thereof and detection method.
Background technology
Quantum dot (QDs) is a kind of common narrow bandwidth semiconductive light active material, is obtained in optical electro-chemistry field
Extensive use, especially biomarker context of detection (Zhang, K.Y., Lv, S.Z., Lin, Z.Z., Tang,
D.P.Biosens.Bioelectron.2017,95,34.).However individually quantum dot has some inevitable defects,
Such as there is stronger self-quenching and more sensitive to thermally and chemically interfering, and cannot be carried out directly as electrode material
Modification, which greatly limits the applications of quantum dot.Therefore we apply quantum dot as signal amplification factor in optical electro-chemistry
Field makes it preferably be applied.
Photoelectrochemical process is that generation is electrically separated with transfer in electricity after light-sensitive material absorbs photon under illumination condition
Pole surface formed electron hole pair thus redox reaction occurs and generates the process of photoelectric current.Whole process is presented as light
Can, conversion between electric energy and chemical energy (Da, H.M., Liu, H.Y., Zheng, Y.N., Yuan, R., Chai, Y.Q.,
Biosens.Bioelectron.2018,101,213.).Entire analysis method background signal is low, high sensitivity, response quickly,
The advantages that instrument is simple, cheap and analyst coverage is wide.In recent years, photoelectrochemical assay technology is as a kind of novel biology
Analysis method get the attention and quickly grow (Wang, Y., Zhou, Y.L., Xu, L., Han, Z.W., Yin, H.S.,
Ai,S.Y.,Sensors and Actuators B 2014,257,237.).However traditional optical electro-chemistry material light-use
The problems such as rate is low, poor biocompatibility limits its application in Analytical Chemistry in Life Science, therefore, find it is new and effective, inexpensive,
The photoelectric chemical electrode material of good biocompatibility is still the important research target for building optical electro-chemistry immunosensor.
A variety of biomarkers in clinical diagnosis in super sensitivity detection complex biological structure are very important.More
In kind biomarker, specific DNA or RNA sequence are in molecular diagnostics, pathogeny detection, gene therapy and cancer early stage
Application in diagnosis is more and more important.So far, a variety of with specific DNA or a variety of sides of RNA sequence in order to detect
Method is developed and is applied, such as the methods of fluorescence probe, electrogenerated chemiluminescence, chemiluminescence and electrochemistry etc..But it is existing
These methods having still have many defects, such as expensive equipment, complicated for operation, sensitivity is low and time-consuming, all unfavorable
In Site Detection.Therefore, it is badly in need of a kind of simple, quick and highly sensitive analysis method of development and realizes specific DNA sequences
Quickly detection.
Titania nanoparticles are combined with gold nano grain, layer assembly modified electrode is prepared, can fully send out
The two advantage is waved, application range is expanded, improving the efficiency of light energy utilization simultaneously enhances stability, is development optical electro-chemistry DNA sensor
Novel strategy will have broad application prospects in the fields such as immuno-biology and clinical diagnosis.Currently based on titanium dioxide/gold
The optical electro-chemistry DNA sensor of composite nano materials also there is no report.
Invention content
The present invention is in order to solve above-mentioned existing deficiencies in the technology, it is desirable to provide a kind of detection DNA sequence dna
Optical electro-chemistry DNA sensor and preparation method thereof and detection method, to can be with high photoelectrochemical current, high-biocompatibility
Optical electro-chemistry DNA sensor realize simple, quick detection to specific DNA sequences.
The present invention detects the optical electro-chemistry DNA sensor of DNA sequence dna, is covered on the surface of FTO conductive glass electrodes
Titanium dioxide/gold composite nano materials is fixed on the surface of the titanium dioxide/gold composite nano materials by gold-sulfide linkage
Basic base sequence, the basis base sequence are connect with signal amplification factor.
The basis base sequence is 5 '-NH2-CGG AGT TCT GCA CAC CTC TTG ACA CTC CGT TT-
SH-3 ', the basic sequence can occur specific base complementation with target dna sequence and be combined.
The signal amplification factor is to pass through 1- ethyls -3- (3- dimethylaminopropyls) carbodiimide hydrochlorides and N- hydroxyls
Base succinimide treated CdSe quantum dots.
The titanium dioxide/gold composite nano materials are modified in titania nanoparticles by absorption-calcination reaction
The area load gold nano grain of electrode and obtain.
The present invention detects the preparation method of the optical electro-chemistry DNA sensor of DNA sequence dna, includes the following steps：
Step 1：FTO electro-conductive glass is cleaned with acetone, water and EtOH Sonicate respectively, it is then dried under the conditions of 60 DEG C
Night obtains FTO conductive glass electrodes, spare, and effective work area is 45mm2；
Step 2：200mg titanium dioxide is dispersed in 100mL deionized waters, ultrasonic disperse is uniform, obtains titanium dioxide
Solution；
Step 3：The surface for the FTO conductive glass electrodes that step 1 obtains drops evenly the titanium dioxide of 30 μ L steps 2 preparation
Solution, room temperature are dried, and then calcine 0.5h in 450 DEG C of Muffle furnace, obtain titania nanoparticles modified electrode；
Step 4：The sodium hydroxide solution for preparing the chlorauric acid solution and 0.2mol/L of 10mg/mL respectively, by preparation
It is 4.5 that 10mg/mL chlorauric acid solutions adjust pH value with the sodium hydroxide solution of 0.2mol/L, is then immersed in the two of step 3 preparation
Titanium dioxide nanoparticle modified electrode, impregnates 0.5h, then ultra-pure water cleaning calcines 2h in 300 DEG C of Muffle furnace, obtains two
Titanium oxide/gold composite nanometer material modified electrode；
Step 5：The base of 1 μM of 30 μ L is added dropwise on titanium dioxide/gold composite nanometer material modified electrode prepared by step 4
Plinth base sequence (purchased in market, work biology is given birth in Shanghai), react 12~14 hours at 4 DEG C, and 10mmol/L, pH value 8.0 are used after taking-up
TE buffer solutions rinse；Then 30 μ L signal amplification factors are added dropwise, are reacted 1 hour at 37 DEG C, with 10mmol/L, pH value after taking-up
7.4 phosphate buffer solution rinses, and 30 μ L is then added dropwise, the mercaptoacetic acid solution of 1~5mmol/L is closed 1~2 hour, taking-up
Afterwards use 10mmol/L, pH value 7.4 phosphate buffer solution rinse to get to detection DNA sequence dna optical electro-chemistry DNA sensor.
The signal amplification factor is to pass through 1- ethyls -3- (3- dimethylaminopropyls) carbodiimide hydrochlorides and N- hydroxyls
Base succinimide treated CdSe quantum dots, preparation method include the following steps：
5a, 840 μ L thioacetic acid be added in 60mL ultra-pure waters stir evenly, then with the chlorine of a concentration of 0.1M of 50mL
Cadmium aqueous solution is uniformly mixed, and adjusts pH value to 10 with 0.1M sodium hydroxides, and stir 30min under High Purity Nitrogen protection, then
The sodium hydrogen selenide solution of 12mL a concentration of 0.2M is added, reacts 4h at 75 DEG C, natural cooling, reaction product with ethyl alcohol settle from
The heart, and constant volume obtains CdSe quantum dots solution to primitive reaction volume；
5b, preparation 1- ethyls -3- (3- dimethylaminopropyls) carbodiimide hydrochlorides and 5 containing 10~20mg/mL~
The mixed solution of the n-hydroxysuccinimide of 10mg/mL, according to the volume ratio 5 of CdSe quantum dots solution and mixed solution:1
Ratio mixed, under the conditions of 37 DEG C react 1h to activate CdSe quantum dots, that is, obtain signal amplification factor.
The present invention detects the detection method of the optical electro-chemistry DNA sensor of DNA sequence dna, includes the following steps：
1,30 μ L DNA sequence dna samples to be measured are added dropwise to the surface of optical electro-chemistry DNA sensor of the present invention, are incubated at 37 DEG C
50min is rinsed with the TE buffer solutions of 10mmol/L, pH value 8.0 after taking-up, obtains immunosensor to be measured；
2, the immunosensor to be measured for obtaining step 1 is in the 0.1mol/L containing 0.1mol/L AA, the phosphorus of pH value 7.4
In acid buffering solution carry out optical electro-chemistry test, obtain the photo-current intensity of DNA sequence dna sample to be measured, using photo-current intensity with
The standard relationship curve of DNA sequence dna sample concentration obtains the concentration of DNA sequence dna sample to be measured.
The standard relationship curve of the photo-current intensity and DNA sequence dna sample concentration obtains by the following method：
30 μ L DNA sequence dna standard samples to be measured are added dropwise to the surface of optical electro-chemistry DNA sensor of the present invention, are incubated at 37 DEG C
50min is rinsed after taking-up with the TE buffer solutions of 10mmol/L, pH value 8.0, and get standard samples immunosensor；By gained
Standard sample immunosensor the 0.1mol/L containing 0.1mol/L AA, pH value 7.4 phosphate buffer solution in carry out photoelectricity
Test chemical obtains the photo-current intensity corresponding to each concentration DNA sequence dna standard sample, then with DNA sequence dna standard sample
The logarithm of concentration is abscissa, is fitted using its corresponding photo-current intensity as ordinate, you can obtains photo-current intensity
With the standard relationship curve of DNA sequence dna sample concentration.
A concentration of 10fmol/L to 100nmol/L of the DNA sequence dna standard sample to be measured, at least takes 6 various concentrations
Point value.
As shown in Figure 2, concentration corresponding to each lines is from a left side for photo-current intensity corresponding to each concentration DNA sequence dna standard sample
It is respectively to right each lines：10fmol/LL、100fmol/L、1pmol/L、10pmol/L、100pmol/L、1nmol/L、
10nmol/L and 100nmol/L；Then using the logarithm of the concentration of DNA sequence dna standard sample as abscissa, with its corresponding light
Current strength is fitted for ordinate, and as shown in the illustration of Fig. 2, standard relationship curve is I (μ A)=90.0934-
2.5529log CDNA/pM.Detection shows to work as DNA sequence dna sample concentration in 10fmol/L to 100nmol/L ranges, photoelectric current
Intensity is reduced with the increase of DNA sequence dna sample concentration, linear with concentration, and detection limit reaches 3fmol/L.
It is to electrode, with saturation that the optical electro-chemistry test, which is using immunosensor to be measured as working electrode, with Pt electrodes,
Silver chloride electrode is the three-electrode system of reference electrode, and using 250W xenon lamps as light source, wave-length coverage is 280~1000nm, additional
Voltage is 0V, is changed with CHI660D type electrochemical workstation record currents.
Compared with the prior art, beneficial effects of the present invention are embodied in：
1, the present invention realizes the detection to target dna sequence by optical electro-chemistry DNA sensor, and method is simple, sensitive
Degree is high, easily operated；
2, sample size needed for detection method of the present invention to target dna sequence is few, and testing cost is low；
3, the present invention prepares optical electro-chemistry immunosensor, optical electro-chemistry electricity by titanium dioxide/gold composite nano materials
Stream is high, good biocompatibility, and has fabulous stability.
Description of the drawings
Fig. 1 is the preparation flow schematic diagram of the optical electro-chemistry DNA sensor of present invention detection target dna sequence.
Fig. 2 is that the present invention is respectively 10fmol/LL, 100fmol/L, 1pmol/L, 10pmol/L, 100pmol/ to concentration
L, the target dna sequence standard sample of 1nmol/L, 10nmol/L and 100nmol/L carry out the test result of optical electro-chemistry conversion,
Illustration is standard relationship curve.
Fig. 3 is scanning electron microscope (SEM) characterization result of titania nanoparticles modified electrode in the present invention.
Fig. 4 is scanning electron microscope (SEM) characterization of titanium dioxide/gold composite nanometer material modified electrode in the present invention
As a result.
Specific implementation mode
Embodiment 1：
The preparation method that the optical electro-chemistry DNA sensor of DNA sequence dna is detected in the present embodiment is as follows：
1, FTO electro-conductive glass is cleaned with acetone, water and EtOH Sonicate respectively, is then dried overnight under the conditions of 60 DEG C,
FTO conductive glass electrodes are obtained, spare, effective work area is 45mm2；
2,200mg titanium dioxide is dispersed in 100mL deionized waters, ultrasonic disperse 0.5h, obtains titania solution；
3, it is molten to drop evenly titanium dioxide prepared by 30 μ L steps 2 for the surface for the FTO conductive glass electrodes that step 1 obtains
Liquid, room temperature are dried, and then calcine 0.5h in 450 DEG C of Muffle furnace, obtain titania nanoparticles modified electrode；
4, the sodium hydroxide solution for preparing the chlorauric acid solution and 0.2mol/L of 10mg/mL respectively, by the 10mg/mL of preparation
It is 4.5 that chlorauric acid solution adjusts pH value with the sodium hydroxide solution of 0.2mol/L, and the titanium dioxide for being then immersed in step 3 preparation is received
Rice grain modified electrode, impregnate 0.5h, ultra-pure water cleaning, then calcine 2h in 300 DEG C of Muffle furnace, obtain titanium dioxide/
Golden composite nanometer material modified electrode；
5, the basic alkali of 1 μM of 30 μ L is added dropwise on titanium dioxide/gold composite nanometer material modified electrode prepared by step 4
Basic sequence reacts 12 hours at 4 DEG C, is rinsed with the TE buffer solutions of 10mmol/L, pH value 8.0 after taking-up；Then 30 μ L are added dropwise
Signal amplification factor is reacted 1 hour at 37 DEG C, is rinsed with the phosphate buffer solution of 10mmol/L, pH value 7.4 after taking-up, then
30 μ L are added dropwise, the mercaptoacetic acid solution of 1mmol/L is closed 1 hour, molten with 10mmol/L, the phosphoric acid buffer of pH value 7.4 after taking-up
Liquid rinse to get to detection DNA sequence dna optical electro-chemistry DNA sensor.
The preparation method of the signal amplification factor is as follows：
5a, 840 μ L thioacetic acid be added in 60mL ultra-pure waters stir evenly, then with the chlorine of a concentration of 0.1M of 50mL
Cadmium aqueous solution is uniformly mixed, and adjusts pH value to 10 with 0.1M sodium hydroxides, and stir 30min under High Purity Nitrogen protection, then
The sodium hydrogen selenide solution of 12mL a concentration of 0.2M is added, reacts 4h at 75 DEG C, natural cooling, reaction product with ethyl alcohol settle from
The heart, and constant volume obtains CdSe quantum dots solution to primitive reaction volume；
5b, preparation 1- ethyls -3- (3- dimethylaminopropyls) carbodiimide hydrochlorides and 5mg/mL containing 10mg/mL
The mixed solution of n-hydroxysuccinimide, according to the volume ratio 5 of CdSe quantum dots solution and mixed solution:1 ratio into
Row mixes, and under the conditions of 37 DEG C reaction 1h obtains signal amplification factor to activate CdSe quantum dots.
The method that DNA sequence dna is detected using optical electro-chemistry DNA sensor manufactured in the present embodiment is as follows：
1,30 μ L DNA sequence dna samples to be measured are added dropwise to the surface of optical electro-chemistry DNA sensor of the present invention, are incubated at 37 DEG C
50min is rinsed with the TE buffer solutions of 10mmol/L, pH value 8.0 after taking-up, obtains immunosensor to be measured；
2, the immunosensor to be measured for obtaining step 1 is in the 0.1mol/L containing 0.1mol/L AA, the phosphorus of pH value 7.4
In acid buffering solution carry out optical electro-chemistry test, obtain the photo-current intensity of DNA sequence dna sample to be measured, using photo-current intensity with
The standard relationship curve of DNA sequence dna sample concentration obtains the concentration of DNA sequence dna sample to be measured.
To verify the accuracy of the present embodiment method, it is respectively 10fmol/LL, 100fmol/L, 1pmol/ to take known concentration
L, the DNA sequence dna standard sample to be measured of 10pmol/L, 100pmol/L, 1nmol/L, 10nmol/L and 100nmol/L utilizes this
The optical electro-chemistry DNA sensor of embodiment detects, calculates the concentration of each sample respectively as stated above, is followed successively by 11.96fmol/
LL、97.95fmol/L、1.132pmol/L、10.159pmol/L、114.19pmol/L、1.099nmol/L、11.105nmol/L
And 93.325nmol/L, it can be seen that prepared DNA sensor has quick, sensitive, accurate and efficient inspection to target dna sequence
It surveys.
Embodiment 2：
The present embodiment is to prepare optical electro-chemistry DNA according to the same manner as in Example 1 with the EDC of a concentration of 20mg/mL to pass
Sensor, gained optical electro-chemistry DNA sensor and the pattern of 1 gained optical electro-chemistry DNA sensor of embodiment are similar with property, pass through
Detection to identical DNA sequence dna sample to be measured, obtains the testing result consistent with embodiment 1.
Embodiment 3：
The present embodiment is to prepare optical electro-chemistry DNA according to the same manner as in Example 1 with the NHS of a concentration of 10mg/mL to pass
Sensor, gained optical electro-chemistry DNA sensor and the pattern of 1 gained optical electro-chemistry DNA sensor of embodiment are similar with property, pass through
Detection to identical DNA sequence dna sample to be measured, obtains the testing result consistent with embodiment 1.
Embodiment 4：
The preparation process of the present embodiment with embodiment 1, unlike in step 5, in titanium dioxide/gold prepared by step 4
1 μM of 30 bases μ L base sequence is added dropwise on composite nanometer material modified electrode, reacts 14 hours.Remaining condition step and implementation
Example 1 is identical, and gained optical electro-chemistry DNA sensor and the pattern of 1 gained optical electro-chemistry DNA sensor of embodiment are similar with property,
By the detection to identical DNA sequence dna sample, the testing result consistent with embodiment 1 is obtained.
Embodiment 5：
With embodiment 1, the difference is that in step 5, " the sulfydryl second of 30 μ L, 1mmol/L is added dropwise in the preparation process of the present embodiment
Acid solution is closed 1 hour " it is adjusted to " 30 μ L being added dropwise, the mercaptoacetic acid solution of 5mmol/L is closed 1 hour ", remaining condition step
It is same as Example 1, the pattern and property of gained optical electro-chemistry DNA sensor and 1 gained optical electro-chemistry DNA sensor of embodiment
It is similar, by the detection to identical DNA sequence dna sample to be measured, obtain the testing result consistent with embodiment 1.
Embodiment 6：
With embodiment 1, the difference is that in step 5, " the sulfydryl second of 30 μ L, 1mmol/L is added dropwise in the preparation process of the present embodiment
Acid solution is closed 1 hour " it is adjusted to " 30 μ L being added dropwise, the mercaptoacetic acid solution of 1mmol/L is closed 2 hours ", remaining condition step
It is same as Example 1, the pattern and property of gained optical electro-chemistry DNA sensor and 1 gained optical electro-chemistry DNA sensor of embodiment
It is similar, by the detection to identical DNA sequence dna sample to be measured, obtain the testing result consistent with embodiment 1.
Claims (10)
1. a kind of optical electro-chemistry DNA sensor of detection DNA sequence dna, it is characterised in that：The optical electro-chemistry DNA sensor be
The surface of FTO conductive glass electrodes is covered with titanium dioxide/gold composite nano materials, in the titanium dioxide/gold composite Nano
The surface of material is fixed with basic base sequence by gold-sulfide linkage, and the basis base sequence is connect with signal amplification factor.
2. optical electro-chemistry DNA sensor according to claim 1, it is characterised in that：
The basis base sequence is 5 '-NH2- CGG AGT TCT GCA CAC CTC TTG ACA CTC CGT TT-SH-3 ',
The basic sequence can occur specific base complementation with target dna sequence and be combined.
3. optical electro-chemistry DNA sensor according to claim 1, it is characterised in that：
The signal amplification factor is to pass through 1- ethyls -3- (3- dimethylaminopropyls) carbodiimide hydrochlorides and N- hydroxyl ambers
Amber acid imide treated CdSe quantum dots.
4. optical electro-chemistry DNA sensor according to claim 1, it is characterised in that：
The titanium dioxide/gold composite nano materials are by absorption-calcination reaction in titania nanoparticles modified electrode
Area load gold nano grain and obtain.
5. a kind of preparation method of the optical electro-chemistry DNA sensor of detection DNA sequence dna described in claim 1, it is characterised in that
Include the following steps：
Step 1：FTO electro-conductive glass is cleaned with acetone, water and EtOH Sonicate respectively, is then dried overnight under the conditions of 60 DEG C,
FTO conductive glass electrodes are obtained, spare, effective work area is 45mm2；
Step 2：200mg titanium dioxide is dispersed in 100mL deionized waters, ultrasonic disperse is uniform, obtains titania solution；
Step 3：The titanium dioxide that the surface for the FTO conductive glass electrodes that step 1 obtains drops evenly the preparation of 30 μ L steps 2 is molten
Liquid, room temperature are dried, and then calcine 0.5h in 450 DEG C of Muffle furnace, obtain titania nanoparticles modified electrode；
Step 4：The sodium hydroxide solution for preparing the chlorauric acid solution and 0.2mol/L of 10mg/mL respectively, by the 10mg/mL of preparation
It is 4.5 that chlorauric acid solution adjusts pH value with the sodium hydroxide solution of 0.2mol/L, and the titanium dioxide for being then immersed in step 3 preparation is received
Rice grain modified electrode, impregnate 0.5h, ultra-pure water cleaning, then calcine 2h in 300 DEG C of Muffle furnace, obtain titanium dioxide/
Golden composite nanometer material modified electrode；
Step 5：The basic alkali of 1 μM of 30 μ L is added dropwise on titanium dioxide/gold composite nanometer material modified electrode prepared by step 4
Basic sequence reacts 12~14 hours at 4 DEG C, is rinsed with the TE buffer solutions of 10mmol/L, pH value 8.0 after taking-up；Then it is added dropwise
30 μ L signal amplification factors are reacted 1 hour at 37 DEG C, are rinsed with the phosphate buffer solution of 10mmol/L, pH value 7.4 after taking-up,
Then 30 μ L are added dropwise, the mercaptoacetic acid solution of 1~5mmol/L is closed 1~2 hour, with 10mmol/L, pH value 7.4 after taking-up
Phosphate buffer solution rinse to get to detection DNA sequence dna optical electro-chemistry DNA sensor.
6. preparation method according to claim 5, it is characterised in that：
The signal amplification factor is to pass through 1- ethyls -3- (3- dimethylaminopropyls) carbodiimide hydrochlorides and N- hydroxyl ambers
Amber acid imide treated CdSe quantum dots, preparation method include the following steps：
5a, 840 μ L thioacetic acid be added in 60mL ultra-pure waters stir evenly, then with the caddy of a concentration of 0.1M of 50mL
Aqueous solution is uniformly mixed, and is adjusted pH value to 10 with 0.1M sodium hydroxides, and stir 30min under High Purity Nitrogen protection, is then added
The sodium hydrogen selenide solution of a concentration of 0.2M of 12mL reacts 4h, natural cooling at 75 DEG C, and reaction product is settled with ethyl alcohol and centrifuged,
And constant volume obtains CdSe quantum dots solution to primitive reaction volume；
5b, 1- ethyls -3- (3- dimethylaminopropyls) carbodiimide hydrochlorides and 5~10mg/ containing 10~20mg/mL are prepared
The mixed solution of the n-hydroxysuccinimide of mL, according to the volume ratio 5 of CdSe quantum dots solution and mixed solution:1 ratio
Example is mixed, and 1h is reacted under the conditions of 37 DEG C to activate CdSe quantum dots, that is, obtains signal amplification factor.
7. a kind of detection method of the optical electro-chemistry DNA sensor of detection DNA sequence dna described in claim 1, it is characterised in that
Include the following steps：
Step 1：30 μ L DNA sequence dna samples to be measured are added dropwise to the surface of optical electro-chemistry DNA sensor of the present invention, are incubated at 37 DEG C
50min is rinsed with the TE buffer solutions of 10mmol/L, pH value 8.0 after taking-up, obtains immunosensor to be measured；
Step 2：The immunosensor to be measured that step 1 is obtained is in the 0.1mol/L containing 0.1mol/L AA, the phosphorus of pH value 7.4
In acid buffering solution carry out optical electro-chemistry test, obtain the photo-current intensity of DNA sequence dna sample to be measured, using photo-current intensity with
The standard relationship curve of DNA sequence dna sample concentration obtains the concentration of DNA sequence dna sample to be measured.
8. detection method according to claim 7, it is characterised in that：The photo-current intensity and DNA sequence dna sample concentration
Standard relationship curve obtain by the following method：
30 μ L DNA sequence dna standard samples to be measured are added dropwise to the surface of optical electro-chemistry DNA sensor of the present invention, are incubated at 37 DEG C
50min is rinsed after taking-up with the TE buffer solutions of 10mmol/L, pH value 8.0, and get standard samples immunosensor；By gained
Standard sample immunosensor the 0.1mol/L containing 0.1mol/L AA, pH value 7.4 phosphate buffer solution in carry out photoelectricity
Test chemical obtains the photo-current intensity corresponding to each concentration DNA sequence dna standard sample, then with DNA sequence dna standard sample
The logarithm of concentration is abscissa, is fitted using its corresponding photo-current intensity as ordinate, you can obtains photo-current intensity
With the standard relationship curve of DNA sequence dna sample concentration.
9. detection method according to claim 8, it is characterised in that：
A concentration of 10fmol/L to 100nmol/L of the DNA sequence dna standard sample to be measured, at least takes the point of 6 various concentrations
Value.
10. detection method according to claim 7, it is characterised in that：
It is to electrode, to be saturated chlorination that the optical electro-chemistry test, which is using immunosensor to be measured as working electrode, with Pt electrodes,
Silver electrode is the three-electrode system of reference electrode, and using 250W xenon lamps as light source, wave-length coverage is 280~1000nm, applied voltage
For 0V, changed with CHI660D type electrochemical workstation record currents.
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| CN111272848A (en) * | 2020-03-06 | 2020-06-12 | 安徽大学 | High-sensitivity photoelectrochemical biosensor for detecting miRNA159c and preparation and detection methods thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111272848A (en) * | 2020-03-06 | 2020-06-12 | 安徽大学 | High-sensitivity photoelectrochemical biosensor for detecting miRNA159c and preparation and detection methods thereof |
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