CN110412097A - A kind of preparation method of the optical electro-chemistry sensor of super sensitivity detection microRNA - Google Patents
A kind of preparation method of the optical electro-chemistry sensor of super sensitivity detection microRNA Download PDFInfo
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- 108700011259 MicroRNAs Proteins 0.000 title claims abstract description 39
- 239000002679 microRNA Substances 0.000 title claims abstract description 39
- 238000001514 detection method Methods 0.000 title claims abstract description 32
- 230000005518 electrochemistry Effects 0.000 title claims abstract description 21
- 230000003287 optical effect Effects 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 230000035945 sensitivity Effects 0.000 title claims description 13
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims abstract description 44
- 108020004414 DNA Proteins 0.000 claims abstract description 38
- 238000000034 method Methods 0.000 claims abstract description 25
- 229910052697 platinum Inorganic materials 0.000 claims abstract description 22
- 239000002245 particle Substances 0.000 claims abstract description 19
- 238000006243 chemical reaction Methods 0.000 claims abstract description 15
- BTIJJDXEELBZFS-QDUVMHSLSA-K hemin Chemical compound CC1=C(CCC(O)=O)C(C=C2C(CCC(O)=O)=C(C)\C(N2[Fe](Cl)N23)=C\4)=N\C1=C/C2=C(C)C(C=C)=C3\C=C/1C(C)=C(C=C)C/4=N\1 BTIJJDXEELBZFS-QDUVMHSLSA-K 0.000 claims abstract description 12
- 229940025294 hemin Drugs 0.000 claims abstract description 12
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 11
- 229910052737 gold Inorganic materials 0.000 claims abstract description 11
- 239000010931 gold Substances 0.000 claims abstract description 11
- 238000007639 printing Methods 0.000 claims abstract description 10
- 101710163270 Nuclease Proteins 0.000 claims abstract description 9
- BERDEBHAJNAUOM-UHFFFAOYSA-N copper(I) oxide Inorganic materials [Cu]O[Cu] BERDEBHAJNAUOM-UHFFFAOYSA-N 0.000 claims abstract description 9
- KRFJLUBVMFXRPN-UHFFFAOYSA-N cuprous oxide Chemical compound [O-2].[Cu+].[Cu+] KRFJLUBVMFXRPN-UHFFFAOYSA-N 0.000 claims abstract description 9
- 108091081406 G-quadruplex Proteins 0.000 claims abstract description 7
- 230000004048 modification Effects 0.000 claims abstract description 7
- 238000012986 modification Methods 0.000 claims abstract description 7
- NNLOHLDVJGPUFR-UHFFFAOYSA-L calcium;3,4,5,6-tetrahydroxy-2-oxohexanoate Chemical compound [Ca+2].OCC(O)C(O)C(O)C(=O)C([O-])=O.OCC(O)C(O)C(O)C(=O)C([O-])=O NNLOHLDVJGPUFR-UHFFFAOYSA-L 0.000 claims abstract description 6
- 230000000694 effects Effects 0.000 claims abstract description 6
- 238000011065 in-situ storage Methods 0.000 claims abstract description 4
- 229940112669 cuprous oxide Drugs 0.000 claims abstract description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 51
- 229910052799 carbon Inorganic materials 0.000 claims description 51
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
- 229910002915 BiVO4 Inorganic materials 0.000 claims description 23
- 239000010949 copper Substances 0.000 claims description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 15
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- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 claims description 9
- 238000004458 analytical method Methods 0.000 claims description 8
- 230000015572 biosynthetic process Effects 0.000 claims description 8
- 238000004070 electrodeposition Methods 0.000 claims description 8
- ZSIAUFGUXNUGDI-UHFFFAOYSA-N hexan-1-ol Chemical compound CCCCCCO ZSIAUFGUXNUGDI-UHFFFAOYSA-N 0.000 claims description 8
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- WTDHULULXKLSOZ-UHFFFAOYSA-N Hydroxylamine hydrochloride Chemical compound Cl.ON WTDHULULXKLSOZ-UHFFFAOYSA-N 0.000 claims description 6
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- 150000001875 compounds Chemical class 0.000 claims description 5
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 4
- 238000003786 synthesis reaction Methods 0.000 claims description 4
- 239000003643 water by type Substances 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 3
- BDJYZEWQEALFKK-UHFFFAOYSA-N bismuth;hydrate Chemical compound O.[Bi] BDJYZEWQEALFKK-UHFFFAOYSA-N 0.000 claims description 3
- 230000000903 blocking effect Effects 0.000 claims description 3
- OPQARKPSCNTWTJ-UHFFFAOYSA-L copper(ii) acetate Chemical compound [Cu+2].CC([O-])=O.CC([O-])=O OPQARKPSCNTWTJ-UHFFFAOYSA-L 0.000 claims description 3
- 238000001548 drop coating Methods 0.000 claims description 3
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- 238000003760 magnetic stirring Methods 0.000 claims description 3
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- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 3
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- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 3
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- 239000001509 sodium citrate Substances 0.000 claims description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 3
- CMZUMMUJMWNLFH-UHFFFAOYSA-N sodium metavanadate Chemical compound [Na+].[O-][V](=O)=O CMZUMMUJMWNLFH-UHFFFAOYSA-N 0.000 claims description 3
- 238000001179 sorption measurement Methods 0.000 claims description 3
- YUKQRDCYNOVPGJ-UHFFFAOYSA-N thioacetamide Chemical compound CC(N)=S YUKQRDCYNOVPGJ-UHFFFAOYSA-N 0.000 claims description 3
- DLFVBJFMPXGRIB-UHFFFAOYSA-N thioacetamide Natural products CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 claims description 3
- 229910052724 xenon Inorganic materials 0.000 claims description 3
- FHNFHKCVQCLJFQ-UHFFFAOYSA-N xenon atom Chemical compound [Xe] FHNFHKCVQCLJFQ-UHFFFAOYSA-N 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 2
- 229910052760 oxygen Inorganic materials 0.000 claims description 2
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- 238000010992 reflux Methods 0.000 claims description 2
- APSBXTVYXVQYAB-UHFFFAOYSA-M sodium docusate Chemical compound [Na+].CCCCC(CC)COC(=O)CC(S([O-])(=O)=O)C(=O)OCC(CC)CCCC APSBXTVYXVQYAB-UHFFFAOYSA-M 0.000 claims description 2
- 238000001291 vacuum drying Methods 0.000 claims description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims 1
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- UGZAJZLUKVKCBM-UHFFFAOYSA-N 6-sulfanylhexan-1-ol Chemical compound OCCCCCCS UGZAJZLUKVKCBM-UHFFFAOYSA-N 0.000 description 4
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
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- 235000009566 rice Nutrition 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
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- 229910052801 chlorine Inorganic materials 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
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- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 description 1
- 238000000018 DNA microarray Methods 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 1
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- 229910052739 hydrogen Inorganic materials 0.000 description 1
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- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
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- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- JKQOBWVOAYFWKG-UHFFFAOYSA-N molybdenum trioxide Chemical compound O=[Mo](=O)=O JKQOBWVOAYFWKG-UHFFFAOYSA-N 0.000 description 1
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- 231100000252 nontoxic Toxicity 0.000 description 1
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- 229910000077 silane Inorganic materials 0.000 description 1
- KKCBUQHMOMHUOY-UHFFFAOYSA-N sodium oxide Chemical compound [O-2].[Na+].[Na+] KKCBUQHMOMHUOY-UHFFFAOYSA-N 0.000 description 1
- 229910001948 sodium oxide Inorganic materials 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
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Classifications
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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
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Electrochemistry (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Nanotechnology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses the paper base optical electro-chemistry sensors of highly sensitive detection microRNA a kind of.Paper chip is prepared using wax printing technique, and the functionalization of paper chip is realized in its hydrophilic working region growth in situ gold nano grain, then modification cuprous oxide/bismuth sulfide/pucherite three-level sensitizer, microRNA is captured by fixed hair clip DNA chain, pass through the identification and digestion effect of the specificity of double-stranded specific nuclease, it realizes and the signal of microRNA is amplified, then pass through more cross chain reactions, nano platinum particle is embedded in the trunk portion of chain, limb part forms hemin/G- tetrad structure, form the DNA concatermer with catalase-like biological nature, further realize the amplification of signal, to complete the preparation of optical electro-chemistry sensor, realize the hypersensitive to microRNA, accurate detection.
Description
Technical field
The present invention relates to microRNA analysis and detection technologies, more hybridization chain signal amplification techniques, DNA mimetic enzyme catalysis skill
Art, core chip technology and composite nano materials technical field, it is more specifically a kind of for super sensitivity detection microRNA's
The preparation of optical electro-chemistry sensor.
Background technique
In recent years, global cancer morbidity steeply rises, and greatly threatens human health, cancer early detection is increasingly
It attracts people's attention.The variation of microRNA can be a kind of potential tumor markers, therefore with the expression of controlling gene
The detection of microRNA causes the extensive concern of people.Highly sensitive, highly selective measurement microRNA is to genescreen, something lost
The early diagnosis and therapy for passing disease all has very important significance.Up to the present, some detection methods are as real-time fluorescence
Quantitative polyase chain reaction, the hybridization of promise plucked instrument, DNA microarray method have been developed and have been used for the detection of cancer cell.But these
Method is time-consuming, needs complicated sample pre-treatments and expensive instrument, thus be still badly in need of developing it is a kind of it is overdelicate, portable,
The analysis method of cheap detection microRNA.
Cuprous oxide (Cu2O it) is used as a kind of common photoelectric material, due to its non-toxic, good catalytic activity, well
Biocompatibility and unique light and electrical property, be widely used in optical electro-chemistry sensor.Optical electro-chemistry is improved to pass
The key of sensor sensitivity is to improve the optical electro-chemistry response of photoelectric material.By pucherite (BiVO4)/bismuth sulfide (Bi2S3) with
Cu2The ternary complex that O is compounded to form can reduce the recombination rate of electrons and holes, more effectively enhance photosignal.
Currently, DNA analogue enztme has a wide range of applications in biocatalysis field, and compared with other analogue enztmes, DNA analogue enztme
Have the characteristics that stablize, be readily synthesized duplication, not facile hydrolysis, so by favor.Hemin/G- tetrad peroxidating
Object analogue enztme is the emerging DNA analogue enztme developed rapidly in recent years, it can be catalyzed H2O2The many reactions participated in, so
Signal amplification technique based on this is widely utilized in bioanalysis.In addition, more hybridization chain signal amplification techniques
Extensive concern is also resulted in recent years, by more cross chain reactions, can obtain the longer DNA with a plurality of branch
Double-spiral structure.In order to which analysis detection signal is further amplified, we combine both signal amplification techniques, are received using platinum
More nano platinum particles are adsorbed in electrostatic adsorption between rice corpuscles and DNA chain in DNA double spiral skeleton, and more miscellaneous
Branch on interlinkage can form hemin/G- tetrad structure in the presence of hemin, higher enhancing light
The optical electro-chemistry of electric material responds, and improves the sensitivity of analysis detection.
Summary of the invention
The purpose of the present invention is have big specific surface area, good biocompatibility and photoelectricity by electrodeposition process preparation
The Cu of performance2O/Bi2S3/BiVO4Hair clip DNA chain is fixed on its surface by paper electrode, is captured target microRNA using it, is led to
The specific recognition and digestion effect for crossing double-stranded specific nuclease, realize the circulation amplification of signal, then by cascade more
Branch cross chain reaction is embedded in nano platinum particle in the trunk portion of chain, and branch stem portion forms hemin/G- tetrad knot
Structure further realizes the amplification of signal, completes the preparation of optical electro-chemistry sensor, realizes the super sensitivity detection to microRNA.
In order to solve the above-mentioned technical problem, the present invention is realized by following measures:
(1) the hydrophobic wax printed drawings of the micro-fluidic paper chip of Adobe illustrator CS4 software design are utilized on computers
Designed print pattern is printed upon on chromatographic paper by wax printer, the chromatographic paper printed is then placed on baking oven by case
In, the thickness for melting wax in 50 seconds and permeating entire chromatographic paper is heated at 130 DEG C, forms hydrophobic region and hydrophilic workspace
Domain；
(2) working electrode to match with the wax print pattern obtained in step (1) is designed on computers, to electrode and reference
The printed patterns of electrode, and carbon work electricity is printed on the wax printing chromatographic paper obtained in step (1) using screen printing technique
Pole, carbon are to electrode and Ag/AgCl reference electrode；
(3) working region of the carbon working electrode obtained in step (2) using in situ synthesis grows gold nano grain, tool
Body step are as follows: synthesize gold nanoparticle first: it measures 60-90 mL secondary water and is placed in three-necked flask and is heated to 90 DEG C, so
The gold chloride that 0.5-0.9 mL mass fraction is 1% is added afterwards, continues heating water bath to 96 DEG C, after reacting and carrying out 1 min, stands
The sodium citrate that 2.5-2.9 mL mass fraction is 1% is added, reacts 15-20 min under magnetic stirring, obtains Jenner's grain of rice
Sub- solution；Then using " drop coating-drying " method first in drop coating 10-20 μ L Jenner's grain of rice of the working region of carbon working electrode
Sub- solution, spontaneously dries at room temperature, repeats the operation 3-5 times, and taking the freshly prepared mass fraction of 10-20 μ L is 1% chlorine
The mixed solution of the hydroxylamine hydrochloride of auric acid and 0.2 M is added drop-wise to working region, and two kinds of liquor capacity ratios are 1:1, in room
After the lower growth 20-40 min of temperature, 30 min are spontaneously dried with secondary water cleaning region and at room temperature；
(4) utilize electro-deposition method by the Cu of small planar2The working region for the carbon working electrode that O modification obtains in step (3)
And by BiVO4/Bi2S3Compound is modified in Cu2On O；
(5) hair clip DNA chain S1 is fixed on to the working region of the carbon working electrode obtained in step (4), then uses 6- sulfydryl-
Then 1- hexanol closing activity site captures microRNA using S1 chain；
(6) the carbon working electrode for successively obtaining target detection thing microRNA and double-stranded specific nuclease in step (5)
Working region carry out be incubated for and washed with the phosphate buffer of pH 7.4 (PBS)；
(7) formation of DNA concatermer: it will be dissolved in pH's 7.4 on hair clip DNA molecular 1(H1) and hair clip DNA molecular 2(H2)
In PBS, it is added dropwise to the working region that carbon working electrode is obtained in step (6), Proliferation forms more hybridization chains, continues to be added dropwise
Hemin, formed hemin/G- tetrad structure, using electrostatic adsorption by nano platinum particle be embedded in more it is miscellaneous
In the main chain of interlinkage, the DNA concatermer with biocatalysis is formed, H can be catalyzed2O2It decomposes and generates O2；
(8) working region of the carbon working electrode obtained in step (7) a metallic, it includes 0.01 M H that 10 μ L, which are added dropwise,2O2PH 7.4
PBS solution, under the irradiation of xenon lamp, using the Ag/AgCl electrode of printing as reference electrode, the carbon electrode of printing is used as to electricity
Pole, the carbon working electrode obtained in step (3) is as working electrode, using three-electrode system, by time current curve method,
Optical electro-chemistry signal detection is carried out at 0.0 V of voltage, draws the standard curve of photo-current intensity and microRNA concentration, is realized
Detection to microRNA.
It is of the present invention using electro-deposition method by the Cu of small planar2O modification the working region of carbon working electrode simultaneously
By BiVO4/Bi2S3Compound is modified in Cu2Specific steps on O are as follows:
A. the working region of the carbon working electrode obtained in step (3) deposits Cu2O: electric using Ag/AgCl electrode as reference
Pole, platinum electrode are used as to electrode, and the middle carbon working electrode obtained of step (3) is passed through as working electrode using three-electrode system
On Potentiometric Stripping Analysis, the working region of the carbon working electrode obtained in step (3) deposit the Cu of small planar2O, deposit electrolyte
Liquid is made of the lactic acid that the copper acetate that concentration is 0.02 M and concentration are 0.3-0.5 M, the hydrogen that the pH of electrolyte is 2 M by concentration
Sodium oxide molybdena is adjusted between 9-11, and deposition voltage is between -0.3 V and -0.5 V, and depositing temperature is 60 DEG C, sedimentation time
For 25-45 min, after the completion of deposition, working region surface is washed with secondary water and is spontaneously dried at room temperature；
B. BiVO is synthesized4/Bi2S3Compound: BiVO is synthesized by hydro-thermal method first4, 1 mmol, five nitric hydrate bismuth is dissolved in
In the mixed solution of 40-60 mL secondary water and ethylene glycol, wherein secondary water and the volume ratio of ethylene glycol are 1:1, sequentially add 1
Mmol sodium metavanadate and 2.5-3.5 mmol Aerosol OT, are vigorously stirred after 5-10 min that move into 50 mL anti-
Kettle is answered, is reacted 16-18 hours under the conditions of 160 DEG C, uses secondary water and dehydrated alcohol eccentric cleaning 3- after being cooled to room temperature respectively
It 5 times, is then dried in vacuo 4 hours at 60 DEG C, obtains BiVO4；The BiVO for taking 0.25 mmol to prepare4It is dissolved in 40 mL bis- times
Water is added 0.05-0.2 mmol thioacetamide and is transferred to 50 mL reaction kettles after stirring 5-10 min, in 120 DEG C of condition
After lower reaction 8-10 hours, secondary water and dehydrated alcohol eccentric cleaning 3-5 times are used after being cooled to room temperature respectively, then at 60 DEG C
Vacuum drying 4 hours obtains BiVO4/Bi2S3Compound；
C. by BiVO4/Bi2S3Compound and Cu2O is compound: the 3- aminopropyl triethoxy that 10 μ L mass fractions are 2% is added dropwise
Silane is in Cu2The surface O after being washed working region with the PBS of pH 7.4 and dried, is added dropwise 10 μ L and contains BiVO4/Bi2S3It is compound
Object and mass fraction are the mixed liquor of 2.5% glutaraldehyde, are washed after dry with the PBS of pH 7.4.
The working region of the present invention that hair clip DNA chain S1 is fixed on to the carbon working electrode obtained in step (4), with
6- sulfydryl -1- hexanol closing activity site is used afterwards, then utilizes the specific steps of S1 capture microRNA are as follows: contain 10 μ L
There is the mixed liquor for the glutaraldehyde and DNA chain S1 that mass fraction is 2.5% to be added dropwise to the work of the carbon working electrode obtained in step (4)
Make region, after being incubated for 2 hours, it is 2 mM's that 10 μ L concentration are added dropwise after being washed with the PBS of the pH 7.4 containing 0.05% polysorbas20
6- sulfydryl -1- hexanol washs removing with 7.4 PBS of pH after 30 min of incubation and is not associated with for blocking nonspecific binding site
6- sulfydryl -1- hexanol and spontaneously dry.
It is of the present invention successively to obtain target detection thing microRNA and double-stranded specific nuclease in step (5)
The working region of carbon working electrode be incubated for and the specific steps washed with the PBS of pH 7.4 are as follows: 10 μ L are added dropwise not
With the working region of the microRNA-141 of the concentration carbon working electrode obtained into step (5), hatch 40 at 37 DEG C
Min is then washed with 7.4 PBS of pH and is removed unbonded microRNA-141, and the double-strand for continuing to be added dropwise 10 μ L, 0.05 U is special
Specific nuclease enzyme solutions are hatched after 40 min at 37 DEG C and are washed with the PBS of pH 7.4.
It is of the present invention to form comprising the concrete steps that for DNA concatermer:
A. synthesize nano platinum particle: taking 500-1000 μ L concentration is that the chloroplatinic acid of 20 mM is added to 20-50mL water and ethyl alcohol
Mixed solution, wherein the volume ratio of water and ethyl alcohol is 1:1, and 0.4-0.9 mol polyvinylpyrrolidone is added, in 80 DEG C of reflux 4
Hour obtains nano platinum particle solution；
The preparation of b.DNA concatermer: the carbon working electrode that step (6) obtain is added in the mixed liquor for taking 10 μ L to contain H1 and H2
Working region, H1 the and H2 concentration are 5 μM, are incubated at room temperature after 80 min and wash removing not with the PBS of pH 7.4
The H1 and H2 of association reaction；The hemin solution that 10 μ L concentration are 0.2 mM is then added dropwise, after being incubated for 50 min at room temperature
It is washed using the PBS of pH 7.4, continues the nano platinum particle that 10 μ L preparation is added dropwise after dry, finally washed with the PBS of pH 7.4
Remove extra nano platinum particle.
Beneficial effects of the present invention:
(1) Cu prepared by simple electrodeposition process2O has big surface area, can load more BiVO4/Bi2S3It is multiple
Object is closed, the three-level sensitization structure of formation can effectively enhance photosignal, to be also simultaneously that fixed a large amount of hair clip DNA chain mentions
A good platform has been supplied, amplification detection signal is conducive to, has enhanced the sensitivity of analysis.
(2) using double-stranded specific nuclease can be highly selective identification and digestion DNA/RNA hybridization chain in DNA,
Realization recycles target microRNA, further realizes the amplification to signal.
(3) the longer more hybridization chains with a plurality of branch obtained using more cross chain reactions, longer branch
DNA provides a large amount of active site for load nano platinum particle, more hybridization chain components can be formed a large amount of hemin/
G- tetrad structure, can be catalytically decomposed more H2O2Generate O2As electron acceptor, enhances cathode photo current, greatly mention
High analyte detects signal.
(4) multiple signal amplifying technique is utilized, the overdelicate optical electro-chemistry sensor for detecting microRNA is constructed,
May be implemented it is simple, quickly and accurately detect microRNA, there is weight in the early detection of clinical cancer, transfer and treatment
Big meaning.
Specific embodiment:
For a better understanding of the present invention, below with reference to the embodiment content that the present invention is furture elucidated, but the contents of the present invention
It is not limited solely to the following examples.
Embodiment 1: application of the overdelicate optical electro-chemistry sensor in detection microRNA-141
(1) the hydrophobic wax printed drawings of the micro-fluidic paper chip of Adobe illustrator CS4 software design are utilized on computers
Designed print pattern is printed upon on chromatographic paper by wax printer, the chromatographic paper printed is then placed on baking oven by case
In, the thickness for melting wax in 50 seconds and permeating entire chromatographic paper is heated at 130 DEG C, forms hydrophobic region and hydrophilic workspace
Domain；
(2) working electrode to match with the wax print pattern obtained in step (1) is designed on computers, to electrode and reference
The printed patterns of electrode, and carbon work electricity is printed on the wax printing chromatographic paper obtained in step (1) using screen printing technique
Pole, carbon are to electrode and Ag/AgCl reference electrode；
(3) working region of the carbon working electrode obtained in step (2) using in situ synthesis grows gold nano grain, tool
Body step are as follows: first synthesis gold nanoparticle: measure 80 mL secondary waters be placed in three-necked flask and be heated to 90 DEG C, then plus
Enter 0.8 mL, 1% gold chloride, continues heating water bath to 96 DEG C, after reacting and carrying out 1 min, 2.8 mL mass point are added immediately
The sodium citrate that number is 1% reacts 15 min under magnetic stirring, obtains solution of gold nanoparticles；Then using " drop coating-is dry
It is dry " method in the 10 μ L solution of gold nanoparticles of drop coating of the working region of carbon working electrode, spontaneously dries at room temperature first, weight
Multiple the operation 3 times, freshly prepared 1% gold chloride of 10 μ L and the mixed solution of the hydroxylamine hydrochloride of 0.2 M is taken to be added drop-wise to workspace
Domain, two kinds of liquor capacity ratios are 1:1, after growing 30 min at room temperature, with secondary water cleaning region and in room
Temperature is lower to spontaneously dry 30 min；
(4) utilize electro-deposition method by the Cu of small planar2The working region for the carbon working electrode that O modification obtains in step (3)
And by BiVO4/Bi2S3Compound is modified in Cu2On O, Cu is prepared first2O material: using Ag/AgCl electrode as reference electrode, platinum
Electrode is used as to electrode, and the middle carbon working electrode obtained of step (3) passes through current potential using three-electrode system as working electrode
Stripping Analysis Method For Simultaneous, the working region of the carbon working electrode obtained in step (3) deposit the Cu of small planar2O, deposited electrolyte by
The lactic acid composition that the copper acetate and concentration that concentration is 0.02 M are 0.4 M, the sodium hydroxide tune that the pH of electrolyte is 2 M by concentration
Section is 11, and deposition voltage is -0.4 V, and depositing temperature is 60 DEG C, and sedimentation time is 27 min, after the completion of deposition, uses secondary water
Washing working region surface simultaneously spontaneously dries at room temperature；Then preparation BiVO4/Bi2S3Compound: it is closed first by hydro-thermal method
At BiVO4, 1 mmol, five nitric hydrate bismuth is dissolved in the mixed solution of 40 mL secondary waters and ethylene glycol, wherein secondary water and
The volume ratio of ethylene glycol is 1:1, sequentially adds 1 mmol sodium metavanadate and 3 mmol Aerosol OTs, acutely stirs
Move into 50 mL reaction kettles after mixing 5 min, reacted 18 hours under the conditions of 160 DEG C, after being cooled to room temperature respectively with secondary water and
It dehydrated alcohol eccentric cleaning 3 times, is then dried in vacuo 4 hours at 60 DEG C, obtains BiVO4；0.25 mmol is taken to prepare
BiVO440 mL secondary waters are dissolved in, 0.1 mmol thioacetamide is added and are transferred to 50 mL reaction kettles, In after stirring 5 min
After being reacted 8 hours under the conditions of 120 DEG C, secondary water and dehydrated alcohol eccentric cleaning 3 times are used after being cooled to room temperature respectively, are then existed
60 DEG C are dried in vacuo 4 hours, obtain BiVO4/Bi2S3Compound；Finally by BiVO4/Bi2S3Compound and Cu2O is compound: being added dropwise
The 3-aminopropyltriethoxysilane that 10 μ L mass fractions are 2% is in Cu2Working region is washed with the PBS of pH 7.4 in the surface O
And after drying, 10 μ L are added dropwise and contain BiVO4/Bi2S3Compound and mass fraction are the mixed liquor of 2.5% glutaraldehyde, are used after dry
The PBS of pH 7.4 is washed；
(5) the 10 μ L mixed liquor for containing glutaraldehyde and DNA chain S1 that mass fraction is 2.5% is added dropwise in step (4) and is obtained
Carbon working electrode working region, be incubated for 2 hours after, be added dropwise 10 after being washed with the PBS of the pH 7.4 containing 0.05% polysorbas20
6- sulfydryl -1- the hexanol that μ L concentration is 2 mM uses 7.4 PBS of pH after being incubated for 30 min for blocking nonspecific binding site
Washing removes unbonded 6- sulfydryl -1- hexanol and spontaneously dries；
(6) workspace of microRNA-141 carbon working electrode of acquisition into step (5) of 10 μ L various concentrations is then added dropwise
40 min are hatched in domain at 37 DEG C, are then washed with the PBS of pH 7.4 and are removed unbonded microRNA-141, continue to be added dropwise
The double-stranded specific nucleic acid enzyme solutions of 10 μ L, 0.05 U are hatched after 40 min at 37 DEG C and are washed with the PBS of pH 7.4
It washs；
(7) formation of DNA concatermer: prepare nano platinum particle first: taking 750 μ L concentration is that the chloroplatinic acid of 20 mM is added to 30
The mixed solution of mL water and ethyl alcohol, wherein the volume ratio of water and ethyl alcohol is 1:1,0.6 mol polyvinylpyrrolidone is added, 80
It flows back 4 hours under the conditions of DEG C and obtains nano platinum particle solution；Then DNA concatermer is prepared: the mixing for taking 10 μ L to contain H1 and H2
The working region for the carbon working electrode that step (6) obtain is added in liquid, H1 the and H2 concentration is 5 μM, is incubated at room temperature
The H1 and H2 for removing unbonded reaction are washed after 80 min with the PBS of pH 7.4；The chlorine that 10 μ L concentration are 0.2 mM is then added dropwise
Change haemachrome solution, washed after being incubated for 50 min at room temperature using the PBS of pH 7.4, continues that 10 μ L preparation is added dropwise after dry
Nano platinum particle is finally washed with the PBS of pH 7.4 and removes extra nano platinum particle；
(8) working region of the carbon working electrode obtained in step (7) a metallic is added dropwise 10 μ L and contains 0.01 M H2O2PH 7.4
PBS, under the irradiation of xenon lamp, using the Ag/AgCl electrode of printing as reference electrode, the carbon electrode of printing is used as to electrode,
The carbon working electrode obtained in step (3) is as working electrode, using three-electrode system, by time current curve method, in electricity
Progress optical electro-chemistry signal detection under 0.0 V is pressed, the standard curve of photo-current intensity and microRNA concentration, realization pair are drawn
The detection of microRNA.
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Claims (5)
1. a kind of preparation method of the optical electro-chemistry sensor of super sensitivity detection microRNA, it is characterized in that the following steps are included:
(1) the hydrophobic wax printed drawings of the micro-fluidic paper chip of Adobe illustrator CS4 software design are utilized on computers
Designed print pattern is printed upon on chromatographic paper by wax printer, the chromatographic paper printed is then placed on baking oven by case
In, heating at 130 DEG C melts wax in 60 seconds and permeates entire chromatographic paper, forms hydrophobic region and hydrophilic working region；
(2) working electrode to match with the wax print pattern obtained in step (1) is designed on computers, to electrode and reference
The printed patterns of electrode, and carbon work electricity is printed on the wax printing chromatographic paper obtained in step (1) using screen printing technique
Pole, carbon are to electrode and Ag/AgCl reference electrode；
(3) working region of the carbon working electrode obtained in step (2) using in situ synthesis grows gold nano grain, tool
Body step are as follows: synthesize gold nanoparticle first: it measures 60-90 mL secondary water and is placed in three-necked flask and is heated to 90 DEG C, so
The gold chloride that 0.5-0.9 mL mass fraction is 1% is added afterwards, continues heating water bath to 96 DEG C, after reacting and carrying out 1 min, stands
The sodium citrate that 2.5-2.9 mL mass fraction is 1% is added, reacts 15-20 min under magnetic stirring, obtains gold nano
Particle solution；Then using " drop coating-drying " method first in the drop coating 10-20 μ L gold nano of the working region of carbon working electrode
Particle solution spontaneously dries at room temperature, repeats the operation 3-5 times, and taking the freshly prepared mass fraction of 10-20 μ L is 1%
The mixed solution of the hydroxylamine hydrochloride of gold chloride and 0.2 M is added drop-wise to working region, and two kinds of liquor capacity ratios are 1:1, In
After growing 20-40 min at room temperature, 30 min are spontaneously dried with secondary water cleaning region and at room temperature；
(4) utilize electro-deposition method by the cuprous oxide (Cu of small planar2O the carbon working electrode obtained in step (3)) is modified
Working region and by pucherite (BiVO4)/bismuth sulfide (Bi2S3) compound modification in Cu2On O；
(5) hair clip DNA chain S1 is fixed on to the working region of the carbon working electrode obtained in step (4), then uses 6- sulfydryl-
Then 1- hexanol closing activity site captures microRNA using S1 chain；
(6) the carbon working electrode for successively obtaining target detection thing microRNA and double-stranded specific nuclease in step (5)
Working region carry out be incubated for and washed with the phosphate buffer of pH 7.4 (PBS)；
(7) formation of DNA concatermer: it will be dissolved in pH's 7.4 on hair clip DNA molecular 1(H1) and hair clip DNA molecular 2(H2)
In PBS, it is added dropwise to the working region that carbon working electrode is obtained in step (6), Proliferation forms more hybridization chains, continues to be added dropwise
Hemin, formed hemin/G- tetrad structure, using electrostatic adsorption by nano platinum particle be embedded in more it is miscellaneous
In the main chain of interlinkage, the DNA concatermer with biocatalysis is formed, H can be catalyzed2O2It decomposes and generates O2；
(8) working region of the carbon working electrode obtained in step (7) a metallic is added dropwise 10 μ L and contains 0.01 M H2O2PH 7.4
PBS, under the irradiation of xenon lamp, using the Ag/AgCl electrode of printing as reference electrode, the carbon electrode of printing is used as to electrode,
The carbon working electrode obtained in step (3) is as working electrode, using three-electrode system, by time current curve method, in electricity
Progress optical electro-chemistry signal detection under 0.0 V is pressed, the standard curve of photo-current intensity and microRNA concentration, realization pair are drawn
The detection of microRNA.
2. a kind of preparation method of the optical electro-chemistry sensor of super sensitivity detection microRNA according to claims 1,
It is characterized in, using electro-deposition method by the Cu of small planar2O modification is in electrode working zone and by BiVO4/Bi2S3Compound modification exists
Cu2Specific steps on O are as follows:
A. the working region of the carbon working electrode obtained in step (3) deposits Cu2O: using Ag/AgCl electrode as reference electrode,
Platinum electrode is used as to electrode, and the middle carbon working electrode obtained of step (3) passes through electricity using three-electrode system as working electrode
Position Stripping Analysis Method For Simultaneous, the working region of the carbon working electrode obtained in step (3) deposits the Cu of small planar2O, deposited electrolyte
It is made of the lactic acid that copper acetate and concentration that concentration is 0.02 M are 0.3-0.5 M, the hydrogen-oxygen that the pH of electrolyte is 2 M by concentration
Change sodium to be adjusted between 9-11, deposition voltage is between -0.3 V and -0.5 V, and depositing temperature is 60 DEG C, and sedimentation time is
25-45 min after the completion of deposition, washs working region surface with secondary water and spontaneously dries at room temperature；
B. BiVO is synthesized4/Bi2S3Compound: BiVO is synthesized by hydro-thermal method first4, 1 mmol, five nitric hydrate bismuth is dissolved in 40-
In the mixed solution of 60 mL secondary waters and ethylene glycol, wherein secondary water and the volume ratio of ethylene glycol are 1:1, sequentially add 1
Mmol sodium metavanadate and 2.5-3.5 mmol Aerosol OT, are vigorously stirred after 5-10 min that move into 50 mL anti-
Kettle is answered, is reacted 16-18 hours under the conditions of 160 DEG C, uses secondary water and dehydrated alcohol eccentric cleaning 3- after being cooled to room temperature respectively
It 5 times, is then dried in vacuo 4 hours at 60 DEG C, obtains BiVO4；The BiVO for taking 0.25 mmol to prepare4It is dissolved in 40 mL bis- times
Water is added 0.05-0.2 mmol thioacetamide and is transferred to 50 mL reaction kettles after stirring 5-10 min, in 120 DEG C of condition
After lower reaction 8-10 hours, secondary water and dehydrated alcohol eccentric cleaning 3-5 times are used after being cooled to room temperature respectively, then at 60 DEG C
Vacuum drying 4 hours obtains BiVO4/Bi2S3Compound；
C. by BiVO4/Bi2S3Compound and Cu2O is compound: the 3- aminopropyl triethoxysilicane that 10 μ L mass fractions are 2% is added dropwise
Alkane is in Cu2The surface O after being washed working region with the PBS of pH 7.4 and dried, is added dropwise 10 μ L and contains BiVO4/Bi2S3Compound
It is the mixed liquor of 2.5% glutaraldehyde with mass fraction, is washed after dry with the PBS of pH 7.4.
3. a kind of preparation method of the optical electro-chemistry sensor of super sensitivity detection microRNA according to claims 1,
It is characterized in, the working region that hair clip DNA chain S1 is fixed on to the resulting carbon working electrode of step (4), then uses 6-
Then sulfydryl -1- hexanol closing activity site utilizes the specific steps of S1 chain capture microRNA are as follows: 10 μ L are contained quality
The mixed liquor of glutaraldehyde and DNA chain S1 that score is 2.5% is added dropwise to the working region of the carbon working electrode obtained in step (4),
After being incubated for 2 hours, the 6- sulfydryl-that 10 μ L concentration are 2 mM is added dropwise after being washed with the PBS of the pH 7.4 containing 0.05% polysorbas20
1- hexanol is incubated for after 30 min for blocking nonspecific binding site and washs the 6- mercapto for removing and being not associated with 7.4 PBS of pH
Base -1- hexanol simultaneously spontaneously dries.
4. a kind of preparation method of the optical electro-chemistry sensor of super sensitivity detection microRNA according to claims 1,
It is characterized in, the carbon working electrode that target detection thing microRNA and double-stranded specific nuclease are successively obtained in step (5)
Working region be incubated for and comprised the concrete steps that with what the PBS of pH 7.4 was washed: 10 μ L various concentrations are added dropwise
Electrode working zone domain described in microRNA to step (5), hatches 40 min at 37 DEG C, utilizes 7.4 PBS buffer solution of pH
Unbonded microRNA is washed away, the double-stranded specific nuclease that 10 μ L, 0.05 U is then added dropwise is incubated for 40 min, utilizes pH
It is dried at room temperature for after 7.4 PBS cleaning.
5. a kind of preparation method of the optical electro-chemistry sensor of super sensitivity detection microRNA according to claims 1,
It is characterized in, forms comprising the concrete steps that for DNA concatermer:
A. synthesize nano platinum particle: taking 500-1000 μ L concentration is that the chloroplatinic acid of 20 mM is added to 20-50mL water and ethyl alcohol
Mixed solution, wherein the volume ratio of water and ethyl alcohol is 1:1, and 0.4-0.9 mol polyvinylpyrrolidone is added, in 80 DEG C of reflux 4
Hour obtains nano platinum particle solution；
The preparation of b.DNA concatermer: the carbon working electrode that step (6) obtain is added in the mixed liquor for taking 10 μ L to contain H1 and H2
Working region, H1 the and H2 concentration are 5 μM, are incubated at room temperature after 80 min and wash removing not with the PBS of pH 7.4
The H1 and H2 of association reaction；The hemin solution that 10 μ L concentration are 0.2 mM is then added dropwise, after being incubated for 50 min at room temperature
It is washed using the PBS of pH 7.4, continues the nano platinum particle that 10 μ L preparation is added dropwise after dry, finally washed with the PBS of pH 7.4
Remove extra nano platinum particle.
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| CN113061649A (en) * | 2021-04-02 | 2021-07-02 | 福州大学 | Surface enhanced Raman spectrum sensor for detecting microRNA and preparation method thereof |
| CN113418898A (en) * | 2021-06-18 | 2021-09-21 | 贵州医科大学 | Integrated device of PIFO platform and composed of micro-fluidic chip and fluorescence sensor |
| CN113418898B (en) * | 2021-06-18 | 2022-10-28 | 贵州医科大学 | Integrated device of PIFO platform and composed of micro-fluidic chip and fluorescence sensor |
| CN114965638A (en) * | 2022-04-29 | 2022-08-30 | 南京邮电大学 | Ratio type biosensor with internal reference signal and preparation method |
| CN114965638B (en) * | 2022-04-29 | 2024-05-24 | 南京邮电大学 | Ratio type biosensor with internal reference signal and preparation method |
| CN116893206A (en) * | 2023-09-11 | 2023-10-17 | 南方电网数字电网研究院有限公司 | Copper oxide/bismuth sulfide heterojunction material, gas sensor, gas detection device, preparation method and application |
| CN116893206B (en) * | 2023-09-11 | 2024-01-23 | 南方电网数字电网研究院有限公司 | Copper oxide/bismuth sulfide heterojunction material, gas sensor, gas detection device, preparation method and application |
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