CN106442673B - A kind of preparation method of biocompatible body sensor - Google Patents
A kind of preparation method of biocompatible body sensor Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 108091023037 Aptamer Proteins 0.000 claims abstract description 100
- 239000007853 buffer solution Substances 0.000 claims abstract description 43
- SZVJSHCCFOBDDC-UHFFFAOYSA-N iron(II,III) oxide Inorganic materials O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims abstract description 39
- 108010081589 Becaplermin Proteins 0.000 claims abstract description 30
- 239000002105 nanoparticle Substances 0.000 claims abstract description 29
- 230000004048 modification Effects 0.000 claims abstract description 14
- 238000012986 modification Methods 0.000 claims abstract description 14
- 108020003215 DNA Probes Proteins 0.000 claims abstract description 13
- 239000003298 DNA probe Substances 0.000 claims abstract description 13
- 239000000243 solution Substances 0.000 claims description 70
- 108020004414 DNA Proteins 0.000 claims description 44
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 27
- 239000000758 substrate Substances 0.000 claims description 20
- 238000006243 chemical reaction Methods 0.000 claims description 19
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 18
- 235000019441 ethanol Nutrition 0.000 claims description 17
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 16
- 239000010931 gold Substances 0.000 claims description 16
- 229910052737 gold Inorganic materials 0.000 claims description 16
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- 108010090804 Streptavidin Proteins 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 8
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 7
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- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical class [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 6
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- 238000001027 hydrothermal synthesis Methods 0.000 claims description 6
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 claims description 6
- 229920001223 polyethylene glycol Polymers 0.000 claims description 6
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- BEAZKUGSCHFXIQ-UHFFFAOYSA-L zinc;diacetate;dihydrate Chemical compound O.O.[Zn+2].CC([O-])=O.CC([O-])=O BEAZKUGSCHFXIQ-UHFFFAOYSA-L 0.000 claims description 6
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 4
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- 101710134784 Agnoprotein Proteins 0.000 claims description 2
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 abstract description 22
- 239000008103 glucose Substances 0.000 abstract description 22
- 238000001514 detection method Methods 0.000 abstract description 13
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- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 abstract description 7
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- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 4
- 239000004411 aluminium Substances 0.000 description 4
- 229910052782 aluminium Inorganic materials 0.000 description 4
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- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(I) nitrate Inorganic materials [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 2
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
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- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 1
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- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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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
- G01N27/416—Systems
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Abstract
The present invention relates to a kind of preparation methods of biocompatible body sensor, include the following steps：Prepare buffer solution；ZnS nano-clusters are synthesized, aptamers 1 is prepared and modifies ZnS nano-clusters；Synthesize Fe3O4Nano-particle prepares DNA probe modification Fe3O4Nano-particle；Prepare 2 modified electrode of aptamers；Platelet-derived growth factor BB is added, builds biosensor.The present invention builds biosensor using widely used portable glucose meter combination aptamers, it is intended to provide a kind of new method for quick, the quantitative glucose content by converting out for indirect detection platelet derived growth factor.
Description
Technical field
The present invention relates to biosensor technology fields, and in particular to a kind of preparation method of biocompatible body sensor and
The method for detecting platelet-derived growth factor-BB, may be implemented the Sensitive Detection to platelet-derived growth factor-BB.
Background technology
Platelet derived growth factor is a kind of peptide growth factor containing sugar chain of vascular endothelial growth factor family,
With preferable thermal stability, for heat resistance up to 100 DEG C, isoelectric point 9.8, relative molecular weight is 30kD or so.Blood platelet
There are at least three kinds of isomers in derivative growth factor；Platelet-derived growth factor-AA, platelet-derived growth factor-AB and
Platelet-derived growth factor-BB, wherein PDGF-AA and platelet derived growth factor are homodimer, and PDGF-AB is
Heterodimer.Platelet derived growth factor either all plays an important role under normal physiological or pathological state.
Under normal physiological condition, platelet derived growth factor can promote a variety of interstitial cells (such as glomerulus vascular cell, at fibre
Tie up cell, Deiter's cells, vascular smooth muscle cells and endothelial cell etc.) chemotactic, division and proliferation, and to body
Growth and development has important role.But the excessive activation of the unconventionality expression of platelet derived growth factor and its receptor can
Induced synthesis tumor neogenetic blood vessels and proliferation and the migration for directly or indirectly promoting tumour cell.Wherein, platelet-derived life
The growth of the long factor-BB and tumour cell has direct relationship with conversion, can be over-expressed in cancer cell.Therefore, it is
A kind of important tumor marker has great significance for the diagnosis, treatment and prognosis of cancer, detects platelet-derived life
The long factor-BB has important scientific research value.
In recent years due to the development of aptamer and its relative to many advantages of antibody, use aptamers former for identification
The biosensor detection protein of part is widely used.Aptamer is referred to by a kind of in-vitro screening technology-
A kind of single stranded DNA that can be specifically bound with protein and small-molecule substance that number enrichment Fas lignand system evolution technology screens
Or RNA oligonucleotide segment, generally by 25-80 base composition.Aptamers have high specific and affinity, and target molecule
Range is wide, can not only be combined with macromolecular (such as nucleic acid, protein, polypeptide), also can with small molecule (such as amino acid, metal from
Son etc.) it combines, or even can also be with entire cell combination.This indicates that aptamers are recognition component and can significantly widen related transducer
The application range of device.Aptamers are easier to modify than antibody, and can be modified in the exact site of aptamers, and not
Its affinity is influenced, such as modifies some electrochemical probes, fluorophor and quencher, this allows for the structure of biosensor
It builds more convenient.In recent years, with being further appreciated that aptamer, the development of the biosensor based on aptamers and
Using having obtained widely paying close attention to, and construct a series of biosensors based on aptamers for protein detection,
Detection, Pharmaceutical Analysis, the environmental monitoring of small molecule, the fields such as food inspection.
The development of biosensor technique has greatly pushed application of the Electroanalytical Chemistry in biochemical field with progress.
However electrochemical method instrument is more heavy, thus a kind of portable detection device of application becomes the new development side of analytical chemistry
Exhibition.Blood glucose meter is a kind of electronic instrument measuring blood glucose level.Since its is small, at low cost, easy to operate, standard can be obtained
True quantitative result, has been obtained for extensive use in life, improves the quality of life of diabetic.However, blood glucose
Instrument can only detect a kind of glucose this substance, and detection range is 0.6~33mmol/L (10~600mg/dL).Currently with
Blood glucose meter binding antibody or DNA quantitatively detect the method for non-glucose substance it has been reported that as used blood glucose meter binding function
DNA sensor quantitatively detects plurality of target molecule, using the portable quantitative DNA molecular of commercialized personal blood glucose meter, using invading
Enter that formula DNA method combination blood glucose meters are portable quantitatively to detect metal ion.In actually detected, often content is extremely low for object,
And the purpose of detection when detecting the extremely low substance of these concentration, is extremely difficult to using conventional biosensor technique.
Invention content
For the above prior art problem, the purpose of the present invention is to provide a kind of preparation sides of biocompatible body sensor
Method quickly and easily detects target substance by DNA based on composable, foldable property using blood glucose meter as detecting instrument,
Zn2+, which is released, using cation exchange carries out DNA enzymatic shearing cycle to carry out the sensitivity that signal amplification improves experiment, structure
The aptamers biosensor of signal amplification is built, sensitive, quick detection platelet-derived growth factor-BB method is built.
A kind of preparation method of biocompatible body sensor, includes the following steps：
(1) buffer solution is prepared；
(2) ZnS nano-clusters are synthesized, aptamers 1 is prepared and modifies ZnS nano-clusters；
(3) Fe is synthesized3O4Nano-particle prepares DNA probe modification Fe3O4Nano-particle；
(4) 2 modified electrode of aptamers is prepared；
(5) platelet-derived growth factor-BB is added, builds biosensor.
Further, buffer solution described in step (1) includes that buffer solution used in experimental system and/or DNA bufferings are molten
Liquid.
Further, step (1) includes the following steps：By the aptamers 1 of respectively 2.5OD, aptamers 2, DNA probe,
8-17DNA enzyme sequences are dissolved in respectively in 20mM pH=7.4PBS buffer solutions, respectively obtain multiple a concentration of 100 μM of DNA
Buffer solution saves backup at 4 DEG C.
Further, DNA sequence dna is respectively：
Further, ZnS nano-clusters are synthesized using hydro-thermal method in step (2), included the following steps：
(2-1) thiocarbamide of the Zinc diacetate dihydrate of 0.1756g and 1.5224g is dissolved in the ultra-pure water of 20mL formed it is clear
Clear solution；
(2-2) is stirred 30 minutes at room temperature；
(2-3) is fitted into reaction kettle and heats 90 minutes for 140 DEG C；
After (2-4) is cooled to room temperature, centrifugation obtains white precipitate substance；
60 DEG C of drying obtain ZnS nano-clusters in 6 hours after (2-5) 20mL twice of 20mL ethyl alcohol of ultrapure washing washes twice.
Further, aptamers 1 are prepared in step (2) and modify ZnS nano-clusters, include the following steps：
The ZnS nano-clusters of 1mg are dissolved in the PBS buffer solutions of 1mL by (2-6)；
Streptavidin 6mg of 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides of (2-7) 25 μ L 1mg/mL
The N- hydroxy thiosuccinimides of hydrochloride and 5mg are dissolved in 1mL PBS buffer solutions；
(2-8) shakes 15 minutes；
The ZnS nano-clusters of 20 μ L 1mg/mL are added in (2-9)；
(2-10) room temperature shakes the ZnS nano-cluster solution for obtaining Streptavidin modification for 3 hours；
(2-11) aptamers 1 are added in above-mentioned solution, realize 5 μM of 1 final concentration of aptamers；
(2-12) stirs 30 minutes synthesis aptamers 1 and modifies ZnS nano-clusters at room temperature.
Further, step (3) synthesizes Fe3O4Nano-particle includes the following steps：
The Iron(III) chloride hexahydrate of (3-1) 1.35g, which is dissolved in 40mL ethylene glycol, forms clear solution；
3.6g sodium acetates and 1.0g polyethylene glycol is added in (3-2)；
(3-3) mixture pours into after stirring 30 minutes in reaction kettle；
200 DEG C of (3-4) heating 8 hours；
(3-5) is cooled to room temperature；
(3-6) is cleaned with second alcohol and water；
Fe is obtained after 60 DEG C of (3-7) drying 6 hours3O4Nano-particle.
Further, step (4) includes the following steps：
(4-1) is added in 100 μM of 2 solution of aptamers of 100 μ L in the 100mM thio-alcohol reducing agents TCEP of 0.1 μ L；
(4-2) reacts 1 hour at room temperature, prevents DNA itself from forming disulfide bond；
Aptamers 2 derived above are diluted to 0.5 μM by (4-3) with PBS buffer solutions；
The aptamers 2 of 0.5 μM of 5 μ L are added drop-wise to clean gold electrode surfaces by (4-4)；
(4-5) is reacted at room temperature 2 hours under dark surrounds；
(4-6) is cleaned with ultra-pure water；
The gold electrode that aptamers 2 are modified is stored in the 20mM PBS containing 1.0mM 6- mercaptoethanols to close by (4-7)
Remaining hole.
Further, in step (4-7) PBS pH=7.4.
Further, step (5) includes the following steps：
Electrode is immersed in 1.0 × 10 by (5-1)-12In M platelet-derived growth factor-BB solution；
Electrode is immersed in 200 μ L and contains 14 μ L 1.0mM AgNO by (5-2)3PBS buffer solutions in；
(5-3) reacts 10 minutes at room temperature；
150nM Substrate DNAs are added in (5-4) in above-mentioned solution, and 75nM 8-17DNAzyme react 60 minutes at 37 DEG C；
(5-5) Substrate DNA is sheared, and by Magnetic Isolation, the part Substrate DNA after being sheared connects converting Enzyme；
The sucrose solution of (5-6) 20 μ L 1.0M is added in isolated above-mentioned solution；
(5-7) reacts 20 minutes at room temperature.
Compared with currently available technology, the present invention is using widely used portable glucose meter combination aptamers structure biology
Sensor, it is intended to be provided for indirect detection platelet derived growth factor for quick, the quantitative glucose content by converting out
A kind of new method, building some, there is highly sensitive, signal scale-up version electrochemica biological sensor to have become people day
The center of gravity of benefit research.Wherein, most effective and most common method is exactly to amplify strategy by certain signal to make detection signal make
Signal is enhanced, to improve the sensitivity of analysis.
Description of the drawings
Fig. 1 is to utilize the portable detection platelet-derived growth factor-BB schematic diagram of blood glucose meter based on the triple amplifications of DNA enzymatic；
Fig. 2A is that the transmission electron microscope of ZnS nano-clusters characterizes；
Fig. 2 B are the transmission electron microscope characterization that ZnS nano-clusters modify aptamers 1；
Fig. 3 A are that the scanning electron microscope of Fe3O4 nano-particles characterizes；
Fig. 3 B are the scanning electron microscope characterization of the Fe3O4 nano-particles of Streptavidin modification；
Fig. 3 C are the scanning electron microscope characterization of the Fe3O4 nano-particles of Substrate DNA modification；
Fig. 4 is experimental feasibility figure.(a) object is added without object (b)；
The experimental condition optimization figure in Fig. 5 A reaction time between aptamers and platelet-derived growth factor-BB；
Fig. 5 B are the experimental condition optimization figure of converting Enzyme and sucrose reaction time；
Fig. 5 C are the experimental condition optimization figure for testing pH；
Fig. 6 is the blood glucose meter signal for the platelet-derived growth factor-BB for detecting various concentration；Concentration is followed successively by from a to f
1.0×10-15M,3.16×10-15M,1.0×10-14M,3.16×10-14M,1.0×10-13M,3.16×10-13M,
1.0×10-12M,3.16×10-12M；
Specific implementation mode
Below according to attached drawing, the present invention will be described in detail, is a kind of preferred reality in numerous embodiments of the present invention
Apply example.
In a preferred embodiment, a kind of preparation method of biocompatible body sensor, includes the following steps：
(1) buffer solution used in experimental system and DNA buffer solutions are prepared；
(2) ZnS nano-clusters are synthesized, aptamers 1 is prepared and modifies ZnS nano-clusters；Synthesize Fe3O4Nano-particle prepares probe
DNA modification Fe3O4Nano-particle；
(3) 2 modified electrode of aptamers is prepared；
(4) platelet-derived growth factor-BB is added, build biosensor and detects.
Step (1) includes the following steps：By respectively aptamers 1, the aptamers 2 of 2.5OD of purchase, DNA probe, 8-
17DNA enzyme sequences are dissolved in respectively in 20mM pH=7.4PBS buffer solutions, and it is slow to respectively obtain multiple a concentration of 100 μM of DNA
Solution is rushed, is saved backup at 4 DEG C.DNA sequence dna is respectively：
Step (2) includes the following steps：Hydro-thermal method synthesizes ZnS nano-clusters.By the Zinc diacetate dihydrate of 0.1756g and
The thiocarbamide of 1.5224g, which is dissolved in the ultra-pure water of 20mL, forms clear solution, stirs 30 minutes at room temperature.Then, it is packed into reaction
In kettle, 140 DEG C are heated 90 minutes.After being cooled to room temperature, centrifugation obtains white precipitate substance, 20mL twice of 20mL second of ultrapure washing
60 DEG C of drying obtain ZnS nano-clusters in 6 hours after alcohol washes twice.Synthesize Fe3O4Nano-particle.The Iron(III) chloride hexahydrate of 1.35g is molten
Solution forms clear solution in 40mL ethylene glycol, adds 3.6g sodium acetates and 1.0g polyethylene glycol, and mixture stirs 30 minutes
After pour into reaction kettle, 200 DEG C heat 8 hours, be cooled to room temperature, clean repeatedly with second alcohol and water, 60 DEG C dry 6 hours after must
To Fe3O4Nano-particle.
The ZnS nano-clusters of 1mg are dissolved in the PBS buffer solutions of 1mL.The Streptavidin 6mg of 25 μ L 1mg/mL
The N- hydroxy thiosuccinimides of of1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and 5mg are dissolved in 1mL
In PBS buffer solutions, after shaking 15 minutes, the ZnS nano-clusters of 20 μ L 1mg/mL are added, room temperature, which is shaken, obtains strepto- parent for 3 hours
With the ZnS nano-cluster solution of element modification.Aptamers 1 are added in above-mentioned solution, realize 5 μM of 1 final concentration of aptamers, stir at room temperature
Synthesis aptamers 1 modify ZnS nano-clusters within 30 minutes.Same step prepares DNA probe modification Fe3O4Nano-particle.
Step (3) includes the following steps：The 100mM thio-alcohols of 0.1 μ L are added in 100 μM of 2 solution of aptamers of 100 μ L
It in reducing agent TCEP, reacts 1 hour at room temperature, prevents DNA itself from forming disulfide bond, PBS buffer solutions is then used to be obtained above
To aptamers 2 be diluted to 0.5 μM, 5 μ L, 0.5 μM of aptamers 2 are added drop-wise to clean gold electrode surfaces, in dark surrounds
Lower room temperature reaction 2 hours, is cleaned, then the gold electrode that aptamers 2 are modified is stored in containing 1.0mM6- mercaptoethanols with ultra-pure water
20mM PBS (pH=7.4) to close remaining hole.
The polishing treatment is specially：Gold electrode is first to be processed by shot blasting with the aluminium powder of 0.3 and 0.5mm successively, then successively
It is put into volume ratio HNO3:H2O=1:In 1 solution, ethanol solution, ultra-pure water, ultrasonic cleaning, the time point of ultrasonic cleaning are carried out
It Wei not 3~5min.
Step (4) is：Electrode is immersed in 1.0 × 10-12In M platelet-derived growth factor-BB solution, due to adaptation
The aptamers 2 of the effect of body-object-aptamers, platelet-derived growth factor-BB identification step (3) are connected on electrode,
Then, the aptamers 1 that step (2) obtains modify ZnS nano-clusters identification platelet-derived growth factor-BB and are connected further to electricity
On extremely, 1/ platelet-derived growth factor-BB of aptamers/aptamers, 2 modified electrode is obtained.Then, electrode is immersed in 200 μ L
Contain 14 μ L 1.0mM AgNO3PBS buffer solutions in, at room temperature react 10 minutes.Then, it is added in above-mentioned solution
150nM Substrate DNAs, 75nM 8-17DNAzyme react 60 minutes at 37 DEG C.Then, Substrate DNA is sheared, and passes through magnetism point
From the part Substrate DNA after being sheared connects converting Enzyme.Finally, the sucrose solution of 20 μ L 1.0M is added to isolated
Above-mentioned solution in, at room temperature react 20 minutes, be detected from taking 2 μ L to be added drop-wise on blood glucose meter test paper in this solution.
In a further advantageous embodiment, in order to it is more efficient it is sensitive easily detection target substance platelet derived growth because
The concentration and content of son, in this experiment based on our quasi- properties composable, foldable by DNA, using blood glucose meter as inspection
Survey instrument quickly and easily detect target substance, using cation exchange release Zn2+ carry out DNA enzymatic shearing cycle into
The amplification of row signal improves the sensitivity of experiment, and the aptamers biosensor of structure signal amplification builds sensitive, quick detection
Platelet-derived growth factor-BB method.A kind of preparation method of the biocompatible body sensor provided, includes the following steps：
(1) buffer solution used in experimental system and DNA buffer solutions are prepared；
(2) ZnS nano-clusters are synthesized, aptamers 1 is prepared and modifies ZnS nano-clusters；Fe3O4 nano-particles are synthesized, probe is prepared
DNA modification Fe3O4 nano-particles；
(3) 2 modified electrode of aptamers is prepared；
(4) platelet-derived growth factor-BB is added, build biosensor and detects.
Step (1) includes the following steps：By respectively aptamers 1, the aptamers 2 of 2.5OD of purchase, DNA probe, 8-
17DNA enzyme sequences are dissolved in respectively in 20mM pH=7.4PBS buffer solutions, and it is slow to respectively obtain multiple a concentration of 100 μM of DNA
Solution is rushed, is saved backup at 4 DEG C.DNA sequence dna is respectively：
Step (2) includes the following steps：Hydro-thermal method synthesizes ZnS nano-clusters.By the Zinc diacetate dihydrate of 0.1756g and
The thiocarbamide of 1.5224g, which is dissolved in the ultra-pure water of 20mL, forms clear solution, stirs 30 minutes at room temperature.Then, it is packed into reaction
In kettle, 140 DEG C are heated 90 minutes.After being cooled to room temperature, centrifugation obtains white precipitate substance, 20mL twice of 20mL second of ultrapure washing
60 DEG C of drying obtain ZnS nano-clusters in 6 hours after alcohol washes twice.Synthesize Fe3O4 nano-particles.1.35g Iron(III) chloride hexahydrate
It is dissolved in 40mL ethylene glycol and forms clear solution, add 3.6g sodium acetates and 1.0g polyethylene glycol, mixture stirs 30 points
It is poured into reaction kettle after clock, 200 DEG C are heated 8 hours, are cooled to room temperature, are cleaned repeatedly with second alcohol and water, after 60 DEG C dry 6 hours
Obtain Fe3O4 nano-particles.
The ZnS nano-clusters of 1mg are dissolved in the PBS buffer solutions of 1mL.The Streptavidin 6mg of 25 μ L 1mg/mL
The N- hydroxy thiosuccinimides of of1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and 5mg are dissolved in 1mL
In PBS buffer solutions, after shaking 15 minutes, the ZnS nano-clusters of 20 μ L 1mg/mL are added, room temperature, which is shaken, obtains strepto- parent for 3 hours
With the ZnS nano-cluster solution of element modification.Aptamers 1 are added in above-mentioned solution, realize 5 μM of 1 final concentration of aptamers, stir at room temperature
Synthesis aptamers 1 modify ZnS nano-clusters within 30 minutes.Same step prepares DNA probe modification Fe3O4 nano-particles.
Step (3) includes the following steps：The 100mM thio-alcohols of 0.1 μ L are added in 100 μM of 2 solution of aptamers of 100 μ L
It in reducing agent TCEP, reacts 1 hour at room temperature, prevents DNA itself from forming disulfide bond, PBS buffer solutions is then used to be obtained above
To aptamers 2 be diluted to 0.5 μM, 5 μ L, 0.5 μM of aptamers 2 are added drop-wise to clean gold electrode surfaces, in dark surrounds
Lower room temperature reaction 2 hours, is cleaned, then the gold electrode that aptamers 2 are modified is stored in containing 1.0mM6- mercaptoethanols with ultra-pure water
20mM PBS (pH=7.4) to close remaining hole.
The polishing treatment is specially：Gold electrode is first to be processed by shot blasting with the aluminium powder of 0.3 and 0.5mm successively, then successively
It is put into volume ratio HNO3:H2O=1:In 1 solution, ethanol solution, ultra-pure water, ultrasonic cleaning, the time point of ultrasonic cleaning are carried out
It Wei not 3~5min.
Step (4) is：Electrode is immersed in 1.0 × 10-12M platelet-derived growth factor-BB solution, due to adaptation
The aptamers 2 of the effect of body-object-aptamers, platelet-derived growth factor-BB identification step (3) are connected on electrode,
Then, the aptamers 1 that step (2) obtains modify ZnS nano-clusters identification platelet-derived growth factor-BB and are connected further to electricity
On extremely, 1/ platelet-derived growth factor-BB of aptamers/aptamers, 2 modified electrode is obtained.Then, electrode is immersed in 200 μ L
In PBS buffer solutions containing 14 μ L1.0mMAgNO3, react 10 minutes at room temperature.Then, 150nM is added in above-mentioned solution
Substrate DNA, 75nM 8-17DNAzyme react 60 minutes at 37 DEG C.Then, Substrate DNA is sheared, and by Magnetic Isolation, can be obtained
Part Substrate DNA after to shearing connects converting Enzyme.Finally, the sucrose solution of 20 μ L 1.0M is added to isolated above-mentioned
In solution, reacts 20 minutes, be detected from taking 2 μ L to be added drop-wise on blood glucose meter test paper in this solution at room temperature.
Preferred embodiment 1
A method of utilizing the portable detection platelet-derived growth factor-BB of blood glucose meter, packet based on the triple amplifications of DNA enzymatic
Include following steps：
Step (1) includes the following steps：By respectively aptamers 1, the aptamers 2 of 2.5OD of purchase, DNA probe, 8-
17DNA enzyme sequences are dissolved in respectively in 20mM pH=5~10PBS buffer solutions, respectively obtain multiple a concentration of 100 μM of DNA
Buffer solution saves backup at 4 DEG C.DNA sequence dna is respectively：
Step (2) includes the following steps：Hydro-thermal method synthesizes ZnS nano-clusters.By the Zinc diacetate dihydrate of 0.1756g and
The thiocarbamide of 1.5224g, which is dissolved in the ultra-pure water of 20mL, forms clear solution, stirs 30 minutes at room temperature.Then, it is packed into reaction
In kettle, 140 DEG C are heated 90 minutes.After being cooled to room temperature, centrifugation obtains white precipitate substance, 20mL twice of 20mL second of ultrapure washing
60 DEG C of drying obtain ZnS nano-clusters in 6 hours after alcohol washes twice.Synthesize Fe3O4 nano-particles.1.35g Iron(III) chloride hexahydrate
It is dissolved in 40mL ethylene glycol and forms clear solution, add 3.6g sodium acetates and 1.0g polyethylene glycol, mixture stirs 30 points
It is poured into reaction kettle after clock, 200 DEG C are heated 8 hours, are cooled to room temperature, are cleaned repeatedly with second alcohol and water, after 60 DEG C dry 6 hours
Obtain Fe3O4 nano-particles.
The ZnS nano-clusters of 1mg are dissolved in pH=5~10PBS buffer solutions of 1mL.The strepto- parent of 25 μ L 1mg/mL
With the N- hydroxy thiosuccinimides of plain 6mg of 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and 5mg
It is dissolved in 1mL pH=5~10PBS buffer solutions, after shaking 15 minutes, the ZnS nano-clusters of 20 μ L 1mg/mL, room temperature is added
Concussion obtains the ZnS nano-cluster solution of Streptavidin modification for 3 hours.Aptamers 1 are added in above-mentioned solution, realize aptamers 1
5 μM of final concentration stirs 30 minutes synthesis aptamers 1 and modifies ZnS nano-clusters at room temperature.Same step prepares DNA probe modification
Fe3O4 nano-particles.
Step (3) includes the following steps：The 100mM thio-alcohols of 0.1 μ L are added in 100 μM of 2 solution of aptamers of 100 μ L
It in reducing agent TCEP, reacts 1 hour at room temperature, prevents DNA itself from forming disulfide bond, PBS buffer solutions is then used to be obtained above
To aptamers 2 be diluted to 0.5 μM, 5 μ L, 0.5 μM of aptamers 2 are added drop-wise to clean gold electrode surfaces, in dark surrounds
Lower room temperature reaction 2 hours, is cleaned, then the gold electrode that aptamers 2 are modified is stored in containing 1.0mM 6- sulfydryl second with ultra-pure water
The 20mM PBS (pH=5~10) of alcohol are to close remaining hole.
The polishing treatment is specially：Gold electrode is first to be processed by shot blasting with the aluminium powder of 0.3 and 0.5mm successively, then successively
It is put into volume ratio HNO3:H2O=1:In 1 solution, ethanol solution, ultra-pure water, ultrasonic cleaning, the time point of ultrasonic cleaning are carried out
It Wei not 3~5min.
Step (4) is：Electrode is immersed in 1.0 × 10-12M platelet-derived growth factor-BB solution, due to adaptation
The aptamers 2 of the effect of body-object-aptamers, platelet-derived growth factor-BB identification step (3) are connected on electrode,
Then, the aptamers 1 that step (2) obtains modify ZnS nano-clusters identification platelet-derived growth factor-BB and are connected further to electricity
On extremely, 1/ platelet-derived growth factor-BB of aptamers/aptamers, 2 modified electrode is obtained.Then, electrode is immersed in 200 μ L
In pH=5~10PBS buffer solutions containing 14 μ L1.0mMAgNO3, react 10 minutes at room temperature.Then, in above-mentioned solution
150nM Substrate DNAs are added, 75nM 8-17DNAzyme react 60 minutes at 37 DEG C.Then, Substrate DNA is sheared, and passes through magnetism
Separation, the part Substrate DNA after being sheared connect converting Enzyme.Finally, the sucrose solution of 20 μ L 1.0M, which is added to, detaches
To above-mentioned solution in, at room temperature react 20 minutes, be detected from taking 2 μ L to be added drop-wise on blood glucose meter test paper in this solution.
Preferred embodiment 2
Step (1) includes the following steps：By respectively aptamers 1, the aptamers 2 of 2.5OD of purchase, DNA probe, 8-
17DNA enzyme sequences are dissolved in respectively in 20mM pH=7.4PBS buffer solutions, and it is slow to respectively obtain multiple a concentration of 100 μM of DNA
Solution is rushed, is saved backup at 4 DEG C.DNA sequence dna is respectively：
Step (2) includes the following steps：Hydro-thermal method synthesizes ZnS nano-clusters.By the Zinc diacetate dihydrate of 0.1756g and
The thiocarbamide of 1.5224g, which is dissolved in the ultra-pure water of 20mL, forms clear solution, stirs 30 minutes at room temperature.Then, it is packed into reaction
In kettle, 140 DEG C are heated 90 minutes.After being cooled to room temperature, centrifugation obtains white precipitate substance, 20mL twice of 20mL second of ultrapure washing
60 DEG C of drying obtain ZnS nano-clusters in 6 hours after alcohol washes twice.Synthesize Fe3O4 nano-particles.1.35g Iron(III) chloride hexahydrate
It is dissolved in 40mL ethylene glycol and forms clear solution, add 3.6g sodium acetates and 1.0g polyethylene glycol, mixture stirs 30 points
It is poured into reaction kettle after clock, 200 DEG C are heated 8 hours, are cooled to room temperature, are cleaned repeatedly with second alcohol and water, after 60 DEG C dry 6 hours
Obtain Fe3O4 nano-particles.
The ZnS nano-clusters of 1mg are dissolved in the PBS buffer solutions of 1mL.The Streptavidin 6mg of 25 μ L 1mg/mL
The N- hydroxy thiosuccinimides of of 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and 5mg are dissolved in
In 1mL PBS buffer solutions, after shaking 15 minutes, the ZnS nano-clusters of 20 μ L 1mg/mL are added, room temperature, which is shaken 3 hours, obtains chain
The ZnS nano-cluster solution of mould Avidin modification.Aptamers 1 are added in above-mentioned solution, realize 5 μM of 1 final concentration of aptamers, at room temperature
30 minutes synthesis aptamers 1 of stirring modify ZnS nano-clusters.Same step prepares DNA probe modification Fe3O4 nano-particles.
Step (3) includes the following steps：The 100mM thio-alcohols of 0.1 μ L are added in 100 μM of 2 solution of aptamers of 100 μ L
It in reducing agent TCEP, reacts 1 hour at room temperature, prevents DNA itself from forming disulfide bond, PBS buffer solutions is then used to be obtained above
To aptamers 2 be diluted to 0.5 μM, 5 μ L, 0.5 μM of aptamers 2 are added drop-wise to clean gold electrode surfaces, in dark surrounds
Lower room temperature reaction 2 hours, is cleaned, then the gold electrode that aptamers 2 are modified is stored in containing 1.0mM6- mercaptoethanols with ultra-pure water
20mM PBS (pH=7.4) to close remaining hole.
The polishing treatment is specially：Gold electrode is first to be processed by shot blasting with the aluminium powder of 0.3 and 0.5mm successively, then successively
It is put into volume ratio HNO3:H2O=1:In 1 solution, ethanol solution, ultra-pure water, ultrasonic cleaning, the time point of ultrasonic cleaning are carried out
It Wei not 3~5min.
Step (4) is：Several electrodes are immersed in the platelet-derived growth factor-BB solution of several various concentrations respectively
In, concentration is followed successively by 1.0 × 10-15M, 3.16 × 10-15M, 1.0 × 10-14M, 3.16 × 10-14M, 1.0 × 10-13M,
3.16×10-13M,1.0×10-12M,3.16×10-12M.Due to the effect of aptamers-object-aptamers, blood platelet spreads out
The aptamers 2 of raw growth factor-BB identification step (3) are connected on electrode, and then, the aptamers 1 that step (2) obtains are modified
ZnS nano-clusters identification platelet-derived growth factor-BB is connected further on electrode, obtains 1/ platelet-derived life of aptamers
Long 2 modified electrode of the factor-BB/ aptamers.Then, that electrode is immersed in the PBS bufferings that 200 μ L contain 14 μ L1.0mMAgNO3 is molten
In liquid, react 10 minutes at room temperature.Then, 150nM Substrate DNAs are added in above-mentioned solution, 75nM8-17DNAzyme is at 37 DEG C
Reaction 60 minutes.Then, Substrate DNA is sheared, by Magnetic Isolation, the part Substrate DNA connection conversion after being sheared
Enzyme.Finally, the sucrose solution of 20 μ L 1.0M is added in isolated above-mentioned solution, is reacted 20 minutes at room temperature, from this
It takes 2 μ L to be added drop-wise on blood glucose meter test paper in solution to be detected.
The present invention is exemplarily described above in conjunction with attached drawing, it is clear that the present invention implements not by aforesaid way
Limitation, as long as using the various improvement of inventive concept and technical scheme of the present invention progress, or not improved direct application
In other occasions, within protection scope of the present invention.
Claims (10)
1. a kind of preparation method of biocompatible body sensor, which is characterized in that include the following steps：
(1) buffer solution is prepared；
(2) ZnS nano-clusters are synthesized, aptamers 1 is prepared and modifies ZnS nano-clusters；
(3) Fe is synthesized3O4Nano-particle prepares DNA probe modification Fe3O4Nano-particle；
(4) 2 modified electrode of aptamers is prepared；
(5) platelet-derived growth factor-BB is added, builds biosensor.
2. the preparation method of biocompatible body sensor as described in claim 1, which is characterized in that delay described in step (1)
It includes buffer solution used in experimental system and/or DNA buffer solutions to rush solution.
3. the preparation method of biocompatible body sensor as claimed in claim 1 or 2, which is characterized in that step (1) include with
Lower step：By the aptamers 1 of respectively 2.5OD, aptamers 2, DNA probe, 8-17DNA enzyme sequences are dissolved in 20mM pH respectively
In=7.4PBS buffer solutions, multiple a concentration of 100 μM of DNA buffer solutions are respectively obtained, are saved backup at 4 DEG C.
4. the preparation method of biocompatible body sensor as claimed in claim 3, which is characterized in that DNA sequence dna is respectively：
Aptamers 1:CAGGCTACGGCACGTAGAGCATCACCATGATC CTGTTTTTT-biotin；Aptamers 2:
CAGGCTACGGCACGTAGAGCATCACCATGATCCTGTTTT-(CH2)3-HS；Substrate DNA：biotin–
CCACCACAATGTTATACAGGTACTATrAG GAAGTTGAGTTACGAGGCGGTGGTGG-(CH2)3-SH；8-17DNA enzymes：
CTCAACTTCTCCGAGCCGGTCGAAATAGTACCT。
5. the preparation method of biocompatible body sensor as claimed in claim 1 or 2, which is characterized in that used in step (2)
Hydro-thermal method synthesizes ZnS nano-clusters, includes the following steps：
(2-1) by the thiocarbamide of the Zinc diacetate dihydrate of 0.1756g and 1.5224g be dissolved in the ultra-pure water of 20mL formed clarification it is molten
Liquid；
(2-2) is stirred 30 minutes at room temperature；
(2-3) is fitted into reaction kettle and heats 90 minutes for 140 DEG C；
After (2-4) is cooled to room temperature, centrifugation obtains white precipitate substance；
60 DEG C of drying obtain ZnS nano-clusters in 6 hours after (2-5) 20mL twice of 20mL ethyl alcohol of ultrapure washing washes twice.
6. the preparation method of biocompatible body sensor as claimed in claim 1 or 2, which is characterized in that prepared in step (2)
Aptamers 1 modify ZnS nano-clusters, include the following steps：
The ZnS nano-clusters of 1mg are dissolved in the PBS buffer solutions of 1mL by (2-6)；
Streptavidin 6mg of 1- (3- dimethylamino-propyls) -3- ethyl carbodiimide hydrochlorides of (2-7) 25 μ L 1mg/mL
The N- hydroxy thiosuccinimides of salt and 5mg are dissolved in 1mL PBS buffer solutions；
(2-8) shakes 15 minutes；
The ZnS nano-clusters of 20 μ L 1mg/mL are added in (2-9)；
(2-10) room temperature shakes the ZnS nano-cluster solution for obtaining Streptavidin modification for 3 hours；
(2-11) aptamers 1 are added in above-mentioned solution, realize 5 μM of 1 final concentration of aptamers；
(2-12) stirs 30 minutes synthesis aptamers 1 and modifies ZnS nano-clusters at room temperature.
7. the preparation method of biocompatible body sensor as claimed in claim 1 or 2, which is characterized in that step (3) synthesizes
Fe3O4Nano-particle includes the following steps：
The Iron(III) chloride hexahydrate of (3-1) 1.35g, which is dissolved in 40mL ethylene glycol, forms clear solution；
3.6g sodium acetates and 1.0g polyethylene glycol is added in (3-2)；
(3-3) mixture pours into after stirring 30 minutes in reaction kettle；
200 DEG C of (3-4) heating 8 hours；
(3-5) is cooled to room temperature；
(3-6) is cleaned with second alcohol and water；
Fe is obtained after 60 DEG C of (3-7) drying 6 hours3O4Nano-particle.
8. the preparation method of biocompatible body sensor as claimed in claim 1 or 2, which is characterized in that step (4) includes such as
Lower step：
(4-1) is added in 100 μM of 2 solution of aptamers of 100 μ L in the 100mM thio-alcohol reducing agents TCEP of 0.1 μ L；
(4-2) reacts 1 hour at room temperature, prevents DNA itself from forming disulfide bond；
Aptamers 2 derived above are diluted to 0.5 μM by (4-3) with PBS buffer solutions；
The aptamers 2 of 0.5 μM of 5 μ L are added drop-wise to clean gold electrode surfaces by (4-4)；
(4-5) is reacted at room temperature 2 hours under dark surrounds；
(4-6) is cleaned with ultra-pure water；
The gold electrode that aptamers 2 are modified is stored in the 20mM PBS containing 1.0mM 6- mercaptoethanols to close residue by (4-7)
Hole.
9. the preparation method of biocompatible body sensor as claimed in claim 8, which is characterized in that PBS in step (4-7)
PH=7.4.
10. the preparation method of biocompatible body sensor as claimed in claim 1 or 2, which is characterized in that step (5) includes
Following steps：
Electrode is immersed in 1.0 × 10 by (5-1)-12In M platelet-derived growth factor-BB solution；
Electrode is immersed in 200 μ L and contains 14 μ L 1.0mM AgNO by (5-2)3PBS buffer solutions in；
(5-3) reacts 10 minutes at room temperature；
150nM Substrate DNAs are added in (5-4) in above-mentioned solution, and 75nM 8-17DNA enzymes react 60 minutes at 37 DEG C；
(5-5) Substrate DNA is sheared, by Magnetic Isolation, the part Substrate DNA converting Enzyme after being sheared；
The sucrose solution of (5-6) 20 μ L 1.0M is added in isolated above-mentioned solution；
(5-7) reacts 20 minutes at room temperature.
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