CN101738425A - Method for manufacturing aptamer biosensor used for quickly detecting antibiotic and heart disease markers - Google Patents
Method for manufacturing aptamer biosensor used for quickly detecting antibiotic and heart disease markers Download PDFInfo
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- CN101738425A CN101738425A CN201010300063A CN201010300063A CN101738425A CN 101738425 A CN101738425 A CN 101738425A CN 201010300063 A CN201010300063 A CN 201010300063A CN 201010300063 A CN201010300063 A CN 201010300063A CN 101738425 A CN101738425 A CN 101738425A
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Abstract
The invention relates to a method for manufacturing an aptamer biosensor used for quickly detecting antibiotic and heart disease markers, which comprises the following steps: 1, modifying an electrode; and 2, fixing an aptamer. In the aptamer biosensor of the invention, an antibiotic and heart disease marker (cardiac troponin I) aptamer serving as a recognition molecule is fixed on the surface of a glassy carbon electrode to specifically combine and quickly detect an antibiotic or an cardiac troponin I (cTnI) in a specimen, and an electrochemical signal generated can reflect the concentration of the antibiotic or cTnI in solution of the specimen. The biosensor has the advantages of high specificity, easy carrying, quickness, high stability, low cost and the like.
Description
Technical field
The invention belongs to technical field of biological, especially a kind of method for making that is used for the aptamer biosensor of microbiotic and heart disease markers fast detecting.
Background technology
Adaptive son has that target molecule is wide, high specificity, advantage such as stable, can be applicable to biology sensor, and can solve some shortcomings in the sensor, adaptive son has been successfully applied to multiple bio-sensing applicator platform as the identification molecule, demonstrates good prospects for application.
According to the retrieval, existing market does not have the aptamer biosensor about tetracycline, terramycin and penicillin and cTnI detection.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art part, a kind of preparation method is provided the method for making of the aptamer biosensor that is used for microbiotic and heart disease markers fast detecting simple, easy to use.
The objective of the invention is to be achieved through the following technical solutions:
A kind of method for making that is used for the aptamer biosensor of microbiotic and heart disease markers fast detecting, the step of making is:
(1) electrode modification: after glass-carbon electrode cleaned, electrode is carried out oxidation processes, put into 3-aminopropyl triethoxysilane hydrochloric acid solution silanization then, again electrode is immersed in 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides and the N-hydroxy-succinamide solution and activate;
(2) adaptive son is fixed: with adaptive son drip to the activation after electrode surface, crosslinked under the irradiation of ultraviolet light, fix in oven for baking again, use the redistilled water cleaning electrode then, dry up electrode surface with nitrogen, use not binding site of bovine serum albumin(BSA) enclosed-electrode surface at last, clean with distilled water again and remove free bovine serum albumin(BSA).
And, described electrode carry out oxidation processes be with working electrode in pH=6.0PBS solution, make scan round 8-9 circle in-0.8-+1.8V potential range.
And described 3-aminopropyl triethoxysilane hydrochloric acid solution is 3-aminopropyl triethoxysilane: HCl: water=1: 15: 10.
And described adaptive son is tetracycline, terramycin and penicillin and the adaptive son of cTnI.
And the concentration and the consumption of described fixed adaptation are respectively: 25ng/ μ L and 10 μ L.
Advantage of the present invention and good effect are:
1, aptamer biosensor involved in the present invention is adaptive sub as the identification molecule with microbiotic and heart disease markers (cardiac muscle troponin I), be fixed in the glass-carbon electrode surface, microbiotic in the sample or cardiac muscle troponin I (cTnI) are carried out specificity combination and fast detecting, the electrochemical signals that is produced can reflect the concentration of microbiotic in the sample solution or cTnI, and this biology sensor has specificity height, portable, quick, good stability, advantage such as with low cost.
2, the present invention combines microbiotic or the adaptive son of cTnI with biology sensor, by the adaptive son of antibiotics to antibiotic specificity combination, through the oxidation of electrode sun, hydrophiling, amination, a series of modifications of activation, utilizing, the amino and the adaptive sub-phosphate group covalent bond on modified electrode surface form the phosphorylated amino ester bond, can be for detecting antibiotic content in the samples such as aqueous solution and milk, do non-specific contrast simultaneously, increased the reliability that detects.
Description of drawings
Fig. 1 is that the adaptive sub-sensor of the present invention is to detection of antibiotics in PBS-K3Fe (CN) 6 solution; Wherein: the typical curve that a detects tetracycline, the typical curve that b detects terramycin (the detection range of linearity: 1-0.1mg/L);
Fig. 2 is the detection of the adaptive sub-sensor of the present invention to Tetracyclines in milk K3Fe (CN) 6 solution; Wherein: the typical curve that a detects tetracycline, the typical curve that b detects terramycin (the detection range of linearity: 1-0.1mg/L).
Embodiment
The invention will be further described below by specific embodiment, below real be descriptive from example, be not determinate, can not limit protection scope of the present invention with this.
A kind of method for making that is used for the aptamer biosensor of microbiotic and heart disease markers fast detecting, step is:
Present embodiment is the preparation method of example narration aptamer biosensor with microbiotic and the adaptive son of cTnI
(1) electrode modification: after glass-carbon electrode cleaned, in pH=6.0PBS solution, make scan round 8-9 circle in-0.8-+1.8V potential range, electrode is carried out oxidation place amount, put into 3-aminopropyl triethoxysilane hydrochloric acid solution (3-aminopropyl triethoxysilane: HCl: water=1: 15: 10) then, silanization was carried out in 53 ℃ of water-baths in 3 hours, promptly at electrode surface importing-NH
2, then electrode is immersed 100 μ L and contains in 0.05M 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) and 0.03M N-hydroxy-succinamide (NHS) solution, 50 ℃ of water-bath activation 90min;
(2) adaptive son is fixed: specific recognition molecule fixing on the glass-carbon electrode is microbiotic or the adaptive son of cTnI, earlier the adaptive sub 10 μ L of 25ng/ μ L are dripped to electrode surface, and under the irradiation of ultraviolet light, crosslinked 1h, fix in 80 ℃ of oven for baking 30min again, then with the thorough cleaning electrode of redistilled water, remove unnecessary loose adaptive son, dry up electrode surface with nitrogen again, use 5 μ L at last, 1% bovine serum albumin(BSA) (BSA) enclosed-electrode surface is binding site 2h not, cleans with distilled water and removes free BSA.
The application example of this aptamer biosensor:
1, sample determination: join tetracycline, terramycin, penicillin, the cTnI solution of variable concentrations, every 5 minutes, do the cyclic voltammetry test in-0.1-0.6 electromotive force interval, sweep speed is 0.05V/s.
2 results and discussion: the responding range that adaptive son detects tetracycline, terramycin, penicillin and cTnI.
Under selected optimal conditions, with modify the working electrode of getting well respectively with a series of variable concentrations (0mg/L, 0.001mg/L of containing, 0.005mg/L, 0.01mg/L, 0.015mg/L, 0.02mg/L, 0.03mg/L, 0.05mg/L, 0.08mg/L, tetracycline/terramycin 0.1mg/L)/penicillin/cTnI potassium ferricyanide PBS solution or milk soln reaction, measure cyclic voltammetry curve, and make the typical curve (as Fig. 1 and Fig. 2) of concentration the cutting edge of a knife or a sword electric current.The concentration same methods of tetracycline in the sample, terramycin, penicillin or cTnI detects, and calculates by typical curve.
Claims (5)
1. method for making that is used for the aptamer biosensor of microbiotic and heart disease markers fast detecting, it is characterized in that: the step of making is:
(1) electrode modification: after glass-carbon electrode cleaned, electrode is carried out oxidation processes, put into 3-aminopropyl triethoxysilane hydrochloric acid solution silanization then, again electrode is immersed in 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides and the N N-Hydroxysuccinimide solution and activate;
(2) adaptive son is fixed: with adaptive son drip to the activation after electrode surface, crosslinked under the irradiation of ultraviolet light, fix in oven for baking again, use the redistilled water cleaning electrode then, dry up electrode surface with nitrogen, use not binding site of bovine serum albumin(BSA) enclosed-electrode surface at last, clean with distilled water again and remove free bovine serum albumin(BSA).
2. the method for making that is used for the aptamer biosensor of microbiotic and heart disease markers fast detecting according to claim 1, it is characterized in that: described electrode carry out oxidation processes be with working electrode in pH=6.0PBS solution, make scan round 8-9 circle in-0.8-+1.8V potential range.
3. the method for making that is used for the aptamer biosensor of microbiotic and heart disease markers fast detecting according to claim 1 is characterized in that: described 3-aminopropyl triethoxysilane hydrochloric acid solution is 3-aminopropyl triethoxysilane: HCl: water=1: 15: 10.
4. the method for making that is used for the aptamer biosensor of microbiotic and heart disease markers fast detecting according to claim 1 is characterized in that: described adaptive son is tetracycline, terramycin and penicillin and the adaptive son of cTnI.
5. according to claim 1 or the 4 described method for makings that are used for the aptamer biosensor of microbiotic and heart disease markers fast detecting, it is characterized in that: the concentration and the consumption of described fixed adaptation are respectively: 25ng/ μ L and 10 μ L.
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Cited By (10)
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| CN102175742A (en) * | 2010-12-23 | 2011-09-07 | 天津前方科技有限公司 | Method for preparing novel antibiotic nanometer biosensor |
| CN102816764A (en) * | 2011-06-07 | 2012-12-12 | 浦项工科大学校产学协力团 | DNA aptamer specifically binding to human cardiac troponin I |
| CN103387991A (en) * | 2013-08-05 | 2013-11-13 | 上海交通大学 | Improved penicillin antibiotic aptamer without fixed point target substance and application thereof |
| CN103499620A (en) * | 2013-10-18 | 2014-01-08 | 山东理工大学 | Method for preparing current type aptamer sensor for detecting tetracycline residue |
| CN103616423A (en) * | 2013-12-02 | 2014-03-05 | 济南大学 | Preparation method and application of competitive type aptamer sensor for detecting oxytetracycline |
| CN104849331A (en) * | 2015-05-17 | 2015-08-19 | 济南大学 | Preparing method of photoelectrochemical sensor based on sandwich cardiac troponin T marked by Ag2Se@CdSe and application |
| CN105424787A (en) * | 2015-11-20 | 2016-03-23 | 济南大学 | Preparation method of silk screen printing sensor for detecting tetracycline and oxytetracycline at same time |
| CN107831198A (en) * | 2017-12-08 | 2018-03-23 | 济南大学 | A kind of preparation method and application of the optical electro-chemistry cTnI sensor based on multistage micron cube zinc stannate composite |
| CN110241119A (en) * | 2018-03-07 | 2019-09-17 | 中国科学技术大学 | Cardiac muscle troponin I specific nucleic acid aptamers and its screening technique and application |
| CN110257472A (en) * | 2019-06-06 | 2019-09-20 | 南京农业大学 | Utilize the test strips and method of tetracycline antibiotics in biosensor detection water |
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| CN102175742A (en) * | 2010-12-23 | 2011-09-07 | 天津前方科技有限公司 | Method for preparing novel antibiotic nanometer biosensor |
| CN102816764A (en) * | 2011-06-07 | 2012-12-12 | 浦项工科大学校产学协力团 | DNA aptamer specifically binding to human cardiac troponin I |
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| CN103387991A (en) * | 2013-08-05 | 2013-11-13 | 上海交通大学 | Improved penicillin antibiotic aptamer without fixed point target substance and application thereof |
| CN103499620B (en) * | 2013-10-18 | 2015-12-09 | 山东理工大学 | A kind of preparation method detecting the current type aptamer sensor of tetracycline residue |
| CN103499620A (en) * | 2013-10-18 | 2014-01-08 | 山东理工大学 | Method for preparing current type aptamer sensor for detecting tetracycline residue |
| CN103616423A (en) * | 2013-12-02 | 2014-03-05 | 济南大学 | Preparation method and application of competitive type aptamer sensor for detecting oxytetracycline |
| CN104849331A (en) * | 2015-05-17 | 2015-08-19 | 济南大学 | Preparing method of photoelectrochemical sensor based on sandwich cardiac troponin T marked by Ag2Se@CdSe and application |
| CN104849331B (en) * | 2015-05-17 | 2016-04-20 | 济南大学 | A kind of based on Ag 2the preparation method of the Optical Electro-Chemistry sensor of the sandwich type serum cardiac troponin T of SeCdSe mark and application |
| CN105424787A (en) * | 2015-11-20 | 2016-03-23 | 济南大学 | Preparation method of silk screen printing sensor for detecting tetracycline and oxytetracycline at same time |
| CN107831198A (en) * | 2017-12-08 | 2018-03-23 | 济南大学 | A kind of preparation method and application of the optical electro-chemistry cTnI sensor based on multistage micron cube zinc stannate composite |
| CN110241119A (en) * | 2018-03-07 | 2019-09-17 | 中国科学技术大学 | Cardiac muscle troponin I specific nucleic acid aptamers and its screening technique and application |
| CN110241119B (en) * | 2018-03-07 | 2022-07-15 | 中国科学技术大学 | Cardiac troponin I specific aptamer, and screening method and application thereof |
| CN110257472A (en) * | 2019-06-06 | 2019-09-20 | 南京农业大学 | Utilize the test strips and method of tetracycline antibiotics in biosensor detection water |
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