CN103499620A - Method for preparing current type aptamer sensor for detecting tetracycline residue - Google Patents
Method for preparing current type aptamer sensor for detecting tetracycline residue Download PDFInfo
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- CN103499620A CN103499620A CN201310494699.7A CN201310494699A CN103499620A CN 103499620 A CN103499620 A CN 103499620A CN 201310494699 A CN201310494699 A CN 201310494699A CN 103499620 A CN103499620 A CN 103499620A
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- aptamers
- shitosan
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- glutaraldehyde
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Abstract
The invention relates to a method for preparing a current type aptamer sensor for detecting tetracycline residue. The method is characterized in that a novel compound of chitosan, glutaraldehyde and potassium ferricyanide is prepared, a first-layer substance for decorating an electrode can be used as a molecular wire, so that transmission of electrons on an electron surface is accelerated, the chitosan is good in adsorbability, stability and biocompatibility, abundant amidogen and the porous structure of the chitosan are beneficial to the improvement of the performance of the sensor, and the abundant amidogen on the chitosan can further provide position points for covalent binding of colloidal gold nanoparticle of a second-layer decorating substance. The aptamer sensor prepared through the steps is short in detection time, high in sensitivity, good in specificity and stability and capable of meeting the requirement for the recycling rate.
Description
Technical field
The invention provides a kind of preparation method who detects the residual current mode aptamers sensor of tetracycline, belong to the biosensor technology field.
Background technology
In recent years, along with developing rapidly of China's livestock breeding industry, milk yield increases substantially, and fresh milk and dairy produce have become the important component part in the people (especially old man and children) life food.Yet the widespread use of microbiotic in Modern Animal Husbandry, inevitably cause Residue of Antibiotics in Milk.If long-term drinking contains antibiotic milk, equal beyond doubt the microbiotic of long-term taking low dose, can cause allergic reaction after the people of microbiotic allergic constitution being taken to the milk of residual antibiotic.Normal drinking person, can cause some the conditionality pathogenic bacteria in body easily to produce drug resistance, once ill, with antibiotic therapy of the same race, is difficult to prove effective again.Antibiotic residue is harm humans health not only, also affects the quality of milk simultaneously, causes economic loss.Because the mastadenitis of cow incidence of disease is higher, usually treat obstetrics' diseases such as mastitis with Fourth Ring still more general, this easily causes the residual of in milk tetracycline.Excessive use TCs unavoidably can make the associated antibiotics such as parent metabolic product residue in the muscle, egg, milk, organs and tissues of animal, and then affects health by food chain.Visible, strengthen the detection residual to tetracycline in milk, especially ensure that human health has very profound significance.
Traditional method for antibiotic residue detection mainly contains: gas chromatography (GC), high performance liquid chromatography (HPLC), Chromatography/Mass Spectrometry coupling technique (GC/LC-MS), capillary electrophoresis (CE), fluorescence analysis, euzymelinked immunosorbent assay (ELISA) (ELISA).Although these method selectivity are good, highly sensitive, accuracy is high, detection limit is low, can detect multiple element or compound, but it needs expensive instrument and equipment, sample pretreatment process is loaded down with trivial details, time-consuming simultaneously, and the technical merit requirement to the analyst is very high, is unsuitable for field quick detection.Aptamer is artificial synthetic nucleotide sequence, through aptamer and the target molecules that repeatedly screening obtains, very high specificity and affinity is arranged.With traditional antibody, compare, aptamers has following several advantage: (1) prepares aptamers without the immune supervisor that carries out animal, can be screened in vitro and carry out a large amount of, preparation fast; (2) for the compatibility of target molecule higher than traditional antibody, and can combine with one-level, secondary and the tertiary structure of nucleic acid, for the screening of target material in a big way, have necessarily and have superiority clinically; (3) more stable than traditional antibody, the holding time is long, can at room temperature be transported.Biosensor technique based on aptamers, for the fast detecting of livestock products antibiotic residue, is contributed to improve stability, reappearance and the accuracy of antibiotic residue fast detecting, be expected to become the new tool of antibiotic residue fast detecting.
The purpose of invention is to provide a kind of preparation method that can overcome above-mentioned defect and the residual current mode aptamers sensor of detection tetracycline simple to operate, highly sensitive, that selectivity is good.
Its technical scheme is: a kind of preparation method who detects the residual current mode aptamers sensor of tetracycline, it is characterized in that: the responsive interface of current mode aptamers sensor forms and comprises compound and the nanometer gold size (AuNPs) prepared by shitosan, glutaraldehyde and three kinds of materials of the potassium ferricyanide, and then fixing aptamers corresponding to tetracycline.
Described a kind of preparation method who detects the residual current mode aptamers sensor of tetracycline, it is characterized in that: the cleaning of glass-carbon electrode (d=3mm), the structure at aptamers sensor sensing interface and process characterize, the foundation of aptamers working sensor curve, the detection of aptamers sensor performance, the detection of aptamers sensor to actual sample.
Described a kind of preparation method who detects the residual current mode aptamers sensor of tetracycline, it is characterized in that: the optimization of experiment condition mainly comprises amount, aptamers concentration and the incubation time of shitosan in the compound of shitosan, glutaraldehyde and the potassium ferricyanide; The working curve of prepared current mode immunosensor is: Δ I (μ A)=5.738+0.386 LogC (M) (R
2=0.994,1 * 10
-9m-1 * 10
-5m), Δ I (μ A)=14.464+2.094 LogC (M) (R
2=0.992,1 * 10
-5m-1 * 10
-2m); The aptamers sensor performance detects and comprises specificity stability, reappearance and the aptamers sensor mensuration to the milk sample recovery.
Its preparation principle is: the aptamers biology sensor is usingd aptamers as recognition component, by immobilization technology, aptamers is attached to susceptor surface, aptamers is after object is combined, the compound formed is associated with physics or the chemical signal of generation, by transducer be translated into relevant with test substance concentration (or activity) can quantitative or accessible physical chemistry signal, amplify and output signal by secondary instrument again, thereby realize the detection to test substance.The present invention adopts and with compound and the nanometer gold size of shitosan, glutaraldehyde and the potassium ferricyanide, glass-carbon electrode is modified.The compound of shitosan, glutaraldehyde and the potassium ferricyanide not only can be used as molecular wire as the ground floor material of modified electrode and accelerates the transmission of electronics in electron surface, and on shitosan a large amount of amino can also modify that material nm of gold glue provides for the second layer can covalently bound site.Shitosan belongs to polysaccharide, it has excellent film forming, adsorbability, gas penetration potential and perviousness, have good adsorbability, stability and good biocompatibility after film forming, its abundant amino, cellular structure make it be widely used in the preparation of the fixing and modified electrode of biomolecule.The aminoterminal of shitosan connects the nanometer gold size by golden ammonia key, for aptamers fixedly providing of electrode surface a good bioelectric interface, the biologically active that has kept aptamers is to be used for and tetracycline carries out effective combination.In addition, the specific surface area that the nanometer gold size is higher, stability and electric conductivity can increase transmissibility and the biocompatibility of electrode surface electronics, and then increase the response current of aptamers sensor.The current mode aptamers sensor that adopts the present invention to make can be before the milk listing, carry out the residual Fast Measurement of tetracycline, directly whether Tetracycline Residues is exceeded standard and detected, avoid causing serious consequence because the edible milk that contains tetracycline causes tetracycline accumulating in human body, for livestock products, keep the safety in production the technical support of residue detection is provided with consumption.
For reaching above purpose, take following technical scheme to realize: a kind of preparation method who detects the residual current mode aptamers sensor of tetracycline, it is characterized in that: cleaning, activation and the performance test of naked glass-carbon electrode before the preparation of (1) current mode aptamers sensor, if the spike potential in the test loop volt-ampere curve is poor below 120 mV, oxidation peak and reduction peak symmetry, described glass-carbon electrode can be used, otherwise will return in cleaning step, until meet the requirements.(2) cleaned naked glass-carbon electrode surface is dripped and is coated with finely dispersed shitosan-glutaraldehyde-potassium ferricyanide compound, decorated nanometer gold size then, and then the fixed adaptation body, finally seal nonspecific binding site with mercaptoethanol.After current mode aptamers sensor preparation finishes, put in refrigerator 4 ℃ and save backup.
For reaching above purpose, take following technical scheme to realize: a kind of preparation method who detects the residual current mode aptamers sensor of tetracycline, it is characterized in that: (1) does cyclic voltammetric method scanning with the different speed of sweeping by the above-mentioned current mode aptamers sensor prepared in work end liquid, can obtain peak value and sweep fast relation, obtaining being subject to diffusion control.(2) configure a series of tetracycline titer, carry out differential pulse voltammetry scanning, the front and back that obtain electric current change, and further draw working curve, sensing range and the detectability of the current mode aptamers sensor of above-mentioned preparation; (3) configure the antibiotic solution that a series of frequent mixing are used, to detect the specificity of prepared current mode aptamers sensor; (4) store the stability of variation checking the sensor of differential pulse voltammetry of the aptamers sensor of 7-21 days by detection, it reappears performance 5 different electrode detection of modifying by the contrast same procedure; (5) actual milk sample analysis is drawn to the recovery of this aptamers sensor.
The present invention adopts the potassium ferricyanide to be built in the ground floor material that is used as modified electrode in the compound of shitosan and glutaraldehyde, shitosan with good biocompatibility and film forming can promote the transmission of electronics in electrochemical reaction as the complexes membrane of prepared shitosan, glutaraldehyde and the potassium ferricyanide become of spreading agent, improve the response current on electrode, improve the microenvironment of electrode surface, thereby can be used as carrier material, in order to prepare the strong and highly sensitive aptamers sensor of response signal; There is the nano composite membrane of a large amount of amino and the golden ammono-system of nanometer gold size formation and there is higher stability and high-sequential, increase the current-responsive of aptamers sensor and improved the effectively fixing of sensor surface aptamers, thereby increased accuracy of detection.
The preparation technology of described current mode aptamers sensor is as follows: the compound that (1) gets shitosan, glutaraldehyde and the potassium ferricyanide that 5 μ L prepare drips and is coated in the glass-carbon electrode surface that pre-service is good, air drying, rinse electrode surface with the PBS damping fluid of PH=7.5; (2), after electrode surface dries, get 5 μ L nanometer gold sizes and drip and be coated in electrode surface, standing to dry under normal temperature, then with ultrapure water, rinse the nm of gold of surface physics absorption, nitrogen dries up; (3) then the aptamers of 5 μ L 5mM is dropped in above-mentionedly on nanometer-material-modified good electrode, cover the evaporation that prevents moisture with rubber cap, after 4h, with ultrapure water, rinse surface, dry stand-by; (4) the last above-mentioned electrode prepared is dipped in the mercaptoethanol sealing 1h with 1M, and with the sealing nonspecific binding site, the aptamers sensor production completes, and is kept under 4 ° of C conditions standby.
Embodiment
Embodiment: the preparation of (1) shitosan, glutaraldehyde and potassium ferricyanide compound: at first configure the acetum of 1% (1 ml/100 ml water) and prepare the chitosan solution of 50 ml 1% (1 g/100 ml) as solvent with the acetum prepared.The K of 5 mM of 500 μ l
3[Fe (CN)
6] mix ultrasonic concussion with the chitosan solution (400 μ l 1% shitosans+600 μ l water) of 1 mL and within 5 minutes, obtain limpid yellow solution.To appealing the glutaraldehyde standing 24h after ultrasonic 10 minutes that adds 100 μ l 5% in solution, prepared by shitosan, glutaraldehyde and potassium ferricyanide compound.(2) preparation of nanometer gold size adopts trisodium citrate thermal reduction gold chloride (HAuCl
4) method.All glasswares all soak with chloroazotic acid, and washes clean is standby.Configure the chlorauric acid solution of 100 mL 0.01%, be heated to boiling under vigorous stirring, then add rapidly 2.5 mL 1% citric acid three sodium solutions, continue to stir 20min (around here solution by faint yellow transfer to colourless, then become grey black, finally become claret), remove after thermal source continue to be stirred under room temperature slowly cooling.Cooled gold size solution is placed in to clean Brown Glass Brown glass bottles and jars only and keeps in Dark Place standbyly in 4 ° of C refrigerators, the several months is effective.(3) cleaning of glass-carbon electrode: before glass-carbon electrode is modified, at first immerse " piranha " solution (H of heat
2sO
4: 30% H
2o
2=3:1) soak 15min in, water cleans up, and next uses the Al of 0.3 μ m, 30nm
2o
3slurry is polished to minute surface on chamois leather, washes away except surface contaminants with deionized water after polishing, then moves in ultrasonic water bath and clean, and each 5min, repeat secondary, then uses successively the HNO of 6mol/L
3, absolute ethyl alcohol and deionized water ultrasonic cleaning, dry under nitrogen environment.(4) activation of glass-carbon electrode: after thoroughly cleaning, electrode is at 0.5mol/L H
2sO
4in solution, by cyclic voltammetry, activate, sweep limit 1.0V～-1.0V, scanning is until reach stable cyclic voltammogram repeatedly.(5) test of the glass-carbon electrode that pre-service is good: containing 1 * 10
-3mol/L K
3[Fe (CN)
6] 0.20 mol/L KNO
3run cyclic voltammetry curve in solution, to test the performance of described glass-carbon electrode, sweep velocity 50mV/S, sweep limit is-0.1V～0.6V; Spike potential in described cyclic voltammetry curve is poor below 80mV, and approaches as far as possible 64mV, and described glass-carbon electrode can be used, otherwise will return in step (3), processes described glass-carbon electrode, until meet the requirements.(6) compound of getting shitosan, glutaraldehyde and the potassium ferricyanide that 5 μ L prepare drips and is coated in the glass-carbon electrode surface that pre-service is good, and air drying, rinse electrode surface with the PBS damping fluid of PH=7.5.(7), after electrode surface dries, get 5 μ L nanometer gold sizes and drip and be coated in electrode surface, standing to dry under normal temperature, then with ultrapure water, rinse the nm of gold of surface physics absorption, nitrogen dries up.(8) then the aptamers of 5 μ L 5mM is dropped in above-mentionedly on nanometer-material-modified good electrode, cover the evaporation that prevents moisture with rubber cap, after 4h, with ultrapure water, rinse surface, dry stand-by.(9) the last above-mentioned electrode prepared is dipped in the mercaptoethanol sealing 1h with 1M, and with the sealing nonspecific binding site, the aptamers sensor production completes, and is kept under 4 ° of C conditions standby.(10) from the compound of shitosan, glutaraldehyde and the potassium ferricyanide, the amount of shitosan, aptamers concentration and incubation time three aspects: are optimized the experiment condition of prepared immunosensor, in the compound of shitosan, glutaraldehyde and the potassium ferricyanide, the amount of shitosan is 0.2-0.9 (example: 0.2 proportioning is 200 μ l 1% shitosans+800 μ l water), the aptamers concentration range is 2-6 μ M, and the scope of incubation time is 15-90min.(11) configure 1 * 10
-9m-1 * 10
-2the tetracycline standard solution of M, detect the above-mentioned aptamers sensor prepared respectively the tetracycline standard solution of variable concentrations, hatches at normal temperatures 60 min, and before and after detection reaction, curent change obtains its working curve.(12) by the aptamers sensor in the situation that oxytetracycline, kanamycins, gentamicin sulphate chaff interference exist 10
-5the tetracycline of M is tested, to detect its specificity; Select 5 aptamers sensors that prepare under the same terms to detect its reappearance; Within 7-21 days, detect successively the tetracycline of same concentrations to detect its stability continuously.(13) recovery of method detection tetracycline in milk sample of utilizing mark-on to reclaim is 92-106 %.
It is simple that this kind of current mode aptamers sensor detects the residual detection method operating procedure of tetracycline, detection time is shorter, detect the tetracycline concentration range wide, highly sensitive, good stability, power of regeneration is high and analysis has the recovery and reappearance preferably to actual sample, meets the residual Fast Detection Technique development of China's tetracycline and internationalization requirement.
Claims (2)
1. a preparation method who detects the residual current mode aptamers sensor of tetracycline, it is characterized in that: the compound that has prepared a kind of novel shitosan, glutaraldehyde and potassium ferricyanide, ground floor material as modified electrode not only can be used as the transmission of molecular wire acceleration electronics in electron surface, and on shitosan a large amount of amino can also modify that material nm of gold glue provides for the second layer can covalently bound site, the preparation process of the compound of shitosan, glutaraldehyde and the potassium ferricyanide is:
At first configure the acetum of 1% (1 ml/100 ml water) and prepare the chitosan solution of 50 ml 1% (1 g/100 ml) as solvent with the acetum prepared, the K of 5 mM of 500 μ l
3[Fe (CN)
6] mix ultrasonic concussion with the chitosan solution (400 μ l 1% shitosans+600 μ l water) of 1 mL and within 5 minutes, obtain limpid yellow solution, to appealing glutaraldehyde standing 24 h after ultrasonic 10 minutes that add 100 μ l 5% in solution, prepared by shitosan, glutaraldehyde and potassium ferricyanide compound.
2. a kind of preparation method who detects the residual current mode aptamers sensor of tetracycline as claimed in claim 1, it is characterized in that: the aminoterminal of shitosan connects the nanometer gold size by golden ammonia key, utilize shitosan, the common modified electrode of synergy of glutaraldehyde and potassium ferricyanide compound and nanometer gold size bi-material, strengthened significantly current-responsive, and the golden ammono-system formed has higher stability and high-sequential, for aptamers fixedly providing of electrode surface a good bioelectric interface, enlarged effective fixed-area of electrode, improved effective fixed amount of sensor surface aptamers, and the biologically active that has kept aptamers, and then accuracy of detection and sensitivity have been increased, the preparation process that detects the residual current mode aptamers biology sensor of tetracycline is:
The compound of getting shitosan, glutaraldehyde and the potassium ferricyanide that 5 μ L prepare drips and is coated in the glass-carbon electrode surface that pre-service is good, and air drying rinses electrode surface with the PBS damping fluid of PH=7.5; After electrode surface dries, get 5 μ L nanometer gold sizes and drip and be coated in electrode surface, standing to dry under normal temperature, then with ultrapure water, rinse the nm of gold of surface physics absorption, nitrogen dries up; Then the aptamers of 5 μ L 5 mM is dropped in above-mentionedly on nanometer-material-modified good electrode, cover the evaporation that prevents moisture with rubber cap, after 4 h, with ultrapure water, rinse surface, dry stand-by; The last above-mentioned electrode prepared is dipped in the mercaptoethanol of 1M and seals 1 h, and with the sealing nonspecific binding site, the aptamers sensor production completes, and is kept under 4 ° of C conditions standby.
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| CN104931544A (en) * | 2015-06-11 | 2015-09-23 | 山东理工大学 | Method of producing microarray aptamer sensor used for antibiotic residue detection |
| CN106990212A (en) * | 2017-04-01 | 2017-07-28 | 深圳万发创新进出口贸易有限公司 | It is a kind of to upload the Detecting Pesticide system of data in real time |
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