CN103558272A - Rapid detector for antibiotic residue of aptamer sensor - Google Patents
Rapid detector for antibiotic residue of aptamer sensor Download PDFInfo
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- CN103558272A CN103558272A CN201310576568.3A CN201310576568A CN103558272A CN 103558272 A CN103558272 A CN 103558272A CN 201310576568 A CN201310576568 A CN 201310576568A CN 103558272 A CN103558272 A CN 103558272A
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Abstract
The invention relates to a rapid detector for antibiotic residue of an aptamer sensor. The rapid detector comprises an aptamer sensor, a signal detecting and processing system, a display and print storage system and a power supply system. A working electrode is modified by chitosan, glutaraldehyde and potassium ferricyanide compound, and then aptamer is fixed on the surface of the electrode by using nano colloidal gold to prepare the aptamer sensor. The concentration of antibiotics is determined by detecting the current change before and after the aptamer sensor is in contact with the antibiotics. An acquired weak current signal is subjected to I/V conversion, amplification, filtration and A/D (Analog/Digital) conversion by a signal processing circuit, so that a digital signal is sent to a microprocessor for performing program processing. The rapid detector is capable of displaying, storing and printing the concentration value of the antibiotic residue and whether the antibiotic residue in a detected sample exceeds the standard. The rapid detector for antibiotic residue of the aptamer sensor, provided by the invention, is capable of accurately and rapidly detecting information of whether certain antibiotics and the like are contained in the sample, and is very suitable for an occasion at which the antibiotic residue is rapidly detected at site.
Description
Technical field
The present invention relates to a kind of antibiotic residue fast detector, relate in particular to a kind of aptamers sensor antibiotic residue fast detector, belong to agricultural product security detection technique 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 causes 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, is difficult to prove effective again with antibiotic therapy of the same race.Antibiotic residue not only harm humans is healthy, also affects the quality of milk simultaneously, causes economic loss.Because the mastadenitis of cow incidence of disease is higher, with antibiosis, usually treat obstetrics' diseases such as mastitis still more general, this easily causes the residual of in milk tetracycline.Excessive use microbiotic 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 to antibiotic residue in the agricultural product such as 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 analyst is very high, is unsuitable for field quick detection.Compare with traditional analytical approach, biology sensor has following features: the selectivity that (1) is higher, therefore do not need tested component to carry out separation, and need not carry out pre-service to sample.(2) simple in structure, volume is little, easy to use, and portable immunosensor is particularly very beneficial for the Fast Measurement of agricultural product security quality.(3) can realize continuous on-line detection, make the quality control of food processing process become easy.(4) fast response time, amount of samples is few, compares with other large-sized analytic instruments, and immunosensor cost of manufacture is low, and can Reusability.
The object of invention is for the deficiencies in the prior art, and design provides a kind of aptamers sensor antibiotics leftover detection instrument, have easy to carry, detect quick and precisely, simple to operate, the features such as online qualitative, quantitative measurement that can realize antibiotic residue.
The ultimate principle of a kind of aptamers sensor of the present invention antibiotic residue fast detector is: three-electrode system will gather signal by testing circuit because immune response causes the variation of low current signal before and after aptamers sensor contact microbiotic.Testing circuit carries out I/V conversion by the low current signal collecting, amplifies, and filtering, A/D conversion the most at last digital quantity signal is sent into single-chip microcomputer and is carried out routine processes.Finally, show and print storage system by parameter displays such as the concentration of antibiotic residue, inhibition degree out, and data being stored so that user checks in time and carries out data analysis.Detector of the present invention includes mini-printer, data can be carried out to corresponding printout.The single-chip microcomputer of input and disposal system inside carries out programming Control to showing with the unification of printing storage system.
For realizing above function, the preparation method of the aptamers sensor of a kind of aptamers sensor antibiotic residue fast detector of the present invention is: the preparation of (1) shitosan, glutaraldehyde and potassium ferricyanide compound: first configure the acetum of 1% (1 ml/100 ml water) and as solvent, prepare the chitosan solution of 50 ml 1% (1 g/100 ml) with the acetum preparing.The K3[Fe (CN) 6 of 5 mM of 500 μ l] mix ultrasonic concussion with the chitosan solution (400 μ l 1% shitosan+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) method of preparing employing trisodium citrate thermal reduction gold chloride (HAuCl4) of nanometer gold size.All glasswares all soak with chloroazotic acid, and washes clean is standby.Configure the chlorauric acid solution of 100 mL 0.01%, under vigorous stirring, be heated to boiling, 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 continue to be stirred under room temperature after thermal source slowly cooling.Cooled gold size solution is placed in to clean Brown Glass Brown glass bottles and jars only and in 4 ° of C refrigerators, keeps in Dark Place standbyly, the several months is effective.(3) 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 silver orthophosphate damping fluid of pH7.5.(4), 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, with rubber cap, cover the evaporation that prevents moisture, after 4h, with ultrapure water, rinse surface, dry stand-by.(9) the last above-mentioned electrode preparing is dipped in the mercaptoethanol sealing 1h with 1M, and to seal nonspecific binding site, aptamers sensor production completes, and is kept under 4 ° of C conditions standby.
Described a kind of aptamers sensor antibiotic residue fast detector, is characterized in that: three-electrode system circuit is by three electrode aptamers sensors, three electrode detection circuit, and potentiostatic circuit forms; Three electrode immunosensors gather current signal and are transmitted to input and disposal system circuit by three electrode detection circuit.Potentiostatic circuit provides the burning voltage of 500mV to reaction solution environment.
Input and disposal system circuit have I/V conversion, amplify, and filter function, includes A/D conversion chip and convert the analog signals through enzyme sensor collection after I/V conversion to digital quantity signal and send into single-chip microcomputer and carry out routine processes.The singlechip microcontroller of input and disposal system is STC89C52.The trace routine of single-chip microcomputer comprises data-printing and storage, time showing, and operation instruction and the dialog box to important step prompting, help user to grasp fast the using method of instrument and carry out data-printing and convenient storage carries out inspection and the processing of data later.Input and disposal system circuit adopt TLC272CP chip multichannel discharge circuit to have I/V conversion, amplify, and filtering also provides the function of 500mv constant voltage to three electrode reaction environment.
Show and print storage system circuit by display circuit, print circuit and memory circuitry form.Display controls for showing data the interface of controlling as user by operation push-button.Printing storage system circuit is connected and carries out data-printing and storage with mini-printer and storer respectively.Display screen has adopted the general display screen of 3.5 cun, and mini-printer adopts micro embedded printer, and its size and power consumption are satisfied to be convenient for carrying and portable power consumption requirement.
For reaching above object, take following technical scheme to realize: electric power system electric routing switch power supply and voltage conversion circuit form, Switching Power Supply converts alternating current to direct current and to each circuit system and mini-printer, powers respectively through voltage conversion circuit.Switching Power Supply is+12V, 1A ,-12V, 1A ,+5V ，5A tri-tunnels outputs.Power supply is connected with the Switching Power Supply in electric power system.
accompanying drawing explanation
Fig. 1 is the structured flowchart of a kind of aptamers sensor of the present invention antibiotic residue fast detector,
Fig. 2 is the present invention's three electrodes and potentiostatic circuit and A/D conversion treatment circuit in early stage.
Embodiment
First, structural representation block diagram of the present invention is illustrated; Secondly, input and included potentiostatic circuit, the A/D conversion treatment circuit in early stage of disposal system are described in detail; Finally, the application software of whole system and testing process are illustrated.
Fig. 1 is the structured flowchart of a kind of aptamers sensor of the present invention antibiotic residue fast detector.As shown in Figure 1, the present invention is by three-electrode system, and input and disposal system, show and print storage system, electric power system forms.Three-electrode system gathers the low current signal in reaction solution by immunosensor; The low current signal that input and disposal system collect three electrodes carries out I/V conversion, amplifies, and filtering, A/D conversion the most at last digital quantity signal is sent into single-chip microcomputer and is carried out routine processes; Show and print storage system by parameter displays such as residual antibiotic concentration, inhibition degree out, and data being stored so that user checks in time and carries out data analysis.Detector of the present invention includes mini-printer, data can be carried out to corresponding printout.The single-chip microcomputer of input and disposal system inside carries out programming Control to showing with the unification of printing storage system.Electric power system converts alternating current to three road direct currents for each circuit system and mini-printer power supply.Described microcontroller adopts the STC89C52RC+ microcontroller that kernel is MCS-51, loads trace routine on it.
In the present invention, the potentiostatic circuit in testing circuit provides stable operating voltage, the embodiment that Fig. 2 is potentiostat circuit for three-electrode system current/voltage transforms testing circuit, by a two-way operational amplifier U10 TLC272CP and R44, R46, R4, C35, C16, R48 forms.The pin 6 of amplifier B is connected with 7 and is connected with 3 pin of A, 2 pin of A are connected and have connected C16 with the working electrode of three-electrode system, strengthen antijamming capability, R48 be as feedback resistance on amplifier A, the external potentiometer R46 of 5 pins of B, by positive 5V, supply electric excitation, R44, R46, R4 supplies with 500mv voltage to forming potentiostatic circuit with 5 pins of B by voltage dividing potential drop, initial voltage be 500mv. amplifier A and connect+5V of pin 8 power supply, connect-5V of pin 4, the amplifier B pin 7 of U9 TLC272CP is directly connected as voltage follower with pin 6, ADE pin 3 is connected with the pin 5 of B, common ground, play jamproof effect.The pin 2 of A is directly connected with the contrast electrode of three-electrode system, pin 1 is directly connected with the auxiliary electrode of three-electrode system, between connect electric capacity 14, object has been the effect of buffer circuit, can improve load-carrying ability simultaneously, therefore U9 and U10 have formed the three-electrode system of biology sensor jointly, and test side used, by drawing here, gathers and the low current signal that detects electrochemical reaction generation.
The aptamers transducer production method of a kind of aptamers sensor antibiotic residue fast detector of the present invention is: the preparation of (1) shitosan, glutaraldehyde and potassium ferricyanide compound: first configure the acetum of 1% (1 ml/100 ml water) and as solvent, prepare the chitosan solution of 50 ml 1% (1 g/100 ml) with the acetum preparing.The K3[Fe (CN) 6 of 5 mM of 500 μ l] mix ultrasonic concussion with the chitosan solution (400 μ l 1% shitosan+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) method of preparing employing trisodium citrate thermal reduction gold chloride (HAuCl4) of nanometer gold size.All glasswares all soak with chloroazotic acid, and washes clean is standby.Configure the chlorauric acid solution of 100 mL 0.01%, under vigorous stirring, be heated to boiling, 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 continue to be stirred under room temperature after thermal source slowly cooling.Cooled gold size solution is placed in to clean Brown Glass Brown glass bottles and jars only and in 4 ° of C refrigerators, keeps in Dark Place standbyly, the several months is effective.(3) 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 silver orthophosphate damping fluid of pH7.5.(4), 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, with rubber cap, cover the evaporation that prevents moisture, after 4h, with ultrapure water, rinse surface, dry stand-by.(9) the last above-mentioned electrode preparing is dipped in the mercaptoethanol sealing 1h with 1M, and to seal nonspecific binding site, aptamers sensor production completes, and is kept under 4 ° of C conditions standby.
Testing process of the present invention is: first, the aptamers sensor preparing is put into not containing liquid at the bottom of antibiotic test to the electric current I while recording not containing microbiotic
1, electric current before suppressing (default value that before suppressing, electric current is set in also can employing system); Afterwards aptamers sensor is removed, with after distilled water flushing, aptamers working sensor electrode is put into detected sample liquid and hatch 10 minutes, again aptamers sensor is put into test end liquid, record the electric current I after inhibition
2; Finally by the trace routine of moving on microcontroller, automatically calculate inhibiting rate (I%), computing formula is as follows:
I%
=( I
1- I
2)/ I
1 ×100%
If conventionally detect in sample liquid and contain microbiotic, because producing immune complex, aptamers and microbiotic generation immune response hindered the electronics transfer between test fluid and electrode, electric current I
2be less than I
1.I
1with I
2difference more inhibiting rate is larger, the concentration that shows to detect antibiotic residue in sample liquid is higher; I
1with I
2the less inhibiting rate of difference less, the concentration that shows to detect antibiotic residue in sample liquid is lower.
Think at present when inhibiting rate be greater than, while equaling 50%, antibiotic residue exceeds standard; When inhibiting rate is less than 50%, antibiotic residue does not exceed standard.
Microcontroller is made qualitative conclusion according to the inhibiting rate obtaining, and result is delivered to display screen and show, and by end product printout, data is stored later the searching and studying for related personnel simultaneously in the lump.
The trace routine of moving on microcontroller of the present invention also comprises screen redirect demonstration and dialog box, print prompt to important step prompting, can help user to grasp fast the using method of instrument.
The embodiment of trace routine: first, immunosensor is put into substrate solution and detect preparation.Then, press instrument switch key, enter homepage after system initialization, display screen side has test button, reset and three of printings to broadcast mill button.Click for the first time test button, start to detect and show reading I
1; After clicking detection for the second time, observe prompting, show result I
2; Click and print, print result is also preserved.Opening power key, screen display " START detection ", zero clearing.Crocodile clip is stepped up respectively to three columnar electrodes that three-electrode system is corresponding, Pen-down detection key in the phosphate buffered solution of preparing before the immunosensor of sessile antibody is immersed in, screen display starts to detect, after about 1min, baseline reaches maintenance level, after about 30s, obtain first complete current data, be designated as I
1.Testing sample test: immunosensor is put into the sample solution having extracted, about 10min, is immersed in quantification of phosphate buffer solution afterwards again, presses measurement key, clicks detection key and measures for the second time, shows second current data I simultaneously
2.By two current data, automatically calculate the maximum inhibition of this sample liquid to immobilised enzymes.Select suitable relation curve to calculate the target antibiotic concentration of extract, and be converted to the antibiotic content of target in sample, the report of the last measurement result of generation automatically.Press printing key, the data that detect voltage, microbiotic inhibiting rate and microbiotic detectable concentration before and after being printed on for twice are printed to output.
Of the present invention a kind of be aptamers sensor antibiotic residue fast detector, operating procedure is simple, detection time is shorter, can realize the qualitative, quantitative of antibiotic residue measures, have highly sensitive, good stability, high repeatability and other advantages, meet China's antibiotic residue Fast Detection Technique development and internationalization requirement.
Claims (5)
1. an aptamers sensor antibiotic residue fast detector, is characterized in that: this detector is by three electrode aptamers sensors, input and treatment circuit, show and print memory circuit, constant potential feed circuit, operation push-button, data-carrier store, mini-printer, electric power generating composition; The current signal that immunosensor produces is transmitted to input and disposal system circuit by testing circuit; Potentiostatic circuit is to the stable operating voltage that 500mV is provided between working electrode and contrast electrode; Described input and treatment circuit have I/V conversion, amplify, and filter function, includes A/D conversion chip and convert the analog signals of aptamers sensor collection to digital quantity signal and send into single-chip microcomputer and carry out routine processes; Described immunosensor is used after nanometer-material-modified working electrode, and specific microbiotic aptamers is fixed to electrode surface, can in the measurement sample liquid of specificity, whether contain certain microbiotic.
2. a kind of aptamers sensor antibiotic residue fast detector as claimed in claim 1, is characterized in that described input and the singlechip microcontroller of disposal system are STC89C52.
3. a kind of aptamers sensor antibiotic residue fast detector as claimed in claim 1, what it is characterized in that this detector use is immunosensor, at aptamers working sensor electrode surface, fixed specific microbiotic aptamers, therefore this detector can realize in the working sample of specificity whether containing certain microbiotic, realizes the antibiotic residue qualitative, quantitative of sample and measures.
4. a kind of aptamers sensor antibiotic residue fast detector as claimed in claim 1, it is characterized in that described input and disposal system circuit adopt TLC272CP chip multichannel discharge circuit to have I/V conversion, amplify, filtering also provides the function of 500mv constant voltage to three electrode reaction environment.
5. a kind of aptamers sensor antibiotic residue fast detector as claimed in claim 1, it is characterized in that the working electrode of modifying with shitosan, glutaraldehyde and potassium ferricyanide compound, with nanometer gold size, aptamers is fixed to electrode surface again, has prepared aptamers sensor.
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| CN106483175A (en) * | 2016-12-22 | 2017-03-08 | 山东理工大学 | A kind of palm rapid detector for antibiotic residue based on Internet of Things |
| CN106841346A (en) * | 2016-12-22 | 2017-06-13 | 山东理工大学 | A kind of palm aflatoxin fast detector based on Internet of Things |
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| CN106872539A (en) * | 2017-03-29 | 2017-06-20 | 深圳市赛亿科技开发有限公司 | The intelligent chopsticks of clenbuterol content in a kind of detection meat |
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| CN104535729A (en) * | 2014-12-23 | 2015-04-22 | 温岭市太平高级职业中学 | Portable antibiotic detection pocket |
| 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 |
| CN106841335A (en) * | 2016-09-21 | 2017-06-13 | 青岛农业大学 | A kind of preparation method of antibiotic self energizing aptamer sensor |
| CN106841335B (en) * | 2016-09-21 | 2019-04-30 | 青岛农业大学 | A kind of preparation method of antibiotic self energizing aptamer sensor |
| CN106483175A (en) * | 2016-12-22 | 2017-03-08 | 山东理工大学 | A kind of palm rapid detector for antibiotic residue based on Internet of Things |
| CN106841346A (en) * | 2016-12-22 | 2017-06-13 | 山东理工大学 | A kind of palm aflatoxin fast detector based on Internet of Things |
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| CN106872539A (en) * | 2017-03-29 | 2017-06-20 | 深圳市赛亿科技开发有限公司 | The intelligent chopsticks of clenbuterol content in a kind of detection meat |
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