Study On Rapid Determination Technology For Antibiotic Residue In Milk Based On Aptasensor

Antibiotic residues in milk not only cause great harm to people’s health but also bring heavy economic losses to dairy processing enterprises. So, it is necessary to control antibiotic residues in milk. Besides controlling the use of antibiotics from the source, the timely and accurate detection of antibiotic residues in animal derived food is an effective way to protect the safety of livestock products.The study is intending to conduct direct and rapid detection of antibiotic residues in the milk during production and sales process so that the consumers’ health and the economic interests of the dairy processing enterprises can be guaranteed. By studying the modification and immobilization methods of aptamers on the ordinary electrodes, microarray electrode and screenprinted electrodes, the fabrication method of aptasensors for rapid detection on spot was defined. By studying the weak current signal acquisition and processing technology, weak current detection circuit and the aptasensors were integrated, and the rapid detection system for antibiotic residues rapid detection on site was developed which can conduct rapid detection of antibiotic residues in milk on spot. The concrete research content is as follows:(1)Study on the aptasensor fabrication method based on the ordinary electrode.In this paper, the aptasensors modified by nanogold/methylene blue(Au NPs/MB for short) and multi-walled carbon tubes-chitosan/nano magnetite-chitosan(MWCNTs-CS for short) were developed on the surface of the ordinary electrodes respectively. The parameters affecting performance, such as aptamer concentration, p H of the test solution, incubation time of reaction between aptamers and their antibiotics, were optimized. Meanwhile the optimal determination parameters of aptasensor are defined. The nanogold/methylene blue composite modified aptasensor and multi-walled carbon tubes- chitosan/ nano magnetite-chitosan modified aptasensor were used for detecting actual samples of spiked milk. Their recoveries rates were 94%~108% and 86%~104% respectively. Under the optimal conditions, the detection limits were 0.42×10-11 M and 1×10-9M respectively. Compared with the above-mentioned aptasensors, we can find that detection limit of aptasensor modified with MWCNTs-CS is higher than that of aptasensor modified with Au NPs/MB, but incubation time of aptasensor modified with MWCNTs-CS is smaller than that of aptasensor modified with AuNPs/MB. The incubation time of aptasensor modified with MWCNTs-CS is only 14 minutes and its recovery rate satisfied the detection requirements. So the aptasensor is moresuitable for onsiet detection.(2)Study on the aptasensor fabrication method based on the microarray electrode.Aptasensors modified by chitosan-multi-walled carbon nanotubes and ionic liquid-nano magnetite based on the microarray electrodes were developed respectively. The effects of microarray aptasensors based on the two above-mentioned modification methods were analyzed. Under optimal conditions, different concentration of tetracyclines was detected with the two above-mentioned aptasensors. The recovery rates of the two aptasensors are 95%~105% too, and linear ranges are from 10-9 to 10-5M. Their detection limits are 1×10-9M that are obviously lower than the standard value stipulated by China national standard.Compared with that of the multi-walled carbon nanotubes-chitosan modified electrode aptasensor, impedance changes of the ionic liquid-nano magnetite modified aptasensor before and after exposure to antibiotics are obviously. Although the linear range of the latter aptasensor is narrower than that of the former one. Under the practical application condition, both the linear ranges can satisfy the requirement of actual test. Under the condition of detecting the same concentration of tetracycline, the ionic liquid–nano magnetite modified microarray aptasensor can obtain great impedance changes than the chitosan-multi-walled carbon nanotubes modified aptasensor. This means that the interference of background noise can be reduced under an actual testing environment. From the view of practical application, the ionic liquid-nano magnetite modified microarray aptasensor preparation method is much more practical.(3)Study on the aptasensor fabrication method based on screen-printed electrodesBecause of the simple manufacture process and low cost and single use, screen-printed electrodes(SPEs for short) are suitable for rapid detection of antibiotic residues on spot. Based on preparation methods of the aptasensors fabricated on ordinary electrodes and microarray electrodes, ionic liquid and nano magnetite were coated on SPEs to further study fabrication method of aptasensors based on screen-printed electrodes. The two developed aptasensor were used to detect tetracycline residue in milk.Their linear ranges fall in the range of [10-9,10-2]M and their detection limits are 1×10-9M that are obviously lower than the standard value stipulated by China national standard.Comparsion conducted between the two aptasensors, the apatasensor modified with ionic liquid and nano magnetite is easily made. They have no obvious difference in linear range and detection limit.Taken pratical application into accout, the aptasensor modified with ionic liquid and nano magnetite was chosen to make practical modification element.An aptasensor modified by chitosan-multi-walled carbon nanotubes/chitosan and chitosannano magnetite based on SPEs was developed. Meanwhile an aptasensor modified by ionic liquid and nano magnetite based on screen-printed electrodes was fabricated too. And both aptasensors were adopted in rapid detection of antibiotic residues in actual milk samples.(4)Study on detection technology of weak current signalIn order to achieve good portability of antibiotic residues detection, a weak current signal detection circuit, including I/V conversion circuit, a differential amplifier circuit, filter circuit, A/D conversion circuit, was designed. So weak current signal can be displayed real-timely and curve can be plotted timely. The detection circuit can replace an electrochemical analysis instrument which lay a solid foundation for the process of intelligence and portability for the detection instrument of antibiotic residue.(5)Development of rapid detection of antibiotic residues based on SPEsA rapid detection system of antibiotic residues was developed. A mathematical model between tetracycline concentration and aptasenor sensor current change was established which provide the basis for an accurate calculation of the quantitative detection of tetracycline residues in milk. The developed rapid detection system of antibiotic residues in milk of samples is adopted to detect the actual tetracycline residues. The limit of detection, accuracy, precision and other parameters were defined. The value of limit of detection for the instrumentation is lower than that of the minimum detection limit in developed countries and China which satisfies the detection for conventional antibiotic residues.Due to its excellent performance, single-use and low cost, the aptasensor based on screenprinted electrodes is very suitable for on-site rapid detection of antibiotic residues.