Study On Fabrication And Application Of Milk Detection For Two Antibiotic Electrochemical Aptasensors

Antibiotics have been widely used as veterinary drugs in cattle breeding.There often contains a certain amount of residue in milk because of metabolism.Longterm consumption of milk with excessive levels of antibiotics can have toxic side effects on the human body,causing allergies or drug resistance for light cases and leading to shock or even death for severe cases.In recent years,with the emergence of new kinds of drugs,the antibiotic residue issue is becoming more and more complex,which brings new problems and challenges to food safety constantly.It is urgent and necessary to develop new rapid on-site analytical technology to meet the increasing detection requirements.As one of the important development directions of drug detection in the future,electrochemical aptasensor technology has been developed rapidly.It has the general features of electrochemical devices and unique advantages of no sample pretreatment procedure and good anti-fouling detection ability,which can effectively overcome the disadvantages of traditional methods,for example,chromatography,with high operational requirements,complicated steps and unportableness,showing the feasibility of being developed into the rapid on-site analysis technology.In this paper,using two antibiotics as target molecules,two different sensor detection methods were established with good performance helpful for on-site analysis.1.Using a newly reported aptamer as the recognition probe,a folding-type penicillin electrochemical aptasensor based-on electrode-bound interface reaction was fabricated.The molecular simulation technology was used to reveal the molecular mechanism of aptamer/target complex.Electrochemical measurement methods were applied to characterize the sensing process and optimize its influencing parameters,and the comprehensive analytical performance was also investigated systematically.The research results show that hydrogen bonding is the main force that maintains the stability of the aptamer/target complex,and the sensing system follows the signal transduction mechanism of reaction-induced the conformation change of aptamer recognition probe.Under the optimal conditions,when applied for the detection of practical milk samples,the proposed method showed good analytical performances with the linear range of 5.0 n M-5.0 μM,the detection limit of 1.7 n M,and the signal response equilibrium time of ～270 s.When the method was validated as a quantitative analysis method,the mean standard recoveries were 80.2-92.3% with the relative standard deviation(RSD)<10.0%.All detection process can be finished within 30 minutes.The method performs excellently in analyzing speed,sensitivity,anti-fouling ability and operability,which is beneficial for being developed into the on-site detection method.2.Aiming at the disadvantages of large steric resistance and low reaction efficiency for the traditional electrode-bound interface reactions,a solution-type displacement reaction-based kanamycin electrochemical aptasensor was fabricated.The molecular simulation technology was used to reveal the displacement reaction mechanism of double-aptamer/target complex.Electrochemical measurement methods were applied to characterize the sensing process and optimize its influencing parameters,and the comprehensive analytical performance was also investigated systematically.The research results show that the hydrogen bonding and the electrostatic interaction are main forces between the aptamer duplex and the target.Their perturbation can induce the breaking of aptamer duplex.Correspondingly,the sensing system follows the signal transduction mechanism of signal probe shifting drived by solution-type displacement reaction.Under the same conditions,the absolute detection current increased by 1.6 times compared with electrode-bound interface reaction.When applied for the detection of practical milk samples,the proposed method showed good analytical performances with the linear range of 100.0 n M-0.5 m M,the detection limit of 26.3 n M,and the signal response equilibrium time of ～90 min.When the method was validated as a quantitative analysis method,the mean standard recoveries were 79.3-98.4% with the relative standard deviation(RSD)<10.0%.All detection process can be finished within 120 minutes.The method performs excellently in sensitivity and operability,showing a potential for being developed into the on-site detection method.