Study On Electrochemical Immunosensor For The Detection Of Norfloxacin In Animal-derived Foods

Norfloxacin as a kind of quinolone antimicrobial agents has been widely used in the treatment of enteritis and dysentery.And its excessive intake can cause dizziness,headache,drowsiness or insomnia,and can also affect the growth and development of adolescent.There are many methods for the detection of norfloxacin such as high performance liquid chromatography,liquid chromatography-mass spectrometry,enzyme-linked immunosorbent assay,and microbiological method.However,these methods have obvious demerits such as time-consuming and laborious,complicated sample preparation,and expensive experimental equipment.Therefore,a rapid,simple,and sensitive method for detecting norfloxacin is highly desired.In this paper,dendrimer-encapsulated gold nanoparticles composites(PAMAM-Au)were prepared by formic acid reduction using dendrimer and chloroauric acid as substri.By transmission electron microscopy(TEM),it was found PAMAM-Au were uniform with the average size 5 nm diameter.The surface of PAMAM-Au are rich in carboxyl groups and it contains numerous of gold nanoparticles inside which can not only provide anchors to connect more substances but also can enhance the electrochemical signal and improve the sensitivity of the detection.In the experiment,p-aminobenzoic acid was firstly electropolymerized on the electrode to modify carboxyl group on the electrode surface,and PAMAM-Au was firmly connected to the electrode through an amide reaction between the carboxyl group and the amino group on PAMAM.And then substances like antigens or antibodies were dropped on the electrode to construct two methods for the detection of norfloxacin in animal-derived foods.By immobilizing the norfloxacin antibody on PAMAM-Au modified electrode and adding an equal volume of horseradish peroxidase-labeled antigen(HRP-Ag)and the target antigen(Ag)mixture,a competitive-type electrochemical immunosensor was constructed.The quantitative detection was realized by the competitive effect of HRP-Ag and Ag.The experimental conditions were optimized and under the optimal condition,the linear range of the constructed sensor was 1 ?g L-1-10 mg L-1 with a detection limit of 0.38 ?g L-1.In the recovery study of the spiked samples,the range of 91.6 to 106.1%were obtained which demonstrated the feasibility of proposed immunosensor.The norfloxacin coating antigen was immobilized on the electrode modified with PAMAM-Au,and then a mixed solution containing equal volume of antibody and target antigen(Ag)was added on the electrode surface.An inhibition electrochemical impedance immunosensor was constructed.The electrical signal was inhibited by the reaction of antigen to antibody,so that the inhibition rate can be calculated based on the change of the impedance value to realize the quantitative detection of norfloxacin.The experimental conditions were optimized and under the optimal condition,the proposed immunosensor shows a wide linear range from 0.5 ?g L-,-5 mg L-1,low limit of detection(IC15:0.12?g L-1)and high sensitivity(ICso:0.30 ?g L-1).The proposed method displayed good stability,selectivity and reproducibility.And the acceptable recovery(90.9%-104.8%)in practical sample analysis indicate this strategy has the great potential in practical application of norfloxacin monitoring.