An Electrochemical Immunosensor Based On Nitrocellulose Membrane For Rapid Determination Of Chloramphenicol

Chloramphenicol(CAP) is the first artificial broad-spectrum antibiotic, which is active against both Gram-positive and Gram-negative bacteria. Because of its low price, it was widely used in husbandry and aquaculture industry. But it have been found that chloramphenicol have toxic and side effects to human body. The chloramphenicol residues can inhibit human hematopoietic function and lead to renewable obstacles anemia. Chromatographic, microbiological, enzymatic, and immunological methods have been reported for the detection of chloramphenicol residues in food-producing animals. However, most of detection methods require long response time or high cost, which are limited suitable for practical applications. Therefore, a sensitive, rapid and accurate method for determination of CAP is necessary.Electrochemical immunosensors, combined the high specificity of traditional immunoassay methods with the low detection limits and low costs of electrochemical measurement system, have gained more and more attention by researchers. The method has been used by investigators for determination of various analytes with great sensitivity and specificity. Several electrochemical immunosensors have been successfully developed for CAP detection with different types of electrodes. A key issue for electrochemical immunosensor is the immobilization of immunological sensitivity compounds on the electrodes. The immunological sensitivity compounds immobilized in films on electrode surfaces should exhibit their specific immunoactivity, retain their native structure and demonstrate good stability. We reported the employment of nitrocellulose membrane, a high mechanical strength, ideal membrane for blotting of proteins with good biocompatibility, as a novel immobilization matrix for development of electrochemical immunosensors.In the present study, a novel, label-free electrochemical immunosensor based on a competitive mechanism was constructed for the determination of chloramphenicol. The complete antigens were immobilized on NC membrane modified on a gold electrode by electrophoresis. Cyclic voltammetry and differential pulse voltammetry were employed to observe the change of the electrode response of the redox marker K3[Fe(CN)6] during different modified stages. Due to the existence of antibody, it inhibited electron transfer of K3[Fe(CN)6]. Antibdies in incubation liquid combine with the immobilized antigen.The method based on the competitive immuno-interaction was used to detect the free chloramphenicol in the PBS. The selected monoclonal antibody showed very high sensitivity and specificity for chloramphenicol. This immunosensor had good specificity. The detective range of this electrochemical immunosensor was20～1000ng/mL and the detection limit was4ng/mL.There are two creative points in this study. One is electrochemical method combined with the electrophoresis to immobilize immunological sensitivity compounds. The other one is nitrocellulose membrane was used for a novel immobilization matrix.