Detection of Fluoroquinolone Antibiotics in Milk via a Labeless Immunoassay Based upon an Alternating Current Impedance Protocol

Abstract

This paper describes the construction of a labeless immunosensor for the antibiotic ciprofloxacin in milk and its interrogation using an ac impedance protocol. Commercial screen-printed carbon electrodes were used as the basis for the sensor. Polyaniline was electrodeposited onto the sensors and then utilized to immobilize a biotinylated antibody for ciprofloxacin using classical avidin-biotin interactions. Antibody loaded electrodes were exposed to solutions of antigen in milk and interrogated using an ac impedance protocol. The faradaic component of the impedance of the electrodes was found to increase with increasing concentration of antigen. Control samples containing a nonspecific IgG antibody were also studied but were found to display large nonspecific responses, probably due to the antibody binding some of the large number of components found in milk. Control sensors could, however, be fabricated using antibodies specific for species not found in milk. Calibration curves could be obtained by subtraction of the responses for specific and control antibody-based sensors, thereby eliminating the effects of nonspecific adsorption of antigen. Sensors exposed to ciprofloxacin in milk gave increases in impedance whereas ciprofloxacin in phosphate buffer led to decreases, indicating the possibility of developing sensors which can both detect and differentiate between free and chelated antigen.