Label-free simultaneous detection of quinolone antibiotic residues using an origami paper–based electrochemical immunosensor

Label-free electrochemical sensors have gained popularity as point-of-care devices because they do not require complicated labeling procedures. However, they have difficulties detecting multiple analytes simultaneously. In this study, we developed a novel label-free electrochemical immunosensor on an origami paper–based analytical device (oPAD) for the simultaneous detection of quinolone antibiotics, in which norfloxacin (Nor) and enrofloxacin (Enr) were used as a model. The oPAD was designed with two antibody zones, each zone contains different antibodies. The oPAD has two thionine- and ferrocenecarboxylic acid-coated reagent zones, as redox species, which will generate the response current signals for the detection of quinolone antibiotics. The simultaneous current responses of thionine and ferrocenecarboxylic acid decreased proportionately when exposed to Nor and Enr concentrations, respectively. Under optimal conditions, this device provided linear range at concentrations of 0.01–10 µg/mL for the detection of Nor and Enr and limit of detection was calculated as 2.02 and 1.70 ng/mL, respectively. Moreover, this device demonstrates performance comparable to standard methods for the simultaneous detection of Nor and Enr in milk, honey, and fish. Therefore, label-free electrochemical immunosensors provide a highly potent, inexpensive, and portable method for detecting multiple quinolone antibiotics simultaneously on a single device.