Direct surface plasmon resonance immunosensor for in situ detection of benzoylecgonine, the major cocaine metabolite

In this paper the development of the first direct surface plasmon resonance (SPR) immunoassay for the detection of benzoylecgonine (BZE) is described. Immunosensor chips consisting of a high affinity monoclonal anti-BZE-antibody (anti-BZE–Ab) immobilized at high density to a sensor chip were prepared. First, BZE detection in Hepes buffer was achieved by direct, real time monitoring of the binding between BZE in solution and the surface bound antibody. The detection protocol was based on calibration curves obtained from reaction rate data and end point data analysis of sensorgrams registered after injection of a series of known BZE concentrations over the chips. Moreover, immunosensor accuracy, reproducibility, stability and robustness were tested to demonstrate their good performance as reusable devices. The immunosensor was used for BZE detection in oral fluid (OF) showing that, within 180 s, our immunoassay detects BZE concentrations as low as 4 μg/L in filtered OF-buffer (1:4) samples. This value is remarkably lower than current cut off levels established by the Substance Abuse and Mental Health Services Administration. These results manifest the potential use of this direct SPR immunoassay for the in situ sensitive detection of recent cocaine abuse, of utility in roadside drug OF testing. Moreover, it exemplifies the high potential of direct SPR immunoassays for the rapid, sensitive detection of small molecules in contrast with the more established indirect methods.