Continuous-flow fluoro-immunosensor for paclitaxel measurement

A fluorescence-based continuous-flow immunosensor for sensitive, precise, a ccurate and fast determination of paclitaxel was developed. The sensor util izes anti-paclitaxel antibody immobilized through its Fe region and crossli nked by dimethylpimelimidate to protein A attached covalently onto the sila nized inner walls of a glass capillary column followed by saturation of the paclitaxel-binding sites with rhodamine-labeled paclitaxel. The assay is b ased on the displacement and detection downstream of the rhodamine-labeled paclitaxel, by a flow-through spectrofluorometer, as a result of the compet ition with paclitaxel introduced as a pulse into the stream of carrier buff er flowing through the system. The peak height of the fluorescence intensit y profile of the displaced rhodamine-labeled paclitaxel was directly propor tional to the concentration of paclitaxel applied and was a function of the carrier buffer flow rate. The sensitivity of the immunosensor response ran ged from 0.31 relative fluorescence units (RFU)/ng/ml at a flow rate 0.1 ml /min to 0.52 RFU/ng/ml at 1 ml/min, while the lower detection limit ranged from 1 ng/ ml at 0.1 ml/min to 4 ng/ml at 1 ml/min. The immunosensor respon se was very reproducible (RSD = 4.8%; n = 10) and linear up to 100 ng;/ml. The assay time ranged from 2 min at 1 ml/min to 8 min at 0.1 ml/min. A tech nique developed to resaturate the antigen binding sites of the immobilized antibody with rhodamine-labeled paclitaxel was successful in regenerating t he capillary column without affecting its performance, thus enhancing the e conomic viability of the immunosensor. The immunosensor was successfully ap plied for the determination of paclitaxel in human plasma. (C) 2001 Elsevie r Science B.V. All rights reserved.