In situ antigen immobilization for stable organic-phase immunoelectrodes

A new method based on enzymatic single-step in situ synthesis of hapten-carrier conjugates on electrodes is described yielding stable, reproducible, and reusable organic-phase immunoelectrodes (OPIEs). The electrodes developed were tailored for analyte detection in organic solvents and allow for the analysis of soil extracts without further sample processing and cleanup. Catalyzed by transglutaminase from a variant of Streptoverticillium mobaraense, the reaction proceeds in aqueous solution with and without addition of organic media in only 1.5 hours. In this study, the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was chosen as model compound and chemically amino-functionalized prior to its enzymatic immobilization. The high reproducibility of the immobilization procedure allowed for batch calibration of the immunoelectrodes. Moreover, pure methanol or treatment with diluted sulfuric acid used for regeneration studies did not disturb the hapten layer. The OPIE consists of screen-printed carbon electrodes, monoclonal anti-2,4-D antibodies, and the immunochemical recognition reaction and was optimized with regard to a high stability in organic media. For electrochemical detection, horseradish peroxidase was used as enzyme label together with H2O2 as substrate and hexacyanoferrate (II)/(III) as mediator. The OPIE showed high stability upon storage over 93 days. Response times of 17 s (t95) were found to be advantageous compared to those of other biosensors. Including the immunochemical reactions, the complete assay takes 30 min. A calibration curve for 2,4-D in 30% methanol/buffer obtained with 70 electrodes within 4 weeks revealed a detection limit of 9 mg/L, a sensitivity of 1.3 nA L mg-1 cm-2, and a repeatability of 6.8%. Although we calculated a lowered repeatability for reused electrodes of 13.4% and a slightly decreased sensitivity of 0.9 nA L mg-1 cm-2, multiple-used OPIEs could also be applied for calibration.