Exploring the scope of an electrochemical immunosensor for the direct detection of small molecules in complex matrices

A user intervention free electrochemical immunosensor is developed that allows a direct detection of small molecules (i.e., veterinary drug residue, namely enrofloxacin) in complex matrices, with no washing or rinsing steps via a displacement assay. This is achieved using an interface comprised of a mixed layer of oligo(phenylethynylene) molecular wire (MW), to allow electrochemical communication and oligo(ethylene glycol) (OEG) to control the interaction of proteins and electroactive species with the electrode surface. The mixed layer is formed from in situ-generated aryl diazonium cations. To the distal end of the MW, a redox probe 1,1’-di(aminomethyl)ferrocene is attached followed by an epitope (the structural feature the antibody selectively recognises) to which an antibody would specifically bind (current suppression). When placed in a sample containing the small target analyte, transduction is achieved by antibody leaving the electrode surface due to the competition for the antibody between the surface bound epitope and the small analyte (current increase). This type of assay is referred to as a displacement assay.In a displacement assay, the ratio of the surface components (MW/OEG) and the use of electric field both are shown to have strong impact on sensor sensitivity enhancement. The former controls the antibody-epitope specific binding whilst the latter perturbs the thermodynamic equilibrium and hence enhances the effectiveness of antibody-epitope dissociation. In addition, the apparent affinity of the antibody to the surface bound epitope is shown to be another important key factor to have direct influence on sensor sensitivity. The magnitude of current being suppressed (after the antibody-epitope complexation) has strong impact on the magnitude of the possible current increase (analytical signal) in a displacement assay. A compromise between these two variables is shown crucial to achieve optimum sensor performance. To control the antibody-epitope affinity, the strategy used was by electrode surface modification. Six different analogues of enrofloxacin which have different affinities to antibody were employed as the surface bound epitopes. Interestingly with this strategy, not only the sensor sensitivity can be enhanced, also the sensor specificity can be in control. Besides experiments, modelling of a displacement assay was performed based on a simple equilibrium theoretical model to determine the optimum conditions. The experimental results were shown similar trends and were consistent to the simulated plots for the calibration curves obtained for six different epitopes. The trend from small to large according to binding strength was ofloxacin 88 %) with little loss of analyte attributed to binding with humic substances