Development of an electrochemical immunosensor for direct detection of interferon-gamma at the attomolar level.

An electrochemical immunosensor for direct detection of the 15.5-kDa protein interferon-gamma (IFN-gamma) at attomolar level has been developed. Self-assembled monolayers (SAMs) of cysteine or acetylcysteine are formed on electropolished polycrystalline Au electrodes. IFN-gamma adsorbs physically to each of these SAMs. With injections of 100 mM KCl, IFN-gamma can be removed in the flow without damaging the acetylcysteine SAM. However, the cysteine SAM is affected by these KCl injections. In an on-line procedure in the flow, a specific antibody (MD-2) against IFN-gamma is covalently attached following carbodiimide/succinimide activation of the SAM. The activation of the carboxylic groups, attachment of MD-2, and deactivation of the remaining succinimide groups with ethanolamine are monitored impedimetrically at a frequency of 113 Hz, a potential of +0.2 V versus SCE, and an ac modulation amplitude of 10 mV. Plots of the real (Z') and imaginary (Z") component of the impedance versus time provide the information to control these processes. In the thermostated setup (23.0 degrees C), samples of unlabeled IFN-gamma (in phosphate buffer pH 7.4) are injected and the binding with immobilized MD-2 is monitored with ac impedance or potential-step methods. While the chronoamperometric results are rather poor, the ac impedance approach provides unsurpassed detection limits, as low as 0.02 fg mL-1 (approximately 1 aM) IFN-gamma. From a calibration curve (i.e. Z" versus the amount injected), recorded by multiple 50-microL injections of 2 pg mL(-1) of IFN-gamma, a dynamic range of 0-12 pg mL(-1) could be derived. However, when nonspecific adsorption is taken into account, which has been found to be largely reduced through injections of 100 mM KCl, a much smaller dynamic range of 0-0.14 fg mL(-1) remains. The immunosensor can be regenerated by using a sequence of potential pulses in the flow by which the SAM with attached MD-2 and bound IFN-gamma is completely removed. When the developed procedures described above are repeated, the response of the immunosensor is reproducible within 10%.

Electrochemical pretreatment of polycrystalline gold electrodes to produce a reproducible surface roughness for self-assembly: a study in phosphate buffer pH 7.4

It has been emphasized in several studies that the state of the surface, including the surface roughness, is very important for the reproducible formation of high-quality self-assembled monolayers on gold. The pulsed-potential pretreatment procedure described in this paper can, in a reproducible way, reduce the surface roughness of mechanically polished polycrystalline gold electrodes by a factor 2. The developed procedure, in which the gold is alternately oxidized and reduced, has been optimized for use in a flow system (100 mM phosphate buffer pH 7.4). The influence of the pretreatment procedure on the surface roughness of the electrodes has been studied by in-situ oxygen adsorption measurements using cyclic voltammetry. The most effective pulse regime in producing a gold surface with a reproducible and relatively low surface roughness is a triple-potential pulse waveform, with potentials of +1.6, 0.0, and -0.8 Vvs SCE and pulse widths of 100 ms for each potential. Prolonged pulsing for 2000-5000 s with the gold working electrode in a flow-through cell showed an electropolishing effect, i.e., a decrease of the roughness in time. Flow conditions are very important: the roughness decreased faster at higher flow rates, while an increase was observed without flow. A process of reconstruction and dissolution of gold during application of the potential pulses under flow conditions is assumed to account for the observed phenomena. A self-assembled monolayer of thioctic acid with reproducible characteristics, determined with impedance measurements, could be formed on a pretreated gold surface.