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.
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%.
The change in characteristics of self-assembled monolayers (SAMs) of thioctic acid (TA) and 11-mercaptoundecanoic acid (MUA) with time in buffered hexacyanoferrate(II/III) solutions of pH 7.4 has been studied with electrochemical impedance spectroscopy. Frequency scans were recorded at regular time intervals at +0.2 V versus SCE and a superimposed sinusoidal potential with an amplitude of 10 mV, in the frequency range from 10 kHz to 50 mHz. The stability of the SAMs was studied in the dark and during subsequent exposure of the solution to light. A TA SAM, a MUA SAM, and clean gold can be modeled with the same equivalent circuit. From this model, it was found that, as expected, charge transfer and diffusion of the redox probe through the MUA SAM are more inhibited than through the TA SAM. Further, the MUA SAM has a larger mean monolayer thickness, as is revealed from the lower value of the capacitive component. After exposure of the solution to light, a relatively rapid decrease in the resistances and increase in the double-layer capacitance are observed. Despite the racemic mixture of TA used, the characteristics of the MUA SAM change much faster than those of the TA SAM, and after 6 h of exposure, the equivalent circuit has to be modified to give an appropriate fit. The manner in which the values of the elements describing the TA SAM change suggests that TA molecules are gradually removed. A possible mechanism is etching of the gold substrate by CN- ions through pinholes in the SAM. Support of this mechanism is obtained from the large difference in characteristics of the gold electrode before SAM formation and after long-term incubation in illuminated buffered hexacyanoferrate(II/III) solution, pointing to a change in the structure of the gold, and by the observation that thin gold layers are etched in this solution.
Formation of self-assembled monolayers (SAMs) of thioctic acid (TA) on polycrystalline gold electrodes has been investigated. The Au electrodes are polished, first mechanically and subsequently electrochemically, until the roughness of the surface is minimal. The best TA SAMs are formed onto these electropolished electrodes from stirred solutions, while a potential E = 0.0 V or E = +0.2 V versus saturated calomel reference electrode (SCE) is applied during the adsorption process. Alternating current (ac) impedance methods are used to monitor the formation of the SAMs and to characterize these. Potentiostatically formed TA SAMs have better characteristics than those formed at open circuit. The TA SAMs are stable in 100 mM phosphate buffer pH 7.4, and when protected from light, also in buffered 2 mM hexacyanoferrate(II/III) solution. However, when the hexacyanoferrate(II/III) solutions are exposed to light, the TA SAMs are not stable. Probably, damaging of the SAM proceeds via etching of the gold surface by photochemically released cyanide ions. Using potential pulses (+1.6, 0.0, −0.8 V versus SCE, each 0.1 s) for 15 min in the flow (0.5 mL min-1), the TA SAM can be removed completely, recovering the initial state of the gold electrode, as deduced from potential-step experiments. This way TA SAMs can be formed and removed repeatedly and reproducibly on a single electrode.
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