The advantages of the presented nIDAs and the potential of their use in bioelectrochemistry were studied utilizing cyclic voltammetry (CV) and chronoamperometry. For the electrochemical characterization ferrocenemethanol (FcMeOH) and p-aminophenol (pAP) were selected as redox couples. Cyclovoltammetric measurements were performed for the determination of the redox potentials of both FcMeOH and pAP. The potential windows from 0.0 V to +0.3 V and from -0.3 to +0.4 V (vs. Ag/AgCl as reference electrode) were defined as the optimal scan ranges for ferrocenemethanol and p-aminophenol respectively. The cyclovoltammetric measurement in generator/collector mode was applied to obtain information about electron transfer. In this mode, one working electrode (generator) is scanned while the second working electrode (collector) is fixed at the constant potential (for the applied redox couples at the reduction potential). The measurement diagrams obtained by generator/collector experiments point out that the electron transfer of pAP is diffusion inhibited in contrast to FcMeOH. Furthermore, chronoamperometric measurements were carried out to determine the signal amplification and collection efficiency. In these measurements, constant potentials were applied to both generator and collector electrodes. On working electrode 1 (WE1) the oxidation potential and on working electrode 2 (WE2) the reduction potential was applied respectively. Compared to a single electrode configuration, a current amplification factor above 100 was achieved.
The maximum, 161-fold, amplification was obtained with FcMeOH (Figure 1b). To the best of our knowledge that is the highest reported amplification achieved by chronoamperometric measurements in bulk solutions (the sensor was dipped into the solution). For the same nIDA a significantly lower amplification factor was observed for p-aminophenol compared to FcMeOH. We assume that the formation of a polymer film takes place during redox cycling of pAP, this leads to an electron transfer inhibition and hence to the decrease of amplification. SEM images support this assumption and show a polymer film on the electrode surface. It has been verified that the cleanness of the IDA surface and measuring sequence of cyclovoltammetric experiments are strongly influencing the signal amplification of pAP. Figure 1c shows the correlation between gap size and amplification factor. The gap size is relevant for the amplification factor and with a gap size of 100 nm an amplification of 161 has been achieved. The results show that not only high signal amplification but also high current efficiency above 99 % has been reached for both tested redox couples. Moreover, we demonstrated that our fabricated nIDA has the potential of high signal amplification by chronoamperometric measurements of the generally used redox couples. Therefore, it is an excellent candidate for the further ELISA experiments.
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