We describe a novel instrument and electrical circuit for sensitive electrochemical measurements at simultaneous direct electrode heating. The new measuring principle can be applied to working electrodes of various designs featuring two end contacts. In our experiments, the contacts were connected to a 100 kHz AC heating power supply and the potentiostat via the new inductor bridge circuit. A compact heating-generator housing contains all components necessary for sine wave generation as well as amplification and transformation of the heating power. The new arrangement yields high temperature cyclovoltammetric signals for the [Fe(CN) 6 ] 3À/4À redox system with a noise level superior to the earlier symmetrically branched wire electrode designs. Noise and disturbances are dramatically suppressed especially for high resistance electrodes such as glassy carbon electrodes. Without a center contact, the working electrode design is greatly simplified. This opens new opportunities for the design of a great variety of heated electrodes that may be arranged in arrays or consist of materials with relatively high resistivity such as carbon and conducting polymers.
Single stranded DNA-targets from asymmetric polymerase chain reaction (PCR) of a sequence of the gram positive, spore forming bacterium Clostridium acetobutylicum were detected by square-wave voltammetry after labeling with osmium tetroxide bipyridine and hybridization with DNA capture probes immobilized on gold electrodes. The asymmetric PCR, performed with a 10-fold excess of the forward-primer, was used without any further purification for hybridization with protective strands and covalent labeling with osmium tetroxide bipyridine. Square-wave voltammetric signals of 20 nmol/L targets were significantly higher at 50 8C compared with 23 8C hybridization temperature. A fully noncomplementary protective strand yielded thoroughly modified targets unable for further hybridization. Coupling this with thermal discrimination opens new opportunities for sequence specific DNA detection.
In this report, we present sequence-specific DNA detection by means of a competitive hybridization assay with osmium tetroxide-labelled signalling strands. The labelling of the signalling strands has been performed using protective strands to preserve the recognition site of these single strands for hybridization with the immobilized capture probes. At optimized measuring conditions and especially assay temperature, we could detect the presence of 25 nM target DNA within 30 minutes, whereas the non-complementary target sequence did not yield any signal. The latter was observed as a decrease in square-wave voltammetric response of the signalling probes. Single base mismatches could be detected at a stringent 35 degrees C electrolyte temperature. Moreover, the concentration dependency of the signal was investigated. A time-consuming labelling procedure of the target, as typically used before, is not necessary. Upon application of the new protocol, there is no need for handling osmium(VIII) compounds during sample treatment. The signalling strands containing Os(VI) are prepared separately and can be stored over several months.
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