Electrochemical product detection of an asymmetric convective polymerase chain reaction.

For the first time, we describe the application of heated microwires for an asymmetric convective polymerase chain reaction (PCR) in a modified PCR tube in a small volume. The partly single-stranded product was labeled with the electrochemically active compound osmium tetroxide bipyridine using a partially complementary protective strand with five mismatches compared to the single-stranded product. The labeled product could be successfully detected at a gold electrode modified with a complementary single-stranded capture probe immobilized via a thiol-linker. Our simple thermo-convective PCR yielded electrochemically detectable products after only 5-10 min. A significant discrimination between complementary and non-complementary target was possible using different immobilized capture probes. The total product yield was approx. half the amount of the classical thermocycler PCR. Numerical simulations describing the thermally driven convective PCR explain the received data. Discrimination between complementary capture probes and non-complementary capture probes was performed using square-wave voltammetry. The coupling of asymmetric thermo-convective PCR with electrochemical detection is very promising for future compact DNA sensor devices.

Electrochemical competitive hybridization assay for DNA detection using osmium tetroxide-labelled signalling strands.

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.

Thermally induced electrode protection against biofouling.

This paper demonstrates that a combined thermal and electrochemical conditioning step can greatly minimize electrode blocking. We detected 50 ppm dopamine after a blocking step in 1000 ppm gelatine solution. Only a treatment of the electrode at -1.5 V and 61.5 degrees C can reveal the voltammetric dopamine signals to 82%. The increase of the peak separation of the cyclic voltammograms obtained in 50 ppm dopamine is limited to 14%, whereas negative polarization (-1.5 V) alone leads to a 31% increase compared to 109% upon thermal and 105% without any conditioning. The positive effects can be addressed to an enforced reductive degradation and accelerated removal of the blocking agents. Also the formation of hydrogen bubbles might play a significant role. Thermo-electrochemical treatment holds great promise for electrochemical sensors and detectors which are applied for long-term monitoring of samples that contain blocking matrices.