Fabrication and application of Micro and Nanoelectrodes

A biosensor is defined as an analytical device consisting of a biological sensing element either integrated within or in close vicinity of a transducer element which transforms the selective information of the presence of an analyte of interest into a quantifiable signal. A typical biosensor architecture comprises three features: a biological recognition element which is usually immobilised on the surface of a suitable transducer for converting the primary signal into a proportional change of a physical or chemical property, and an amplification or processing element. The design of appropriate sensor architectures for a given analyte depends on the specific demands arising from the analytical problem which has to be solved such as selectivity, sensitivity, dynamic range, detection limit, response time, precision, reproducibility, stability, and cost of the measurement. In our research group, the focus is directed towards electron-transfer reactions subsequent to redox reactions catalysed by suitable redox enzymes. In this case, the transducer is usually a noble metal or carbon electrode and often dynamic voltammetric methods such as cyclic voltammetry (CV), differential pulse voltammetry (DPV), differential pulse amperometry (DPA) are used to elucidate the chosen biosensor architecture and the properties of the immobilised redox proteins. In addition, constant-potential amperometry is frequently used to obtain correlations between substrate concentration and sensor output. The most simple design of an amperometric biosensors is the direct measurement of either an enzymatically generated product or of an electron-transfer mediator naturally involved in the biocatalytic process. A typical example for this design is the basic set-up of glucose sensors comprising the enzyme glucose oxidase as biorecognition element and recording either the enzymatically produced product H2O2 or the decrease of the concentration of the co-substrate O2 for collecting information about the glucose concentration in a sample. The corresponding glucose sensors are typical examples of so-called ‘first generation biosensors’. However, since this detection principle may lead to a poor reproducibility of the overall sensing process due to varying O2 concentrations in the sample under investigation, the application of artificial redox mediators was introduced in order to avoid the interference-prone oxygen dependence. Moreover, it is essential to decrease the unfavourable working electrode potential necessary for either the reduction of O2 or the oxidation of H2O2. Sensors realising a design with artificial mediators at known/constant concentration are addressed as ‘second generation biosensors’. In second generation biosensors redox enzymes donate or accept electrons to or from electrochemically active redox mediators having a redox potential adjusted to that of the enzyme’s cofactor. Ideally the mediator is otherwise inactive, i.e. highly specific only for the desired electron transfer process between the recognition element and the transducer. As a matter of fact, free-diffusing low-molecular weight redox mediators are prone to leak from the electrode surface thus imposing an overall decreased long-term operation stability to this type of enzyme electrodes. This inherent problem, however, is not preventing the successful application of such sensors in one-shot devices, an application field which is especially important for self-monitoring of blood glucose levels. Another alternative approach is seen in immobilising the redox enzyme on a suitable electrode surface in such a way, that the protein-integrated redox site can directly exchange electrons with the electrode avoiding any free-diffusing redox mediator. This sensor design was named ‘third-generation biosensors’. Based on these considerations present research is directed towards the development of reagentless amperometric biosensors which are designed in such a way that all components necessary for the complementary biological recognition, the biocatalytic reaction, and the signal transduction are securely immobilized in a specifically designed sensor architecture. In addition, main work is focused on the development of immobilization strategies enabling the controlled and specific deposition of complex sensor architectures exclusively on the surface of electrodes. This implies the application of electrochemically induced deposition schemes using conducting polymers, electrodeposition paints, redox-modified electrodeposition paints.