Early and accurate diagnosis of a specific disease plays a decisive role for its effective treatment. However, in many cases the clinical findings, based on a single biomarker detection alone, are not sufficient for the appropriate diagnosis as well as monitoring of its treatment. Furthermore, it is highly desirable to screen multi-analytes (e.g. various diseases and drugs) at the same time enabling a low-cost, quick and reliable quantification. Thus, multiplexing, simultaneous detection of different analytes from a single sample, has become in recent years essential for diagnostics, especially for point-of-care testing (POCT). This thesis focuses on the scientific issue regarding the sensitivity enhancement of microfluidic biosensor platforms. Simulations, design studies and experiments are employed to investigate the interplay between the immobilization area and the resulting sensitivity. Thereby, a novel concept comprising design rules for microfluidic biosensors using the stop-flow technique has been introduced. In combination with different technical measures it allows the realization of an electrochemical lab-on-a-chip (LOC) platform for the fast, sensitive and simultaneous POCT in clinically relevant samples. This system employs a universally applicable, bioaffinity based biomolecule immobilization along with an amperometric readout. By means of the dry film photoresist technology, the fabrication of disposable microfluidic biosensors is enabled with high yield on wafer-level. The presented LOC platform offers three different biosensors with a microfluidic channel network of two, four or eight discrete immobilization sections, each with a volume of 680 nl. They can be actuated by individual channel inlets allowing a high flexibility in the assay design with respect to its format (e.g. competitive) and its technology (e.g. genomics). The feasibility for multiplexing is successfully demonstrated with DNA-based antibiotic assays for tetracycline and streptogramin, both important growth promoters in livestock breeding. The extensive usage of antibiotics is one of the major causes of the multi-drug-resistant bacteria and so, it has to be kept under surveillance. This platform allows the simultaneous POCT of different antibiotics from human plasma along with a limit of detection of less than 10 ng ml⁻¹, a wide working range up to 1,600 ng ml⁻¹ and inter-assay precisions of about 10 %. Moreover, the microfluidic LOC system provides a low consumption of reagent and sample, reduces the total assay time drastically with a sample-to-result time of only 10 min. The shelf-life of the biosensors is proven to be at least 3 months at +4 °C. The introduced design concept with specific technical measures facilitates the implementation of microfluidic multiplexed biosensors in a low-cost, compact, and at the same time sensitive manner. This platform targets the POCT in the first place, yet, owing to its multiplexing approach it can be expanded for in vitro diagnostics
