Extremely sensitive protein detection is crucial for early diagnosis and monitoring of diseases. Present day’s limit of detection for an antibody-antigen recognition-based immunoassay in serum without having to use special target antigen amplification techniques, like immuno-linked polymerase chain reaction (PCR), is 10 fg/mL, a sensitivity achieved thanks to the use of relatively large sample volumes (40-100 μL). In this thesis, a new method is developed to analyze rare protein biomarkers in a serum sample using a magnetic bead surface coverage assay. The assay protocols are realized on a microfluidic chip having channels, valves, an integrated mixer, and a protein micro-array. The microfluidic chip provides automated analysis, minute sample consumption (5 μL), resulting in an unprecedented 2 fg/mL detection performance for Tumor Necrosis Factor-α (TNF-α) in serum. A first part of the thesis consists in fabricating protein micro-arrays using the surface of immobilized ‘small’ (1.0 μm size) superparamagnetic beads that are placed in microfluidic flow channels. The small beads are immobilized on a glass chip with pre-patterned aminopropyl- trietoxysilane (APTES) structures using an electrostatic self-assembly method. Different target antigens are captured from a flow passing over the micro-array inside the microfluidic channel, and are specifically bound on the small bead surfaces. The presence of target antigens is detected as a fluorescent signal by realizing a sandwich immunocomplex on the capture antibody-functionalized surfaces of the small beads. However, the fluorescent detection method is abandoned due to high fluorescence background levels, resulting in only a 0.25 ng/mL detection limit for mouse immunoglobulin in phosphate-buffered saline. In a second part of the thesis, a polydimethylsiloxane (PDMS) chip having microfluidic channels, valves and an integrated mixer is designed. A new active mixing strategy is performed in the chip using multiple source-sink microfluidic flows. To do so, four different pressure- controlled actuation chambers are arranged on top of the 5 μL volume of the mixing chamber. After microfluidic valves, realized using ‘microplumbing technology’, seal the mixing volume, a virtual source-sink pair is created by pressurizing one of the membranes and, at the same time, releasing the pressure of a neighboring one. This creates microfluidic flows from the squeezed region (source) to the released region (sink) where the PDMS membrane is turned into the initial state. Perfect mixing is 7000-fold faster than achievable with pure diffusion-based mixing. This excellent performance is possible thanks to the implementation of chaotic advection by inducing vortices inside the mixing chamber. A DNA isolation and purification protocol using magnetic beads in the mixing chamber is also performed on-chip, showing the enhanced DNA binding performance due to active mixing. Finally, a new protein detection method is realized in a microfluidic chip. ‘Large’ (2.8 μm size) functionalized superparamagnetic beads specifically capture target antigens from a serum matrix under active microfluidic mixing. Subsequently, the large beads loaded with the antigens gently expose antibody-functionalized small bead patterns on the glass detection chip. During the exposure, attractive magnetic bead dipole-dipole interactions improve the contact between the two bead types and help the antigen-antibody immunocomplex formation, while non- specific large bead adsorption is limited by exploiting viscous drag forces in the microfluidic channel on the detection chip. This efficient transport mimics a biological process, where leukocytes roll and slow down on blood vessel walls by selectin-mediated adhesion for rapid recognition of tissue molecules. Afterwards, the antigen concentration is detected by simply counting the surface pattern-bound large magnetic beads. The new technique allows detection of only a few hundreds of TNF-α molecules (600 yoctomole) spiked in the 5 μL serum sample. This performance, in terms of detectable number of molecules, is at least 20 times better than today’s most-sensitive PCR-free antibody-based methods. Furthermore, the method has the supplementary advantage of being rapid, resulting from the automated assay protocols and the extremely fast (1 minute) antigen capture.