Microfluidic biosensors which incorporate both sample preparation and analyte detection, also referred to as lab-on-a-chip (LOC) devices, are a promising means of providing low cost, rapid, and portable analyte detection in point-of-care, rural, and developing world applications1- 3 . However, despite numerous reports of LOC devices capable of detecting a range of clinical analytes1-3, there are several key challenges that face the development of true LOC devices. First, due to the small size of these miniaturized systems, it is often necessary to significantly concentrate the sample volume to the nL-[MICRO SIGN]L range 4. Additionally, samples must be purified to remove particulates and impurities that may impede analyte detection. Finally, fluid flow in microfluidic devices is generally laminar, which limits the amount of fluid mixing that occurs within the channels5-7. Because rapid fluid mixing is typically required to facilitate chemical reactions and ensure access of analytes to functional surfaces within the microchannels, micromixers need to be incorporated into the design of a LOC device. This research aims to address the need for both better sample preparation and fluid mixing within microfluidic assays through the use of functionalized electrospun nanofibers. Electrospinning is a fiber formation process in which electrical forces are used to form ultrathin fibers from viscous polymer spinning solutions8. The nonwoven fiber mats produced during electrospinning are characterized by extremely large surface-area-to-volume ratios and high porosities. Additionally, electrospun nanofibers can easily be functionalized either through the inclusion of nanoscale materials into the polymer spinning dope, or through post-spinning modifications. In this work, positively and negatively charged poly(vinyl alcohol) (PVA) nanofibers were created through the addition of hexadimethrine bromide (polybrene) and poly(methyl vinyl ether-alt-maleic anhydride) (poly(MVE/MA), respectively, into a 10% w/v PVA spinning solution. Additionally, larger diameter polystyrene (PS) microfibers with a range of morphologies were spun using 12.5, 15, and 17% w/v PS spinning solutions. Previously, gold microelectrodes patterned onto poly(methyl methacrylate) (PMMA) were used to incorporate the nanofibers into microfluidic channels9,10. However, in this work, fibers were bonded into microchannels without the use of a gold electrode, resulting in simple, inexpensive device fabrication. Both PVA and PS fibers were spun onto metal collector plates and manually transferred to pieces of PMMA that had undergone UV-Ozone treatment. In order to produce nanofiber mats with uniform fiber distributions along their height, thin nanofiber mats were stacked together to create multilayered mats 11,12. Positively charged PVA mats were shown to successfully bind and concentrate E. coli cells, while negatively charged PVA mats repelled the cells and were used to minimize nonspecific retention within the channels. The 3D morphology of the PVA nanofiber mats was optimized to eliminate nonspecific mechanical retention of the E. coli while also providing sufficient surface area for E. coli capture. Finally, anti-E. coli antibodies were immobilized on negatively charged PVA fibers to allow for successful specific capture of the analyte. Fluid mixing within Y-shaped microchannels was enhanced through the incorporation of both PVA nanofibers and PS microfibers, though the PVA fibers produced the most significant mixing. We assume that mixing within the PVA nanofiber mats is caused by the inhomogeneity of pore size and pore distribution within the mats rather than by the individual nanofibers. Statistical analysis of mixing within the nanofiber mats indicates that mixing is dependent on the height of the nanofiber mat (i.e. the number of layers) but is independent of the length of the nanofiber mat. As expected, the amount of mixing observed increased with decreasing fluid flow rate. The results of this study can be used to provide both enhanced sample preparation and fluid mixing with microfluidic biosensors. In addition, further functionalization of the nanofiber surfaces can be used to allow for detection of a wide range of analytes.