In order to detect pathogens in complex food matrices, concentration and separation of the bacteria from the sample matrix is necessary. Electrospun nanofibers are ideal for use in filtration because they provide a higher surface area as compared to traditional membranes. Functionalized nanofibers carrying positive and negative charges were spun by the Frey Group at Cornell University and incorporated into PMMA microfluidic devices. The fibers were investigated for their ability to separate E.coli from buffer samples and actual apple juice samples. This separation is based on the knowledge that E.coli and gram negative bacteria have a strong net negative charge at neutral pH and a net positive charge at low pH caused by deprotonation and protonation of carboxyl and ammonium groups on the cell surface. Filtration was evaluated using two methods 1) collecting effluent and enumerating cells using standard plate counts 2) by staining the cells with Syto9 green fluorescent dye and measuring fluorescence intensity in the channel. In the first method, positive fibers were shown to retain 98 ± 1.6% of E.coli that passed through the microfluidic channels whereas negative fibers only retained 35 ± 1.1%. In the second method, E.coli in pH7 buffer was filtered onto positive fibers producing a strong fluorescence. Elution of the cells was possible by introducing a pH4 washing buffer producing an 84% drop in fluorescence intensity. E.coli in pH4 buffer attached better to negative fibers producing average fluorescence intensity 1.6 times higher than the channels with positive fibers. The results were repeated with apple juice spiked with E.coli producing similar results. These results show the potential for electrospun nanofibers to be used on-chip as bioseparators.