Fabrication of hydrogel-micropatterned nanofibers for highly sensitive microarray-based immunosensors having additional enzyme-based sensing capability

Nanofiber-based protein microarrays were fabricated through a combination of electrospinning and hydrogel lithography. Electrospinning generated polystyrene (PS)/poly(styrene-alt-maleic anhydride) (PSMA) fibers with diameters ranging from 0.5 to 1.0 mu m and photopatterning of poly(ethylene glycol) (PEG) hydrogel on the electrospun fibers created clearly defined hydrogel microstructures with incorporated nanofibers. The resultant micropatterned nanofibrous substrates were obtained as freestanding and bidirectionally porous sheets, where most of the nanofibers were inserted through the side walls of the hydrogel microstructures. Because of the protein-repellent nature of PEG hydrogels, IgG was selectively immobilized only within the nanofibrous region, creating an IgG microarray. Due to increased surface area, IgG loading in nanofibrous substrates was about six times greater than on planar substrates, which consequently yielded a higher fluorescence signal and faster reaction rate in immunoassays. The capability of encapsulating enzymes made it possible for PEG hydrogels to be used not only for defining protein micropatterns but also for additional biosensor elements. Based on this result, micropatterned nanofibrous substrates consisting of IgG-immobilized nanofibers and enzyme-entrapping PEG hydrogels were fabricated, and their potential to simultaneously carry out both immunoassays and enzyme-based assays was successfully demonstrated.