This paper describes the formation of highly defined two-dimensional protein arrays onto a polymeric substrate expressing biotin functionalities at its surface. Micron-scale arrays of avidin were created by exploiting its interaction with biotin, thereby creating a versatile patterned surface onto which any biotinylated species can be subsequently immobilized. The patterning process utilized the technique of microfluidic networks (mu FN) to spatially confine the flow of protein solution on a substrate. We describe methods developed to control protein deposition with nanometer-scale precision. Utilizing atomic force microscopy (AFM), these patterned surfaces were analyzed to the molecular level. In addition, the applicability of this technique to a wider range of substrates has been investigated. In particular, developments within the mu FN technique have enabled the successful formation of protein micropatterns onto a highly hydrophilic surface, a substrate which is generally not suited to the technique of mu FN.