We functionalized nanowires with three different probe peptide nucleic acid (PNA) sequences, and assembled the three populations onto a lithographically patterned chip. Electrofluidic assembly enabled positioning each set of nanowires to span a different pair of guiding electrodes. Fluorescence imaging was used to probe whether the PNA on the individual nanowires remained able to selectively bind complementary DNA targets following assembly and integration of the positioned nanowires onto the chip surface.