Nanospheres lithographic (NSL) method has been used to fabricate nano-structured arrays (NAs) of hexagonally close-packed gold (Au) using polystyrene beads [PS, diameter similar to 300 nm] as mask. The developed NA was incorporated with a customized and cheap microfluidics system to demonstrate its applicability as an alternative easy and efficient platform for multiplex analysis and Lab-on-a-Chip applications. The chip functionality was demonstrated with horseradish peroxidase (HRP) and anti-HRP antibody as model for recognition system. The enzyme-linked immunosorbent assay (ELISA) performed on fabricated protein biochip had a detection limit 100 pg/mL for HRP. The antibody chip was also checked for the shelf-life and it was found that these chips could be stored for 50 days when stored at 4 degrees C without any significant loss of activity. Therefore, NAs based protein biochip with the correct microfluidics could find huge potential application in diagnostics and biosensing technology. (C) 2012 Elsevier Inc. All rights reserved.