This paper describes a single-molecule sandwich immunoassay method that utilizes total internal reflection fluorescence microscopy (TIRFM) at the single-molecule level for nanoarray protein chip applications. Nanoarray patterning of a biotin-probe with a spot diameter of 179 +/- 1 nm was performed successfully on a (3-mercaptopropyl)trimethoxysilane (MPTMS)-coated glass substrate by atomic force microscopy (AFM). The formation of biotin patterns was confirmed directly by observing the heights of bound streptavidin and biotin-antibody on glass substrates using an AFM in contact mode. Target protein molecules (or antigen) at the zepto-molar (zM) concentration level (x 10(-21) M) were detected on MPTMS-coated glass nanoarray protein chips by TIRFM. Finally, cytokine clinical samples (i.e. TNF-alpha and IL-1alpha) as cancer marker protein molecules were applied to nanoarray protein chips, and detection limits were at 600 zM.