An ultra-sensitive nanoarray chip based on single-molecule sandwich immunoassay and TIRFM for protein detection in biologic fluids.
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.
Lee
Seungah
S
Department of Chemistry and Research Institute of Physics and Chemistry (RINPAC), Chonbuk National University, Jeonju 561-756, South Korea.
Cho
Nam-Pyo
NP
Kim
Jung Dong
JD
Jung
Hyungil
H
Kang
Seong Ho
SH
D056656:Lab-On-A-Chip Devices; D046208:Microchip Analytical Procedures / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D036103:Nanotechnology; D017646:Organosilicon Compounds; D040081:Protein Array Analysis / Q000295:instrumentation* / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D012821:Silanes / Q000737:chemistry
D017646:Organosilicon Compounds; D011506:Proteins; D012821:Silanes; C102833:(3-mercaptopropyl)trimethoxysilane