FRET detection of Octamer-4 on a protein nanoarray made by size-dependent self-assembly
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An alternative approach for fabricating a protein array at nanoscale is suggested with a capability of characterization and/or localization of multiple components on a nanoarray. Fluorescent micro- and nanobeads each conjugated with different antibodies are assembled by size-dependent self-assembly (SDSA) onto nanometer wells that were created on a polymethyl methacrylate (PMMA) substrate by electron beam lithography (EBL). Antibody-conjugated beads of different diameters are added serially and electrostatically attached to corresponding wells through electrostatic attraction between the charged beads (confirmed by zeta potential analysis) and exposed p-doped silicon substrate underneath the PMMA layer. This SDSA method is enhanced by vibrated-wire-guide manipulation of droplets on the PMMA surface containing nanometer wells. Saturation rates of antibody-conjugated beads to the nanometer patterns are up to 97% under one component and 58–70% under two components nanoarrays. High-density arrays (up to 40,000 wells) could be fabricated, which can also be multi-component. Target detection utilizes fluorescence resonance energy transfer (FRET) from fluorescent beads to fluorescent-tagged secondary antibodies to Octamer-4 (Oct4), which eliminates the need for multiple steps of rinsing. The 100 nm green beads are covalently conjugated with anti-Oct4 to capture Oct4 peptides (39 kDa); where the secondary anti-Oct4 and F(ab)2 fragment of anti-gIgG tagged with phycoerythrin are then added to function as an indicator of Oct4 detection. FRET signals are detected through confocal microscopes, and further confirmed by Fluorolog3 spectrofluorometer. The success rates of detecting Oct4 are 32% and 14% of the beads in right place under one and two component nanoarrays, respectively. Ratiometric FRET is used to quantify the amount of Oct4 peptides per each bead, which is estimated about 2 molecules per bead.
KeywordsE-beam lithography Nanometer pattern generation system Fluorescence resonance energy transfer (FRET) Wire-guide droplet manipulation
The authors thank Dr. Brooke Beam and Paul Lee of the Keck’s facility for their assists in AFM, SEM, and confocal imaging, and Dr. Urs Utzinger and Ronnie George for the use and assistance of Fluorolog3 spectrofluorometer. In addition, authors would like to acknowledge Yee Tchao for her early contribution in this project and Vincent Wong for assistance in measuring zeta potential of beads. This work was supported by U.S. National Institute of Health under grant R03EB006754, as well as fellowship supports for Phat L. Tran (NIH-Initiative for Maximizing Student Diversity Fellowship and NIH-Cardiovascular Training Grant HL007955).
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