It has recently identified that the transcription factors Oct3/4 and Cdx2 play an important role for the embryonic stem cells to maintain pluripotency. Detection of such factors in this complicated expression system is believed to be very challenging, primarily, or at least partly, due to their extremely low concentrations. Performing a 2-D gel analysis often requires large amount of sample, which is rather impractical in the stem cell experiments. In addition, the signal from the common proteins large in their quantity typically overwhelms the other low-quantity proteins, presumably including Oct3/4 and Cdx2. This will dramatically limit the signal-to-noise (S/N) ratio from such target molecules. Antibody-based assay, such as ELISA, may eliminate such problems, but its reproducibility is traditionally known to be inferior to the nucleotide-based assays. This is primarily due to the denaturation issues and the variance in the binding capacity. Here, we propose to detect Oct3/4 and Cdx2 with an antibody nanoarray at the level of single molecule. This protein nanoarray will be constructed through self-assembling several gold nanoparticles (AuNPs), conjugated with several different antibodies, to the desired locations of nanopatterns etched by e- beam. Each array grid will hold a single AuNP conjugated with roughly a single antibody. AuNP-antibody conjugates will be immobilized to the specific location only when their sizes match to the e-beam nano- patterns. Multiple duplicates of the same antibody will be immobilized. Although not all of them will exhibit antibody-antigen binding signal, similar to the ELISA, but the signal will be identified individually, i.e., at the level of single molecule detection. This will lead to the greatly enhanced S/N ratio, since the binding signal will simply be yes or no (1 bit). In ELISA, however, the signal will be ensemble-averaged, typically generating indiscernable dim signals, or erratic irreproducible results, depending on the activity and subsequently binding capacity of the antibodies. We hope this new method can make a major contribution to the pluripotency research with the stem cells, and possibly the more general cell differentiation study as well. This, in turn, may lead to the development of new stem-cell-based therapy, including tissue-engineered grafts. Overall, the proposed work will demonstrate one perfect example integrating physical and engineering (AuNPs and e-beam etching) with the life science (cell differentiation), which is the mission of NIBIB. ? ? ?
| Tran, Phat L; Gamboa, Jessica R; You, David J et al. (2010) FRET detection of Octamer-4 on a protein nanoarray made by size-dependent self-assembly. Anal Bioanal Chem 398:759-68 |
| Powell, Tremaine B; Tran, Phat L; Kim, Keesung et al. (2009) Size-dependent self-assembly of submicron/nano beads-protein conjugates for construction of a protein nanoarray. Mater Sci Eng C Mater Biol Appl 29:2459-2463 |
