Abstract
Surface-enhanced Raman spectroscopy (SERS) is an optical device that is complicated but fast, that deals with high precision cells, noninvasive and label-free. Tumor necrosis factor-alpha (TNF-α) is primarily produced by macrophages an inflammatory cytokine. When TNF-α binds to TNFR, it activates nuclear factor kappa B (NF-kB) transcript factors. This will lead to oscillation in nuclear NF-kB and steering gene expression by negative feedback loops. We applied biotin anti-human TNF-α antibody to bind with TNF-α proceeded by chemical bonding for streptavidin- Phycoerythrin (PE) to detect the space structure between TNF-α and TNFR. The intracellular p65 was labeled with GFP for NF-kB tracking. We indicated the probing of spatiotemporal interaction between binding of TNF-α to TNF-receptor. PE interacted with TNF-α on a ratio of 1:1 and 1:4. Quantum dots (QDs) with TNF-α at 1:1. The signal strength had decreased in the ratio 1:4. There was low bright field in QD interaction to TNF-α. We used real-time imaging for single TNF-α molecule on the living NIH-3T3 cell surface. The result showed the ligand-induced receptor dimerization and trimerization. The bioconjugation and diameter of fluorescent probes both affected the spatial binding of TNF-α to its receptor. Using real-time imaging of GFP-P65, we successfully visualized the intracellular NF-kB nuclear translocation. Our goal is to compare the results from fluorescence imaging to SERS experiment. SERS has the potential to analyze NF-kB. With this image, label-free SERS space detection and with potentially patentable novel gold nano-particles (Au NPs) it can be seen NF-kB activation in two hours. In addition, we can conduct a 96 or 384 test in parallel and increase the throughput by two or three orders by 100-1000 faults. This will be the ultimate goal to move this device into clinical application.
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