Quantitative auto-fluorescence quenching of free and bound NADH in HeLa cell line model with Carbonyl cyanide-p-Trifluoromethoxy phenylhydrazone (FCCP) as quenching agent

Abstract

The autofluorescence of endogenous biomolecules (Nicotinamide adenine dinucleotide (NAD, its reduced form NADH and the phosphorylated form NAD(P)H take part in cellular metabolic pathways and has vital importance for in vivo and ex vivo photo diagnostic applications of biological tissues. We present a detailed quenching analysis of Carbonyl cyanide-p-Trifluoromethoxy phenylhydrazone (FCCP) 50-1000 µM and analyzed the fluorescence signal from NADH/ NAD(P)H in vitro (in solution) and in vivo (HeLa cell suspension).The in vitro samples of pure NADH/ NAD(P)H were excited at λ=340±1 nm while the fluorescence signal was collected in the range of 400-550 nm. The quenching process was characterized using excitation emission matrix (EEM) fluorescence spectroscopy and Stern- Volmer plots. The experimental results illustrated maximum fluorescence emission for the control NADH samples (i.e., no FCCP), while the fluorescence signal from the solution progressively decreased with the increasing concentration of the FCCP, until it reaches the base line (i.e., no fluorescence signal) at 1000 µM of FCCP. In vitro study shows that the fluorescence quenching of free NADH was found to be lower than the bound NAD(P)H with similar diminishing trend. The quenching of bound NAD(P)H in cells is attenuated compared to solution quenching possibly due to a contribution from the metabolic/antioxidant response in cells and fluorescence exponential decay curve lies between plated and suspended HeLa cells. A two-fold increase in the fluorescence intensity of NAD(P)H was observed after the bond formation with L-Malate Dehydrogenase (L-MDH, Sigma Aldrich #10127248001) protein This work has applications for sharp tumor demarcation during sensitive surgical procedures as well as to enhance fluorescence based diagnosis of biological tissues.

Keywords: Carbonyl cyanide-p-Trifluoro methoxy phenylhydrazone FCCP; Endogenous fluorophore; Excitation Emission Matrix (EEM); HeLa cells; NADH/NAD(P)H fluorescence quenching.