'All that glisters is not gold'

NBD-Cl is a fluorescent labeling reagent widely used to track biomolecules such as lipids and proteins or to monitor enzyme activities. However, the potential impact of NBD labeling on cellular metabolism remains a concern. The Optical Nanospectroscopy Group (Janina Kneipp) and the Organic and Bioorganic Chemistry Group (Christoph Arenz) investigated the molecular changes induced by NBD-Cl and NBD-ceramide (NBDCER) in the endolysosomal compartments of 3T3 fibroblast cells and J774 macrophage cells to identify specific molecular alterations and understand their implications for cellular function. Find out more about the 'hidden' contributions of the NBD group in their The Journal of Physical Chemistry C Article!

Abstract

7-Nitrobenz-2-oxa-1,3-diazole (NBD) is a widely used fluorescent label for proteins, peptides, and lipids. Its chloride derivative, NBD-Cl, can be highly reactive toward thiol and amine groups, forming stable fluorescent adducts. When labeling the ubiquitous lipid molecule ceramide, NBD-ceramide (NBDCER) aids in visualizing sphingolipid metabolism in cells. This study investigates intracellular molecular changes induced by NBD-Cl and NBDCER using surface-enhanced Raman scattering (SERS). SERS spectra from the endolysosomal compartment of two cell lines, 3T3 fibroblast cells and J774 macrophage cells, obtained with gold nanoparticles as probes, reveal changes in the molecular composition and interactions under different incubation conditions. Applying the random forest (RF)-based algorithm surrogate minimal depth (SMD) to the SERS data to identify important spectral classifiers and their relations, both NBD-Cl and NBDCER are found to alter the biochemical makeup of the endolysosomal compartment. The data indicate significant structural and interaction changes in the molecular constituents of the cells that are in agreement with possible interference of the labels in the cellular metabolism and the reaction of NBD-Cl with functional groups of cellular molecules.