Single Gold Nanoparticles as Real‐Time Optical Probes for the Detection of NADH‐Dependent Intracellular Metabolic Enzymatic Pathways

Plasmonics, is an emerging subfield of nanophontonics, and it attracts increasing attention because of its potential applications in controlling and manipulating light at nanoscale dimensions. The advent of dark-field microscopy (DFM) has enabled the study of plasmonic nanoparticles, especially the coinage metals and the effects of their size, shape, composition as well as the local environment, which further facilitate its use in biological-labeling and detection. DFM provides a direct means to probe chemical reactions, real-time optical sensing with high sensitivity, and the in vivo imaging of cancer cells. Recently, redox reactions were directly monitored on single gold nanocrystals using DFM. Actually, every individual nanoparticle (NP) in the assembly could potentially act as an independent probe. Single-nanoparticle sensing platforms offer advantages since they are readily implemented in multiplex detection. Single nanoparticle probes offer improved absolute detection limits and also enable higher spatial resolution. Single nanoparticles have promising applications for measurements in vitro and in vivo events that are non-reachable by fixed solid array. However, the use of plasmonic nanoparticles for the detection of biomolecules or biological processes is still scarce. Nicotinamide adenine dinucleotide/reduced nicotinamide adenine dinucleotide (NAD/NADH) plays an important role as cofactor in numerous biocatalyzed processes, including energy metabolism, mitochondrial responses, immunological functions, aging and cell death. The catalytic deposition of copper on gold nanoparticles (AuNPs) by the NADH cofactor has been applied for the optical and electrochemical detection of NADH and NAD-dependent biocatalytic processes. Herein, we describe a novel method to detect enzymatic activity at the single particle level inside and outside cells by DFM. To our knowledge, it is the first time to monitor the intracellular metabolism and the effect of anticancer drugs on the cell metabolism using copper growth on the AuNP probes. To investigate the application of single Au@Cu nanoparticles for nano-sensing, the plasmon resonance Rayleigh scattering (PRRS) spectra lmax of a single particle was used to probe the gold-catalyzed reduction of Cu ions on AuNPs by NADH or by NAD-cofactor-dependent enzyme/substrate system that generates NADH (Scheme 1). Compared with the scattering spectra in the absence of NADH, the scattering spectra acquired with NADH exhibit a distinct peak shift