Hydrogen-bond-assisted meta-nitrogen-doped graphyne enables real-time electrocatalytic NADH tracking in single cells

Nicotinamide adenine dinucleotide (NADH), a crucial biomarker for cellular redox homeostasis, is intrinsically linked to mitochondrial function and neurodegenerative diseases. However, electrochemical detection of intracellular NADH faces dual challenges: low abundance and biofouling, as well as high overpotential. Herein, we designed a hydrogen-bond-assisted <i>meta</i>-nitrogen/oxygen co-doped graphyne (3NGYO) nanoelectrode for high-performance NADH sensing. Precise tri-N doping in graphyne (3NGY) generates sp²-N atoms associated with hydrogen atoms. Specifically, pyrrolic N-H forms a 2.502 Å N-H⋯O[double bond, length as m-dash]P hydrogen bond with NADH's PO₄ groups, boosting adsorption energy to -5.48 eV and reducing NADH oxidation potential to 0 V. This achieves a 59-fold higher catalytic current response compared to pristine graphyne. Subsequent acid oxidation introduces oxygen-containing functional groups (<i>e.g.</i>, -COOH, C[double bond, length as m-dash]O), increasing hydrophilicity (contact angle: 54.0°) and anti-fouling performance (80% current retention after 2 h BSA exposure). Electrodeposited 3NGYO nanotips attain a sensitivity of 0.419 pA µM^-1 and a linear range of 0-20 µM at +0.2 V <i>vs.</i> Ag/AgCl. Real-time amperometry in SH-SY5Y cells demonstrates that 1-methyl-4-phenylpyridinium (MPP⁺)-induced mitochondrial dysfunction triggers NADH release. In contrast, hydrogen sulfide (H₂S) pretreatment reduces NADH leakage by 95.4%, correlating with suppressed calcium ion (Ca²⁺) influx and reactive oxygen species (ROS) generation. This work provides a tool for studying mitochondrial dysfunction and establishes a new paradigm for <i>in situ</i> electrocatalytic biosensing.