Nanopore Identification of Nicotinamide Adenine Nucleotide and Its Derivatives

Abstract Nicotinamide adenine dinucleotide (NAD), existing primarily in two interconvertible forms: oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) and reduced nicotinamide adenine dinucleotide (NADH), is an essential coenzyme in cellular energy metabolism and redox reactions. NAD related derivatives, such as oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), reduced nicotinamide adenine dinucleotide phosphate (NADPH), nicotinic acid adenine dinucleotide (NAAD), nicotinic acid mononucleotide (NAMN), nicotinamide mononucleotide (NMN), and nicotinamide riboside (NR), are involved in the biosynthesis, metabolism, and salvage pathways of NAD ⁺ . Their significance in cellular metabolism evaluation and disease diagnosis has led to an urgent need for the detection. Here, an engineered Mycobacterium smegmatis porin A (MspA) nanopore sensor, which is capable of rapidly and simultaneously identifying NAD ⁺ and a variety of its derivatives, is developed. With the aid of a customized machine learning algorithm, a 99.9% accuracy is achieved. Additionally, this sensor is applied to investigate the catalytic process of glucose‐6‐phosphate dehydrogenase (G‐6‐PD) by real‐time monitoring of the dynamic transformations of NAD ⁺ to NADH and NADP ⁺ to NADPH, providing a single‐molecule tool for the diagnosis of G‐6‐PD‐related disorders.