Cobalt-doped MoS₂ nanocomposite with NADH oxidase mimetic activity and its application in colorimetric biosensing of NADH

Highlights

• Co-MoS₂ nanocomposite exhibiting NADH oxidase mimic activity.

• The mechanism of the NADH oxidase mimics was studied.

• Sensitive and selective colorimetric sensor for NADH developed.

Abstract

NADH oxidase (NOX) is an oxidoreductase that catalyzes the oxidation of NADH to NAD⁺ with the participation of oxygen. NOX can artificially regulate metabolic processes as an important regulatory enzyme for NAD⁺ regeneration. In this study, cobalt-doped molybdenum sulfide (Co-MoS₂) nanocomposite was prepared by hydrothermal synthesis, which exhibited NOX mimics. The catalytic mechanism of NOX mimics was studied. The NOX-like activity of Co-MoS₂ is optimal at pH 4.5 and in the temperature range of 20–45℃. Further, its catalytic activity maintained almost the same after 5 cycles of repetitive addition of NADH, suggesting that Co-MoS₂ nanozyme possessed significant catalytic persistence. The produced H₂O₂ catalyzed by Co-MoS₂ can be detected by horseradish peroxidase (HRP) and 3,3′,5,5′-tetramethylbenzidine at pH4.5. Thus, interestingly, a colorimetric NADH sensor was proposed on the basis of the enzymatic cascade reaction of NADH oxidase-like conjugated with HRP. This as-established method had wide detection range (0.5–800 μM) with the detection limit of 0.25 μM NADH, capable of detecting NADH in real sample.

Introduction

Enzyme, as a protein with specific catalysis and high efficiency, is involved in almost all metabolic reactions in vivo and controls substance metabolism mainly through the regulation of enzymatic activity [1,2]. However, for most enzymes, they experience in-stability, low repeatability and high-cost [[3], [4], [5]]. In 2007, Yan et al. discovered that Fe₃O₄ nanoparticles can simulate peroxidase [1,6]. Since then, a great deal of work has begun to investigate nanomaterials to simulate natural enzymes [7,8], which have been applied in detection [9,10,79], pollution control and cancer therapy [[11], [12], [13]]. Most nanozyme designs are based on the active center of natural enzymes [14]. Nanozymes have become research hotspot, because they have high catalytic activity and stable characteristics as well as scale-up synthesis [4,15,16]. To date, a wide range of nanozymes, for example, noble metals including gold nanoparticles (NPs) [[17], [18], [19], [20]], AgNPs [21] and PtNPs [22], gold nanoplates [19] and Au@Ag heterogeneous nanorods [23], metal oxide NPs (Co₃O₄ NPs [24,25], V₂O₃ NPs [26], NiO NPs [27], MnO₂ [[28], [29], [30]], Copper-based nanoclusters [31,32]), functionalized porous carbon [33,34] and nanocomposites [35] were reported. Nanozymes can be roughly divided into oxidordeuctase (e.g., peroxidase [36,38], oxidase [39], superoxide dismutase (SOD) [40,41]and catalase [42]), hydrolase (e.g., phosphatase [43]) and lyase (photolyase [44]) as well as synthetase [45].

Nicotinamide adenine dinucleotide (NAD⁺) is an important redox cofactor that participates in a variety of biotransformation systems and drive decomposition and synthesis reactions. The catabolism of main nutrients (sugar, fat and protein) is inseparable from NAD⁺. On one hand, it requires NAD⁺ (polyadenylate-ribosylation) to repair DNA damage. Thus, the bioactive NAD⁺ regeneration is of great value to most dehydrogenase mediated reactions [20,[46], [47], [48], [49], [50], [51], [52], [53]]. The higher NAD(H) (the reduced form of nicotinamide adenine dinucleotide) or NADH/NAD⁺ ratio indicates that the cell respiration consumes more oxygen and is in a state of peroxidation. A higher ratio of NADH/NAD⁺ can also inhibit glycolysis and tricarboxylic acid cycling. On the other hand, many diseases are closely related to NADH levels. Therefore, the detection NADH holds significance. The traditional colorimetric NADH detection method, based on the characteristic peak wavelength at 340 nm, has great limitations, which is vulnerable to the interference from similar substances with ultraviolet absorption in the sample solution [54]. NADH detection kits are convenient and rapid, nevertheless, they are relatively expensive. To date, a variety of nanocomposites based electrochemical sensors have realized the detection of NADH [[55], [56], [57], [58], [59]], however, they are time-consuming. To this end, it has great potential to develop nanozymes based colorimetric sensor for NADH.

NADH oxidase (NOX) is an oxidoreductase that can catalyze the oxidation of NADH [60]. In the presence of O₂, the NOX is capable of catalyzing the oxidation of NADH to NAD⁺ and reducing O₂ to H₂O or H₂O₂ [[61], [62], [63]]. That is, NOX could be an important target for regulating the proportion of NADH/NAD⁺ [[64], [65], [66]]. Natural NOX is widely present in lactic acid bacteria [67,68]. Nevertheless, the process of isolation and purification NOX from microorganisms is complex and cost-ineffective. To overcome this challenge, it is of great advantage to simulate NOX activity by simple synthesizing nanozyme. MoS₂ nanoparticles can mimics peroxidase [69,70]. The doping of P, Al, Ni and other elements in MoS₂ was reported to enhance the activity of mimic enzymes [71,72]. Further, MoS₂ is an excellent catalyst for hydrogen evolution reaction [73]. However, there is no report on cobalt-doped MoS₂ (Co-MoS₂) nanomaterials exhibiting NADH oxidase activity.

In this work, we report on the Co-MoS₂ nanocomposite to simulate NADH oxidase. In addition, the catalytic mechanism of the nanozyme is investigated in detail. Finally, by conjugating cascade reaction of NOX mimics and horseradish peroxidase (HRP), a sensitive and selective colorimetric sensor for NADH is proposed based on the characteristic peak of 652 nm.