Specific and Rapid Glucose Detection Using NAD‐dependent Glucose Dehydrogenase, Diaphorase, and Osmium Complex

Abstract

Selective glucose measurement in serum and blood and rapid glucose measurement using nicotinamide adenine dinucleotide (NAD)‐dependent glucose dehydrogenase (NAD‐GDH) are still very challenging. Here, we report a selective and rapid glucose sensor, based on electrochemical‐enzymatic‐enzymatic (ENN) redox cycling involving bis(2,2‐bipyridyl)dichloroosmium(II) [Os(bpy)2Cl2], diaphorase (DI), NAD+, NAD‐GDH, and glucose. DI and Os(bpy)2Cl2 are used to obtain fast mediated oxidation of NADH that is generated as a result of glucose oxidation by NAD‐GDH. DI and NAD‐GDH are co‐immobilized via affinity binding on an avidin‐modified indium tin oxide electrode to obtain fast and stable ENN redox cycling. Two enzymes (DI and NAD‐GDH) and two electron mediators [Os(bpy)2Cl2 and NAD+] are insensitive to oxygen. The applied potential (0.0 V vs Ag/AgCl) is low enough to minimize interfering electrochemical reactions, and the redox reactions of Os(bpy)2Cl2 with interfering species are slow. NAD‐GDH is much less reactive to problematic monosaccharides such as xylose, fructose, galactose, and mannose than glucose. Artificial serum containing 5 % (w/v) human serum albumin shows a similar electrochemical background level in serum. All results enable us to obtain selective and reproducible glucose detection. The fast ENN redox cycling allows sensitive glucose detection with a wide range of concentrations in artificial serum with a short measuring time (5 s) without an incubation period.

Electrochemical Detection of Glucose and Lactate Dehydrogenase Using a Zwitterionic Phenazine Compound as an Electron Mediator for NADH Oxidation

“…The detection limit of glucose was approximately 0.02 mM. Considering that the normal concentration range of glucose in human serum is 3.9–6.6 mM [2e], the detection limit is sufficiently low to detect glucose in human serum.…”

“…Many metabolites are detected by measuring the concentration of NADH (or NAD + ) that is generated when NAD + (or NADH) is added to a sample solution either with redox enzyme(s) associated with a target metabolite (case i) or with the metabolite(s) associated with a target redox enzyme (case ii) [2]. For sensitive and selective measurements, NADH (or NAD + ) is converted into a highly fluorescent or electroactive signaling species in the presence of an enzyme such as diaphorase [2e, 3]. In electrochemical measurements, this enzymatic conversion is of great importance because the electrochemical oxidation of NADH (or reduction of NAD + ) requires a large overpotential even at a highly electrocatalytic electrode [4].…”

Addressing the Selectivity of Enzyme Biosensors: Solutions and Perspectives

“…Thus, the diaphorases both recycles NAD(P) + /NAD(P)H that is useful to shift the equilibrium in the desired direction and also facilitate the electrochemical detection. One typical example of a combination dehydrogenase with diaphorase is the detection of glucose using NAD-dependent glucose dehydrogenase co-immobilized with diaphorase from Bacillus stearothermophilus (EC 1.6.99.-) and an osmium complex used as an electrochemical mediator between the electrode and diaphorase [ 60 ]. Nevertheless, the advantages provided by diaphorase must be balanced against the inherent complications due to a bienzymatic enzyme and one should also take into consideration the NAD(P)H can be electrochemically detected using various electrode-mediators [ 61 ].…”

Development of a Glucose Sensor Based on Glucose Dehydrogenase Using Polydopamine-Functionalized Nanotubes

“…Within second-generation glucose sensors, glucose is oxidized by GOx to produce gluconic acid and H 2 O 2 [ 6 , 11 ], and electrons generated by glucose oxidation are transferred to the electrode via an electron transfer mediator such as iron or ruthenium—a sensing concept which has been employed in commercial glucose biosensors [ 9 , 10 , 12 ]. GOx has a high temperature and pH stability along with excellent glucose substrate selectivity [ 13 , 14 , 15 ]; however, GOx uses O 2 as an external electron acceptor in the oxidation reaction so device performance is sensitive to and variable depending on the atmospheric oxygen level [ 16 , 17 , 18 ]. To overcome this challenge and improve sensing reliability, glucose dehydrogenase (GDH) does not require O 2 and is hence being used in various types of glucose sensors together with pyrroloquinoline (PQQ), nicotinamide adenine dinucleotide (NAD), and flavin adenine dinucleotide (FAD) redox cofactors [ 19 , 20 , 21 ].…”