A Self‐Assembled Respiratory Chain that Catalyzes NADH Oxidation by Ubiquinone‐10 Cycling between Complex I and the Alternative Oxidase

Complex I is a crucial respiratory enzyme that conserves the energy from NADH oxidation by ubiquinone‐10 (Q₁₀) in proton transport across a membrane. Studies of its energy transduction mechanism are hindered by the extreme hydrophobicity of Q₁₀, and they have so far relied on native membranes with many components or on hydrophilic Q₁₀ analogues that partition into membranes and undergo side reactions. Herein, we present a self‐assembled system without these limitations: proteoliposomes containing mammalian complex I, Q₁₀, and a quinol oxidase (the alternative oxidase, AOX) to recycle Q₁₀H₂ to Q₁₀. AOX is present in excess, so complex I is completely rate determining and the Q₁₀ pool is kept oxidized under steady‐state catalysis. The system was used to measure a fully‐defined K_M value for Q₁₀. The strategy is suitable for any enzyme with a hydrophobic quinone/quinol substrate, and could be used to characterize hydrophobic inhibitors with potential applications as pharmaceuticals, pesticides, or fungicides.     

Exploring Redox Behavior of Neutral pH Active Poly(melamine) and its Electrocatalytic Origination towards NADH Detection

We report here in detail the redox properties and catalytic origination of poly(melamine). Gradual change in redox behavior of the monomer from polymer is explained by grafting monomer and different amounts of polymer on screen printed carbon electrode. The redox peak of the poly(melamine) is pH‐dependent with a slope of ?60 mV/pH, representing a Nernstian type proton‐coupled electron‐transfer process. Catalytic origination from the ? NH‐NH? bond formed in polymer is proved using NADH as a probe. This polymer is effective to catalyze NADH oxidation with a wide linear range of 1 μM–10 mM and a detection limit (S/N=3) of 0.67 μM. Most importantly, unlike most neutral pH active polymer dyes, it shows high stability even after 15 days.     

Horseradish Peroxidase Enzyme Electrodes for Nadh and Nadph

Abstract

The aerobic oxidation of reduced pyridine phosphonucleotides catalyzed by horseradish peroxidase (E.C.1.11.1.7) (HRP) in combination with Mn²⁺-ions, phenolics and redox dyes was used to perform NADH and NADPH determinations with an amperometric HRP enzyme electrode. Optimal operational conditions have been elaborated for such a sensor and a linear relationship between derivative current (dI/dt) and NADH-concentration was obtained between 2.10^?5 and at least 2.10^?4 mol/l. The coefficient of variation was equal to or smaller than 4 %. The time for one measurement was less than 10 minutes including equilibration of the electrode.     

Fluorescence Properties of Carba Nicotinamide Adenine Dinucleotide for Glucose Sensing

Carba nicotinamide adenine dinucleotide (cNAD) may serve as a stable cofactor for the enzyme‐based detection of glucose. Many characteristics of cNAD and its reduced form cNADH resemble those of NAD and NADH, respectively. The fluorescence lifetimes of cNADH are determined to be 0.32(2) ns and 0.66(3) ns compared to 0.28(2) ns and 0.60(3) ns for NADH, and the temperature dependence of these lifetimes hints towards identical processes for quenching. The maximum emission occurs at 464 nm for both cNADH and NADH and absorbance maxima are found at 360 nm and 340 nm, respectively. In contrast to previous suggestions the respective maximum extinction coefficient of cNADH equals that of NADH and amounts to 6.2(2) mM ^?1 cm^?1. When changing from NADH to cNADH we observe a ～50 % increase in quantum efficiency, which—together with the larger excitation wavelength and the higher stability—should make cNAD a well suited alternative as coenzyme for robust glucose detection.     

Sensitive Detection of NADH by Ferrocenylalkanethiol Functionalized Multiwall Carbon Nanotubes Electrodes

Abstract

The 6-ferrocenylhexanethiol (FcC₆SH) functionalized multiwall carbon nanotubes (MWNTs) modified glassy carbon electrode (FcC₆SH/MWNTs/GCE) was easily fabricated and used for the sensitive detection of NADH. Cyclic voltammetric and amperometric methods were used to study the behavior of NADH on the FcC₆SH/MWNTs/GCE. A broader linear response range to the NADH concentration from 5 µM to 1.5 mM with a correlation coefficient of 0.9982 was obtained. The detection limit was 0.54 µM. The synergetic effects of FcC₆SH and MWNTs make the modified electrode highly sensitive to NADH. In addition, the modified electrode can decrease the fouling of the electrode surface.     

Chemical Cross-Linking of a Redox Mediator Thionin for Electrocatalytic Oxidation of Reduced β-Nicotinamide Adenine Dinucleotide

Abstract

Thionin, a redox mediator that has been used to study the electrochemical behavior of reduced β-nicotinamide adenine dinucleotide (NADH), was chemically cross-linked on the surface of a spectroscopic graphite electrode by using a triisocyanate cross-linking agent. The electrodes modified in this manner had a purple film with an additional reversible redox couple a t E° of +73 mV vs. Ag/AgCl compared to uncross-linked thionin. The thionin modified electrode mediated oxidation of NADH with response to NADH between 7.0 × 10–7 to 1.8 × 10–3 M, a sensitivity of 113 pAJcmWmM, and a detection limit of 0.5 μM.     

NADH Oxidation Catalyzed by Electropolymerized Azines on Carbon Nanotube Modified Electrodes

Electropolymerizing azines on a carbon nanotube (CNT) modified electrode yields a high‐surface area interface with excellent electrocatalytic activity towards NADH oxidation. Electrodeposition of poly(methylene green) (PMG) and poly(toluidine blue) (PTBO) on the carboxylated CNT‐modified electrodes was achieved by cyclic voltammetry. The PMG‐CNT interface demonstrates 5.0 mA cm^?2 current density for NADH oxidation at 50 mV vs. Ag|AgCl in 20 mM NADH solution. The kinetics of NADH electrocatalysis were analyzed using a quantitative mass‐transport‐corrected model with NADH bulk concentration and applied potential as independent variables. This high‐rate poly(azine)‐CNT interface is potentially applicable to high‐performance bioconversion, bioenergy and biosensors involving NADH‐dependent dehydrogenases.     

Dehydrogenase‐Based Reagentless Biosensors: Electrochemically Assisted Deposition of Sol‐Gel Thin Films on Functionalized Carbon Nanotubes

Multiwalled carbon nanotubes (MWCNT) have been functionalized, for the electrocatalytic detection of NADH, by microwave treatment, electrochemical deposition of poly(methylene green) or wrapping with an Os‐complex modified polymer. Sol‐gel thin films have been then electrodeposited on the carbon nanotube layers for co‐immobilization of D ‐sorbitol dehydrogenase and diaphorase when necessary and NAD⁺ via covalent linkage using glycidoxypropyltrimethoxysilane. The comparison of these systems shows that the electrodeposited sol‐gel matrix can significantly affect the operational behavior of functionalized MWCNT. Only MWCNT wrapped with the Os‐complex modified polymer and covered with a sol‐gel biocomposite allowed the electrochemical detection of D ‐sorbitol in a reagentless configuration.     

Electrodeposition of Catechol on Glassy Carbon Electrode and Its Electrocatalytic Activity Toward NADH Oxidation

Catechol can be oxidized electrochemically to its corresponding o‐benzoquinone. The electrogenerated quinone can be deposited by cycling the potential at the surface of glassy carbon electrodes. We have studied the electrochemical features of films derived from catechol by cyclic voltammetry. The electrodeposited film shows stable reversible redox response, dependent on pH as anticipated for quinone/catechol functionalities. Glassy carbon electrodes covered with a film derived from catechol exhibit catalytic activity in the electrooxidation of NADH at a low potential. The catalytic current is proportional to the concentration of NADH over the range 0.02–0.34 mM.     

Fabrication of Redox‐Mediator Supported Potentiometric Nitrate Biosensor with Nitrate Reductase

Fabrication of a more superior nitrate potentiometric biosensor than previously achieved with NaR and NADH has been accomplished by co‐entrapment of redox mediators and NaR into polypyrrole (PPy) film during galvanostatic polymerization of pyrrole. The replacement of NADH with redox mediators such as thionin acetate (ThAc), safranin (Saf), and azure A (AzA) gave more sensitive potentiometric responses, better minimum detectable concentration, linear concentration range and response time for nitrate than possible with NADH. The co‐entrapment of ThAc, Saf, AzA and methyl viologen (MV) with NaR into PPy films also improved the Nernstian behavior of the electrode process beyond the capability of the PPy‐NaR‐NADH biosensor. Substantial reduction in volume and quantity of cofactor/mediator and, hence cost, was achieved by the replacement of NADH with a redox mediator. Only 50 μM of AzA was required to form a PPy‐NaR‐AzA biosensor which gave the most sensitive potentiometric response for nitrate, achieving a minimum detectable concentration of 10 μM, a linear concentration range of 50–5000 μM and a response time of 2–4 s.