Fluorescence Quenching-Based Evaluation of Glucose Oxidase Composite with Conducting Polymer, Polypyrrole

Abstract

Glucose oxidase (GOx) composites with conducting polymers (e.g., polypyrrole (Ppy)) are excellent nanobiomaterials suitable for the design of bioelectronic devices such as biosensors and biofuel cells. Here, we address the spectroscopic properties of GOx, flavin adenine dinucleotide (FAD), and composites of these compounds with polypyrrole (Ppy). The exploration of native GOx and FAD solutions confirmed that about 5% of FAD dissociated from GOx during the period of solution preparation, and this fraction remained constant for 1 month. It has been found that the Ppy, which formed composites with FAD and GOx, facilitated the removal of FAD molecules from GOx and twice reduced the fluorescence decay rate. Differences in the FAD and Ppy average fluorescence relaxation times showed that the FAD composite with Ppy and Ppy effectively quenched the FAD fluorescence and FAD could not freely unfold. The intramolecular electron transfer took place between adenine and isoalloxazine moieties over the first 5 ps after the excitation. The findings are very useful in the selection and adaptation of enzyme immobilization strategies, which are applied in the development of biosensors and biofuel cells.

Calculation of the Geometries and Infrared Spectra of the Stacked Cofactor Flavin Adenine Dinucleotide (FAD) as the Prerequisite for Studies of Light-Triggered Proton and Electron Transfer

“…The majority of computational studies on FAD in the past have been dedicated to flavoproteins with FAD adopting T-shaped and other "open" geometries, the "stacked" geometry of FAD has not yet been the subject of those studies. The recent publication of Bubniene et al [16] reflects this gap. According to Bubniene et al differences in the FAD and polypyrrole average fluorescence relaxation time showed that the FAD composite with polypyrrole effectively quenched the FAD fluorescence since FAD could not freely unfold in this environment.…”

“…The model of Li et al [13,14] has been adopted by Sengupta et al [15] in their investigation of pH-dependent dynamic behavior of FMN and FAD on a femtosecond to nanosecond time scale. More recently the pH-depending prevalence of the stacked form of FAD proposed by Li et al [13] has been used as a model concept in the publication of Bubniene et al [16] about the fluorescence quenching-based evaluation of a glucose oxidase composite with a conducting polymer-polypyrrole: The stacked form of FAD is thought to be responsible for the fast quenching of the FAD fluorescence, which could not unfold freely in this specific environment as the authors stated.Despite theoretical studies which include lumiflavin, riboflavin and FMN as a model of FAD-flavoprotein interactions in proteins [17][18][19][20], theoretical studies of the stacked form of FAD as well as studies of the unfolding process from the closed to the open conformer are still missing. The present work aims to fill this gap.…”

“…The model of Li et al [13,14] has been adopted by Sengupta et al [15] in their investigation of pH-dependent dynamic behavior of FMN and FAD on a femtosecond to nanosecond time scale. More recently the pH-depending prevalence of the stacked form of FAD proposed by Li et al [13] has been used as a model concept in the publication of Bubniene et al [16] about the fluorescence quenching-based evaluation of a glucose oxidase composite with a conducting polymer-polypyrrole: The stacked form of FAD is thought to be responsible for the fast quenching of the FAD fluorescence, which could not unfold freely in this specific environment as the authors stated.…”

The Energy Consumed by Electrochemical Molecular Machines as Self‐Sensor of the Reaction Conditions: Origin of Sensing Nervous Pulses and Asymmetry in Biological Functions

“…The sensing features of the reaction rate have been used from the very beginning of the electroanalytical methodologies (polarographic, voltammetric, chronoamperometric, etcetera) to determine the concentration of the analyte reacting either, directly on any non‐reactive electrode or indirectly through an enzyme or an catalyst adsorbed on the electrode …”

“…The sensing features of the reaction rate have been used from the very beginning of the electroanalytical methodologies (polarographic, voltammetric, chronoamperometric, etcetera) to determine the concentration of the analyte reacting either, directly on any non-reactive electrode [8] or indirectly through an enzyme [9] or an catalyst adsorbed on the electrode. [10][11][12] Recently, this sensing principle has been reconsidered and generalized by our research group substituting the passive electrodes by electroactive electrodic materials constituted by multistep molecular machines and studying how the energy consumed by the electrodic reaction is influenced by the different physical or chemical working conditions.…”