Substrate Specificity and Interferences of a Direct-Electron-Transfer-Based Glucose Biosensor

Abstract

Abbreviations: (Ag/AgCl) silver/silver chloride, (CDH) cellobiose dehydrogenase, (CGMS) continuous glucose monitoring system, (CLSI) Clinical and Laboratory Standards Institute, (DET) direct electron transfer, (FAD) flavin adenine dinucleotide, (GDH) glucose dehydrogenase, (GOx) glucose oxidase, (PBS) phosphate-buffered saline, (PQQ) pyrroloquinoline quinone Electrochemical sensors for glucose monitoring employ different signal transduction strategies for electron transfer from the biorecognition element to the electrode surface. We present a biosensor that employs direct electron transfer and evaluate its response to various interfering substances known to affect glucose biosensors. Methods:The enzyme cellobiose dehydrogenase (CDH) was adsorbed on the surface of a carbon working electrode and covalently bound by cross linking. The response of CDH-modified electrodes to glucose and possible interfering compounds was measured by flow-injection analysis, linear sweep, and chronoamperometry. Results:Chronoamperometry showed initial swelling/wetting of the electrode. After stabilization, the signal was stable and a sensitivity of 0.21 µA mM -1 cm -2 was obtained. To investigate the influence of the interfering substances on the biorecognition element, the simplest possible sensor architecture was used. The biosensor showed little (<5% signal deviation) or no response to various reported electroactive or otherwise interfering substances. Conclusions:Direct electron transfer from the biorecognition element to the electrode is a new principle applied to glucose biosensors, which can be operated at a low polarization potential of -100 mV versus silver/silver chloride. The reduction of interferences by electrochemically active substances is an attractive feature of this promising technology for the development of continuous glucose biosensors.

Transforming the blood glucose meter into a general healthcare meter for in vitro diagnostics in mobile health

“…The efficiency of DET depends not only on the distance between the active site of the enzyme and the electrode but also on the properties of the electrode material and on the immobilization technique (Tellechea et al, 2012, Ludwig et al, 2013). For the limited number of GOx and GDH that are capable of DET, the efficient electron transfer still requires conformational change of the enzyme, which may result in an obvious loss of activity (Felice et al, 2013, Seehuber and Dahint, 2013). An appropriate balance between the enzymatic and electrochemical activities is vital for DET glucose biosensors, and more innovation is still necessary to reach that balance and a commercially viable product.…”

Molecular and catalytic properties of fungal extracellular cellobiose dehydrogenase produced in prokaryotic and eukaryotic expression systems

“…Class III contains a different branch of so far uncharacterized CDHs [ 9 ]. Due to its electron transferring properties, CDH has been widely recognized as a versatile biorecognition element in electrochemical biosensors, which is capable of detecting a wide variety of carbohydrates (cellobiose, cellodextrins, lactose, maltose, glucose) as well as quinones and catecholamines [ 10 , 11 ]. This ability is also exploited for the development of CDH-based anodes in enzymatic biofuel cells [ 12 ].…”

Engineering of Cellobiose Dehydrogenases for Improved Glucose Sensitivity and Reduced Maltose Affinity

“…wt Ct CDH and wt Hi CDH were recombinantly overexpressed in Pichia pastoris and Aspergillus oryzae , respectively. Variants with a single mutation in the active site were constructed for each enzyme.…”

“…The resulting plasmid was linearized with Sac I and transformed into electrocompetent Pichia pastoris X‐33 cells. Recombinant wild‐type (wt Ct CDH) and mutated Ct CDH ( Ct CDH C291Y) were heterologously expressed in the methylotropic yeast P. pastoris and purified as described previously …”

“…Alternatives to GOx have been studied, where PQQ‐dependent glucose dehydrogenase (PQQ‐GDH) has been the main competitor . In the last few years other dehydrogenases have appeared that have the advantage of having a low or none activity towards O 2 , making them real alternatives to GOx; among them FAD‐dependent GDH, NAD + dependent GDH, pyranose dehydrogenase (PDH) from wood degrading fungi and cellobiose dehydrogenase (CDH) …”