Potentiometric Glycerol Biosensor Based On Immobilization Of Glycerol-Dehydrogenase On Au Using A Molecular Wiring System

An intensive interest in glycerol determination exists due to its extensive applications in major industries including medical, food and biofuels. This has led to a quest for a simple, specific, rapid and highly sensitive method for glycerol quantification. This study attempts to develop an enzyme-based electrochemical biosensor for glycerol quantification using NAD⁺-dependent enzyme, Glycerol dehydrogenase (GlDH) immobilized on a modified gold (Au) electrode. The objective of this study is to fabricate a molecular wiring system to anchor GlDH enzyme onto a Au electrode and to evaluate the efficacy of the proposed wiring system to effectively transport generated charges (as a result of glycerol oxidation) from the enzyme active site to the supporting electrode. A novel concatenation of molecules was utilized to attach the enzyme onto the Au surface via layer-by-layer (LBL) self-assembly method. Surface characterization of the enzyme electrodes using cyclic voltammetry and SEM techniques confirmed attachment of enzyme on the Au electrode with the assistance of the tethering molecules. The enzymatic biosensor was assessed for its potentiometric response to glycerol concentrations ranging from low (0.001M) to very high (10 M). The effects of supporting electrolyte(s) with varying buffers and presence of enzyme stimulators on the electrical potential generated were analyzed and compared. Current sensing-Atomic force microscopy (CS-AFM) was used to elucidate the overall surface electrical conductance of the electrodes. The carrier electrolytes and glycerol concentrations had a significant impact on open circuit potentials produced by the cell (p-value<0.0001). The glycerol vs electrode open circuit voltage was highly correlated with a logarithmic relationship with a correlation coefficient of R² = 0.9921 in a water solution with enzyme stimulants and R²=0.9643 in pure water (both at a (pH ~ 4.5). Water along with enzyme stimulators was found to be the suitable carrier electrolyte for producing high potentials. The potentiometric electrode showed high reproducibility and repeatability for the determination of glycerol concentrations ranging from 0.01 – 10M in 1M resolution. It was observed that attachment of the enzyme to the NAD⁺ layer caused a significant reduction in current flow, and thus the electron transport. The crosslinking agent, glutaraldehyde also seemed to negatively affect charge transport between the enzyme active site. The CS-AFM images obtained showed that the surface charge distribution in the electrode was unhindered as a result of enzyme attachment.  Simple fabrication, high speed of measurement, high sensitivity and possibility of miniaturization are the main advantages of this biosensor.  Additionally, glycerol being the backbone of triglycerides, the long-term goal of the study is to fabricate a lipid sensor using the concentration of glycerol produced during lipid hydrolysis as an indirect modality for measuring lipids. Sensing glycerol accurately with a simple set-up is the first step toward this longer-term goal.