Glycerol Oxidation By NAD+-Dependent Enzymatic Systems
Authors: Narváez Villarrubia Claudia W. , Atanassov Plamen 
Published: February 2020

The development of lactate biosensors have been considered to monitor the gauge time the human body can exercise in anaerobic conditions1, after passing aerobic conditions, and before the metabolic process produces the body to collapse due to lack of oxygen consumed during the exercise. Current technology requires the invasive collection of blood samples to accurately determine the lactate level during exercise which make the monitor process inconvenient for general use outside the lab. In order to overcome the sampling process limitation, an external non-invasive and reagentless biosensor design needs to be considered. As the lactate and glucose level in sweat is needed to correlate to the gauge time the body has been exercising under anaerobic conditions, and glucose is not present in sweat, the biosensor device needs to be integrated to a biofuel cell capable to power and induce a reverse-iontophoretic process for glucose/lactate sampling. The biofuel powering the device need to be introduced into the biosensor-integrating-biofuel cell design and accomplish biocompatibility, storage-in-device and power output requirements to successfully collect glucose from subcutaneous fluids by reverse-iontophoresis. In this research, the use of glycerol, a high energy density fuel, available mainly from biodiesel manufacture, has been considered as biofuel to be employed in a reagentless air-breathing biofuel cell design.
This paper will focus in the development of the bioanode to fully oxidize glycerol. The glycerol conversion to CO2 could be done by a sequential oxidation with nicotinamide adenine dinucleotide (NAD+/NADH)-dependent enzymes such as alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AlDH) and oxalate oxidase (OxOx), lignin peroxidase (Lig) or Pt-Ru nanostructured catalyst.2 The anode design makes use of electrodeposited methylene green (MG) as catalyst toward the oxidation of NADH to NAD+ in order to minimize the overpotential of this reaction and provide the cofactor to the enzymes.3 Also, the NAD+/NADH cofactor will be tethered on the electrode surface by pyrene butanoic acid succinimidyl ester (PBSE) linker to the carbon nanotube-based material.4 Multiwalled carbon nanotube (MWCNTs)-based bucky papers are promising electrode materials for biofuel cells because of the combination of the MWCNTs high electrical conductivity, mechanical strength, thermal stability and chemical stability.5 On the bucky paper surface, the enzymes are immobilized in a polymeric chitosan-carbon nanotubes (CNTs) matrix after MG electrodeposition. In the end, two bioanode designs, a fully enzymatic and a hybrid (employing Pt-Ru catalyst) could be constructed, characterized and compared for full glycerol oxidation to CO2.
The glycerol-based bioanode can later be integrated to the lactate/glucose sensor design to induce reverse-iontophoresis of glucose from interstitial fluids. The utilization of glycerol in a lactate sensor will facilitate the storage of the biofuel in the patch-sensor design due to its low evaporation rate and high viscosity. The sensor/biofuel cell will be user-friendly and non-invasive lactate/glucose sampling.

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