When redox enzymes are wired to electrodes outside a living cell (ex vivo), their ability to produce a sufficiently powerful electrical current diminishes significantly due to the thermodynamic and kinetic limitations associated with the wiring systems. Therefore, we are yet to harness the full potential of redox enzymes for the development of self-powering bioelectronics devices (such as sensors and fuel cells). Interestingly, nature uses iron-sulfur complexes ([Fe-S]), to circumvent these issues in vivo. Yet, we have not been able to utilize [Fe-S]-based chains ex vivo, primarily due to their instability in aqueous media. Here, a simple technique to attach iron (II) sulfide (FeS) to a gold surface in ethanol media and then complete the attachment of the enzyme in aqueous media is reported. Cyclic voltammetry and spectroscopy techniques confirmed the concatenation of FeS and glycerol-dehydrogenase/nicotinamide-adenine-dinucleotide (GlDH-NAD(+)) apoenzyme-coenzyme molecular wiring system on the base gold electrode. The resultant FeS-based enzyme electrode reached an open circuit voltage closer to its standard potential under a wide range of glycerol concentrations (0.001-1M). When probed under constant potential conditions, the FeS-based electrode was able to amplify current by over 10 fold as compared to electrodes fabricated with the conventional pyrroloquinoline quinone-based composite molecular wiring system. These improvements in current/voltage responses open up a wide range of possibilities for fabricating self-powering, bio-electronic devices.

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An electrochemical biosensor for glycerol was obtained by using a novel concatenation of molecules to immobilize glycerol dehydrogenase (GlDH) on a gold electrode via layer-by-layer (LBL) self-assembly. The surface of the enzyme electrodes was characterized by cyclic voltammetry and scanning electron microscopy which confirmed the attachment of enzyme on the gold electrode with the assistance of the tethering molecules. The biosensor was assessed for its potentiometric and amperometric response to glycerol in the presence of the enzyme stimulants, ammonium sulfate and manganese chloride. The electrodes demonstrated good selectivity and reproducibility, with a amperometric response at a working voltage of 1.3 V in the 0.001 to 1 M glycerol concentration range, a 12.07 μA·M −1 sensitivity, and a 6.8 μM lower limit of detection. The average diffusion coefficient of glycerol is 8.63×10 −6 cm 2 s −1 as determined by chronoamperometry.

An improved glycerol biosensor was developed via direct attachment of NAD-glycerol dehydrogenase coenzyme-apoenzyme complex onto supporting gold electrodes, using novel inorganic iron (II) sulfide (FeS)-based single molecular wires. Sensing performance factors, i.e., sensitivity, a detection limit and response time of the FeS and conventional pyrroloquinoline quinone (PQQ)-based biosensor were evaluated by dynamic constant potential amperometry at 1.3V under non-buffered conditions. For glycerol concentrations ranging from 1 to 25mM, a 77% increase in sensitivity and a 53% decrease in detection limit were observed for the FeS-based biosensor when compared to the conventional PQQ-based counterpart. The electrochemical behavior of the FeS-based glycerol biosensor was analyzed at different concentrations of glycerol, accompanied by an investigation into the effects of applied potential and scan rate on the current response. Effects of enzyme stimulants ((NH)SO and MnCl·4HO) concentrations and buffers/pH (potassium phosphate buffer pH 6-8, Tris buffer pH 8-10) on the current responses generated by the FeS-based glycerol biosensor were also studied. The optimal detection conditions were 0.03M (NH)SO and 0.3µm MnCl·4HO in non-buffered aqueous electrolyte under stirring whereas under non-stirring, Tris buffer at pH 10 with 0.03M (NH)SO and 30µm MnCl·4HO were found to be optimal detection conditions. Interference by glucose, fructose, ethanol, and acetic acid in glycerol detection was studied. The observations indicated a promising enhancement in glycerol detection using the novel FeS-based glycerol sensing electrode compared to the conventional PQQ-based one. These findings support the premise that FeS-based bioanodes are capable of biosensing glycerol successfully and may be applicable for other enzymatic biosensors.