Potentiometric vs amperometric sensing of glycerol using glycerol dehydrogenase immobilized via layer-by-layer self-assembly

2018

Microchim Acta

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Wiring the active site of an enzyme directly to an electrode is the key to ensuring efficient electron transfer for the proper performance of enzyme-based bioelectronic systems. Iron-sulfur complexes, the first link between proteins and mediating molecules in the biological electron transport chain(s), possess an intrinsic electron transport capability. The authors demonstrate the application of inorganic iron-sulfur clusters (Fe-S) viz. FeS, FeS2, Fe2S3, and Fe3S4, as molecular wires to mediate electron transport between a glucose-selective redox enzyme and the gold electrode. It is shown that Fe-S can emulate the functionality of the natural electron transport chain. Voltammetric studies indicate a significant improvement in electron transport, surface coverage, and resilience achieved by the Fe-S-based glucose anodes when compared to a conventional pyrroloquinoline quinone (PQQ)-based electrode. The Fe-S-based glucose anodes showed glucose oxidation at a potential of +0.5 V vs. Ag/AgCl with Tris-HCl buffer (pH 8) acting as a carrier. The current densities positively correlated with the concentrations of glucose in the range 0.1–100 mM displaying detection limits of 0.77 mM (FeS), 1.22 mM (FeS2), 2.95 mM (Fe2S3), and 14.57 mM (Fe3S4). The metal-anchorable sulfur atom, the strong π-coordinating iron atom, the favorable redox properties, low cost, and natural abundance make Fe-S an excellent electron-mediating relay capable of wiring redox active sites to electrode surfaces. Graphical abstractSchematic representation of inorganic iron-sulfur clusters used as molecular wires to facilitate direct electron transfer between NAD-glucose dehydrogenase and the gold electrode. The iron-sulfur based glucose anodes improve current response to selectively sense glucose concentrations in the range 0.1–100 mM. Electronic supplementary materialThe online version of this article (10.1007/s00604-018-2871-x) contains supplementary material, which is available to authorized users.

Section: Resultsmentioning

confidence: 99%

Inorganic iron-sulfur clusters enhance electron transport when used for wiring the NAD-glucose dehydrogenase based redox system

2018

Microchim Acta

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“…Water containing the enzyme stimulants viz. (NH 4 ) 2 SO 4 and MnCl 2 .4H 2 O was used as the point of reference carrier electrolyte as described previously (Mahadevan et al 2015). Molecular-biology-grade water (termed "pure water" in this paper) obtained from Sigma-Aldrich was used to prepare all the aqueous-based solutions and for rinsing and cleaning throughout this study.…”

Section: Reagents and Apparatusmentioning

confidence: 99%

An improved glycerol biosensor with an Au-FeS-NAD-glycerol-dehydrogenase anode

Biosensors and Bioelectronics

2017

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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.

“…First, the gold (Au) electrodes were prepared for modification by following the three-step rigorous cleaning procedure as described earlier (Mahadevan et al 2014). The clean Au electrodes were immersed in a 0.3 M FeS solution suspended in ethanol for 2 h. Then the FeS-modified electrodes were immersed in 1 mM of ß-NAD + for 2 h followed by 1 mg mL -1 of GlDH for 2 h for formation of NAD+ and GlDH monolayers respectively.…”

Section: Fabrication Of the Bioanodementioning

confidence: 99%

“…As a working model, a glycerol-sensitive gold bioanode is described based on direct attachment of the glycerol-dehydrogenase (GlDH)-NAD + apoenzyme-coenzyme complex onto the supporting gold surface using iron (II) sulfide (FeS) mediation. A conventional PQQ-based electrode, described previously (Mahadevan et al 2014), was used as the control. The performances of the two electrode systems were compared using amperometric and potentiometric studies.…”

Section: Introductionmentioning

confidence: 99%

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Iron–sulfur-based single molecular wires for enhancing charge transport in enzyme-based bioelectronic systems

Biosensors and Bioelectronics

2016

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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.