Laccase Biocathode Research Articles

Design and Evaluation of a Microfluidic Device Such As a Self-Powered Glucose Biosensor Using a Solution of Glucose and Human Blood

Glucose sensors have been seen as a greater research interest because they are related to type I diabetes disease and because individuals diagnosed with this disease are more susceptible to complications, such as high blood pressure, high cholesterol levels, eye problems, and even death. Self-powered glucose biosensor (SPGB) based on biofuel cells (BFCs) combine the operational systems of a BFC and a biosensor in where the energy could be used for glucose quantification as an SPGB and operate the sensor without the need for any external energy source. This strategy could have certain advantages as the sensor is limited to two electrodes without applying an external voltage, it can be fed with the same biological fluid with the prospect of being implanted, the interferers can be decreased by not applying a potential, and a single concentration of the substrate is sufficient to calibrate the system. In this context, the microfluidics has proven to be an important technology in the area of biosensors and has been used to transport or mix the sample, this device using different and independent electrolytes depends on the enzyme to improve performance, reported in the work group before. The glucose oxidase (GOx) bioanode was prepared using dimethylferrocene-modified linear polyethyleneimine (FcMe2-LPEI). On other hand, the laccase (Lc) biocathode was prepared using anthracene-modified MWCNTs (AnMWCNTs). The microfluidic device consisted of two parts fabricated from PMMA using a micromilling system CNC (Computer Numerical Control). For the calibration curve, it was used as anolyte PBS pH 7.4 (0.1 M) using 0, 1, 3, 5, 7, 10, and 20 mM glucose and PBS pH 5.6 (0.1 M). In the case of the real samples, the anolyte was blood or serum, and the catholyte was PBS pH 5.6. The electrolytes were fed using a syringe pump (Kd Scientific) at a flow rate of 3 mL h-1. Polarization curves for microfluidic self-powered glucose biosensing (m-SPGB) were obtained by linear polarization from OCP to 0 V at 2 mV s-1. All experiments were performed in triplicate, and the average ± standard deviation was reported. The interferers evaluated in the m-SPGB were 0.025 mM ascorbic acid (AA), 0.1 mM uric acid (UA) and 0.5 mM dopamine (DA) and arranged in the anolyte prepared with PBS pH 7.4 (0.1 M). The architecture of the m-SPGB device is defined mainly by two modules: the sensor unit and the Wi-Fi transmission/reception unit. The electrochemical characterization of the FcMe2-LPEI/GOx bioanode and AnMWCNT/Lc biocathode immobilized on TCP was performed by cyclic voltammetry (CV), showing activity towards glucose oxidation and oxygen reduction respectively. Confirming the electrocatalytic activity of the bioelectrodes, both were used in the m-SPGB using PBS pH 7.4 and different concentrations of glucose in the anolyte and PBS pH 5.6 as the catholyte. Under these operating conditions, the average OCP was 0.81 ± 0.021 V constant voltage at the different glucose concentrations evaluated. The result is important because the system is sensitive to low glucose concentrations of 1 mM and 3 mM, and an increase in the current is observed, differentiating very well in each concentration from 0 to 20 mM. Plots of the comparative responses to the increase in current and power in the m-SPGB versus glucose concentration. The linear ranges of both parameters were 0–10 mM with correlation coefficients (R2) of 0.9907 and 0.9913, respectively; in both cases, they were within the range to measure blood or serum glucose. On the other hand, the sensitivity was higher, at a current of 1.24 μA mM-1, while the power was 0.19 μW mM-1, with a better limit of detection (LOD) of 0.48 mM vs. 0.76 mM of the power. The electronic device connected to the m-SPGB was used for the quantification of the different glucose concentrations, sending the wireless-type information to a laptop for processing and more efficient interpretation without the need for a potentiostat/galvanostat and the connection between the electronic device for sending and processing the information and the microfluidic device where the enzymatic reactions are carried out. In summary, the results, in general, are comparable and superior to other SPGBs published by other authors and show their importance in using a microfluidic system.

Spider nest shaped multi-scale three-dimensional enzymatic electrodes for glucose/oxygen biofuel cells

A spider nest shaped multi-scale three-dimensional substrate consisting of reduced graphene oxide (RGO) and nickel foam is fabricated for enzymatic electrodes and biofuel cells. According to the excellent conductivity and large electroactive surface area of this special structure, the enzymatic electrodes show large enzymatic loading density, low electron transfer resistance and high electrocatalytic efficiency. The strong forces in the spider nest shaped structure and the excellent enzymatic embedding method ensure the stability of the glucose oxidase bioanodes and laccase biocathodes. The Michaelis–Menten constant value for the bioanodes to glucose is calculated to be 2.24 mM, which is close to the Michaelis–Menten constant for free glucose oxidase, implying a remarkably high enzymatic activity. Employed the obtained bioelectrodes with great properties, the relative glucose/oxygen biofuel cell has an open-circuit voltage of 0.70 V, with a high output power performance, and a maximum output power of 7.05 ± 0.05 mW cm−2, owe to the high enzyme loading and low electron transfer resistance of the electrode based on the spider nest shaped structure. After 60 days of periodic storage experiments, the performance of the biofuel cell still maintained 84.2%, showing a good long-term stability.

A Novel and Enhanced Membrane-Free Performance of Glucose/O2 Biofuel Cell, Integrated With Biocompatible Laccase Nanoflower Biocathode and Glucose Dehydrogenase Bioanode

In this research, a novel glucose/O2 biofuel cell was developed with glucose dehydrogenase bioande and laccase biocathode. For the preparation of a bioanode, glassy carbon electrode was modified in a layer by layer structure of graphene oxide nano-sheets, glucose dehydrogenase, and nicotine amide adenine dinucleotide by electrostatic affinities and the capping layer consist of glutaraldehyde, bovine serum albumin and glucose dehydrogenase. The proposed system depicted a high bioelectrocatalytic activity toward glucose oxidation and the oxidation commenced at −0.30 V (vs. Ag/ AgCl). For implementation in a biofuel cell, a biocompatible enzyme-based cathode was developed. Therefore, laccase nanoflowers were immobilized onto gold nanoparticles, which were electrodeposited on a gold electrode and polydopamine biofilm was employed to keep the immobilized laccase nanoflowers in place. The biocathode illustrated a high electrocatalytic activity toward oxygen reduction reaction with a direct electron transfer at a positive potential of +0.61 V (vs. Ag/AgCl). The prepared bioelectrodes were integrated into a glucose/O2 biofuel cell system, without a membrane and the performance and the stability of the biofuel cell were evaluated in vitro . The obtained operating circuit voltage and maximum current densities were 0.86 V and 1.11 mA $\cdot $ cm−2 and maximum power density of 0.40 mW $\cdot $ cm−2 was achieved at 0.67 V (vs. Ag/AgCl). The output power of the biofuel cell is almost persistent after 24 days of sustained operation. The achieved results suggest that the proposed modifications provide a high-performance and very stable biofuel cell for application in implantable medical devices.

Membraneless ethanol,O2 enzymatic biofuel cell based on laccase and ADH/NAD+ bioelectrodes

This work describes EtOH,O2 membraneless enzymatic biofuel cells (EtOH,O2 MlessEBFCs) that employ laccase-based biocathodes and ADH/NAD+ bioanode. Laccase biocathodes were prepared by immobilizing a polypyrrole film containing different redox mediators (ruthenium and osmium complexes). The bioanode for EtOH,O2 MlessEBFCs was fabricated by immobilizing multiwalled carbon nanotubes, NAD+-dependent alcohol dehydrogenase enzyme (ADH), poly-methylene green, and poly(amidoamine) (PAMAM) dendrimer onto a carbon cloth platform. Maximum power density and current density were 21.0 ± 0.2 mW cm-2 and 0.15 ± 0.07 mA cm-2, respectively, in PBS (pH 6.5). Lifetime tests conducted for EtOH,O2 MlessEBFCs showed promising perspectives for their future application in miniaturized devices.

(Invited) Confocal Raman Microscopy in the Study of Membrane Materials for Energy Conversion

Confocal Raman microscopy techniques are being adapted for quantitation, structural characterization and spatial profiling in the development of materials for electrochemical energy conversion. This presentation will discuss applications to fuel cell catalytic membranes and bipolar membranes and strategies for estimation of the limiting spatial resolution in depth profiling.1,2 In the study of catalytic membranes, experiments have focused on steps in the processing of electrodes containing biocatalyst. Confocal Raman microscope measurements confirm the presence of entrapped enzymes within dispersion cast membranes and enable charge compensating mobile ions and protein to be quantified. For the enzyme laccase, a common biofuel cell O2reduction catalyst, features associated with ligands coordinating the T1 copper center are present between 400 cm-1- 500 cm-1 and provide a possible route to interrogate mechanistic chemistry at the active site of laccase biocathodes under fuel cell reaction conditions. Using a similar experimental approach, the junction connecting the anion exchange and cation exchange phases of a bipolar membrane was spatially profiled. The transition region separating the phases was broader than the limiting axial spatial resolution of the instrument, consistent with known roughness and inhomogeneity at the boundary on the order of a few micrometers. Strong Raman scattering from perchlorate enabled its use as a probe for mobile ions in the anion-exchange phase. Self-modeling curve resolution applied to spectral datasets within a depth profile enhanced spatial resolution and confirmed perchlorate ions track the spatial distribution of the AEM phase. 1Cai, R.; Abdellaouia, S.; Kitt, J.P.; Harris, J.M.; Minteer, S.D.; Korzeniewski, C. “Confocal Raman Microscopy for the Determination of Protein and Quaternary Ammonium Ion Loadings in Biocatalytic Membranes for Electrochemical Energy Conversion and Storage” Anal. Chem.(2017) 89, 13290-13298. DOI: 10.1021/acs.analchem.7b03380. 2Korzeniewski, C.; Kitt, J.P.; Bukola, S.; Creager, S.E.; Minteer, S.D.; Harris, J.M. ”Single layer graphene for estimation of axial spatial resolution in confocal Raman microscopy depth profiling” Anal. Chem. (DOI:DOI: 10.1021/acs.analchem.8b04390).

Direct electron transfer of laccase enzyme based on RGO/AuNPs/PNR

Biofuel cell has been received much attention in recent years because the global energy demand increases every year. This paper presents a design of Mytheliophthora thermophile laccase on the electrode as a biocathode for biofuel cells based on direct electron transfer (DET) between the active site of the enzyme and reduced graphene oxide-gold nanoparticles-poly neutral red (RGO/AuNPs/PNR). The RGO/AuNPs/PNR/laccase biocathode was characterized by cyclic voltammetry (CV) method. The CV experiments demonstrated the activity, direct electron transfer, and stability of immobilized enzyme on the nanocomposite. The results showed the immobilized enzyme had good stability and performance on the nanocomposite after 10 days. Therefore, the presented method would be used in the design of biosensors or biocathode of biofuel cells.

Fuel‐Free Bio‐photoelectrochemical Cells Based on a Water/Oxygen Circulation System with a Ni:FeOOH/BiVO4 Photoanode

AbstractA bio‐photoelectrochemical cell (BPEC) based on a fuel‐free self‐circulation water–oxygen–water system was fabricated. It consists of Ni:FeOOH modified n‐type bismuth vanadate (BiVO4) photoanode and laccase catalyzed biocathode. In this BPEC, irradiation of the photoanode generates photocurrent for photo‐oxidation of water to oxygen, which is reduced to water again at the laccase biocathode. Of note, the by‐products of two electrode reactions could continue to be reacted, which means the H2O and O2 molecules are retained in an infinite loop of water–oxygen–water without any sacrificial chemical components. As a result, the assembled fuel‐free BPEC exhibits good performance with an open‐circuit potential of 0.97 V and a maximum power density of 205 μW cm−2 at 0.44 V. This BPEC based on a self‐circulation system offers a fuel‐free model to enhance multiple energy conversion and application in reality.

Fuel-Free Bio-photoelectrochemical Cells Based on a Water/Oxygen Circulation System with a Ni:FeOOH/BiVO4 Photoanode.

A bio-photoelectrochemical cell (BPEC) based on a fuel-free self-circulation water-oxygen-water system was fabricated. It consists of Ni:FeOOH modified n-type bismuth vanadate (BiVO4 ) photoanode and laccase catalyzed biocathode. In this BPEC, irradiation of the photoanode generates photocurrent for photo-oxidation of water to oxygen, which is reduced to water again at the laccase biocathode. Of note, the by-products of two electrode reactions could continue to be reacted, which means the H2 O and O2 molecules are retained in an infinite loop of water-oxygen-water without any sacrificial chemical components. As a result, the assembled fuel-free BPEC exhibits good performance with an open-circuit potential of 0.97 V and a maximum power density of 205 μW cm-2 at 0.44 V. This BPEC based on a self-circulation system offers a fuel-free model to enhance multiple energy conversion and application in reality.

Characteristics of Two Self-Powered Glucose Biosensors

Here, we describe the characterization of a self-powered glucose biosensor that is capable of generating electrical power from the biochemical energy stored in glucose to serve as the primary source of power for microelectronic devices. One self-powered glucose biosensor is based on multi-walled carbon nanotubes modified with pyroquinoline quinone glucose dehydrogenase (PQQ-GDH) and laccase at the bioanode and biocathode, respectively, whereas the other employed bilirubin oxidase at the biocathode. The self-power glucose biosensor employing the bilirubin oxidase biocathode operated at physiological condition and produced an enhanced peak power and current densities as compared with the self-powered glucose biosensor comprising of PQQ-GDH bioanode and laccase biocathode. The self-powered glucose biosensor employing bilirubin oxidase produced an average open circuit voltage of 0.480 V and delivered an average short circuit current density of 0.64 mA/cm2 with a peak power density of 0.089 mW/cm2. In addition, this self-powered glucose biosensor exhibited a linear dynamic range of 0.5–35 mM with a sensitivity of 12.221 Hz/mM $\cdot $ cm2. The use of bilirubin oxidase as the biocathodic enzyme makes it a viable candidate as a potential power source for in vivo applications.

One-year stability for a glucose/oxygen biofuel cell combined with pH reactivation of the laccase/carbon nanotube biocathode

This study reports a mixed operational/storage stability of a MWCNT-based glucose biofuel cell (GBFC) over one year. The latter was examined by performing a one hour discharge every day during one month followed by several discharges over a period of 11months. Under continuous discharge in physiological conditions (5mM glucose, 37°, pH7), the GBFC exhibits a 25% power decrease after 1h of operation. This decrease is mainly due to the deactivation of laccase biocathodes at neutral pH. Nevertheless, the biocathodes can be reversibly reactivated via storage in phosphate buffer (pH5). Under these conditions, the GBFC finally exhibits 22% of its initial maximum power density after one year at intermittent reactivation/discharge cycles. Although both GBFC electrodes can exhibit one year stability, short-term experiments show that biocathodes are limited by hydroxide inhibition while long-term experiments indicate that bioanodes are likely limited by the stability of the GOx itself. While most of the GBFCs in the literature present stability in the range of several weeks, these results demonstrate the viability of a GBFC for industrial applications in a long period of time.

Recent progress in oxygen-reducing laccase biocathodes for enzymatic biofuel cells.

This review summarizes different approaches and breakthroughs in the development of laccase-based biocathodes for bioelectrocatalytic oxygen reduction. The use of advanced electrode materials, such as nanoparticles and nanowires is underlined. The applications of recently developed laccase electrodes for enzymatic biofuel cells are reviewed with an emphasis on in vivo application of biofuel cells.

Design of an enzymatic biofuel cell with large power output

Based on a graphene–AuNP–GOx bioanode and graphene–AuNP–laccase biocathode, a novel enzymatic biofuel cell with large power output was successfully designed.

Biofuel cell for generating power from methanol substrate using alcohol oxidase bioanode and air-breathed laccase biocathode

We report here an alcohol oxidase (AOx) based third generation bioanode for generating power from methanol substrate in a fuel cell setup using air breathed laccase biocathode. A composite three dimensional microporous matrix containing multiwalled carbon nanotubes, carbon paste and nafion was used as electroactive support for immobilization of the enzymes on toray carbon paper as supporting electrode in the fabrication of the bioelectrodes. Polyethylenimine was used to electrostatically stabilize the AOx (pI 4.3) on the anode operating on direct electrochemistry principle. Osmium tetroxide on poly (4-vinylpyridine) was used to wire the laccase for electron transfer in the biocathode. The enzymatic biofuel cell (EFC) generated an open circuit potential of 0.61 (±0.02) V with a maximum power density of 46 (±0.002)µWcm−2 at an optimum of 1M methanol, 25°C and an internal resistance of 0.024µΩ. The operation and storage half life (t1/2) of the EFC were 17.22h and 52 days, respectively at a fixed load of 1.85Ω. The findings have demonstrated the feasibility of developing EFC using AOx based bioanode and laccase based biocathode without applying any toxic free mediator and metal electrode supports for generating electricity.

Operational Stability Assays for Bioelectrodes for Biofuel Cells: Effect of Immobilization Matrix on Laccase Biocathode Stability

One of the main issues of enzymatic biofuel cells is the need to develop stable bioelectrodes. However, there is no standard method to study bioelectrode stability. In this study, laccase and anthracene modified multiwall carbon nanotubes (anth-MWCNTs) were used in conjunction with different immobilization polymers in order to increase the stability of the biocathodes. A series of stability assays were used to understand the operational stability of the biocathode in a biofuel cell environment. The immobilization matrices used in this study were tetrabutyl ammonium bromide modified Nafion (TBAB modified Nafion), octyl modified linear polyethylenimine (C8-LPEI), and vapor deposited tetramethyl orthosilicate (TMOS) gels. The decrease in activity of the galvanostatic and potentiostatic measurements over a 24 hour period of the TBAB modified Nafion were 4.0 ± 0.6% and 4.1 ± 0.4%; C8-LPEI were 0.7 ± 0.1% and 9.6 ± 0.5%; and TMOS were 4.1 ± 0.1% and 10 ± 2.0%, respectively. This data show that potentiostatic measurements provide a harsher environment for the enzyme and result in lower stability, as well as showing that the TBAB modified Nafion offers a more stabilizing immobilization strategy compared to the C8-LPEI or TMOS matrices.

A highly efficient gold/electrospun PAN fiber material for improved laccase biocathodes for biofuel cell applications

We explore for the first time the ability of a three-dimensional polyacrylonitrile/gold material prepared by a low-cost and scalable synthesis method, based on the combination of electrospinning and sputtering, as a new material with large surface area to provide high loadings of enzymes to enhance the electrochemical performances of enzyme electrodes in biofuel cells (BFCs). An ethanol/O2 BFC has been developed based on enzymatic reactions performed at both the cathode and anode with immobilization of the respective enzymes and mediators on the three-dimensional nanostructured electrodes. The power density delivered is 1.6 mW cm−2 at 0.75 V, which is five times the power density delivered by the BFC built on flat bioelectrodes. The greatly improved performance of these synthesized nanostructured electrodes makes them exciting materials for their implantation in biofuel cell applications.

Carbon Nanotube Covalently Attached Laccase Biocathode for Biofuel Cell

Biocathode for biofuel cell was prepared by covalently immobilizedLaccaseon CNT (CNT-Laccase) using glutaraldehyde as conjugates. Successful laccase immobilization was confirmed by Fourier Transform Infrared (FTIR) Spectrophotometry, Surface Electron Microscopy (SEM) and Thermogravimetric Analysis (TGA). Immobilization affected Laccase enzymatic activity where it boosts the stability at high temperature and neutral pH. At temperature 65ºC, free Laccase completely loss its activity, while CNT-Laccase still retaining 57.12% of its activity at 45ºC. The activity of CNT-Laccase at pH 7 was 7.04% of activity at pH 5 which was higher than that of free Laccase. CNT-Laccase was able to perform oxygen electroreduction with addition ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as mediator. Performance of oxygen electroreduction activity was also determined by type and composition of binding polymer. Nafion was able to provide better environment for oxygen electroreduction activity compare to polyvinyl alcohol (PVA). Current density resulted in using Nafion in ratio 1:10 to buffer volume was 1.31 mA/cm 2 , which was higher than that of PVA (1.01 mA/cm 2 ). Increasing binding polymer ratio into 1:2 and 1:1 undermined oxygen electroreduction activity.

High performance thylakoid bio-solar cell using laccase enzymatic biocathodes

Thylakoid membranes have previously been used for electrochemical solar energy conversion, but the current output and open circuit voltage are low, in part due to limitations of the cathode. In this paper, a thylakoid bioanode and laccase biocathode were combined in the construction of a bio-solar cell capable of light-induced generation of electrical power. This two-compartment cell showed a greater than 5-fold increase in short circuit current density and an open circuit voltage 0.275 V larger than that of a thylakoid bio-solar cell incorporating an air-breathing Pt cathode. The electrodes were then tested in several solutions of varying pH to evaluate the possibility of constructing a compartment-less bio-solar cell. This membrane-less cell, operating at pH 5.5, generated a short circuit photocurrent density of 14.0 ± 1.8 μA cm(-2) which is 25% larger than the two-compartment cell and a similar open circuit voltage of 0.720 ± 0.018 V.

Glucose oxidase nanotube-based enzymatic biofuel cells with improved laccase biocathodes

Glucose/O(2) biofuel cells (BFCs) with an improved power density and stability were developed, using glucose oxidase (GOD) nanotubes with polypyrrole (PPy)-carbon nanotubes (CNTs)-GOD layers deposited on their surface as an anode and a PPy-laccase-2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS) film type cathode. The GOD nanotubes were fabricated within the nanopores of an anodized aluminum oxide membrane using a template-assisted layer-by-layer deposition method. These BFCs exhibited a higher volumetric power than the best performance reported previously; this was likely due to an increase in enzyme loading of GOD nanotubes and improved electrochemical properties of the PPy-CNTs-GOD layers. The stability of BFCs was closely related to the leakage of ABTS from the cathode. When the leakage of ABTS was suppressed, the power density of BFCs was nearly unchanged for at least 8 days under physiological conditions.

Electrochemical characterization of methanol/O2 biofuel cell: Use of laccase biocathode immobilized with polypyrrole film and PAMAM dendrimers

This paper describes the performance of a mediated electron transfer (MET) biocathode for a methanol/O2 biofuel cell. To this end, we employed PAMAM (polyamidoamine) dendrimers for the immobilization of laccase using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS) in solution or entrapped into polypyrrole films. We used the enzyme immobilized onto the carbon platform obtained either in the presence or in the absence of the electropolymerized film to determine kinetic parameters. The results point to a very similar kinetic rate conversion in both situations; however, substrate affinity seems to increase in the bioelectrode containing the entrapped substrate molecules. The electrochemical characterization tests confirmed that the electropolymerized polypyrrole film was able to retain entrapped ABTS molecules. Additionally, laccase provides enhanced catalytic oxidation current for the mediator compared with the control sample containing PAMAM dendrimer only. Compared to the control sample, which gave power density values around 0.7μWcm−2, tests employing ABTS as mediator furnished 6μWcm−2 when the mediator was added in solution and around 25μWcm−2 when it was entrapped into the biocathode layers. Overall, the developed biocathode is environmentally friendly for immobilization of the enzyme laccase, being satisfactorily stable in the kinetic tests and affording good power data in the biofuel cell tests.

High Current Density Air-Breathing Laccase Biocathode

Although biofuel cell research has progressed over the past decade, there were still problems with employing enzymes at air-breathing cathodes, because enzymes need to remain hydrated and most enzyme reactions occur in solution and not in gas phase. This research details an approach to the development of an air-breathing biocathode employing direct electron transfer. This laccase biocathode is studied in two different fuel cell configurations: a proton exchange membrane hydrogen/air fuel cell and a direct methanol fuel cell (DMFC) with an anion exchange membrane. The laccase from the Rhus vernificera biocathode with an enzyme loading of provides fuel crossover tolerance and provides a high operational current density of and a maximum power density of in a 40% methanol DMFC. The laccase biocathode shows a lifetime of 290 h in a DMFC. The hydrogen/air fuel cell provides a stable current for a total of 350 discontinuous hours when operated for 8 h daily.