Autores
Honorio Franco, J. (UNIVERSIDADE DE SÃO PAULO - USP) ; Bonaldo, J.V. (UNIVERSIDADE DE SÃO PAULO - USP) ; Minteer, S. (UNIVERSITY OF UTAH) ; Rodrigues de Andrade, A. (UNIVERSIDADE DE SÃO PAULO - USP)
Resumo
The combination of a metallic catalyst (Ni@Pt-CNT) and an oxalate oxidase (OxOx)
enzyme immobilized on the electrode surface was reported as an Enzymatic Fuel
Cell to completely oxidize glucose. The hybrid Ni@Pt-CNT/OxOx electrode showed
an outstanding catalytic activity of 1.3 mA cm-2, provided by the synergetic
effect between the metallic catalyst and the OxOx. In addition, the
electrochemical impedance spectroscopy results for the hybrid system provided an
increase in the charge transfer resistance and the capacitance of the double
layer. Notably, the bulk electrolysis for 18 hours revealed that the hybrid
electrode presented improved current density and achieved complete glucose
oxidation. Remarkably, the improved bioelectrode can be used as promising small
electronic devices.
Palavras chaves
enzymatic biofuel cell; hybrid bi-catalytic ; metallic catalyst
Introdução
The main objective of an EFC is the generation of energy through the collection
of electrons produced in the bioelectrocatalytic reaction by the action of
enzymes to achieve the oxidation of the chosen fuel (NETO et al, p. 1891-1912,
2013). Although enzymes have promising characteristics to act in the oxidation
of a range of substrates such as high specificity and turnover rate (PRYA et al,
p. 1-8, 2014), many difficulties have been encountered to provide an improved
catalytic activity by harvesting electrons from fuel through the enzymatic route
(NETO et al, p. 153-158, 2018). Recent studies have proven the efficiency of the
enzymatic fuel cell (EFC) in generating energy through the oxidation of several
fuels (FRANCO et al, p. H575-H579, 2018). Glucose is an important energy source
for living organisms where is easily oxidized to CO2 and water via aerobic
metabolic pathways releasing 24 electrons. If one can mimetize the biological
pathway makes glucose a is very promising fuel to apply in EFC, another
advantage of glucose are it is abundant in nature, renewable, low cost, non-
toxic, and highly safety for storage and distribution (LI et al, p. 107983,
2022). There are few studies for glucose in hybrid systems, however different
biocatalytic proposals to oxidize glucose and enhance the stability and the
energy production rates of EFC have been reported. Minteer and Xu developed a
six-enzyme cascade for complete glucose oxidation and generated a power density
of 6.74 ± 1.43 μW cm-2. However, the stability and production/storage energy
rates have not been deepened (XU et al, p. 91-94, 2012). The only hybrid
catalyst system for electrochemical oxidation of glucose was recently developed
by Hu et al. (LI et al, p. 107983, 2022), which consisted of an organic
oxidation catalyst, 2,2,6,6-tetramethyl-1-piperidine N-oxyl (TEMPO), and an
enzyme, glucose oxidase (GOx). The bi-catalytic TEMPO/GOx anode exhibited a
maximum power density of 38.1 μW cm-2, which can be attributed to the enhanced
energetic efficiency that occurred due to the promising performance of the
organic catalyst and the enzyme acting together.The electrocatalytic strategies
provided in recent years for glucose/EFC showed satisfactory current density
results and revealed an improvement in stability, however, the power density
achieved is still very low compared to EFC related to other fuels (FRANCO et
al, p. 138044, 2021). It was concluded that one way to increase electrode
stability and improve the fuel oxidation rate is to prepare a simple and
efficient system containing a minimum of components on the electrode surface to
avoid loss of EFC performance (FRANCO et al, p. 112077, 2020). The continuous
search for catalysts that act synergistically with enzymes to generate high
energy production in a system assembled on an electrode surface is essential for
the development of promising power systems as wearable electronic devices (HUANG
et al, p. 124-125, 2019), and glucose sensors (CHANSAENPAK et al, p. 16, 2021).
On the other hand, to circumvent these problems, new materials with singular
characteristics are being developed focusing on bimetallic electrocatalysts
(COBOS et al, p. 7155-7164, 2016), which combine metallic atoms with different
proportions and unique morphologies to act in the efficient oxidation of short-
chain molecules (WANG et al, p. 1507-1534, 2015). A successful example of a
bimetallic electrocatalyst was described by De Andrade et al. (DA SILVA et al,
p. 174-182, 2017), which reports the preparation and characterization of the
Ni@Pt bimetallic electrocatalyst supported on MWCNTs for glycerol oxidation.
Besides the high catalytic activity, the bulk electrolysis of the metallic
catalyst in the presence of the fuel reached 60 % of glycerol electrooxidation,
allowing the formation of several high value-added products, with glyceric acid
as the main species formed. Furthermore, to form a competitive and promising
catalytic system for oxidation of fuels, the addition of decarboxylase enzymes
able to cleave carbon-carbon bonds has been proposed successfully in EFC (FRANCO
et al, p. 107331, 2019) by combining through a hybrid elegant and efficient way
to achieve deep oxidation of fuels (HICKEY et al, p. 15917-15920, 2014). In this
study, we report the complete glucose electrooxidation through a bi-catalytic
hybrid system containing the metallic catalyst Ni@Pt-CNT, and the immobilized
oxalate oxidase (OxOx) enzyme to achieve an efficient EFC with high energy
production and stability. Electrochemical experiments were performed to
demonstrate and evaluate the catalytic activity achieved by the hybrid system.
Furthermore, high-performance liquid chromatography (HPLC) was responsible to
confirm the complete electrochemical oxidation of glucose by the detection of
the oxidation products formed after bulk electrolysis.
Material e métodos
Glucose, glucuronic acid, hydroxypyruvic acid, acetic acid, and sodium carbonate
were all purchased from Sigma-Aldrich and used as received. Citric acid-
phosphate buffer (150 mM, pH = 5.5) and acetate buffer (50 mM, pH 4.0) were
prepared by dissolving the appropriate amounts of salts in ultrapure water. The
oxalate oxidase (OxOx) enzyme was expressed and purified in the lab (vide infra)
and stored in - 80 °C in 50 µL Eppendorf's separately until use. The OxOx was
obtained by our research group through the expression and purification of the
cloned OxOx gene in the pPICZαA vector. The LabscaleTM TFF System was used in
the enzyme ultrafiltration process accompanied by a 50-cm2 Biomax® Membrane able
to retain proteins greater than 5 kDa. The obtained enzyme was stored at -80 ºC
in separate 500 μL Eppendorf® tubes until use. The cyclic voltammetric
experiments (CV) were carried out on an AUTOLAB potentiostat/galvanostat
(software NOVA 1.11) at a scan rate of 10 mV s-1, step potential of 0.001 V,
potential range of 0.00–1.0 V (vs Ag/AgCl), and 25° C by using a single-
compartment electrochemical cell containing the Ag/AgCl and Pt mesh as reference
and counter electrode, respectively. The long-term electrolysis was performed in
a homemade two-compartment cell separated by a Nafion® membrane pressed to a
gaseous diffusion cathode containing 20 % platinum (A6ELAT/BASF). For the
experiments, the cathode compartment was kept in direct contact with air and the
anode compartment was filled with 10 mL of 150 mM citric acid-phosphate buffer
(pH = 5.5) containing 3 mg of Ni@Pt-CNT, 0.03 U mL-1 OxOx immobilized onto
electrode surface in the absence and presence of 100 mmol L-1 glucose. A
potential of 0.75 V (vs Ag/AgCl) was applied at 25.0 °C for 18 h.
Electrochemical impedance spectroscopy (EIS) was performed on a Autolab 302N
with a FRA module. The EIS measurements provided the frequency ranged from 0.1
Hz to 10 kHz and the amplitude of 0.1 mV. The capacitance of the system was
calculated through the constant-phase element (CPE), which is also employed to
improve the model fitting of the impedance data. The chromatograph system
consisted to an ultraviolet ( = 210 nm) and a refraction (RID = 10) detector.
The sample injection volume was 20 μL. The Aminex® HPX-87H column (300 x 7.8 mm,
9 μm, 8 % cross-linkage, pH range 5-9) (column temperature of 30 °C) with a
mobile phase of 3.33 mmol L-1 sulfuric acid (H2SO4) operating in the isocratic
mode at a constant flow rate of 0.6 mL min-1 was performed to detect the
products from the electrolysis. Products were identified by comparison with the
retention time of standard samples. To confirm the complete glucose oxidation
after bulk electrolysis, the formed CO2 was reacted with 0.5 mol L-1 NaOH at a
1:1 ratio to generate sodium carbonate (Na2CO3), which can be detected by the
RID detector.
Resultado e discussão
The hybrid system was first evaluated by the cyclic voltammetric experiments
(CV). The blank experiment it was performed using the glassy carbon without any
modification on the surface. The hybrid system in the absence and in the
presence of 100 mM glucose. The onset potential of the glucose at the Ni@Pt-
CNT/OxOx electrode is observed at 0.75 V vs SCE. An oxidation peak around 0.75
V it was observed in the presence of glucose (1.3 mA cm-2). Furthermore, a 2.5-
fold increase in current density was achieved in relation to the hybrid system
in the absence of substrate (0.4 mA cm-2), indicating the efficiency of the bi-
catalytic electrode in oxidizing glucose and generating energy. To understand
more concisely the catalytic activity of the hybrid system and evaluate the
current density value provided on different scan rates, we plotted the peak
current against the scan rate for the system containing only Ni@Pt-CNT (black
squares) and the bi-catalytic anode Ni@Pt-CNT/OxOx (red spheres) in the presence
of glucose. The peak current and square root of the scan rate displayed a
satisfactory linear relationship of R2 > 0.9998. Besides that, the hybrid
system showed a considerable enhancement in peak current as the scan rate
increased, reaching its plateau at 27 (mV/s)1/2. Based on these results, it is
possible to confirm that the action of the enzyme together with the metallic
catalyst Ni@Pt-CNT improves the current density as the scan rate also increases.
Long-term electrolysis was performed to calculate the total charge mass-produced
by the anode after 18 hours and also to identify the glucose oxidation products
through the proposal of a catalytic route for each electrocatabolic step. In the
absence of glucose no current density was observed during the electrolysis,
confirming that the current generated was totally delivered by glucose
oxidation. The hybrid system furnished a higher current density compared to the
system containing only the metallic catalyst during the electrolysis, achieving
more glucose oxidized. The charge mass-produced after 18 hours electrolysis by
the bi-catalytic electrode (1.58 C) was four-fold higher than the Ni@Pt-CNT
system (0.38 C). This confirms without any doubt that the hybrid system with the
two catalysts is more active than a single catalyst for glucose
electrooxidation. To confirm that the higher catalytic activity is due the
collection of more electrons from the glucose molecule we perform electrolysis
and follow the products obtained by HPLC. The identified glucose oxidation
products were confirmed by comparison with commercial standards for the Ni@Pt-
CNT and Ni@Pt-CNT/OxOx systems at t = 0 h (before electrolysis) and t = 18 h
(after electrolysis). The electrode containing only Ni@Pt-CNT presented only two
glucose oxidation products after 18 h of electrolysis, glucuronic acid (4.65 ±
0.30 mmol L-1), and hydroxypyruvic acid (4.20 ± 0.10 mmol L-1). The glucose
electrooxidation at the hybrid anode provided glyoxylic acid (5.10 ± 0.20 mmol
L-1), and CO2 (14.65 ± 1.10 mmol L-1) as mais product at the end of the
electrolysis. Moreover, the hybrid electrode allowed a high yield of oxidation,
as evidenced by the complete oxidation of glucose achieved by the detection of
CO2. Moreover, several studies focused on the oxidation of ethanol [30] and
ethylene glycol [12] revealed an increase in current density after the addition
of the enzyme to the electrode, indicating the high efficiency of the OxOx to
cleave the carbon-carbon bonds, in addition to the efficient enzymatic
performance when combined with another type of catalyst. In this way, the
evaluation of the oxidation products formed after long-term electrolysis is
essential to further understand the role of each catalyst in the mechanism
involved in the glucose oxidation steps. The power density test was performed to
evaluate the stability and reproducibility of the Ni@Pt-CNT and Ni@Pt-CNT/OxOx
hybrid system before and after long-term electrolysis. The system containing
only the metallic catalyst (solid black line) presented 215 ± 13 µA cm-2 and 140
± 8 µW cm-2 of maximum current and power density, respectively. On the other
hand, the Ni@Pt-CNT/OxOx bi-catalytic electrode (solid red line) showed an
outstanding maximum current, 625 ± 32 µA cm-2 and power density, 400 ± 27 µW cm-
2. The power density values obtained for the hybrid system was 3-fold higher
compared to the simple system (Ni@Pt-CNT). Notably, the hybrid electrode
developed herein exhibited outstanding maximum power density never achieved
before for a glucose BFC. Given the opportunities offered by the newly developed
BFC system, the hybrid bi-catalyst electrode provides an efficient performance
that allows the production of promising devices with high stability and
production/management of energy through the complete glucose oxidation. The
impedance values for the Ni@Pt-CNT in the absence and presence of glucose
displayed discrepant impedance spectra values when compared to the hybrid system
for the two analyzed potentials, allowing to affirm that the composition of the
double layer and the oxidation mechanism exhibits different behaviors. An
important point evaluated for the Ni@Pt-CNT and Ni@Pt-CNT/OxOx electrodes is
that in the presence of glucose there is no significant increase in Rs (solution
resistance), suggesting that both catalytic systems did not become more
resistive, which could impair the final potency of the cell. Notably, it is
possible to observe a higher impedance values generated for both systems at
0.8V, which allow us to correlate with the results obtained by CV,
chronoamperometric and bulk electrolysis experiments. The Ni@Pt-CNT and the bi-
catalytic system Ni@Pt-CNT/OxOx in the presence of glucose at 0.4 V showed
different impedance curves. The hybrid system exhibited a double layer
capacitance 60 % (87.4 mF) higher compared to the system containing only Ni@Pt-
CNT (53.7 mF), which represents a benefit in the operational part, since glucose
acts more actively in the Helmontz inner plane of the double layer, that is,
close to the electrode surface. For the hybrid system, the charge transfer
resistance (R_ct) decreased 4-fold in the glucose oxidation potential, 0.8 V vs
Ag/AgCl, (10.1 Ω) compared to the OCP analysis (42.1 Ω). Furthermore, both
circuits present a Warburg diffusion component and the presence of Rs which is
related to electrolyte resistance. However, the addition of the enzyme OxOx to
the electrode creates a constant phase element CPE associated with resistance in
parallel. The existence of a CPE element in parallel with the charge transfer
resistance in the Ni@Pt-CNT/OxOx hybrid system is associated with the
interaction of the enzyme with the metallic nanoparticles anchored in the CNT,
forming a highly heterogeneous surface. Therefore, the heterogeneous surface
confirms the low value of α parameter (0.534), which is related to how much a
CPE surface diverges from an ideal capacitor. The hybrid system showed an
exponential increase in capacitance at potential of 0.8 V (486 mF). This value
is 18 times higher than that achieved in the system containing only metallic
catalyst and carbon nanotubes (27.7 mF). Furthermore, the capacitance of the
hybrid system at the glucose oxidation potential is 5 times higher than the same
system analyzed in OCP, indicating the ability of the bi-catalytic electrode to
store energy and improve the total charge produced through the glucose
oxidation.
Conclusões
The present study showed the total oxidation of glucose through the action of a
hybrid system containing a metallic catalyst and an immobilized enzyme oxalate
oxidase. The bi-catalytic electrode furnished a high catalytic activity, 1.3 mA
cm-2, and power density, 400 µW cm-2, confirming that the metallic catalyst can
replace a large number of enzymes in the cascade multi-step reactions.
Furthermore, these results provide evidence of the synergistic action between
the catalysts evidencing the efficient approaches employed in the promising EFC
to achieve excellent electrocatalytic performance. Furthermore, the increase in
charge in the hybrid system can be evaluated as an improvement in the value of
the modeled C elements followed by the presence of a CPE element in the presence
of the enzyme. Notwithstanding, the enhanced electrocatalytic performance of the
bioanode Ni@Pt-CNT /OxOx for glucose electrochemical oxidation by R_ct and
capacitance experiments helped to understand how the hybrid electrode functions
after long time of applied current, acting as a charge/discharge management
system with high stability. The detection of the glucose products by HPLC-UV/RID
confirmed the complete oxidation of the fuel through high yield of CO2 by
achieving the harvest of all electrons (24 electrons) from the glucose molecule.
The demand for a smaller amount of both catalysts makes the hybrid system a
simple, low-cost, and fast fabrication dispositive to the generation of energy
by the oxidation of fuel through the construction of an efficient EFC. Overall,
the improved bi-catalytic architecture provided a promising advance in the
development of new glucose/O2 EFCs, allowing the implementation of this
bifunctional hybrid device as an outstanding alternative energy conversion and
management source for long-term practical applications.
Agradecimentos
We acknowledge the financial support from the Brazilian research funding agencies
FAPESP (2014/50924-4, 2017/20431-7, 2018/24180-1, 2021-01134-7),(CAPES) Finance
Code 001, and the Army Research Office MURI (W911NF-14-1-0263).
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