Oxygen Enzymatic Biofuel Cell Using Laccase and NAD+-

Subscriber access provided by READING UNIV

Article

A Methanol/Oxygen Enzymatic Biofuel Cell Using Laccase and NAD+-dependent Dehydrogenase Cascades as Biocatalysts on Carbon Nanodots Electrodes Guozhi Wu, Yue Gao, Dan Zhao, Pinghua Ling, and Feng Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b12295 • Publication Date (Web): 31 Oct 2017 Downloaded from http://pubs.acs.org on November 1, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

A Methanol/Oxygen Enzymatic Biofuel Cell Using Laccase and NAD+-dependent Dehydrogenase Cascades as Biocatalysts on Carbon Nanodots Electrodes

Guozhi Wu, Yue Gao, Dan Zhao, Pinghua Ling, Feng Gao*

Laboratory of Functionalized Molecular Solids, Ministry of Education, Anhui Key Laboratory of Chemo/Biosensing, Laboratory of Optical Probes and Bioelectrocatalysis (LOPAB), College of Chemistry and Materials Science, Anhui Normal University, Wuhu 241000, P. R. China

*

To whom correspondence should be addressed.

Dr. Prof. Feng Gao, Tel./Fax: +86-553-3937137. E-mail: [email protected].

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract The efficient immobilization of enzymes on favorable supporting materials to design enzyme electrodes endowed with specific catalysis performances such as deep oxidation of biofuels, and direct electron transfer (DET)-type bioelectrocatalysis is highly desired for fabricating enzymatic biofuel cells (BFCs). In this study, carbon nanodots (CNDs) have been used as the immobilizing matrixes and electron relays of enzymes to construct NAD+-dependent dehydrogenase cascades-based bioanode for the deep oxidation of methanol and DET-type laccase-based biocathode for oxygen reduction to water. At the bioanode, multiplex enzymes including alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and formate dehydrogenase (FDH) are co-immobilized on CNDs electrode which is previously coated with in situ polymerized methylene blue (polyMB) as the electrocatalyst for oxidizing NADH to NAD+. At the biocathode, fungal laccase is directly cast on CNDs and facilitated DET reaction is allowed. As a result, a novel membrane-less methanol/O2 BFC has been assembled and displays a high open-circuit voltage (OCV) of 0.71(± 0.02) V and a maximum power density of 68.7 (±0.4) µWcm-2. These investigated features imply that CNDs may act as new conductive architectures to elaborate enzyme electrodes for further bioelectrochemical applications. Keywords: enzymatic biofuel cell, laccase, NAD+-dependent dehydrogenase cascades, carbon nanodots, complete oxidation of methanol, oxygen reduction, direct electron transfer

2


Page 2 of 34

Page 3 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Enzymatic biofuel cells (BFCs), a type of energy-converting devices harvesting electrical energies from chemical energies of fuels by means of enzymatic bioelectrocatalysis mechanism, have been envisaged as the attractive candidates of potential green power sources for implantable biomedicine electric devices and micro-sized electric products1-9. Comparing with conventional fuel cells, BFCs display remarkable advantages including diverse biocatalysts and fuels, mild working conditions, good biocompatibility, and easy miniaturization1-9. However, the present developments and studies on BFCs are still in their early stages and focus on the fundamental research due to some key issues on BFCs desired to be solved including construction of enzyme electrodes with high catalytic performances, low power density and stability, etc1-12. Fabrication of enzymatic electrodes is bottle-neck point to improve the performances of BFCs10-12, although the developments of enzymatic electrodes for BFCs applications have been extensively studied. The most significant issues include developing new immobilizing materials, enzyme immobilization methods, simplified enzyme electrode structures, facilitated electron shuttle between enzymes and electrode, high stability of enzyme electrodes, improved enzyme biocatalytic activity, and multi-enzymes modified electrode for deep oxidation of fuels10-12, which are the current tendency to design enzyme electrodes for constructing BFCs10-12. Carbon-based nanomaterials have attracted particular attention as immobilizing supports of enzymes to construct enzymatic electrodes, owing to their inherent properties including good biocompatibility, good electrical conductivity, easy functionalization, diverse forms, and so on12-17. In present study, we demonstrate the preparation and performance of enzyme-based biocathode and bioanode using carbon nanodots (CNDs) as immobilization supports for fabrication of a

3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 34

methanol/O2 BFC (Figure 1). As a new type of nanocarbons-based nanomaterilas, carbon nanodots constitute of disperse and quasi-spherical nanoparticles18-22. Up to now, a few studies on CNDs for immobilization supports of enzymes in bioelectrochemistry area have been reported21-23. Such limited attention is not proportional to the remarkable properties and potential merits of CNDs. In this study, a laccase-based biocathode for oxygen reduction on the basis of direct electron transfer (DET) of laccase immobilized in CNDs film coated on conventional glassy carbon electrode (denoted as laccase-CNDs/GC electrode) has been fabricated. For the assembly of a methanol/O2 BFC, a multidehydrogenases-based bioanode, which is also based on CNDs-modified glassy carbon electrode immobilized with multiplex dehydrogenases including alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and formate dehydrogenlyase (FDH) for the complete oxidation of methanol, has been further constructed. In the dehydrogenase-based catalytic system, in situ polymerized methylene blue (polyMB) is first coated on CNDs/GC electrode as electrocatalyst to oxidize NADH to NAD+ prior to immobilizing multiplex dehydrogenases. The assembled methanol/O2 BFC based on carbon nanodots displays good performances including high open-circuit voltage and facilitated DET of laccase, suggesting that carbon nanodots are favorable electrode materials to immobilize enzymes to construct enzyme electrodes for bioelectrochemical applications.

EXPERIMENTAL SECTION Materials and chemicals. Alcohol dehydrogenase (ADH, E.C.1.1.3.4) from baker's yeast, Saccharomyces

cerevisiae,

aldehyde

dehydrogenase

(ALDH,

E.C.1.2.1.3)

and

formate

dehydrogenase (FDH, E.C.1.2.1. 2) from Candida boidinii, fungal laccase (EC 1.10.3.2) from

4


Page 5 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Trametes Versicolor, and 2, 2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were all purchased from Sigma. Dihydronicotinamide adenine dinucleotide (NADH), and nicotinamide adenine dinucleotide (NAD+, disodium hydrate) were provided by Fluka. Methylene blue (MB) and other chemicals were all obtained from Sinopharm Chemicals Co., Ltd. (Shanghai, China). All the chemicals were of at least analytical grade and used without further purification. The 0.10 M, pH 6.0 citrate buffer solution was used for the electrochemical measurements of methanol oxidation and oxygen reduction. Milli-Q purified water (>18.0 MQ) was used to prepare all solutions. In this study, candle soots were used to prepare CNDs as starting materials on the basis of the methods reported in literature 18-22. Typically, 20 mg candle soot was suspended in 20 mL of mixed solvent composed of water and ethanol with the volume ratio of 1:1 with the aid of sonicating. After sonicating for three hours, the obtained black suspension was centrifuged for 4 min at 3000 rpm to get rid of large-size particles. The suspension was then collected and centrifuged for 15 min at 6000 rpm to get black precipitate. After drying, ca. 5 mg black powder was obtained for further use. The activity of laccase was measured by spectrophotometry with 5 mM ABTS as the substrate for laccase in citrate buffer (0.10 M, pH 6.0) with a maximum absorption coefficient (εmax) of 29300 mM-1 cm-1at λmax = 436 nm.24,25 As normal, the amount of laccase that oxidized 1µmol of ABTS per minute is defined to be one activity unit of laccase. The activity of laccase used in this study was determinated to be 220 U mg-1. Fabrication of the biocathode and bioanode. Glassy carbon disk electrodes (GC, CHI Instruments, Shanghai) with a diameter of 3 mm were employed to be the underlying electrodes for fabricating enzyme electrodes and the methanol/O2 BFC. Prior to use, the GC electrodes were

5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

polished on a polishing cloth using 0.3 and 0.05 µm alumina slurry, respectively, and then carefully cleaned in acetone and water for 3 min with the aid of sonication, respectively, and then let them dried at room temperature. In this study, different modified electrodes were fabricated with conventional drop-cast method. In preparing process, 0.5% Nafion solution was spread on the surfaces of different modified-electrodes as binders to maintain the stability of the film modified on electrode. CNDs were dispersed into N, N-dimethyleneformamide (DMF) to obtain a 0.40 mg mL-1 homogeneous suspension with the aid of sonication. A 4 µL of the obtained suspension was carefully cast onto the GC electrode and then let the suspension dried under lamp by evaporating the solvents. The resultant CNDs-modified GC electrode with a uniform CNDs film is denoted as CNDs/GC electrode. Laccase solution was obtained by dissolving 4.5 mg laccase in 1.0 mL 0.10 M pH 6.0 citrate buffer solution. 20 µL aliquot of laccase solution was cast on CNDs/GC electrode to obtain laccase-CNDs/GC electrode. The resultant electrode was dried in fridge (4 oC), and carefully rinsed with distilled water before use. For preparing bioanode, the CNDs modified electrodes, CNDs/GC electrode was immersed in 0.15 mM MB solution for 5 h, and then thoroughly rinsed with distilled water to get rid of the adsorbed MB. The as-prepared electrode was poised at + 0.90 V (vs. Ag/AgCl) in 0.10 M pH 6.0 citrate solution for 60 min for the polymerization of MB on CNDs, and then polyMB-CNDs nanocomposites modified electrode (denoted as polyMB-CNDs/GC) was obtained. The 5 mg mL-1 enzymatic solutions of different dehydrogenases, ADH, ALDH, and FDH were obtained by dissolving 5 mg enzyme in 1.0 mL 0.10 M pH 6.0 citrate buffer. A 20 µL of aqueous solution of ADH (5 mg mL-1) was cast onto the polyMB-CNDs/GC electrode, and then kept in the refridge (4 oC) to let it dried. The obtained enzymatic electrode was denoted as ADH-polyMB-CNDs/GC electrode and washed with distilled water prior to use. Similarly,

6


Page 6 of 34

Page 7 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


bi-enzyme (ADH and ALDH), and tri-enzyme (ADH, ALDH, and FDH) modified electrodes were fabricated

using

the

same

procedures

to

get

ADH-ALDH-polyMB-CNDs/GC

and

ADH-ALDH-FDH-polyMB-CNDs/GC electrodes, respectively. Apparatus and measurements. All the electrochemical measurements were carried out on a CHI 760D potentiostat (CHI Instruments, Shanghai). The rotating disk electrochemical experiments were performed on rotating disk electrode system by attaching the working electrodes to the shaft of an electrode rotator (Pine Inc., USA). S-4800 field emission scanning electron microscopy (SEM) (Hitachi, Japan), Hitachi H-600 transmission electron microscopy (TEM) (Tokyo, Japan), and JEOL-2010F high-resolution transmission electron microscope (HRTEM) (JEOL, Japan) with an acceleration voltage of 200 KV were used for characterizing the morphologies of CNDs. The activity of laccase was measure by spectrophotometry on a Lambda 35 spectrophotometer (PerkinElmer, USA). The electrochemical impedance spectroscopy (EIS) measurements were achieved in 20 mM, pH 7.0 phosphate buffer with 5 mM K3[Fe(CN)6], 5 mM K4[Fe(CN)6], and 0.1M KCl within the frequency range 0.01-1.0×105 Hz under a perturbation signal of 10 mV. In electrochemical experiments, different modified electrodes or bare GC electrode, platinum spiral wire, and KCl-saturated Ag/AgCl electrode were used as working electrode, counter electrode, and reference electrode, respectively. The methanol/O2 BFC was fabricated by co-immersing the bioanode and the biocathode in a 10 mL electrolytic cell and the performances of the BFC were investigated in 0.10 M pH 6.0 citrate buffer solution with 100 mM methanol and 20 mM NAD+ under O2 atmosphere. The electrochemical measurements were carried out at ambient temperature and repeated minimum three times.

7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

RESULTS AND DISCUSSION Preparation and properties of CNDs. As the cousin of popular carbon nanomaterials, CNDs have displayed several distinguished electrochemical features of carbon-based nanomaterials such as high electrical conductivity, good biocompatibility, high specific surface area20-23. And also, the CNDs film cast on glassy carbon electrode shows almost ideally polarizable interface between CNDs film and electrolyte, strong tendency to resist deactivation when exposed to laboratory air for several hours21,22. These remarkable characteristics of CNDs make them suitable for supports of biocatalysts and electrochemical signal transduction. In this study, CNDs were obtained using candle soots as starting materials on the basis of previous studies19-22. Figure 2 shows the SEM and TEM morphologies with low and high magnifications of the prepared carbon nanodots, respectively. From the SEM morphologies displayed in Figure 2A and 2B, we can see that the prepared products constitute of lots of nanodots. The TEM morphologies further reveal that these nanodots are almost nanostructured spheres with uniform diameters between 50 and 60 nm, as shown in Figure 2C and D. These results suggest that the desired carbon nanodots are successfully prepared. Biocathode for oxygen reduction based on DET of laccase on CNDs. Oxygen is an ambient and natural fuel of biocathode and its four-electron reduction (Eo = 1.23 V at 25 oC) at low overpotentials through enzyme-mediated bioelectrocatalysis mechanisms is highly desired for fabricating BFCs26. DET-type bioelectrocatalysis for four-electron oxygen reduction is a very attractive mean and provides some advantages over widely-used mediated electron transfer (MET)-type bioelectrocatalysis reaction27-30. First, the onset reduction potential at DET-based enzyme electrode generally occurs at the intrinsic redox potential of the enzyme, which is in favour of reducing the potential loss and therefore improve the open-circuit cell voltage (OCV) of

8


Page 8 of 34

Page 9 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


BFCs27-30. Second, DET-based enzyme electrodes can avoid the problems originated from the usage of redox mediators in MET-based enzyme electrodes, such as poor stability, potential toxicity, and high cost 27-30. And at last, DET-based enzyme electrodes can offer relatively simple electrode architectures to miniaturize BFCs27-30. However, the dominant hindrance to achieve DET between enzyme and the electrode is the insulation of the surrounding globular proteins structure of enzymes, which restricts DET due to the long electron transfer distance or tunneling event27-30. Laccase (EC 1.10.3.2 ), belonging to the family of multi-copper oxidases in which the catalytic sites contain four coppers categorized into three types including Type 1 (T1), Type 2 (T2), and Type3 (T3) sites based on their spectroscopic characteristics, is a promising and efficient biocatalyst for the electro-reduction of oxygen into water25,31-35. The catalytic mechanism of the laccase begins with the rapid one-electron oxidation reaction of substrates (electron donors) to radical products that dissociate before following reactions at π-electron-rich T1 Cu site which is generally regarded as the electrochemical control center in oxygen reduction into water and located near a wide, hydrophobic, and substrate-binding pocket-like site. Subsequently, an intra-molecular electron transfer takes place from the reduced T1 through a His-Cys-His bridge to the T2-T3 Cu tri-nuclear cluster site formed by one T2 Cu and two T3 Cu, in which oxygen is bound with the two T3 copper nuclear and reduced into water25,31-35. The thermodynamic redox potential of most fungal laccases is as high as 0.78 V vs. NHE (i.e., redox potential of T1 Cu center), which therefore endows them with high biocatalytic ability to O2 reduction into water at very low overpotentials with 100% efficiency and high turnover rates32. This intrinsic characteristic of laccase makes it to be a highly desired bioelectrocatalyst to construct O2-consuming biocathodes of BFCs25,31-35. Immobilizing laccase on appropriate supports to integrate into DET-type O2-consuming enzyme

9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

biocathodes of BFCs is a key step to fabricate mediator-free BFCs, and many attempts have been devoted into this field36-50. The use of nanomaterials to immobilize enzyme to fabricate DET-type enzyme electrodes has attracted much attention36-50. The DET can be significantly facilitated by using conductive nanostructured materials because nanostructured materials may act as electron-mediating functions12-15,

21, 22, 36-50

. In this study, a laccase-based DET-type enzyme

biocathode was demonstrated by directly immobilizing laccase on CNDs film coated on glassy carbon electrode. The behaviors of electron transfer kinetics of different surface-modified electrodes including CNDs/GC, laccase/GC, and laccase-CNDs/GC electrodes were investigated with electrochemical impedance spectroscopy (EIS) in 0.1 M KCl solution with 5 mM [Fe(CN)6]3−/4−. As shown in the EIS of Figure 3A, the electron-transfer resistance values (Ret) are estimated to be about 5 (±0.5) Ω for bare GC electrode (curve a) and 25(±1) Ω for CNDs/GC electrode (curve b), respectively. In addition, both the bare GC and CNDs/GC electrodes as shown in Figure 3A display almost straight lines. These results are characteristics of a diffusion-limited step in the electrochemical process, and also indicate that CNDs possess excellent electric conductivity and the CNDs film-electrolyte interface is almost ideally polarizable. When laccase was cast on bare GC electrode, the resulting laccase/GC electrode shows the largest semicircle with a Ret value of about 2362(±10) Ω which is corresponding to the largest interfacial electron transfer resistance (curve d), suggesting that laccase film hinders the electron exchange between the redox probes, [Fe(CN)6]3−/4− and electrode surface due to the insulativity of enzyme molecules. However, upon introducing CNDs into the laccase film, the diameter of semicircle decreased obviously (curve c) and the Ret value is about 1145(±10) Ω, suggesting that the resistance of the laccase-CNDs film to probing molecules is decreased and CNDs in the film play an important role in enhancing

10


Page 10 of 34

Page 11 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


electron transfer. The cyclic voltammograms (CVs) of different modified electrodes in a 5 mM [Fe(CN)6]3−/4− solution containing 0.1 M KCl are also employed to monitor the electron transfer behaviors of modified electrodes. As shown in Figure 3B, a pair of well-shaped and reversible redox peak with a peak potential difference (∆Ep) value of 72 mV and a Ipox/Ipred value of 1.0 was observed at bare (curve a) and CNDs-modified (curve b) GC electrodes, indicating that the CNDs film exhibits reversible electron transfer kinetics for redox probe of Fe(CN)63-/4-. We also can find that the peak currents obtained at CNDs/GC electrode (curve b) are increased by 13.5 % comparing to those obtained at bare GC electrode (curve a). The increased peak currents are ascribed to the increased effective surface area of the CNDs film electrode. The surface area of CNDs film electrode was measured to be 0.62 cm2 by the classical Randles-Sevcik equation,22 while the geometrical area of the bare flat glassy carbon electrode was only 0.0707 cm2. Comparing to the CV obtained at laccase/GC electrode (curve d), the peak potential difference at the laccase-CNDs/GC electrode (curve c) gets smaller and closer to the situation of reversible electron transfer accompanying with the increased redox peak currents, also indicating that CNDs can facilitate electron transfer. The DET reaction of laccase at laccase-CNDs/GC electrode was studied by investigating the bioelectrocatalytic reduction of substrate (oxygen). The CVs for electrocatalytic oxygen reduction at CNDs/GC and laccase-CNDs/GC electrode were collected in pH 6.0 citrate buffer solution at room temperature. As shown in Figure 4A, CVs at CNDs/GC electrode under N2 (curve a) and O2 (curve b) atmosphere are almost same in the potential window from -0.2 to 0.8 V, suggesting no electrocatalytic features of oxygen reduction at CNDs/GC electrode. The CVs of laccase-CNDs/GC electrode in N2, air and O2 atmosphere are shown in Figure 4B (curve a, b, and c), respectively.

11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Pronounced and oxygen concentration-dependent catalytic currents under air (curve b) and O2 (curve c) atmosphere are observed, while no such current is observed under N2 atmosphere (curve a), as displayed in Figure 4B. At the meantime, the control experiment performed with denatured laccase immobilized on CNDs/GC electrode was also carried out and the CV was shown in Figure 4B (curve d). It is clear that the CV obtained at denatured laccase modified CNDs/GC electrode did not show any obvious change comparing to the CV at CNDs/GC electrode, implying that no catalytic current was obtained with denatured laccase. These observations essentially suggest that the voltammetric responses are resulted from the DET-type bioelectrocatalytic feature of laccase for oxygen reduction at laccase-CNDs/GC electrode. As demonstrated in Figure 4B, at laccase-CNDs/GC electrode, the onset potential for O2 reduction starts around 0.60 V vs. Ag/AgCl (0.80 V vs. NHE), which is close to the redox potential of the T1 Cu of laccase (0.78V vs. NHE) and well matched with the assumption that the type I Cu site of laccase acts as the electron-accepting site from electrodes in direct electrochemistry of the laccase31-35. Considering the DET mechanism of laccase at CNDs/GC electrode, this result may provide a convincing opinion that the T1 Cu site is involved in the electron transfer process44. Based on the bio-catalyzed reduction of O2 at laccase-CNDs/GC electrode, it is speculated that O2 is reduced to water with participating of four-electron in the electron transfer process, agreeing with the most common phenomenon that the T1 Cu site acts as the primary electron-acceptor in electron transfer process44. This observation further demonstrates that O2 reduction at a low overpotential based on the DET of laccase can be successfully achieved at the constructed laccase-CNDs/GC electrode, and therefore this electrode can be used as the new kind of biocathode for the fabrication of BFCs. The DET behaviors of laccase was further evaluated with the rotating disk electrode (RDE)

12


Page 12 of 34

Page 13 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


voltammetry technique by measuring the electrocatalytic currents of O2 reduction in the O2-saturated citrate buffer(0.1 M, pH 6.0). Figure 4C describes the background current-subtracted linear sweep voltammograms (LSV) of the laccase-CNDs/GC electrode at various rotation rates. As shown in Figure 4C, obvious electrocatalytic currents ascribed to O2 reduction were observed at potentials below +0.6 V at different rotations rates. Furthermore, the current densities start to increase at around +0.6 V with the increasing in the rotation rates because of the enhanced mass transfer of O2 to the electrode.37 The voltammograms at different rotation rates display hump-like peak rather than typical sigmoidal wave shapes with true limiting currents starting from +0.6 V to more negative potential (i.e., potential-independent plateau region), suggesting that DET reaction of laccase in CNDs film is controlled by dynamics of electron transfer between laccase and GC electrode although the electron transfer between them can be facilitated by the CNDs. Figure 4D displays the catalytic currents recorded at laccase-CNDs/GC electrode poised at 0.20 V with a rotating rate of 500 rpm in citrate buffer solution (0.1 M, pH 6.0) saturated with oxygen over a continuous period of 8000 s. The good stability of current was observed at the laccase adsorbed CNDs modified electrode. The current density retained about 80% of its initial value after 8000 s. This high stability may be ascribed to the CNDs film which provides better biocompatibility and stability along with higher bioactivity for the immobilization of laccase. Bioanode for complete oxidation of methanol based on NAD+-dependent dehydrogenase cascades-modified CNDs electrode. Currently, low power density is one of the plagued challenges of BFCs technology for further applications1-9. Typically, the biocatalysts used in BFCs include enzymes, organells, and microbes. For the enzymatic bioanodes, the fuels are generally incompletely oxidized by a single enzyme (e.g., dehydrogenases, oxidases), which generally

13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

harnesses a two-electron partial oxidation of the biofuel and therefore leads to severe loss of the theoretical energy density of BFCs51, 52. The research using multi-enzymes for complete oxidation of fuels in BFCs has been pioneered by Palmore et al. group in 199853. In that study, three NAD+-dependent dehydrogenases including alcohol dehydrogenase, aldehyde dehydrogenase, and formate dehydrogenase were used to completely oxidize methanol into CO2 at graphite plate using benzylviologen to regenerate NAD+ cofactor and diaphorase as catalyst for electron transfer in working solution53. However, the proposed biocatalytic system is complicated and the mediators used in solution rather than immobilized on electrode. Recently, Minteer group has developed different multi-enzymatic biocatalytic systems to completely oxidize fuels in BFCs52-67, such as the complete oxidation of glycerol using PQQ-ADH, PQQ-AldDH and oxalate oxidase co-entrapped into a Nafion membrane modified with tetrabutylammonium 54, the ethanol oxidation with NAD-ADH and NAD-ALDH at the anode55, the complete oxidation of glucose to CO2 using a six-enzyme cascades including pyrroloquinoline quinone-dependent enzymes extracted from Gluconobacter sp., aldolase from Sulfolobus solfataricus and oxalate oxidase from barley by a synthetic metabolic pathway56, the complete oxidation of pyruvate using enzymes including pyruvate dehydrogenase, citrate synthase, aconitase, isocitric dehydrogenase, α-ketoglutarate dehydrogenase, succinyl CoA synthetase, fumarase, and malate dehydrogenase57. Much attention has been focused on constructing NAD+-dependent dehydrogenase-based bioanodes for fabrication BFCs because over 300 dehydrogenases have been used today and therefore different substrates of dehydrogenases can be employed for biofuels40, 41, 51-70. In respect to NAD+-dependent dehydrogenase-based bioanodes, the oxidation of dihydronicotinamide adenine

14


Page 14 of 34

Page 15 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dinucleotide (NADH) at a low overpotential to its oxidized form, nicotinamide adenine dinucleotide (NAD+) is a key precondition to develop bioanodes of BFCs due to its participation in the enzymes-catalyzed cycle reactions68. Methanol is abundant and a commonly used fuel of bioanode in BFCs. In this study, we focused on the complete oxidation of methanol via co-immobilizing NAD+-dependent dehydrogenase cascades and electrocatalyst to NADH oxidation on CNDs electrode. The electrocatalyst for NADH oxidation, ploy(methylene blue) (polyMB) was first electro-polymerized on CNDs electrode, which exhibits an excellent electrocatalytic activity toward NADH oxidation40. And then, three dehydrogenases including alcohol dehydrogenase(ADH), aldehyde dehydrogenase(ALDH), and formate dehydrogenase (FDH) were further immobilized on the

CNDs

coated

with

polyMB,

respectively,

to

get

multienzymes

bioanode,

ADH-ALDH-FDH-polyMB-CNDs/GC electrode, which is envisaged to complete oxidize methanol. Figure 5A shows the CVs obtained at polyMB-CNDs/GC electrode for the oxidation of NADH with various concentrations in 0.10 M pH 6.0 citrate buffer solution. From curve a, we can see that a pair of redox wave appeared at -0.10 V, which is originated from the electropolymerization of MB on CNDs and good agreement with previous studies40. Upon introducing 1.0 mM NADH to the solution, it is clearly observed that the oxidation peak current of polyMB increases while its reversed reduction peak current decreases at -0.10 V(curve b). Furthermore, the more NADH are added, the more increasing in oxidation peak currents and decreasing in reduction peak currents can be observed, as shown in curve c and d in Figure 5A. It has to be noted that such phenomenon of NADH oxidation is not observed at bare CNDs/GC electrode from the control experiment (data not shown). These features are the typical electrocatalytic oxidation characterization and suggest that

15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

NADH can be electrocatalytically oxidized at the polyMB-CNDs/GC electrode40. The prepared polyMB-CNDs nanocomposite for NADH oxidation was further used to construct dehydrogenases-based bioanode for methanol oxidation based on the mechanism illustrated in Figure 1. Figure 5B shows the polarization curves of different dehydrogenase-based electrodes including ADH -polyMB-CNDs/GC (curve a), ADH-ALDH-polyMB-CNDs/GC (curve b) and ADH-ALDH-FDH -polyMB-CNDs/GC (curve c) electrodes for the oxidation of methanol with a concentration of 100 mM. As shown in Figure 5B, the oxidation of methanol at these three kinds of dehydrogenase-based electrodes starts at a threshold potential as low as -0.10 V vs. Ag/AgCl, which is in the vicinty of that for the oxidation of NADH (Figure 5A). However, no obvious oxidation current was observed at polyMB-CNDs/GC electrode modified with three denatured dehydrogenases from the control experiment shown in Figure 5B (curve d). At the prepared ADH-ALDH-FDH-CNDs/GC electrode, the current reached its maximum of 120 µA cm-2 at 0.3 V in the presence of 100 mM methanol, as displayed in Figure 5B (curve c), which is much larger than those recorded at the single-dehydrogenase (curve a) and two-dehydrogenase (curve b)-modified enzyme electrodes, indicating that methanol can be deeply oxidized at the enzyme electrode modified with enzyme cascades including ADH, ALDH, and FDH. The steady-state amperometric responses recorded at the polyMB-CNDs/GC electrodes decorated with single-, two-, threedehydrogenase polarized at 0.3 V upon successive addition of methanol to the stirring citrate buffer aqueous solution (0.10 M, pH 6.0) are displayed in Figure 5C, respectively. For these three kinds of enzyme electrodes, a subsequent addition of methanol results in a remarkable enhancement of oxidation current, and the steady-state current arrives within 5 s, indicating that these enzymatic electrodes respond significantly and rapidly to the changes of the concentration of methanol. The

16


Page 16 of 34

Page 17 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


catalytic

currents

for

each

concentration

of

methanol

obtained

at

the

ADH-ALDH-FDH-polyMB-CNDs/GC electrode are clearly larger than those obtained at ADH-ALDH-polyMB-CNDs/GC and ADH-polyMB-CNDs/GC electrode due to the deep oxidation of methanol by the successive catalytic activities of three dehydrogenases. Fabrication and performance of the methanol/O2 BFC. By using the multi-enzyme cascade to completely oxidize methanol at the bioanode and laccase to catalyze oxygen reduction at the biocathode, a membrane-less methanol/O2 BFC was successfully fabricated. The steady-state polarization curves of methanol oxidation at ADH-ALDH-FDH-polyMB-CNDs/GC electrode in 0.1 M, pH 6.0 quiescent citrate buffer with 20 mM NAD+ and 100 mM methanol, and oxygen reduction at laccase-CNDs/GC electrode in quiescent buffer saturated with oxygen are shown in Figure 6A, respectively. The steady-state current density of ethanol oxidation is much larger than that of oxygen reduction, implying that the power output of the assembled methanol/oxygen BFC will be limited by oxygen reduction at the biocathode. Figure 6B describes the polarization curve (left panel) and the power density (P) (right panel) of the constructed methanol/O2 BFC as a function of the current density (j) in citrate buffer solution (0.1 M, pH 6.0), respectively. As described in this figure, the open-circuit voltage (OCV) of the fabricated methanol/O2 BFC is about 0.71 V (± 0.02) and a power density of 68.7 (±0.4) µW/cm2 at 0.34 V is obtained. These performances of the present methanol/O2 BFC are quite comparable to those of methanol/ O2 BFCs studied recently25. The stability of the constructed methanol/O2 BFC loaded with an external resistance of 1 MΩ were investigated under the condition of continious operation in a quiescent citrate buffer solution with 100 mM methanol and 20 mM NAD+ under ambient air at room temperature. The power density lost ca. 8.3 % of its original power in the first day and remained ca.

17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 34

67.1% of its original power after a week continuous work. It has to be noted that the power density of the present methanol/O2 BFC is dominated by the current density of the DET-type laccase-CNDs/GC biocathode. Although the DET-type enzyme electrode is favorable for simplifying the structure of electrode, the current density is still much lower than those of MET-based enzyme electrodes, which is desired to be solved for further studies on DET-type enzyme electrodes.

CONCLUSIONS In conclusion, multienzymes-biocatalyzing ADH-ALDH-FDH-polyMB-CNDs/GC electrode for deep oxidation of methanol, and DET-type laccase-CNDs /GC electrode for four-electron reduction of oxygen were constructed, respectively using carbon nanodots as supporting matrixes. The bioelectrocatalytic performances of the prepared enzyme electrodes were studied systematically and a

novel

methanol/O2

BFC

was

subsequently

assembled

by

using

ADH-ALDH-FDH-polyMB-CNDs/GC electrode as bioanode and laccase-CNDs/GC as biocathode. In all, as demonstrated in this work, carbon nanodots are believed to be the promising immobilizing matrixes of enzymes to fabricate enzyme electrodes.

ADDITIONAL INFORMATION Competing Financial Interests statement: These authors declare no competing financial interests.

ACKNOWLEDGEMENTS The financial supports from the Natural Science Foundation of China (Grant Nos. 21575004, 21175002), Program for New Century Excellent Talents in University (NCET-12-0599), the project 18


Page 19 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


sponsored by SRF for ROCS, SEM, and the fund for innovation group of bioanalytical chemistry of Anhui Province are deeply acknowledged by all contributors of this study.

REFERENCES (1) Barton, S. C.; Gallaway, J.; Atanassov, P. Enzymatic Biofuel Cells for Implantable and Microscale Devices. Chem. Rev. 2004, 104, 4867-4886. (2)Wu, F.; Yu, P.; Mao, L. Self-powered electrochemical systems as neurochemical sensors: toward self-triggered in vivo analysis of brain chemistry. Chem. Soc. Rev. 2017, 46, 2692-2704. (3)Bandodkar, A. J.; Wang, J. Wearable Biofuel Cells: A Review. Electroanalysis 2016, 28, 1188-1200. (4)Zhou, M.; Dong, S. Bioelectrochemical Interface Engineering: Toward the Fabrication of Electrochemical Biosensors, Biofuel Cells, and Self-Powered Logic Biosensors. Acc. Chem. Res. 2011, 44, 1232-1243. (5)Rasmussen, M.; Abdellaoui, S.; Minteer, S. D. Enzymatic biofuel cells: 30 years of critical advancements. Biosens. Bioelectron. 2016, 76, 91-102. (6) Osman, M. H.; Shah, A. A.; Walsh, F. C. Recent progress and continuing challenges in bio-fuel cells. Part I: Enzymatic cells. Biosens. Bioelectron. 2011, 26, 3087-3102. (7) Mano, N.; Mao, F.; Heller, A. A Miniature Biofuel Cell Operating in A Physiological Buffer. J. Am. Chem. Soc. 2002,124, 12962-12963. (8) Halámková, L.; Halámek, J.; Bocharova, V.; Szczupak, A.; Alfonta, L.; Katz, E. Implanted Biofuel Cell Operating in a Living Snail. J. Am. Chem. Soc. 2012, 134, 5040-5043. (9) Gao, F.; Viry, L.; Maugey, M.; Poulin, P.; Mano, N. Engineering hybrid nanotube wires for high-power biofuel cells. Nat. Commun. 2010, 1, doi: 10.1038/ncomms1000. (10)Milton, R. D.; Wang, T.; Knoche, K. L.; Minteer, S. D. Tailoring Biointerfaces for Electrocatalysis. Langmuir 2016, 32, 2291-2301.

19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(11) Yu, E. H.; Scott, K. Enzymatic Biofuel Cells-Fabrication of Enzyme Electrodes. Energies 2010, 3, 23-42. (12) Zhao, C.; Cai, P.; Song, R.; Chen. Y.; Zhang, J.; Zhu, J. Chem. Soc. Rev. 2017, 46,1545-1564. (13) Majdecka, D.; Bilewicz, R. Nanostructuring carbon supports for optimal electrode performance in biofuel cells and hybrid fuel cells. J. Solid State Electrochem. 2016, 20, 949-955. (14) Wen, D.; Eychmüller, A. Enzymatic Biofuel Cells on Porous Nanostructures. Small 2016, 12, 4649-4661. (15) Babadi, A. A.; Bagheri, S.; Hamid, S. B. A. Progress on implantable biofuel cell: Nano-carbon functionalization for enzyme immobilization enhancement. Biosens. Bioelectron. 2016, 79, 850-860. (16) Minteer, S. D.; Atanassov, P.; Luckarift, H. R.; Glenn R. Johnson, G. R. New materials for biological fuel cells. Mater. Today 2012,15, 166-173. (17)Servat, K.; Tingry, S.; Brunel, L.; Querelle, S.;Cretin, M.; Innocent, C.; Jolivalt, C.; Rolland, M. Modification of porous carbon tubes with enzymes: application for biofuel cells. J. Appl. Electrochem.2007, 37, 121-127. (18) Baker, S. N.; Baker, G. A. Luminescent Carbon Nanodots: Emergent Nanolights. Angew Chem. Int. Ed. 2010, 49, 6726-6744. (19) Li, H.; Zhai, J.; Sun, X. Sensitive and Selective Detection of Silver(I) Ion in Aqueous Solution Using Carbon Nanoparticles as a Cheap, Effective Fluorescent Sensing Platform. Langmuir 2011, 27, 4305-4308. (20) Zhang, L.; Cui, P.; Zhang, B.; Gao, F. Aptamer-Based Turn-on Detection of Thrombin in Biological Fluids Based on Efficient Phosphorescence Energy Transfer from Mn-Doped ZnS Quantum Dots to Carbon Nanodots. Chem. Eur. J. 2013,19, 9242-9250. (21) Sheng, M.; Gao, Y.; Sun, J.; Gao, F. Carbon nanodots-chitosan composite film: a platform for protein immobilization, direct electrochemistry and bioelectrocatalysis. Biosens. Bioelectron. 2014, 20


Page 20 of 34

Page 21 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


58, 351-358. (22) Zhao, M.; Gao, Y.; Sun, J.; Gao, F. Mediatorless Glucose Biosensor and Direct Electron Transfer Type Glucose/Air Biofuel Cell Enabled with Carbon Nanodots. Anal. Chem. 2015, 87, 2615-2622. (23) Selloum, D.; Tingry, S.; Techer, V.;

Renaud, L.; Innocent, C.; Zouaoui, A. Optimized

electrode arrangement and activation of bioelectrodes activity by carbon nanoparticles for efficient ethanol microfluidic biofuel cells. J. Power Sources 2014, 269, 834-840. (24) Camarero, S.; Garcı́a, O.; Vidal, T.; Colom, J.; del Rı́o, J. C.; Gutiérrez, A.; Gras, J. M.; Monje, R.; Martı́nez, M. J.; Martı́nez, Á. T. Efficient bleaching of non-wood high-quality paper pulp using laccase-mediator system. Enzyme Microb. Technol. 2004, 35,113-120. (25)Aquino Neto, S.; Zimbardi, A. L. R. L.; Cardoso, F. P. Potential application of laccase from Pycnoporus sanguineus in methanol/O2 biofuel cells. J. Electroanal. Chem. 2016,765, 2-7. (26)Gewirth, A. A.; Thorum, M. S. Electroreduction of Dioxygen for Fuel-Cell Applications: Materials and Challenges. Inorg. Chem. 2010, 49, 3557-3566. (27) Nöll, T.; Nöll, G. strategies for “wiring” redox-active proteins to electrodes and applications in biosensors, biofuel cells, and nanotechnology. Chem. Soc. Rev. 2011, 40, 3564-3576. (28) Liu,Y.; Du, Y.; Li, C. M. Direct Electrochemistry Based Biosensors and Biofuel Cells Enabled with Nanostructured Materials. Electroanalysis 2013, 25, 815-831. (29) Gao, F.; Yan, Y.; Su, L.; Wang, L.; Mao, L. An Enzymatic Glucose/O2 Biofuel Cell: Preparation, Characterization and Performance in Serum. Electrochem. Commun. 2007, 9, 989-996. (30) Cracknell, J. A.; Vincent, K. A.; Armstrong, F. A. Enzymes as Working or Inspirational Electrocatalysts for Fuel Cells and Electrolysis. Chem. Rev. 2008, 108, 2439-2461. (31) Fernández-Fernández, M.; Sanromán, M. Á.; Moldes, D. Recent developments and applications of immobilized laccase. Biotechnol. Adv. 2013, 31, 1808-1825. 21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(32) Shleev, S.; Tkac,J.; Christenson, A.; Ruzgas, T.; Yaropolov, A. I.; Whittaker, J. W.; Gorton, L. Direct electron transfer between copper containing proteins and electrodes. Biosens. Bioelectron. 2005, 20, 2517-2554. (33) Goff, A. L.; Holzinger, M.; Cosnier, S. Recent progress in oxygen-reducing laccase biocathodes for enzymatic biofuel cells. Cell. Mol. Life Sci. 2015, 72, 941-952. (34) Qian, Q.; Su, L.; Yu, P.; Cheng, H.; Lin, Y.; Jin, X.; Mao, L. Ionic Liquid-Assisted Preparation of Laccase-Based Biocathodes with Improved Biocompatibility. J. Phys. Chem. B 2012,116, 5185-5191. (35) Mano, N.; Soukharev, V.; Heller, A. A Laccase-Wiring Redox Hydrogel for Efficient Catalysis of O2 Electroreduction. J. Phys. Chem. B 2006, 110, 11180-11187. (36) Qiu, H.; Xu, C.; Huang, X.; Ding, Y.; Qu, Y.; Gao, P. Adsorption of Laccase on the Surface of Nanoporous Gold and the Direct Electron Transfer between Them. J. Phys. Chem. C 2008, 112, 14781-14785. (37)Mano, N; Poulpiquet, A. O2 Reduction in Enzymatic Biofuel Cells. Chem. Rev. 2017, 117, DOI: 10.1021/acs.chemrev.7b00220. (38) Zheng, W.; Li, Q.; Su, L.; Yan, Y.; Zhang, J.; Mao, L. Direct Electrochemistry of Multi-Copper Oxidases at Carbon Nanotubes Noncovalently Functionalized with Cellulose Derivatives. Electroanalysis 2006, 18, 587-594. (39)Wu, F.; Su, L.; Yu, P.; Mao, L. Role of Organic Solvents in Immobilizing Laccase on Single-Walled Carbon Nanotubes for Improved Current Response in Direct Bioelectrocatalysis. J. Am. Chem. Soc. 2017, 139, 1565-1574. (40) Yan, Y.; Zheng, W.; Su, L.; Mao, L. Carbon-Nanotube-Based Glucose/O2 Biofuel Cells. Adv.

22


Page 22 of 34

Page 23 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Mater 2006,18, 2639-2643. (41) Li, X.; Zhou, H.; Yu, P.; Su, L.; Ohsaka, T.; Mao, L. A Miniature glucose/O2 biofuel cell with single-walled carbon nanotubes-modified carbon fiber microelectrodes as the substrate. Electrochem. Commun. 2008,10, 851-854. (42) Lalaoui, N.; Rousselot-Pailley, P.; Robert, V.; Mekmouche, Y.; Villalonga, R.; Holzinger, M.; Cosnier, S.; Tron, T.; Goff, A. L. Direct Electron Transfer between a Site-Specific Pyrene-Modified Laccase

and

Carbon

Nanotube/Gold

Nanoparticle

Supramolecular

Assemblies

for

Bioelectrocatalytic Dioxygen Reduction. ACS Catal. 2016, 6, 1894-1900. (43)Thorum, M. S.; Anderson, C. A.; Hatch, J. J.; Campbell, A. S.; Marshall, N. M.; Zimmerman, S. C.; Lu, Y.; Gewirth, A. A. Direct, Electrocatalytic Oxygen Reduction by Laccase on Anthracene-2-methanethiol-Modified Gold. J. Phys. Chem. Lett. 2010, 1, 2251-2254. (44)Wang, X.; Latonen, R. M.; Sjöberg-Eerola, P.; Eriksson, J. E.; Bobacka, J.; Boer, H.; Bergelin, M. Direct Electron Transfer of Trametes hirsuta Laccase in a Dual-Layer Architecture of Poly(3,4-ethylenedioxythiophene) Films. J. Phys. Chem. C 2011,115, 5919-5929. (45)Pita, M.; Gutierrez-Sanchez, C.; Olea, D.; Velez, M.; Garcia-Diego, C.; Shleev, S.; Fernandez,V. M.; Lacey, A. L. D. High Redox Potential Cathode Based on Laccase Covalently Attached to Gold Electrode. J. Phys. Chem. C 2011, 115, 13420-13428. (46) Agbo, P.; Heath, J. R.; Gray, H. B. Catalysis of Dioxygen Reduction by Thermus thermophilus Strain HB27 Laccase on Ketjen Black Electrodes. J. Phys. Chem. B 2013, 117, 527-534. (47)Tominaga, M.; Togami, M.; Tsushida, M.; Kawai, D. Effect of N-Doping of Single-Walled Carbon Nanotubes on Bioelectrocatalysis of Laccase. Anal. Chem. 2014, 86, 5053-5060. (48) Hou, C.; Yang, D.; Liang, B.; Liu, A. Enhanced Performance of a Glucose/O2 Biofuel Cell

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Assembled with Laccase-Covalently Immobilized Three-Dimensional Macroporous Gold Film-Based Biocathode and Bacterial Surface Displayed Glucose Dehydrogenase-Based Bioanode. Anal. Chem. 2014, 86, 6057-6063. (49) Adam, C.; Scodeller, P.; Grattieri, M.; Villalba, M.; Calvo, E. J. Revisiting direct electron transfer in nanostructured carbon laccase oxygen cathodes. Bioelectrochemistry 2016, 109,101-107. (50)Lalaoui, N.; Goff, A. L.; Holzinger, M.; Mermoux, M.; Cosnier, S. Wiring Laccase on Covalently Modified Graphene: Carbon Nanotube Assemblies for the Direct Bio-electrocatalytic Reduction of Oxygen. Chem. Eur. J. 2015, 21, 3198-3201. (51) Sokic-Lazic, D.; Arechederra, R. L.; Treu, B. L.; Minteer, S. D. Oxidation of Biofuels: Fuel Diversity and Effectiveness of Fuel Oxidation through Multiple Enzyme Cascades. Electroanalysis 2010, 22, 757-764. (52) Addo, P. K.; Arechederra, R. L.; Minteer, S. D. Evaluating Enzyme Cascades for Methanol/Air Biofuel Cells Based on NAD(+)-Dependent Enzymes. Electroanalysis 2010, 22, 807-812. (53) Palmore, G. T. R.; Bertschy, H.; Bergens, S. H.; Whitesides, G. M. A methanol/dioxygen biofuel cell that uses NAD(+)-dependent dehydrogenases as catalysts: application of an electro-enzymatic method to regenerate nicotinamide adenine dinucleotide at low overpotentials. J. Electroanal. Chem. 1998, 443, 155-161. (54) Arechederra, R. L.; Minteer, S. D. Complete Oxidation of Glycerol in an Enzymatic Biofuel Cell. Fuel Cells 2009, 9, 63-69. (55)Topcagic, S.; Minteer, S. D. Development of a membraneless ethanol/oxygen biofuel cell. Electrochim. Acta 2006, 51, 2168-2172. (56) Xu, S.; Minteer, S. D. Enzymatic Biofuel Cell for Oxidation of Glucose to CO2. ACS Catal. 2012, 2, 91-94. (57) Sokic-Lazic, D.; Minteer, S. D. Pyruvate/Air Enzymatic Biofuel Cell Capable of Complete 24


Page 24 of 34

Page 25 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Oxidation. Electrochem. Solid-State Lett. 2009, 12, F26-F28. (58) Sokic-Lazic, D.; de Andrade, A. R.; Minteer, S. D. Utilization of enzyme cascades for complete oxidation of lactate in an enzymatic Biofuel cell. Electrochim. Acta 2011, 56, 10772-10775. (59) Zhang, Y.Y.; Arugula, M. A.; Williams, S.T.; Minteer, S. D.; Simonian, A. L. Layer-by-Layer Assembly of Carbon Nanotubes Modified with Invertase/Glucose Dehydrogenase Cascade for Sucrose/O2 Biofuel Cell. J. Electrochem. Soc. 2016,163, F449-F454. (60) Lau, C., Moehlenbrock, M. J.; Arechederra, R. L.; Falase, A.; Kristen Garcia, K.; Rincon, R.; Minteer, S. D.; Banta, S.; Gupta, G.; Babanova, S.; Atanassov, P. Paper based biofuel cells: Incorporating enzymatic cascades for ethanol and methanol oxidation. Int. J. Hydrogen Energy 2015, 40, 14661-14666. (61) Neto, S. A.; Hickey, D. P.; Milton, R. D.; De Andrade, A. R.; Minteer, S. D. High current density PQQ-dependent alcohol and aldehyde dehydrogenase bioanodes. Biosens. Bioelectron. 2015, 72, 247-254. (62) Hickey, D. P.; McCammant, M. S.; Giroud, F.; Sigman, M. S.; Minteer, S. D. Hybrid Enzymatic and Organic Electrocatalytic Cascade for the Complete Oxidation of Glycerol. J. Am. Chem. Soc. 2014, 136, 15917-15920. (63) Nguyen, K.V.; Giroud, F.; Minteer, S. D. Improved Bioelectrocatalytic Oxidation of Sucrose in a Biofuel Cell with an Enzyme Cascade Assembled on a DNA Scaffold. J. Electrochem. Soc. 2014, 161, H930-H933. (64) Xu, S.; Minteer, S. D. Characterizing Efficiency of Multi-Enzyme Cascade-Based Biofuel Cells by Product Analysis. ECS Electrochem. Lett. 2014, 3, H24-H27. (65) Hickey, D. P.; Giroud, F.; Schmidtke, D. W.; Glatzhofer, D. T.; Minteer, S. D. Enzyme Cascade for Catalyzing Sucrose Oxidation in a Biofuel Cell. ACS Catal. 2013, 3, 2729-2737. (66) Kim, Y.H.; Campbell, E.; Yu, J.; Minteer, S. D.; Banta, S. Complete Oxidation of Methanol in Biobattery Devices Using a Hydrogel Created from Three Modified Dehydrogenases. Angew Chem.

25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 34

Int. Ed. 2013, 52, 1437-1440. (67)Gorton, L.; Domínguez, E. Electrochemistry of NAD(P)+/NAD(P)H in Encyclopedia of Electrochemistry; Bard, A. J.,

Stratmann, M.,

Eds.; Wiley-VCH: Weinheim, 2002; pp 67-143.

(68) Gao, F.; Guo, X.; Yin, J.; Zhao, D.; Li, M.; Wang, L. Electrocatalytic activity of carbon spheres towards NADH oxidation at low overpotential and its applications in biosensors and biofuel cells. RSC Adv. 2011, 1, 1301-1309. (69) Ratautas, D.; Tetianec, L.; Marcinkeviciene, L.; Meskys, R.; Kulysa, J. Bioanode with alcohol dehydrogenase undergoing a direct electron transfer on functionalized gold nanoparticles for an application in biofuel cells for glycerol conversion. Biosens. Bioelectron. 2017, 98, 215-221. (70) Wu, G.; Yao, Z.; Fei, B.; Gao, F. An Enzymatic Ethanol Biosensor and Ethanol/Air Biofuel Cell Using Liquid-Crystalline Cubic Phases as Hosting Matrices to Co-Entrap Enzymes and Mediators. J. Electrochem. Soc. 2017,164, G82-G86.

Figure Legends Figure 1. Schematic illustration of membraneless methanol/O2 BFC at CNDs electrodes. Figure 2. Low (A) and high (B) magnification SEM and Low (C) and high (D) magnification TEM images of the obtained CNDs. Figure 3. Nyquist plots (A) in the frequency range from 105 to 10-2 Hz under a perturbation signal of 10 mV and CVs (B) with a scan rate of 50 mV s-1 obtained at bare GC (a) CNDs/GC(b), laccase-CNDs/GC(c), and laccase/GC (d) electrode in 5.0 mM Fe(CN)

6

3-

/Fe(CN)

6

4-

solution

containing 0.1 M KCl. Figure 4. (A) CVs at CNDs/GC electrodes in 0.1 M pH 6.0 citrate buffer solution saturated with N2(curve a) and O2 (curve b) with a scan rate of 20 mV s-1. (B) CVs at laccase-CNDs/GC electrodes in 0.1 M pH 6.0 citrate buffer solution saturated with N2 (curve a), air (curve b), and O2 (curve b), and CV at denatured laccase modified CNDs/GC electrode in buffer solution saturated with O2 26


Page 27 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(curve d) with a scan rate of 20 mV s-1. (C) Linear sweep voltammograms of laccase-CNDs/GC electrode in 0.1 M pH 6.0 citrate buffer solution saturated with O2 at 250, 300, 500, 750, 1000, and 8 000 rpm with a scan rate of 20 mV s-1. (D) Amperometric responses for oxygen reduction at laccase-CNDs/GC electrode over a continuous 8000 s in 0.1 M pH 6.0 citrate buffer saturated with O2 electrode poised at 0.20 V with a rotate rate of 500 rpm. Figure 5. (A) CVs obtained at the polyMB-CNDs/GC bioanode in 0.10 M pH 6.0 citrate buffer solution containing 0 (curve a), 1.0 (curve b), 2.0 (curve c), 3.0 (curve d) mM NADH with a scan rate of 20 mV s-1. (B) Polarization curves for methanol oxidation at ADH-polyMB-CNDs/GC (curve a), ADH-ALDH-polyMB-CNDs/GC (curve b), ADH-ALDH-FDH -polyMB-CNDs/GC (curve c), and three denatured dehydrogenases modified polyMB-CNDs/GC (curve d) electrodes 0.1 M pH 6.0 quiescent citrate buffer solution containing 20 mM NAD + and 100 mM methanol with a scan rate of 20 mV s-1. (C) Amperometric responses of ADH-polyMB-CNDs/GC (curve a), ADH-ALDH-polyMB-CNDs/GC (curve b) and ADH-ALDH-FDH-CNDs-polyMB/GC(curve c) electrodes at an applied potential of +0.30 V to successive addition of different concentrations

of

methanol (20mM) in 0.1 M pH 6.0 stirring citrate buffer solution containing 20 mM NAD+. Figure 6. (A) Steady-state polarization curves of methanol oxidation at ADH-ALDH-FDH -polyMB-CNDs/GC electrode in quiescent 0.1 M pH 6.0 citrate buffer containing 20 mM NAD+ and 100 mM methanol, and oxygen reduction at laccase-CNDs/GC electrode in quiescent 0.1 M pH 6.0 citrate buffer saturated with oxygen. (B)Polarization curve (square dots) and the dependence of power density of the assembled DET-based glucose/air BFC on current density (circle dots) in quiescent 0.1 M pH 6.0 citrate buffer solution containing 20 mM NAD+ and 100 mM methanol under O2 atmosphere.

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1-fgao

28


Page 28 of 34

Page 29 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2-fgao

29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3-fgao

30


Page 30 of 34

Page 31 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4-fgao

31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5-fgao

32


Page 32 of 34

Page 33 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6-fgao

33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For “TOC” only

34


Page 34 of 34

Oxygen Enzymatic Biofuel Cell Using Laccase and NAD+-

Recommend Documents