Tailoring Biointerfaces for Electrocatalysis - Langmuir (ACS

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Tailoring Biointerfaces for Electrocatalysis Ross D. Milton, Tao Wang, Krysti L. Knoche, and Shelley D. Minteer Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b04742 • Publication Date (Web): 21 Feb 2016 Downloaded from http://pubs.acs.org on February 27, 2016

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Tailoring Biointerfaces for Electrocatalysis Ross D. Milton, Tao Wang, Krysti L. Knoche, and Shelley D. Minteer* Departments of Chemistry and Materials Engineering, University of Utah, 315 S 1400 E, Room 2020, Salt Lake City, Utah, USA

KEYWORDS. Bio-interfaces, bioelectrocatalysis, biosensors, biofuel cells

ABSTRACT. Bioelectrocatalysis is an expanding research area, due to the use of this type of electrocatalysis in electrochemical biosensors, biofuel cells, bioelectrochemical cells, and bio-solar cells. This feature article discusses recent advancements in tailoring the bio-interface between electrodes and biocatalysts for facile electrocatalysis. This includes the design of pyrene moieties for directing orientation of biocatalysts on electrode surfaces and mediation, as well as rational design of redox polymers for selfexchange based electron transport to/from biocatalysts and the electrode and the use of bioscaffolding techniques for designing the bioelectrode structure. However, the recent advances of the last decade have shown the importance of hybrid bioelectrocatalytic systems and future work will be needed to use these same pyrene, redox polymer, and bioscaffolding techniques for hybrid bioelectrocatalysis. 1 ACS Paragon Plus Environment

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1. Introduction to bioelectrocatalysis Bioelectrocatalysis is the process of electrochemical catalysis performed via biological catalysts. Those biological catalysts could be whole living organisms, organelles, protein enzymes, or nucleic acid enzymes. Bioelectrocatalysis is the critical electrochemical mechanism in electrochemical biosensors, biofuel cells, and other bioelectrochemical systems. In this article, we review recent advances in the field of bioelectrocatalysis, mainly focused on different electrochemical mechanisms of bioelectrocatalysis processes while also covering an introduction of different types of bioelectrocatalysis, as well as their mechanisms and applications. The three main advances that will be discussed include utilization of π-π stacking for immobilization of bioelectrocatalysts or mediators, redox hydrogels for facile electron transfer between bioelectrocatalysts and electrodes, and the use of bioscaffolding to immobilize bioelectrocatalysts. 1.1. Types of bioelectrocatalysts There are four main types of bioelectrocatalysts used in biological fuel cells today. Table 1 summarizes various combinations these bioelectrocatalytic systems, detailing substrates, substrates and catalysts. Microbial bioelectrocatalysis The discovery of microbial fuel cells dates back to 1910, although attention surrounding this technology has increased dramatically over the past 10 years.1 Utilizing living bacteria as catalysts, microbial fuel cells are able to oxidize both organic and inorganic substrates as fuels. The most common application for microbial bioelectrocatalysis is the microbial fuel cell, which has been proposed for wastewater treatment applications, 2 ACS Paragon Plus Environment

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although there has been substantial research in the last 5 years in bioelectrochemical systems utilizing microbial bioelectrocatalysis for electrosynthesis. Compared with other types of biofuel cells, microbial fuel cells have outstanding lifetimes, which could be up to 5 years.1 MFCs are now commonly studied for wastewater treatment due to the great adaptation capability of bacteria against different environments, utilizing both anaerobic and aerobic bacteria.1 Moreover, continuous operation of MFCs in wastewater is gaining more attention as it is a more economical way to operate MFCs in real applications. Additionally, stacking MFCs also allows for improved power output.2, 3, 4 Operations of MFCs on different types of real wastewater are also commonly seen.2,

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Different

substrates, electrochemical cell configurations and electrode materials are widely studied, so there is no standard in the field.1, 6, 7 Organelles Organelles

contain

membranes

that

allow

for

subcellular

microcompartmentalization in living cells. The two main types of organelles used in bioelectrocatalysis are mitochondria and thylakoids. Mitochondria contain a whole set of metabolic enzymes for the Krebs cycle whereby pyruvate is completely oxidized to carbon dioxide. Therefore, mitochondria are capable of anodic electrocatalysis. The electron transport chain inside mitochondria contains electrochemically-active species that can communicate with the electrode (such as ubiquinone or cytochrome c). More recent studies suggest that ubiquinone located inside the mitochondrial inner membrane is the most common electrochemically-active active species that communicates with the electrode.8 Mitochondria immobilized on electrodes have been utilized within biofuel cells and biosensors. Mitochondria are capable of metabolizing pyruvate, succinate, fatty

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acids and amino acids9 and have shown ability to completely oxidize fuels (similar to microbial fuel cells) while maintaining high theoretical energy efficiency and producing high catalytic current densities (similar to enzymatic fuel cells), because they do not have issues of transport across cell walls. Other organelles commonly used in bioelectrocatalysis are thylakoids, which are the organelles responsible for photoenergy conversion in plants. In a similar fashion to mitochondria, thylakoids are capable of direct electron transfer (DET);10 DET and mediated electron transfer (MET) will be discussed in Section 1.2. In thylakoids, both photosystems I and II are involved in the electron transfer chain mechanism, as well as plastoquinone, the cytochrome b6f complex and plastocyanin. Moreover, a study on different membrane structures of thylakoids, grana and stroma were performed and it was concluded that stroma thylakoids are capable of generating four times the photocurrents produced by grana thylakoids.11 Thylakoids are commonly used in bio-solar cells, as well as biosensors. Being the target organelle of many herbicides, thylakoids have also been utilized as a universal sensor for different pesticides, such as atrazine, bromacil and diuron, with detection limits lower than EPA limits.12, 13

Nucleic acids Ribozymes and deoxyribozymes are nucleic acids that are catalytically-active. Deoxyribozymes are nucleic acid enzymes that are completely artificial. They have been extensively studied as catalysts for cleaving nucleic acids. A significant advantage of deoxyribozymes is that DNA is more stable at high ionic strength than proteins. Additionally, being smaller in size than proteins results in more active sites that can be


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immobilized per given electrode surface area. Moreover, as with DNA, deoxyribozymes are easily modified with thiols thus their attachment to a gold surface is easily achieved. Deoxyribozymes are able to bind many different cofactors via specific design, therefore, obtaining the ability to catalyze various kinds of reactions, for example, one of the commonly used deoxyribozymes are those mimicking horseradish peroxidase using hemin as a cofactor.14 Our research group in collaboration with the Baum research group has shown deoxyribozymes catalyze H2O2 electroreduction and have incorporated them within actual biofuel cells.15 A higher open circuit potential is obtained when compared to enzymatic fuel cells. While the enzymatic catalytic activity is not usually comparable to the original enzyme, deoxyribozymes possess the advantage of specifically binding to corresponding DNA or RNA sequences and therefore have high analytical specificity.

Enzymes Enzymes are proteins that catalyze a specific type of biological reaction. A redox active enzyme either contains redox active cofactors or metal centers that allow the enzyme to directly communicate with the electrode (DET) or utilize a small redox active electron mediator (MET) to transfer the electrons to/from the electrode. Among all of the above types of bioelectrocatalyst categories, enzymes possess the best specificities, selectivities and efficiencies but are generally not comparable to bacteria in terms of functional lifetime or the ability to adapt to different environments. There are two main categories of enzymatic bioelectrocatalytic applications: enzymatic biofuel cells (EFC) and enzymatic biosensors. EFCs are galvanic devices that convert chemical energy into electrical energy via enzymatic bioelectrocatalysis, while


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enzymatic biosensors are electrolytic devices that essentially make use of the same bioelectrocatalytic process (in the case of amperometric biosensors) for detection of analyte. The most significant advantage of EFCs over microbial fuel cell is the higher power density. Recent performance of 1.5-2.7 mW/cm2 at room temperature have been reported.16, 17 EFCs have also been made for implantable devices and those devices have been tested on different animals and plants.18 Future medical applications of biofuel cells include powering implantable devices (such as pacemakers) and wearable biosensors (such as smart contact lens systems), whereby a miniature biofuel cell placed onto a contact lens is able to generate electricity from lactate found in tears.19 At this moment in time, more than 85% of the biosensor market is comprised of glucose biosensors that utilize glucose oxidase (GOx).20 Other than glucose, many other analytes related to human health have been evaluated using enzymatic biosensors such as lactate, pyruvate,21 and urea.22 In recent years, there has been significant research in transitioning amperometric biosensors to self-powered biosensors,23, 24, 25 whereby the biosensors don’t need an external power source to operate, which is beneficial for portable platforms. These self-powered sensors are essentially EFCs that can utilize the analyte as fuel or as an inhibitor or activator of the enzyme.


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Type of bioelectroc atalysis Microbial

Organelle

Enzymatic

Anodic vs. Cathodic Anodic

Electron transfer mechanisms DET

Materials

Reactant

Advantage

Refs

Geobacter, Shewanella

Fe(III), U(VI) etc.

1, 6, 26, 27

MET

Carbohydrates and byproducts Os3+

Adaptable to various electron donors. Wide fuel use and high efficiency Does not require anaerobic environment Broad fuel choice and high efficiency biofuel completely oxidation O2 generating bioelectrocatalysis system Higher current density with mediator present Broad alcohol substrate specificity and O2 insensitive Utilizes one of the most common substrates found in human body O2 insensitive, does not generate H2O2 Utilizes common alcoholic fuels and fermentation broth High activity that results in high current density High promiscuity

Broad substrate specificity and deep oxidation of fuel Broad substrate specificity and deep oxidation of fuel Substrate is easily found in human body Substrate is commonly found

30

Cathodic

MET

Geobacter, Shewanella, Enterococcus, Pseudomonas, etc. Rhodobacter

Anodic

DET

Mitochondria

Pyruvate Amino acids

Anodic

DET

Thylakoid

Water

Anodic

MET

Thylakoid

Water

MET

PQQ-dependent Alcohol Dehydrogenase

Ethanol

FAD-dependent glucose oxidase

Glucose

FAD-dependent glucose dehydrogenase

Glucose

FAD-dependent alcohol oxidase

Methanol/Ethanol/ 1-Propanol Fructose

Fructose dehydrogenase Cellobiose Dehydrogenase

FAD-dependent cholesterol oxidase

Cellobiose (primarily) and other hexoses and polysaccharides Hexoses and polysaccharides Hexoses and poly saccharides Cholesterol

FAD-dependent lactate oxidase

Lactate

Pyranose oxidase Pyranose dehydrogenase

1, 6, 26, 27

1, 6

9

10, 11, 12, 31 31

29, 30

28, 30

48, 67

30

29, 30

30, 62

30, 63

31

68

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DET

Cathodic

MET DET

Nucleic acid

Cathodic

DET

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Hydrogenase

H2

PQQ-dependent glucose dehydrogenase

Glucose

NAD-dependent glucose dehydrogenase

Glucose

NAD-dependent alcohol dehydrogenase

Ethanol methanol Ethanol

PQQ-dependent alcohol dehydrogenase FAD-dependent glucose dehydrogenase (bacterial) Hydrogenase

Glucose

PQQ-dependent lactate dehydrogenase

Lactate

Multi-copper oxidase bilirubin oxidase) Multi-copper oxidase bilirubin oxidase)

H2

(Laccase

and

O2

(Laccase

and

O2

Deoxyribozyme-hemin complex

H2O2

and

in human body Hydrogen oxidation under mild conditions Utilize one of the most common substrates found in human body, O2 insensitive Substrate is commonly found in the human body, O2 insensitive Common fuels for traditional fuel cells Broad alcohol substrate specificity and O2 insensitive Substrate easily found in human body Hydrogen oxidation under mild conditions Substrate is easily found in human body Most commonly used enzymatic cathode Multi-coppers allow for facile DET if properly oriented with the surface Higher power density than normal glucose/H2O2 fuel cells

30, 49

30, 58

30

28, 30

29, 30

30, 47

30, 31

29, 30

28, 31

28, 31, 46

15

Table 1: Summary of common bioelectrocatalytic systems with different catalysts, substrates, and electron transfer mechanisms.


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1.2. Mediated and direct electron transfer Electron transfer between a biocatalyst and an electrode ultimately takes place by mediated electron transfer (MET) or direct electron transfer (DET). In the case of MET, small redox-active moieties shuttle electrons between a biocatalyst and an electrode (Scheme 1). An early example of this was the use of the ferrocene/ferrocinium redox couple dissolved in solution to transport electrons from glucose oxidase (GOx), whereby the flavin adenine dinucleotide (FAD) cofactor of GOx is reduced by the enzymatic oxidation of glucose.32 In turn, ferrocene is reduced at the

Scheme 1 – Reduction of O2 to H2O by bilirubin oxidase (PDB: 2XLL) via DET or MET. enzyme’s active site and the reduced FADH2 cofactor returns to its oxidized state (enabling further glucose oxidation). Finally, ferrocene is then oxidized at an electrode surface. Nowadays, electron mediators commonly used for bioelectrocatalysis are typically based on ferrocene,33, 34 osmium complexes,35 quinones,16, 36 and phenothiazines.37, 38 However, these redox moieties are usually immobilized at the electrode surface in recent years.


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Contrary to MET, DET takes place when electrons are transferred between an enzyme and an electrode without the requirement of an electron mediator.39 This distance-dependent electron transfer mechanism is inherently slow by comparison (especially in the case of large biocatalysts); however, multiple systems have been developed for improved performances (where improved performances refers to improved bioelectrocatalytic turnover by an enzyme, observed as a function of catalytic current densities). The ability of multicopper oxidases (such as laccase and bilirubin oxidase) to undergo DET will be discussed below. Other efficient DET systems have been demonstrated for hydrogenase40 and formate dehydrogenase.41 Although DET and MET studies are perhaps most commonly associated with enzymatic systems, their mechanisms have been extended to understanding how bacteria communicate with electrodes. Many bacteria transfer electrons to the electrodes via direct contact between cell walls and electrodes and this process is completed by redox active proteins present in the specific bacteria, most commonly cytochrome c or through conductive nanowires that are formed between the bacteria and the electrode, like those observed in Geobacter42, 43 or Shewanella.

44

The cytochromes and nanowires formed in DET type MFCs are drawing great attention as very effective ways to increase the powerdensity of those MFCs.26 The second mode of electrical contact, MET, uses small redox active moieties that can either be added into the microbial electrochemical system or synthesized by the bacteria themselves when required. MET in MFCs could either be performed via artificial redox mediators such as quinones or metabolites of that specific bacteria such as 2-Amino-3-carboxy-1,4-naphthoquinone; because of the high power output, MET type MFCs are considered more potent for application and many studies on different mediators, especially metabolite mediators were performed.26


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2. Tailored biointerfaces When considering electron transfer between an electrode and biocatalyst, it is often favorable to rationally-design electrode surfaces to optimize interaction between the biocatalyst and the electrode surface. Nowadays, this is mostly achieved by immobilization of the biocatalyst onto electrode surfaces since this commonly improves the magnitude of bioelectrocatalysis (localized, high concentration of biocatalyst). Methods used to immobilize biocatalysts include simple adsorption,45 entrapment within solid supports,46 chemical crosslinking,32,

47

and covalent

attachment to polymer supports.33, 37, 48, 49 This perspective will focus on tailoring the biointerface for protein-based bioelectrocatalysis. A classic example of a rationally designed biointerface is that of establishing DET between the enzyme laccase (a multi-copper oxidase) and an electrode surface. Typically, DET of laccase is not overly efficient because only a small portion of the enzyme is in the correct orientation for DET, although Blanford et al. initially demonstrated how the chemical modification of electrode surfaces with moieties that somewhat mimic the natural substrate of laccase (i.e. anthracene) can favorably orientate the enzyme for DET and the resulting direct bioelectrocatalytic reduction of O2 to H2O.50 In addition to the authors’ own contributions below, efforts have been made to extend this rationale towards expanded electrode architectures, whereby Bilewicz et al. utilized naphthalene functional groups on carbon nanotubes (CNTs) to orientate laccase for DET.51 In addition to polycyclic aromatic groups, bilirubin has also been demonstrated to efficiently orientate bilirubin oxidase (BOx, another multicopper oxidase than can reduce O2 to H2O by DET).52, 53


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2.1. Pyrene π-π stacking systems Pyrene, a tetracyclic aromatic compound (Scheme 2), is emerging as a useful functional group for bioelectrode modification. Surface modification of insoluble CNTs with biocatalysts is expected to be sluggish, whereas the modification of soluble pyrene moieties not only allows for

Scheme 2 – Pyrene has been used within bioelectrode architectures to (a) immobilize proteins via lysine chemistry, (b) modify electrode surfaces with anthracene moieties to orientate enzymes to afford DET, and to modify bioelectrode surfaces with naphthoquinone to afford (c) DET and (d) MET. Protein crystal structures used: PDB 2XLL and 3A9H.


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more efficient biocatalyst modification but also the ability to purify successfully functionalized moieties. Functionalized pyrenes can then be immobilized onto carbon/CNT surfaces by π orbital overlap (π-π stacking) between the pyrene group and a graphitic basal plane of a carbon electrode architecture. Using this methodology, many pyrene functionalities have been exploited.54 Amine-reactive pyrene (pyrenebutanoic acid n-hydroxysuccinimidyl ester, typically used to fluorescently label proteins) has also been used to immobilized enzymes onto carbonaceous electrode surfaces by covalent modifying lysine amino acid residues (Scheme 2a).48,

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This technique is useful for immobilization, but due to the ability to react with all

surface lysine residues non-specifically, it does not provide control of orientation.

The authors previously demonstrated that the covalent functionalization of multi-walled carbon nanotubes (MWCNTs) with anthracene moieties resulted in the efficient orientation and direct bioelectrocatalytic reduction of O2 by laccase and BOx,46, 56 but minimal loading of enzyme due ot only immobilization at the end of the MWCNTs. Recently, Minteer et al. demonstrated that this could be improved further by chemically modifying pyrene with anthracene groups with varying covalent linkers and spacing (Scheme 2b).57 This approach was extended further in the creation of a intelligently-designed electrode architecture that could be utilized for both the bioanode and biocathode of a glucose/O2 EFC.58 The chemical properties of naphthoquinone (NQ) were used to orientate laccase and BOx at the biocathode (as a substrate mimic of both enzymes, Scheme 2c), whereas the electrochemical properties of NQ (outside of the useful potential window for MET of laccase and BOx) were able to mediate electrons between a pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH, Scheme 2d) and the


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carbon nanotube electrode surface. This resulting electrode architecture increases the simplicity of EFC design, whereby a single bioelectrode support can be used at both the bioanode and biocathode of an EFC (Figure 1).

Figure 1 – Electrodes modified with naphthoquinone-functionalized pyrene/carbon nanotubes are able to facilitate direct electron transfer to laccase and bilirubin oxidase, as well as facilitate mediated electron transfer from PQQ-dependent glucose dehydrogenase. Figure reproduced with permission from Giroud, F.; Milton, R. D.; Tan, B.-X.; Minteer, S. D. ACS Catal. 2015, 5, 12401244. Copyright American Chemical Society.

2.2. Redox polymers A redox polymer is a scaffold to which electrochemically (redox) active species are grafted to. Additionally, the use of chemical crosslinkers (such as amine-reactive species) are mixed with the redox active polymer and an enzyme (making use of their surface-accessible lysine residues), resulting in an extended 3D network whereby protein-protein, protein-polymer and polymer-


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polymer covalent linkages are created. Once crosslinked, these water-soluble polymers remain adhered to an electrode surface resulting in a relatively enhanced surface concentration of enzymes and electron mediators. Organometallic redox polymers Redox polymers are desirable when considering the immobilization of biocatalysts at biointerfaces. Not only does the redox polymer act as a platform for immobilizing a nondiffusive electron mediator (yielding a high localized concentration of electron mediator), but it also enables the covalent immobilization of biomoieties. Additionally, redox polymers increase the loading of biomoieties over typical monolayers, whereby electrons are able to self-exchange across the immobilized network of electron mediators to/from the electrode surface.59 Inorganic redox polymers have been coupled with a range of enzymes,35, 60 including GOx,61 cellobiose dehydrogenase (CDH),62 pyranose dehydrogenase (PDH),63 laccase,64 and BOx.65 In most cases, osmium-based redox polymers are used. Ferrocene-based redox polymers have been coupled with GOx,66 flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH),67 lactate oxidase (LOx),68 PQQ-dependent alcohol and aldehyde dehydrogenases,69 and an engineered glucose oxidase with broader substrate specificity (bGOx).70 The first example combined a dimethylferrocene-modified linear poly(ethylenimine) redox polymer (FcMe2-C3-LPEI) with invertase, GOx, and fructose dehydrogenase, yielding a cascade EFC that was able to deliver 42 µW cm-2 from sucrose.66 The use of FcMe2-C3-LPEI with FADGDH yielded a glucose/O2 EFC that was able to operate on human serum (oxidizing physiological glucose) and deliver 58 µW cm-2. The same ferrocene redox polymer was used to create a self-powered lactate biosensor, whereby the oxidation of lactate by LOx (undergoing MET with the redox polymer) produces power densities (122 µW cm-2) that are sufficient for


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sensing physiological concentrations of lactate. The penultimate use of the FcMe2-C3-LPEI redox polymer incorporates the use of gold-decorated MWCNTs to oxidize ethanol and acetaldehyde by PQQ-ADH and PQQ-AldDH, respectively. An ethanol/O2 cascade EFC was created, whereby ethanol was oxidized to acetic acid with associated power densities of 280 µW cm-2. In the final example, a GOx engineered to oxidize a broader range of substrates (bGOx) was coupled with the ferrocene redox polymer to yield saccharide/O2 EFCs that were able to operate on glucose, galactose, cellobiose, lactose, maltose and xylose. The EFC configuration was also able to produce electrical energy by oxidizing lactose present in untreated dairy milk samples (80.1 µW cm-2 ) along with industrially important milk whey (50.9µW cm-2 ). These examples show the versatility of this one generic redox polymer for anodic MET in EFCs.

Organic redox polymers While the aforementioned self-exchange across an organic redox polymer could be expected to take place at a lower rate (presumably limited by an inner-sphere electron transfer mechanism), multiple high performance organic redox polymers with desirable properties have been designed and reported, because of their favorable interaction with enzymes. Recently, methylviologen was grafted to a poly(ethylenimine) backbone, yielding a redox polymer that was able to undergo MET with oxygen-sensitive hydrogenases while simultaneously protecting the enzyme from oxygen (in oxygen containing solutions).49,

71

A series of phenothiazines have also been

immobilized onto a polymer backbone, yielding low-potential bioanodes operating with both CDH and GOx.37


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Our research group has recently reported the rational design and application of a novel naphthoquinone-based redox polymer (Scheme 3), whereby an epoxide-functionalized naphthoquinone was subtly grafted only a linear poly(ethylenimine) backbone (NQ-LPEI); to date MET has been established between FAD-GDH and nicotinamide adenine dinucleotidedependent GDH (NAD-GDH).48

Scheme 3 – A NQ-LPEI is able to mediate the bioelectrocatalytic oxidation of glucose by (a) FAD-GDH and (b) NAD-GDH, while offering a polymeric backbone for the immobilization of both enzymes.

In the first system, NQ-LPEI was used to immobilize FAD-GDH and undergo biocatalytic MET at a relatively low onset potential.48 A FAD-GDH/NQ-LPEI bioanode was then coupled with a DET-type BOx biocathode to yield a glucose/O2 EFC that possessed a high open circuit potential (OCP) and delivered a high current density of 5.4 mA cm-2. The EFC also delivered a


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large power density of 2.3 mW cm-2 at a large operational potential of 0.55 V (Figure 2). This is very interesting, because the redox polymer does not mediate glucose oxidase even though they have similar FAD-cofactors and the potentials are similar. This shows that rational design of a mediator an enzyme active site is beneficial to bioelectrocatalytic performance, whereby the redox mediator is specifically tailored for optimal bioelectrochemical and chemical properties (i.e. redox potential and immobilization chemistry). In the second system, NAD-GDH was immobilized within the redox polymer to immobilize the enzyme and mediate NADH oxidation.72 Typically, naphthoquinones and anthraquinones coupled with NAD-dependent enzymes additionally require the incorporation of diaphorase as an intermediary enzyme to allow for MET; the above NAD-GDH/NQ-LPEI system was able to undergo mediated bioelectrocatalysis in the absence of diaphorase.

Figure 2 – (A) Grafting naphthoquinone to linear poly(ethylenimine) yields a redox polymer (NQ-LPEI) that is able to mediate electrons from FAD-dependent glucose dehydrogenase. (B) The NQ-LPEI bioanode is coupled with a bilirubin oxidase direct electron transfer-type biocathode to yield an enzymatic fuel cell that produces a maximum power density of 2.3 mW cm-2, at a high operational voltage of 0.55 V. Figure reproduced with permission from Milton, R.


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D.; Hickey, D. P.; Abdellaoui, S.; Lim, K.; Wu, F.; Tan, B.; Minteer, S. D. Chem. Sci. 2015, 6, 4867-4875 – Published by The Royal Society of Chemistry.

2.3. Scaffolding Intracellular multienzyme biocatalytic or biosynthetic reaction pathways have highly efficient yields, no cross-talk between signaling pathways, and sequential cascaded reactions that are often controlled through threshold or feedback mechanisms.73 These unique features come from the precisely controlled physical and spatial ordering of enzymes, mediators, cofactors, and substrates.73, 74 Control is achieved by the use of complex, macromolecular, structured networks that organize the relative positions, orientations, ratios, and distances between enzymes and other enzymes as well as between enzymes and molecules like cofactors or mediators. These highly organized networks facilitate substrate diffusion and electron transfer along the pathways.74 Mimicking

nature’s

organizational

structures

can

help

improve

efficiencies

in

bioelectrocatalysis. DNA and proteins can be used to link enzymes to each other as well as to electrode surfaces.75, 76, 77, 78, 79 This can be in the form of individual protein-protein or DNA base pair interactions or in large entrapping networks of protein or DNA, such as hydrogels. Linking enzymes to the scaffold or to each other provides a way to control relative spatial organization. The entrapment of enzymes in hydrogels has the advantage of maintaining high concentrations of enzymes close to the electrode surface with minimal enzyme leaching while still allowing smaller molecules like substrate and product to permeate the hydrogel. Biohydrogels (made of protein or DNA) are particularly appealing, because they can be rationally designed to be


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compatible with the enzymes, self-assemble, and control the relative spatial organization of multiple enzymes for cascades.80 DNA scaffolds DNA is a self-assembling biopolymer that can be directed by base pairing to form double helix secondary structures stabilized by hydrogen bonds, π-π stacking, and hydrophobic interactions.74 Hydrogel formation and base pairing allow for very specific spatial organization. DNA hydrogels are formed by self-assembly of DNA “monomers” under physiological conditions, making them ideal for in situ entrapment of enzymes and other biomolecules.75, 76, 77 DNA origami is a scaffolding strategy where short oligonucleotides are used to bridge between DNA sites to specify folding. Hybridization of nucleic acids (aptamers) allows organization of multiple enzymes on DNA scaffolds for enhanced relative spatial control.73 Our research group entrapped glucose oxidase (GOx) in a DNA hydrogel for use in an enzymatic biobattery (Scheme 4).80 This was the first use of DNA hydrogels to entrap oxidoreductase enzymes for bioelectrocatalysis. There was a significant increase in bioelectrocatalytic signal for GOx hydrogel electrodes in comparison with control electrodes of physically adsorbed enzymes and of hydrogel only. The biobatteries (GOx and ferrocene carboxylic acid entrapped in a DNA hydrogel anode, coupled with an air breathing cathode) were stable over ten hours and performances were comparable to those of other GOx-based biofuel cells using redox polymer immobilized mediators and air breathing cathodes, reported in the literature.81


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Scheme 4 – Entrapment of glucose oxidase within a DNA hydrogel, adapted from Nguyen and Minteer.80 Previously, a bioelectrocatalytic cascade was activated on a DNA scaffold associated with a Au electrode with thiolated nucleic acids.82 The ordering of the enzymes had a major effect on the efficiency of the cascade. Additionally, spectroscopic studies have shown an increase in catalytic activity of multi-enzyme cascades when a DNA scaffold is employed.83 Our research group made the first enzyme cascade on a DNA scaffold for bioelectrocatalytic purposes.84 RCA templated DNA-enzyme conjugation allowed for the relative spatial organization of the two enzymes, invertase (Inv) and glucose oxidase, in the sucrose oxidation cascade. Amperometric measurements of RCA assembled Inv/GOx anodes showed a 50-100% increase in current density compared to non-RCA assembled Inv/GOx anodes, depending on the concentration of sucrose. RCA assembled sucrose/O2 biofuel cells (Inv/GOx anode and air breathing cathode) showed a 40% increase in maximum current density compared to a sucrose/O2 biofuel cell where the two enzymes were free in solution. Future work is focused on combining DNA hydrogels with DNA scaffolding of sequential enzymes for improved bioelectrocatalytic performance. Protein scaffolds


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Protein engineering can be used to link enzymes to each other,85,

86

attach enzymes to

electrodes by thiol interactions with gold, create protein scaffolds including metalloprotein scaffolds that contain coordinated metal ions and cofactors,78 and form hydrogels.79 Protein hydrogels can be created by cross-linking proteins and peptides.87 Proteins that can be crosslinked to form a hydrogel include globular proteins such as enzymes. Work by our research group has shown that covalently linking enzymes to form artificial metabolons improves bioelectrocatalytic performance versus randomly dispersed multi-enzyme suspensions, although substrate channeling was not observed.85,

88

However, cross-linking

enzymes brings them close enough together for substrate channeling.86 The Banta research group in collaboration with our research group engineered three NAD(H)dependent dehydrogenase enzymes to self-assemble with each other.87 Together, the three enzymes form a synthetic metabolic pathway to oxidize methanol to CO2. When the anode of the methanol/O2 biobattery (air breathing cathode) consisted of a hydrogel (25 wt% protein) of the self-assembled enzymes on carbon paper, the maximum current density was 45 times higher than when the anode was carbon paper with a dilute, non-hydrogel version (2.5 wt% protein) of the self-assembled enzymes. Linking the enzymes to each other by self-assembly optimizes spatial organization while the hydrogel allows for a much higher loading of enzyme. 87

3. Future directions Recent research with pyrene has either focused on covalent binding of enzyme to pyrene without any site-specific orientation or non-covalent binding using orientational moieties to orientate the active site with respect to the electrode. Future research will need to utilize site-specific binding motifs that allow for better orientation for direct electron transfer between enzymes and carbon


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surfaces. These will likely require protein engineering of the enzyme for site-specific binding. As far as redox polymers, the recent work with viologen and quinone redox polymers has shown the role of organic redox polymers in bioelectrocatalysis. However, further research is needed to improve the stability of these systems for long term bioelectronics devices, i.e. biofuel cells. Finally, bioscaffolding has shown us that we can utilize proteins and DNA to increase loading of bioelectrocatalysts and control distances between bioelectrocatalysts, but further research is needed in tailoring those bioscaffolds for trapping mediators and intermediates for high current densities and longer lifetimes. Over the last decade, there has been significant effort focused on improving the bio-interface for bioelectrocatalysis, but as we move forward, there are more and more examples of hybrid bioelectrocatalytic systems. Hybrid systems are important for two reasons: (1) frequently we are unable to engineer an individual enzyme to have the attributes desired (catalytic activity that matches the activity of other enzymes in an enzyme cascade or ability to function in the pH range appropriate for other enzymes), but there may be inorganic and organic electrocatalysts that have the desired properties or (2) two different catalysts work synergistically together for better performance. These hybrid bioelectrocatalytic systems include the combination of metal nanoparticle electrocatalysts with bioelectrocatalysts as well as organocatalysts combined with bioelectrocatalysts. There are many examples of hybrid bioelectrocatalysis with metal nanoparticles. These include using gold nanoparticles and platinum nanoparticles incorporated into the bio-interface. There are also recent examples of combining organocatalysts with enzymatic bioelectrocatalysts, since there are stability and catalytic activity issues with entirely enzymatic catalytic cascades. Recent research utilizing the organocatalyst TEMPO has shown


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that these catalysts can replace PQQ-dependent alcohol dehydrogenase and PQQ-dependent aldehyde dehydrogenase in glycerol bioelectrocatalysis89, as shown in Figure 3. The authors reported the immobilization of an organic catalyst onto an LPEI backbone for the oxidation of multiple energy dense fuels. The immobilization of 2,2,6,6-tetramethyl-1piperidinyloxy (TEMPO) onto LPEI (TEMPO-LPEI) was found to significantly increase the corrected catalytic current density for the oxidation of methanol, ethanol, isopropanol, glycerol, fructose and sucrose.90 Further, the immobilization of TEMPO was found to extend the operational active pH of TEMPO beyond the typical pH range of homogeneous TEMPO, and the complete oxidation of methanol to CO2 was achieved. Finally, the immobilized TEMPO-LPEI anodic catalyst was combined with an enzymatic O2 reducing biocathode (laccase, DET-type) demonstrating the first reported example of a complete TEMPO-based hybrid fuel cell. The hybrid fuel cell was able to produce a current density of 0.38 mA cm-2 when operated in a solution of 2 M methanol; additionally, the immobilization of the TEMPO catalyst enabled the hybrid fuel cell to be operated in a membrane-less configuration. However, over the next few years, these TEMPO-style redox polymers will need to be combined with biocatalytic cascades for better performance, since there are no organocatalysts or metal nanoparticle catalysts that easily break carbon-carbon bonds. However, it is important to note that these combinations of hybrid bioelectrodes will be challenging to ensure that all catalysts can operate in the same environmental conditions (i.e. solvent, pH, temperature).


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Figure 3. Hybrid electrocatalysis of glycerol utilizing an organocatalyst (TEMPO-LPEI) and a biocatalyst oxalate decarboxylase for complete oxidation.

AUTHOR INFORMATION Corresponding Author *[email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources


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National Science Foundation Air Force Office of Scientific Research ACKNOWLEDGMENTS The authors would like to thank the National Science Foundation and the Air Force Office of Scientific Research for funding. REFERENCES 1. Du, Z.; Li, H.; Gu, T. A state of the art review on microbial fuel cells: a promising technology for wastewater treatment and bioenergy. Biotechnol. Adv. 2007, 25 (5), 464-482. 2. Zhuang, L.; Zheng, Y.; Zhou, S.; Yuan, Y.; Yuan, H.; Chen, Y. Scalable microbial fuel cell (MFC) stack for continuous real wastewater treatment. Bioresour. Technol. 2012, 106, 8288. 3. Akman, D.; Cirik, K.; Ozdemir, S.; Ozkaya, B.; Cinar, O. Bioelectricity generation in continuously-fed microbial fuel cell: Effects of anode electrode material and hydraulic retention time. Bioresour. Technol. 2013, 149, 459-464. 4. Zhang, F.; Ge, Z.; Grimaud, J.; Hurst, J.; He, Z. Long-term performance of liter-scale microbial fuel cells treating primary effluent installed in a municipal wastewater treatment facility. Environ. Sci. Technol. 2013, 47 (9), 4941-4948. 5. Kalathil, S.; Lee, J.; Cho, M. H. Efficient decolorization of real dye wastewater and bioelectricity generation using a novel single chamber biocathode-microbial fuel cell. Bioresour. Technol. 2012, 119, 22-27. 6. Pant, D.; Van Bogaert, G.; Diels, L.; Vanbroekhoven, K. A review of the substrates used in microbial fuel cells (MFCs) for sustainable energy production. Bioresour. Technol. 2010, 101 (6), 1533-1543. 7. Logan, B. E. Scaling up microbial fuel cells and other bioelectrochemical systems. Appl. Microbiol. Biotechnol. 2010, 85 (6), 1665-1671. 8. Giroud, F.; Nicolo, T. A.; Koepke, S. J.; Minteer, S. D. Understanding the mechanism of direct electrochemistry of mitochondria-modified electrodes from yeast, potato and bovine sources at carbon paper electrodes. Electrochim. Acta 2013, 110, 112-119. 9. Bhatnagar, D.; Xu, S.; Fischer, C.; Arechederra, R. L.; Minteer, S. D. Mitochondrial biofuel cells: expanding fuel diversity to amino acids. Phys. Chem. Chem. Phys. 2011, 13 (1), 86-92. 10. Rasmussen, M.; Minteer, S. D. Investigating the mechanism of thylakoid direct electron transfer for photocurrent generation. Electrochim. Acta 2014, 126, 68-73. 11. Rasmussen, M.; Minteer, S. D. Thylakoid direct photobioelectrocatalysis: utilizing stroma thylakoids to improve bio-solar cell performance. Phys. Chem. Chem. Phys. 2014, 16 (32), 17327-17331.


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32. Cass, A. E. G.; Davis, G.; Francis, G. D.; Hill, H. A. O.; Aston, W. J.; Higgins, I. J.; Plotkin, E. V.; Scott, L. D. L.; Turner, A. P. F. Ferrocene-mediated enzyme electrode for amperometric determination of glucose. Anal. Chem. 1984, 56 (4), 667-671. 33. Merchant, S. A.; Tran, T. O.; Meredith, M. T.; Cline, T. C.; Glatzhofer, D. T.; Schmidtke, D. W. High-sensitivity amperometric biosensors based on ferrocene-modified linear poly(ethylenimine). Langmuir 2009, 25 (13), 7736-42. 34. Meredith, M. T.; Kao, D. Y.; Hickey, D.; Schmidtke, D. W.; Glatzhofer, D. T. High Current Density Ferrocene-Modified Linear Poly(ethylenimine) Bioanodes and Their Use in Biofuel Cells. J. Electrochem. Soc. 2011, 158 (2), B166-B174. 35. Barton, S. C.; Gallaway, J.; Atanassov, P. Enzymatic biofuel cells for Implantable and microscale devices. Chem. Rev. 2004, 104 (10), 4867-4886. 36. Reuillard, B.; Le Goff, A.; Agnes, C.; Holzinger, M.; Zebda, A.; Gondran, C.; Elouarzaki, K.; Cosnier, S. High power enzymatic biofuel cell based on naphthoquinonemediated oxidation of glucose by glucose oxidase in a carbon nanotube 3D matrix. Phys. Chem. Chem. Phys. 2013, 15 (14), 4892-4896. 37. Pöller, S.; Shao, M.; Sygmund, C.; Ludwig, R.; Schuhmann, W. Low potential biofuel cell anodes based on redox polymers with covalently bound phenothiazine derivatives for wiring flavin adenine dinucleotide-dependent enzymes. Electrochim. Acta 2013, 110 (0), 152-158. 38. Gorton, L.; Dominguez, E. Electrocatalytic oxidation of NAD(P)H at mediator-modified electrodes. Rev. Mol. Biotechnol. 2002, 82 (4), 371-392. 39. Karyakin, A. A. Principles of direct (mediator free) bioelectrocatalysis. Bioelectrochemistry 2012, 88, 70-75. 40. Lojou, E.; Luo, X.; Brugna, M.; Candoni, N.; Dementin, S.; Giudici-Orticoni, M. T. Biocatalysts for fuel cells: efficient hydrogenase orientation for H(2) oxidation at electrodes modified with carbon nanotubes. J. Biol. Inorg. Chem. 2008, 13 (7), 1157-1167. 41. Reda, T.; Plugge, C. M.; Abram, N. J.; Hirst, J. Reversible interconversion of carbon dioxide and formate by an electroactive enzyme. Proc. Natl. Acad. Sci. 2008, 105 (31), 1065410658. 42. Childers, S. E.; Ciufo, S.; Lovley, D. R. Geobacter metallireducens accesses insoluble Fe (III) oxide by chemotaxis. Nature 2002, 416 (6882), 767-769. 43. Yi, H.; Nevin, K. P.; Kim, B.-C.; Franks, A. E.; Klimes, A.; Tender, L. M.; Lovley, D. R. Selection of a variant of Geobacter sulfurreducens with enhanced capacity for current production in microbial fuel cells. Biosens. Bioelectron. 2009, 24 (12), 3498-3503. 44. Gorby, Y. A.; Yanina, S.; McLean, J. S.; Rosso, K. M.; Moyles, D.; Dohnalkova, A.; Beveridge, T. J.; Chang, I. S.; Kim, B. H.; Kim, K. S. Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms. Proc. Nat. Acad. Sci. 2006, 103 (30), 11358-11363. 45. Duca, M.; Weeks, J. R.; Fedor, J. G.; Weiner, J. H.; Vincent, K. A. Combining noble metals and enzymes for relay cascade electrocatalysis of nitrate reduction to ammonia at neutral pH. ChemElectroChem 2015, 2 (8), 1086-1089. 46. Meredith, M. T.; Minson, M.; Hickey, D.; Artyushkova, K.; Glatzhofer, D. T.; Minteer, S. D. Anthracene-Modified Multi-Walled Carbon Nanotubes as Direct Electron Transfer Scaffolds for Enzymatic Oxygen Reduction. ACS Catal. 2011, 1 (12), 1683-1690. 47. Yehezkeli, O.; Tel-Vered, R.; Reichlin, S.; Willner, I. Nano-engineered FlavinDependent Glucose Dehydrogenase/Gold Nanoparticle-Modified Electrodes for Glucose Sensing and Biofuel Cell Applications. ACS Nano 2011, 5 (3), 2385-2391.


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48. Milton, R. D.; Hickey, D. P.; Abdellaoui, S.; Lim, K.; Wu, F.; Tan, B.; Minteer, S. D. Rational design of quinones for high power density biofuel cells. Chem. Sci. 2015, 6 (8), 48674875. 49. Plumeré, N.; Rüdiger, O.; Oughli, A. A.; Williams, R.; Vivekananthan, J.; Pöller, S.; Schuhmann, W.; Lubitz, W. A redox hydrogel protects hydrogenase from high-potential deactivation and oxygen damage. Nat. Chem. 2014, 6 (9), 822-827. 50. Blanford, C. F.; Foster, C. E.; Heath, R. S.; Armstrong, F. A. Efficient electrocatalytic oxygen reduction by the 'blue' copper oxidase, laccase, directly attached to chemically modified carbons. Faraday Discuss. 2008, 140, 319-335. 51. Karaśkiewicz, M.; Nazaruk, E.; Żelechowska, K.; Biernat, J. F.; Rogalski, J.; Bilewicz, R. Fully enzymatic mediatorless fuel cell with efficient naphthylated carbon nanotube–laccase composite cathodes. Electrochem. Commun. 2012, 20 (0), 124-127. 52. Cracknell, J. A.; McNamara, T. P.; Lowe, E. D.; Blanford, C. F. Bilirubin oxidase from Myrothecium verrucaria: X-ray determination of the complete crystal structure and a rational surface modification for enhanced electrocatalytic O(2) reduction. Dalton Trans. 2011, 40 (25), 6668-6675. 53. Lopez, R. J.; Babanova, S.; Ulyanova, Y.; Singhal, S.; Atanassov, P. Improved Interfacial Electron Transfer in Modified Bilirubin Oxidase Biocathodes. ChemElectroChem 2014, 1, 241248. 54. Le Goff, A.; Reuillard, B.; Cosnier, S. A Pyrene-Substituted Tris(bipyridine)osmium(II) Complex as a Versatile Redox Probe for Characterizing and Functionalizing Carbon Nanotubeand Graphene-Based Electrodes. Langmuir 2013, 29 (27), 8736-8742. 55. Brocato, S.; Lau, C.; Atanassov, P. Mechanistic study of direct electron transfer in bilirubin oxidase. Electrochim. Acta 2012, 61, 44-49. 56. Milton, R. D.; Giroud, F.; Thumser, A. E.; Minteer, S. D.; Slade, R. C. T. Bilirubin oxidase bioelectrocatalytic cathodes: the impact of hydrogen peroxide. Chem. Commun. 2014, 50 (1), 94-96. 57. Giroud, F.; Minteer, S. D. Anthracene-modified pyrenes immobilized on carbon nanotubes for direct electroreduction of O2 by laccase. Electrochem. Commun. 2013, 34 (0), 157-160. 58. Giroud, F.; Milton, R. D.; Tan, B.-X.; Minteer, S. D. Simplifying Enzymatic Biofuel Cells: Immobilized Naphthoquinone as a Biocathodic Orientational Moiety and Bioanodic Electron Mediator. ACS Catal. 2015, 5 (2), 1240-1244. 59. Heller, A. Miniature biofuel cells. Phys. Chem. Chem. Phys. 2004, 6 (2), 209-216. 60. Leech, D.; Kavanagh, P.; Schuhmann, W. Enzymatic fuel cells: Recent progress. Electrochim. Acta 2012, 84 (-), 223-234. 61. Mano, N.; Mao, F.; Heller, A. Characteristics of a miniature compartment-less glucoseO-2 biofuel cell and its operation in a living plant. J. Am. Chem. Soc. 2003, 125 (21), 6588-6594. 62. Tasca, F.; Gorton, L.; Harreither, W.; Haltrich, D.; Ludwig, R.; Noll, G. Comparison of Direct and Mediated Electron Transfer for Cellobiose Dehydrogenase from Phanerochaete soridida. Anal. Chem. 2009, 81 (7), 2791-2798. 63. Zafar, M. N.; Tasca, F.; Boland, S.; Kujawa, M.; Patel, I.; Peterbauer, C. K.; Leech, D.; Gorton, L. Wiring of pyranose dehydrogenase with osmium polymers of different redox potentials. Bioelectrochemistry 2010, 80 (1), 38-42.


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64. Soukharev, V.; Mano, N.; Heller, A. A Four-Electron O2-Electroreduction Biocatalyst Superior to Platinum and a Biofuel Cell Operating at 0.88 V. J. Am. Chem. Soc. 2004, 126 (27), 8368-8369. 65. Jenkins, P. A.; Boland, S.; Kavanagh, P.; Leech, D. Evaluation of performance and stability of biocatalytic redox films constructed with different copper oxygenases and osmiumbased redox polymers. Bioelectrochemistry 2009, 76 (1-2, Sp. Iss. SI), 162-168. 66. 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 (3), 2729-2737. 67. Milton, R. D.; Lim, K.; Hickey, D. P.; Minteer, S. D. Employing FAD-dependent glucose dehydrogenase within a glucose/oxygen enzymatic fuel cell operating in human serum. Bioelectrochemistry 2015, 106, Part A, 56-63. 68. Hickey, D. P.; Reid, R. C.; Milton, R. D.; Minteer, S. D. A self-powered amperometric lactate biosensor based on lactate oxidase immobilized in dimethylferrocene-modified LPEI. Biosens. Bioelectron. 2016, 77, 26-31. 69. Aquino Neto, S.; 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. 70. Milton, R. D.; Wu, F.; Lim, K.; Abdellaoui, S.; Hickey, D. P.; Minteer, S. D. Promiscuous Glucose Oxidase: Electrical Energy Conversion of Multiple Polysaccharides Spanning Starch and Dairy Milk. ACS Catal. 2015, 5 (12), 7218-7225. 71. Oughli, A. A.; Conzuelo, F.; Winkler, M.; Happe, T.; Lubitz, W.; Schuhmann, W.; Rüdiger, O.; Plumeré, N. A Redox Hydrogel Protects the O2-Sensitive [FeFe]-Hydrogenase from Chlamydomonas reinhardtii from Oxidative Damage. Angew. Chem. Int. Ed. 2015, 54, 12329-12333. 72. Abdellaoui, S.; Milton, R. D.; Quah, T.; Minteer, S. D. NAD-dependent dehydrogenase bioelectrocatalysis: the ability of a naphthoquinone redox polymer to regenerate NAD. Chem. Commun. 2016, DOI: 10.1039/C5CC09161F. 73. Wang, F.; Lu, C.-H.; Willner, I. From cascaded catalytic nucleic acids to enzyme-DNA nanostructures: controlling reactivity, sensing, logic operations, and assembly of complex structures. Chem. Rev. 2014, 114 (5), 2881-2941. 74. Fu, J.; Liu, M.; Liu, Y.; Yan, H. Spatially-Interactive Biomolecular Networks Organized by Nucleic Acid Nanostructures. Acc. Chem. Res. 2012, 45 (8), 1215-1226. 75. Lilienthal, S.; Shpilt, Z.; Wang, F.; Orbach, R.; Willner, I. Programmed DNAzymeTriggered Dissolution of DNA-Based Hydrogels - Means for Controlled Release of Biocatalysts and for the Activation of Enzyme Cascades. ACS Appl. Mater. Interfaces 2015, 7, 8923-8931. 76. Park, N.; Um, S. H.; Funabashi, H.; Xu, J.; Luo, D. A cell-free protein-producing gel. Nat. Mater. 2009, 8 (5), 432-437. 77. Zhu, Z.; Wu, C.; Liu, H.; Zou, Y.; Zhang, X.; Kang, H.; Yang, C. J.; Tan, W. An aptamer cross-linked hydrogel as a colorimetric platform for visual detection. Angew. Chem. Int. Ed. 2010, 49 (6), 1052-1056. 78. Kennedy, M. Metalloprotein and redox protein design. Curr. Opin. Struct. Biol. 2001, 11 (4), 485-490. 79. Banta, S.; Wheeldon, I. R.; Blenner, M. Protein Engineering in the Development of Functional Hydrogels. Annu. Rev. Biomed. Eng. 2010, 12 (1), 167-186. 80. Van Nguyen, K.; Minteer, S. D. Investigating DNA hydrogels as a new biomaterial for enzyme immobilization in biobatteries. Chem. Commun. 2015, 51 (66), 1-3.


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81. Aquino Neto, S.; Milton, R. D.; Crepaldi, L. B.; Hickey, D. P.; de Andrade, A. R.; Minteer, S. D. Co-immobilization of gold nanoparticles with glucose oxidase to improve bioelectrocatalytic glucose oxidation. J. Power Sources 2015, 285, 493-498. 82. Piperberg, G.; Wilner, O. I.; Yehezkeli, O.; Tel-Vered, R.; Willner, I. Control of bioelectrocatalytic transformations on DNA scaffolds. J. Am. Chem. Soc. 2009, 131 (25), 8724-5. 83. Wilner, O. I.; Weizmann, Y.; Gill, R.; Lioubashevski, O.; Freeman, R.; Willner, I. Enzyme cascades activated on topologically programmed DNA scaffolds. Nat. Nanotechnol. 2009, 4 (4), 249-254. 84. Van Nguyen, K.; 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 (14), H930-H933. 85. Moehlenbrock, M. J.; Toby, T. K.; Waheed, A.; Minteer, S. D. Metabolon Catalyzed Pyruvate/Air Biofuel Cell. J. Am. Chem. Soc. 2010, 132 (18), 6288-6289. 86. Wu, F.; Minteer, S. Krebs Cycle Metabolon: Structural Evidence of Substrate Channeling Revealed by Cross-Linking and Mass Spectrometry. Angew. Chem. Int. Ed. 2015, 54 (6), 18511854. 87. 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. Int. Ed. 2013, 52 (5), 1437-1440. 88. Moehlenbrock, M. J.; Meredith, M. T.; Minteer, S. D. Bioelectrocatalytic Oxidation of Glucose in CNT Impregnated Hydrogels: Advantages of Synthetic Enzymatic Metabolon Formation. ACS Catal. 2012, 2 (1), 17-25. 89. 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 (45), 15917-15920. 90. Hickey, D. P.; Milton, R. D.; Chen, D.; Sigman, M. S.; Minteer, S. D. TEMPO-Modified Linear Poly(ethylenimine) for Immobilization-Enhanced Electrocatalytic Oxidation of Alcohols. ACS Catal. 2015, 5 (9), 5519-5524.


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Table of Contents Entry


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Author Biographies

Ross D. Milton received his B.Sc. (Hons) and his Ph.D. in Chemistry at the University of Surrey (UK). He is currently a Marie Curie Postdoctoral Fellow in Chemistry at the University of Utah. His research interests include novel enzymes for bioelectrocatalysis and the synthesis and characterization of organic redox polymers for bioelectrocatalysis.


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Tao Wang received his Bachelor’s degree in Chemistry and Master’s degree in Physical Chemistry at Wuhan University. He is currently a Ph.D. candidate in chemistry at the University of Utah. His research interests include mitochondrial bioelectrocatalysis and lab-on-a-chip devices.


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Krysti Knoche received her PhD in Chemistry at the University of Iowa. She is currently a postdoctoral research associate in chemistry at the University of Utah. Her research interests include cyanobacteria bioelectrocatalysis and developing novel methods for evaluating mechanisms of bioelectrocatalysis.

Shelley D. Minteer is a USTAR professor of chemistry and materials science & engineering at the University of Utah. She received her B.S. in Chemistry at Western Illinois University, followed by a PhD in Chemistry at University of Iowa. She was a faculty member in the Department of Chemistry at Saint Louis University from 2000 to 2011, before moving to the University of Utah. Her research program focuses on both the fundamental science and application of bioelectrocatalysis.


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Tailoring Biointerfaces for Electrocatalysis - Langmuir (ACS

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