Generating Electricity on Chips: Microfluidic Biofuel Cells in

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Cite This: Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Generating Electricity on Chips: Microfluidic Biofuel Cells in Perspective Yang Yang,*,†,‡ Tianyu Liu,§ Kai Tao,†,‡ and Honglong Chang*,†,‡ †

Ministry of Education Key Laboratory of Micro/Nano Systems for Aerospace, School of Mechanical Engineering and ‡Unmanned System Research Institute, Northwestern Polytechnical University, Xi’an 710072, China § Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, United States of America

ABSTRACT: Microfluidic electrochemical energy systems precisely manipulate the fluid flow pattern in microfluidic channel to generate electrical energy without the need to use physical barriers such as proton-exchange membranes that other fuel cells normally do. Among various microfluidic devices, microfluidic biofuel cells combine the microfluidic technology with biocatalysts (e.g., enzyme and bacteria), representing an emerging membrane-free power generator. In this work, we first present an overview of the recent progress made for microfluidic biofuel cells, including the understanding of fundamental working principles, the designs of state-of-the-art devices, and the solutions to substantial challenges in further refining the technique. Key factors to improve power output, the scaling-up process, and the design of novel-architecture electrodes are thoroughly discussed. At last, we propose potential opportunities and discuss their advantages associated with microfluidic biofuel cells as power sources for miniature electronics, in vivo sensors, and total bioanalysis systems.

1. INTRODUCTION The widespread of microelectronics along with the fast development of microfabrication techniques has led to an era of ever-widening distribution of miniature and portable devices, with typical examples being cellular phones and personal digital assistants. The components of the miniature devices must be lightweight and easy to integrate into small working spaces. Miniature or micro-scale power sources such as batteries, fuel cells, and electrochemical capacitors are indispensable powers for miniature electronics. Among the aforementioned three devices, micro fuel cells represent the energy-generating source with the steadiest performance. Micro fuel cells are typically able to deliver power ranging from 0.5 to 20 W.1 Such a power is in tune with the functional requirement of most portable electronics. A micro fuel cell behaves similarly to internal-combustion engines, which can generate power by combustion reactions of fuels and oxidants. Unlike internal combustion engines that provide mechanical energy as the “power”, micro fuel cells sustain electricity to power electronics, which is produced from redox reactions between fuels and oxidants at the electrode− electrolyte interfaces.2 The oxidation of fuels (e.g., organic © XXXX American Chemical Society

mixture, glucose, hydrogen gas, formic acid, methanol, and ethanol)3 generally demands certain catalysts (e.g., noble metals such as Pt and Ir and their alloys) to facilitate the electron flowing from fuels (at anodes), through the outer electric circuits, and, eventually, to oxidants (at cathodes). To form closed electric circuits, ions in electrolytes are driven to corresponding electrodes by electric fields and concentration gradients. Proposed by Whitesides and his co-workers in 2002,4 microfluidic fuel cells represent a promising power source for portable electronics owing to their highly efficient energy output, low maintenance cost, low noise, good safety, and high volumetric power density. Typically a microfluidic fuel cell is able to deliver power ranging from 0.03 to 450 mW with output voltage varying from 0.5 to 1 V.5 One of the striking features of microfluidic fuel cells is their membrane-free configuration. Conventional milliliter-scale or Received: Revised: Accepted: Published: A

January 3, 2018 February 7, 2018 February 7, 2018 February 7, 2018 DOI: 10.1021/acs.iecr.8b00037 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Review

Industrial & Engineering Chemistry Research

Figure 1. Schematic illustration of the working mechanism of the (a) microfluidic fuel cell and (b) microfluidic biofuel cell. In panel b, the fuel dissolved in anolyte is catalytically decomposed by biocommunities to generate electrons. The oxygen molecules are reduced to water by the metal catalysts deposited on cathode. The anolyte and catholyte flow separately without hydrodynamic mixing in a microscopic channel (with volumes of several microliters to milliliters). A stable mixing zone exists between the catholyte and anolyte. With the consumption of reactants, boundary layers are formed on the surface of electrodes.

new category of microfluidic fuel cells called microfluidic biofuel cells have emerged.6 Microfluidic biofuel cells utilize pure or mixed enzymes of electrochemically active bacteria (EAB) as catalysts to oxidize fuels, which is in contrast to conventional microfluidic fuel cells that rely on chemical oxidants to oxidize fuels. Figure 1b shows a representative configuration of microfluidic biofuel cells with atmospheric oxygen as the oxidant (sometimes, this type of microfluidic biofuel cells is called air-breathing cells). Microfluidic biofuel cells can be readily assembled by incorporating the EAB in vivo onto single or both electrodes (must be biocompatible). They are gaining more support than traditional microfluidic fuel cells, mainly due to two points: (i) biological enzymes are less expensive than noble metal catalysts, and (ii) enzymes with ultrahigh selectivity at ambient temperatures can compete against or even outperform Pt in terms of catalytic activity.7 Equipped with these biocatalysts, microfluidic biofuel cells are particularly suitable for offering microwatt to milliwatt power. Several reviews have already been presented in-depth discussions of microfluidic biofuel cells technology focusing on their basic working principles, mass-transport phenomena, and development progress of electrodes.8−10 Thus, we herein present a prospective targeting at over-viewing the recent progress made since 2010 on the development of the microfluidic biofuel cells as a whole. The article begins with discussions on an overview of microfluidic biofuel cells, proceeds with a discussion of the progress of two subgroups of microfluidic biofuel cells, i.e., microfluidic enzymatic fuel cells and microfluidic microbial fuel cells, and ends with an outline of the challenges facing microfluidic biofuel cells as well as opportunities such as high-density integration and scaling-up to address these challenges.

larger fuel cells require a physical barrier (usually proton exchange membranes) between catholyte (electrolyte in the cathode chamber containing oxidants) and anolyte (electrolyte in the anode chamber containing fuels) to prevent intermixing of fuels and oxidants. Fuel-oxidant mixing is so disastrous that it almost instantly prevents fuel cells from producing electricity. However, the presence of the proton exchange membranes inevitably increases the manufacturing cost, internal resistance and bulkiness of fuel cells, which is especially treated as one of the major hurdles that must be addressed for the wide implementation of highly integrated micro fuel cells. Microfludic fuel cells successfully avoid the adoption of membranes because of their unique electrolyte flow patterns. The functioning of a microfluidic fuel cell is initiated by sending in catholyte and anolyte at two sides of a microchannel (Figure 1a). The flow rate of both electrolyte streams is carefully controlled to achieve a low Reynolds number that leads to a colaminar flow behavior without hydrodynamic mixing. Benefiting from a high Péclet number (i.e., a dimensionless number describing the ratio of the rate of advection and diffusion transports), the transverse diffusion rate is significantly lower than the streamwise convective velocity. The diffusion mixing is severely restricted to a narrow interface in the middle of channel. The naturally formed anolyte−catholyte interface thus serves as a virtual “barrier” that functions identically to the previously mentioned proton exchange membranes, thereby simplifying the cell structure and reducing the fabrication cost. Following the pioneering work demonstrated by the Whitesides’s group in 2002, worldwide researchers have extensively explored the microfluidic fuel cells and published more than 1000 journal papers (according to Web of Science with the keywords of “microfluidic/laminar-flow fuel cells”).5 In 2005, with the rapid development of biocatalysts-related systems, a B

DOI: 10.1021/acs.iecr.8b00037 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Figure 2. Schematics showing the microfluidic enzymatic cells of different types: (a) an enzyme-based glucose-oxygen microfluidic biofuel cell used to study the influence of electrode configuration and microchannel structure on cell performance;15 (b) nanomaghemite (γ-Fe2O3) particle/GOx mixture deposited on the channel surface to trigger the glucose oxidation in microfluidic fuel cells;20 (c) an air-breathing microfluidic biofuel cell with glucose diluted in blood as the nutrient;19 and (d) three biofuel cells embedded in a microchannel device and connected in parallel or in series.18 Panels a−d are reproduced with permission from refs 15 and 18−20, respectively. Copyright 2008, 2016, 2015, and 2012 Elsevier.

2. MICROFLUIDIC BIOFUEL CELLS Microfluidic biofuel cells are a type of microfluidic fuel cells using biocatalysts (e.g., enzymes in EAB or EAB themselves) to convert chemical energy stored in organic compounds to bioelectricity through a series of reactions. As shown in Figure 1b, biocommunities as catalysts are attached to the surface of cathodes, anodes, or both. They are able to “digest” both single fuel molecules and mixed fuels such as human blood, marine sediment and wastewater.11 The electrons extracted from these fuels need to undergo the extracellular electron-transfer (EET) process to be injected from the biocatalysts to electrodes. The EET process involves two pathways; some of the electrons go through the direct electron-transfer process, i.e., via direct outer membrane protein-electrode assembles, conductive pili, or both; some of the electrons are delivered by mediated electrontransfer processes, i.e., through electron mediators such as flavins and quinines. The aforementioned two processes can work concomitantly in the transport of electrons. Depending on the types of biocatalysts used, microfluidic biofuel cells can be categorized into microfluidic enzymatic fuel cells using enzymes as the biocatalysts, and microfluidic microbial fuel cells using microorganisms as the biocatalysts. 2.1. Microfluidic Enzymatic Fuel Cells. Microfluidic enzymatic fuel cells convert chemical energy into bioelectricity with one or more enzyme(s) acting as the biocatalyst(s). Considering the biocompatibility and integratability of biotissues, most research works selected glucose or lactate as the fuel and atmospheric oxygen as the oxidant. Correspondingly, glucose oxidase (GOx) and lactate oxidase (LOx) extracted from bacteria Aspergillusniger and Aerococcus Williams are two predominant enzymes immobilized on anodes. Besides catalyzing the anodic oxidization reactions, enzymes can also be applied to facilitate oxygen-reduction reactions (ORR) taking place at the cathode surface. Pt-based catalysts possess excellent catalytic activity,7 but their scarcity and vulnerability to glucose cross-over poisoning are their notorious disadvantages.12 The use of low-cost enzyme-based ORR catalysts with exceptionally

high selectivity and efficiency could potentially resolves the above drawbacks of the classical Pt-based electro-catalysts. Two representative classes of high-performance enzymatic ORR catalysts are multiple copper oxidases and blue copper oxidases including laccase, bilirubin oxidase and polyphenol oxidases.13 The firm attachment of these oxidases is a crucial prerequisite to minimize the overall resistance of the assembled cells. Sometimes, mediators such as 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) are added as electron shuttles to further accelerate the EET between enzymes and electrodes.14 Both pure enzymes and hybrid materials involving enzymes and inorganic scaffolds are adopted to decorate the electrodes of microfluidic enzymatic fuel cells. For instance, Moore et al. first demonstrated a microfluidic enzymatic cell that could produce a maximum current density of 53.0 ± 9.1 μA cm−2.6 The bioanode contained two layers composed of an alcoholdehydrogenase enzyme (the top layer) and nicotinamide adenine dinucleotide hydride (NADH)-oxidation catalyst (the bottom layer). Ethanol as the fuel dissolved in the anolyte first contacted the top layer and was degraded by the enzyme in the presence of nicotinamide adenine dinucleotide (NAD+). In this case, NAD+ and NADH functioned as an electron shuttle between the top catalytic layer and the immobilized bottom layer. Inspired by the above-mentioned demonstrations, researchers continue fabricating a diverse array of microfluidic enzymatic fuel cells. Most recent research efforts focus on the construction of electrodes,15−18 microfluidic channels15 and novel materials.19,20 As shown in Figure 2a, the influences of electrode configuration and channel height on the cell performance were first investigated by Togo et al.15 They concluded that the pre-electrolysis at the upstream cathode was beneficial to elevate power density. Similar to the aforementioned study by Togo, Toit et al. separated the microelectrode pairs to three parts, which was located at upstream, mid-stream, and downstream locations, respectively (Figure 2d).18 Through the stacking of the components, the systems were much more C


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Figure 3. Schematics of the microfluidic microbial fuel cells of different types: (a) a microfluidic microbial fuel cell with graphite electrodes was proposed and investigated by varying substrate concentration and flow rates;28 (b) a microfluidic microbial fuel cell equipped with the 3D porous nickel foam delivered a maximum power density of 104 W m−3 during the continuous mode operation;27 (c) three pairs of Au electrodes were arranged along the flow direction to real-time visualize the effects of fuel/oxidant cross-over on bacterial colonization, which was proposed by Choi’s group;31 and (d) a microfluidic microbial three-electrode cell for biosensing the electrochemical activity of bacteria.32 Panels a−d are reproduced with permission from refs 27, 28, 31, and 32, respectively. Copyright 2016 and 2013 Elsevier, 2013 Wiley, and 2016 Elsevier.

flexible in switching between the electrical connection mode and the substrate-feeding mode. Besides the system optimization, functional materials have been gradually participated in the design of enzymatic fuel cells. Nanomaterials were selected to modify the surface of the electrodes, such as maghemite nanoparticles/GOx-coated anodes (Figure 2b)20 and multiwalled carbon nanotubes modified electrodes (Figure 2c)19 to boost the current output. These nanomaterials are biocompatible and can serve as bimolecular immobilizing carriers or electron collectors. In the case of maghemite nanoparticles/ GOx modification, a microfluidic enzymatic fuel cell delivered a maximum current density of 175 μA cm−2 at a power density of 31 μW cm−2. To date, the record-high current density achieved by a microfluidic enzymatic fuel cell is 2.1 mA cm−2, a value nearly 40 times higher than that of the first-generation microfluidic biofuel cells.21 2.2. Microfluidic Microbial Fuel Cells. Microfluidic microbial fuel cells exploit bacteria or exoelectrogens (altogether named microorganisms here after) to produce bioelectricity from organic matters.22,23 Different from enzymes, microorganisms are living cells and the multistep oxidization processes can be easily achieved with the help of the diverse bacterial communities. More importantly, microorganisms are competent in degrading complex organic mixtures (such as wastewater) and are more durable than enzymes.9 Demonstrated microorganisms include Geobacter sulf urreducens,24−26 Shewanella oneidensis,24 Escherichia coli (E. coli),27 and mixed communities (e.g., activated sludge) having two or more exoelectrogenic bacteria.28,29 Research on microfluidic microbial fuel cells was pioneered by Li et al., who presented an on-chip biological energy system with a total volume of 0.3 μL in 2011.24 A pair of microorganisms, G. sulf urreducens and S. oneidensis, were inoculated together on the surface of a gold electrode immersed in a phosphate buffer solution, using fumarate as the fuel. Angenent et al. proposed a bioelectrochemical system (BES) with a Y-type microfluidic channel.30 This system utilized G.

sulf urreducens and was characterized by a potentiostatic mode to study the relationship between the magnitude of bioelectricity and some external stimuli (e.g., oxygen and anthraquinone disulfide). The authors claimed that the laminarflow based BES was a vital tool for studying the microbial activity. A similar BES was also built to characterize the bacterial electrochemical activity by Wang et al.29 The principle was based on the varied open circuit voltage (OCV) from the unbalanced anolyte and catholyte composition. Ye et al. reported a microfluidic microbial fuel cell equipped with graphite plates as two electrodes (Figure 3a).28 The microchannel dimension of this cell was 40 mm × 2 mm × 1 mm, and the total volume of the working chamber was 80 μL. Mixed bacteria were inoculated on the anode surface and respired in the acetate-rich anolyte. The effects of volumetric flow rates and reactant concentration on the power output performance were explored, and a peak power density of 618 mW m−2 was achieved at an internal resistance of 2350 Ω under the flow rate of 10 mL h−1 and the nutrient concentration of 3.39 g L−1.

3. CHALLENGES AND POTENTIAL SOLUTIONS OF MICROFLUIDIC BIOFUEL CELLS 3.1. Cell Performance. Although the highest volumetric power density of microfluidic biofuel cells has reached ∼2.5 kW m−3 at a current density of ∼5000 A m−3 thus far,33 this power performance still cannot compete with other microbial fuel cell (maximum reported volumetric power density of 11.22 kW m−3),34 microfluidic redox batteries (maximum reported volumetric power density of 3000 kW m−3),35 and Li ion batteries (maximum reported volumetric power density of 74 000 kW m−3).36 Previous computational studies elucidate that the power generation limitation of microfluidic biofuel cells lies at the sluggish reactant-transportation kinetics in the boundary layers37 as well as limited biocatalytic reaction rate.38 Reactant is transferred in the convective and diffusion manner from the bulk to the active sites and consumed at the electrode. Both factors are caused by high internal resistance of the entire D


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Figure 4. Schematics showing the microfluidic microbial fuel cells with (a) a straight channel, (b) a diverging channel, and (c) a multi-inlet microfluidic flow channel. The term “OP” in panels a and b is the abbreviation of observation position. Panels a and b are reproduced with the permission from refs 33 and 40. Copyright 2015 and 2016 Elsevier.

two paragraphs will present two general strategies, namely design novel flow pattern and structured electrodes, to lessen RCT. Designing novel electrolyte flow patterns is a dominating approach to enhancing the power output. The flow pattern could be optimized by modifying the microchannel architecture. The first aspect is to avoid unfavorable catholyte crossover. If the mixing region formed at the liquid junction extends to regions close to the anodes, the usually biohazardous catholyte will contact the biocatalysts, decreasing or even completely suppressing the biocatalytic activity. A number of engineering efforts have been devoted to dealing with the harmful cross-over. Yang et al. proposed a diverging channel with the same type as the mixing zone,33 as shown in Figure 4b. This configuration can prevent the catholyte from crossing over due to the increased crossing-over distance along the flow direction. The second aspect is to compensate the concentration depletion of both fuels and oxidants by installing multiple reactant inlets along the microscopic channels. Lim et al. studied the influence of the diffusion layer thickness on cell performance by placing the anodes at different positions along a microfluidic channel.44 If unperturbed, the width of diffusion layer would increase along the direction toward the stream effluent, as already depicted in Figure 1b. They observed that the power output decreased along the flow direction, and this observation evinced the concept that the continuously increased thickness of boundary layer retards the fuel diffusion to the reaction area. To decrease the thickness of the anodic boundary layer, a microchannel with multiple anolyte inlets was installed (Figure 4c). Compared to the conventional single inlet configuration (Figure 4a), this microfluidic flow channel equipped with multiple inlets allowed the growth of moreuniform and denser bacterial communities across the anode surface. The extra inlets hindered the development of boundary layers and enhanced the mass transfer of fuels for the inoculation of biocatalysts.40 Besides dealing with electrolyte flow pattern, developing electrodes able to facilitate biocatalysts loading and subsequently to improve EET efficiency is another method to lower RCT. The three-dimensional (3D) electrodes with wide open pores are promising electrode architectures. They provide ample surface area for attaching and straight diffusion channels for biocatalysts. For example, nickel foam coated with reduced graphene oxide (rGO) possesses a high electrochemically accessible surface area (∼13.6 m2 g−1) and has served as the bioanode.45 Due to the good biocompatibility and hierarchically

cells. Intuitively, reducing internal resistance becomes crucial to further advance the performance of microfluidic fuel cells. The internal resistance is the sum of resistances from the ohmic resistance (RΩ), mass-transfer resistance (RD), and charge-transfer resistance (RCT). RΩ refers to the electrical resistances of the electrodes, membrane, electrolyte and interfaces between each two of them. RΩ can be generally minimized by reducing the microfluidic channel width because it brings two electrodes close to each other and minimizes the distance between the two electrodes. Because microfluidic biofuel cells are membrane-free, it is not too challenging to bring down the value of RΩ. Therefore, RΩ is usually not the major contributor to the internal resistance. RD is defined as the transfer resistance associated with the convection and diffusion transfer process of reactants, protons, other ions, or a combination of these three. To reduce RD, a two-pronged strategy can be applied. On the one hand, because RD is primarily and directly related to the characteristic length (the distance a fluid flows through), reducing the characteristic length can effectively decrease RD. As demonstrated by Ren et al., the mass-transfer coefficient of a microfluidic fuel cell increased dramatically from 1.7 × 10−6 to 1.66 × 10−4 m s−1 when the characteristic length was downsized from 1 cm to 100 nm.39 On the other hand, increasing the mass loading of biocatalysts by supplying sufficient nutrition can reduce RD as well.28 Increasing the reactant concentrations and mean flow velocity, and reducing the cross-stream distance can also facilitate the reactant transportation. RD of a microscale fuel cell is typically insignificant. The major contributor and hence deserves more attention is RCT. RCT involves the EET resistance at the anode (RCT,a) and cathode (RCT,c) sides resulted from the sluggish biocatalytic and chemical reaction kinetics. In microfluidic biofuel cells, electron transportation in the biocatalysts and transfer at interfaces between the biocatalysts and electrodes are two processes that lead to resistance. Typically, RCT can account for as much as ∼96% of the total internal resistance, as experimentally determined by a previous work published in 2016.40 For microfluidic enzymatic fuel cells, ensuring the firm and seamless connection between enzymes and electrodes with the aid of electrostatic interactions or chemical bonds is crucial to mitigating RCT and also beneficial to elongate the devices’ lifetime. To date, a number of attaching methods depending on noncovalent adsorption,41 covalent bond,46 polymeric gel entrapment,42 and cross-linking of an enzyme to anchor enzymes onto electrodes43 have been realized. The following E


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Figure 5. Typical fabrication process of microstructures by photo lithography: (a) aluminum is sputtered on the layer and then patterned to form mask; (b) the photoresist is patterned on the handle layer; (c) deep reactive ion etching (DRIE) is performed on the handle layer and removes the photoresist mask; (d) DRIE is performed on the handle layer again to reach the buried oxide layer, and a 50 μm height difference is created, after which the aluminum mask and the exposed silica are removed; and (e) patterning the photoresist on the device layer and etching through the device layer are performed. A support wafer with grooves is temporarily bonded in this step to support the wafer; (f) after the removal of the photoresist layer, the wafer is soaked in HF solution to etch the oxide inside the releasing holes. Reproduced with permission from ref 49. Copyright 2017 IEEE.

interconnected porous structure, the RCT of the rGO-coated nickel foam was only 458.5 Ω, which was 2.5 times lower than that of previously reported graphite plate bioanode.12 Nanomaterial-decorated 3D structures are also favorable to enzymatic catalysts. Babadi et al. summarized the progress of nanocarbon (e.g., carbon nanotubes and carbon dots) functionalization for enzymatic immobilization enhancement on the electrode surface.46 Carbon nanomaterials with sizes matching the active sites of enzymes can accelerate EET and redox mediators transfer between enzymes and the electrodes. These materials also decreased the overpotential of the redox reactions of the mediators (e.g., NADH ↔ NAD+ + e−). 3.2. Fabrication Techniques. The chambers and electrodes of microsized fuel cells are generally fabricated by photo lithography29,47 and soft lithography.24,30,44,48 In a common synthesis protocol of photo lithography reported by our group,49 a silicon wafer is first spin-coated with a layer of photoresist (Figure 5a,b). The thickness of this layer is tailored by the intrinsic properties of the applied photoresist (e.g., composition, viscosity, and photosensitivity) and spin-coating rotational speed. The electrode structure is patterned using computer-aided design software and printed as a photomask. Then, the masked substrate is exposed to ultraviolet light and immersed in developer liquids to remove the unexposed parts (Figure 5c,d). The remainder is used as a mold, and poly(dimethylsiloxane) (PDMS) is poured into the mold and cured to obtain the desired microstructures (Figure 5e,f). The obtained structure is usually post-treated with plasma to reduce pressure drop and increase wettability. More details are available in a previous article on photo lithography.49 Though the lithography techniques are well-developed for various applications, their mass production is still limited by the lengthy steps. The majority of PDMS-based devices still need intensive

manual labor and bulky control systems for operation. At the same time, automatic manufacturing using PDMS calls for welltrained operators.50 Besides, PDMS is a material with high oxygen permeability, which may unfavorably lower the biocatalytic performance. Development of convenient fabrication approach and more friendly material is vital to speeding up the devices’ mass production. For the past decade, a printing technique has emerged as an encouraging technique to substitute the conventional lithography in applications such as microfluidic electrochemical devices, microchannels, bioreactors, microvalves, switches and pumps.51 The technique is mainly based on three methods including inkjet printing, screenprinting, and solid-wax printing. Polymers, thermoplastics, glass, and metal are employed in the fabrications of various device and electrodes. Among these techniques, direct-ink writing (DIW) is one of the most successful 3D printing techniques and the most-promising approach due to its flexibility, low cost, and ability to create the sophisticated and functional devices. Furthermore, it is particularly suitable for the construction of 3D electrode architectures with periodic distributed and wide penetrating pores. DIW utilizes ink materials with shear-thinning rheological behavior (i.e., viscosity of a fluid decreases when the fluid experiences shear force) and computer-controlled printing nozzles and printing stages. Upon printing, the ink is loaded into the printing nozzle and a high pressure is applied atop of the nozzle. The ink is then extruded from the bottom tip of the nozzle and deposited on the printing stage without spreading, sagging or collapsing.52,53 By moving the printing nozzle (or the stage), the ink filament can be directed to deposit layer-by-layer and eventually construct the 3D macrostructures preprogrammed into the controlling computer. At the time of writing, there are some successful applications on F


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Figure 6. Schematic of the DIW and post-treatment processes for manufacturing woodpile-shaped graphene aerogel electrochemical capacitor electrodes. Reproduced with permission from ref 54.

Figure 7. Schematics for microfluidic biofuel cell array fabricated by the xurography technique: (a) serial connection of electrodes, (b) parallel connection of electrodes, (c) reactant-supply mode, and (d) the assembled microfluidic chip. Reproduced with permission from ref 59. Copyright 2015 Springer.

rate capability (i.e., how much charge can be retained when increasing charging rates). We anticipate that these patterned macropores are especially beneficial to provide bacterial cells or enzymes because their large pore width can provide access for them to diffuse into the interior portion of the anodes, thus increasing the utilization efficiency and improving power density.55 Nevertheless, the application of DIW in constructing microfluidic biofuel cells is still incipient and deserves extensive research efforts in future engineering 3D microelectronics. 3.3. Scaling-Up. A viable way to scale-up microfluidic biofuel cells is to link multiple cells in series or in parallel to assemble microfluidic arrays. Figure 7 depicts microfluidic

DIW for fabricating electrochemical devices. For example, Zhu et al. printed a 3D porous woodpile-shaped graphene aerogel for electrochemical capacitors.54 As illustrated in Figure 6, the ink consists of fumed silica powder, graphene nanoplates, and resorcinol-formaldehyde polymers with GO colloidal suspensions. The woodpile-shaped graphene aerogel electrode was obtained by DIW, supercritical drying, and high-temperature annealing in nitrogen atmosphere. It possesses periodic distributed macropores that can support ion diffusion for charge storage. Experimental results showed that the 3D woodpile graphene lattice outperformed other 2D graphene electrodes with thickness 10 to 100 times thinner in terms of G


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communities, and to zoom in a single bacterium (Figure 8). Future perspectives are specifically discussed below.

enzymatic fuel cell arrays with a multilayered structure: the electrode layer at bottom, the microfluidic channel in middle, and the cover-plate containing two electrolyte inlets and one outlet on top. The electrodes can be connected in series (Figure 7a) or in parallel (Figure 7b) for different purposes. The OCV for this multilayered cell array reached 1.07 V, and the current output at the maximum power density (MPD) of the in-parallel configuration was 77.51 μA, values that were 3.24 and 3.13 times higher than those of a single cell with same electrodes and electrolytes, respectively. Another similar design combining four microfluidic microbial fuel cells was proposed by Yang et al.56 Constructing the network configuration as described in the above paragraph is considered as the most straightforward way for fuel cell scaling-up because of its inherent convenience of microfabrication for integration. However, there are still several scientific challenges in building the network configuration. One of them is the behavior inconsistency of single cell unit used to fabricate the networks. In situations in which the current is pushed to ultrahigh values, the voltage generated from one unit may not equal to other units. It causes a fuel starvation of other units with lower output potential and leads to the voltage reverse to negative values, a phenomenon called voltage reversal.57 If voltage reversal occurs, the overall cell assemble becomes an electrolysis cell that needs the input of electricity instead of a galvanic cell that outputs electricity, so the whole system is dead. Besides the unfavorable voltage reversal, inconsistent performance of each cell will lead to a poor reproducibility, a problem that will be detrimental for the largescale commercialization of miniature biofuel cells.58 The passive bypass method is one of the major methods developed to combat the voltage reversal. This method is based on the prevention of the current density from surpassing the critical value that leads to the voltage reversal. The current density can be controlled by installing external resistors between individual fuel cells60 or tracking the MPD point.61 However, this method is challenging to perform for microfluidic biofuel cells due to the need of ponderous voltage controller, magnets, and switches that can hardly be installed within a miniature space. Instead, an applicable strategy is to compensate voltage to cells with low voltage. The use of electric capacitors, which is capable of generating high voltage instantly, is current approach for stacked microfluidic biofuel cells.62,63,65,66 For example, a capacitor connected in parallel to a stacked microfluidic biofuel cell was charged by the biofuel cells initially and then used to maintain the constant voltage output of each cell by distributing charges to cells with low potential. Therefore, the capacitor could boost the whole cellstack’s voltage and avoid voltage reversal. Experimental tests confirmed that the columbic efficiency of the fuel cell stack coupled with a capacitor was largely enhanced compared to the counterpart without capacitor compensation. Its peak power was boosted to 2.6 times higher.63

Figure 8. Potential applications of microfluidic biofuel cells in the perspective. Figures of the MEMS chip and cell analysis were reproduced with permission from refs 64 and 65. Copyright 2012 and 2015, IEEE.

4.1. Power Sources. The MPD benchmark of 2.8 W m−2 from a single microfluidic microbial fuel cell33 and 6.0 W m−2 for microfluidic enzymatic fuel cell66 have already been achieved. Significantly, the microfluidic enzymatic fuel cell successfully produced a maximum volumetric power output of 83.3 kW m−3 calculated based on the volume of the anode chamber, as shown in Table 1.14 These values are ∼10−100 times higher than those achieved 10 years ago, as presented in Figure 9. These encouraging results suggest a bright future of microfluidic biofuel cells as attractive power sources for microelectronics with power demand less than 1 mW or voltage lower than 0.7 V. Heart pacemakers, in vivo blood or urine metabolites analysis systems, and portable environmental monitors are all possible options (Table 2). Specifically, a single-channel microfluidic biofuel cell with an air-breathing cathode is the most promising configuration owing to its simplified structure, small volume, light weight, and biocompatible anode. Integration of microfluidic biofuel cells into arrays is a practical way for powering microelectronics requiring high voltage, power, or both. Herein, we propose a unique treebranch-shaped microfluidic biofuel cell array mimicking the trees’ bulk-branch structures (Figure 10). Solutions containing animal, brewery, or winery wastewater (or a combination of sources) serve as the fuels. The solutions are driven by capillary force exerted from the hydrophilic microchannels as well as water evaporation, similar to the respiration process of trees. The fuels are fed to each unit and oxidized by biocatalysts inoculated on the channel walls to produce bioelectricity from each unit. Microorganisms with better durability than enzymes can be chosen as the biocatalysts in this case. All the electrons are collected as bioelectricity via the external circuit and eventually scavenged by atmospheric oxygen to close the electric circuit. Each “tree” module can be interconnected together to amplify the power and voltage. In conjunction with flow capacitors or flow batteries, the proposed tree-branch shaped system can be engineered into an independent energy-

4. FUTURE PERSPECTIVES Because the first microfluidic biofuel cell is proposed, various types of cell architecture have been demonstrated. More importantly, improvement in the energy output has gradually narrowed the gap between microfluidic biofuel cells and other micro power sources. It is also successfully demonstrated to be free-standing biosensors and opens the doors to effective platforms for studying the self-characteristics of bacterial H


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Industrial & Engineering Chemistry Research Table 1. Overview of the Selected Microfluidic Biofuel Cells Reported in the Literature bio-electrode

biocatalyst/nutrient

graphite nickel gold graphite AuAg/C [anode] + Vulcan XC-72 [cathode] gold reduced graphene oxide-nickel gold a

volume (μL)

Pmax‑volumetric (W m−3)

80 50 12a 3.1a

618a 104 312.5b 4000a

0.3a 3.75 50 1.44

mixed bacteria /acetate E. coli/butrient broth GOD-laccase enzymes/glucose P. aeruginosa PAO1/L-broth medium laccase /glucose laccase/glucose mixed bacteria/acetate Geobacter /acetate

Imax‑volumetric (A m−3)

Pmax‑areal (W m−2)

Imax‑areal (A m−2)

ref

2250a 1400b 4167b 3550b

0.618 NA NA 0.605

2.25b NA NA 0.55a

28 27 59 56

83 300a

3.27 × 105

5

19.6

14

5870a 1181.4 NA

37 300a 4000 148.75a

1.1 NA NA

7 NA 0.042a

37 12 26

Value based on the reported data. bValues estimated from the reported figures.

Figure 9. Summary of areal power density output from microfluidic (a) enzymatic fuel cells and (b) microbial fuel cells.

generation-storage 2-in-1 system. Conversely, gaseous organic compounds (e.g., toluene vapor) and liquid oxidants (e.g., hydrogen peroxide) can be chosen as the fuels and oxidants, respectively, depending on the working environment or other customer concerns. 4.2. Micrototal Analysis Systems. The confluence of microfluidic technology and biofuel cells has been deemed particularly appropriate for micrototal analysis systems (μTASs).30,73 A μTAS is a mini-sized self-driving device equipped with all the necessary accessories for chemical analysis. First, the system can be used to study the behaviors of biocatalysts or directly applied as biosensors or bioassays. For instance, microfluidic biofuel cells, owing to their

Table 2. Overview of the Energy Demand for Microelectronics Reported in the Literature

a

device

current (mA)

voltage (V)

power (μW)

ref

pacemaker pacemakera cardiac defibrillator drug pump digital thermometer switch

1 0.1 − − − −

0.54 3 − − 0.5 1.35

160 300 30−100 100−2000 40 15

67 68 69 70 71 72

Model: Affinity DR 5330L, St. Jude Medical.

Figure 10. Schematics for a tree network consisting of microfluidic biofuel cells. Each gray line represents a microfluidic fuel cell. Black solid dots and open circles are cell−cell nodes and external circuit contacts, respectively. The schematic for the unit cell is showed in the dashed box. In the anode, the organics (e.g., CH3COO−) are catalyzed by biocatalysts to release the electrons. The electrons are passed through the external circuit and consumed at the cathode with oxygen. I


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Figure 11. Schematics of a human (a) lung-on-a-chip and (b) gut-on-a-chip microfluidic devices. Both “organs on chips” were reported by Ingber’s group.86,87 A pair of microfluidic channels (made of PDMS elastomer) were separated by a porous membrane coated with extracellular matrix and lined by human intestinal epithelial. The role of membrane was to serve as a support for the growth of human alveolar epithelial cells and pulmonary microvascular endothelial cells. The organ’s microenvironment was created by the feeding fluids (blue and red dyes) and maintained by a vacuum controller. Different from the above membrane-separated “organ-on-a-chip”, the reactants (e.g., glucose and blood oxygen) are driven by breathing movements and operated in a laminar-flow mode without physical membranes. Copyright 2010 Royal Society of Chemistry and 2012 American Association for the Advancement of Science.

sulf urreducens increased near-linearly with increasing the ferric citrate concentration from 250 μM to 1000 μM or the formaldehyde concentration from 0.0001% to 0.1%.32 Compared with large-scale biosensors, the involved labor work and material cost of microsensors can be largely reduced given their miniature size. In addition to bioapplications, microfluidic biofuel cells are possible to perform as lateral flow test strips for in situ monitoring of water quality by probing factors such as toxicity, oxygen dissolution, and biochemical oxygen demand (BOD) because the activity of biocatalysts are extremely sensitive to these factors. The microsensor devices can be further simplified by utilizing paper-based microfluidics (PM) to assemble layer-by-layer architectures.21,77−85 Porous and hydrophilic paper is placed at the top of hydrophobic patterns to form a microfluidic diffusion channel. This platform is environmental friendly as all components are biodegradable. They can, therefore, be manufactured into disposable test kits with low capital costs. These biosensors can probe the fluctuation of the reactant concentration in real time based on the observed current signals. Once the concentration exceeds the safe threshold, an abnormally high current density will be yielded and initiate alert that is integrated into the testing kit. Besides serving as test strips, microfluidic biofuel cells can also be implanted in vivo as power sources for artificial organs and signal generators (with two examples shown in Figure 11).86,87 Physiological breathing movement (Figure 11a) or gut motility (Figure 11b) creates vacuum force that pumps the glucose-containing reactants in the microfluidic channel. The microfluidic biofuel cells in vivo eliminate the need of external pumps to drive the laminar flow. Enzymes fixed on both electrode walls catalyze gluconeogenesis to release electrons. The probed magnitude of current is then sent to actuators that adjust the rate of breathing or gut motility to maintain it in an

appreciable current output and miniature size, have the potential for functioning as bioelectricity online recording devices. Because the size of a microfluidic fuel cell is comparable to the size of several bacterial cells or even a single bacterium, these devices are possible to gauge the growth processes of individual bacterium cell or several bacterial cells at the interfaces between biocatalyst and electrodes by monitoring the corresponding electricity generated.74 In addition, a number of parameters associated with the miniature ecosystems such as pH, temperature and pressure can be altered to study their effects on cell growth.75 For example, some exoelectrogens (e.g., S. oneidensis MR-1) conduct electrons through DET with their molecular-scale proteins (e.g., extracellular polymer substrates, electron shuttles, and multiheme cytochromes). Second, microfluidic fuel cells can also serve as quantitative analysis platforms on these molecular-scale proteins. It is advantageous to in situ analysis of these proteins with little influence from the external environment. Third, the identification of EAB can also be realized by measuring the charges carried on cell surface. Dekker et al. presented a relevant review on the developed microfluidic tools on investigating bacterial diversity and physiologies, and interested readers are referred to this review for more details.76 Taking together, microfluidic biofuel cells are promising lab-on-a-chip tools for fundamental research on the bioassisted electron-transfer processes. Moreover, microfluidic biofuel cells can generate observable electrical signals shortly after successful inoculation of biocatalysts. The magnitude of the generated bioelectricity is, in principle, linear with the concentration of the feeding species (e.g., fuels). These two characteristics enable the use of microfluidic biofuel cells as functional microbiosensors or microbioassays. For example, Li et al. constructed a microfluidic three-electrode system with the structure shown in Figure 3d. They concluded that the bioelectricity produced by G. J


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(3) Morse, J. D. Micro-fuel Cell Power Sources. Int. J. Energy Res. 2007, 31, 576. (4) Ferrigno, R.; Stroock, A. D.; Clark, T. D.; Mayer, M.; Whitesides, G. M. Membraneless Vanadium Redox Fuel Cell Using Laminar Flow. J. Am. Chem. Soc. 2002, 124, 12930. (5) Goulet, M.-A.; Kjeang, E. Co-laminar Flow Cells for Electrochemical Energy Conversion. J. Power Sources 2014, 260, 186. (6) Moore, C. M.; Minteer, S. D.; Martin, R. S. Microchip-based Ethanol/oxygen Biofuel Cell. Lab Chip 2005, 5, 218. (7) Soukharev, V.; Mano, N.; Heller, A. A Four-electron O2electroreduction Biocatalyst Superior to Platinum and A Biofuel Cell Operating at 0.88 V. J. Am. Chem. Soc. 2004, 126, 8368. (8) Yang, Y.; Ye, D.; Li, J.; Zhu, X.; Liao, Q.; Zhang, B. Microfluidic Microbial Fuel Cells: From Membrane to Membrane Free. J. Power Sources 2016, 324, 113. (9) Lee, J. W.; Kjeang, E. A Perspective on Microfluidic Biofuel Cells. Biomicrofluidics 2010, 4, 41301. (10) Safdar, M.; Janis, J.; Sanchez, S. Microfluidic Fuel Cells for Energy Generation. Lab Chip 2016, 16, 2754. (11) Xie, X.; Yu, G.; Liu, N.; Bao, Z.; Criddle, C. S.; Cui, Y. Graphene−sponges as High-Performance Low-cost Anodes for Microbial Fuel Cells. Energy Environ. Sci. 2012, 5, 6862. (12) Yang, Y.; Liu, T.; Liao, Q.; Ye, D.; Zhu, X.; Li, J.; Zhang, P.; Peng, Y.; Chen, S.; Li, Y. A Three-dimensional Nitrogen-doped Graphene Aerogel-activated Carbon Composite Catalyst That Enables Low-cost Microfluidic Microbial Fuel Cells with Superior Performance. J. Mater. Chem. A 2016, 4, 15913. (13) Leech, D.; Kavanagh, P.; Schuhmann, W. Enzymatic Fuel Cells: Recent Progress. Electrochim. Acta 2012, 84, 223. (14) López-González, B.; Dector, A.; Cuevas-Muñiz, F.; Arjona, N.; Cruz-Madrid, C.; Arana-Cuenca, A.; Guerra-Balcázar, M.; Arriaga, L.; Ledesma-García, J. Hybrid Microfluidic Fuel Cell Based on Laccase/C and AuAg/C Electrodes. Biosens. Bioelectron. 2014, 62, 221. (15) Togo, M.; Takamura, A.; Asai, T.; Kaji, H.; Nishizawa, M. Structural Studies of Enzyme-based Microfluidic Biofuel Cells. J. Power Sources 2008, 178, 53. (16) González-Guerrero, M. J.; Esquivel, J. P.; Sánchez-Molas, D.; Godignon, P.; Muñoz, F. X.; Del Campo, F. J.; Giroud, F.; Minteer, S. D.; Sabaté, N. Membraneless Glucose/O2 Microfluidic Enzymatic Biofuel Cell Using Pyrolyzed Photoresist Film Electrodes. Lab Chip 2013, 13, 2972. (17) Zebda, A.; Renaud, L.; Cretin, M.; Innocent, C.; Ferrigno, R.; Tingry, S. Membraneless Microchannel Glucose Biofuel Cell with Improved Electrical Performances. Sens. Actuators, B 2010, 149, 44. (18) Du Toit, H.; Rashidi, R.; Ferdani, D. W.; Delgado-Charro, M. B.; Sangan, C. M.; Di Lorenzo, M. Generating Power from Transdermal Extracts Using A Multi-electrode Miniature Enzymatic Fuel Cell. Biosens. Bioelectron. 2016, 78, 411. (19) Dector, A.; Escalona-Villalpando, R.; Dector, D.; VallejoBecerra, V.; Chávez-Ramírez, A.; Arriaga, L.; Ledesma-García, J. Perspective Use of Direct Human Blood as An Energy Source in Airbreathing Hybrid Microfluidic Fuel Cells. J. Power Sources 2015, 288, 70. (20) Galindo, R.; Dector, A.; Arriaga, L. G.; Gutiérrez, S.; Herrasti, P. Maghemite as A Catalyst for Glucose Oxidation in A Microfluidic Fuel Cell. J. Electroanal. Chem. 2012, 671, 38. (21) Escalona-Villalpando, R. A.; Reid, R. C.; Milton, R. D.; Arriaga, L.; Minteer, S. D.; Ledesma-García, J. Improving the Performance of Lactate/oxygen Biofuel Cells Using A Microfluidic Design. J. Power Sources 2017, 342, 546. (22) Logan, B. E.; Regan, J. M. Microbial Fuel Cells-Challenges and Applications. Environ. Sci. Technol. 2006, 40, 5172. (23) Qian, F.; Morse, D. E. Miniaturizing Microbial Fuel Cells. Trends Biotechnol. 2011, 29, 62. (24) Li, Z.; Zhang, Y.; LeDuc, P. R.; Gregory, K. B. Microbial Electricity Generation via Microfluidic Flow Control. Biotechnol. Bioeng. 2011, 108, 2061.

optimal range. When the microfluidic biofuel cell served as an analysis platform or power generator in vitro, it can use an external pump to drive the flow. Zebda et al., compared the pumping power to sustain the laminar flow with the delivered power.48 They found that the pumping power consumed only 1.5% of total delivered power at a relatively low flow rate (lower than 700 μL min−1). The volumetric flow rates of microfluidic devices in the lab are typically lower than 500 μL min−1. It illustrates the possibility of microfluidic biofuel cell to serve as a μTAS in vivo or in vitro.

5. CONCLUSIONS The perspective article presents an overview of the recent progress of a family of fuel cells coined microfluidic biofuel cells. Basic working principles, challenges related to reducing internal resistance, constructing 3D electrodes as well as scaling-up toward commercialization, together with potential proof-of-concept applications, are summarized. Possible combinations of the advanced fluid-control techniques, applicationson-a-chip techniques, and their advantages and feasibility have been specifically prospected and discussed in the last section. Considering that the demonstrations on the practical applications of microfluidic fuel cells as power sources and analysis systems for which we aim at in this prospective are not abundant (

Generating Electricity on Chips: Microfluidic Biofuel Cells in

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