Anal. Chem. 2009, 81, 9538–9545
Analytical Techniques for Characterizing Enzymatic Biofuel Cells Michael J. Moehlenbrock, Robert L. Arechederra, Kyle H. Sjo¨holm, and Shelley D. Minteer Saint Louis University
The rapid depletion in global nonrenewable energy stores has prompted a dramatic increase in both academic and industrial research toward alternate means of energy conversion. Although improbable as a single solution to the problem, fuel cells provide many potential contributions in the form of performance combined with scalability. Fuel cells have demonstrated high levels of power production through the consumption of renewable resources in an easily engineered small scale device that operates under mild conditions and could potentially replace today’s ubiquitous batteries. Many evaluate energy conversion devices on the basis of the amount of energy converted per unit volume [energy density (Whr/L)] or per unit mass [specific energy (Whr/kg)]. Fuel cells not only rival battery performance in these terms, but also do not require time-consuming charging and do not suffer from the severe hysteresis effects seen in secondary batteries. However, many of these devices use expensive precious metal catalysts that often limit their commercial viability. Passivation by carbon monoxide or other byproducts causes losses in power production over time, and these devices often require high temperatures and harsh conditions to operate efficiently. Thus, many researchers have looked to nature to assist in our current energy conversion needs. Because of biological versatility and efficiency, organisms are able to convert enormous amounts of energy from an incomparable range of chemical substrates. Many researchers have examined ways of harnessing this ability using biofuel cells. Biofuel cells replace the metal catalyst with a biological catalyst: a microbe, enzyme, or even organelle interacting with an electrode surface.1-3 These types of catalysts offer 9538
Analytical Chemistry, Vol. 81, No. 23, December 1, 2009
ROBERT GATES
Enzymatic biofuel cells, which replace expensive metal catalysts with enzymes, are still in an early stage of development. This article details the analytical techniques that are often employed for evaluating and characterizing enzymatic biofuel cells and their corresponding bioanodes and biocathodes. (To listen to a podcast about this feature, please go to the Analytical Chemistry multimedia page at pubs.acs.org/page/ancham/audio/index.html.)
great benefits in catalytic activity, specificity, and cost. However, development and full evaluation of these dynamic and often sensitive bioelectrochemical systems require a diverse range of expertise. This article will focus on the current techniques used to evaluate the integrity, kinetics, and performance of enzymatic biofuel cells and the applications of the devices. The techniques described span not only the obvious electroanalytical characterization methods needed to evaluate a power source or analytical device, but also common biological and materials characterization techniques. Enzymatic biofuel cells are in an early stage of development, so new analytical techniques are being employed to understand the advantages and limitations of this technology and the engineering design envelope for applications. SPECTROSCOPIC TECHNIQUES Enzymatic biofuel cells employ oxidoreductase enzymes capable of catalyzing redox reactions. Enzymes that are free in solution 10.1021/ac901243s CCC: $0.00 2009 American Chemical Society Published on Web 11/04/2009
often have short lifetimes, limiting their use in biofuel cell applications, so many researchers have focused on immobilization strategies to both stabilize the protein structure and put the enzyme closer to the electrode surface. When immobilizing enzymes to an electrode surface, one must carefully evaluate the chemistry and accurately monitor the enzymes’ catalytic activity. Traditional spectroscopic enzyme activity assays are often the choice for evaluating the bioelectrode viability. Simultaneous spectroscopic analysis of enzyme activity with electrochemical characterization can also provide powerful insight into the limitations of the catalyst or transfer of electrons to the electrode surface. Spectroscopic techniques can also determine the reduction potentials of the enzymatic reactions. The most common spectrophotometric method used is UV-vis spectroscopy. Oxidoreductase enzymes often either require an electron-carrying cofactor or produce/deplete another substrate whose generation/consumption can be detected through a change in absorbance. Most commonly, NAD(P)-dependent dehydrogenase enzyme activity is assayed via absorbance of NAD(P)H at 340 nm. When the cofactor is bound to the enzyme or otherwise unavailable to be assayed through typical UV-vis absorbance, an indirect method of spectroscopy using dyes such as DCIP (2,6dichloroindophenol) and PMS (N-methylphenazonium methosulfate) can be employed. These dyes are reduced by the enzyme’s catalytic oxidation of a substrate.4 Many fluorescence assays also are performed similarly. For instance, a dehydrogenase enzyme catalyzing the oxidation of fuel produces NADH, which can be quantified with fluorescence. Although usually simple, fluorescence spectroscopy in these systems is often complicated by the intrinsic fluorescence of the immobilized proteins. Spectroscopic assays are also complicated by the extremely high concentration of catalyst within the film; not only high intrinsic fluorescence background, but also non-uniform absorbance of the protein causes a high background in assays. Similarly, examining enzymes that have been used to serially transform a substrate (i.e., that are part of metabolic pathways) but do not oxidize or reduce a substrate is important. These cannot be monitored electrochemically, but for enzymes that generate ATP, a luciferin/luciferase chemiluminescent assay can be used. However, as improving energy density of the biofuel cell has become an important issue, NMR and MS have become popular techniques for studying reaction intermediates and products of isotopically labeled substrates/fuel. With the mass specificity of MS, one can use single ion mode monitoring to examine the consumption of a substrate or the formation of a product of catalysis by sampling the fuel cell compartment in real time. Using NMR, one could simultaneously examine substrate consumption and product formation to determine the rate of reaction and demonstrate the level of oxidation of an isotopically labeled fuel.5-7 X-ray photoelectron spectroscopy (XPS) is very useful for characterizing materials in biofuel cells and surface composition of electrodes.8 XPS directs a beam of monochromatic X-rays onto a sample, and the resulting photoelectrons are focused into a detector that measures their energies. The surface composition is mapped, and in the case of nanomaterials, their compositions and up to 10 nm of their surface strata can be analyzed. XPS can study the chemical binding between elements in materials and
spatially resolve and determine protein density on a bioelectrode surface.8,9 ELECTROCHEMICAL CHARACTERIZATION A biofuel cell is composed of an anode and a cathode, which are connected to a load (i.e., a resistor) and examined using a wide range of electroanalytical techniques. Either a polymer electrolyte membrane or a salt bridge can separate the electrodes. These electroanalytical techniques are the backbone of the analytical tools used for evaluating biofuel cells and their electrodes. Common techniques include linear sweep voltammetry, cyclic voltammetry, amperometry, and galvanostatic and potentiostatic coulometry.1,10-13 One of the most common and simplest evaluation tools is the measurement of the electrochemical potential at open circuit (i ) 0). This is similar to measuring potentials with a voltmeter and provides information about the thermodynamicssbut not the kineticssof the cell. Early on, this was considered an important evaluation tool, but researchers have now realized that kinetics and transport are typically the limiting issues in a biofuel cell. Voltammetry is a very common method for characterizing enzyme-modified electrodes. Cyclic voltammetry scans a potential window in the forward and reverse directions and measures the resulting current. This technique can determine the reduction potential of the enzyme or co-enzyme and the overpotential for the system, which corresponds to its efficiency. Voltammetry can also show the enzyme’s ability to catalyze fuel oxidation or oxygen reduction by either direct (to the electrode) or mediated electron transfer (DET and MET, respectively). MET is compatible with almost all naturally occurring oxidoreductase enzymes and co-enzymes. But because the enzyme cannot efficiently transfer the electrons to the electrode, it requires additional components (either small molecule redox mediators or redox polymers), which make the system more complex and less stable.14 The vast majority of oxidoreductase enzymes that require MET are NAD+ dependent; the most common of these used in biofuel cell anodes are glucose dehydrogenase and alcohol dehydrogenase. The half-cell electrochemistry and biofuel cell operation of these enzymes have been thoroughly characterized. DET is a more straightforward process resulting in a simpler system but is limited to enzymes capable of transferring electrons directly from the enzyme to the current collector. Pyrroloquinoline quinone (PQQ)-dependent, heme containing, and flavin adenine dinucleotide (FAD)-dependent enzymes can undergo DET. As examples, PQQ-dependent alcohol dehydrogenase and FADdependent glucose oxidase have both had their DET thoroughly characterized with cyclic voltammetry and their anodic operation demonstrated in biofuel cells. One disadvantage of some FADdependent enzymes such as glucose oxidase is that they produce highly reactive hydrogen peroxide, which can damage biofuel cell components. Polarization curves, which are a common method of evaluation for cell performance, are developed by applying a variable load and plotting potential as a function of current density for the fuel cell.15 Figure 1 is an example polarization curve showing the contributions to deviations from ideal behavior, which result from many factors including concentration, activation, transfer, resistance, and earlier or simultaneous reactions within the system. Analytical Chemistry, Vol. 81, No. 23, December 1, 2009
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Figure 1. Representative polarization curve that illustrates deviations from ideality, which include increased electrochemical cell resistance, slow electron transfer kinetics, overpotential losses due to cofactors and/or mediators, and losses due to slow mass transport at bioelectrode surfaces. ηo represents the overpotential of the system, ηR represents the resistance of the cell, and ηkin represents performance losses due to the kinetics of the catalysts.
Polarization curves can be used to generate power curves by plotting the power produced by the system versus the current density or potential. This allows for the determination of the maximum power density that the biofuel cell can generate at the optimal potential. Methods of obtaining a polarization curve (and thus a power curve) include very slow scan (
