Enzymatic Fuel Cells: From Fundamentals to Applications -- Contents -- Preface -- Contributors -- 1 Introduction -- List of Abbreviations -- 2 Electrochemical Evaluation of Enzymatic Fuel Cells and Figures of Merit -- 2.1 Introduction -- 2.2 Electrochemical Characterization -- 2.2.1 Open-Circuit Measurements -- 2.2.2 Cyclic Voltammetry -- 2.2.3 Electron Transfer -- 2.2.4 Polarization Curves -- 2.2.5 Power Curves -- 2.2.6 Electrochemical Impedance Spectroscopy -- 2.2.7 Multienzyme Cascades -- 2.2.8 Rotating Disk Electrode Voltammetry -- 2.3 Outlook -- Acknowledgment -- List of Abbreviations -- References -- 3 Direct Bioelectrocatalysis: Oxygen Reduction for Biological Fuel Cells -- 3.1 Introduction -- 3.2 Mechanistic Studies of Intramolecular Electron Transfer -- 3.2.1 Determining the Redox Potential of MCO -- 3.2.2 Effect of pHand Inhibitors on the Electrochemistry of MCO -- 3.3 Achieving DET of MCO by Rational Design -- 3.3.1 Surface Analysis of Enzyme-Modified Electrodes -- 3.3.2 Design of MCO-Modified Biocathodes Based on Direct Bioelectrocatalysis -- 3.3.3 Design of MCO-Modified "Air-Breathing" Biocathodes -- 3.4 Outlook -- Acknowledgments -- List of Abbreviations -- References -- 4 Anodic Catalysts for Oxidation of Carbon-Containing Fuels -- 4.1 Introduction -- 4.2 Oxidases -- 4.2.1 Electron Transfer Mechanisms of Glucose Oxidase -- 4.3 Dehydrogenases -- 4.3.1 The NADH Reoxidation Issue -- 4.3.2 Mediators for Electrochemical Oxidation of NADH -- 4.3.3 Electropolymerization of Azines -- 4.3.4 Alcohol Dehydrogenase as a Model System -- 4.4 PQQ-Dependent Enzymes -- 4.5 Outlook -- Acknowledgment -- List of Abbreviations -- References -- 5 Anodic Bioelectrocatalysis: From Metabolic Pathways to Metabolons -- 5.1 Introduction -- 5.2 Biological Fuels -- 5.3 Promiscuous Enzymes Versus Multienzyme Cascades Versus Metabolons.

5.3.1 Promiscuous Enzymes -- 5.3.2 Multienzyme Cascades -- 5.3.3 Metabolons -- 5.4 Direct and Mediated Electron Transfer -- 5.5 Fuels -- 5.5.1 Hydrogen -- 5.5.2 Ethanol -- 5.5.2.1 Protein Engineering -- 5.5.3 Methanol -- 5.5.4 Methane -- 5.5.5 Glucose -- 5.5.5.1 Early Work -- 5.5.5.2 Recent Work -- 5.5.5.3 Miniature and Implantable Glucose BFCs -- 5.5.5.4 Promiscuous Enzymes for Glucose Anodes -- 5.5.6 Sucrose -- 5.5.7 Trehalose -- 5.5.8 Fructose -- 5.5.9 Lactose -- 5.5.10 Lactate -- 5.5.11 Pyruvate -- 5.5.12 Glycerol -- 5.5.13 Fatty Acids -- 5.6 Outlook -- Acknowledgment -- List of Abbreviations -- References -- 6 Bioelectrocatalysis of Hydrogen Oxidation/Reduction by Hydrogenases -- 6.1 Introduction -- 6.2 Hydrogenases -- 6.3 Biological Fuel Cells Using Hydrogenases: Electrocatalysis -- 6.4 Electrocatalysis by Functional Mimics of Hydrogenases -- 6.4.1 [FeFe]-Hydrogenase Models -- 6.4.2 [NiFe]-Hydrogenase Models -- 6.4.3 Incorporation of Outer Coordination Sphere Features -- 6.5 Outlook -- Acknowledgments -- List of Abbreviations -- References -- 7 Protein Engineering for Enzymatic Fuel Cells -- 7.1 Engineering Enzymes for Catalysis -- 7.2 Engineering Other Properties of Enzymes -- 7.2.1 Stability -- 7.2.2 Size -- 7.2.3 Cofactor Specificity -- 7.3 Enzyme Immobilization and Self-Assembly -- 7.3.1 Engineering for Supermolecular Assembly -- 7.4 Artificial Metabolons -- 7.4.1 DNA-Templated Metabolons -- 7.5 Outlook -- List of Abbreviations -- References -- 8 Purification and Characterization of Multicopper Oxidases for Enzyme Electrodes -- 8.1 Introduction -- 8.2 General Considerations for MCO Expression and Purification -- 8.3 MCO Production and Expression Systems -- 8.4 MCO Purification -- 8.5 Copper Stability and Specific Considerations for MCO Production -- 8.6 Spectroscopic Monitoring and Characterization of Copper Centers -- 8.7 Outlook.

Acknowledgment -- List of Abbreviations -- References -- 9 Mediated Enzyme Electrodes -- 9.1 Introduction -- 9.2 Fundamentals -- 9.2.1 Electron Transfer Overpotentials -- 9.2.2 Electron Transfer Rate -- 9.2.3 Enzyme Kinetics -- 9.3 Types of Mediation -- 9.3.1 Freely Diffusing Mediator in Solution -- 9.3.2 Mediation in Cross-Linked Redox Polymers -- 9.3.2.1 The "Wired" Glucose Oxidase Anode -- 9.3.3 Further Redox Polymer Mediation -- 9.3.4 Mediation in Other Immobilized Layers -- 9.4 Aspects of Mediator Design I: Mediator Overpotentials -- 9.4.1 Considering Species Potentials in a Methanol-Oxygen BFC -- 9.4.2 The Earliest Methanol-Oxidizing BFC Anodes -- 9.4.3 A Four-Enzyme Methanol-Oxidizing Anode -- 9.5 Aspects of Mediator Design II: Saturated Mediator Kinetics -- 9.5.1 An Immobilized Laccase Cathode -- 9.5.2 Potential of the Osmium Redox Polymer -- 9.5.3 Concentration of Redox Sites in the Mediator Film -- 9.6 Outlook -- List of Abbreviations -- References -- 10 Hierarchical Materials Architectures for Enzymatic Fuel Cells -- 10.1 Introduction -- 10.2 Carbon Nanomaterials and the Construction of the Bio-Nano Interface -- 10.2.1 Carbon Black Nanomaterials -- 10.2.2 Carbon Nanotubes -- 10.2.3 Graphene -- 10.2.4 CNT-Decorated Porous Carbon Architectures -- 10.2.5 Buckypaper -- 10.3 Biotemplating: The Assembly of Nanostructured Biological-Inorganic Materials -- 10.3.1 Protein-Mediated 3D Biotemplating -- 10.4 Fabrication of Hierarchically Ordered 3D Materials for Enzyme and Microbial Electrodes -- 10.4.1 Chitosan-CNT Conductive Porous Scaffolds -- 10.4.2 Polymer/Carbon Architectures Fabricated Using Solid Templates -- 10.5 Incorporating Conductive Polymers into Bioelectrodes for Fuel Cell Applications -- 10.5.1 Conductive Polymer-Facilitated DET Between Laccase and a Conductive Surface -- 10.5.2 Materials Design for MFC -- 10.6 Outlook.

Acknowledgment -- List of Abbreviations -- References -- 11 Enzyme Immobilization for Biological Fuel Cell Applications -- 11.1 Introduction -- 11.2 Immobilization by Physical Methods -- 11.2.1 Adsorption -- 11.3 Entrapment as a Pre- and Post-Immobilization Strategy -- 11.3.1 Stabilization via Encapsulation -- 11.3.2 Redox Hydrogels -- 11.4 Enzyme Immobilization via Chemical Methods -- 11.4.1 Covalent Immobilization -- 11.4.2 Molecular Tethering -- 11.4.3 Self-Assembly -- 11.5 Orientation Matters -- 11.6 Outlook -- Acknowledgment -- List of Abbreviations -- References -- 12 Interrogating Immobilized Enzymes in Hierarchical Structures -- 12.1 Introduction -- 12.2 Estimating the Bound Active (Redox) Enzyme -- 12.2.1 Modeling the Performance of Immobilized Redox Enzymes in Flow-Through Mode to Estimate the Concentration of Substrate at the Enzyme Surface -- 12.3 Probing the Distribution of Immobilized Enzyme Within Hierarchical Structures -- 12.4 Probing the Immediate Chemical Microenvironments of Enzymes in Hierarchical Structures -- 12.5 Enzyme Aggregation in a Hierarchical Structure -- 12.6 Outlook -- Acknowledgment -- List of Abbreviations -- References -- 13 Imaging and Characterization of the Bio-Nano Interface -- 13.1 Introduction -- 13.2 Imaging the Bio-Nano Interface -- 13.2.1 Scanning Electron Microscopy -- 13.2.1.1 Backscattered Electrons -- 13.2.1.2 Three-Dimensional Imaging -- 13.2.2 Transmission Electron Microscopy -- 13.3 Characterizing the Bio-Nano Interface -- 13.3.1 X-Ray Photoelectron Spectroscopy -- 13.3.1.1 Specific Considerations for Analysis of Enzymes Using XPS -- 13.3.1.2 Instrumentation and Experimental Details for XPS of Biomolecules -- 13.3.1.3 Elemental Quantification for Fingerprinting Enzymes -- 13.3.1.4 High-Resolution Analysis for Fingerprinting Enzymes -- 13.3.1.5 Probing Molecular Interactions.

13.3.1.6 Probing Physical Architecture of Thin Films Using ARXPS -- 13.3.2 Surface Plasmon Resonance -- 13.4 Interrogating the Bio-Nano Interface -- 13.4.1 Atomic Force Microscopy -- 13.4.1.1 Basic Principles of AFM -- 13.4.1.2 AFM Techniques -- 13.4.1.3 Examples of AFM Analysis and Applications -- 13.5 Outlook -- Acknowledgment -- List of Abbreviations -- References -- 14 Scanning Electrochemical Microscopy for Biological Fuel Cell Characterization -- 14.1 Introduction -- 14.2 Theory and Operation -- 14.3 Ultramicroelectrodes -- 14.3.1 Approach Curve Method of Analysis -- 14.4 Modes of SECM Operation -- 14.4.1 Negative Feedback Mode -- 14.4.2 Positive Feedback Mode -- 14.4.3 Generation-Collection Mode -- 14.4.4 Induced Transfer Mode -- 14.5 SECM for BFC Anodes -- 14.5.1 Enzyme-Mediated Feedback Imaging -- 14.5.1.1 Imaging Glucose Oxidase Activity Using FB Mode -- 14.5.2 Generation-Collection Mode Imaging -- 14.5.2.1 Imaging GOx Using SG/TC Mode -- 14.6 SECM for BFC Cathodes -- 14.6.1 Tip Generation-Substrate Collection Mode -- 14.6.1.1 Imaging ORR by TG/SC Mode -- 14.6.1.2 Imaging Laccase by SG/TC Mode -- 14.6.2 Redox Competition Mode -- 14.6.2.1 Imaging ORR by RC Mode -- 14.7 Catalyst Screening Using SECM -- 14.8 SECM for Membranes -- 14.9 Probing Single Enzyme Molecules Using SECM -- 14.10 Combining SECM with Other Techniques -- 14.10.1 Atomic Force Microscopy -- 14.10.2 Confocal Laser Scanning Microscopy -- 14.11 Outlook -- List of Abbreviations -- References -- 15 In Situ X-Ray Spectroscopy of Enzymatic Catalysis: Laccase-Catalyzed Oxygen Reduction -- 15.1 Introduction -- 15.2 Defining the Enzyme/Electrode Interface -- 15.3 Direct Electron Transfer Versus Mediated Electron Transfer -- 15.3.1 Mediated Electron Transfer -- 15.4 The Blue Copper Oxidases -- 15.4.1 Laccase -- 15.5 In Situ XAS -- 15.5.1 Os L3-Edge -- 15.5.2 uMET.

15.5.3 Mediated Electron Transfer.