A combined electrochemical and optical trapping platform for measuring single cell respiration rates at electrode interfaces Benjamin J. Gross and Mohamed Y. El-Naggar Citation: Review of Scientific Instruments 86, 064301 (2015); doi: 10.1063/1.4922853 View online: http://dx.doi.org/10.1063/1.4922853 View Table of Contents: http://scitation.aip.org/content/aip/journal/rsi/86/6?ver=pdfcov Published by the AIP Publishing Articles you may be interested in An electrochemical surface plasmon resonance imaging system targeting cell analysis Rev. Sci. Instrum. 84, 085005 (2013); 10.1063/1.4819027 A microfluidic platform for measuring electrical activity across cells Biomicrofluidics 6, 034121 (2012); 10.1063/1.4754599 An electroactive microwell array for trapping and lysing single-bacterial cells Biomicrofluidics 5, 024114 (2011); 10.1063/1.3605508 Combining multiple optical trapping with microflow manipulation for the rapid bioanalytics on microparticles in a chip Rev. Sci. Instrum. 78, 116101 (2007); 10.1063/1.2804768 Confocal micro-Raman spectroscopy of single biological cells using optical trapping and shifted excitation difference techniques J. Appl. Phys. 93, 2982 (2003); 10.1063/1.1542654
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REVIEW OF SCIENTIFIC INSTRUMENTS 86, 064301 (2015)
A combined electrochemical and optical trapping platform for measuring single cell respiration rates at electrode interfaces Benjamin J. Gross1 and Mohamed Y. El-Naggar1,2,3,a) 1
Department of Physics and Astronomy, University of Southern California, 920 Bloom Walk, Los Angeles, California 90089-0484, USA 2 Molecular and Computational Biology Section, Department of Biological Sciences, University of Southern California, Los Angeles, California 90089-0484, USA 3 Department of Chemistry, University of Southern California, Los Angeles, California 90089-0484, USA
(Received 12 February 2015; accepted 7 June 2015; published online 23 June 2015) Metal-reducing bacteria gain energy by extracellular electron transfer to external solids, such as naturally abundant minerals, which substitute for oxygen or the other common soluble electron acceptors of respiration. This process is one of the earliest forms of respiration on earth and has significant environmental and technological implications. By performing electron transfer to electrodes instead of minerals, these microbes can be used as biocatalysts for conversion of diverse chemical fuels to electricity. Understanding such a complex biotic-abiotic interaction necessitates the development of tools capable of probing extracellular electron transfer down to the level of single cells. Here, we describe an experimental platform for single cell respiration measurements. The design integrates an infrared optical trap, perfusion chamber, and lithographically fabricated electrochemical chips containing potentiostatically controlled transparent indium tin oxide microelectrodes. Individual bacteria are manipulated using the optical trap and placed on the microelectrodes, which are biased at a suitable oxidizing potential in the absence of any chemical electron acceptor. The potentiostat is used to detect the respiration current correlated with cell-electrode contact. We demonstrate the system with single cell measurements of the dissimilatory-metal reducing bacterium Shewanella oneidensis MR-1, which resulted in respiration currents ranging from 15 fA to 100 fA per cell under our measurement conditions. Mutants lacking the outer-membrane cytochromes necessary for extracellular respiration did not result in any measurable current output upon contact. In addition to the application for extracellular electron transfer studies, the ability to electronically measure cell-specific respiration rates may provide answers for a variety of fundamental microbial physiology questions. C 2015 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4922853]
I. INTRODUCTION
Electron transfer is a fundamental process that governs the reduction-oxidation (redox) reactions critical for all cellular energy conversion pathways, including photosynthesis and respiration.1,2 Respiratory organisms extract free energy from their environment by coupling the oxidation of electron donors (food) to the reduction of electron acceptors (oxidants). While oxygen is the familiar electron acceptor of aerobic respiration, many anaerobic microbes are capable of performing electron transfer to alternative soluble acceptors, such as nitrates and sulfates, which can also diffuse to the electron transport chain inside living cells. In contrast to these intracellular electron transfer processes, we now know that a class of anaerobic microbes called dissimilatory metal-reducing bacteria (DMRB), including Shewanella, are capable of extracellular electron transfer (EET) to external surfaces.3–5 This respiratory strategy is attracting considerable fundamental and applied interest, considering that the external electron acceptors can range from natural oxidized metals in the environment to anodes.5
a)Author to whom correspondence should be addressed. Electronic mail:
[email protected]
From an environmental perspective, microbes performing EET are major players in elemental cycles, including the carbon cycle, occurring at a global earth scale.4,5 From a technological perspective, microbial EET is heavily pursued for interfacing redox reactions to electrodes in multiple renewable energy technologies. These technologies include microbial fuel cells (MFCs),6 where microbial biofilms oxidize diverse fuels and route the resulting electrons to energy-harvesting anodes, and the reverse process of microbial electrosynthesis,7 where renewable electricity drives reductive microbial metabolism for synthesis of high value fuels. The success of these technologies hinges on efficient electron exchange mechanisms between microbes and electrode surfaces. Two decades of research on the most studied DMRB model organism, Shewanella oneidensis MR-1, have revealed multiple mechanisms that can be categorized as either direct or indirect.8 Indirect mechanisms rely on biogenic or naturally occurring molecules, including flavins,9,10 that diffusively shuttle electrons from cells to electrodes. Direct mechanisms route electrons through multiheme cytochromes that are either located on the cell surface11–13 or along micrometer-long membrane extensions known as bacterial nanowires.14–18 Optimization of microbial fuel cells requires detailed knowledge of the contribution from each EET mechanism
0034-6748/2015/86(6)/064301/8/$30.00 86, 064301-1 © 2015 AIP Publishing LLC This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP: 129.115.103.99 On: Mon, 24 Aug 2015 01:46:53
064301-2
B. J. Gross and M. Y. El-Naggar
within biofilms.19 This includes an understanding of the biological, mass transport, and electrochemical factors that can limit performance,20 down to the fundamental respiration rate by individual cells. Much of our mechanistic knowledge of microbe-to-electrode EET is derived from a combination of electrochemical techniques and genetic approaches. Microbial fuel cells and anodic half cells have been used to quantify and compare the electricity production from wild-type S. oneidensis MR-1 and mutant strains lacking specific proteins that are hypothesized to affect EET.21 The impact of specific mutations on EET, especially when combined with additional biochemical, structural, and spectrometric techniques, can be used to reconstruct the cellular EET pathways.22 One difficulty, however, stems from the bulk nature of most electrochemical techniques, which measure the total current from a large microbial population (e.g., ≫107 cells). While it is tempting to think of an average cell-specific EET rate, obtained by normalizing with (often qualitative) cell density measurements performed at the end of an experiment, such an approach ignores the microbial heterogeneity present in any population.23 For instance, the same total anodic current can result from all cells respiring uniformly at a specific rate, or from only 10% of cells respiring at ten times that specific rate; these two scenarios clearly require completely different strategies for optimization. The effect of heterogeneity is particularly important in microbial fuel cells that contain a mixture of biofilm and planktonic cells, keeping in mind that a wide statistical distribution of cellular respiration current is possible even within each of these two sub-groups. The causes of microbial heterogeneity include basic genotypic variability resulting from mutations, as well as phenotypic variability resulting from progression through the cell cycle or as a physiological response modulated by the local environment and its history.24 As we seek a more complete understanding of the maximum power densities possible from microbial technologies such as microbial fuel and electrosynthesis cells, there is a clear need for techniques that quantify both the expected per-cell and statistical distribution of EET in microbial cultures. Single cell techniques will also allow more direct comparisons between experiments utilizing different microbes, which may result in different biofilms properties or cell densities, even under similar growth conditions. Perhaps more importantly, given the wealth of knowledge that can be gained from genetic studies, single cell techniques are also critical for studies that compare wild-type to mutant strains lacking putative EET proteins. Gene products hypothesized to perform EET (e.g., surface cytochromes or type IV pili) may also impact the cellular surface charge, attachment ability, and biofilm forming properties. Bulk techniques therefore cannot distinguish whether a decrease in anodic current is due to diminished EET directly through a specific protein, or indirectly by disrupting cellular attachment to electrodes and the subsequent biofilm development. Only a handful of recent studies address these issues surrounding bulk electrochemical techniques. McLean et al. quantified the average per-cell EET to graphite electrodes by combining live noninvasive imaging with an optically accessible MFC, allowing an accurate cellular count as
Rev. Sci. Instrum. 86, 064301 (2015)
S. oneidensis MR-1 population of the anode developed from separate cells to mature biofilms.25 This approach revealed a current per cell ranges from 50 to 200 fA, depending on the MFC resistance and growth phase. Another study obtained single cell measurements of EET from a different DMRB, Geobacter sulfurreducens DL-1, using microscale Ti/Au electrode arrays.26 When active cells transiently came into contact with these electrodes, a short circuit current of 92(±33) fA per cell was observed in about 30% of the contact events. Finally, Liu et al. reported an innovative approach taking advantage of optical tweezers to attach single S. oneidensis MR-1 cells to electrodes, revealing a current output of 200 fA per cell at a working electrode potential of +200 mV vs. Ag/AgCl.27 To date, however, there is no technical description of a complete instrument or standardized measurement procedure for detecting single cell extracellular respiration. Here, we present the first complete description of a system inspired by, and combining elements from, the abovementioned studies of specific respiration rates. The integrated system combines live imaging, an infrared optical trap, and a new electrochemical chip concept containing an array of indium tin oxide (ITO) microelectrodes that serve as ‘landing pads’ for individually trapped bacteria. The microelectrode potentials are controlled by a potentiostat, allowing the detection of the respiration current concomitant with landing a cell on a specific microelectrode. The entire system can be built from standard commercially available optical and electrochemical components, in combination with standard lithographic microfabrication techniques. The microfabrication procedure was optimized to address several challenges stemming from the biocompatibility requirement and high current sensitivity needed to detect single cells. Furthermore, the biological growth conditions and measurement protocol are described in detail. In addition to allowing single cell measurements of EET from a variety of organisms and mutants, the reported system may find wider applicability for general microbiology studies, including the analysis of how respiration rates in heterogeneous microbial cultures are impacted by specific environmental factors. II. EXPERIMENTAL SETUP
The single cell extracellular respiration platform integrates three main components: (1) an infrared optical tweezers system for positioning individual cells, (2) a custom transparent electrochemical chip containing an array of ITO microelectrodes on standard coverslips, and (3) a vacuumsealed perfusion chamber for loading and handling of bacterial cultures in physiological media. In what follows, we detail the design criteria and construction of each of these components. A. Infrared optical trap
Optical trapping stemmed from a series of pioneering experiments by Ashkin,28 demonstrating the effect of laserinduced optical forces on controlling micrometer-sized particles in both air and liquid. The technique has been extensively used to characterize biological samples, ranging from DNA and single protein molecules to whole cells.29
This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP: 129.115.103.99 On: Mon, 24 Aug 2015 01:46:53
064301-3
B. J. Gross and M. Y. El-Naggar
Rev. Sci. Instrum. 86, 064301 (2015)
background levels.31 This is ideal for our purposes, since our goal is to perform EET measurements under anaerobic conditions to exclude oxygen as an alternate electron acceptor. By taking appropriate precautions including a near-IR laser, anaerobic conditions, and
This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP: 129.115.103.99 On: Mon, 24 Aug 2015 01:46:53
REVIEW OF SCIENTIFIC INSTRUMENTS 86, 064301 (2015)
A combined electrochemical and optical trapping platform for measuring single cell respiration rates at electrode interfaces Benjamin J. Gross1 and Mohamed Y. El-Naggar1,2,3,a) 1
Department of Physics and Astronomy, University of Southern California, 920 Bloom Walk, Los Angeles, California 90089-0484, USA 2 Molecular and Computational Biology Section, Department of Biological Sciences, University of Southern California, Los Angeles, California 90089-0484, USA 3 Department of Chemistry, University of Southern California, Los Angeles, California 90089-0484, USA
(Received 12 February 2015; accepted 7 June 2015; published online 23 June 2015) Metal-reducing bacteria gain energy by extracellular electron transfer to external solids, such as naturally abundant minerals, which substitute for oxygen or the other common soluble electron acceptors of respiration. This process is one of the earliest forms of respiration on earth and has significant environmental and technological implications. By performing electron transfer to electrodes instead of minerals, these microbes can be used as biocatalysts for conversion of diverse chemical fuels to electricity. Understanding such a complex biotic-abiotic interaction necessitates the development of tools capable of probing extracellular electron transfer down to the level of single cells. Here, we describe an experimental platform for single cell respiration measurements. The design integrates an infrared optical trap, perfusion chamber, and lithographically fabricated electrochemical chips containing potentiostatically controlled transparent indium tin oxide microelectrodes. Individual bacteria are manipulated using the optical trap and placed on the microelectrodes, which are biased at a suitable oxidizing potential in the absence of any chemical electron acceptor. The potentiostat is used to detect the respiration current correlated with cell-electrode contact. We demonstrate the system with single cell measurements of the dissimilatory-metal reducing bacterium Shewanella oneidensis MR-1, which resulted in respiration currents ranging from 15 fA to 100 fA per cell under our measurement conditions. Mutants lacking the outer-membrane cytochromes necessary for extracellular respiration did not result in any measurable current output upon contact. In addition to the application for extracellular electron transfer studies, the ability to electronically measure cell-specific respiration rates may provide answers for a variety of fundamental microbial physiology questions. C 2015 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4922853]
I. INTRODUCTION
Electron transfer is a fundamental process that governs the reduction-oxidation (redox) reactions critical for all cellular energy conversion pathways, including photosynthesis and respiration.1,2 Respiratory organisms extract free energy from their environment by coupling the oxidation of electron donors (food) to the reduction of electron acceptors (oxidants). While oxygen is the familiar electron acceptor of aerobic respiration, many anaerobic microbes are capable of performing electron transfer to alternative soluble acceptors, such as nitrates and sulfates, which can also diffuse to the electron transport chain inside living cells. In contrast to these intracellular electron transfer processes, we now know that a class of anaerobic microbes called dissimilatory metal-reducing bacteria (DMRB), including Shewanella, are capable of extracellular electron transfer (EET) to external surfaces.3–5 This respiratory strategy is attracting considerable fundamental and applied interest, considering that the external electron acceptors can range from natural oxidized metals in the environment to anodes.5
a)Author to whom correspondence should be addressed. Electronic mail:
[email protected]
From an environmental perspective, microbes performing EET are major players in elemental cycles, including the carbon cycle, occurring at a global earth scale.4,5 From a technological perspective, microbial EET is heavily pursued for interfacing redox reactions to electrodes in multiple renewable energy technologies. These technologies include microbial fuel cells (MFCs),6 where microbial biofilms oxidize diverse fuels and route the resulting electrons to energy-harvesting anodes, and the reverse process of microbial electrosynthesis,7 where renewable electricity drives reductive microbial metabolism for synthesis of high value fuels. The success of these technologies hinges on efficient electron exchange mechanisms between microbes and electrode surfaces. Two decades of research on the most studied DMRB model organism, Shewanella oneidensis MR-1, have revealed multiple mechanisms that can be categorized as either direct or indirect.8 Indirect mechanisms rely on biogenic or naturally occurring molecules, including flavins,9,10 that diffusively shuttle electrons from cells to electrodes. Direct mechanisms route electrons through multiheme cytochromes that are either located on the cell surface11–13 or along micrometer-long membrane extensions known as bacterial nanowires.14–18 Optimization of microbial fuel cells requires detailed knowledge of the contribution from each EET mechanism
0034-6748/2015/86(6)/064301/8/$30.00 86, 064301-1 © 2015 AIP Publishing LLC This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP: 129.115.103.99 On: Mon, 24 Aug 2015 01:46:53
064301-2
B. J. Gross and M. Y. El-Naggar
within biofilms.19 This includes an understanding of the biological, mass transport, and electrochemical factors that can limit performance,20 down to the fundamental respiration rate by individual cells. Much of our mechanistic knowledge of microbe-to-electrode EET is derived from a combination of electrochemical techniques and genetic approaches. Microbial fuel cells and anodic half cells have been used to quantify and compare the electricity production from wild-type S. oneidensis MR-1 and mutant strains lacking specific proteins that are hypothesized to affect EET.21 The impact of specific mutations on EET, especially when combined with additional biochemical, structural, and spectrometric techniques, can be used to reconstruct the cellular EET pathways.22 One difficulty, however, stems from the bulk nature of most electrochemical techniques, which measure the total current from a large microbial population (e.g., ≫107 cells). While it is tempting to think of an average cell-specific EET rate, obtained by normalizing with (often qualitative) cell density measurements performed at the end of an experiment, such an approach ignores the microbial heterogeneity present in any population.23 For instance, the same total anodic current can result from all cells respiring uniformly at a specific rate, or from only 10% of cells respiring at ten times that specific rate; these two scenarios clearly require completely different strategies for optimization. The effect of heterogeneity is particularly important in microbial fuel cells that contain a mixture of biofilm and planktonic cells, keeping in mind that a wide statistical distribution of cellular respiration current is possible even within each of these two sub-groups. The causes of microbial heterogeneity include basic genotypic variability resulting from mutations, as well as phenotypic variability resulting from progression through the cell cycle or as a physiological response modulated by the local environment and its history.24 As we seek a more complete understanding of the maximum power densities possible from microbial technologies such as microbial fuel and electrosynthesis cells, there is a clear need for techniques that quantify both the expected per-cell and statistical distribution of EET in microbial cultures. Single cell techniques will also allow more direct comparisons between experiments utilizing different microbes, which may result in different biofilms properties or cell densities, even under similar growth conditions. Perhaps more importantly, given the wealth of knowledge that can be gained from genetic studies, single cell techniques are also critical for studies that compare wild-type to mutant strains lacking putative EET proteins. Gene products hypothesized to perform EET (e.g., surface cytochromes or type IV pili) may also impact the cellular surface charge, attachment ability, and biofilm forming properties. Bulk techniques therefore cannot distinguish whether a decrease in anodic current is due to diminished EET directly through a specific protein, or indirectly by disrupting cellular attachment to electrodes and the subsequent biofilm development. Only a handful of recent studies address these issues surrounding bulk electrochemical techniques. McLean et al. quantified the average per-cell EET to graphite electrodes by combining live noninvasive imaging with an optically accessible MFC, allowing an accurate cellular count as
Rev. Sci. Instrum. 86, 064301 (2015)
S. oneidensis MR-1 population of the anode developed from separate cells to mature biofilms.25 This approach revealed a current per cell ranges from 50 to 200 fA, depending on the MFC resistance and growth phase. Another study obtained single cell measurements of EET from a different DMRB, Geobacter sulfurreducens DL-1, using microscale Ti/Au electrode arrays.26 When active cells transiently came into contact with these electrodes, a short circuit current of 92(±33) fA per cell was observed in about 30% of the contact events. Finally, Liu et al. reported an innovative approach taking advantage of optical tweezers to attach single S. oneidensis MR-1 cells to electrodes, revealing a current output of 200 fA per cell at a working electrode potential of +200 mV vs. Ag/AgCl.27 To date, however, there is no technical description of a complete instrument or standardized measurement procedure for detecting single cell extracellular respiration. Here, we present the first complete description of a system inspired by, and combining elements from, the abovementioned studies of specific respiration rates. The integrated system combines live imaging, an infrared optical trap, and a new electrochemical chip concept containing an array of indium tin oxide (ITO) microelectrodes that serve as ‘landing pads’ for individually trapped bacteria. The microelectrode potentials are controlled by a potentiostat, allowing the detection of the respiration current concomitant with landing a cell on a specific microelectrode. The entire system can be built from standard commercially available optical and electrochemical components, in combination with standard lithographic microfabrication techniques. The microfabrication procedure was optimized to address several challenges stemming from the biocompatibility requirement and high current sensitivity needed to detect single cells. Furthermore, the biological growth conditions and measurement protocol are described in detail. In addition to allowing single cell measurements of EET from a variety of organisms and mutants, the reported system may find wider applicability for general microbiology studies, including the analysis of how respiration rates in heterogeneous microbial cultures are impacted by specific environmental factors. II. EXPERIMENTAL SETUP
The single cell extracellular respiration platform integrates three main components: (1) an infrared optical tweezers system for positioning individual cells, (2) a custom transparent electrochemical chip containing an array of ITO microelectrodes on standard coverslips, and (3) a vacuumsealed perfusion chamber for loading and handling of bacterial cultures in physiological media. In what follows, we detail the design criteria and construction of each of these components. A. Infrared optical trap
Optical trapping stemmed from a series of pioneering experiments by Ashkin,28 demonstrating the effect of laserinduced optical forces on controlling micrometer-sized particles in both air and liquid. The technique has been extensively used to characterize biological samples, ranging from DNA and single protein molecules to whole cells.29
This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP: 129.115.103.99 On: Mon, 24 Aug 2015 01:46:53
064301-3
B. J. Gross and M. Y. El-Naggar
Rev. Sci. Instrum. 86, 064301 (2015)
background levels.31 This is ideal for our purposes, since our goal is to perform EET measurements under anaerobic conditions to exclude oxygen as an alternate electron acceptor. By taking appropriate precautions including a near-IR laser, anaerobic conditions, and
