In Copyright - Non-Commercial Use Permitted Rights ......Diss. ETHNo. 23773 Biofuel cell operating on activated THP-1 cells Athesissubmittedtoattainthedegreeof DOCTOROFSCIENCESofETHZURICH

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Doctoral Thesis

Biofuel cell operating on activated THP-1 cells

Author(s): Javor, Kristina

Publication Date: 2016

Permanent Link: https://doi.org/10.3929/ethz-a-010795225

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Biofuel cell operating on activatedTHP-1 cells

Diss. ETH No. 23773

Diss. ETH No. 23773

Biofuel cell operating on activatedTHP-1 cells

A thesis submitted to attain the degree ofDOCTOR OF SCIENCES of ETH ZURICH

(Dr. sc. ETH Zurich)

presented byKRISTINA JAVOR

Master degree in Bioengineering and BiotechnologyEPFL, Switzerland

born on 04.01.1985citizen of Saint-Prex, VD

accepted on the recommendation ofProf. Dr. Andreas Stemmer, examiner

Dr. Emmanuel Delamarche, co-examiner

2016

Pour Tastouze

Abstract

Energy supply is currently a significant challenge in the developmentof biomedical implants. The emergence of implantable medical de-vices in the last decades has led to a quest for alternative powersources that could replace conventional batteries. These are oftenheavy and bulky compared to the rest of the medical device andrequire surgery for their replacement. The necessity of finding moresuitable energy sources seamlessly integrated within the tissues andcapable of harvesting energy from the body's own resources has ledresearchers to consider biofuel cells as an alternative to batteries. Re-cently, several biofuel cells operating on mammalian cells have beenstudied. These showed the possibility of harvesting electrochemi-cal energy from white blood cells (WBC). This work focuses on animproved biofuel cell operating on activated THP-1 human mono-cytic cells. THP-1 monocytes were differentiated into macrophagesand activation stimuli were applied in order to trigger respiratoryburst, during which great quantities of free radicals, including super-oxide, were released through the NADPH oxidase transmembranecomplex. Superoxide has a short lifespan and quickly undergoesspontaneous dismutation to form hydrogen peroxide. Electrochemi-cal investigation showed strong evidence pointing towards hydrogenperoxide being the primary source of electrical current, confirmingthat the current originates from NADPH oxidase activity. Addi-tionally, an adequate substrate for differentiation and activation ofTHP-1 cells was examined. ITO, gold, platinum, and glass weretested and the amount of superoxide anion produced by NADPHoxidase was measured by spectrophotometry through WST-1 reduc-tion at 450 nm and used as an indicator of cellular activity andviability. These substrates were subsequently used in a conventionaltwo-compartment biofuel cell and the different biofuel cells were

characterized by recording polarization and power curves. The ma-terial showing the highest cell activity compared to the reference cellculture plate and the highest power output was ITO. The maximumpower density reached was 4.5 µW/cm2. This value is in the orderof magnitude required to power contemporary pacemakers. Further-more, to understand the role of NADPH oxidase and its products inthe generation of electrical current, the overall system was studied byrecording power density output in the presence of enzymes catalyz-ing the degradation of superoxide and hydrogen peroxide, on the onehand, and after incubation of the cells with several NADPH oxidaseinhibitors, including diphenylene iodonium (DPI) and staurosporine,on the other hand.

ii

Résumé

L'approvisionnement en énergie constitue à l'heure actuelle un défiimportant dans le développement d'implants biomédicaux. Face àleur généralisation dans les dernières decénnies, notamment dansle domaine de la stimulation cardiaque, la recherche s'est focaliséesur l'exploration de nouvelles sources d'énergie internes au corpsqui remplaceraient les batteries conventionnelles, souvent lourdes,encombrantes et de courte durée de vie par rapport au reste dudispositif. Ces batteries conventionnelles nécessitent par ailleursune intervention chirurgicale pour leur remplacement. La recherchede sources d'énergie plus appropriées, parfaitement intégrées dansles tissus vivants et capables de récupérer l'énergie générée par lesressources propres de l'organisme a conduit à considérer les piles àcombustible biologiques (piles biologiques) comme une alternativecrédible aux batteries conventionnelles. Récemment, plusieurs mod-èles de piles biologiques opérant grâce à des cellules immunitairesissues de mammifères ont été proposés. La possibilité de récolter del'énergie électrochimique à partir des globules blancs a égalementété démontrée. L'objet de cette thèse est l'étude d'un modèle de pilebiologique amélioré fonctionnant grâce à des cellules monocytaireshumaines (THP-1) activées. Pour cela, les monocytes ont été dif-férenciés en macrophages et activés afin de stimuler leur métabolismeoxydatif, une réaction immunitaire au cours de laquelle de grandesquantités de radicaux libres, dont l'anion superoxyde, sont libérés parle complexe protéique transmembranaire appelé NADPH oxydase.Le superoxyde produit est instable en solution et subit rapidementune dismutation spontanée pour former du peroxyde d’hydrogène.L'étude electrochimique du système par voltammétrie cyclique nouspermet d'affirmer que le peroxyde d'hydrogène est la source princi-pale de courant, confirmant que celui-ci provient bien de l'activité dela NADPH oxydase. En outre, la recherche d'un substrat adéquatpour la différenciation et l’activation des cellules monocytaires a étéconduite. L'oxyde d'indium-étain (ITO), l'or, le platine et le verre

ont été testés, et la quantité de superoxyde produite par la NADPHoxydase a été mesurée par spectrophotométrie et a pu être corréléeà la viabilité et à l'activité cellulaire. Ces substrats ont ensuiteété utilisés dans une pile à combustible classique à deux comparti-ments et les différentes piles biologiques ont été caractérisées par lamesure de leurs courbes de polarisation et de puissance. ITO estle matériau qui présente l'activité cellulaire la plus élevée en com-paraison à la plaque de culture cellulaire de référence ainsi que lapuissance électrique fournie la plus élevée. La densité de puissancemaximale atteint 4.5 µW/cm2, ce qui correspond à la consomma-tion de certains modèles de stimulateurs caridaques. Pour prouverl'implication de la NADPH oxydase et de ses produits sur le courantgénéré, le système cellulaire a été étudié en enregistrant la densitéde puissance en présence d’enzymes qui catalysent la dégradation dusuperoxyde et du peroxyde d'hydrogène, ainsi qu'après l'incubationdes cellules avec plusieurs inhibiteurs de la NADPH oxydase, dontle diphénylène iodonium (DPI) et la staurosporine.

iv

Acknowledgments

First and foremost, I would like to thank Prof. Dr. Andreas Stem-mer, head of the Nanotechnology Group at ETH Zürich, for givingme the opportunity to conduct my PhD thesis in his group, for al-ways being present, for his sense of humor and for always believingin me.I would also like to thank Dr. Emmanuel Delamarche at IBM Re-search in Rüschlikon, Switzerland, for giving me the honor of beingthe co-examiner of this thesis, for his interest in the project and hisencouragement.I would like to thank all the members of the Nanotechnology Group:Dr. Antje Rey, Blerim Veselaj, Dr. Carlos Ruiz-Vargas, Dr. FabianMenges, Hannes Beyeler, Dr. Jean-Nicolas Tisserant, Dr. Jing-Hua Tian, Katharina Herkendell, Dr. Khaled Kaja, Dr. KrithikaVenkataramani, Lea Nowack, Dr. Nassir Mojarad, Patrick Reissner,Ralph Friedlos, Dr. Raoul Enning and Dr. Tino Wagner for allthe great moments spent together, both inside and outside of thelab, enriching discussions, continuous support, and for the friendlyatmosphere.Special thanks go to Dr. Jean-Nicolas Tisserant and Dr. KhaledKaja for the unforgettable moments we shared and because they aresimply the best.Many thanks go to my students, Andreas Schiller and Derk Wild,for their boundless motivation, excellent work and for all the great

moments and discussions shared in the lab.My gratitude also goes to Dr. Robert Lovchick and Ute Drechslerat IBM Research, Rüschlikon for their invaluable help in microfab-rication and motivating conversations.I am also deeply grateful to my family, my brothers, my parents,and my awesome husband, for their love and unconditional support.

vi

Acronyms

BOD: bilirubin oxidaseCV: cyclic voltammetryCyt C: cytochrome CDMSO: dimethylsulphoxideDPI: diphenylene iodoniumFBS: fetal bovine serumGOX: glucose oxidaseHBSS: Hanks balanced salt solutionIFN-γ: interferon γITO: indium tin oxideKO2: potassium superoxideLPS: lipopolysaccharideNADPH oxidase: nicotinamide adenine dinucleotide phosphate-oxidasePBMC: peripheral blood mononuclear cellsPBS: phosphate-buffered salinePET: poly(ethyleneterephthalate)PMA: phorbol myristate acetatePMMA: poly(methylmethacrylate)ROS: reactive oxygen speciesSOD: superoxide dismutaseTEMPO: (2,2,6,6-tetramethylpiperidin-1-yl)oxylTNF-α: tumor necrosis factor αWBC: white blood cellsWST-1: water-soluble tetrazolium salts-1

Contents

Abstract i

Résumé iii

Acknowledgments v

Acronyms vi

1 Introduction 1

2 Theory 72.1 Biofuel cell history and electron transfer mechanisms 72.2 Biofuel cells operating on human cells: development

and current state of the art . . . . . . . . . . . . . . 102.3 Motivation . . . . . . . . . . . . . . . . . . . . . . . 132.4 NADPH oxidase: a brief history, description and

mechanism . . . . . . . . . . . . . . . . . . . . . . . 132.5 THP- 1 cell line . . . . . . . . . . . . . . . . . . . . . 162.6 Biofuel cells: theoretical aspects . . . . . . . . . . . . 18

2.6.1 Electrochemical fuel cell . . . . . . . . . . . . 192.6.2 Open circuit voltage . . . . . . . . . . . . . . 202.6.3 Biofuel cell thermodynamics . . . . . . . . . . 222.6.4 Factors decreasing cell voltage . . . . . . . . 232.6.5 Biofuel cell performance . . . . . . . . . . . . 25

ix

CONTENTS

2.6.6 Cathodic compartment . . . . . . . . . . . . 26

3 Materials and Methods 273.1 Cell culture and differentiation . . . . . . . . . . . . 273.2 Substrates . . . . . . . . . . . . . . . . . . . . . . . . 283.3 Superoxide anion dismutation . . . . . . . . . . . . . 283.4 Cellular activity and superoxide anion production . . 293.5 Biofuel cell setup . . . . . . . . . . . . . . . . . . . . 293.6 Polarization curves and power curves . . . . . . . . . 303.7 Long-term measurement of the biofuel cell . . . . . . 313.8 Biofuel cell measurement using inhibitors . . . . . . 313.9 Effect of enzymes catalyzing reactive oxygen species

degradation . . . . . . . . . . . . . . . . . . . . . . . 313.10 Electrochemical measurements . . . . . . . . . . . . 323.11 THP-1 cell images on different substrates . . . . . . 33

4 Results and Discussion 354.1 Biofuel cell feasibility . . . . . . . . . . . . . . . . . . 354.2 First biofuel cell setup . . . . . . . . . . . . . . . . . 364.3 Power density using different substrates . . . . . . . 394.4 THP-1 cells activity and viability on different substrates 414.5 Quantity of superoxide produced by THP-1 cells . . 454.6 Biofuel cell characterization . . . . . . . . . . . . . . 464.7 Investigation of the origin of the current . . . . . . . 514.8 Superoxide and hydrogen peroxide contribution to

the current . . . . . . . . . . . . . . . . . . . . . . . 554.9 Analysis of electron transfer between cells and electrode 594.10 Attempts at biofuel cell optimization . . . . . . . . . 60

5 Future perspectives 63

6 Summary 65

Bibliography 66

x

Chapter 1

Introduction

Progress in the medical sciences over the past decades has led to thedevelopment of a large number of electrically operated implantabledevices. These biomedical implants have become increasingly preva-lent in medicine and can be found in almost all areas, includingcardiology, drug delivery, neurology, audiology, diagnostic and mon-itoring (Figure 1.1) [1].

Biomedical implants can be divided in two categories: devices devel-oped for long-term application, such as retinal implants capable ofrestoring sight in visually impaired patients [2, 3], cochlear implantsallowing to restore impaired hearing [4], artificial urinary sphinc-ters for incontinent patients [5], neural implants for diagnostic andtissue stimulations [6], artificial pancreata that combine a glucosesensor, the associated insulin pump and pump control system forthe treatment of diabetes [7], as well as a large variety of cardiacimplants, including intravascular pressure monitoring systems [8]and pacemakers [9, 10]. The second category includes devices de-veloped for acute and temporary applications, such as systems fordrug delivery [11], telemetric measurement systems for monitoringintracranial pressure and temperature [12, 13], capsule endoscopy

1

CHAPTER 1. INTRODUCTION

systems allowing gastrointestinal tract diagnostic [14], numerouscardiovascular sensors [15, 16], and imaging systems [17] for lessinvasive diagnostics.

Device type Power consumption References

Pacemaker 30 to 100 µW [18, 19, 20, 21]Neurological stimulator 300 µW to several mW [18, 19, 20, 21]

Drug pump 100 µW to 2 mW [18, 19, 20, 21]Cochlear implant several µW to 10 mW [18, 19, 21]

Table 1.1: Power consumption for different types of implantablemedical devices.

A common challenge in this area of technology is to find reliablepower sources. Implantable devices that have a physical connectionto the outside of the body do not pose problems in terms of powersupply. On the other hand, autonomous biomedical implants requirethe use of embedded power sources that are small, biocompatible andhave sufficient autonomy [1]. Hence the importance of developingenergy sources specially targeted for biomedical implants. Table1.1 presents a summary of the most common biomedical devicesand their respective power consumption. Those devices display arelatively low power consumption in comparison to other devicescommonly used in daily life, such as smartphones, which powerconsumption can reach several Watts [22].So far, the power supplies for medical devices have relied on batteries,some of which are rechargeable, as is the case for cochlear implants[23] or hearing aids [24]. However, the majority of medical devicesrely on primary cell technology, mostly lithium iodine batteries [25].Such is the case for pacemakers, which are probably the best-knownbio-implantable devices.

2


Figure 1.1: Biomedical devices have become increasingly prevalentand are found in numerous area of medicine including neurology, au-diology, cardiology, drug delivery, diagnostic and monitoring. Manysuch devices are developed to replace impaired body functions othersfor diagnostic purposes.

3


The first fully implantable artificial pacemaker was tested in 1958[9, 10] and powered by a rechargeable nickel-cadmium battery. Be-fore that, many models of transcutaneous and wearable pacemakershad been tested [9, 10]. One major challenge faced with fully im-plantable pacemakers was the lack of a reliable, safe and lastingsource of energy. Poor battery reliability was indeed the principlecause of concern in pacemaker development, as battery failure wasthe major cause of pacemaker replacement [10, 26]. Therefore, sincethe development of the first implantable pacemaker, researchershave sought to develop lasting, safe and reliable implantable powersources.

In the 1960s, several strategies were investigated, including the de-velopment of glucose fuel cells [27, 28] and the use of zinc-mercuryprimary batteries, which proved to be poorly reliable and potentiallytoxic due to their mercury content [25, 26]. Efforts were also madeto develop nuclear batteries using plutonium 238 isotopes, but theadvent of lithium-iodide batteries on the market in the early 1970srapidly supplanted all other types of energy sources [26]. Lithiumiodine batteries are still used today to power pacemakers. Eventhough primary batteries have proven to be an acceptable source ofenergy, there are still opportunities for alternative power suppliesfor medical devices. Primary cells have a limited lifespan and, there-fore, regular replacement of the battery is necessary, which requiressurgery. Furthermore, they are often bulky and heavy compared toother device components, thus posing challenges in terms of devicedesign and size.The ideal power supply should have the following characteristics[29]: it should be small enough as not to impair the overall size andweight of the apparatus, should require low maintenance and have anadequate lifespan, to limit the need for surgery. Consequently, thereis an increasing demand for better integrated power sources thatwould allow biomedical implants to work autonomously. Implantable

4


devices with an embedded power supply capable of harvesting energyfrom surrounding tissues or fluids would drastically lower the needfor regular replacement, as the receiver's body would continuouslysupply the device with fuel.In an effort to replace batteries and develop a sustainable sourceof energy more naturally embedded in the human body, researchhas focused on the development of biofuel cells, electrochemicaldevices capable of harvesting energy from the patient's body withoutrequiring external refueling.Recently, in an effort to investigate alternative routes for harvestingenergy from the human body, several research groups have devel-oped biofuel cells operating on white blood cells [30, 31, 32, 33, 34].They have shown the possibility of harvesting electrochemical energygenerated by leukocytes during physiological processes. Biofuel cellsoperating on white blood cells are the focus of the present thesis[35].

5

Chapter 2

Theory

2.1 Biofuel cell history and electron trans-fer mechanisms

In 1911, Potter et al. pioneered the field of biofuel cells by buildinga galvanic cell in which the decomposition of organic compoundsby microorganisms was accompanied by the liberation of electricalenergy [36]. This was the first work demonstrating the possibilityof using microorganisms to produce electricity, but it received onlylittle coverage [37]. It is only in 1931, when Cohen et al. presenteda number of microbial half-cells connected in series capable of pro-ducing over 35 V and 2 mA of current, that the topic really emerged[38].In the following decades, the field of microbial fuel cells saw muchprogress and is nowadays very developed. A great number of bacte-rial strains, such as Escherichia coli [39, 40], Klebsiella pneumonaie[41], Proteus vulgaris [42, 43, 44] or Actinobacillus succinogenes [40],have been investigated for generation of electricity via microbialmetabolism.In a microbial fuel cell, protons and electrons are liberated during the

7

CHAPTER 2. THEORY

biological process through which organic materials are metabolizedby microbes [37]. The electrons produced through the oxidation oforganic compounds are then transferred extracellularly to the anode.The mechanisms for electron transfer are numerous. Bacteria, suchas Escherichia coli or Actinobacillus succinogenes, require the addi-tion of a mediator, such as methylene blue or neutral red, in order toachieve the electron transfer [45, 46]. Others are capable of produc-ing their own mediator, which is the case for Clostridium butyricumand Pseudomonas aeruginosa [47]. Moreover, bacteria belonging tothe Geobacter species are capable of direct electron transfer whereelectrons are transferred to the anode directly, without the use ofmediators.Several mechanisms explaining direct electron transfer have beensuggested. Electrons may be transferred through direct contact ofthe anode with c-type cytochrome displayed on the surface of thecell or through the anode biofilm (layer of bacteria) via electricallyconductive pili found on the surface of the bacteria or via looselybound c-type cytochromes found in the biofilm [48, 49].

The advent of the first implantable pacemaker has triggered a majorinterest in developing biocompatible, implantable and autonomouspower sources that are capable of harvesting energy directly fromthe human body. However, microbial biofuel cells are not suitablepower sources for bio-implantable devices, due to the widespread useof pathogenic microorganisms for the catalytic oxidation of the fuel.A way to counteract this problem was to extract and use only thecatalytic sites of the microorganisms, rather than using the wholemicrobe. This idea led, in 1963, to the development of the firstenzymatic fuel cell, which was capable of catalyzing the oxidationof glucose using glucose oxidase (GOX) as biocatalyst [50].Nowadays, research in glucose fuel cells is divided into two maincategories: enzymatic and abiotic glucose fuel cells. In both cases,chemical energy from glucose is converted to electrical energy by

8

CHAPTER 2. THEORY

oxidizing glucose at the anode and reducing oxygen to water at thecathode [51]. Enzyme-based fuel cells operate using enzymes suchas glucose oxidase (GOX) and bilirubin oxidase (BOD) in orderto catalyze the oxidation of glucose and the reduction of oxygen,respectively [52], whereas abiotic fuel cells use metal catalysts, suchas platinum [27, 53].This technology has led to the recent development of implantabledevices operating seamlessly within living organisms, which so farhave been tested in snails [54], rats [55, 56], insects [57], lobsters[58], and rabbits [59].Nevertheless, enzymatic fuel cells suffer from limited lifespan dueto their lack of stability [60, 61]. Indeed, enzymes are macromolec-ular biological catalysts that possess a unique three-dimensionalstructure, which determines their catalytic activity [62]. Changesor denaturation of this specific three-dimensional structure, due tovariations in temperature, pH or damages occasioned by the immo-bilization of the enzymes, cause the loss of activity [63]. Abiotic fuelcells also are subject to electrode fouling [64].

In this work, an alternative route for the development of an im-plantable biofuel cell was chosen and focuses on energy harvestingfrom human cells. Therefore, the presently investigated biofuel cellshares more similarities with microbial fuel cells, described earlier,than with glucose fuel cells. Indeed, the metabolism of whole cellsis involved in the electrochemical process: microbes in the case ofmicrobial fuel cell and white blood cells in the present case.

9

CHAPTER 2. THEORY

2.2 Biofuel cells operating on human cells:development and current state of theart

Electron transport across the biological membrane is a processknown to exist in mitochondria, bacteria and chloroplasts, but isgenerally known to be absent from plasma membrane of eukaryoticcells with the exception of the nicotinamide adenine dinucleotidephosphate-oxidase (NADPH oxidase), capable of generating super-oxide [65, 66, 67, 68]. It has been suggested that the process inwhich NADPH oxidase transfers electrons across the plasma mem-brane before coupling them to molecular oxygen is electrogenic [69].In 1998, Schrenzel et al. observed electron currents generated acrossthe plasma membrane of eukaryotic cells by the phagocyte NADPHoxidase using patch clamp experiments [70]. Following this observa-tion, several other studies have supported the idea of electrogeniccurrents produced by leukocytes [71, 72].Based on this concept, in 2004, Justin et al. published a workpresenting a biofuel cell using white blood cells as electron donors, asopposed to using microorganisms, with the goal of investigating thepossibility of using human cells for energy harvesting. The biofuelcell thus built was basically identical to a standard microbial biofuelcell with the exception that the anodic compartment was filled withwhite blood cells purified from human blood (mostly neutrophils)stimulated with phorbol myristate acetate (PMA) and ionomycinin order to activate the NADPH oxidase transmembrane complex[73]. This first leukocyte-based fuel cell showed stable currents overan extended period of time. The exact mechanism through whichelectrons were transferred from the neutrophils to the electrode wasnot elucidated. The possibility that electron transfer might haveoccurred via the superoxide produced by the active NADPH oxidasewas suggested. It was also suggested that superoxide played the

10

CHAPTER 2. THEORY

role of mediator. Furthermore, the possibility that NADPH oxidasemight have transferred electrons to the electrode directly was alsoconsidered.Several other biofuel cells operating on white blood cells were inves-tigated since then. The next year, in a follow up study, the sameresearch group expressed doubts about the origin of the currentsobserved, particularly about the involvement of the NADPH oxidasecomplex [31].In 2006, in an attempt to further investigate the electron transferfrom the leukocytes to the electrode and shed light on the originof the current observed, Justin et al. performed electrochemicalmeasurements of WBC isolated from venous blood [32]. The resultssuggested that, upon activation, white blood cells release redoxactive species, which allow the indirect electron transfer between theleukocytes and the electrode. It has been proposed that serotonin,which was suggested to be released from certain types of leukocytes,was possibly responsible for the oxidation current observed in theelectrochemical measurements, therefore suggesting that serotonincould be the redox species oxidized at the anode of the WBC biofuelcell. As to the role of the NADPH oxidase in the generation ofcurrent, they were neither able to confirm nor to refute that theelectron transfer occurs directly through the enzyme complex [32].A few years later, Sakai et al. presented a WBC biofuel cell operatingon macrophages where the cells were allowed to attach directly ontoa gold anode [34]. In their work, they confirmed the possibilityof harvesting electrical energy from leucocytes and demonstrated,through a series of inhibitory experiments, that the current generatedwas linked to NADPH oxidase activity. Moreover, superoxide anionwas hypothesized as a possible, but not necessarily unique, sourceof current.In 2011, Justin et al. observed that significant currents were mea-sured only in the presence of white blood cells. Additionally, in anexperiment comparing the current outputs from peripheral blood

11

CHAPTER 2. THEORY

mononuclear cells (PBMC) and WBC isolated from venous blood,which contains several other cell types like neutrophils, in additionto PBMC, WBC showed a higher average power output than PBWC.This observation implies that within the general WBC population,certain cells are capable of releasing electrochemically active com-pounds in variable amounts and that either these compounds orredox active species that are present in the cell membrane, such assubunits of the NADPH oxidase complex, may be oxidized at theelectrode surface [33].In 2013, Güven et al. published the latest known work regardingWBC biofuel cell. Their study focused on WBC biofuel cell perfor-mance. A biofuel cell similar to the one used by Justin et al. wascharacterized for the first time, obtaining a power density outputof 1.5 µW/cm2 [30]. Such a power density is however below thethreshold required to power a small medical device [25].

The field of WBC biofuel cells is quite young and thus far, has notbeen extensively studied. At the present stage, many aspects of theoperating mechanism of WBC biofuel cells have yet to be unraveled,especially those concerning the origin of the current. Is there adirect electron transfer from the flavocytochrome transmembranecomplex of the NADPH oxidase to the electrode? Or could thecurrent originate from oxidation at the anode of one or several speciessecreted by leukocytes and acting as mediators? If this is the case,what are these compounds? Can they be identified? If the primarycompound secreted from the cells that is oxidized at the anode can beidentified, more targeted efforts could be made in order to improvethe performance of such a biofuel cell and potentially reach powerdensity outputs capable of powering small implantable biosensors.This includes the development of better electrode catalysts and moreadvanced biofuel cell designs.

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CHAPTER 2. THEORY

2.3 Motivation

A byproduct generated by NADPH oxidase that can possibly par-ticipate in the electrochemical process of energy harvesting fromleukocytes is hydrogen peroxide. Several studies have shown the fea-sibility of using hydrogen peroxide in fuel cells [74, 75] and enzymaticbiofuel cells [76]. However, no previous work on WBC biofuel hasinvestigated the role played by hydrogen peroxide in such a system.The aim of this work was to study the questions raised in the previoussection and possibly shine light on the operating mechanism of WBCbiofuel cells in order to improve their performance. The experimentspresented in this thesis use elements of previous studies as well as newfeatures in order to better understand and unravel the underlyingmechanisms and attempt to provide answers to unresolved questionsregarding the origin of the current.To this end, different fuel cell setups were tested, including differentmaterials for the differentiation and activation of monocytic cells.Several biofuel cells operating on activated THP-1 monocytes werecharacterized and their power output density was compared to thepreviously published data by Güven et al. [30]. Here, for the firsttime, the possibility of electro-oxidation of hydrogen peroxide at theanode of a biofuel cell operating on THP-1 cells was evaluated.

2.4 NADPH oxidase: a brief history, de-scription and mechanism

Phagocytes constitute part of the immune system and are composedof many types of white blood cells, including neutrophils, mono-cytes, macrophages, mast cells and dendritic cells [77, 78]. Phago-cytes are capable of producing large quantities of reactive oxygenspecies through NADPH oxidase in a process commonly known asphagocytosis [79].NADPH oxidase is a transmembrane enzyme complex that generates

13

CHAPTER 2. THEORY

superoxide anion (O−2 ·) by transporting an electron from cytosolic

NADPH across the cell membrane and coupling it to molecularoxygen, thus producing the free oxygen radical [80] (Figure 2.1).The overall reaction is the following:

2O2 +NADPH −→ 2O−2 ·+NADP+ +H+ (2.1)

The rapid release of reactive oxygen species (ROS) from immunecells, known as respiratory burst, was initially characterized earlyin the 20th century. However, it is only decades later that theunderlying molecular mechanism was elucidated [81]. In 1961, Iyer etal. observed the formation of hydrogen peroxide in leukocytes duringphagocytosis [82]. In 1973, Babior et al. showed that superoxideanion was the initial product of the respiratory burst by incubatingleukocytes with cytochrome C and latex particles. The reduction ofcytochrome C was inhibited by the addition of superoxide dismutase,an enzyme capable of catalyzing the degradation of superoxide tooxygen and hydrogen peroxide, thus proving that the reduction wasaccomplished through superoxide [83]. Later Segal et al. showedthat cytochrome b558, also known as NOX2, was responsible for theproduction of reactive oxygen species [84, 85].In the following years, the different subunits composing the NOXsystem were characterized, establishing a global picture of the en-zyme complex and its biochemical role [86, 87, 88, 89]. NADPHoxidase is composed of multiple subunits, five phagocytic oxidases(phox) and two Rho guanosine triphosphatases, that interact to formthe active enzyme complex capable of releasing ROS [80] (Figure2.2). The complex is latent under normal circumstances and is acti-vated during the respiratory burst, where it assembles in the plasmamembrane and releases a great quantity of superoxide in order tokill bacteria and fungi [80]. The large flavocytochrome subunit b558(cyt b558), is composed of two NOX subunits, known as gp91phox

and p22phox, which are membrane proteins. Under resting condi-tions, three other phagocytic oxidases, p40phox, p47phox, p67phox,

14

CHAPTER 2. THEORY

Figure 2.1: Production of reactive oxygen species during phagocy-tosis. Reactive oxygen species are produced in order to kill pathogens.Phagocytes are capable of producing large quantities of reactive oxy-gen species through the transmembrane enzyme complex, NADPHoxidase. Upon activation, NADPH oxidase generates superoxide an-ion (O−

2 ·) by transporting electrons from cytosolic NADPH acrossthe cell membrane and coupling them to molecular oxygen. Superox-ide either quickly reacts with nitric oxide, generating peroxynitrite,or undergoes spontaneous dismutation, leading to the formation ofhydrogen peroxide.

are found in the cytosol as a complex [90]. The separation of thesetwo groups of subunits allows the NADPH oxidase to be inactiveif no stimuli are received. Upon stimulation, p47phox undergoesphosphorylation, following which the entire complex migrates to themembrane where it binds to cyt b588 to form an active oxidase[80]. The activation also requires the participation of two guaninenucleotide-binding proteins, Rac2, found in the cytosol, and Rap1A,which is a membrane protein [91, 92, 93]. The activated oxidasecomplex transfers electrons to molecular oxygen through heme andflavin groups, which are carrying electrons [81]. The electrons arethen coupled to molecular oxygen forming superoxide anion.The exact mechanism by which superoxide acts on harmful microor-

15

CHAPTER 2. THEORY

ganisms is not fully understood, but it has been shown to be biolog-ically toxic and used by the immune system as a defense mechanismagainst pathogens [94].Superoxide is a free radical that possesses an unpaired electron asa result of a one-electron reduction of molecular oxygen carryingtwo unpaired electrons. Consequently it is highly reactive and hasa short half-life. Superoxide either quickly reacts with nitric oxidegenerating peroxynitrite (6.7 ×109 M−1s−1) [95] or undergoes spon-taneously dismutation (1× 105 M−1 s−1 at pH=7 [96], leading tothe formation of hydrogen peroxide.Although NADPH oxidase is primarily found in leukocytes, it isalso present in various other cell types, such as endothelial cells[97], vascular smooth muscle cells [98], fibroblasts [99], renal mesan-gial cells [100], and corneal stromal cells [101]. Cells that processNADPH oxidase that is not primarily used for phagocytosis arecalled non-phagocytic cells. The NADPH oxidase of the later cate-gory is structurally very similar to the phagocytic NADPH, despitebeing functionally distinct. However, activation mechanisms of thenon-phagocytic NADPH oxidase differ markedly from the phagocyticcounterpart, in the sense that the former is constitutively active,whereas the later is activated upon stimulation [102, 97]. Althoughone is not involved in host defense, they both share the capacity totransport electrons across the cell membrane.

2.5 THP- 1 cell line

The cell type used throughout this thesis is the monocytic cell lineTHP-1, which is a human cell line derived from acute monocyticleukemia patients. THP-1 cells are widely used as a model to studymonocyte/macrophage functions, mechanisms, signaling pathways,nutrient and drug transport [103].Monocytes are part of the phagocytes family. In the human body,monocytes circulate in the blood stream and differentiate to differ-

16

CHAPTER 2. THEORY

Figure 2.2: NADPH oxidase is composed of multiple subunits, fivephagocytic oxidases (phox) and two Rho guanosine triphosphatases.(A) Under normal circumstances, the complex is latent and the sub-units are separated. The large flavocytochrome subunit located inthe plasma membrane is composed of two NOX subunits, known asgp91phox and p22phox. The other subunits, including the three otherphagocytic oxidases, p40phox, p47phox and p67phox, are found in thecytosol as a complex [90]. Additionally, the complex is composed oftwo small guanine nucleotide-binding proteins, Rap1A a membraneprotein and Rac2 found in the cytosol. (B) During respiratory burst,the subunits can interact to form an active enzyme complex capableof releasing ROS. Upon stimulation, the entire complex migrates tothe membrane where it binds to the transmembrane subunits to forman active oxidase [80]. The separation of these two groups of sub-units, the one found in the cytosol and the one located in the plasmamembrane, acts as a switch and guarantees that the NADPH oxidaseis inactive if no stimuli are received.

17

CHAPTER 2. THEORY

ent types of macrophages upon migration to the different tissueswhere they serve to protect the tissues from foreign bodies [104]. Asdescribed earlier, macrophages display the ability to release largequantities of reactive oxygen species during respiratory burst.The advantage of the use of THP-1 is the fact that THP-1 cellsare monocytes. Therefore, they are non-adherent cells that can,upon exposure to the right stimuli, differentiate to adherent cellscalled macrophages. Monocytes can be seeded and subsequentlydifferentiated to macrophages directly on a chosen substrate, whichcan either be an electrode or any biocompatible material. Thisfeature is important for several reasons. It enables the investigationof possible direct electron transfer from the cell membrane to theelectrode and it also enables one to study the viability and activityof the cells on different materials.Whether monocytes are differentiated directly on the anode or on abiocompatible substrate, this feature allows a closer contact betweenthe cells and the electrode material, rather than having leukocytesisolated from venous blood in suspension in the anodic compart-ment in which case cells are likely to sediment at the bottom of thecompartment over time.Additionally, the use of a cell line offers several advantages over usingcells isolated from venous blood, such as continuous access to cellsneeded for experiments and reduced variations that can arise fromcells isolated from patients. Indeed, cell behavior can vary fromone patient to another or even over time within the same patient.THP-1 cells provide a uniform and stable cell population to studysubstrate-related effects.

2.6 Biofuel cells: theoretical aspects

The field of WBC biofuel cells is very young and lack establishedmethodology for the analysis of system performances. Consequently,a comparison between devices on an equivalent basis is difficult. In

18

CHAPTER 2. THEORY

an effort to provide comparable results and since WBC biofuel cellsshare many similarities with microbial fuel cells, the methodologyused in this work is based on the standards proposed by Logan etal. in their work Microbial fuel cell: Methodology and Technology[105]. Furthermore, the study of WBC biofuel cells requires a generalknowledge of fuel cells. Thus, the present section presents insights onfundamental aspects of biofuel cells including some thermodynamicconsiderations and factors decreasing cell voltage that are furtherdetailed by Logan et al. [105].

2.6.1 Electrochemical fuel cell

The principle of an electrochemical cell, also known as galvanic cell,is the following. A salt bridge separates two half-cells by provid-ing ionic contact between the two compartments but at the sametime prevents the solutions from mixing, eliminating unwanted sidereactions. Each half-cell is composed of an electrolyte and an elec-trode. One half-cell contains the negative electrode (anode) whereoxidation takes place, whereas the other one contains the positiveelectrode (cathode) where reduction takes place. The combinationof both half-cells forms the full cell, allowing electrons and protonsto flow from the anode to the cathode. While the electrons flowin an outside circuit, generating a charge difference, the protonsflowing through the salt bridge reestablish the charge balance, thusmaintaining a steady state charge distribution.The electrochemical reactions may involve the electrodes, the elec-trolytes or even a fuel, as is the case for biofuel cells. Each half-cellhas a characteristic voltage, which can be predicted through theuse of standard electrode potentials defined relative to the 0 V ofa standard hydrogen electrode (SHE). The difference between theanode and the cathode electrode potential equals the cell potential[106]:

Ecell = Ecathode − Eanode (2.2)

19

CHAPTER 2. THEORY

A biofuel cell is basically a galvanic cell that uses bio-catalyzedelectrochemical reactions in order to generate electricity. Biofuelcells are commonly classified according to the biocatalyst employed,such as microbial and enzymatic fuel cells [107]. Despite the lack ofbiocatalysts, abiotic glucose fuel cells also belong to the biofuel cellcategory, as they are developed for powering biomedical implantsusing blood glucose as a fuel [108].A standard biofuel cell is composed of an anodic and a cathodic com-partment separated by a cation exchange membrane. In the anodiccompartment, a fuel is oxidized at the electrode, producing electronsand protons. Electrons generated at the anode are transferred tothe cathode through an external electric circuit, whereas protonsare transferred to the cathode through the membrane, in the sameway as in a galvanic cell. Protons and electrons are then consumedat the cathode by combining to a terminal electron acceptor, suchas molecular oxygen, which is reduced to water. The resulting dif-ference in electrochemical potential between the cathode and theanode redox pair constitutes the driving force for electron flow [109].

2.6.2 Open circuit voltage

The cell potential is a thermodynamic value, under standard con-ditions, that does not take internal losses into account. Voc is theactual maximum voltage reached by the biofuel cell. It is measuredafter stabilization of the cell and in the absence of current.In theory, the values of Voc should be close to that of the ideal voltagevalue (Figure 2.3). In practice, this value can be substantially lower,due to various potential losses including mixed potentials due to sidereactions as well as losses caused by measurements performed undernon-standard conditions [110].

20

CHAPTER 2. THEORY

Figure 2.3: Ideal polarization curve. The performance of a biofuelcell in an ideal system is determined by its thermodynamic values.For an ideal process under standard conditions, the cell voltage dis-plays the theoretical maximum value of the voltage a biofuel cell canreach. However, in practice this value is lower than the theoreticalthermodynamic value of the voltage due the formation of mixed po-tential and measurements performed under non-standard conditions.Additionally, during biofuel cell operation three different overpoten-tials, located at different current ranges, can be identified. At lowcurrents activation losses prevail, at intermediate currents Ohmicoverpotentials predominate and at high currents losses due to masstransport become dominant.

21

CHAPTER 2. THEORY

2.6.3 Biofuel cell thermodynamics

Electricity can be harvested from biofuel cells only if the overallreaction is thermodynamically favorable. The reaction can be eval-uated by the measure of the maximum work that can be derived,also called Gibbs free energy, which is expressed in Joules (J) andcalculated as follows [111, 112]:

4Gr = 4G0r +RTln(Π) (2.3)

where 4G0r (J) is the Gibbs free energy under standard conditions

namely 298.15 K, 1 bar pressure, and 1 M concentration for allspecies, R (8.31447 Jmol−1K−1) being the universal gas constant,T (K) the absolute temperature, and Π (dimensionless) the reactionquotient given as the activities of the products (here reducing agents)divided by those of the reactants (oxidizing agents) Π = aox/ared.4Gr (J) is the Gibbs free energy at the specific conditions andneeds to be positive in order to have a thermodynamically favorablereaction. Standard Gibbs free energy can be calculated from theenergies of formation, which are available from numerous sources[113, 114, 115].Commonly, reactions occurring in biofuel cells are rather evaluatedin terms of overall cell electromotive force (EMF), Eemf (V ), alsoknown as cell potential, which is defined as the potential differencebetween the cathode and the anode. Eemf is related to Gibbs freeenergy and work W (J) in the following manner:

W = EemfQ = −4Gr (2.4)

where Q represents the charge transferred in the reaction definedas the number of electrons exchanged in the reaction, n beingthe number of electron per reaction mole, F , Faraday's constant(9.64853 × 104 C/mol), and thus Q = nF , which is expressed inCoulomb (C). Combining the two last equations gives:

22

CHAPTER 2. THEORY

Eemf = −4GrnF

(2.5)

For standard conditions Π = 1 and from equation 2.3

E0emf = −4G

0r

nF(2.6)

where E0emf represents the standard cell electromotive force.

By combining the above equation with equation 2.3 the overallreaction can be expressed in terms of potentials:

Eemf = E0emf −

RT

nF×Π (2.7)

Equation 2.7 provides directly a value for the electromotive force ofthe reaction that is positive and favorable.In the present case, the value of the potential difference at stan-dard conditions for a biofuel cell using reduction of ferricyanide atthe cathode and the oxidation of superoxide and its derivative atthe anode should reach 0.766 V . This calculated value representsthe maximum theoretical cell voltage that can be derived from thebiofuel cell. However, during fuel cell operation the actual valueof voltage will be lower due to the occurrence of numerous lossesincluding activation, concentration and Ohmic losses. These lossesoccur only when current is flowing and are discussed in detail in thefollowing paragraph.

2.6.4 Factors decreasing cell voltage

The difference between the measured cell voltage and thermody-namic value of the cell potential is called overpotential and rep-resents the difference between the thermodynamically determinedreduction potential and the potential at which the redox reaction isexperimentally observed. This difference can be defined as the sumof the overpotentials of the anode, the cathode and all Ohmic lossesof the system. The cell voltage can then be expressed as follows:

Ecell = Eemf − (Σηa + Σηc + IRΩ) (2.8)

23

CHAPTER 2. THEORY

Where Σηa and Σηc are the losses of the anode and cathode respec-tively and the total of Ohmic losses IRΩ are proportional to thecurrent (I) and the Ohmic resistance RΩ.The measured cell voltage is a linear function of the current and canbe obtained from Ohm's law, Ecell = IRext, using the assumptionthat open circuit voltage (Voc) is the sum of voltage drops at theinternal and external resistor, Rext:

Voc = I(Rint +Rext) = IRint + IRext = IRint + Ecell (2.9)

Thus:Ecell = Voc − IRint (2.10)

Where IRint is the sum of all the internal losses, which are pro-portional to the internal resistance of the system and the currentgenerated.The principal application of thermodynamic calculation for biofuelcells is to identify the nature and the magnitude of losses. In aoperating biofuel cell, three major types of losses can be identified[105].

Activation losses

Activation losses are linked to the electron transfer to or from thespecies reacting at the electrode surface depending on whether itoccurs at the anode or cathode. The overpotentials are caused bythe activation energy needed for the oxidation/reduction to happen.Increasing the surface area of the electrodes and using catalysts canhelp achieve lower activation losses.

Ohmic (resistance) losses

In a biofuel cell, the Ohmic losses are tied to the cell design and in-clude the following resistances: resistance to the flow of ions throughthe anodic and cathodic electrolyte, as well as the cation exchangemembrane, and resistance to electron flow through the electrode and

24

CHAPTER 2. THEORY

its connections [116, 117]. Increasing the electrolyte conductivity,minimizing the electrode spacing and choosing a membrane with alower resistivity can reduce Ohmic losses.

Concentration losses

Concentration losses are linked to the rate of mass transport of thecompound to or from the electrode [116, 117]. A limited mass trans-fer due to a slow diffusion of the chemical compound to the electrodewill limit current production. Structure and geometry of the elec-trode as well as the nature of the electrolyte affect overpotentialsdue to mass transport. Moreover, in an unstirred system, diffusionalgradients may occur in the bulk solution, increasing concentrationlosses.

2.6.5 Biofuel cell performance

The overall performance of a biofuel cell can be evaluated in termsof power output:

P = IEcell (2.11)

The current, I, is obtained using Ohm's law, Ecell = IRext, whilethe voltage is measured across an external resistor. Therefore, thepower can be expressed in the following manner:

P =Ecell

2

Rext(2.12)

The power output is commonly normalized to the anode surfacearea, because the biological reaction occurs at the anode [118, 119]:

Pan =Ecell

2

ARext(2.13)

where Pan is the power density per cm2 (W/cm2) and A is thesurface area of the anode.

25

CHAPTER 2. THEORY

2.6.6 Cathodic compartment

Potassium ferricyanide is a very efficient electron acceptor commonlyused in experimental microbial fuel cells [119]. The greatest advan-tage of ferricyanide lies in its ability to allow negligible overpotential,therefore, allowing one to reach cathode working potential close toopen circuit potential [105]. Due to its good performance, potas-sium ferrricyanide was used as the electron acceptor throughout thiswork.

26

Chapter 3

Materials and Methods

3.1 Cell culture and differentiation

A THP-1 cell line was purchased from DSMZ (Braunschweig, Ger-many). Cells were maintained in RPMI1640 media containing Gluta-max (Invitrogen) supplemented with 10% fetal bovine serum (FBS,Invitrogen) and kept at 37 C in a humidified atmosphere with 5%

CO2. The cells were passaged twice a week and maintained at a celldensity between 0.1-1×106 cells/ml. 2×106 cells were seeded eitherin custom-made incubation containers holding different substrates(Au, Pt, indium tin oxide (ITO) and glass) or in culture wells. Theincubation containers were built out of poly(methylmethacrylate)(PMMA). Adding 300 ng/ml lipopolysaccharide from E. coli (LPS,Sigma-Aldrich), 20 ng/ml human IFN-γ (Sigma-Aldrich) and 20ng/ml human TNF-α (Life Technologies) to the complete mediafollowed by 2 days incubation resulted in cell differentiation. Finally,50 nM phorbol myristate acetate (PMA, Sigma-Aldrich) were addedto initiate cell activation.

27

CHAPTER 3. MATERIALS AND METHODS

3.2 Substrates

Following processing steps, each substrate was placed in a custom-made incubation container and the entire assembly was autoclavedprior to cell seeding.

Glass

100 µl of poly(l-lysine) 0.01% solution (Sigma-Aldrich) was dropcast on a clean glass slide and allowed to dry. Subsequently, thesurface was thoroughly rinsed with DI water.

Platinum and gold

Clean glass slides were covered by metal using thermal evaporation(BAK 501, Evatec) at a pressure of 10−6 mbar. 10 nm of Cr weredeposited as an adhesion layer, followed by 100 nm of either Pt orAu.

ITO

ITO-coated poly(ethyleneterephthalate) (PET) sheets were pur-chased from Sigma-Aldrich and used as received.

3.3 Superoxide anion dismutation

Superoxide samples were prepared according to a known protocol[120]. Briefly, 1 mg/ml of KO2 (Sigma Aldrich) was dissolved in ex-tra dry dimethysulphoxide (DMSO, Acros Organics) under nitrogenatmosphere. After sonication, the vial was spun in a mini centrifuge(Model GMC-060, LMS Co.) to precipitate the remaining solid KO2.Unless otherwise stated, 200 µl of KO2 in DMSO were added to1x PBS (pH=7.4) (Invitrogen) followed by the addition of 50 µMof cytochrome C (Cyt C, Sigma-Aldrich) at different times (varied

28


from 10 s to 60 s), the final volume was 2 ml. The 0 s point wasset by adding 200 µl of KO2 in DMSO to PBS already containing50 µM Cyt C. Spontaneous dismutation of superoxide anion overtime was determined by the reduction of Cyt C by superoxide anionmeasured by UV-vis spectroscopy (Bio-Rad Laboratories, Inc.). Allmeasurements were carried out at 550 nm relative to a blank con-taining 50 µM of Cyt C and 200 µl of extra dry DMSO in 1x PBS,final volume 2 ml.

3.4 Cellular activity and superoxide an-ion production

Cellular activity on different substrates was assessed through super-oxide anion measurement using WST-1 colorimetric assay (Biochem-ica). After cell differentiation, the media was replaced by HBSScontaining 100 µM WST-1 and 50 nM PMA. Following 1-hour incu-bation, the supernatant was collected, spun down to remove possiblecellular debris using a mini centrifuge and transferred to cuvettes.The amount of reduced WST-1 was determined by measuring thesample's absorbance at 450 nm using UV-Vis spectroscopy. All mea-surements were performed relative to a blank containing 100 µM ofWST-1 in Hanks balanced salt solution (HBSS, Invitrogen).

3.5 Biofuel cell setup

A two-compartment custom-made biofuel cell was constructed out ofpoly(methylmethacrylate) (PMMA); the anodic and cathodic com-partments both had a volume of 5 ml and were separated by a cationexchange membrane (BDH, VWR International Ltd.).

29


First part: THP-1 cells differentiated on the anode

The cathodic compartment was filled with 0.1 M potassium ferri-cyanide (Sigma-Aldrich) diluted in 1x PBS (pH=7.4). A platinumelectrode produced as described in section 2.2 was used as cathode.The anode was composed of either an Au or Pt electrodes carry-ing 2x106 THP-1 cells and was introduced, prior to the biofuel cellmeasurement, in the anodic compartment filled with PBS. In orderto induce the activation of the macrophages, 50 nM of PMA wereadded to the anodic compartment and the fuel cell was allowed tostabilize for at least 1 hour. Control measurements were performedin parallel using bare Au and Pt electrodes as anodes.

Second part: THP-1 cells separated from the anode

The cathodic compartment was filled with 0.1 M potassium ferri-cyanide (Sigma-Aldrich) diluted in HBSS. The anodic compartmentwas only filled with HBSS. Carbon cloth electrodes purchased fromthe National Center for Education at the University of Reading(Reading, UK) were used both as cathode and anode. The geomet-ric electrode size was 2.5 cm2. Prior to measurement, the substrate(ITO, glass, Au or Pt) carrying 2x106 THP-1 cells was placed insidethe fuel cell, next to the anode. Subsequently, 50 nM of PMA wereadded to the anodic compartment and the fuel cell was allowed tostabilize for at least 1 hour.

3.6 Polarization curves and power curves

The biofuel cell was characterized by recording the current andthe voltage across a set of variable external load resistances usingtwo Keithley multimeters (Model 2000). Resistances were variedincrementally from 10 MΩ to 10 Ω. Polarization and power curveswere extracted from these measurements.

30


3.7 Long-term measurement of the bio-fuel cell

The long-term measurement of the biofuel cell was obtained byrecording the current and voltage across a resistance of 15 KΩ during16 hours.

3.8 Biofuel cell measurement using in-hibitors

Prior to the biofuel cell measurements, ITO-coated PET sheets car-rying 2x106 differentiated THP-1 cells were incubated with either5 µM of diphenylene iodonium (DPI, Sigma-Aldrich) or 10 nM ofstaurosporine (Sigma-Aldrich) for 1 hour. Subsequently the sub-strate was placed in the anodic compartment of the fuel cell and 50nM PMA was added in order to trigger cell activation. The biofuelcell was allowed to stabilized for an hour before power curves wererecorded. Control experiments were carried out in parallel withoutthe addition of DPI or staurosporine.

3.9 Effect of enzymes catalyzing reactiveoxygen species degradation

Following the differentiation of 2x106 THP-1 cells on a glass sub-strate, the cells were placed in the anodic compartment and 50 nMPMA was added. After 1 hour of stabilization, a first power curvewas recorded, then 100 U/ml of superoxide dismutase (SOD, Sigma-Aldrich) were added and a second power curve was recorded. Finally,100 U/ml of catalase purified from bovine liver (Sigma-Aldrich) wereadded and a third power curve was recorded.

31


3.10 Electrochemical measurements

Electrochemical measurements were obtained using a PalmSens3potentiostat (PlamSens BV). All cyclic voltammograms (CV) wererecorded at a scan rate of 10 mV/s using an Ag/AgCl 1 M KCl ref-erence electrode (CH Instruments, Inc), a 6 mm carbon rod (AgarScientific Ltd.) as counter electrode and carbon cloth as a work-ing electrode unless stated otherwise. The measurements were per-formed at room temperature.

THP-1 cells, H2O2 Versus 5-HT

Prior to the CVs measurements, both an ITO-coated PET sheetcontaining 2x106 THP-1 cells and 50nM PMA or 100 nM H2O2

were added to 1x PBS pH=7.4. For the serotonin measurement, 500µM of 5-HT (Sigma-Aldrich) were added to the PBS solution.

TEMPO: THP-1 and H2O2

For the electrochemical measurement involving the redox cata-lyst (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl (TEMPO), a CV wasrecorded after placing an ITO substrate carrying 2x106 THP-1 cellsin a solution of 3 mM of TEMPO in PBS and after subsequentaddition of 50 nM PMA. Another CV was performed after adding30 mM of H2O2 to a solution of 3 mM of TEMPO in PBS. Forthe measurement with hydrogen peroxide, a glassy carbon electrode(CH Instruments, Inc.) was used as working electrode. For eachcase, a control measurement was carried out without the additionof the THP- 1 cells and without hydrogen peroxide.

32


3.11 THP-1 cell images on different sub-strates

After successful differentiation of 2x106 THP-1 cells on Au, Pt, glassand ITO substrates, images of the cells were acquired using a ZeissAxiovert 40 CFL light microscope with a 10X objective.

33

Chapter 4

Results and Discussion

4.1 Biofuel cell feasibility

Strong evidence suggests that electrical energy harvested throughactivated macrophage biofuel cells originates from the NADPH ox-idase complex. This implies that a product of NADPH oxidase isoxidized at the anode suggesting two molecules as major candidatesfor the generation of current, superoxide and hydrogen peroxide.Superoxide is the direct product of a single-electron reduction ofmolecular oxygen, peroxide results from the spontaneous dismuta-tion of superoxide [80, 121]. In order to evaluate the thermodynamicfeasibility of such a biofuel cell, one should consider the standardcell potential (Ecell) calculated using the reduction half-cell stan-dard potentials of the electro-active compounds of the anode andthe cathode. Potassium ferricyanide is reduced at the cathode andits standard reduction potential at pH=7 is [114, 122]:

[Fe(CN)6]3− + e− −→ [Fe(CN)6]4− E0 = 0.436V (4.1)

At the anode side, the two previously mentioned candidates are

35

CHAPTER 4. RESULTS AND DISCUSSION

considered and their standard reduction potentials are the following:

O2 + e− −→ O−2 · E0 = −0.33V [123] (4.2)

O2 + 2H+ + 2e− −→ H2O2 E0 = 0.281V [115] (4.3)

Both superoxide and hydrogen peroxide have a lower standard half-cell potential than the half-cell potential of ferricyanide, thus yieldingan Eemf > 0. Therefore, in both cases, the reaction can happenspontaneously.

4.2 First biofuel cell setup

As detailed earlier, several hypotheses describing the origin of thecurrent harvested in WBC biofuel cells have been formulated, butthe question was left open [34, 73, 32]. A current originating fromactivated macrophages and associated with the activity of NADPHoxidase has been observed by Sakai et al.. The activity of thetransmembrane complex has been evaluated through the amountof superoxide produced upon cell stimulation in combination withthe assessment of the level of expression of the different NADPHsubunits, as well as through the use of different NADPH oxidaseinhibitors [34]. Moreover, in their work, they observed that multipleelectron transport pathways exist. One discovered pathway wassuperoxide anion while the others remained unidentified [34].Whether the current is generated through direct electron transfer orvia a compound secreted from the leukocytes, placing the leukocytesas close as possible to the electrode seems to be a logical choice.Therefore, the first part of this work presents a characterizationof the performance of a WBC biofuel cell following the strategyemployed by Sakai et al., whereby THP-1 monocytes were seeded anddifferentiated to macrophages directly on the anode [34]. The setupused is composed of a standard two-compartment fuel cell separatedby a cation exchange membrane and is presented in Figure 4.1 A.

36


Figure 4.1: Standard two-compartment biofuel cell setups. The an-ode compartment was filled with HBSS or PBS. The cathode compart-ment contained 0.1 M of ferricyanide. (A) The anode was composedof either a gold or platinum electrode carrying 2×106 macrophages.Platinum was used for the cathode. (B) Carbon fiber was used forboth the cathode and anode. A substrate composed of either; ITO,glass, gold or platinum carrying 2×106 macrophages was introducednext to the anode. (C) Image of the biofuel cell presented in (B).

The power output from activated macrophages was measured andcompared to a control in which the activator, PMA, was added tothe anodic compartment in the absence of cells.Figure 4.2 shows the power density outputs obtained for 2x106

THP-1 monocytes seeded and differentiated either on a gold or plat-inum anode. After 2 days of incubation with LPS, TNF-α and IFN-γmonocytes attached to the electrodes showed successful differentia-tion to macrophages. The electrodes were then placed in the anodiccompartment and PMA was added for the macrophage activationprior to the fuel cell measurement. Power density outputs observedfor the controls were on the order of nW/cm2, which constitutes a

37


Figure 4.2: Biofuel cell performance using either a gold or a plat-inum electrode with or without THP-1 seeded on the anode. 2×106

cells were differentiated on either a gold or a platinum anode. Sub-sequently, the electrodes were placed in the anodic compartment and50 nM PMA was added prior to the biofuel cell measurement. Inparallel the same experiments were carried out without the presenceof cells on the anode. In all experiments a platinum electrode wasused as cathode. Standard deviations are shown for each substrate,Au (n=6), Pt (n=6), Au without cells (n=5), Pt without cells (n=3).

very small background possibly originating from the activator. In-deed, Justin et al. have previously reported small currents, allegedlydue to PMA [73].For both gold and platinum, the difference between the experimentalresults using activated macrophages and the control was small. Eventhough the biofuel cells containing activated THP-1 cells showedslightly better power output than the control, the difference remainsnegligible when compared to similar biofuel cells reported by Güvenet al., where power densities of 1.5 µW/cm2 have been reported.

38


These results show that, under these experimental conditions, theactivated THP-1 cells hardly produce any current. At this stageof the study, the reason for the extremely poor performance of thepresent biofuel cell was not clear. The choice of electrode materialmight have played a role, possibly because of poor viability and/oractivity of the macrophages on gold and platinum or because of poorelectron transfer from the cells or solution to the anode materials.Indeed, it is interesting to note that all reported successful WBCbiofuel cells use carbon cloth electrodes [30, 73, 31, 32, 33].Therefore, in an attempt to understand and optimize biofuel cellsoperating on activated THP-1, carbon cloth electrodes were used asboth anode and cathode for the rest of this thesis. As a consequenceof this choice, THP-1 cells were no longer differentiated on the anodedirectly, but rather on a separate substrate that was subsequentlyplaced in close proximity of the anode, as presented in Figure 4.1B.

4.3 Power density using different sub-strates

Sakai et al. showed that NADPH oxidase was involved in the processof current generation and that energy can be harvested from THP-1cells upon successful differentiation and activation [34].In an effort to find better substrates for the differentiation and ac-tivation of THP-1 cells, in the present work, several materials wereinvestigated and evaluated via the overall performance of the corre-sponding biofuel cells. To this end, biofuel cells were characterizedby recording polarization and power curves. The voltage was plottedagainst current density for a set of resistors. Power output at eachload step was calculated according to the equation P = I × Ecelland plotted against current density.Figure 4.3 presents the average power densities measured using

39


Figure 4.3: Average power densities recorded using biofuel cellsoperating on PMA activated THP-1 monocytes differentiated on dif-ferent substrates. Carbon cloth electrodes were used for both theanode and cathode. ITO (n=15), glass (n=5), Au (n=3) Pt (n=3).

THP-1 monocytes seeded and differentiated either on gold, plat-inum, ITO or glass. After two days of incubation with LPS, TNF-αand IFN-γ, monocytes achieved differentiation to macrophages and,consequently, attached to the substrates. PMA was added to theanode compartment for macrophage activation prior to the fuel cellmeasurement. For each material, at least three measurements wereperformed.When using carbon cloth as electrodes, the power densities observedreached high values, even higher than previously reported by Güvenet al. [30]. This indicated that the choice of electrodes, especiallythe anode, is of paramount importance. Although the biofuel cellsshowed general variability between measurements, on average, ITOand glass achieved better power densities than gold and platinum,which generally remained low.

40


The highest power density was reached using monocytes differenti-ated and activated on the ITO substrate. The difference in powerdensity observed between biofuel cells using different substrates forTHP- 1 cell differentiation may be caused by a difference in theviability and the activity of the cells on the different materials.Considering the fact that some substrates, such as gold and plat-inum, yielded negligible power densities when used as electrodes andshowed poor results when used as cell substrates in conjunction witha carbon cloth anode, the activity of the THP-1 cell on differentsubstrates was studied in order to find a material that would allowfor maximum cell viability and activity.

4.4 THP-1 cells activity and viability ondifferent substrates

Since THP-1 cells produce superoxide anion through NADPH ox-idase [124, 125], the activity and viability of the cells on differentmaterials was assessed through the amount of superoxide generatedfollowing differentiation to macrophages and subsequent activation.Here, the same four materials, namely ITO, glass, gold and platinum,were tested as substrates for the differentiation and activation ofTHP-1. Upon differentiation of monocytes to macrophages andsubsequent activation using PMA, the amount of superoxide anionwas estimated by spectrophotometry through the measurement ofthe amount of reduced water-soluble tetrazolium (WST-1).The reduction of WST-1 is a commonly used method for assessingcell proliferation, growth, chemosensitivity and viability [126]. Thechemical reaction used is the following:

2O−2 ·+WST+ H+

−−→ 2O2 + formazan (4.4)

WST-1 is reduced by superoxide in a two-electron process givinga reduced product called formazan, which exhibits an absorbance

41


Figure 4.4: Activity of THP-1 monocytes differentiated and acti-vated on different substrates. The activity of the cells was assessedthrough the amount of superoxide anion produced, measured throughthe absorbance of reduced WST-1 at 450 nm. For each substrate,at least 7 measurements were recorded and the average was plotted.Standard deviations are shown for each substrate, cell culture plate(CCP) (n=9), ITO (n=7), glass (n=7), Au (n=9), Pt (n=9).

maximum at around 450 nm [127]. For each substrate, the experi-ment was repeated at least seven times and the average is presentedin Figure 4.4. Cell culture plates were used as a reference.Figure 4.4 shows that the highest activity was achieved on ITOand glass, which is in accordance with previous work [128]. It alsoshows that, generally, power densities correlate with the activityof the THP-1 monocytes, showing high values for ITO and glassand low values for gold and platinum. This result is in agreementwith other studies showing the link between current output from thebiofuel cell and the presence of activated human cells [33, 34].Several factors can be responsible for the different absorbance mea-

42


Figure 4.5: Images of 2×106 THP-1 cells following their differen-tiation on different substrates. Cells were incubated with 300 ng/mlLPS, 20 ng/ml TNF-α and 20 ng/ml IFN-γ for 2 days on (A) ITO,(B) glass, (C) gold and (D) platinum, in order to achieve differen-tiation. Images were acquired using a Zeiss Axiovert 40 CFL lightmicroscope with a 10X objective.

surements observed for the substrate materials studied. Cell viabilitycan certainly be one factor, as some substrates might provide a moresuitable environment for human cells than others. Indeed, assumingthat, for every substrate, all the cells attached upon differentiation,the material allowing the highest cell viability will lead to highersuperoxide anion production, which alone might account for somedifferences between the materials. A previous study on biocompati-ble materials for implantable microelectrodes has presented ITO asa material of choice owing to its ability to promote cell growth andlow protein adsorption [129].

43


In reality, the amount of cells attached upon differentiation is verylikely to vary from one material to the other, widening the gapbetween the amounts of activity measured on different materials.To verify this, optical microscopy images of THP-1 differentiated onITO, glass, gold and platinum were taken and are shown in Figure4.5. The same number of cells was seeded on each substrate, namely2x106 cells.It is clear enough that gold and particularly platinum seem to beless suitable substrates for monocyte differentiation. These materi-als show reduced cell attachment compared to glass and especiallycompared to ITO, whose surface appears almost totally covered withcells. These images indicate that cell attachment on the differentsubstrates is a major factor of the differences observed in the overallactivity observed in macrophages. Indeed, ITO, which exhibits thehighest number of cells, also achieves the highest activity; similarly,platinum shows fewer cells attached and the lowest cell activity.Therefore, the activity of THP-1 cells on different materials seemsto be linked to the amount of cells attached upon differentiation.Although gold and platinum showed reduced cell attachment andreduced activity compared to other materials, this observation alone,does not explain the low power outputs observed with the first bio-fuel cell setup (Figure 4.2). A possible explanation might be thefact that leukocytes attached to the anode died as a consequence ofthe biofuel cell operation. Indeed, Güven et al. pointed out that cellviability seems to be closely linked to biofuel cell operation. Theyreported up to 83% of dead cells at the end of fuel cell measure-ments. In contrast, only 15% of cells were found dead on the controlelectrode onto which the same amount of cells was immobilized [30].A drop in cell viability due to the presence of cells on the anode, inaddition to the already low number of cells attached, might accountfor the extremely low power density output.Interestingly, the two substrates exhibiting a low absorbance at 450nm are both metallic substrates, suggesting the possibility that yet

44


an additional factor may play a role in the lower quantity of su-peroxide anion measured on gold and platinum and the very lowpower density output of the first biofuel setup. A possible explana-tion might be that gold and platinum have partially scavenged thesuperoxide anion generated by the activated macrophages, leadingto a lower signal. This especially seems to be the case for platinum.Indeed, radical scavenging by materials containing gold and/or plat-inum has been reported in several other studies [130, 131].

4.5 Quantity of superoxide produced byTHP-1 cells

The spectroscopic measurement of the absorbance of reduced WST-1allows the estimation of its concentration using Beer-Lambert law:

Eλ = εcl (4.5)

where Eλ is the value of the absorbance measured at a given wave-length. This value is directly proportional to c the molar concen-tration of the species in the solution and l the path length of thebeam of light (here the cuvette length was 1 cm). The molar attenu-ation coefficient ε constitutes a measure of how strongly a chemicalcompound attenuates light at a given wavelength. The attenuationcoefficient of formazan at 450 nm is 37 mM−1cm−1 [132].Therefore, using 0.4 (Figure 4.4) as representative value of theabsorbance observed for the activation of 2x106 cells in a volume of1 ml, and knowing that two superoxide molecules reduce one WST-1,the estimated amount of superoxide is around 10 nmol/h/106 cells.This value is in accordance with other studies that use formazan asa measure of the activity of THP-1 cells [133, 125].It is important to note that the rate of reduction of WST-1 by su-peroxide is around 3-4 x 104 M−1 s−1 [96]. This value is by approx-imately one order of magnitude lower than the rate of spontaneous

45


conversion of superoxide to hydrogen peroxide, which occurs at arate around 1x 105 M−1 s−1 at pH=7 [96]. Therefore, due to theslower rate of reaction of WST-1 and superoxide compared to rateof spontaneous degradation of superoxide, the amount of superoxideobserved using the reduction is expected to be an underestimationof the actual amount produced by the macrophages. However, thereduction of WST-1 provides a useful method for the comparisonbetween the amounts of activity and viability of the cells on differentsubstrates.

4.6 Biofuel cell characterization

As presented earlier, the highest power density was reached usingmonocytes differentiated and activated on the ITO substrate.Figure 4.6 shows polarization and power curves typically observed.The maximum short circuit current obtained was around 45 µA/cm2

and the maximum power output reached 4.5 µW/cm2. This is athreefold higher power density output than reported previously forbiofuel cells operating with white blood cells [30]. This improvementmay in part be attributed to the use of a different cell type; in thiscase, the monocytic cell line THP-1, rather than isolated white bloodcells from venous blood. Moreover, as discussed earlier, a drop incell viability due to the presence of the cells on the electrode mightaccount for a lower power density output.Although the power density output reached with the present biofuelcell is higher than that of previous reports, it is small comparedto other types of biofuel cells, such as abiotic glucose fuel cells[53, 64] and especially enzymatic glucose fuel cells that are capableof yielding power densities on the order of mW/cm2 [134, 135].The present WBC biofuel cell nevertheless constitutes a potentialalternative power production method as it reaches power outputsclose to the minimum required for the operation of some modelsof implanted devices, which is as low as around 20 µW [25]. The

46


Figure 4.6: Polarization and power curves of a biofuel cell operat-ing on PMA activated THP-1 monocytes differentiated on an ITOsubstrate. Prior to the addition of 50 nM PMA, the cells were in-cubated for 2 days with 300 ng/ml LPS, 20 ng/ml TNF-α and 20ng/ml IFN-γ on an ITO substrate in order to achieve differentiation.Carbon cloth electrodes were used for both the anode and cathode.

maximum Voc measured using the present biofuel cell reached 0.385V . This value is comparable to another report presenting a similardevice operating on lymphocytes purified from human blood thatdisplayed about 0.4 V of Voc [30].

As detailed earlier, many observations point toward hydrogen perox-ide being the primary source of current, but this does not necessarilyexclude any possible contribution from superoxide. Indeed, bothspecies are present in the solution, as superoxide is produced con-tinuously. The species with the more negative standard reductionpotential is oxidized more favorably. Thus, superoxide oxidation is

47


more favorable than that hydrogen peroxide [114, 115].The value of the potential difference at standard conditions for abiofuel cell using reduction of ferricyanide at the cathode and theoxidation of superoxide and its derivative at the anode should reach0.766 V . In theory, the Voc should approach the Eemf . In practice,however, this is never the case and the observed Voc is usually sub-stantially lower [105]. In the case of microbial fuel cells oxidizingacetate at the anode and reducing oxygen at the cathode, the max-imum theoretical voltage is 1.1 V [105]. However, the maximumopen circuit voltage achieved so far was 0.8 V [136], and duringcurrent generation the value of voltage never exceeded 0.62 V [137].Therefore the presently observed Voc represents a satisfying value,especially given the fact that the system is not highly optimized.

Polarization curves allow the study of losses occurring during biofuelcell operation. In a polarization curve of a typical fuel cell, threedistinct regions, located at different current ranges, can be identified.For microbial fuel cells, these regions usually overlap and the polar-ization curve exhibits a linear relationship [138]. The polarizationcurves presented in Figure 4.6 illustrate typical characteristics ofa fuel cell operating on living cells.At low currents, activation losses prevail and derive from the slowrate of reaction taking place at the electrode. The poor electrontransfer from the reactant to the electrode induces these overpoten-tials. Increasing the electrode surface area, improving the electrodecatalyst and also increasing the operating temperature of the sys-tem yields small activation losses. In the present case, the electrodesize was already fairly large, 2.5 cm2. However, anode materialsexhibiting a micro or nano-structured surface would increase thesurface area without increasing the overall size of the electrode. Fur-thermore, the use of catalysts would decrease the activation lossesleading to better biofuel cell performances. Increasing the operatingtemperature of the system from room temperature to 37 C would

48


be advantageous, both in terms of biofuel cell operation and cellmetabolism. Indeed, it has been reported that superoxide produc-tion by THP1- cells increases gradually from 25 C to 32 C followedby a sharp increase between 32 C and 37 C [139].At intermediate currents, Ohmic overpotentials predominate andinclude both losses due to the ionic resistances of the electrolyte andmembrane as well as electronic resistances of the electrode and theinterconnections. These losses can be reduced by selecting a mem-brane exhibiting low resistivity, minimizing the electrode spacing,increasing the solution conductivity and checking all contacts andinterconnections. Nafion membranes are the most commonly usedcation exchange membranes in microbial fuel cells [105]. In a pre-vious work performed by our group, the conductivity of two typesof cation exchange membranes was compared. A performance testusing a proton exchange membrane fuel cell, where the anodic com-partment was filled with pressured hydrogen and the cathode wasexposed to air, showed that performance of BDH cation exchangemembranes (BDH VWR International Ltd.) reached only 10% ofthose of Nafion membranes. However, the use of Nafion membraneswas quickly excluded following the observation of a fast drop in pHoccurring in the anodic compartment [128]. No pH changes werereported with the BDH cation exchange membrane. Owing to theseissues using Nafion, the membrane selected for this work was a BDHcation exchange membrane.At high currents, losses due to mass transport become dominant andare the results of changes in concentration of the chemical speciesat the interface of the electrode, where the transport is limited bydiffusion. This overpotential is caused by either a limited supplyof reactant to the electrode or a limited discharge of products fromthe electrode. Structure and geometry of the electrode as well asthe nature of the electrolyte affect losses due to mass transport.Moreover, in poorly mixed systems, as is the case here, a concen-tration gradient can also arise in the bulk solution. In the case of

49


Figure 4.7: Long term power output experiment. 2×106 THP-1cells were seeded on glass. Power output was measured across a 15KΩ resistance over 16 hours.

the present biofuel cell, concentration losses are quite significant.This is especially apparent for the polarization presented in pinkin Figure 4.6, where the curve exhibits a steeper slope at highcurrents. Due to the fragility of the attachment of the cells to thesubstrate, stirring of the solution was excluded. The nature of theelectrolyte was imposed by the metabolic needs of the cells. Onlyphosphate-buffered saline solution, such as PBS or HBSS, could beused.Figure 4.7 shows long-term power output from a biofuel cell oper-ating on activated macrophages seeded on glass. Over the courseof the first couple of hours, power stayed constant, then started toincrease, reaching almost double the initial value after 14 hours ofmeasurement, and abruptly dropping to zero afterwards. Güven etal. observed a quasi-identical curve in their long-term power out-put measurement [30]. Apart from observing a sharp increase at

50


the beginning, the curve exhibited a very similar shape showing aprogressive increase and then a drastic drop after 16 hours. Therapid drop in power after 14 hours might be attributed to a swift celldeath due to extended exposure of the cells to suboptimal conditions,such as room temperature, limited food supply, low CO2 level and anon-sterile environment. Nonetheless, 14 hours of continuous powersupplied by a biofuel cell operating on human cells under suboptimalconditions is an encouraging result.

4.7 Investigation of the origin of the cur-rent

Several studies on biofuel cells have shown that activated whiteblood cells are capable of generating a current. Various compoundsproduced by WBCs have been identified as possible, but not nec-essarily unique, sources of current, among which serotonin (5-HT)[32, 33] and superoxide anion [73, 31, 34].This section deals with the investigation of the origin of the currentand aims to gain insight as to the possible mechanisms and moleculesinvolved in the operation of the present biofuel cell.In the alternative biofuel cell setup presented in Figure 4.1 B,given the fact that the cells are separated from the anode by severalmillimeters, the possibility of a direct electron transfer between theactivated macrophages and the electrode can be excluded.

In order to evaluate the role of NADPH oxidase in the power outputobserved, two inhibitors of the enzyme complex were used, dipheny-lene iodonium (DPI) and staurosporine. DPI is the most commonlyused inhibitor of NADPH oxidase [140]. It acts by abstracting anelectron from an electron transporter, forming a radical that will inturn inhibit the electron transporter via a covalent binding step [141].Therefore, DPI is a non-specific inhibitor, which acts on many other

51


Figure 4.8: Power density measured for 2×106 THP-1 cells prein-cubated with either DPI or staurosporine for 1 hour prior to cellactivation using PMA. For each inhibitor, a control experiment wascarried out in parallel using only PMA

electron transporters, including nitric oxide synthase [142], xanthineoxidase [143], mitochondrial complex I [144] and cytochrome P-450reductase [145].Staurosporine is a protein kinase inhibitor that is able to suppresssuperoxide production, by selectively inhibiting protein kinase Cfunction [146]. The role of protein kinase C is to induce the phospho-rylation of cytosolic NADPH oxidase subunits [147]. The presenceof staurosporine suppresses its action, thus preventing the cytosolicsubunits from binding and translocating to the plasma membrane,therefore blocking the assembly of the NADPH oxidase complex[146].

52


Differentiated monocytes were incubated with either DPI or stau-rosporine for 1 hour prior to the transfer of the ITO substrate carry-ing the cells to the anodic compartment and the addition of PMA.Subsequently, power outputs from the biofuel cell were recorded. Inparallel and for each inhibitor, a control experiment using the samesubstrate seeded with the same amount of cells was performed inthe absence of inhibitor. The results are presented in Figure 4.8.In both cases, incubation of the macrophages with the inhibitorefficiently lowered the power output generated. This observationdemonstrates that NADPH oxidase is involved in current produc-tion. Nevertheless, the possibility of another contributor cannot becompletely excluded, as for both inhibitors; a small power outputis still observable. This might be due to the incomplete effect ofthe inhibitors on the NADPH oxidase or to the fact that anotherelectron transfer route exists, independent from NADPH oxidase.However, due to the small magnitude of the residual signal, no fur-ther experiments were carried out to investigate this in more details.Rather, the efforts were focused on the product of NADPH oxidaseand its derivatives.Superoxide anion is known to be the primary product of NADPHoxidase [80, 124, 125] and has been hypothesized as being a possiblesource of the current observed in WBC fuel cells [34]. Althoughsuperoxide acts as a reducing agent and is believed to be able tooxidize at the fuel cell anode, as a free radical, it is known to bevery unstable and to quickly undergo dismutation forming hydrogenperoxide and molecular oxygen [121].Figure 4.9 shows the spontaneous dismutation of superoxide anionover time, under our experimental conditions, namely 1x PBS solu-tion (pH=7.4) and at room temperature. The amount of superoxideis determined by measuring the amount of reduced Cyt C at 550 nm.As expected, the absorbance drops very quickly to zero. After 60seconds, there is almost no superoxide left. This short lifetime sug-gests that only negligible quantities of superoxide would stay active

53


Figure 4.9: Spontaneous dismutation of superoxide anion over timein 1x PBS at room temperature determined by measuring absorbanceof cytochrome C at 550 nm, (n=3).

long enough to reach the electrode, making superoxide unlikely tobe the primary source of current. This is especially the case in thepresent biofuel cell, where the anode is separated from the substratecarrying the human cells by a few millimeters. However, this doesnot mean that any contribution from superoxide is excluded; a smallamount of the superoxide produced can certainly be oxidized at theanode, but the large majority is expected to undergo spontaneousdismutation, forming a more stable product capable of accumulationin the solution. Therefore, the product of spontaneous superoxidedecomposition, hydrogen peroxide, was investigated, as it may playa predominant role in biofuel cell current generation.If hydrogen peroxide participates in the electro-oxidation at the an-ode, an oxidation peak should be visible during electro-chemicalmeasurements. In order to verify this hypothesis, cyclic voltamme-try was performed on activated macrophages. Authors of previous

54


studies have shown oxidation peaks following the activation of leuko-cytes and have suggested serotonin as being the compound oxidized[32, 30]. In order to confirm the participation of H2O2 and excludea possible contribution from serotonin, the recorded CV of activatedTHP-1 cells was compared to the CVs of serotonin and H2O2. Theresults are shown in Figure 4.10.Activated cells show one clear peak at around 100 mV with an anodiccurrent starting at around 20 mV. The H2O2 peak also occurs ataround 100 mV and has similar shape to that of activated THP-1cells. In contrast, the serotonin peak appears at around 450 mV.The absence of this peak from the activated cells demonstrates theabsence of serotonin in the case of THP-1 cells.It is important to note that although all WBC biofuel cells operatewith leukocytes, they do not use the same cell types. Some operatewith lymphocytes [30], neutrophils [31] or a mixture of different whiteblood cell types, including monocytes [33] isolated from venous blood.In the present work, monocytes/macrophages derived from acutemonocytic leukemia (THP-1 cell line) were used. This diversity incell types employed in WBC biofuel cells may lead to diversity inthe substances secreted and, therefore, different compounds may beat the origin of the current measured, depending on the cell type.Under our experimental conditions, primarily H2O2, a product ofsuperoxide dismutation, seems to be involved in current generation.

4.8 Superoxide and hydrogen peroxidecontribution to the current

In order to evaluate the contributions of hydrogen peroxide to thepower density output generated by the biofuel cell operating on ac-tivated THP-1, power curves were recorded following the sequentialaddition of PMA, superoxide dismutase (SOD) and catalase to theanodic compartment.

55


Figure 4.10: Cyclic voltammetry of activated THP-1. CVs wererecorded at 10 mV/s using carbon cloth as a working electrode, acarbon rod as counter electrode and Ag/AgCl (1 M KCl) as referenceelectrode. (A) Blue line: CV of 100 nM H2O2, pink line: CV ofPMA activated THP-1 cells differentiated on ITO-coated PET sheet,grey line: CV of PBS as a control. (B) Violet line: CV of 500 µM5-HT, grey line: CV of PBS as a control.

56


SOD is an enzyme that catalyzes the dismutation of superoxideanion into oxygen and hydrogen peroxide.

2O−2 ·+2H+ SOD−−−→ H2O2 + 2O2 (4.6)

Catalase is an enzyme that catalyzes the decomposition of hydrogenperoxide to oxygen and water.

2H2O2catalase−−−−−→ 2H2O +O2 (4.7)

Figure 4.11 shows the average power densities obtained. The mea-surements were performed twice for each compound. The additionof SOD did not induce any significant changes in the power output.This was expected, as most of the superoxide had probably alreadyundergone spontaneous dismutation before reaching the anode bydiffusion. What is more surprising is that catalase did not appearto lower the power output as expected. Indeed, the rate constantsof SOD and catalase are 2 x 109 M−1 s−1 [95] and 7.9 x 106 M−1s−1

[148], respectively. After the addition of catalase, only water andoxygen should be present in the solution and the signal is expectedto drop quickly. However, the power output remained almost thesame. A possible explanation could be that catalase was less activeor never completely active due to the temperature of the solution,since the catalase used in the present work was purified from bovineliver and reaches its optimum activity at 40 C [149]. In the presentcase, experiments were all performed at room temperature. How-ever, this might not completely explain the absence of drop in thepower output following the addition of catalase. The reason for thecatalase inactivation is further discussed in the following paragraph.

Interestingly, this work is not the only one to have reported the ab-sence of current drop after addition of catalase during experimentsperformed on currents produced by NADPH oxidase. Sakai et al.have suggested that NADPH oxidase is involved in the current gen-erated in their biofuel cell, either via the contribution of superoxide

57


Figure 4.11: Effect of enzymes catalyzing the degradation of reac-tive oxygen species. After the addition of each compound, the poweroutput from the biofuel cell was recorded. PMA (n=2), SOD (n=2),catalase (n=2).

anion or hydrogen peroxide. The addition of catalase did not induceany change in the current observed; they therefore have excluded anycontribution from hydrogen peroxide and have concluded that super-oxide must have been the primary source of current [128]. Moreover,Schrenzel et al. have performed whole-cell patch clamp experimentson eosinophil for the study of current mediated by NADPH oxidase[70]. They have observed inward currents associated with the gen-eration of oxygen radicals by the NADPH oxidase. These currentswere strongly diminished by the pretreatment of the cells by DPI.In contrast, the addition of SOD and catalase did not affect thecurrents measured, which remained constant.

58


In order to test the activity of catalase, further experiments wereperformed. A drop of solution containing catalase was added toa solution of H2O2, and bubbles appeared, demonstrating oxygenformation and activity of the enzyme. However, the formation ofoxygen bubbles stopped after a few seconds. Two reasons may bethe cause: either all hydrogen peroxide was consumed or the enzymelost its activity. To verify this, another drop of catalase was added tothe same solution of hydrogen peroxide. Again, formation of oxygenwas observed for only a short time and then stopped. The sameprocedure was repeated several times, showing identical results. Thisobservation suggests that catalase was not able to sustain its activityfor a period of time longer than a few seconds. The reason for thefast loss of activity of the enzyme is not known and might possiblybe linked to the suboptimal temperature of the solution. Sincerecording of the power curves was carried out over 15-30 min, thefast deactivation of catalase provides an explanation for the absenceof a power drop following its addition to the anodic compartment ofthe biofuel cell.

4.9 Analysis of electron transfer betweencells and electrode

In order to evaluate the contribution of NADPH oxidase and itsproducts on the current generated in the present biofuel cell, theobserved current was compared to the theoretical current that canbe generated by the cells assuming 100 % efficiency of electron trans-fer. The theoretical current was estimated based on the amount ofsuperoxide generated by leukocytes and measured using cytochromeC, reported in the literature. Indeed, cytochrome C exhibits a rateconstant of 1.1 x 106 M−1s−1 at pH=7 [150] and is expected tolead to a more accurate estimation of the real amount of superoxideproduced.

59


Using 10 nmol/min/106cells as reference value for the rate of super-oxide production [70], the current produced by the present systemshould reach a maximum of about 32 µA. The maximum currentrecorded in this work under short circuit was 112 µA. This valueseems plausible, since prior to each cell measurement, the biofuelcell was allowed to stabilize for 1 hour during which the amount ofhydrogen peroxide accumulated in the solution.

4.10 Attempts at biofuel cell optimiza-tion

Abdellaoui et al. have shown the advantage of using TEMPO inenzymatic biofuel cells, utilizing oxalate as a fuel with oxalate oxidase[76]. The ability of TEMPO to mediate the electro-oxidizationof H2O2 produced by oxidases generated a power density severaltimes higher than the reference biofuel cell without oxalate. In thatwork TEMPO was shown to be a promising electrocatalyst for theoxidation of hydrogen peroxide in bioelectronics applications.Based on these reports, the present experiments employed TEMPOas electro-catalyst for hydrogen peroxide, since hydrogen peroxideis suggested as the primary source of current produced by activatedTHP-1 cells. In an effort to optimize power output, a biofuel celloperating on activated THP-1 coupled with the use of TEMPO asredox catalyst for hydrogen peroxide was evaluated.Figure 4.12 shows CVs of TEMPO in the absence and presenceof H2O2 and activated THP-1. In both cases, the oxidation peakappears at 400 mV. This result is also consistent with the hypothesisthat H2O2 is responsible for the current measured from the fuel cell.Regarding power density, the addition of TEMPO did not inducesignificant improvement, as the value of power output remainedalmost the same.

60


Figure 4.12: (A) Cyclic voltammetry of activated THP-1 in thepresence of 3 mM of TEMPO. CVs were recorded at 10 mV/s usinga carbon cloth as working electrode, a carbon rod as counter elec-trode and Ag/AgCl (1 M KCl) as reference electrode. Pink line: CVwas recorded after placing an ITO substrate carrying 2×106 THP-1cells in a solution of 3 mM of TEMPO in PBS and after subsequentaddition of 50 nM PMA. Grey line: control measurement performedusing 3 mM of TEMPO in the absence of cells. (B) Cyclic voltam-metry of H2O2 in the presence of 3 mM of TEMPO. Blue line: CVwas performed after adding 30 mM of H2O2 to a solution of 3 mMTEMPO in PBS. Grey line: control performed using 3 mM TEMPOin the absence of H2O2.

61

Chapter 5

Future perspectives

Even with very little optimization, the present biofuel cell was ableto yield superior power density output compared to other biofuelcells operating with leucocytes, reaching close to the minimum out-put required to power a small medical device. Indeed, this studyhas primarily focused on the activity of human cells and the studyof the system, but no specially engineered electrode was used andthe overall biofuel cell was not optimized. A modified anode, usingimmobilized mediators or catalyst, is very likely to lead to further im-provements. Using a nano or micro structured anode would increasethe surface area and, therefore, lead to further enhancement.Reducing overall biofuel cell size would minimize the distance be-tween the leukocytes and the anode and, therefore, would lower theconcentration losses.In this work, all experiments were carried out under bench top con-ditions, which are not optimal for cells. Indeed, low temperature,low CO2 level, limited food supply and a non-sterile environmentare likely to impair cell metabolism and lead to premature cell death.Providing a more suitable environment for cells would likely leadto improved biofuel cell performance. Furthermore, in vivo experi-

63

CHAPTER 5. FUTURE PERSPECTIVES

mentation would not only provide superior environmental conditionsfor biofuel cell performance but would also provide a more realisticframework in which to test this technology for its intended use.Additionally, thus far, only white blood cells have been used tooperate biofuel cells of the type presented in this work. However,NADPH oxidase is not only found in phagocytic cells but also in awide variety of human tissues, such as endothelial cells [97], vascularsmooth muscle cells [98], fibroblasts [99], renal mesangial cells [100],and corneal stromal cells [101]. This leads to the opportunity todevelop tissue specific biofuel cells operating on the aforementionedcell types, which could greatly expand the potential use cases of thistechnology.

64

Chapter 6

Summary

With the emergence of ever more biomedical implants, there has beena renewal of interest for biocompatible, more naturally embedded,and sustainable sources of energy, capable of supplying power byharvesting energy from the human body without requiring externalrefueling.In this work, several biofuel cells operating on activated THP-1cells differentiated on different substrates were tested. The setupconsisted of a conventional fuel cell design composed of two com-partments separated by a cation exchange membrane. Carbon clothelectrodes were used as anode and cathode. Prior to each biofuelcell measurement, substrates carrying 2x106 cells were introduced tothe anodic compartment, where the cells were activated using PMA.The highest power density output reached was 4.5 µW/cm2 and wasachieved using activated macrophages seeded on ITO. This valueis within less than an order of magnitude of the power required forcontemporary pacemakers [25, 151]. Separating the anode from thesubstrate carrying the cells proved to be advantageous in terms ofcell viability and biofuel cell performance.In addition to fuel cell characterization, the activity of the

CHAPTER 6. SUMMARY

macrophages on different substrates was studied and ITO provedto be the most suitable material for cell differentiation and activa-tion, showing a higher THP-1 activity compared to other materialstested. Moreover, it was shown, through electrochemical measure-ments, that under our experimental conditions, H2O2 is the primarysource of current generated by the biofuel cell. Even if the presentlyintroduced biofuel cell does not yet reach the minimum power out-put required for powering small biomedical implants, it neverthelessprovides encouraging results in the quest to find new energy sourcesfor biomedical implants.

66

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Kristina Javor, Jean-Nicolas Tisserant, and Andreas Stemmer. Bio-fuel cell operating on activated THP-1 cells: a fuel and substratestudy. Biosensors and Bioelectronics, 81:1-6 2017.

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In Copyright - Non-Commercial Use Permitted Rights ......Diss. ETHNo. 23773 Biofuel cell operating on activated THP-1 cells Athesissubmittedtoattainthedegreeof DOCTOROFSCIENCESofETHZURICH