NANOSCALE BIOCATALYSTS FOR BIOELECTROCHEMCIAL APPLICATIONS
A Thesis
Presented to
The Graduate Faculty of The University of Akron
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
Xueyan Zhao
December, 2006
NANOSCALE BIOCATALYSTS FOR BIOELECTROCHEMCIAL APPLICATIONS
Xueyan Zhao
Thesis
Approved: Accepted:
______Advisor Department Chair Ping Wang Lu-Kwang Ju
______Committee Member Dean of the College Lu-Kwang Ju George K. Haritos
______Committee Member Dean of the Graduate School Steven S. C. Chuang George R. Newkome
______Date
ii ABSTRACT
Rapid advances in nanoscale science and engineering are fueling the development of a new area in biotechnology, nanoscale enzymatic biocatalysis. Carbon nanotubes
(CNT) are fascinating supports for enzyme immobilization because their specific surface area is extremely high; in addition, their high conductivity also makes them greatly useful for electrochemical applications. The overall objective of this study is to examine the use of CNT for construction of enzyme-based electrodes for electro-biochemical applications.
Special focus is on the effect of CNT on reaction kinetics and mass and electron transfer processes on the enzyme electrodes.
Glucose oxidase (GOx) was employed as a model enzyme for immobilization on multi-wall carbon nanotubes (MWCNT). Three different immobilization methods were exploited to incorporate GOx onto MWCNT, including Covalent Attachment (CA),
Enzyme Coating (EC) and Cross-Linked Enzyme Aggregate (CLEA). GOx-MWCNT electrodes were constructed by coating the GOx/CNT complex suspended in a solution containing Nafion® on carbon electrodes including carbon felt and carbon papers. The resulted composite electrodes were and investigated for applications including biosensors and biofuel cells with respect to apparent enzyme activity, stability, and overall electrode performance.
iii A self-made glucose biosensor system was prepared using GOx-CNT composite electrode along with 1, 4-Benzoquione (BQ) was as a mediator. The mediator was deployed to speed up the electron transfer between the reaction active sites of GOx and the surface of electrode. The sensor responses in form of electrical current with respect to changes in glucose concentration in sample solutions were monitored for electrodes prepared by using different enzyme immobilization methods, i.e., CA, EC and CLEA.
That reveals the activity of GOx-CNT electrodes. Moreover, the thermal stability of the biosensor was probed under elevated temperatures, up to 50oC. It was found that CLEA provided the highest activity that gave a sensor sensitivity of 13.3 mA/M·cm2. Although all the three methods of enzyme immobilization improved significantly the stability of the enzyme, CLEA again gives the best stabilization effect.
Biofuel cells were also constructed by using GOx-CNT composite electrodes. IN addition to regular lab-size biofuel cells, miniature biofuel cells of sizes (1×1 and 2×2 cm) were also prepared for potential applications in micro-scale devices. In this part of study, we examined the performance of biofuel cells in terms of output power density and potential-current (V-I) relation. The kinetics of reactions were also examined and correlated to cell performance in order to understand governing factors of the cells. The kinetics of native, CNT-immobilized and electrode-mounted GOx were examined by following changes in substrate concentration via UV spectrometry. Electrodes constructed with different support materials such as Toray® carbon paper, carbon cloth and carbon felt were investigated. Both the storage and operational stability of GOx-CNT electrodes in biofuel cells were investigated. Compare to native enzymes, although all the three
iv methods of enzyme immobilization improved the stability significantly, CLEA again gives the best stabilization effect.
This study showed that mass transfer processes on the surface of the composite electrodes is the key limiting factor determining the overall electrode performance.
Enzyme immobilized on CNT gave an enzyme activity that was compatible of native enzyme in solution; however, compared to electrodes With/without CNT, CNT electrodes demonstrated higher electron transfer rate was about 6 times higher.
Studies on biofuel cell performance revealed that the electrode performance in terms of measured electrical current density represent only 0.6% of the reactivity the enzyme mounted on the electrodes, indicating that electric transfer and other in-cell electrical resistance is the determining factor of the fuel cells. This demonstrated that the further improvement the of biofuel cells performance could be achieved through optimal design of the cells. Of the three methods of enzyme immobilization, CLEA provided the best performance in almost all the aspects examined. It might be attributed to the micro-environment surround enzyme was enhanced by a form of multilayer coatings on the high-surface area CNT. It is expected that combined with better cell design with more efficient electrical transfer, the use of nanotubes can eventually afford highly efficient biochemical electrode for biosensing, biofuel cell or electrobiocatalytic applications.
v ACKNOWLEDGEMENTS
I would like to express my deepest gratitude to my advisor, Dr. Ping Wang for his continuous support, guidance, and encouragement throughout my study and research. He has been a great mentor with his enthusiasm, understanding and willingness to help students professionally and personally. I also appreciate Dr. Lu-Kwang Ju and Dr. Steven
S. C. Chuang for serving as my committee members and for their precious suggestions to my study. I would like to express my thanks to Dr Jungbae Kim for his mentoring and kindly help during my research work in Pacific Northwest National Laboratory.
I want to thank Dr. Guangyu Zhu and Dr. Hongfei Jia for their assistance in initiating my project and whenever in need. I appreciate Dr. Byoung-Chan Kim and Ms.
Hye-Kyang Ahn for their efforts in enzyme immobilization and San-Ae Jun for her help in biofuel cell experiment in PNNL. I also want to thank all my group members for their friendship: Mr. Bilal El-Zahab, Ms. Liang Liao, Mr. Ravindrabharati Narayanan, Mr. Jim
Hancock, Mr. Songtao Wu, Mr. Ramazan Komurcu, Ms. Trivedi Archana Harendra
Kumar and Ms. Fadime Suhan Baskaya.
Most of all, I would like to dedicate this work to my beloved husband, Xiaodong, for his love, understanding and all the joys he brought to me. I am eternally grateful to my parents and brother for their supports throughout my life.
vi TABLE OF CONTENTS
Page
LIST OF TABLES...... x
LIST OF FIGURES ...... xi
CHAPTER
I. INTRODUCTION...... 1
1.1 Enzymatic Biocatalysis...... 1
1.2 Bioelectrochemical System...... 4
1.3 Objectives ...... 7
1.4 Approaches ...... 8
1.4.1 Immobilization of glucose oxidase ...... 8
1.4.2 Nano-composite enzymatic electrodes for biosensor ...... 9
1.4.3 Nano-composite enzymatic electrodes for biofuel cells ...... 10
1.5 Scope...... 10
II. LITERATURE REVIEW...... 12
2.1. Biocatalysis...... 12
2.2. Biosensor...... 14
2.2.1 Biosensor with a soluble or immobilized mediator ...... 17
2.2.2 Mediatorless biosensor...... 18 vii 2.3. Biofuel Cells ...... 19
2.3.1 Current status of biofuel cell research ...... 20
2.3.2 Cathodic biocatalysts in biofuel cells ...... 21
2.3.3 Anodic biocatalysts in biofuel cells ...... 22
2.3.4 Overall performance of biofuel cells ...... 23
2.4. Nanotechnology ...... 24
III. NANO-COMPOSITE ENZYMATIC ELECTRODES FOR APPLICATIONS IN BIOSENSOR SYSTEM...... 25 3.1 Introduction...... 25
3.2 Materials and Methods...... 26
3.2.1 Activation of CNT ...... 27
3.2.2 Preparation of GOx-CNT composites...... 27
3.2.3 Protein content measurement of GOx-CNT composites ...... 29
3.2.4 Activity and stability of GOx-CNT composites ...... 29
3.2.5 Preparation of enzyme electrodes ...... 30
3.2.6 Entrapment of mediator on electrodes ...... 31
3.2.7 Electrochemical measurements...... 32
3.3 Results and Discussions...... 32
3.3.1 Immobilization of glucose oxidase (GOx)...... 32
3.3.2 Enzyme loadings and specific activities of GOx-CNT composites..... 38
3.3.3 Performance of GOx-CNT biosensors...... 42
3.3.4 Effect of free mediator on GOx-CNT biosensors...... 42
3.3.5 Effect of immobilized mediator on GOx-CNT biosensors...... 49
3.3.6 Thermal stabilities of GOx-CNT biosensors ...... 51 viii IV. NANO-COMPOSITE ENZYMATIC ELECTRODES FOR BIOFUEL CELLS APPLICATIONS ...... 53 4.1 Introduction...... 53
4.2 Materials and Methods...... 55
4.2.1 Materials ...... 56
4.2.2 Preparation of GOx-CNT composites...... 56
4.2.3 Measurement of GOx-CNT activity ...... 56
4.2.4 Set-up of biofuel cells ...... 58
4.3 Results and Discussions...... 61
4.3.1 Kinetic study of free enzymatic reactions...... 61
4.3.2 Kinetic study of immobilized enzymatic reactions...... 64
4.3.3 Kinetic study of immobilized enzymatic electrode reactions...... 65
4.3.4 Effect of immobilization methods on biofuel cell performance ...... 67
4.3.5 Effects of CNT on biofuel cell performance...... 71
4.3.6 Thermal stability study of biofuel cell electrodes...... 74
4.3.7 Study of the stability of biofuel cell operation...... 76
4.3.8 Comparisons of self-made and miniature biofuel cells...... 77
V. CONCLUSIONS...... 82
BIBLIOGRAPHY...... 84
ix LIST OF TABLES
Table Page
2-1 Glucose biosensor and their performances...... 16
3-1 Enzyme loading and activity of GOx-CNT composites...... 39
3-2 Comparison of current response at [glucose] = 10 mM ...... 45
3-3 Comparison of biosensor sensitivities with vary [BQ]...... 47
4-1. Usual operation temperatures of some noble catalyst fuel cells...... 54
4-2. Comparison of reaction velocities ...... 66
4-3. Characteristics of enzymatic biofuel cells with different GOx-CNT composite electrodes ...... 69
4-4 CNT effects on biofuel cell performances...... 72
4-5. Comparisons of power output in self-made biofuel cell and miniature biofuel cell (1 cm×1 cm)a...... 79
4-6. Comparison of power output in two different size miniature biofuel cells using Toray® carbon paper (thickness 370 µm) electrodes ...... 81
x LIST OF FIGURES
Figure Page
3.1 Enzymatic glucose biosensor setup ...... 31
3.2. Scheme of Immobilization of GOx on CNT (a). Covalent Attachment of GOx on CNT...... 34 (b). Scheme of Enzyme Coating (EC) of GOx on CNT...... 35 (c). Scheme of Cross-linked Enzyme Aggregate of GOx on CNT ...... 37
3.3 The stability of three immobilizations at room temperature...... 40
3.4 Representative amperommetry curves of GOx-CNT biosensor response ...... 41
3.5 GOx-CNT composite electrode biosensor performances with vary BQ concentration (a) GOx-CNT composite electrodes performances in biosensor with a BQ concentration equals to 5 µM...... 43 (b) Different GOx-CNT composite electrode performances in biosensor with BQ concentration equals to 5 mM ...... 44
3.6 Biosensor Reaction Scheme in Reaction Layer ...... 47
3.7 Immobilization processes effects on the mediator entrapment biosensor system .... 50
3.8. Thermal stability of GOx-CNT electrodes for biosensors...... 52
4.1 Biofuel cell setups used in this study. (a). Scheme of self-made biofuel cell ...... 59 (b). Photo of miniature biofuel cell (2cm×2cm) (c). Photo of two different sizes miniature biofuel cells...... 60
4.2. Dependence of initial reaction rate to glucose concentration ...... 63
4.3. Dependence of Km’ and Vmax’ to glucose concentration ...... 64
4.4. V-I curves of biofuel cell with CA-, EC-, and CLEA-GOx composite electrodes ... 68
4.5. P-I curves of biofuel cell with CA-, EC-, and CLEA-GOx composite electrodes ... 70
xi 4.6. The power density relation to the resistance load in out circuit ...... 71
4.7 Power putout of self-made biofuel cell performances with CA-, EC-, and CLEA electrodes ...... 73
4.8. Thermal stability of CA-and CLEA-GOx electrodes in biofuel cells...... 75
4.9. Long term operation of the self-made biofuel cell performances with CA- and CLEA-GOx composite carbon felt electrodes ...... 76
4.10.P-I curves of miniature biofuel cell (1 cm ×1 cm) with CLEA-GOx composite electrodes on different supporting materials ...... 78
4.11.P-I curves of miniature biofuel cell (2 cm ×2 cm) with CA- and CLEA-GOx composite electrodes ...... 80
xii CHAPTER I
INTRODUCTION
1.1 Enzymatic Biocatalysis
Enzymes are defined as biological molecules that speed up chemical reactions without destroying or altering themselves upon completion of the reactions. Usually, enzymes are proteins or protein-based molecules with unique catalytic properties.
Enzymes possess remarkably advantages over chemical catalysts, in more biocompatible and biodegradable, higher efficiency, higher activity under mild conditions, and higher specific selectivity. Those particular natures make enzymes especially suitable for biomedical, pharmaceutical, and food industries. Therefore, as biocatalysts, enzymes have shown their importance in both bio-industry and human’s routine life. According to the statistics of biotechnology industry organization (BIO) in 2000, the global demand of industrial enzymes was approximately two-billion dollars with an annual growth rate of
5-10%. The market for industrial enzymes could be divided into the following fields:
(1) Technical enzymes, involved in the processes of wood pulp processing, textile and leather manufacturing, laundry detergents making and ethanol fermentation;
(2) Food additive enzymes, widely applied in baking, fruit juice manufacture and dairy production;
1 (3) Feeding enzymes.
Enzymes attracted more and more attentions in both research studies and industry applications, because of their special advantages over chemical catalysts, and much broader potential in utilization.
However, by nature, enzymes cannot work efficiently under the artificial conditions of industrial processes. This detains the utilizing of enzymatic catalysis in several aspects. For example, enzymes could be easily denatured when the environmental factors (temperature, pH, salt concentration), and other physical or chemical system features were changed. The denatured enzymes will lose their catalytic activities temporarily or permanently due to the structure changing or the bonding pattern distributing. If the enzyme activity is temporarily lost, in some cases, refolding the enzyme structure in reverse environmental changes or with the aid of some reagents could regain the enzyme activity. But, if enzyme structure is completely destroyed, to restore enzyme activity is impossible.
Another major concern in limiting the use of enzymes in industry is that traditional enzymatic catalysis usually is performed in aqueous buffer solutions; which the industrial processes may not always be. Gas, liquid and sometime solid reactants are more frequently involved in industrial procedures. To overcome the above problems, immobilized enzymes are designed to pursue the catalytic tasks in industrial process. Enzyme immobilization technology has been extensively studied since 1960’s [1-3].
Immobilization methods had been evolved from the physical adsorption, entrapment, and
2 encapsulation in the porous structure of supports to chemically covalent-bonding, cross-linking of the legends on the materials [3-5].
Enzymes could be immobilized on several media, including gold particles [6], polymers [7, 8], magnetic particles [9, 10], and carbon nanotubes [11]. Among the supports for enzyme immobilization, nanostructured materials, which at least one dimension is less than 100 nm, usually are the most preferred. The reason resulted that larger specific surface areas provided more positions for enzyme attachment. Therefore, nanostructured materials make high-loading enzyme immobilization procedure possible.
Furthermore, new enzymatic functionality could be achieved in the present of nano-materials. Intensively successful and interesting enzyme immobilization attempts have been explored on nano-materials, such as nanoparticles [12-17], nanoporous materials [18-21], nano-capsules [22], carbon nanotubes [23-26] and nano-composites
[27].
Upon immobilization, enzymes are confined or localized, which makes enzymes be recovered and reused easily and continuously. Usually, after immobilization, most enzymes could possess better stability because of the entrapment or attachment of enzyme on/in the supports assists the enzymes to retain their protein structures and prevent the denaturation. On the other hand, immobilized enzymes provide lower activities than native enzymes. To compromise the stability and activity from the industrial point, large-scale processes prefer immobilized enzymes because the separating enzymes from the reaction medium will be much easier.
3 Based on the instruction of The International Union of Biochemistry and Molecular
Biology and the International Union of Pure and Applied Chemistry, enzymes were divided by the reactions they catalyzed. The transferases catalyze functional group transferring reaction, such as the peptidyl transferase reaction of protein synthesis in cells[28]. Hydrolases catalyze the hydrolysis of various bonds, and oxidoreductases, catalyze oxidation/reduction reactions. Oxidoreductases referred to a class of enzymes catalyzing oxido-reductions. When the substrate is regarded as an electron donor, the common name of this kind of enzyme catalyst is called dehydrogenase. On the other hand, the enzymatic catalysts are called oxidase, with an electron acceptor substrate. Since the oxidoreductase reactions usually involving electrons in the reaction mechanism, bioelectrochemical systems could be established based on this characteristic.
1.2 Bioelectrochemical System
Bioelectrochemistry is the study of applying electrochemical principles in biology and biological aspects. In bioelectrochemistry reactions, charged particles (ions or electrons) transferring happened in biological (living) systems and/or biological compounds forms electrochemical signal. This unique characteristic is very useful in monitoring the biological reaction kinetics and dynamics. Thus, bioelectrochemistry became a very active research area, which included biological oxidation/reduction, biological accumulation, biological precipitation, minerals/metals dissolution, and antibody-antigen binding reaction.
4 With the exploitation of modern biology and chemistry, biosensor and biofuel cell emerged and became two most intense studied research fields in the whole bioelectrochemical system.
As defined in Wikipedia encyclopedia, a biosensor is a device that combines a biological component with a physicochemical detector component, and be used for analyte detection. That is, biosensor could be used to measure a chemical or physical response.
The studies of biosensor include three major fields: molecular recognition segment
(MREs), biosensor construction and electronics. The biocomponent with specific molecular recognition properties in biosensor will identify analyte and transfer the amount of analyte into electric signal. Considering of the stability and easiness to recycle, the biocomponents are usually immobilized on supports, which are contacted directly to transducer [29]. The transducer is coded as the electrochemical component that could convert the chemical or physical parameter into electrical output signal, which can work electrochemically, optically, thermally in the biosensor system.
The biocomponents used as molecular recognition element in biosensor include enzymes, cells, organelles, tissues, receptors, antibodies and antigens. Enzymes, for their high specific selectivity, are most commonly used to compose the recognition element in biosensors.
Enzymatic biosensors are usually divided into amperometric and potentiometric enzymatic biosensors. Currently, the most investigated biosensor is electrochemical based, amperometric enzymatic biosensor, which works at a fixed potential value. In this type of biosensor, enzymes catalyze the analyte reaction to produce or consume a redox-active
5 compound that will be oxidized or reduced on the electrode surface. Thus, the production of current is proportional to the concentration of analyte.
Recently, applications of immobilized oxidoreductase and dehydrogenase electrodes become more and more attractive for developing biosensors, especially the glucose biosensors for medical purposes and food industry. The market value of sensors is estimated to be $109/year, in which electrochemical sensors occupied $108/year. Enzymes are the core of working biosensors in this multi-million dollar business, enzyme stability issues are always at the forefront of producing a stable and reproducible biosensor.
Various nanoscale materials, which could provide large surface area for the enzyme immobilization than the traditional supporting materials, have been actively utilized for enzyme stabilization. Reducing the size of enzyme-carrying materials could result in the improvement of the immobilized enzymes stability, and lead to higher enzyme loading per unit mass of particles [30]. Other attempts, such as nanofibers[31] and single enzyme nanoparticles [32], have proved that nanoscale supporting materials have advantages in enzyme immobilization.
Biofuel cells are referred to be a class of fuel cells that utilize microbial or enzymatic biocatalysts or manipulate renewable fuels, could convert chemical energy to electricity. For example, biofuel cells could have laccase on the cathode to reduce oxygen with a non-biofuel feeding or use inorganic catalysts such as Pt to catalyze redox reactions with a biofuel feeding. Moreover, biofuel cells could be developed to provide the miniaturization and portability of power sources that are able to sustain operation over long periods of time. Furthermore, biofuel cells, compare to traditional fuel cells, are more
6 environmentally beneficial because both the catalysts and the fuels come from renewable resources. Generally, biofuel cells could be classified as microbial-based biofuel cells and enzymatic biofuel cells. In addition, compared with the traditional fuel cells, biofuel cells could afford much more fuel options, such as hydrogen, carbon hydrates, organic acids and urea [33].
1.3 Objectives
This research work is aimed to develop high activity and stability enzyme composites for electrochemical applications by using nanostructured materials.
Specifically, the research objectives are:
(1) To develop GOx composites by attaching the enzyme to carbon nanotubes. The
effects of immobilization methods in terms of enzyme loading and activity will
be examined;
(2) To investigate the variables and parameters that control the activity and
stability of GOx-CNT composites under various conditions;
(3) To evaluate the use GOx-CNT composites for biosensors, especially study the
effects of material property, enzyme loading and methods of enzyme
immobilization on the lifetime and sensitivity of the biosensors;
(4) To probe the potentials of GOx-CNT composite electrode for biofuel cell
applications, especially study the effects of the use of nanomaterials on the
power density and lifetime of the biofuel cells; and
7 (5) To investigate the reaction kinetics of BQ mediated bioelectrochemical
reactions on the composite electrodes, to reveal the limiting steps that regulate
the overall performance of the electrodes.
1.4 Approaches
In this study, various strategies for fabricating the enzyme-nanomaterial composites have been developed. Enzyme-carrying nano-structure material, carbon nanotubes, was investigated, and glucose oxidase (GOx), as a model enzyme, was used to study the influences of different immobilization methods on enzyme activity. The
GOx-coated carbon nanotubes were coupled on the electrode, and then applied to compare their performances in the applications of biosensors and biofuel cells. Through the comparisons of the different immobilization products, a novel enzyme immobilization strategy, Cross-Linked Enzyme Aggregate (CLEA), shows a promising future for its applications in biosensors and biofuel cells.
1.4.1 Immobilization of glucose oxidase
A significant advantage of immobilized enzyme is that it could maintain the high activity of enzyme catalysis and increase the stability of enzyme, however, the use of various immobilization methods could result in the different performances. In this thesis, glucose oxidase (GOx) was coupled on the carbon nanotubes as described in the following methods: Covalent Attachment (CA), Enzyme Coating (EC), and Cross-Linked Enzyme
Aggregate (CLEA). Kinetic behaviors of free glucose oxidase were examined. Stability of
8 the enzyme activity also was monitored, and the short lifetime issue of enzyme activity was expected to be alleviated. Through the analyses and comparisons, cross linking of enzymes in vicinity between enzyme molecules on the carbon nanotube surface, prepared by CLEA, was expected to be a powerful approach.
1.4.2 Nano-composite enzymatic electrodes for biosensor
Applications of biosensor have been existed widespread in many scientific fields.
Fundamental studies in biosensor mainly focus on molecular recognition elements, biosensor construction techniques and tools, and basic biosensor devices. Most common available biosensor is using enzymes as molecular recognition segment. In our studies, the enzyme-coated carbon nanotubes were prepared by three various immobilization methods.
These resulting GOx-CNT composites were applied in amperometric glucose biosensor, respectively. Effects of mediator on the detection of glucose by these resulting GOx-CNT composites were investigated in this study. The thermal stabilities of those fabricated
GOx-CNT electrodes in biosensor were probed under 50oC, which could be used to predict the electrode performance under actual working conditions. Because the use of CLEA could increase the loading amount of GOx in a form of multilayer coatings on the high-surface area CNT, these highly stable and active enzyme-coated CNT significantly improved the performance of biosensors.
9 1.4.3 Nano-composite enzymatic electrodes for biofuel cells
Biofuel cells represent one of the potential solutions to efficient generation of electricity from renewable resources. Currently, low power density and poor stability limited the practical applications of enzymatic biofuel cells. In this study, enzymatic electrodes were fabricated by incorporating a model enzyme, glucose oxidase (GOx), into carbon nanotubes (CNT)-Nafion composites. The resulting electrodes were characterized by cyclic voltammetry and activity measurements, respectively. A model glucose/oxygen biofuel cell was also constructed to evaluate the performance of the enzymatic composite electrodes. (BQ) was used as a redox mediator to increase the electron transfer speed. It was expected that high surface area of CNT would afford high enzyme loading, which is critical to obtain high current (power) output. It was also expected that covalent immobilization could greatly extend the lifetime of the enzyme thus improve the stability of the electrodes. In addition, two different size miniature fuel cells (Courtesy from
Washington State University) were also tested in our experiments, respectively, which were used to compare with the results from our self-made biofuel cells.
1.5 Scope
In outline of this thesis is given as follows: Chapter II reviews literatures about biocatalysis, biosensor, biofuel cells and nanobiotechnology. Chapter III focused on the developments of three enzyme immobilization methods on the carbon nanotubes (CNT) and the investigations of the application of those CNT nanobiocatalysts in biosensors. The stability and mediator effects also were investigated. Chapter IV explores the construction
10 GOx-CNT nanocomposite electrodes for biofuel cell applications. Effects of different components in biofuel cells on the performances were investigated to optimize the efficiency of biofuel cells. To better understand the biofuel cells theoretically, the relation between the kinetics of free GOx catalyzed reaction rate and current generated in biofuel cells was determined using spectrum methods. Conclusions are summarized in Chapter V.
11 CHAPTER II
LITERATURE REVIEW
2.1. Biocatalysis
Biocatalysis is defined as using microbial, native or modified enzymes to perform the chemical catalysis in organic molecular system [34-36]. Biocatalysis was initiated in production of alcohol by using fermentation process under anaerobic conditions with yeast presence [37]. The most significant biocatalysis process is probably the production of penicillin by using microbial resources during WWII [38], which allow us to live a much healthier life.
As well known, enzymes were first been recognized in 18th century. Since then, enzymes were regarded as native catalysts, and were used in textile, leather and food industry for a fairly long period. Comparing with chemical catalysts, enzymes have shown remarkable advantages in selectivity, efficiency and friendly to the environment. These superiorities allowed enzymatic technology to attract the research attentions for centuries.
Nowadays, enzymes expand their active working area in pharmaceutical and many other fields, although enzymatic technology is facing several challenges in making a broader prospect of industrial application. For example, enzymes are mostly produced from bacteria, yeast and fungi, by controlling the growth environment of enzyme resources
12 using a bioreactor or fermentor. During this production process, enzymes could be easily denatured when the environmental parameters, including temperature, pH, salt concentration, and other physical or chemical properties, were changed. This makes the maintaining the enzyme activity and stability into a great concern.
Either reducing the cost of enzymes or improving the enzyme activity and stability will provide more opportunities for large-scale enzyme applications. Enzyme immobilization has been proved to be the most promising way in lengthening the enzyme life time. Immobilization usually is defined to attach enzyme on a solid support by non-covalent or covalent bonding. This process was believed to stabilize enzymes by make enzyme molecules less flexible and mobile. In most cases, the immobilized enzymes will lose part of their catalytic activities during immobilization [39]. On the other hand, some of the supporting materials could enforce more activity sites exposed to substrates after immobilization [40]. In addition, the immobilized biocatalysts could be recovered easily from the reaction system for reusing, which will decrease the cost of enzymatic catalysis processes.
Enzymes could be immobilized by entrapment [19, 41], physical adsorption [42, 43] and covalent binding [18, 44-48]. A number of support materials [9-11, 20, 27, 49-51], either natural or synthetic, have been examined for enzyme immobilization. The most widely used materials for enzyme encapsulation or entrapment are sol-gel silica and mesoporous silica. Lei et al. [19] studied the entrapment of enzyme with the unfunctionalized and HOOC-functionalized mesoporous silica (FMS) supports using organophosphrus hydrolase as a model enzyme. Comparing to the entrapment process
13 with common porous silica, the amount and activity of the enzyme entrapped in 2%
HOOC-FMS were doubled.
For physical adsorption of enzymes on/in the support, Ferreira et al. [43]developed a Layer-by-Layer (LbL) technique to immobilize glucose oxidase on the indium–tin oxide
(ITO)-coated glass. The resulting enzyme composite was used to construct a glucose biosensor electrode. The relationship between the layer number and the analytical response sensitivity was studied. The sensitivity and the limit of detection in this biosensor are 16 µA/mM·cm2 and 0.2 mM separately.
Enzymes also could be covalently attached to the supports by their surface groups.
Zhang et al. [44] reported to covalently attach the glucose oxidase on an Au electrode, which has gold nanoparticles on the surface. By exploiting the electrode in biosensor, the sensitivity and detection limit are 8.8 µA/mM·cm2 and 8.2 µM individually. The linear dynamic range of this biosensor is from 2.0×10-2 to 5.7 mM.
Considering all the applications in enzyme immobilization, another major issue was believed to be how to efficiently transfer the soluble substrates to the enzyme catalyst on a non-soluble support. Since the support usually is non-solvable, therefore, the whole reaction system was changed from homogeneous to heterogeneous.
2.2. Biosensor
Electrochemical biosensor could be classified as amperometric biosensor and bioaffinity biosensor. The most frequently used biosensor is glucose biosensor which diabetes patients use to monitor their blood sugar content. The fundamental subject in
14 manufacturing the enzyme electrodes is to improve the mechanism of electron exchange between the enzyme active site and the electrode cross the non-conductive protein shell.
Based on this, two subgroups of biosensor were classified by electron transfer methods:
(1) Biosensor using a soluble or immobilized mediator;
(2) Biosensor with direct electron transfer (mediatorless).
Some of the glucose biosensors with their performances are listed in Table 2-1.
15 Table 2-1 Glucose biosensor and their performances
Operation Electron transfer Detection Dynamic Sensitivity voltage enhancement limit range (mM) (mM) 400 mV redox hydrogel - 15 - [52] 1.0 V Conductive polymer 0.1 5 - [53] Conductive 0.9 V 0.2 50 2.33 nA/mM [54] polymer/CNT palladium 0.3 V 0.15 12 - [55] nanoparticles 0.35/0.4V ferrocene - 42 - [56] 0.6 V - 0.07 2 0.2 A/M [57] 0.25 V ferrocene - 16 - [58] 0.32 V ferrocene/gold layer - ~15 - [59] -0.1 V hydroquinone 1×10 -04 2 - [60] -0.4 V CNT 0.03 2 - [61] 0.2/0.3 V CNT 0.08 30 - [62] -0.5/0.0 V Methyl violgen 0.02 1.2 - [63]
- Prussian Blue 0.20 - 16µA/mM·cm2 [43]
poly(vinylimidazole) 0.22 V 0.004 15 - [64] +/2+ / [Os(bpy)2Cl] -0.3 V Prussian blue 0.05 1 - [65]
0.7 V polypyrrole - ~20 103 µA/mM·cm2 [66]
- Conductive polymer 0.05 5 - [67]
16 2.2.1 Biosensor with a soluble or immobilized mediator
Traditionally, glucose biosensors are based on the amperometric detection of hydrogen peroxide produced or oxygen consumed in glucose oxidation reaction, which is catalyzed by glucose oxidase coated on the electrodes. This kind of glucose biosensor has some disadvantages. For example, the fluctuations of dissolved oxygen may cause the deviation of the current response to glucose concentration. The dynamic range of glucose detection could also be limited by depletion of oxygen in the solution [58]. Diffusion mediators, such as quinines and ferrocenes, are commonly used for helping the electron transport of enzyme electrodes to overcome the oxygen dependence [68-71]. However, because these diffusion mediators are small, the leaching issue of the mediator will be a serious concern in clinic applications.
To eliminate mediator leaching, several larger-size novel diffusion mediators, such as 3-methyl-4-hydroxycyclobut-3-ene-1,2-dione (methylsquarate, OHME) and
3-phenyl-4-hydroxycyclobut-3-ene-1,2-dione (phenylsquarate, OHPH), were fabricated and studied for better electrochemical characteristics [72]. Moreover, mediators were also immobilized to reduce the leaching issue [56, 59, 65, 73]. For example, Ghica et al. described a methyl viologen-mediated amperometric biosensor [63]. Enzyme electrode was developed by immobilizing and cross-linking glucose oxidase on carbon film electrode in the presence of bovine serum albumin. The mediator, methyl viologen, was directly entrapped with the enzyme together by Nafion® cation-exchange polymer. The detection limit of this biosensor was 20 µM, and the linear range was up to 1.2 mM. The stability of this biosensor last for weeks, when stored in phosphate buffer at 4oC.
17 2.2.2 Mediatorless biosensor
Mediator entrapment could mild the mediator leaching problem during some of the biosensor operations, but could not completely solve the possibility of leaching harassment.
Further studies have been exploited in several other research areas, such as employ the conducting polymer to directly wire the enzyme active site with the electrode. For their unique properties in electronic, chemical, and mechanical application, the synthesizing nanostructured materials were interested considerably in recent years. Different materials, such as metal, carbon, and polymers, have been used to prepare nanostructured materials, nanoparticles [55, 74], nanotubes [62, 75], and nanofibers [76].
Three major methods have been investigated in recent years:
(1) Carbon nanotubes and conductive nanoparticles
(2) Conductive polymers
(3) Multienzyme system
Carbon nanotubes have been used in electronic applications and chemical and biological detections for the unique structure and property, such as conductivity. Carbon nanotubes could be divided into single-wall and multi-wall carbon nanotubes by the structure. Recent studies indicated that CNT could enhance the biological molecular electrochemical reactivity and promote the electron transfer in protein reactions [77-79].
The remarkable high conductivity and high surface absorbent of carbon nanotubes make it a promising candidate in making nanostructured biosensors. When the enzyme active site was direct wired to the electrode, usually carbon nanotubes or conductive nanoparticle was coated on the electrode after mixing with enzyme. Lim et al. [55, 80] reported co-deposit glucose oxidase with palladium nanoparticles onto a Nafion-solubilized carbon nanotube
18 (CNT) film. The resulting electrode allows for fast and sensitive glucose quantification.
The fabricated glucose biosensor exhibits a dynamic range of 12 mM and a detection limit of 0.15 mM.
When conductive polymer was used to direct wire the enzyme active site and the electrode, polymer could be formed by electrochemical deposition. Shin et al. [81] reported using electrochemical deposition to form polypyrrole/glucose oxidase film. This conductive film could be used in glucose biosensor as electrode. Linke et al. [52] described that cross-link glucose oxidase into a 3-dimensional network. They found that in the presence of redox polymer, the formed redox hydrogel electrode has high glucose sensitivities.
Besides of direct wiring, multi-enzyme working pathway also could be used to replace the mediatored reactions in biosensor. Multi-enzyme biosensor was initiated in the late 70s [82], in which different enzymes were coupled either in sequence or in cycles on the electrode. In this way, not only a wider range of species could be measured by bioelectroanalytical approach, but the selectivity and sensitivity of biosensors could be enhanced [29].
2.3. Biofuel Cells
Biofuel cells have attracted a lot of research efforts, for its potential of applications in renewable resources. The current research status of biofuel cells is described in the following sections.
19 2.3.1 Current status of biofuel cell research
The gap between energy consumption and supplement brought the energy issue into the ever hottest topic. Moreover, clean and efficient approaches were also demanded for the environment concerns. Fossil fuels were considered as non-renewable energy because the storage limitation of fossil fuel sources. Currently, using renewable energy, and use fossil energy more efficiently were regarded as promising approaches to solve the energy short. The most recognized efficient process of using fossil fuels is the convert the chemical fuels stored in fossil fuels directly to electricity. This process could be realized by fuel cells. The theoretical efficiency of fuel cell process could reach 100%. Also, when hydrogen or alcohols are used as fuels in fuel cells, the byproducts will be only water and carbon dioxide.
In early nineteen century, the electrochemical abilities of glucose and other compounds were investigated at the present of microbes [83, 84]. NASA started to study the possibility of fuel cells to generate electricity in space from living wastes. In the meantime, the byproduct in the fuel cells reactions, H2O, could be used by astronauts.
Those efforts made successful progresses, which realized fuel cells by using urea and methane as fuel [85, 86]. While, the first enzymatic biofuel cell was reported in 1964, in which glucose oxidase (GOx) was used as the anodic catalyst [87]. Therefore, a new concept, biofuel cell, was introduced, which was defined as a class of fuel cells which apply microbial or enzymatic biocatalysts.
Usually, biofuel cells include two subunits: (1) Fuel cells using biocatalysts such as laccase and using hydrogen as fuel, called direct biofuel cells. (2) Fuel cells using hydrogen, alcohol or methane generated from biochemicals such as sugars, but the catalyst
20 could still be the noble metals, called indirect biofuel cells. Other than all the advantages of chemical fuel cell, biofuel cells provide much more options in the fuel selections. For example, carbon hydrates, organic acids and urea could be used as fuel in biofuel cells [33].
Biofuel cells are even more environmental friendly because both the catalysts and the fuels are renewable. Nowadays, with the advent of the development of novel materials and the use of new strategies, biofuel cells using the bio-based energy sources are attracting more and more attentions to compete with fuel cells in the face of energy crisis.
2.3.2 Cathodic biocatalysts in biofuel cells
Although many potential applications have been proposed since the invention of biofuel cells, no biofuel cells were commercially available for power generation other than in research labs currently. This is mostly due to the lower performance of biofuel cells, compared to conventional fuel cells. The low power density and stability narrowed the biofuel cells to the areas, such as clinic, health care and biomedical applications, which fuel cells were not considered.
Much more interests had been gathered to improve the performance of the direct biofuel cells, in which biocatalysts are directly involved in the redox reactions for electricity generation. So, how to transfer electrons efficiently from the biocatalysts reaction active sites to the electrodes becomes the critical issue in direct biofuel cells.
Usually, it is difficult for direct electron transfer to the enzymatic electrodes due to the non-conductive protein hull which covers active sites. Direct electron transfer was observed only in cytochrome c, laccase and several peroxidases [88-91]. Methods of chemical modification were also developed to help increasing the enzymes conductivity. 21 For example, glucose oxidase electron transfer could be enhanced by either modify the enzyme surface or directly wire the active site to the electrode [77, 92, 93]. Other than the above approaches, mediator also could be used to shuttle between the active site and the electrode; therefore, the resistance of enzymatic electrode could be reduced. The use of mediators in microbial and enzymatic biofuel cells has been reported [94-97]. When mediator was used, usually much higher cell efficiency could be reached. Considering the price of mediator, which is usually expensive, how to maintain them in the cells during the continuous fuel feeding model should keep in mind.
Oxygen is the most frequently use oxidant in the cathode compartment in biofuel cells. When oxygen was used as oxidant, several enzymes could work as biocatalysts, including bilirubin oxidase (BOD) [98, 99], laccase [100-104] and cytochrome oxidase
[105-107]. Cytochrome c and cytochrome oxidase on a gold electrode have been reported
[108]. These enzymatic cathodes catalyzed four-electron reduction from oxygen to water.
Impressively, BOD catalyzes the fastest reduction of oxygen on the cathode electrode, and the current density could reach 0.5 mA/cm2. Besides oxygen, hydrogen peroxide generated from oxygen also could work as oxidant at cathode. Peroxidases were applied as biocatalysts in biofuel cell to catalyze the H2O2 oxide reactions in cathode [109-111].
Peroxidase from horseradish (HRP) adsorbed [112] and microperoxidase-11 (MP-11)
[109] also were studied and reported.
2.3.3 Anodic biocatalysts in biofuel cells
Biocatalysts are capable of catalyzing the reduction as mentioned above. On the other hand, biocatalysts also could catalyze the oxidation for electricity generation. The
22 biocatalysts working in power generation are diversified, including hydrogenase [100,
113], alcohol dehydrogenases [114], glucose oxidase and glucose dehydrogenase [99, 101,
105, 107, 109, 110, 112, 115-117], lactate dehydrogenase [118]and Ureanase [85].
2.3.4 Overall performance of biofuel cells
In both cathodic and anodic side of biofuel cells, the lifetime of biofuel cells was raised to be the most concerned issue. Most enzymatic fuel cells usually survive for days
[109, 110, 119]. There are several factors have effects on the lifetime of biofuel cells. For example, the degradation of redox mediators and the stability of enzymatic biocatalyst could limit the cell’s lifetime. Enzymes’ lifetime can be extended by immobilized enzymes on supports, narrow the enzyme mobility in an ambient environment allow enzymes to keep their activities. After immobilization, active lifetimes of more than 100 days were achieved. For the operational stability, promising results were reported by using modified
Nafion membranes with tetrabutylammonium bromide [120]. By applying the same technology in electrode fabrication, biofuel cells operate continuously for weeks were built
[121].
Other than stability issue, the power output of biofuel cells also becomes the major concern in the application of biofuel cells. The power density of biofuel cells was determined by defined as the power generation per surface area of electrode or per volume of cell. Comparing with the microbial and enzymatic biofuel cells, the power density of former usually is magnitudes lower than the latter [121-124]. Accordingly, enzymatic fuel cells could be designed to supply power for compact devices.
23 2.4. Nanotechnology
Nanoscience has grown rapidly in the last decade. Recently, more attentions are focused on the applications of nanotechnologies. The areas of nanotechnology applications include: aerospace [125], national security [126], electronics [127], biology
[128]and medicine [129]. The high potential impacts of nanotechnology almost cover all fields of human activity (environmental, economy, industrial, biomedical, health-related, etc). As we discussed in section 2.1, 2.2 and 2.3, nanostructured materials have been used extensively as carrying materials for enzyme immobilization, biosensoring, and biofuel cells. Although the structure or morphology of the nanostructured materials is different, the performance enhancing is proved variously. This made the whole society expecting a higher yielding of nanotechnology to fulfill the rising needs.
24 CHAPTER III
NANO-COMPOSITE ENZYMATIC ELECTRODES FOR APPLICATIONS IN
BIOSENSOR SYSTEM
3.1 Introduction
Functional biomolecules combining the structural and electronic features of carbon nanotubes (CNT) [130, 131] have manifested great potential for bioelectronic applications in many areas [132]. Enzyme-CNT composites are of special interest for applications in enzymatic biosensors [133]. Various strategies for fabricating enzyme-CNT composites have been developed and reported in the literatures [134-139]. However, a major challenge in biosensor studying is to improve the low enzyme activity and short lifetime, which hampers the practical use of biosensors.
Currently, the development of biosensor involves many research areas, including biology, electrochemistry, nanotechnology and engineering. The most available biosensor is to use enzymes as molecular recognition segments (MREs), which is the core of the biosensor demonstration. Different MREs grouped biosensors into sub-families.
Enzymatic biosensor is one of the hottest and most attractive fundamental studies to researchers engaged in biosensoring community. In this chapter, three various enzyme immobilization methods were reported, and GOx-CNT composites were fabricated
25 accordingly. Glucose oxidase (GOx) was chosen as the model enzyme in this methodology study, because of its excellent performance and bright commercial prospective in biosensor applications. GOx-CNT composite electrodes were explored in biosensors in detecting the glucose concentration. To evaluate the electrode performance in various conditions, the thermal stabilities of the fabricated GOx-CNT biosensor electrodes were monitored at room temperature and 50oC. These highly stable and active enzyme-coated CNT composites significantly improved the performance of biosensors.
Through the comparison of those fabrication approaches, we highlighted a novel powerful strategy to strengthen the stabilization of enzyme activity, Cross-linked Enzyme Aggregate
(CLEA). CLEA, which the stable enzyme coatings, were prepared by salting-out enzyme molecules in the presence of CNT, followed by chemical cross-linking between enzyme molecules and the CNT surface. The enzyme-coated CNT manipulated by this method also appeared to be highly active due to the increasing of enzyme loading in a form of multilayer coatings on the high-surface area CNT.
3.2 Materials and Methods
Glucose oxidase (GOx, EC 1.1.3.4 from Aspergillus niger), D-(+)-Glucose, glutaraldehyde (GA), 1,4-benzoquinone (BQ), 1-1' dimethyl ferrocene, ammonium sulfate, o-dianisidine, 5% Nafion® solution, and horseradish peroxidase (HRP) were purchased from Sigma (St Louis, MO, USA). Carbon nanotubes (CNT, multi-walled, 30 ± 15 nm in outer diameter and 1~5 µm in length, specific surface area: 200~400 m2/g, purity > 95%) were supplied from Nanolab Inc. (Newton, MA, USA).
N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and MES buffer
26 were purchased from Pierce (Rockford, IL, USA), while N-Hydroxysulfosuccinimide
(NHS) was from Alfa Aesar (Ward Hill, MA, USA). Toray® papers from Fuelcellstore
(Boulder CO, USA), and carbon felts from Alfa Aesar (Ward Hill, MA, USA) were used for electrode support materials. All other reagents except specified were ACS grade regent.
3.2.1 Activation of CNT
CNT were treated with acids prior to use. In a typical preparation, 20 mg of CNT powder was added to acid solution consisting of H2SO4 (98%, 150 ml) and HNO3 (70%, 50 ml), followed by overnight incubation at room temperature under shaking condition (200 rpm). The resulting CNT were washed with distilled water and dried at 80 °C in a vacuum oven. For surface functionalization, the acid-treated CNT (20 mg) were first suspended in distilled water (10 ml) and then mixed with 4 ml of MES buffer (pH 6.5, 500 mM), 4 ml of
NHS aqueous solution (434 mM), and 2 ml of EDC aqueous solution (53.2 mM). After vigorous stirring at room temperature for one hour, the suspension was centrifuged and washed with 100 mM MES buffer (pH 6.5) for the further enzyme immobilization.
3.2.2 Preparation of GOx-CNT composites
After the pre-treatment for CNT, three strategies of glucose oxidase (GOx) immobilization were applied for the preparation of GOx-CNT composites. The detail protocols were described as follows.
27 (1) Covalent attachment (CA). The covalent attachment of glucose oxidase (GOx) to the CNT surface was achieved by mixing the 2 ml suspension of functionalized CNT (1 mg/ml) with 1 ml of GOx (10 mg/ml), which was then allowed to react for one hour under shaking condition (200 rpm) at room temperature. The CA-GOx sample was prepared by adding 2 ml of sodium phosphate buffer (pH 7.0, 100 mM), followed by incubation at 4 °C overnight. The illustration of this procedure is shown in Figure 3-2(a).
(2) Enzyme Coating (EC). After the GOx immobilized with CNT following the method of covalent attachment as described above, 0.5 wt% glutaraldehyde (GA) was added for cross-linking. After 30 min shaking at 200 rpm in room temperature, the sample was incubated at 4 °C for overnight. The EC-GOx samples were thus obtained. The illustration of this procedure is shown in Figure 3-2(b).
(3) Cross-linked Enzyme Aggregate (CLEA). For the preparation of CLEA-GOx,
2 ml of ammonium sulfate solution (550 mg/ml) was mixed with the suspension solution
(30 min, 200 rpm shaking) to precipitate GOx in the vicinity of CNT. Then, GA (0.5 wt%) was used to crosslink GOx molecules in the enzyme precipitates entangled with CNT.
After the addition of GA, the suspension was first shaken (200 rpm) at room temperature for 30 min, and then incubated at 4°C overnight. The illustration of this procedure is shown in Figure 3-2(c).
Finally, all of immobilized GOx-CNT were treated with 100 mM Tris buffer (pH
7.4) to cap the unreacted aldehyde groups, and washed with 100 mM sodium phosphate buffer (pH 7.0) until no enzyme were detected in the washing solution. All the GOx-CNT was re-dispersed into 100 mM sodium phosphate buffer (pH 7.0) at a CNT concentration of
2 mg/ml for the further use.
28 3.2.3 Protein content measurement of GOx-CNT composites
Enzyme loadings on CNT were determined by using BCA Protein Assay Kit from
Pierce (Rockford, IL). The detail procedure is described as below: A working reagent was mixed by 50 parts of reagent A and 1 part of reagent B. For a typical measurement, 0.05 ml of sample and 1 ml of working reagent was mixed well and incubate at 60oC for 30 minutes which followed by cooling down back to room temperature. After cooling, the absorbance at 562 nm was measured. The standard curve was measured by using Bovine Serum
Albumin (BSA) as protein standard.
3.2.4 Activity and stability of GOx-CNT composites
The GOx activity on CNT composites was determined using a modified protocol obtained from precious literature [140]. In air-saturated solution, the GOx-catalyzed glucose oxidation produced hydrogen peroxide, which reacted with o-dianisidine in the presence of peroxidase. Activity measurements were performed by using UV-Vis spectrophotometer (Shimadzu, Japan. One millimeter of working reagent containing
0.17mM o-dianisidine and 1.72 wt% D-(+)-glucose was added in 1.5 ml disposable cuvette.
Ten milliliters of 0.2 mg/ml HRP and 50 µl of 5 µg/ml GOx were mixed with working solution in the cuvette just before the measurements were initiated. The absorbance increasing was measured and recorded for one minute at a wavelength of 500nm.
To examine the stability of immobilized enzymes, each sample (0.05 mg/ml) was incubated at room temperature. At each time point, a small aliquot was taken for the measurement of residual activity. The relative activity was calculated from the ratio of
29 residual activity to the initial activity of each sample, and used for an easy comparison of the stability results.
3.2.5 Preparation of enzyme electrodes
To demonstrate the utility of GOx-CNT as a highly stable and active enzyme system for bioelectrochemical applications, the GOx-CNT based electrodes were fabricated and used in glucose sensing tests. The enzyme electrodes were prepared by entrapping CA-GOx, EC-GOx and CLEA-GOx into a Nafion® membrane, which is known to have a good proton permeation selectivity and biocompatibility.
The typical preparation procedure was described as follows. Electrodes for glucose sensing were fabricated on glassy carbon electrodes (GCE, 3 mm diameter, CH
Instruments Inc., Austin, TX, USA). A final concentration of 2 mg/ml of GOx-CNT was dispersed in 100 mM sodium phosphate buffer (pH 7.0) containing 0.5 wt% Nafion®. The suspension (20 µl) was cast on the polished electrode surface, and allowed to dry under ambient condition for two hours.
Figure 3.1 demonstrated the configuration of glucose biosensor examined in this study. A 20 ml glass vial was used to construct a three-compartment electrochemical cell.
Three electrodes were included in this biosensor setup: a glassy carbon electrode (GCE) coated with GOx-CNT composite was used as working electrode, a platinum wire electrode worked as counter electrode, and an Ag/AgCl electrode was employed as reference electrode.
30 Pt wire Electrode Ag/AgCl Reference Electrode GOx-CNTs Composite Electrode
Figure 3.1 Enzymatic glucose biosensor setup. Volume of biosensor chamber is 20 ml, usually working solution contains 15 ml of 100 mM phosphate buffer (pH 7.0).
3.2.6 Entrapment of mediator on electrodes
To better improve the electron transfer in biosensoring reaction, 1-1' dimethyl ferrocene was entrapped on working electrodes as mediator. The preparation of the mediator-entrapped electrodes was performed as described in the following revised instruction [58]. First, the GCE was polished with 0.5 and 0.1 µm α-Al2O3 powder, followed by sonicating in DI water for 5 min, washing with water, and ethanol, and finally drying. After fully dried, ten micro-liters of ethanol solution, which contains 0.05 M 1-1' dimethyl ferrocene was dipped onto the GCE surface, and dried in air at room temperature.
The thickness of the mediator film was estimated to be 15-40 µm. After the entrapment of ferrocene on the GCE, the various GOx-CNT composites were applied to the electrode, and dried at room temperature for further use.
31 3.2.7 Electrochemical measurements
Platinum wire electrode and Ag/AgCl reference electrode were purchased from CH instruments (Austin, TX). The composite-coated GCE, Ag/AgCl electrode and platinum wire were used as the working, reference and counter electrodes, respectively. 100 mM phosphate buffer was added into cell as the electrolyte. For the amperometric study, the working electrode was posed at a specific potential vs. Ag/AgCl reference electrode and the current response to the glucose concentration change was measured with gently stir.
Small aliquots of glucose stock solution (2 M, in 100 mM pH 7.0 phosphate buffer) were added typically at a time interval of 60s. The thermal stability of each electrode was assessed by performing the amperometric measurement after the incubation of electrode in
100 mM phosphate buffer (pH 7.0) at 50°C.
3.3 Results and Discussions
The GOx immobilization methods, their activities, stabilities, and application in biosensors are discussed in the following sections.
3.3.1 Immobilization of glucose oxidase (GOx)
To construct highly stable and active enzyme composites for bioelectrochemical applications, we demonstrate the concepts of enzyme immobilizations on CNT by using glucose oxidase (GOx) as a model enzyme since it is well characterized and frequently used in glucose biosensors manufacturing [61, 141]. Three immobilization methods were studied in this thesis, and the performances of the resulting GOx-CNT composites also were examined and characterized. Three immobilization methods were named as:
32 Covalent Attachment (CA), Enzyme Coating (EC) and Cross-Linked Enzyme Aggregate
(CLEA) separately.
Figure 3-2(a) schematically shows the preparation of covalent attachment of GOx
(CA-GOx) on CNT. Firstly, CNT were pretreated with mixed acid to introduce the carboxylic groups and enhance the dispersion of CNT in buffer solution. GOx was covalently attached to the surface of the acid-treated CNT by using
N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and
N-Hydroxysulfosuccinimide (NHS) as linkers [138]. In the scheme of Figure 3-2(a), after immobilization, the enzymes will be aligned one by one on the surface of CNT. From the
SEM image of CA-GOx, we observed that GOx was shown on the surface of the CNT only on limited spots. Since there were sufficient GOx in the solution and the incubation time was long enough to react the GOx surface groups with hydroxyl groups on CNT surface, the reason of this observation could be addressed in that the hydroxyl groups on CNT surface was limited for enzyme attachment.
33 H2SO4/ HNO3
EDC/ 200 nm NHS
CA Glucose E E E E oxidase
200 nm
CA: Covalent Attachment
Figure 3.2. Scheme of Immobilization of GOx on CNT. (a). Covalent Attachment of GOx on CNT. The scale bar at each SEM image denotes 200 nm. (The illustration and SEM were provided by BC Kim, done in PNNL).
34 H2SO4/ HNO3
200 nm EDC/ NHS
Glucose E E E E oxidase
E E E E GA EC200 nm
EC: Enzyme coating
Figure 3-2 Scheme of Immobilization of GOx on CNT. (Continued) (b). Scheme of
Enzyme Coating (EC) of GOx on CNT. (The illustration and SEM were provided by BC
Kim, done in PNNL).
Figure 3.2(b) schematically demonstrated the enzyme coating (EC) of GOx on
CNT. The difference between CA and EC approaches is the applying of glutaraldehyde
(GA) for cross-linking the functional groups on enzyme surface. Briefly, the EC-GOx was fabricated after the covalent attachment of GOx on CNT. GA was used as cross-linker to
35 form GOx networks and enforce the GOx-CNT construction. The illustration indicates that the enzyme loading of EC-GOx and CA-GOx should be the same magnitude, because of the limiting of hydroxyl group availability on CNT surface. Moreover, according to the
EC concept, we could easily predict that the GOx-CNT structure will be enforced by cross-linking the disulfide bonds on the enzyme surface. This indicates the strength between the enzymes would be enhanced due to the existence of more covalent bonds.
The SEM morphology of CA-GOx and EC-GOx (image not shown) did not shown huge differences in enzyme loading, which proved the assumption that the enzyme loadings of CA-GOx and EC-GOx are at the same level. This prediction also was confirmed by the enzyme loading data in Table 3-1. From Table 3-1, the enzyme loading data gave a 0.04 mg/mg-CNT in CA-GOx immobilization and 0.05 mg/mg-CNT in
EC-GOx immobilization.
The comparison of Table 3-1 and Figure 3.3 proved that the cross-linking of enzyme surface groups has positive contribute for maintaining the enzyme activity and stability. Although CA-GOx and EC-GOx have same magnitude of enzyme loading, the
EC-GOx is much stable than CA-GOx (Figure 3.3). Furthermore, the cross-linking exposing more enzyme active sites in the complex, this gave a 20% higher specific activity.
Even though the investigation of CA and EC approaches pointed out that the availability hydroxyl group on CNT surface is a limitation to high loading enzyme immobilization, CLEA-GOx approach was developed to improve the enzyme loading on
CNT. By using the GOx in CA-GOx composite as a base molecule, and providing the cross-linked enzyme aggregates in the vicinity of these base molecules, large amount of
36 enzyme was immobilized on the surface of CNT. The enzyme loading and activity are further enhanced.
H2SO4/ HNO3
EDC/ 200 nm NHS
CA Glucose oxidase
E E E E
200 nm Precipitated Glucose oxidase (enzyme aggregation) GA
200 nm CLEA
CLEA: Cross-linked Enzyme Aggregate
Figure 3-2 Scheme of Immobilization of GOx on CNT. (Continued) (c). Scheme of
Cross-linked Enzyme Aggregate of GOx on CNT. The scale bar at each SEM image denotes 200 nm. (The illustration and SEM were provided by BC Kim, done in PNNL)
37 Figure 3.2(c) illustrated the CLEA-GOx approach of immobilizing GOx on CNT.
CA-GOx composite was incubated in a highly concentrated GOx solution. Then, ammonium sulfate was added to salt-out GOx in the vicinity of CA-GOx complex. Finally, the enzyme cross-linking via GA treatment resulted in large aggregates of enzyme coatings on the surface of CNT. As shown in the SEM image of Figure 3.2(c), more enzymes could be coupled on the surface of the CNT, moreover, the higher stability of enzymes was confirmed in Figure 3.3.
3.3.2 Enzyme loadings and specific activities of GOx-CNT composites
Based on the above discussions, Figures 3.2(a), (b) and (c) schematically illustrate the preparation of three different approaches in the GOx immobilization on CNT:
Covalent attachment (CA-GOx, Figure 3.2(a)), enzyme coatings (EC-GOx, Figure 3.2(b)) and cross-linked enzyme aggregate (CLEA-GOx, Figure 3.2(c)). The SEM images of acid-treated CNT, CA-GOx and CLEA-GOx are presented. Under the SEM observation,
CA-GOx did not show much difference in the surface morphology, compared to acid-treated CNT without enzyme (Fig. 3.2(a)). Since the covalent attachment process cannot develop more than monolayer coverage of enzyme, GOx molecules (5.2×6.0×7.7 nm) [142] in the CA-GOx sample cannot be visualized under the present resolution of
SEM analysis. On the other hand, after salting-out and cross-linking, large aggregates of
CLEA-GOx were observed on the surface of CNT (Fig. 3.2(c)). This suggests that the enzyme loading of CLEA-GOx would be much higher than that of CA-GOx, mostly due to the formation of multilayer enzyme coatings on CNT.
38 Form Table 3-1, we confirmed that the enzyme loading of CLEA-GOx, which is
0.3 mg/mg-CNT, is much higher than the enzyme loadings in CA-GOx and EC-GOx composite (0.04 and 0.05 mg/mg-CNT). CLEA-GOx approach gave around 7-time higher enzyme loading and specific activity than CA-GOx method. In term of apparent enzyme activity, the CLEA-GOx immobilization method provides over 50 times higher catalytic activity. This could be addressed that the micro-environment of GOx become more favorable to enzyme activity, and as a result of higher enzyme loading, majority enzyme molecules are surrounding by other enzyme molecules rather than facing to the severe environment.
Table 3-1 Enzyme loading and activity of GOx-CNT composites
Enzyme Loading of Composites Apparent Enzyme Activity
(mg/mg CNT) (U/mg CNT)
CA-GOx 0.04 6.6
EC-GOx 0.05 9.9
CLEA-GOx 0.3 338
The apparent activity could determine the practical performance in electrochemical applications. As shown in Table 3-1, the apparent activities of CA-GOx, EC-GOx and
CLEA-GOx were 6.6, 9.9 and 338 Unit per milligram of CNT, respectively. This increase in the apparent activity of CLEA-GOx by about 50 times is due to the formation of multilayer enzyme aggregates. The EC-GOx sample was prepared by the GA treatment of
39 the enzyme solution containing CA-GOx/CNT without the salting-out process. The apparent activity of the EC-GOx was 9.9 Unit per mg of CNT, which is much lower than that of CLEA-GOx and rather close to that of CA-GOx. This suggests that salting-out is a critical process for the development of highly-active enzyme coatings on CNT.
1.2
1.0
0.8
0.6
0.4 CA
Relative Activity Relative EC 0.2 CLEA
0.0 0 20406080100 Time (days)
Figure 3.3 The stability of three immobilizations at room temperature. The relative activity is defined as the ratio of residual activity at each time point to the initial activity of each sample. Solution is 100 mM sodium phosphate buffer, pH 7.0. The activities of CA-GOx,
EC-GOx and CLEA-GOx samples were measured by a conventional GOx assay [140], respectively, and converted to the apparent activity (the enzyme activity per unit weight of
CNT). The stability measurement was done by HK Ahn in PNNL.
40 The stabilities of CA-GOx, EC-GOx and CLEA-GOx were determined by measuring the residual GOx activity. The activities were recorded to be a function of storage time at room temperature. The relative activity is defined as the ratio between the initial enzyme activity and residual activity. CA-GOx showed a continuous deactivation while CLEA-GOx exhibited no activity loss more than 80 days (Fig. 3.3). These results indicate that multi-point covalent cross-linking of enzyme molecules effectively protects the enzyme molecules in the coatings from structural denaturation [143-146]. Stability data also convince us that the enforcement effects of cross-linking reagent, and the micro-environment adjusting are the key factors to maintain the enzyme activity.
400 CLEA
300 A) µ
200 Current ( Current 100
0 0 200 400 600 800 1000 1200
Time (sec)
Figure 3.4 Representative amperommetry curves of GOx-CNT biosensor response.
CLEA-GOx composite sample was coated on the glassy carbon electrode, and the result electrode was used as working electrode in biosensor system.
41 3.3.3 Performance of GOx-CNT biosensors
Figure 4-3 shows a typical current response to analyte concentration figure in biosensor operation, in which glucose concentration is increased gradually by adding aliquots of concentrate glucose solution into biosensor system.
3.3.4 Effect of free mediator on GOx-CNT biosensors
To overcome the biocatalysts-electrode electron conduction resistance, the common strategy is to use mediators shuttling the electrons. In this research, benzoquinone (BQ) was used as mediator in this enzymatic glucose biosensor system to ensure the efficiency of electron transfer.
Typically, BQ was added in the 100 mM phosphate buffer solution (pH 7.0) to study the influence of mediator on the performance of various GOx-CNT composite electrodes in biosensors. To study the BQ effects on current response to glucose concentration, in the experiments, two different concentrations of BQ (5 µM and 5 mM) were executed in three kinds of biosensor systems.
42 25 CA 20 EC CLEA A)
µ 15
10 Current ( Current
5
0 0 5 10 15 20 Glucose Conc (mM)
Figure 3.5 GOx-CNT composite electrode biosensor performances with vary BQ concentration. (a) GOx-CNT composite electrodes performances in biosensor with a BQ concentration equals to 5 µM. Working potential is 500 mV, and the working solution is
100 mM phosphate buffer (pH 7.0).
As shown in Figure 3-5(a), when the mediator concentration equals to 5 µM, comparing the performances of CA-GOx, EC-GOx and CLEA-GOx composite electrodes, the CLEA-GOx composite electrode gave the highest current response at each specific glucose concentration. Furthermore, the current response of CLEA-GOx composite electrode is almost 10 times higher than that of CA-GOx and EC-GOx electrodes. In
43 addition, the current responses of CA-, EC- and CLEA-GOx composite electrodes are almost negligible without existence of BQ (Data not shown here).
350 CA 300 EC 250 CLEA A) µ 200
150
Current ( 100
50
0 0 50 100 150 Glucose Conc.(mM)
Figure 3.5 GOx-CNT composite electrode biosensor performances with vary BQ concentration (Continued). (b) Different GOx-CNT composite electrode performances in biosensor with BQ concentration equals to 5 mM. Working potential is 600 mV, and the working solution is 100 mM phosphate buffer (pH 7.0).
While Figure 3.5(b) shows the performances of three enzyme electrodes in biosensor system at a higher BQ concentration (5 mM), we observed similar phenomenon as shown in Figure 3.5(a). That is, at the same potential, the biosensor with CLEA-GOx
44 composite electrode provides a higher current response at each specific glucose concentration in the present of free mediator. Moreover, the current response gap increases with the glucose concentration. Furthermore, the current response of CLEA-GOx composite electrode is as 37 times as higher than the other two immobilization biosensors
(Figure 3.5(b)).
It was also seen from Figures 3.5(a) and 3.5(b) that the current response increased with the use of higher BQ concentration. At glucose concentration equals to 10 mM, the current responses of three enzyme glucose biosensor are shown in Table 3-2. Comparing the current responses of CA-GOx to 10 mM glucose in two different BQ concentrations, it is revealed that the current increases only 3% with rising BQ concentration. Meanwhile, the current responding of EC-GOx and CLEA-GOx have been improved both over 200%.
This indicates that the reaction velocities catalyzed by EC-GOx and CLEA-GOx are much higher than the reaction velocity catalyzed by CA-GOx. Even there is a large amount of mediator molecules in biosensor; CA-GOx can not catalyze more electrons for transferring.
Table 3-2 Comparison of current response at [glucose] = 10 mM
CA-GOx EC-GOx CLEA-GOx
[BQ] = 5 µM 1.85 1.63 19.3
[BQ] = 5 mM 1.91 4.29 44.63
Traditionally, dynamic range and sensitivity are the parameters for biosensor evaluation. In this study, the CLEA-GOx composite biosensors could have a higher
45 dynamic range at 5 mM of BQ concentration. That is, the dynamic range of CLEA-GOx biosensor is up to 50 mM, and EC-GOx and CA-GOx biosensor are 25 mM and 20 mM separately. Usually, the sensitivity of biosensor is defined as the current response to the change of unit glucose concentration per unit area of electrode. That is to say, the higher current response means the higher sensitivity of detection for the specific electrode. The sensitivity CLEA-GOx electrode was measured to be 13.3 mA/M·cm2, which is 25 times higher than the sensitivity of CA-GOx electrode (0.52 mA/M·cm2). This sensitivity of the is similar to that of multilayer assembled biosensor [147]. Therefore, the above data in
Figure 3.5(a) and 3.5(b) could demonstrate the use of mediator could improve the efficiency of electron transfer, and with subsequent could absolutely increase the sensitivity of glucose biosensor. Moreover, the detection limits of the CA-GOx and
CLEA-GOx electrodes were observed to be 0.07 mM and 0.03 mM, respectively, based on the signal-to-noise ratio of 3.
In this biosensor study, the enzyme was immobilized on electrode, and mediator was dissolved in the solution. We could define the surface of electrode was covered by
2 2 enzyme at a concentration of ΓGOx (mol/cm ) with surface area A (cm ). In the reaction layer of electrode, the reaction scheme is shown as Figure 3.6:
46
Figure 3.6 Biosensor Reaction Scheme in Reaction Layer
The results of biosensor operation are shown in Table 3-3.
Table 3-3 Comparison of biosensor sensitivities with vary [BQ]
Sensitivity [BQ] (mA/M·cm2) 0.5 5 µM CA-GOx 0.5 5 mM 0.5 5 µM EC-GOx 1.1 5 mM 5.8 5 µM CLEA-GOx 13.3 5 mM
Sensitivity is a reflection of current, when substrate concentration, working potential and electrode area are fixed. The limiting steady-state current (ip) at a given potential is dependent on the concentrations of substrate and mediator. As shown in
Equation (3-1) [148].
47 1 1 1 1 K = ( + + M ) (3-1) i p 2FAΓGOx k3[M ] k2 k2[S]
In which,
ΓGOx A is enzyme loading on electrode;
F: Faraday constant;
k3 and k2 are reaction equilibrium constants;
[M] is mediator concentration in the solution;
[S] is substrate concentration in the solution, i.e., glucose concentration.
1 In Equation (3-1), term of represent the rate of enzyme-mediator reaction, k3[M ]
1 which becomes a limiting factor at a low mediator concentration. Term of gives the k2
K breakdown rate of enzyme-substrate complex. The term of M implies that the system k2[S] becoming rate-limiting at low substrate concentration ([S] < The following results could be concluded: (1) Since the enzyme loading on electrode: ΓGOx A (CLEA) > ΓGOx A (EC) > ΓGOx A (CA), we could predict that ip (CLEA) > ip (EC) > ip (CA) by using equation (1). The results in Table 1 showed this trend at a benzoquinone concentration of 5 mM. 48 (2) In the case of CA electrode, since the enzyme loading are so limited (1.6 K mg/electrode), the system is rate-limiting. This makes M a dominant term in k2[S] equation (1). In Table 1, even [BQ] is increased from 5 µM to 5 mM, the sensitivity does not increase. (3) The enzyme loadings on CA and EC electrodes are very similar, which is 1.6 mg/electrode and 2.0 mg/electrode separately. This makes their sensitivities at lower benzoquinone concentration (5 µM) similar to each other. 3.3.5 Effect of immobilized mediator on GOx-CNT biosensors Free mediators usually cause so many problems in the clinic applications of biosensors because of their toxicity and easy to leach to the vacancy of the biosensor. Moreover, the bigger challenge in using free mediators, which are usually expensive, is how to maintain them in the system when the continuous feeding is required. So, the immobilized mediator had been introduced in the applications of biosensors to solve the above concerns. Therefore, biosensor performance with entrapped 1-1' dimethyl ferrocene was examined in our study. 49 4.5 M-CLEA 4 M-EC 3.5 M-CA 3 A) µ 2.5 2 1.5 Current ( 1 0.5 0 0.0 0.5 1.0 1.5 2.0 [Glucose] (mM) Figure 3.7 Immobilization processes effects on the mediator entrapment biosensor system. Working potential is 600 mV, and the working solution is 100 mM phosphate buffer (pH 7.0). The mediator entrapped is 1-1' dimethyl ferrocene. With the use of entrapped mediator, the performances of GOx-CNT prepared by all three enzyme immobilization processes were investigated, respectively, as shown in Figure 3.7. In Figure 3.7, the similar results were obtained with those of using free mediator (BQ) in the biosensor systems (Figures 3.4(a) and (b)). CLEA-GOx composite electrode shows the highest current response at each glucose concentration. When glucose concentration is 1.25 mM, the current response of CLEA-GOx electrode is 2.5 times higher than CA-GOx electrode and 1.85 times higher than EC-GOx electrode. It indicates that CLEA 50 immobilization method is significant advantageous over the two other immobilization methods. However, we also observed the dynamic ranges of these biosensors shrink to 1%, comparing to the free mediator biosensor. The dropping might be contributed that the lower mediator amount during the entrapment caused the lower efficiency of electron transfer rate. 3.3.6 Thermal stabilities of GOx-CNT biosensors Considering the enzyme loading and activity result of CLEA-GOx over EC-GOx and CA-GOx, CLEA-GOx composite electrode and CA-GOx composite electrode are selected as the model composite electrodes for thermal stability study. As stated above, the sensitivities of the CA-GOx and CLEA-GOx electrodes were 0.52×10-3 A/ (M·cm2) and 13.3×10-3 A/ (M·cm2), respectively. The detection limits of the CA-GOx and CLEA-GOx electrodes were about 0.07 mM and 0.03 mM, respectively, based on the signal-to-noise ratio of 3. Since the preliminary study of the composites stability shows that CLEA-GOx has magnificent advantage over CA-GOx after 20 days. In order to study the stability of enzymatic biosensors in a short period of time, the thermal stability was studied by incubating two model electrodes at 50oC and measuring the current responses. The stability of each electrode was tested by checking the performance after the thermal treatment at 50°C. The sensitivity of the CA-GOx electrode was reduced by more than 80% after four-hour thermal treatment, while the CLEA-GOx electrode showed a negligible decrease in its sensitivity after the thermal treatment in the same condition 51 (Figure 3-8). The enhanced apparent activity of CLEA-GOx significantly improves the performance of biosensors. 8 CA t=0h 7 CA t=1h CA t=2h CA t=3h 6 CA t=4h CLEA t=0h 5 CLEA t=1h A) µ CLEA t=2h I ( 4 CLEA t=3h ∆ CLEA t=4h 3 2 1 0 12345678910 [Glucose] (mM) Figure 3.8. Thermal stability of GOx-CNT electrodes for biosensors. The current responding of CA- and CLEA-GOx electrode biosensors to the successive additions of glucose aliquots. The ∆I value is the current difference between the response at each glucose concentration and the background signal when the glucose concentration equals zero. The current responses were also checked after thermal treatment at 50 °C for the specified time span. 52 CHAPTER IV NANO-COMPOSITE ENZYMATIC ELECTRODES FOR BIOFUEL CELLS APPLICATIONS 4.1 Introduction Because of the rapid energy demanding in form of electricity, the development of the efficient electricity generation techniques was highly preferred. Fuel cells were considered to be the most promising ways to convert chemical fuels to electricity. Fuel cells could directly convert hydrogen or alcohols, into electricity, and the byproducts of these cells usually are water and carbon dioxide, which is biocompatible. However, fuel cells which use noble metals as catalysts, such as platinum, generally need to be operated at high temperature, except proton exchange membrane fuel cell, as listed in Table 4-1 [149]. Moreover, the operation conditions of some fuel cells also are in extreme pH values, such as phosphoric acid fuel cell and alkali fuel cell, which raised the concerns on the environmental problems. With the advent of more and more concerns on the environment impacts, green approaches for electricity generation have attracted an extensive interest in both research and industry. Therefore, biofuel cells were introduced. Biofuel cell use biomass as fuels to generate electricity in mild conditions, in which biocatalysts, enzymes or a whole organism, 53 were used to replace the conventional inorganic catalyst. Other than the benefit of working in ambient conditions, comparing with the traditional fuel cells, biofuel cell has a number of advantages. For example, biofuel cells, from catalysts, fuel to byproducts, are biocompatible and renewable, and are even more environmentally beneficial. The fuel options for biofuel cells are diversified, which could be hydrogen, carbon hydrates, organic acids and urea [33]. In addition, the operation conditions needed in biofuel cells are friendly and easy to control. Up to now, the most of biofuel cell studies is focused on using enzyme as the biocatalyst. For instance, the technique of the oxygen-glucose cell had been widely applied in an electrochemical process to convert chemical energy to electricity, when glucose is oxidized at the anode and molecular oxygen is reduced at the cathode. Table 4-1. Usual operation temperatures of some noble catalyst fuel cells Fuel Cell Type Working Temperature (oC) Phosphoric Acid Fuel Cell ~200 Alkali Fuel Cell ~250 Molten Carbonate Fuel Cell ~650 - 1200 Solid Oxide Fuel Cell ~900 Polymer Electrolyte Membrane ~80 Although the concept of enzymatic biofuel cells have been introduced for nearly half a century, they have not been utilized in practical use due to the insufficient power 54 outputs. The main reason for low energy production might be attributed that the enzymes using in biofuel cells are very sensitive to the changes of pH and temperature. Even little variations from the proper environment will lead to inactivation of the enzyme, which results in the reduction of the catalysis efficiency. In this study, to solve the current drawbacks of enzymatic biofuel cells, we explored some novel strategies to construct GOx-CNT composite electrodes and examined their applications in biofuel cells. Carbon nanotubes (CNT), for its property of higher specific surface area, were expected to provide high-quality immobilization supports of glucose oxidase. In our self-made biofuel cells, BQ was used as a redox mediator to increase the electron transfer rate. The kinetics of the redox chain reactions were studied using native glucose oxidase, GOx-CNT, and GOx-CNT coated electrode respectively. The different support materials: Toray® carbon paper, carbon cloth and carbon felt also were investigated in biofuel cells, separately. To estimate the potential applications in the actual working conditions, the thermal stability and operation stability of GOx-CNT electrodes in biofuel cells were measured in this study, individually. Finally, two different sizes of miniature fuel cells were used to test the maximum power density in our experiments, respectively, and compare with our self-made biofuel cells. 4.2 Materials and Methods The materials and methods involved in biofuel cells study are described in the following sections. 55 4.2.1 Materials Glucose oxidase (GOx, Type X-S, from Aspergillus niger, 157,500 units/g), Peroxidase (HRP, Type VI, from horseradish, 298 Purpurogallin units/g), β-D (+)-glucose and o-dianisidine were obtained from Sigma (St. Louis, MO). 1,4-Benzoquione (BQ, 98%) was supplied by Aldrich (Milwaukee, WI). Customized membrane cathodes were purchased from FuelCellStore.Com, Inc. (Boulder, CO). Graphite rod (1/16" × 6") was ordered from Poco Graphite, Inc. (Decatur, TX). Unless specially mentioned, all other reagents used in the experiments are ACS grade. UV-1601 UV-Visible spectrophotometer (Shimadzu, Japan) was used in glucose oxidase activity assay and kinetic study, and DLK-60 Electrochemical analyzer (Analytical Instrument Systems Inc., Flemington, NJ) was employed in electrochemical measurements. 4.2.2 Preparation of GOx-CNT composites GOx-CNT composites were synthesized following various immobilization methods, Covalent Attachment (CA), Enzyme Coating (EC) and Cross-linked Enzyme Aggregate (CLEA), respectively, which were same as described in Chapter III. All the GOx-CNT was re-dispersed into 100 mM sodium phosphate buffer (pH 7.0) at a CNT concentration of 0.5 mg/ml for the further use and analysis. 4.2.3 Measurement of GOx-CNT activity Glucose oxidase activity assay was done as stated in a revised protocol based on previous reference [140]. Briefly, a working solution containing 6 ml of o-dianisidine 56 (0.42 mM), 6 ml of Sodium Acetate Buffer (50 mM, pH 5.1) and 2.5 ml of D-(+)-glucose (10 wt %) were mixed before the measurement. Also, HRP (0.2 mg/ml) was prepared freshly using DI water. In each measurement, 2.9 ml of working solution and 0.1 ml of HRP was mixed well in a 3 ml cuvette. 0.1 ml of 0.005 mg-CNT/ml was added for sample and 0.1 ml of acetate buffer was added instead for the blank measurement. After well mixing, absorbance increase at 500 nm was recorded for 5 min, and the maximum linear rate for both the sample test and blank were recorded as mABS/min. The specific activities of the resulting electrodes towards glucose oxidation were measured by UV-visible spectrophotometry using oxygen and BQ as the electron acceptors, respectively. When oxygen was used as the oxidant, the method was based on testing the generation of hydrogen peroxide during the reaction. The electrode with immobilized enzyme was then incubated in 15 ml of working solution in a 20 ml glass vial, and the concentration of oxidation product of o-dianisidine was monitored at a wavelength of 460 nm for 30 min. When BQ was used as the electron acceptor, same protocol was followed except that the working solution was replaced with the same buffer supplemented with 200 mM glucose and 5 mM BQ. The formation of HQ was monitored at 290 nm. The oxidation reactions using O2 and BQ could be described as below equation 4-1 and 4-2, respectively. GOx Glu cose + O2 ⎯⎯→⎯ Gluconolactone + H 2O2 (4 −1) Glu cose + BQ ⎯GOx⎯→⎯ Gluconlactone + HQ (4 − 2) In which, Gluconolactone is the oxidation product, and HQ is the redox form of BQ. 57 4.2.4 Set-up of biofuel cells To prepare the electrodes for use in biofuel cells, Toray® paper (280nm or 370 nm in thickness; Fuelcellstore, Boulder, CO, USA), carbon cloth and carbon felt were used as electrode supporting materials, respectively. Typically, a casting suspension was firstly prepared by dispersing CA-GOx, EC-GOx and CLEA-GOx (3 mg/ml) in the buffer solution containing 0.5 % (v/v) Nafion®. A piece of backing material (geometric surface area = 0.33 cm2) was immersed into the suspension for 10 min and dried in ambient conditions for overnight. All the resulting electrodes were washed and stored in the phosphate buffer at room temperature until use. Figure 4.1(a) illustrates an example of carbon felt GOx-CNT composite electrode was used in the model of glucose/O2 biofuel cells as the anode. The electrode was pressed against the membrane with a graphite rod. A customized gas diffusion membrane-cathode consists of a gas diffusion electrode (Pt loading: 4.0 mg/cm2) hot-pressed onto Nafion® 117 proton exchange membrane purchase from Fuelcellstore (Boulder, CO). As shown in Figure 4.1(a), the anodic electrolyte, 100 mM phosphate buffer (pH 7.0) contains 200 mM glucose as a fuel, and BQ (10 mM) as the redox mediator. The fuel was circulated by a peristaltic pump (ColeParmer, Vernon Hills, IL) at the designated circulate rate to mild the diffusion effects. Oxygen was supplied to the cathode chamber at a flow rate of 100 ml/min as the oxidant. Voltage and current characteristic curves were obtained using two multimeters (EXTECH Multipro 510), and the data was collected and recoded through R232 connection to a computer. The potentials of the anode and cathode vs. the reference electrode were measured by the DLK-60 Electrochemical analyzer. 58 V Ag/AgCl Reference Electrode A V Enzyme Anode Load Cathode Open to Air Figure 4.1 Biofuel cell setups used in this study. (a). Scheme of self-made biofuel cell. The main body of biofuel cell was fabricated by modifying a 47 mm in-line filter holder (Pall life sciences, MI, USA). The total volume of the system is approximately 20 ml, while the anode chamber occupies 7 ml. The carbon rod and reference electrodes are inserted into the anode chamber, and the Pt-loaded cathode is sandwiched by the mesh of stainless steel. Anode electrode size is 0.33 cm2. Besides the self-made biofuel cells, two miniature fuel cells (Figure 4.1(b), courtesy from Washington State University, 2 cm×2 cm and 1 cm×1 cm) were also utilized to examine the performances of GOx-CNT composite electrodes, respectively. It is noted that the electrodes, as shown in Figure 4.1(b) and Figure 4.1(c), were pressed by using titanium as electron collector separately. 59 Figure 4.1 Biofuel cell setups used in this study (Continued). (b). Photo of miniature biofuel cell (2cm×2cm). Anode electrode size is 8 cm2 (2cm×2cm, and anode chamber on both side of cathode). Courtesy from Washington State University. Figure 4.1 Biofuel cell setups used in this study (Continued). (c). Photo of two different sizes miniature biofuel cells. Courtesy from Washington State University. 60 4.3 Results and Discussions The enzyme immobilization methods (CA, EC and CLEA) were characterized in different biofuel cells setups, and the results are shown in the following sections. 4.3.1 Kinetic study of free enzymatic reactions To better theoretically understand the limiting factors in enzymatic biofuel cells, the kinetic parameters of enzymatic reaction were determined using free GOx in biocatalysis. In this study, the GOx concentration was fixed to be 0.1µg/ml, in order to obtain a linear initial reaction rate. The glucose concentration was selected in the range from 0 to 180 mM (0, 45, 90 and 180 mM), and keep constant during one measurement. To simplify the study, the oxidation reaction by O2 had been blocked by purging reaction system with nitrogen for 20 min. By varying the BQ concentration (0.1, 0.5, 1, 2, 5 mM), the change of absorbance at 290 nm was recorded by UV-Visible spectrophotometer. To obtain the kinetic parameters of the biocatalysis reactions, Michaelis-Menten equation was employed to simulate the experimental data, as shown bellows: The mechanism this two substrates reaction is regarded as Ping-Pong Bi Bi mechanism [150], and as shown in Eq. (4-3). In this mechanism, the enzyme binds with glucose first, followed by the release of gluconolactone, electron and the formation of FADH2. Then, BQ is binding to FADH2 to free the glucose oxidase and form the second product HQ. v [BQ] ' = ' (4 − 3) Vmax K + [BQ] 61 V’max: apparent maximum reaction rate (mM/s); K’: apparent Michaelis-Menten constant (mM) ; v: initial reaction rate (mM/s) at the specific BQ conditions. In addition, V’max and K’ could be mathematically defined as bellows Eq. (4-4) and Eq. (4-5): ' Vmax [G] Vmax = G (4 − 4) αK m + [G] BQ ' K m [G] K = G (4 − 5) αK m + [G] In which [G] is the concentration of glucose [150]. 62 7 0 M 6 0.045M 0.09M 5 0.18M 4 (mM/s) 4 3 V x 10 x V 2 1 0 0246 [BQ] (mM) Figure 4.2. Dependence of initial reaction rate to glucose concentration. Dots: data points; curve: simulation. [GOx] was 0.1 µg/ml, and BQ was used as mediator. A function of the initial reaction rate and BQ concentration was plotted as Figure 4.2. As shown in Figure 4.2, with the increasing of the glucose concentration used, the initial reaction rate subsequently increased. Therefore, under each glucose concentration, BQ the responding V’max and K’ could be obtained according to Eq. (4-3). Therefore, the Km , G -3 Vmax and αKm could be simulated to be 2.96 mM, 1.15×10 mM/s and 92.94 mM, BQ G respectively, where Km and αKm are Michaelis constants, and Vmax is the maximum reaction rate. 63 4.3.2 Kinetic study of immobilized enzymatic reactions In this section of study, EC-GOx immobilization sample was selected to use as the model sample to study the kinetics of immobilized enzyme reactions, because of its higher stable than CA-GOx. Moreover, to determine the initial reaction rate, the enzyme amount in system should be kept in a low scale; EC-GOx was indicated to be ideal sample for this purpose. The EC-GOx concentration was 0.2 µg-GOx/ml, and the glucose concentration was in the range from 0 to 150 mM (0, 10, 25, 45, 90 and 150 mM). By varying the BQ concentration (0, 0.1, 0.5, 1, 2, 5 mM), the change of absorbance at 290 nm was recorded by UV-Visible spectrophotometer. 1.5 9.0 8.0 7.0 1.0 6.0 (mM/s) 4 5.0 4.0 0.5 ' x10 3.0 Km' (mM) max 2.0 V 1.0 0.0 0.0 0.00 0.10 0.20 0.00 0.10 0.20 [glucose] (M) [glucose] (M) Figure 4.3. Dependence of Km’ and Vmax’ to glucose concentration. Dots: data points; curve: simulation. EC-GOx suspension concentration was 0.2 µg-GOx/ml, and BQ was used as mediator. 64 Use the same procedure as discussed in native enzyme kinetic study. At each glucose concentration, Km and V’max were calculated and plotted in Figure 4.3. Coincidently, when EC-GOx sample doubled the enzyme amount of the native enzyme in BQ G section 4.3.1, the Km , Vmax and αKm also were obtained to be similar value as in native enzyme kinetic study. 4.3.3 Kinetic study of immobilized enzymatic electrode reactions EC-GOx immobilization sample was coated on carbon felt electrode, and the reaction kinetics catalyzed by EC-GOx electrode was studied. The EC-GOx amount was 10 µg-GOx/electrode, and the glucose concentration was in the range from 0 to 200 mM (0, 50, 100 and 200 mM). The BQ concentration in this study was kept in a constant value of 5 mM, the change of absorbance at 290 nm was recorded in UV-Visible spectrophotometer. The initial reaction rate was converted from absorbance changing rate with time. The enzyme reaction velocities of native enzyme, EC-GOx and enzyme composite coating electrode, are tabled in Table 4-2. The current calculation is based on the electron generated in the glucose reduction reaction. After GOx being immobilized on CNT, the enzyme amount needs to be doubled, in order to approach the same current generation rate. Whereas, the reaction velocity decreased several magnitudes, after the coating EC-GOx composite on carbon felt electrode. 65 Table 4-2. Comparison of reaction velocities GOx Amount Vmax (mM/s) Current (mA) Free Enzyme Reaction 0.1 µg/ml 1.15x10-3 220 EC-GOx Enzyme Reaction 0.2 µg/ml 1.15x10-3 220 Electrode 10 µg 3.14x10-6 0.6 Compare the reaction velocity data, we found out that the reaction velocity after immobilization decreased to 50%, this could be resulted in the activity lose during immobilization. After the immobilized EC-GOx was coated on electrode, we observed the reaction velocity shrink into only 0.3% of native enzyme velocity. This huge decreasing is partially from the activity losing in process of coating, but the majority cause was regarded to be the mass and electron transfer limitation in the structure of carbon felt electrode, although the carbon felt surface area was reported to be 0.3-0.7 m2/g [151]. Further comparison of the current calculation and current measurement in biofuel cell experiment is based on same enzyme loading. The current measured from biofuel cell experiment is 32.6 µA, which is only 5.4% of the above calculation value (0.6 mA) of EC-GOx coated carbon felt electrode. This indicates that the enzyme catalyst could catalyze much higher reaction rate to generate electrons, if possible, the electron transfer and mass transfer rate are improved for several magnitudes. Therefore, having a much more desired porous electrode material and enhancing the mass transfer and electron transfer rate are the key approaches to improve the performance of biofuel cells. 66 4.3.4 Effect of immobilization methods on biofuel cell performance According to the previous studies of biosensors in Chapter III, we found that the different enzyme immobilization methods could result in current response difference for detecting of glucose. Similarly to Chapter III, the studies in self-made GOx biofuel cells began from the comparisons of the performances in different immobilization methods. According to the conclusion obtained in the literature [152], carbon felt was chosen as the backing materials in the self-made biofuel cells. The voltage-current curves of three immobilization methods are shown in Figure 4.4. As shown in Figure 4.4, the potentials of the biofuel cells with CA-GOx, EC-GOx and CLEA-GOx composites decrease with the decreasing of resistance load. Moreover, the current generated at the same resistance load are quite different from one another when the open circuit potentials of all three composites equal to 600 mV. Typical V-I curve of fuel cell shows that the initial potential drop in low current range is come from the activation losses; the following potential decrease is contributed by ohmic losses; the final potential loss in high current area is because of the mass transportation. Compare to the typical V-I curve, we observed that CA-GOx and EC-GOx electrodes suffer their potential decreasing in mass transport losses region. While, the reduction slope for CLEA-GOx composites is not as steep as the other two composites, majority potential decrease is located in ohmic losses part. In Figure 4.4, CLEA-GOx composite biofuel cell was observed having the ability to provide the highest responding current. When the resistance loading is equal to 210 Ω, the corresponding currents were summarized in Table 4-3. 67 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.3 0.2 0.4 Voltage (V) 0.1 0.0 0.3 000 2 Voltage (V) Current (mA/cm ) 0.2 CA 0.1 EC CLEA 0.0 02468 Current (mA/cm2) Figure 4.4. V-I curves of biofuel cell with CA-, EC-, and CLEA-GOx composite electrodes. The biofuel cell setup was shown in Figure 4-1(a), the supporting materials used were carbon felt and the circuit flow rate of electrolyte is 20 ml/min. The apparent enzymatic activities of GOx-CNT were also summarized in Table 4-3. Analyzing those activities and responding current, we could easily figure out that the current ratio of EC-GOx to CA-GOx is equal to 1.68, and the activity ratio of them is 1.5. This result indicated that the current is correlated with the activity of the catalysts. In the case of EC and CA, the electron produced by chemical reaction could be efficiently transferred to electrode so that the current is executed. 68 Table 4-3. Characteristics of enzymatic biofuel cells with different GOx-CNT composite electrodes Anode Electrode Current (mA)a Enzymatic activity Type (U per mg of CNT) CA-GOx 0.2 6.6 EC-GOx 0.337 9.9 CLEA-GOx 3.25 338 a Current at resistance loading equals to 210 Ω However, when the same calculation strategy is applied to CLEA-GOx and CA-GOx, we found out that the current ratio of CLEA to CA is 16.2, which is much lower than that of the activity ratio, 51.2. It could be explained that the electrons produced in the reaction are much more than the portion which could be converted into current and measured apparently, in case of CLEA-GOx was applied to be the electrode in anode. 69 3.0 0.4 ) 2 0.3 ) 2.5 2 0.2 2.0 (mW/cm 0.1 Power Density 0.0 1.5 0.0 0.2 0.4 Current (mA/cm2) 1.0 CA Power Density (mW/cm Power Density 0.5 EC CLEA 0.0 0246810 Current (mA/cm2) Figure 4.5. P-I curves of biofuel cell with CA-, EC-, and CLEA-GOx composite electrodes. The biofuel cell setup was shown in Figure 4.1(a), the supporting materials used were carbon felt and the circuit flow rate of electrolyte is 20 ml/min. In addition, the P-I curves were plotted in Figure 4.5, with CA-GOx, EC-GOx and CLEA-GOx electrodes work as anode in biofuel cells. The maximum power densities are 0.26, 0.38 and 2.46 µW/cm2, individually. Figure 4.6 represents the relations among the power densities of CA-, EC- and CLEA-GOx electrodes and the out circuit resistance loading. The trend of curves indicates that CLEA-GOx electrode could provide higher reaction rate in self-made biofuel cell reaction, which is a promising immobilization materials in application of biofuel cells. The maximum power densities appeared at the resistance of 11,110 Ω, 7110 Ω and 410 Ω, respectively. By comparing the Figure 4.5 and 70 Figure 4.6, we concluded that the power generated in the biofuel cells is more related to the current rather than biofuel cell voltage. That is, the reaction rate in anode will be the key element in optimizing the biofuel cell power output. Therefore, the property of catalyst is essential. 3.0 ) CA 2 2.5 EC CLEA 2.0 1.5 1.0 Power Density (mW/cm Density Power 0.5 0.0 0 5000 10000 15000 20000 Resistance (ohm) Figure 4.6. The power density relation to the resistance load in out circuit. Experimental conditions are same as Figure 4.5. 4.3.5 Effects of CNT on biofuel cell performance To study the CNT effects on electrode performance in biofuel cells, bare glassy carbon electrode and CNT coated glassy carbon electrode were applied in biofuel cell setup 71 (Figure 4.1(a)) with a fuel (glucose) concentration of 200 mM and BQ concentration of 5 mM. Native GOx was used as catalyst in biofuel cell with a concentration of 0.1 mg/ml. The maximum current and power densities values are listed in Table 4-4. With the same operation conditions, CNT glassy carbon gives 2 times higher current than bare glassy carbon electrode. This means, the electron transfer rate is doubled at the present of CNT, since all other parameters in the experiment are identical. Also, we observed that the maximum power density is more than sextupled by using CNT electrode in biofuel cell. Table 4-4 CNT effects on biofuel cell performances Maximum Current Density Maximum Power Density (µA/cm2) (mW/cm2) Bare Glassy Carbon Electrode 2.9 38.6 CNT Glassy Carbon Electrode 77.5 19.3 To further examine the effects of carbon nanotubes (CNT) on carbon felt electrode and the biofuel cell performances afterwards, similar immobilization process with the above paragraphs was conducted, except that GOx was immobilized on the carbon felt directly instead of CNT. Firstly, carbon felt was treated by sulfuric acid and nitric acid to increase the hydrophilicity and add –COOH group on the fiber surface. According to the immobilization methods, the carbon felt was used as electrode directly, named as D-CA, D-EC and D-CLEA, respectively. Figure 4.7 illustrates that the directly immobilized carbon felt electrode was applied in the self-made biofuel cell system as indicated in Figure 72 4.1(a). As shown in Figure 4.7 and Figure 4.5, it could be seen that the maximum power density of D-CLEA decreased to be over 25% of that with the existence of carbon nanotubes, which could be explained by the less surface area during the glucose oxidase immobilization. 3.0 ) 2 Direct 2.5 Immobilization W/cm GOx-CNT m 2.0 1.5 1.0 0.5 Maximum Power Density ( Density Power Maximum 0.0 CA EC CLEA Figure 4.7 Power putout of self-made biofuel cell performances with CA-, EC-, and CLEA electrodes. The biofuel cell setup was shown in Figure 4-2(a). Circulation rate of electrolyte is 20 ml/min. On the other hand, the electrodes were coated with GOx-CNT composites of with and without CNT during immobilization process, respectively. In the biofuel cell, EC and 73 CLEA have higher power density than CA regardless the presenting of CNT or not. The growing of power density could be attributed to the increase of conductivity in present of CNT. Compare the performances without circulation rate (data not shown here); the power density in direct immobilized CA (D-CA) system is decreasing. This might due to the interfering of circulation on electron transfer between the active site of GOx and the electrode surface. It is also partially confirmed by the fact that the D-CA biofuel cells have lower power density, when circulation of electrolyte was applied in the system, which could enhance the mass transfer. Thus, in the D-CA system, the lower reaction rate is the bottleneck of the biocatalysis, while the mass transfer might be the limitation step in the direct immobilized EC (D-EC) and direct immobilized CLEA (D-CLEA) system. In addition, due to the fact that we applied the pump to circulate the fuel solution in the anode chamber, which was regarded as the way to alleviate the mass transfer limitation. This result agrees with our assumption that the transfer process, mass transfer or electron transfer is limited the whole current generation route in this kind of biofuel cell setup, when the CNT were applied as enzyme immobilization materials. 4.3.6 Thermal stability study of biofuel cell electrodes To further study the glucose oxidase immobilization properties and the electrode performances at different working temperature, experiments similar to section 3.3.6 was conducted. The Toray® carbon paper electrodes coated with CA-GOx and CLEA-GOx composite were incubated at 50oC, and the power density-time courses was measured, respectively. 74 CA-GOx and CLEA-GOx were selected as representatives in the following experiments because the executions of CA-GOx and EC-GOx composites in previous studies showed similar consequences. After 1 hour and 2 hour, the self-made electrodes were put back to biofuel cell to measure the maximum power density. As shown in Figure 4.9, after 2 hours thermal treatment, the power density of CA-GOx biofuel cell decreased 90%. However, in the same time span, the power density of CLEA-GOx biofuel cell decreased only 3%. In our further observation, the power density of CLEA-GOx biofuel cell stays stable in 5 hours. ) 2 1000 0h 1h 2h W/cm µ 100 0h 1h 10 2h 1 Maximum Power Density ( Density Power Maximum CA CLEA Figure 4.8. Thermal stability of CA-and CLEA-GOx electrodes in biofuel cells. The power densities of biofuel cells with CA-GOx and CLEA-GOx electrodes, and their stabilities after thermal treatment at 50°C. The supporting materials used here is Toray® carbon paper (thickness is 370 µm). 75 4.3.7 Study of the stability of biofuel cell operation Besides the thermal stability study of GOx-CNT electrode in biofuel cell functions, we further investigated the operation stability of the entire biofuel cell setup in long term running. From the previous studies, we realized that the reaction rate which for current generation is the key element in analyzing the system optimization. So, in this long term operation, the current was recorded continuously for data evaluation. 3.5 3 2.5 2 CLEA 1.5 CA Current (mA) 1 0.5 0 0 6 12 18 24 Time (hr) Figure 4.9. Long term operation of the self-made biofuel cell performances with CA- and CLEA-GOx composite carbon felt electrodes. The biofuel cell setup was shown in Figure 4-1(a), and the operation potential was 350 mV with the flow rate of 20 ml/min of electrolyte. The resistance load in the circuit is 410 Ω. 76 During the operation, although there is fluctuation in current, the current differences between CA-GOx and CLEA-GOx is still obviously to be observed. The biofuel cell system was operated by circulating the electrolyte with peristaltic pump, and the working voltage was applied at 350 mV. In Figure 4-8, the mounting of current up to 2 hours could be regarded as the initial stabilization stage and the declining trend of CLEA after 2 hours should result in the consumption of the substrate---glucose. On the other hand, the CA-GOx operation curve started to drop off only after 15 hours. 4.3.8 Comparisons of self-made and miniature biofuel cells To demonstrate the better performances of the self-made biofuel cell setup, miniature fuel cells (Courtesy from Washington State University, 2 cm×2 cm and 1 cm×1 cm) were used in our experiments. Firstly, the miniature biofuel cell setup (1 cm ×1 cm) was examined. Four kinds of supporting materials, Toray® carbon paper (thickness 100 µm and 370 µm), carbon cloth and carbon felt, were coated by CLEA-GOx composite to make electrodes, and the effect of supporting material and the biofuel cell performances were studied, respectively. As shown in Figure 4.10, we observed that in term of power density, carbon felt gave the highest value, followed by carbon cloth and 100 µm Toray® carbon paper, and the least one is the 370 µm Toray® carbon paper. Since the composite loading in both thicknesses Toray® carbon paper is similar, the higher power density in thinner carbon paper could be explained as the thinner carbon paper provide more porous structure for mass transfer. 77 300 Toray 100 ) 2 250 Toray 370 Carbon cloth W/cm 200 Carbon felt µ 150 100 50 Power Density ( Density Power 0 0.0 1.0 2.0 3.0 4.0 Current Density (mA/cm2) Figure 4.10. P-I curves of miniature biofuel cell (1 cm ×1 cm) with CLEA-GOx composite electrodes on different supporting materials. The biofuel cell setup was shown in Figure 4-1(b), the supporting materials used were Toray® carbon paper (thickness: 100 µm and 370 µm) carbon cloth and carbon felt. No circuit of electrolyte. (This data was done by SA Jun in PNNL.) Table 4-5 listed the comparison of power output in self-made biofuel cell setup and miniature biofuel cell setup (1 cm ×1 cm). When using same carbon felt as supporting material, the overall power output in self-made biofuel cell and miniature biofuel cell (1 cm×1 cm) could reach 0.82 and 0.06 mW, respectively. In term of power density, based on sectional surface area, it is 2.46 and 0.19 mW/cm2, respectively. While based on the chamber size, power density reached 0.12 and 0.08 mW/cm3. 78 Table 4-5. Comparisons of power output in self-made biofuel cell and miniature biofuel cell (1 cm×1 cm)a. Self-made Biofuel Cell Miniature Biofuel Cell Power output 0.82 0.06 (µW) Size of electrode 0.33 0.33 (cm2) Power Density 2.46 0.19 (mW/cm2) Fuel cell chamber size 7 0.75 (cm3) Power Density (chamber) 0.12 0.08 (mW/cm3) a: Supporting materials is carbon felt Secondly, another 2 cm ×2 cm miniature fuel cell setup also was configured for analyzing the biofuel cell performance. CA-GOx and CLEA-GOx were coated on supporting material, Toray® carbon paper (370 µm), to construct the electrodes. When Toray® carbon paper (thickness 370 µm) was used as supporting electrode; the performances in two different size miniature biofuel cells were investigated. Table 4-5 tabulated the comparison of power density of two miniature biofuel cells. By using the same sectional size of electrode, larger size of miniature biofuel cell approached 20 times higher overall power output and 7 times higher based on chamber capacity. 79 250 ) 2 CA 200 CLEA W/cm µ 150 100 50 Power Density ( Density Power 0 0.00 0.05 0.10 Current (mA/cm2) Figure 4.11. P-I curves of miniature biofuel cell (2 cm ×2 cm) with CA- and CLEA-GOx composite electrodes. The biofuel cell setup was shown in Figure 4.1(c), the supporting materials used were Toray® carbon paper (thickness 370 µm) and no circuit of electrolyte. (This data was done by SA Jun in PNNL.) 80 Table 4-6. Comparison of power output in two different size miniature biofuel cells using Toray® carbon paper (thickness 370 µm) electrodes miniature biofuel cell miniature biofuel cell Biofuel cell system (1 cm ×1 cm) (2 cm ×2 cm) Maximum Power Output (µW) 0.003 0.06 Size of electrode 0.33 0.33 (cm2) Power Density 0.01 0.19 (mW/cm2) Fuel cell chamber size 0.75 2 (cm3) Power Density (chamber) 0.004 0.03 (mW/cm3) 81 CHAPTER V CONCLUSIONS Novel bioelectrochemically active GOx-CNT composites were produced by immobilizing enzymes with nanotubes. Biosensor and biofuel cells were constructed by applying enzymatic electrodes fabricated with the GOx-CNT composites. From the electrochemical behaviors of these systems, we found: (1) The approach of CLEA-GOx increased the effective loading of GOx significantly through the formation of multilayer coatings on surface of CNT. CLEA also significantly enhanced the stability of the immobilized GOx in that no activity loss was observed up to 80 days. Compared to the commonly used CA approach, CLEA gave a 50-ime higher activity. We believe the construction of CLEA-GOx reflect a promising immobilization approach to highly active and stable electrochemical materials. (2) Higher enzyme loading on electrodes leads to better sensitivity for amperometric biosensors. CLEA-GOx electrode gave a sensitivity of 13.3 mA/(M cm2), which is about 25 times the sensitivity of CA-GOx electrode (0.52 mA/(M cm2)). 82 (3) CLEA-GOx composite electrodes also greatly extended the lifetime and enhanced the performance of biofuel cell. In comparison with those have CA- and EC-immobilized GOx, biofuel cells built from CLEA-GOx gave a power density that is about 10 times higher under the same experimental conditions. We believe this is a combined effect of the increased enzyme loading and the high surface area of CNT. 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