University of Ghana http://ugspace.ug.edu.gh INVESTIGATING THE INFLUENCE OF TEMPERATURE ON KA- OLINITE-BASE SYNTHESIS OF ZEOLITE AND UREASE IMMO- BILIZATION FOR POTENTIAL FABRICATION OF ELECTRO- CHEMICAL UREA BIOSENSOR BY DAVID EBO ANDERSON (10552666) A THESIS SUBMITTED TO THE SCHOOL OF GRADUATE STUDIES, UNIVERSITY OF GHANA, LEGON, IN PARTIAL FUL- FILMENT OF THE REQUIREMENT FOR THE AWARD OF DE- GREE OF MASTER OF PHILOSOPHY (MPhil) IN BIOMEDICAL ENGINEERING DEPARTMENT OF BIOMEDICAL ENGINEERING COLLEGE OF BASIC AND APPLIED SCIENCES UNIVERSITY OF GHANA MAY, 2018 i University of Ghana http://ugspace.ug.edu.gh DECLARATION I, DAVID EBO ANDERSON, do hereby declare that I have duly cited references used in this work, the entire work presented in this thesis, titled “Investigating the influence of Temperature on Kaolinite-Base Synthesis of Zeolite and Urease Immobilization for Potential Fabrication of Electrochemical Urea Biosensor” was exclusively done by me, and that, this thesis has never been presented either in part or in whole for any degree in this University or elsewhere. ………………………… ………………………… David Ebo Anderson Date (Student) ………………………… ………………………… Elvis K. Tiburu (PhD) Date (Supervisor) ii University of Ghana http://ugspace.ug.edu.gh ABSTRACT Temperature-dependent zeolite synthesis revealed unique surface morphology, surface area and pore size that influence morphology of zeolite synthesised from kaolin in the immobilization of urease on gold electrode support for biosensor fabrication. XRD char- acterization identified Zeolite X (Na) at all crystallization temperatures tested. However, Nitrogen adsorption and desorption results showed pore size and pore volume of Zeolite X (Na) 60 °C, Zeolite X (Na) 70 °C and Zeolite X (Na) 90 °C range from 1.92 nm – 2.45 nm and 0.012 cm3/g – 0.061 cm3/g respectively, with no significant differences. The spe- cific surface area of Zeolite X (Na) at 60, 70 and 90 °C was 64 m2/g, 67 m2/g, and 113 m2/g respectively. The pore size, specific surface area and pore volumes of Zeolite X (Na) 80 °C and Zeolite X (Na) 100 °C were dramatically increased to 4.21 nm, 295 m2/g, 0.762 cm3/g and 4.92 nm, 389 m2/g, 0.837 cm3/g in that order. The analytical perfor- mance of the embedded urease in Zeolite X (Na) was also investigated using cyclic volt- ammetry measurements and the results showed distinct cathodic and anodic peaks by Zeolite X (Na) 80 °C and Zeolite X (Na) 100 °C. The molar conductance of the modified zeolite biosensors was measured as a function of urea concentration and gave an average exponential decay regression fit of 0.98. The findings in this study suggest crystallization temperature is a critical parameter for electrochemical analysis of zeolites synthesized from natural sources for various biomedical applications. iii University of Ghana http://ugspace.ug.edu.gh DEDICATION The Almighty God and my family, especially my wife and family gave me encourage- ment for this work. I wholeheartedly dedicate this work to them. iv University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT I am grateful to Almighty God for my life and the strength he imbibed in me in accom- plishing this work. I also wish to express my profound gratitude to my supervisor, Dr. Elvis Tiburu, for his mentoring and encouragement during the execution of this thesis. I extend my profound appreciation to all the Lecturers and staff of the Department of Bi- omedical Engineering for the knowledge and support given to me. My earnest indebted- ness goes to the following individuals Srinivasan Shankar, Francis Krampa, Heidimarie N. A. Fleischer and all the staff of WACCBIP (Biochemistry and Biomedical Engineer- ing Department, University of Ghana) for their enormous contribution towards the suc- cessful completion of this work. v University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS ABSTRACT ............................................................................................................... iii DEDICATION ............................................................................................................ iv ACKNOWLEDGEMENT ............................................................................................ v LIST OF FIGURES .................................................................................................... xi LIST OF TABLES ...................................................................................................... xi LIST OF ABBREVIATIONS ................................................................................... xiv CHAPTER ONE .......................................................................................................... 1 INTRODUCTION .................................................................................................... 1 1.1 Background ..................................................................................................... 1 1.2 Problem and Need Statement ........................................................................... 5 1.3 Hypothesis ...................................................................................................... 6 1.4 Aim and Objectives ......................................................................................... 6 CHAPTER TWO ......................................................................................................... 8 LITERATURE REVIEW ......................................................................................... 8 2.1 Introduction ..................................................................................................... 8 2.2 Biosensor ........................................................................................................ 9 2.3 Factors That Affect Immobilization ............................................................... 10 2.4 Methods of Immobilization and Its Effects .................................................... 10 2.4.1 Physical Adsorption ................................................................................... 11 2.4.2 Affinity Immobilization .............................................................................. 12 vi University of Ghana http://ugspace.ug.edu.gh 2.4.3 Cross-Linking ............................................................................................ 12 2.4.4 Entrapment ................................................................................................. 12 2.5 Immobilization of Biological Molecule ......................................................... 13 2.6 Enzyme Biosensors ....................................................................................... 14 2.7 Zeolites for Biosensors .................................................................................. 15 2.7.1 Effect of Zeolite Synthesis on Immobilization ............................................ 15 2.8 Electrochemistry in Biosensing ..................................................................... 16 2.8.1 Electrodes Materials for Biosensing ........................................................... 16 2.9 Types of Biosensors and Their Uses .............................................................. 17 2.10 Rationale ..................................................................................................... 17 CHAPTER THREE ................................................................................................... 19 MATERIALS AND METHODS ............................................................................ 19 3.0 Introduction ................................................................................................... 19 3.1 Materials ....................................................................................................... 19 3.2 Methods ........................................................................................................ 20 3.2.1 Sample Preparation (Zeolite Synthesis) ...................................................... 20 3.3.0 Biophysical Characterization ...................................................................... 24 3.3.1 X-Ray Diffraction (XRD) ........................................................................... 24 3.3.2 Scanning Electron Microscopy (SEM) ........................................................ 25 3.3.3 Energy Dispersive X-Ray (EDX) ............................................................... 26 3.3.4 Brunner Emmett Teller (BET) .................................................................... 26 vii University of Ghana http://ugspace.ug.edu.gh 3.4 Electrode Modification .................................................................................. 26 3.5 Drop Coating................................................................................................. 28 3.5 Immobilization of the Biomolecules .............................................................. 30 3.5.1 Second Drop Coating ................................................................................. 30 3.6 Electrochemical Measurements ..................................................................... 31 3.6.1 Cyclic Voltammetry (CV) .......................................................................... 33 3.7 Exponential decay Regression Fits and Molar Conductance Curves .............. 35 CHAPTER FOUR ...................................................................................................... 36 RESULTS .............................................................................................................. 36 4.0 Introduction ................................................................................................... 36 4.1 Biophysical Characterization ......................................................................... 36 4.1.1 X-Ray Diffraction (XRD) ........................................................................... 36 4.1.2 Scanning Electron Microscopy (SEM) ........................................................ 37 4.1.3 Energy Dispersive X-Ray (EDX) and Brunner Emmett Teller (BET) ......... 38 4.2 Effect of Base Materials ................................................................................ 39 4.2.1 Cyclic behaviour of Zeolite X (Na) at different crystallization temperature. 40 4.2.2 Cyclic voltammogram of bioelectrode with Zeolite X (Na) 60 °C in selected urea concentrations. ............................................................................................ 40 4.2.3 Cyclic voltammogram of bioelectrode with Zeolite X (Na) 70 °C in selected urea concentrations ............................................................................................. 43 4.2.4 Cyclic voltammogram of bioelectrode with Zeolite X (Na) 80 °C in selected urea concentrations ............................................................................................. 45 viii University of Ghana http://ugspace.ug.edu.gh 4.3.6 Cyclic voltammogram of bioelectrode with Zeolite X (Na) 100 °C in selected urea concentrations. ............................................................................................ 49 4.4 Data Analysis ................................................................................................ 51 CHAPTER FIVE ....................................................................................................... 53 DISCUSSION ........................................................................................................ 53 5.1 Discussion ..................................................................................................... 53 CHAPTER SIX .......................................................................................................... 58 CONCLUSION AND RECOMMENTATION ....................................................... 58 6.1 CONCLUSION ............................................................................................. 58 6.2 RECOMMENDATION ................................................................................. 58 REFERENCES .......................................................................................................... 60 APPENDIX A ........................................................................................................ 70 APPENDIX B ........................................................................................................ 71 Exponential decay Fitting Data Generated by Origin Pro 9 Software for Conductance of Zeolite X (Na) 60 °C. ...................................................................................... 71 APPENDIX C ............................................................................................................ 72 Exponential decay Fitting Data Generated by Origin Pro 9 Software for Conductance of Zeolite X (Na) 70 °C. ...................................................................................... 72 APPENDIX D ............................................................................................................ 73 Exponential decay Fitting Data Generated by Origin Pro 9 Software for Conductance of Zeolite X (Na) 80 °C. ...................................................................................... 73 APPENDIX E ............................................................................................................ 74 ix University of Ghana http://ugspace.ug.edu.gh Exponential decay Fitting Data Generated by Origin Pro 9 Software for Conductance of Zeolite X (Na) 90 °C. ...................................................................................... 74 APPENDIX F ............................................................................................................ 75 Exponential decay Fitting Data Generated by Origin Pro 9 Software for Conductance of Zeolite X (Na) 100 °C. .................................................................................... 75 APPENDIX G ............................................................................................................ 76 PLAGIARISM REPORT ........................................................................................ 76 x University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES Figure 1: Basic Architecture of a Biosensor. ................................................................. 1 Figure 2: Harvesting of the kaolin from Amanfrom village in the eastern part of Ghana. ....................................................................................................................... 20 Figure 3: (a) Depicts sieving of grounded kaolin, (b) Fine grains of kaolin sieved with 63 µm. ............................................................................................................................ 21 Figure 4: (a) Shows crucibles filled with kaolin and placed in a furnace, (b) shows the furnace used to heat the kaolin to the desired temperature to obtain metakaolin. ......... 21 Figure 5: Samples of alkaline fused metakaolin for the five different crystallization temperatures. .............................................................................................................. 22 Figure 6: Alkaline fused metakaolin added to deionized water. .................................. 23 Figure 7: (a) Synthesized zeolites at different crystallization temperatures, (b) Packaged crystalline material for biophysical analysis. .............................................................. 24 Figure 8: XRD equipment used for diffraction analysis at the University of Ghana, Department of Physics. .............................................................................................. 25 Figure 9: Modified Griffin gold electrode. .................................................................. 27 Figure 10: Active zone of gold electrode dipped into the zeolite paste. ....................... 28 Figure 11: Zeolite modified gold electrode placed on heat block set at 200 °C for dehydration. ............................................................................................................... 29 Figure 12: Thin layer of zeolite deposited on the gold electrode active zone after dehydration. ............................................................................................................... 29 Figure 13: Cyclic voltammetry setup, AutoLab PGSTAT 204, magnetic stirrer and bare gold electrodes. .......................................................................................................... 31 xi University of Ghana http://ugspace.ug.edu.gh Figure 14: Phywe concentration & conductivity meter for analysing the conductance of the biosensor thus developed. ..................................................................................... 32 Figure 15: A voltammogram of a reversible analyte .................................................. 33 Figure 16: XRD of graphs of Zeolite X (Na) synthesized at various temperatures. ..... 36 Figure 17: SEM images of Zeolite X (Na) synthesized at 60 °C, (b) 70 °C, (c) 80 °C, (d) 90 °C and (e) 100 °C with scale bar = 50 micron. ....................................................... 37 Figure 18: Shows the voltammetric behaviour of electrodes and reagents. .................. 39 Figure 19: (a) Cyclic voltammogram of 0.1 mM urea. ................................................ 41 Figure 20: (a) Cyclic voltammogram of 0.1 mM urea. ................................................ 43 Figure 21: (a) Cyclic voltammogram of 0.1 mM urea ................................................. 45 Figure 22: (a) Cyclic voltammogram of 0.1 mM urea. ................................................ 47 Figure 23: (a) Cyclic voltammogram of 0.1 mM urea. ................................................ 49 Figure 24: Molar conductance curve of Zeolite X (Na) modified electrodes and the response of the biosensor as a function of urea concentration. .................................... 51 Figure 25: An exponential decay fit (type 2) of the conductance curve as a function of urea concentration by Zeolite X (Na) 100°C and Zeolite X (Na) 80°C respectively. ... 52 Figure 26: Cyclic Voltammogram of Kaolinite with selected Concentrations (0.1, 0.5, 0.8, 1.0 mM) of Urea .................................................................................................. 70 xii University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 1: The parameter set used to generate the Cyclic Voltammetry (CV) measurements. .................................................................................................................................. 31 Table 2: Characteristics of Zeolite Synthesized at Different Crystallization Temperature ............................................................................................................... 38 Table 3: Shows the peak anodic current for Zeolite X (Na) 60 °C - Zeolite X (Na) 100 °C obtained using EC-Lab® software. ........................................................................ 51 xiii University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS Brunner Emmett Teller……………………………………………………BET Carbon nanotubes…………………………………………………………CNTs Counter Electrode…………………………………………………………CE Cyclic Voltammetry………………………………………………………CV Energy Dispersive X-ray …………………………………………………EDX Polypyrrole ………………………………………………………………. PPy Field Effect Transistors …………………………………………………. FET Horseradish peroxidase …………………………………………………. HRP Poly l-lysine………………………………………………………………PLL Rapid Diagnostic Test ……………………………………………………RDT Reference Electrode ……………………………………………………...RE Scanning Electron Microscopy…...………………………………………SEM Screen Printed Electrodes………………………………………………...SPE Surface Acoustic Wave…………………………………………………..SAW Working Electrode ……………………………………………………….WE X-ray Diffraction…………………………………………………………XRD Zeolite Modified Electrode ……………………………………………...ZME xiv University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE INTRODUCTION 1.1 Background A biosensor is a device that converts biological information into a signal for meaningful purposes. A classic example of a biosensor is the glucometer which is used in our health care centres. The basic biosensor is made up of an analyte, transducer and an electronic system. The analyte (target) is the product under investigation such as cultured cells, human, food and environmental samples. The transducer consists of the biologically ac- tive molecule (bio-receptor) coupled to the electrode (electrical interface). A bio-receptor is the probe molecule (DNA, enzymes and many more) that is specifically bound to the target analyte. The electrode is the conductive material that recognises changes in activ- ities of the working cell. This measures the changes in activity on the electrode surface (heat output, distribution of charges and light output). Then finally an electronic system that converts the reaction determined by the transducer into meaningful electrical signals [1]. Figure 1: Basic Architecture of a Biosensor. 1 University of Ghana http://ugspace.ug.edu.gh Electrode surfaces of the sensors must be modified with different nano-technological products such as mercury, carbon, nanotubes and polymers to enable easy immobilisation of the biological molecule to improve its analytical characteristics and reproducibility. Over the years, mercury has been used as the most suitable material for the modification of electrode surfaces due to its high reproducibility and smooth surface. Although haz- ardous, the properties of mercury drop electrode led to the Nobel Peace Prize in Chem- istry being awarded to Heyrovsky in 1959. Zeolites have shown great promise, compared to other alumina-silicates and crystalline materials for the modification of electrode surfaces [2]. A particular feature of zeolites is the existence of a network of cavities linked by channels, which provides a large specific surface area, enabling it to trap the biologically active molecules used for biosensor de- velopment. Zeolites are distinguished from other porous materials by its pore size variety and ion exchange capacity [2]. The greatest challenge of biosensor fabrication is the immobilization of the biological molecules (for example, antibodies, cells, and enzymes) on the surface of the transducer. Biologically active molecule (for example enzymes) can be immobilized by either cova- lent binding, entrapment, microencapsulation or cross-linking onto the transducer sur- face. The disadvantages of these methods are; loss of molecule activity, instability, poor reproducibility of biosensor signals, and toxicity of the compounds which induce the binding [3]. 2 University of Ghana http://ugspace.ug.edu.gh The sensitivity and overall performance of enzymatic biosensors have improved tremen- dously over the years as a result of the synthesis and incorporation of nanomaterials in their fabrication [4-7]. These materials have excited engineers due to their unique struc- tural, electrical, chemical and mechanical properties [8-11]. In the field of medical device fabrication, there are currently numerous studies describing the use of gold nanoparticles, graphene, zeolites, quantum dots, silver nanomaterials, nanocomposites as well as carbon nanotubes (CNTs) for enzyme immobilization due to certain characteristics ranging from their biocompatibility to fast response times [12-15]. One of the oldest known materials is clay minerals which have been used predominately for curative, industrial and medical applications [16, 17]. They have been widely applied in such fields because of their high colloidal and swelling capacity, surface reactivity, high adsorption, and cation exchange capacity [18-20]. Among the most studied biosensors, are those developed for urea detection using the different sensing methods mentioned earlier [21-25]. Urea is an organic molecule with chemical formula CO(NH2)2 and is the end product of protein metabolism in the liver. Urea biosensors are of great importance because urea levels in our body are used to di- agnose the early stage of renal failure or disorder. Urea undergoes filtration and a series of processes before it is finally excreted. It is, therefore, an important biomarker for de- tecting renal dysfunction using different testing methods including biosensors. 3 University of Ghana http://ugspace.ug.edu.gh As a well-studied model system, urea biosensors function on the basis that urea is de- composed by urease to form electroactive species (ammonium ion), accruing to the reac- tion in equation (1). The electrochemical monitoring of the enzymatically produced NH +4 at an appropriate transducer generates the analytical response. There are currently concerted efforts by the scientific community tailored towards iden- tifying materials that can enhance enzyme immobilization, due to the high demand of biosensors for rapid point-of-care testing. Biosensors have transducers that are categorised as electrochemical, field effect transis- tors, optical, piezoelectric surface acoustic and thermal sensors. The type usually depends on the application. Electrochemical transducers can be potentiometric, voltammetric and conductometric. All these measure changes in electrical conductivity and voltage differ- ences occurring at the working electrode (we) surface in comparison to a reference elec- trode (re). Electrochemical sensors are made of two or three electrode configurations, with the working electrode (we) and the reference electrode (re), being common to both configu- rations and the latter having a third electrode called the counter (ce). The two-electrode system is mostly used in areas where prolonged stability of the reference electrode is not needed, hence lowering cost of development. The actual reaction occurs at the working electrode surface where the biological molecule interacts closely with the target analyte and the transducer. 4 University of Ghana http://ugspace.ug.edu.gh The counter electrode is usually used as a support for the reference electrode to carry charges from the electrolysis reaction to maintain the half-cell potential of the reference electrode. When the biological molecules are immobilised onto the electrode surface, the technique of immobilization determines the working lifespan due to instability, recovery and response time. Although many polymers are good couplers for electrode surfaces, yet their inherent properties denature the biological materials. To overcome these diffi- culties, zeolites were proposed as carriers for enzyme adsorption. Zeolites are slightly toxic and hardly affected by mechanical, chemical, and thermal effects making zeolite- based biosensors a better option for multicomponent biological samples. In this work, the effect of crystallization temperature which has significant influence on the pore volume and pore size of zeolite is studied with the immobilization of urease enzyme for the detection of urea. 1.2 Problem and Need Statement Biosensors have gained a lot of attention due to the demand for the rapid diagnostic test (RDT) which addresses the inconvenience of having to move to a well-established med- ical laboratory for diseases diagnosis. Test kits are expected to be defined, cost-effective and robust in the diagnoses of diseases. In the development of biosensors, the biological molecule needs to be incorporated in a matrix or on a substrate before being accessed by the analyte. This matrix should be able to retain the biological activity of the biomolecule to enable detection, hence the demand to investigate materials (matrix) such as carbon nanotubes, graphene, polymer, nano-wire and clay minerals which are used as substrates. 5 University of Ghana http://ugspace.ug.edu.gh Zeolites have proven to have the abilities to keep the biological element in its active state and also improve the analytical sensor response due to some key inherit properties. Zeo- lites from the commercial markets are expensive and not easy to come by. , However, it can also be synthesized from kaolin; a natural mineral which is readily available in our local settings. The procedure of obtaining zeolite from kaolin is well established, but the parameters that affect its immobilisation characteristics are not well defined, hence the need to in- vestigate the parameters that affect the syntheses of zeolite from kaolin and study the analytical characteristics of the product using urea as a proof of concept. 1.3 Hypothesis Zeolites can be synthesized from kaolin deposits in Ghana at different crystallization temperatures. Zeolite-based biosensors can be developed as an electrochemical biosensor with an improved analytical response to the rapid diagnostic test. 1.4 Aim and Objectives The aim of the project is to develop electrochemical biosensors with zeolite modified electrode (ZME) surfaces, for the detection of urea as a proof of concept. The develop- ment process will utilize zeolite synthesized from kaolin deposits in Ghana through al- kaline fusion before hydrothermal reaction. The zeolite will then be used to modify the surface of the electrode by drop coating as reported by Kucherenko et al [26]. This would be achieved by carrying out the following activities: 6 University of Ghana http://ugspace.ug.edu.gh a) Synthesize zeolite from kaolin deposits in Amanfrom, Eastern part of Ghana by crys- tallizing at different temperatures. b) Explore the adsorption properties of the zeolite modified electrode. c) Test the electrochemical behaviour of the zeolite modified electrode using urease enzyme. The thesis is apportioned into six chapters; chapter one gives a brief explanation of bio- sensors and materials for its fabrication; chapter two focuses on literature review in the fabrication of a biosensor and techniques employed in biosensor analysis; chapter three vividly explains the materials and methods employed in executing this work; chapter four shows results obtained while chapter five summarizes the results with follow-up discus- sions; and the concluding part of the thesis is found in chapter six with recommendation for further studies. 7 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO LITERATURE REVIEW 2.1 Introduction This chapter describes the review of the literature with respect to the development of biosensors and also highlights subjects that are related to the biosensor development which is being reviewed in this respect. Electrochemical means of detecting target molecules have been shown to have projec- tions in creating miniaturized devices for use in field evaluations. An important consid- eration towards this development is the immobilisation of different biological molecules on various electrodes such as gold and silver nanoparticles to facilitate their conductivity and linkage to antibodies [27]. Zhengpeng et al established a sensor that braced an analytically useful wide detection range from 0.5 µM to 3.0 mM, with a short response time and an adequate storage sta- bility. This method immobilised urease onto piezoelectric alumina membranes using con- ductometric response [28]. Zhao and Zhan demonstrated the detection of Shigella flexneri using screen-printed elec- trode (SPE) biosensors. In this work, Horseradish peroxidase (HRP) labeled antibodies to S. flexneri was entrapped into a multi-wall carbon nanotubes/chitosan matrix for the capture of S. flexneri. The assay showed satisfactory performance with a detection limit 8 University of Ghana http://ugspace.ug.edu.gh of 2.3×103 µmL−1. A particular feature of the device was safe since it did not involve culture of the pathogen, it proved to be very useful, cost-effective and robust [29]. Also, a urea biosensor was reported where urease was covalently immobilized in a film of electroinactive polypyrrole (PPy) with polyions polyacrylate (PAA) and poly l-lysine (PLL) to make a composite PPy/PAA-urease-PLL film. The doping of PPy with anions rendered an enhanced stability to the electrode. The PPy/PAA electrode showed a selec- tive Nernstian response to change in H+ ions (pH), which forms the potentiometric basis of the biosensor. The sensor showed a good operational stability and urea detection in the range 0.018–1.8 mg/dl [30]. A potentiometric array-based biosensor has been reported for the detection of urea in the appearance of interferents such as ammonium, potassium, sodium and hydrogen found in a biological matrix. The sensor is an all solid-state type employing carboxylated PVC as the polymeric membrane, having a linear range detection of 10−4 to 10−2 M urea with a short response time. The bio-electronic tongue was subjected to the analysis of urine samples. Although some discrepancies were observed between real-time and test sam- ples, a similar array-based urea biosensor was further developed by the same group of workers, with creatinine analyte [31]. 2.2 Biosensor Biosensors are known as analytical devices that consist of a biological sensing compo- nent; an immobilized biological element comprising of enzymes [32], whole cells, anti- bodies, tissues, microorganisms, DNA, protein or RNA, and a transducer comprising of either amperometry, potentiometric, thermometric, piezoelectric, surface acoustic wave 9 University of Ghana http://ugspace.ug.edu.gh (SAW) and optical transducers among others [33]. This changes the biochemical signal into a quantifiable electrical signal such as temperature adjustment, current, potential, absorption of light or change in mass [34]. 2.3 Factors That Affect Immobilization Immobilizing a biological element means fixing the molecule in place so as to avoid movement of that component or the confinement of enzymes to a matrix. There are nu- merous requirements for successful immobilization of biological elements used for con- structing or making biosensors. The method used for immobilization is a requirement that is essential to successful immobilization of biological elements. Each chosen method of immobilization should; not be complex but simple to use, be effectively stable for the immobilization of biological elements on the surface of its transducer, constitute non-toxic environmental factors so as to not disrupt the activity of the biological element, and a chemically inert and highly reproducible host structure during large-scale produc- tion [35, 36]. 2.4 Methods of Immobilization and Its Effects Immobilization methods of the biological element include covalent binding, entrapment and encapsulation, crosslinking and adsorption [37-39] or binding to the transducer sur- face among others [40, 41]. The effective use of these methods depend on the nature of the biological element to be , used, the kind of transducer to be considered, the chemical and physical properties of the analyte and operational conditions of the biosensor. Most importantly, these considerations must permit the biological element to demonstrate ef- ficient action in its immobilized microenvironment [33]. 10 University of Ghana http://ugspace.ug.edu.gh Conditions like toxicity and chemical inertness may directly affect the analytical features of the biosensors, such as sensitivity, reproducibility, selectivity and detection limit, in- fluence on enzyme positioning, entrapment, motion, stability, arrangement and biologi- cal activity [42, 43]. Thus, improving the methods of enzyme immobilization is a critical step in biosensor development [44, 45]. Immobilization is a very delicate process and must be well tailored to ensure the efficacy of the biosensor. The option of the most suitable technique depends on the nature of the immobilized material, the transducer and its related detection mode. The finest method of immobilization will depend on where the biosensor would be applied and if it requires maximum sensitivity or stability [46]. The various immobilization methods are well ex- plained below. 2.4.1 Physical Adsorption Physical adsorption involves the modest deposition of an enzyme on a surface and its attachment through weak bonds. This method has commonly been used to make enzy- matic biosensors, because it is simple, with limited loss of immobilized material but its desorption is non-specific [47]. Hence biological elements are denatured after a short while and reproducibility is feasible. 11 University of Ghana http://ugspace.ug.edu.gh 2.4.2 Affinity Immobilization Affinity immobilization technique is also used to make biological affinity bonds be- tween a stimulated support and a particular protein sequence (for example, biotin, car- bohydrate residue, histidine and many others). It permits the biological element to orient in order to evade the deactivation of an enzyme or the blocking of the active site. This method has been elaborated to immobilize enzymes using processes such as strept, avidin-biotin, and many more [48]. 2.4.3 Cross-Linking Cross-linking method is attractive because it is simple and has the ability to effectively bind biological element chemically. The major setback is its ability to lose activity due to the alteration of the immobilized conformation and the chemical changes of the active site during the process [49-51]. 2.4.4 Entrapment Entrapment method involves the incorporation of an immobilized material in a gel or polymer. This method requires no chemical reaction but rather a high concentration of an immobilized molecule. In this method, polysaccharide-based gel entrapment en- zymes (such as chitosan, alginate or agarose) are presumed in contrast to synthetic pol- ymers such as polyacrylamide, because they are biocompatible, nontoxic, provide nor- mal microenvironment to the enzyme and also provide adequate availability to electrons to travel between the enzyme and the electrode [52-53]. 12 University of Ghana http://ugspace.ug.edu.gh Covalent coupling technique is also another prevalent chemical immobilization method that is employed to create enzymatic biosensors. In this method, there is a chemical bond between functional groups of the immobilized material (the enzyme) and those on the support. This method produces no diffusion barrier, has a short response time, is stable, and has a high enzymatic activity. Nonetheless, its demerits are that matrices don’t re- generate and are coupled with toxic products [54]. 2.5 Immobilization of Biological Molecule There are numerous sorts of biological molecules that are used for immobilization in developing biosensors. These molecules include complementary DNA, probe, enzymes, antigens, antibodies, oligonucleotides and others. Even though antibodies and oligonu- cleotides are usually exploited as molecules for immobilization, enzymes are the most frequently used molecular recognition elements in biosensor development. Numerous books, articles and reviews have penned on enzyme immobilization, thus broadcasting thousands of protocols. , in some cases, enzyme immobilization protocols are also based on diverse immobilization techniques. For instance, one can pre-immobilize an enzyme on beads by adsorption, affinity or covalence before entrapping in a porous polymer substrate. Enzyme immobilization is essential in developing competent biosensors with exceptional performances in operational and storage stability, high selectivity, short re- sponse time, high sensitivity and high reproducibility [55-59]. Materials for constructing substrate or supports for immobilization include natural and inorganic polymers. Natural polymer supports for immobilization include alginate, pec- 13 University of Ghana http://ugspace.ug.edu.gh tin, cellulose, gelatin, chitosan, chitin, starch, collagen and many others, whiles inor- ganic polymer supports for immobilization include zeolite, glass, silica, ceramics, acti- vated carbon, charcoal and many others [60]. Materials used for the immobilization of biological elements include glutaraldehyde and hexamethyl diisocyanate. These form crosslinks between the biocatalytic species, or proteins [61]. 2.6 Enzyme Biosensors Enzyme biosensors are electrochemical biosensors that employ enzymes as the probe molecules and have become part of the most adopted type of biosensor for health diag- nosis, food industry, and environmental monitoring. The biomedical field is the foremost area in biosensor applications, such as monitoring blood glucose levels in diabetics with glucose biosensors (glucometer). Also, dependable recognition of urea has prospective tenders for detecting renal failure or disorders. The scientific community has focused on the development of biological molecule immobilisation because the procedure for the immobilization of the probe molecule has great influence on the analytical response (limit of detection, sensitivity, reproducibility, selectivity and many more) of the sensor. Enzyme immobilization on the transducer sur- face is the most essential stage in biosensor fabrication because the enzyme is the probe molecule [62]. 14 University of Ghana http://ugspace.ug.edu.gh 2.7 Zeolites for Biosensors Zeolite-enzymes biosensors have gained immense popularity due to their peculiar prop- erties such as high specific surface area that can be altered using organic and inorganic groups, their shape, charges, and size selectivity, their chemical stability, insolubility in organic solvents, ability to tolerate to microorganisms and resistance to harsh experi- mental factors [63-65]. 2.7.1 Effect of Zeolite Synthesis on Immobilization Zeolite synthesis is a very sensitive method, in that, controlling variables such as tem- perature, aging, mode of crystallization, conditions are critical to obtaining a pure crys- talline zeolite material and can affect its morphology. These synthetic zeolites may have different properties based on all the variables listed above. The pore diameter, the pore volume and surface area may affect the amount of biological element immobilized and the release of that biological element. Furthermore, even the elemental composition, the atomic structure of the zeolite formed and the morphology could greatly influence its effectiveness in electrochemistry. Vari- ous protocols have been explored in zeolite synthesis with the most common being fu- sion and hydrothermal methods [66] using controlled crystallization temperature and media [67]. These methods have effectively been used in making zeolite nanoparticle from kaolin [68, 69], coal fly ash [70-74], lignite fly ash [75] and rice husk ash [76, 77] among others. 15 University of Ghana http://ugspace.ug.edu.gh 2.8 Electrochemistry in Biosensing Measuring electrical signals from activities of biological reaction is electrochemistry. Here, a bioelectrochemical component works as the main transduction element. Electro- chemical sensing in biosensors normally needs a working electrode, a reference elec- trode and a counter or auxiliary electrode. Over the past decades, several sensing theories and devices have been established with the most widely used traditional techniques being cyclic voltammetry, chromo potenti- ometry, chrono-amperometry and others. Cyclic voltammetry is one of the most gener- ally used forms of electrochemical sensing. This is normally employed in obtaining in- formation about the redox potential and electrochemical reaction rates of analyte solu- tions. In this process, voltage is measured between the reference electrode and the work- ing electrode, whereas current measured is between the working electrode and the coun- ter electrode. Measurements acquired are then plotted as current versus voltage, to gen- erate wave form referred to as a voltammogram [78]. The measured values of current and voltage are used in the estimation of analyte concentration. 2.8.1 Electrodes Materials for Biosensing There are numerous electrodes incorporated with transducers for biosensing, such as gold, silver, stainless steel, platinum, nickel, carbon nanofiber, diamond and others. Plat- inum, gold, silver, stainless steel and carbon are commonly used to prepare electrode structures and supporting substrates. These metals have long been incorporated in elec- trochemical electrodes due to their superb electrical and mechanical properties [79]. They do not have direct intimacy with the biological molecule but are able to transduce 16 University of Ghana http://ugspace.ug.edu.gh the reaction through the incorporated substrate which are normally polymers, nanotubes and zeolite material with inert properties. These materials exhibit good electrical properties which makes them ideal for biosensor fabrication, by the act of transmitting the chemical reaction occurring at their surface to measurable electrical signals that are used in diagnosis. 2.9 Types of Biosensors and Their Uses Types of biosensors such as conductometric, potentiometric, electrochemical, amperom- etry, optical, calorimetric biosensors and others [68, 80], can be used to determine the amount of glucose in the blood, to detect renal diseases by monitoring urea concentra- tions in the body and sensing hybridized or single-stranded DNA. Also, cholesterol biosensors have become indispensable in the clinical diagnoses for estimating cholesterol in patients, whiles environmental biosensors show variations in alarm when harmful gases such as ammonia or formalin are present in high or low con- centrations in the environment. Also, food biosensors are used for detecting compounds such as glucose, sucrose, citric and ascorbic acid and many more in food for quality control purposes [81]. 2.10 Rationale The recent rise in epidemics has affirmed the need for the public health authority to gain interest in the readiness for a solution to curb disease occurrences, especially in the de- veloping world. Many advanced techniques are still not feasible in low and middle- 17 University of Ghana http://ugspace.ug.edu.gh income countries (LMIC) where they are most needed in the past few decades. Clearly, these performance parameters would offer an alternative diagnostic approach to healthcare in developing countries where resource constraints hinder the implementation and accessibility of sensitive but expensive diagnostic testing management. This work, therefore, seeks to develop easy-to-use and affordable diagnostic tools to help support the fight by the public health authority in confronting the challenges of low and middle-income countries. Electrochemical devices that are based on the simple principle would improve the qual- ity of laboratory diagnosis of diseases and afford desired clinical information in a sim- pler, faster, and cheaper manner such as the glucometer for sugar test. 18 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE MATERIALS AND METHODS 3.0 Introduction This chapter gives information on the basic materials used in the execution of the work as well as the equipment used and sample preparation procedures. 3.1 Materials Kaolin deposits from Amanfrom in the eastern part of Ghana was collected as the basic material for the production of the proposed zeolite as shown in figure 2. Sodium Hydrox- ide (NaOH) Pellets with 99% purity was used as the base to fuse the kaolin in order to form the zeolite. Phosphate buffered saline (PBS), pH 7.4, was obtained from Sigma- Aldrich, St. Louis, USA and used as a medium for the zeolite synthesis. The urease enzyme (urease active meal from Jack beans) was obtained from Sigma-Aldrich, St. Louis, USA and is used for the hydrolysis of urea. The target analyte; urea was obtained from Department of Chemistry, University of Ghana. Deionized water was also obtained from Millipore, Bedford, MA, USA purposely for the preparation of all solutions in the execution of the research. A pair of gold electrodes was obtained from Griffin and was used to create the bioelec- trode. A hot plate was obtained from IKAMAG RCT to be used to dehydrate the zeolite paste that was deposited on the gold electrodes surface. A furnace was also purchased from Thermo-Scientific, Waltham, MA, USA and used for high-temperature heating of 19 University of Ghana http://ugspace.ug.edu.gh the samples. Finally, a Metrohm AutoLab PGSTAT 204 electrochemical device was used to detect the working cells’ current. Figure 2: Harvesting of the kaolin from Amanfrom village in the eastern part of Ghana. 3.2 Methods The following describes the procedure used in the synthesis of the zeolite from kaolin and the steps followed in modifying the surface of the gold electrodes for the immobili- zation of the biological molecule (urease enzyme). Also, a stepwise procedure of impreg- nating the urease enzyme in the monolayers of the zeolite films are described. 3.2.1 Sample Preparation (Zeolite Synthesis) The zeolite synthesis was carried out using a combined approach involving alkaline fu- sion followed by the hydrothermal reaction as stated by Ríos et al. 2007 [68]. Briefly, 20 g of kaolin was ground into granules and sieved with 100 µm and 63 µm sieves simulta- neously (with 100 µm placed on top of the 63 µm). The residue in the 100 µm was then disposed of and the fine particles after the 63 µm sieve were weighed on a scale to obtain 15 g as depicted in figure 3a & b. 20 University of Ghana http://ugspace.ug.edu.gh (a) (b) Figure 3: (a) Depicts sieving of grounded kaolin, (b) Fine grains of kaolin sieved with 63 µm. The fine grade particles obtained and weighed after sieving was then poured into a cru- cible and calcined in a Thermo-Scientific furnace set at 650 °C for 120 minutes to obtain metakaolin as depicted in figure 4 [68]. (a) (b) Figure 4: (a) Shows crucibles filled with kaolin and placed in a furnace, (b) shows the furnace used to heat the kaolin to the desired temperature to obtain metakaolin. 21 University of Ghana http://ugspace.ug.edu.gh In order to have equal working and external conditions, all the kaolin samples were cal- cined in the same furnace at the same time. The metakaolin which appears in lumps as a result of decomposition as shown in figure 5 was ground and mixed with sodium hydrox- ide (NaOH) powder in a mass ratio of 2:3 (Kaolin: NaOH, w/w). Figure 5: Samples of alkaline fused metakaolin for the five different crystallization tem- peratures. The ensuing mixture was fused at 600 °C for 120 minutes in a furnace. The product obtained as a result of the alkaline fusion followed by the hydrothermal reaction was then ready to be mixed into a measured amount of deionized water. The mixture was then crushed into fine powder in a mortar to increase its reactivity with distilled water. The fine powder was then mixed with distilled (deionized) water in the mass ratio of 1:5 (powder: water, wt/wt) in a beaker and stirred to obtain a homogeneous mixture as shown in figure 6. The homogeneous mixture was kept at room temperature for 24 hours for gelation to take place since nucleation is highly affected by the aging time and tempera- ture. 22 University of Ghana http://ugspace.ug.edu.gh Figure 6: Alkaline fused metakaolin added to deionized water. The resulting gel was then crystallized at temperatures, 60 °C, 70 °C, 80 °C, 90 °C and 100 °C for 24 hours in an oven. The newly formed crystalline material was then removed from the oven. The reaction process was terminated by cooling in cold water. All the materials crystallized at these different temperatures had an average pH of 13.3, which makes them hazardous to handle. The pH was then reduced by constant filtration with deionised water. The samples were then dried in an oven at 60 °C for 24 hours. They were weighed, packed and labelled by their respective crystallization temperatures in falcon tubes as shown in figure 7a, with figure 7b depicting how the samples were packaged for bio- physical characterisation, namely, X-Ray Diffraction (XRD), Scanning Electron Mi- croscopy (SEM), Energy Dispersive X-Ray (EDX) and Brunner Emmett Teller (BET ). These were used in the characterisation of the crystalline materials formed and the ele- mental compositions of the end products were also identified. 23 University of Ghana http://ugspace.ug.edu.gh (a) (b) Figure 7: (a) Synthesized zeolites at different crystallization temperatures, (b) Packaged crystalline material for biophysical analysis. 3.3.0 Biophysical Characterization 3.3.1 X-Ray Diffraction (XRD) XRD is used in the identification of unknown crystalline materials or composites by gen- erating information on the crystalline components present in the material. The XRD was performed with a PanAnalytical Diffractometer as shown in figure 8 using CuKα radia- tion at 2θ, scanning from 5 to 50 degrees in steps of 0.05 degrees, with a tube voltage of 45 kV and a current of 40 mA. The d-spacing (ds) of the material is generated and an 24 University of Ghana http://ugspace.ug.edu.gh automated search engine compares the unknown ds to known ds, since each mineral has a unique set of d-spacing. This provides an identification of the unknown sample that is shown in Chapter 4. Figure 8: XRD equipment used for diffraction analysis at the University of Ghana, De- partment of Physics. 3.3.2 Scanning Electron Microscopy (SEM) SEM involves the interaction of electrons with materials of interest to produce various signals which are then used to obtain information about the surface morphology. SEM micrographs in this study were obtained at an accelerating voltage of 10 kV using a JEOL JSM-7100F field emission scanning electron microscope. Approximately 50 μm of col- loidal graphite (Agar Scientific Ltd, Stansted, UK) was uniformly spread on an aluminum stub. Each powdered sample was then deposited on the sticky colloidal graphite and ex- cess particles were removed by tapping the stub sideways followed by blowing with an aerosol duster. In this work, the topography of the crystalline material was observed. 25 University of Ghana http://ugspace.ug.edu.gh 3.3.3 Energy Dispersive X-Ray (EDX) This is an x-ray technique used for the elemental analysis or chemical characterization of a sample. Data from this technique is then used to analyse the energy spectrum in order to determine the abundance of specific elements. The EDX spectra and the elemental composition of each sample were obtained at an accelerating voltage of 10 kV using a JEOL JSM-7100F field emission scanning electron microscope equipped with a Thermo- Scientific UltraDry EDX detector. Each sample was then removed from the scanning electron microscope (SEM) machine after the EDX analysis and sputter-coated with three nanometres of gold. For the scope of this work, this technique was used to estimate Si: Al ratio by indicating the amount of each element present in the material of interest. 3.3.4 Brunner Emmett Teller (BET) BET entails the physical adsorption of gas molecules on a solid surface and serves as the basis for measuring the specific surface area, pore size distribution, particle size and dis- solution rate. This information is obtained and used to predict the number of biomole- cules that could be entrapped in the voids of the zeolite. Nitrogen adsorption measure- ments were performed at −196 °C on a micrometric ASAP 2020 volumetric adsorption analyser. Before the analysis, the samples were degassed under vacuum at 200 °C for 120 minutes in the port of the adsorption analyser. The result of this test is tabulated in the ensuing chapter. 3.4 Electrode Modification A pair of gold electrodes with a dimension of 3 mm x 40 mm and identical electrical characteristics were modified locally at the University of Ghana, Department of Physics. 26 University of Ghana http://ugspace.ug.edu.gh A thin film of zeolite was deposited on the gold electrode surface after heating the thin film of the paste on the electrode surface. The gold electrode was acting as the transducer, with one open-ended cylindrical dimen- sion obtained from Griffin. The active zone of the gold transducer was 3 mm x 40 mm (one open-ended cylinder) which gave an active surface area of 384.06 mm2 as depicted in figure 9 below. This modification was done in order to bake the transducer on a hot plate. Two identical gold electrodes were used to bring half-cell potential to zero. The surface of the transducer was enhanced to enable the adsorption of the biomolecule (ure- ase) onto its surface and for subsequent bio-recognition of the substrate (urea). Figure 9: Modified Griffin gold electrode. 27 University of Ghana http://ugspace.ug.edu.gh 3.5 Drop Coating To modify the surface of the gold electrode with the zeolite, the following procedure was followed to ensure the deposition of the zeolite membrane on the active zone of the sur- face of the gold electrode. This was achieved by mixing 0.5 g of Zeolite X (Na) in 0.25 ml of 0.1 mM phosphate buffered saline (PBS), pH 7.4 to obtain a paste. The active surface areas on the pair of electrodes were dipped in the paste for a maximum of 90 minutes to enable adhesion of the paste onto the surface of the electrode under room temperature as shown in figure 10 below. Figure 10: Active zone of gold electrode dipped into the zeolite paste. The zeolite modified gold electrode (GE) surface was placed on a heat block set at 200 °C for five minutes as shown in figure 11. This was done to dehydrate the layer of zeolite on the surface of the gold electrode without changing the orientation and the characteris- tics of the electrode. 28 University of Ghana http://ugspace.ug.edu.gh Figure 11: Zeolite modified gold electrode placed on heat block set at 200 °C for dehy- dration. This left a thin layer of zeolite spread over the active zone of the electrode as shown in figure 12 below. Both the working electrode and the reference electrode were dipped in the zeolite paste for all the crystallization temperatures. Figure 12: Thin layer of zeolite deposited on the gold electrode active zone after dehy- dration. 29 University of Ghana http://ugspace.ug.edu.gh 3.5 Immobilization of the Biomolecules One of the two zeolite modified gold electrodes was then dipped into urease and bovine serum albumin (BSA) solutions respectively for optimal absorption. The reference elec- trode (RE) pre-coated with Zeolite X (Na) at a particular crystallized temperature was dipped into 10% BSA solution for absorption onto the pore structure of the zeolite. The working electrode (WE) which was also pre-coated with Zeolite X (Na) of the same crys- tallization temperature was dipped into 2 ml of 10% urease solution (1 g to 10 g of PBS). The two electrodes were air dried for 20 minutes at room temperature in order to maintain the stability of the proteins. The inertness of the zeolite maintained the biomolecules in a stable state. The electrodes were then washed off in a working buffer to remove all adsorbed biomolecules from the surface of the electrode. 3.5.1 Second Drop Coating The modified gold electrode with a layer of zeolite spread over it, as shown in figure 12 above, was subsequently dipped into urease solution and again dipped into the same ze- olite paste, to have the enzyme covered with another layer of zeolite. After this coating, the electrode was then air dried at a temperature of 24 °C for 12 hours to keep the enzyme in its active state, since higher temperatures above 40 °C will dena- ture the enzyme. The electrodes were then washed off in a working buffer to remove all adsorbed biomolecules from the surface of the electrode. 30 University of Ghana http://ugspace.ug.edu.gh 3.6 Electrochemical Measurements Cyclic voltammetry (CV) measurements were taken using Metrohm AutoLab PGSTAT 204 interfacing a computer with NOVA 2.0 as the control setup in figure 13 below and operating software using Microsoft Windows 10 operating system with operational pa- rameters as displayed in table 1 below. Table 1: The parameter set used to generate the Cyclic Voltammetry (CV) measure- ments. Parameter Value Unit Step voltage 0.00244 Incremental V Start Potential 0 V (volts) Upper vertex potential -1 Vref Lower vertex Potential 1 Vref Scan rate 100 mV/s Working electrode (WE) 1 mA–100 nA A current Figure 13: Cyclic voltammetry setup, AutoLab PGSTAT 204, magnetic stirrer and bare gold electrodes. 31 University of Ghana http://ugspace.ug.edu.gh The zeolite modified gold electrodes (ZME) were dipped into the working cell containing 20 ml of 0.1 mM PBS solution. 1 mL of different concentrations of urea solution namely, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 mM were added to 10 different beakers containing 20 ml of 0.1 mM PBS at pH 7.4 in 25 ml working cells respectively. The resulting mixtures were homogenized with a magnetic stirrer for 180 seconds. The elec- trodes had similar treatments for each concentration of urea. The 10 different concentra- tions were chosen in order to plot a standard curve from their conductivity behaviour. The standard curve could be used to determine the concentration of unknown urea solu- tions. For the scope of this work, the electroanalytical method used to study the analyte is cyclic voltammetry. In this technique, the working cell current is measured while an activity that is occurring in the working cell between the urea and the urease alters the working cell’s potential. This causes potential changes that are transduced by the electrode into a current signal that is measured and interpreted as equivalent concentration. Figure 14: Phywe concentration & conductivity meter for analysing the conductance of the biosensor thus developed. 32 University of Ghana http://ugspace.ug.edu.gh The molar conductivity (ohm-1cm2mol-1) is also measured with Phywe conductivity meter as shown in figure 14. Conductivity depends on the availability of ions and the mobility of ions. As concentration increases, there is an increased number of ions but their mobility is restricted by the little available space to travel. This makes molar con- ductivity as described by Debye-Huckel-Onsager equation for weak electrolytes such as ammonia, acetic acid, tartaric acid and many more decrease with increasing analyte con- centration. The molar conductivity test was performed in order to obtain a standard curve that could be extrapolated to determine the concentration of unknown urea solution. 3.6.1 Cyclic Voltammetry (CV) Cyclic voltammetry is a very popular technique for electrochemical studies. It is useful for obtaining information about fairly complicated electrode reactions. It is also used to analyse the current at the working electrode which is used to estimate the analyte con- centration. The data from a CV is used to plot a graph called voltammogram as shown in figure 15 below. Figure 15: A voltammogram of a reversible analyte 33 University of Ghana http://ugspace.ug.edu.gh All Electrochemical experiments were conducted using VSP-300 Multichannel Potenti- ostat/Galvan Stat/EIS with a two-electrode system consisting of identical modified gold electrode. A BioLogic electrochemical analyser with EC-Lab® software was used for the acquisition of data and experimental control. The data analysis was carried out using the EC-Lab® software to identify the peak anodic and cathodic currents which was then used to estimate the concentration of the analyte with respect to Randles-Sevcik Equation (Equation 2). The peak anodic (ipa) and cathodic (ipc) currents were used to estimate the concentration of the analyte under study. The solution was subjected to a redox reaction which then causes electrical changes (resistance or current) near the surfaces of the electrode. The cyclic voltammetry behaviour of the various zeolite modified gold electrodes (ZME) was studied using urease enzyme as the probe specie against urea in a PBS solution. The bio- recognition signal, in this case, current (I), is proportional the concentration of the analyte by the Randles-Sevcik Equation (Equation 2). ip = (2.68 x 105) ACn3/2(DV) 1/2 ……………………….……. (2) “Where, i = Peak Current value (Anode & Cathode) p A = Electroactive Area of the Electrode C = Concentration of probe molecules in the solution D = Diffusion Coefficient of the molecules V = Scan Rate (Potential) n = number of electrons involved in the Redox Reaction” 34 University of Ghana http://ugspace.ug.edu.gh The molar conductivity of urea hydrolysis by urease was obtained from the cyclic volt- ammogram (CV) as depicted. The plot of the current (A) at the working electrode versus the potential applied (V) gives the cyclic voltammogram of the reaction, when the probe molecule (urease) reacts with the substrate (urea) at the surface of the electrode. The resulting electrical changes generate data that is used to plot a voltammogram. In this work Origin Pro 9.0 64-bit software was used in the analysis of the data and the plotting of the voltammogram. 3.7 Exponential Regression Fits and Molar Conductance Curves The peak current and concentration of urea was used to plot exponential decay regression fits and molar conductance curves using Origin Pro 9.0 64 Bit software. This was done to find a relationship between the conductance of each zeolite modified electrode against their molar concentration. And use to determine the zeolite that gave the best analytical response. 35 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR RESULTS 4.0 Introduction This chapter shows data acquisition from the biophysical characterisation and cyclic volt- ammetry experiments performed by the already mentioned equipment. 4.1 Biophysical Characterization 4.1.1 X-Ray Diffraction (XRD) The results obtained from the X-Ray Diffraction (XRD) confirmed Zeolite X (Na) as the crystalline material that was formed. This was because the signature peaks obtained from the XRD matched with that of Zeolite X (Na) for all the different crystallization temper- atures. The crystalline materials were labeled according to each crystallization temperature; this was because the overall yield of the crystalline material was Zeolite X (Na). It was labelled as follows, Zeolite X (Na) 60 °C, Zeolite X (Na) 70 °C, Zeolite X (Na) 80 °C, Zeolite X (Na) 90 °C and Zeolite X (Na) 100 °C respectively. Hence, they will be referred to as indicated above. Figure 16: XRD of graphs of Zeolite X (Na) synthesized at various temperatures. 36 University of Ghana http://ugspace.ug.edu.gh “K” and “Q” denote kaolinite and β-quartz peaks respectively with signature peaks of Zeolite X (Na) indicated in red as “x”. The graphs labelled a-f represents kaolin, Zeolite X (Na) 60 °C, Zeolite X (Na) 70 °C, Zeolite X (Na) 80 °C, Zeolite X (Na) 90 °C, Zeolite X (Na) 100 °C respectively. All the peaks labeled “x” in figure 16 are confirmed signature peaks of Zeolite X (Na). The overall yield of zeolite from the different crystallization temperatures was the same with a significant difference only being the peak intensities of the fingerprint peaks of the Zeolite X (Na). It can be concluded that crystallization temperature did not have an effect on the overall yield of zeolite formed, but rather the intensity of the peaks (area under the peaks) as demonstrated by C. Belviso et al [82]. 4.1.2 Scanning Electron Microscopy (SEM) The result of SEM displayed in Figure 17 below at a scale of 50 microns. Figure 17: SEM images of Zeolite X (Na) synthesized at 60 °C, (b) 70 °C, (c) 80 °C, (d) 90 °C and (e) 100 °C with scale bar = 50 micron. 37 University of Ghana http://ugspace.ug.edu.gh Most nano-porous materials such as zeolites are insulators and are particularly suscepti- ble to electron-beam damage and charging. For this reason, no accurate surface observa- tions have yet been performed. If the accelerating voltage is increased in an effort to acquire more detailed information, although the resolution increases, primary electrons penetrate the sample, yielding little surface information. However, our samples had iden- tical crystal structures, differing only in the aluminum content. 4.1.3 Energy Dispersive X-Ray (EDX) and Brunner Emmett Teller (BET) The Energy Dispersive X-Ray (EDX) results were compared and the respective Si: Al ratios were calculated and shown in the table below. The Brunner Emmett Teller (BET) results revealed distinct pore sizes, diameters and surface area for Zeolite X (Na) crys- tallized at different temperatures. Table 2: Characteristics of Zeolite Synthesized at Different Crystallization Temperature Pore Pore Average Si:Al Surface area Zeolite ratio diameter (m2/g) Volume Crystallite (nm) (cm3/g) size (nm) Zeolite X (Na) 60 °c 1.25 2.23 64.32 0.012 44.32 Zeolite X (Na) 70 °c 2.00 2.45 67.77 0.017 39.77 Zeolite X (Na) 80 °c 1.23 4.21 295.25 0.762 35.30 Zeolite X (Na) 90 °c 2.25 1.92 113.14 0.061 36.10 Zeolite X (Na) 100 °c 1.23 4.92 389.43 0.837 44.30 38 University of Ghana http://ugspace.ug.edu.gh 4.2 Effect of Base Materials The voltammetric behaviour of the bare electrode in the buffer solution with all the rea- gents w studied and shown in figure 18, this was performed in order to observe their effect on the electrochemical reaction using the setup from figure 12 in chapter 3. Figure 18: Shows the voltammetric behaviour of electrodes and reagents. The voltammogram of the buffer solution, urea and urease enzyme with the bare elec- trode were studied to ascertain its effect on the experiment. The buffer solution did not give any significant anodic peak current after the runtime. Also, urease enzyme on the bare gold electrode gave a peak current of an approximately 0.0014 µA after urea was added to the working cell. After the modification of the surface of the gold electrode with Zeolite X (Na), there was an indication of an increase in peak anodic current of 0.0021 µA as shown in figure 18. This compared with the biomolecule that was adsorbed on the bare electrode, indicated an electrocatalytic activity on the electrode surface. Zeolite X (Na) also permitted increased levels of the bio-receptor to be immobilised, which resulted in high-level electron transmission and improved analytical response. 39 University of Ghana http://ugspace.ug.edu.gh 4.2.1 Cyclic behaviour of Zeolite X (Na) at different crystallization temperature. The cyclic voltammetry (CV) behaviour of the Zeolite X (Na) 60 - 100 °C was then studied with urea concentrations from 0.1 mM - 1.0 mM. The figures 19-23 below show the unique behaviour of the zeolite prepared with urea concentrations from 0.1 mM to 1.0 mM. In this work, cyclic voltammogram results for urea 0.1, 0.5 and 1.0 mM and the overall behaviour of the 10 concentrations of the biosensor are depicted in the ensuing pages. The voltammogram shows the analytic behaviour of the biosensor that was developed for all the different crystallisation temperatures. The peak anodic current was calculated from the baseline of zero to the highest peak of the reaction within the runtime. This was indicated in black as a trace line from to the highest point (peak) of the plateau to the x- axis. 4.2.2 Cyclic voltammogram of bioelectrode with Zeolite X (Na) 60 °C in selected urea concentrations. The peak anodic current which is proportional to the concentration of urea that is hy- drolyzed by the urease enzyme on the bioelectrode surface with Zeolite X (Na) 60 °C for selected urea concentrations of 0.1 mM, 0.5 and 1.0 mM are as shown in figure 19 a-c respectively. All the 10 concentrations (0.1 mM – 1.0 mM) of urea for the same ze- olite are depicted in figure 19 d, with the various determined anodic currents tabulated in table 3. 40 University of Ghana http://ugspace.ug.edu.gh 0.00010 0.00005 0.00000 -1.0 -0.8 -0.7 -0.5 -0.3 -0.2 0.0 0.2 0.3 0.5 0.7 0.8 1.0 -0.00005 -0.00010 Potential applied (V) Figure 19: (a) Cyclic voltammogram of 0.1 mM urea. 0.00005 0.00000 -1.0 -0.5 0.0 0.5 1.0 -0.00005 Potential Applied (V) Figure 19: (b) Cyclic voltammogram of 0.5 mM urea. 41 Current Response (A) Current Response (A) University of Ghana http://ugspace.ug.edu.gh 0.00005 0.00000 -1.0 -0.5 0.0 0.5 1.0 -0.00005 Potential Applied(V) Figure 19: (c) Cyclic voltammogram of 1.0 mM urea. 0.0001 Urea (mM) 0.1 0.2 0.0000 0.4 0.5 0.6 0.7 0.8 0.9 1.0 -0.0001 -1.0 -0.5 0.0 0.5 1.0 Potential Applied (V) Figure 19: (d) Cyclic voltammogram of 0.1-1.0 mM urea superimposed for Zeolite X (Na) 60 °C modified biosensor. 42 Current Response (A) Current Response (A) University of Ghana http://ugspace.ug.edu.gh 4.2.3 Cyclic voltammogram of bioelectrode with Zeolite X (Na) 70 °C in selected urea concentrations The peak anodic current which is proportional to the concentration of urea that is hydro- lyzed by the urease enzyme on the bioelectrode surface with Zeolite X (Na) 70 °C for selected urea concentrations of 0.1 mM, 0.5 and 1.0 mM are as shown in figure 20 a-c respectively. All the 10 concentrations (0.1 mM – 1.0 mM) of urea for the same zeolite are depicted in figure 20 d, with the various determined anodic currents tabulated in table 3. 0.00005 0.00000 -1.0 -0.5 0.0 0.5 1.0 -0.00005 Potential Applied (V) Figure 20: (a) Cyclic voltammogram of 0.1 mM urea. 0.00005 0.00000 -1.0 -0.5 0.0 0.5 1.0 -0.00005 Potential Applied (V) Figure 20: (b) Cyclic voltammogram of 0.5 mM urea 43 Current Response (A) Current Response (A) University of Ghana http://ugspace.ug.edu.gh 0.00005 0.00000 -1.0 -0.5 0.0 0.5 1.0 -0.00005 Potential Applied(V) Figure 20: (c) Cyclic voltammogram of 1.0 mM urea. 0.0001 Urea mM 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.0000 0.8 0.9 1.0 -0.0001 -1.0 -0.5 0.0 0.5 1.0 Potential Appied (V) Figure 20: (d) Cyclic voltammogram of 0.1-1.0 mM urea superimposed for Zeolite X (Na) 70 °C modified biosensor. 44 Current Response (A) Current Resonse (A) University of Ghana http://ugspace.ug.edu.gh 4.2.4 Cyclic voltammogram of bioelectrode with Zeolite X (Na) 80 °C in selected urea concentrations The peak anodic current which is proportional to the concentration of urea that is hydro- lyzed by the urease enzyme on the bioelectrode surface with Zeolite X (Na) 80 °C for selected urea concentrations of 0.1 mM, 0.5 and 1.0 mM are as shown in figure 21 a-c respectively. All the 10 concentrations (0.1 mM – 1.0 mM) of urea for the same zeolite are depicted in figure 21 d, with the various determined anodic currents tabulated in table 3. Figure 21: (a) Cyclic voltammogram of 0.1 mM urea 0.0001 0.0000 -1.0 -0.5 0.0 0.5 1.0 -0.0001 -0.0002 Potential Applied (V) Figure 21: (b) Cyclic voltammogram of 0.5 mM urea. 45 Current Response (A) University of Ghana http://ugspace.ug.edu.gh 0.00005 0.00000 -1.0 -0.5 0.0 0.5 1.0 -0.00005 -0.00010 -0.00015 Potential Applied (V) Figure 21: (c) Cyclic voltammogram of 1.0 mM urea. 0.0004 Urea mM 0.1 0.2 0.0002 0.3 0.4 0.5 0.6 0.0000 0.7 0.8 0.9 1.0 -0.0002 -0.0004 -1.0 -0.5 0.0 0.5 1.0 Potential Applied (V) Figure 21: (d) Cyclic voltammogram of 0.1-1.0 mM urea superimposed for Zeolite X (Na) 80 °C modified biosensor. 46 Current Response (A) Current Response (A) University of Ghana http://ugspace.ug.edu.gh 4.3.5 Cyclic voltammogram of bioelectrode with Zeolite X (Na) 90 °C in selected urea concentrations. The peak anodic current which is proportional to the concentration of urea that is hydro- lyzed by the urease enzyme on the bioelectrode surface with Zeolite X (Na) 90 °C for selected urea concentrations of 0.1 mM, 0.5 and 1.0 mM are as shown in figure 22 a-c respectively. All the 10 concentrations (0.1 mM – 1.0 mM) of urea for the same zeolite are depicted in figure 22 d, with the various determined anodic currents tabulated in table 3. 0.00005 0.00000 -1.0 -0.5 0.0 0.5 1.0 -0.00005 Potential Applied (V) Figure 22: (a) Cyclic voltammogram of 0.1 mM urea. 0.00005 0.00000 -1.0 -0.5 0.0 0.5 1.0 -0.00005 Potential Applied (V) Figure 22: (b) Cyclic voltammogram of 0.5 mM urea. 47 Current Response (A) Current Response (A) University of Ghana http://ugspace.ug.edu.gh 0.00005 0.00000 -1.0 -0.5 0.0 0.5 1.0 -0.00005 -0.00010 Potential Applied (V) Figure 22: (c) Cyclic voltammogram of 1.0 mM urea. 0.00010 Urea mM 0.1 0.00005 0.2 0.3 0.4 0.5 0.6 0.00000 0.7 0.8 0.9 1.0 -0.00005 -0.00010 -1.0 -0.5 0.0 0.5 1.0 Potential Applied (V) Figure 22: (d) Cyclic voltammogram of 0.1-1.0 mM urea superimposed for Zeolite X (Na) 90 °C modified biosensor. 48 Current Response (A) Current Response (A) University of Ghana http://ugspace.ug.edu.gh 4.3.6 Cyclic voltammogram of bioelectrode with Zeolite X (Na) 100 °C in selected urea concentrations. The peak anodic current which is proportional to the concentration of urea that is hydro- lyzed by the urease enzyme on the bioelectrode surface with Zeolite X (Na) 100 °C for selected urea concentrations of 0.1 mM, 0.5 and 1.0 mM are as shown in figure 23 a-c respectively. All the 10 concentrations (0.1 mM – 1.0 mM) of urea for the same zeolite are depicted in figure 23 d, with the various determined anodic currents tabulated in table 3. 0.0010 0.0005 0.0000 -1.0 -0.5 0.0 0.5 1.0 -0.0005 -0.0010 Potential Applied (V) Figure 23: (a) Cyclic voltammogram of 0.1 mM urea. 0.0005 0.0000 -1.0 -0.5 0.0 0.5 1.0 -0.0005 Potential Applied (V) Figure 23: (b) Cyclic voltammogram of 0.5 mM urea. 49 Current Response (A) Current Response (A) University of Ghana http://ugspace.ug.edu.gh 0.0004 0.0002 0.0000 -1.0 -0.5 0.0 0.5 1.0 -0.0002 -0.0004 Potential Applied (V) Figure 23: (c) Cyclic voltammogram of 1.0 mM urea. 0.0010 0.2 0.3 0.4 0.5 0.6 0.0005 0.7 0.8 0.9 1.0 0.0000 -0.0005 -0.0010 -1 0 1 Potential applied (V) Figure 23: (d) Cyclic voltammogram of 0.1-1.0 mM urea superimposed Zeolite X (Na) for 100 °C modified biosensor. 50 WE current(A) Current Response (A) University of Ghana http://ugspace.ug.edu.gh 4.4 Data Analysis The peak anodic current was obtained from the various voltammograms; Table 3 depicts urea recognition by enzyme-substrate interaction. Table 3: Shows the peak anodic current for Zeolite X (Na) 60 °C - Zeolite X (Na) 100 °C obtained using EC-Lab® software. Urea in mM 60°C 70°C 80°C 90°C 100°C 0.1 0.00010733 8.87146E-05 0.000370239 7.70874E-05 0.000834656 0.2 8.95386E-05 8.05054E-05 0.000231384 7.34863E-05 0.000724196 0.3 8.95386E-05 8.65479E-05 0.000187012 7.36084E-05 0.000713196 0.4 9.41467E-05 6.93054E-05 0.000130981 7.37305E-05 0.000535828 0.5 8.30994E-05 6.12793E-05 0.000113647 6.47278E-05 0.000449036 0.6 8.28552E-05 5.99945E-05 0.00010788 5.82855E-05 0.000446747 0.7 7.37915E-05 5.99945E-05 0.00010788 6.24695E-05 0.000383118 0.8 6.81763E-05 6.72302E-05 0.0001008 5.90942E-05 0.000357513 0.9 7.15942E-05 6.604E-05 9.61304E-05 6.3446E-05 0.000357513 1 6.80542E-05 6.00891E-05 8.82263E-05 5.88226E-05 0.000278595 18 0 17 100 C 16 900C 15 800C 0 14 70 C 13 600C 12 11 10 9 8 7 6 5 4 3 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 Concentration of Urea (mM) Figure 24: Molar conductance curve of Zeolite X (Na) modified electrodes and the re- sponse of the biosensor as a function of urea concentration. 51 conductance(µS/cm ) University of Ghana http://ugspace.ug.edu.gh On the basis of the linear detection range of 0.1-0.8 and 0.1-0.6 mM of the curves at Zeolite X (Na) 80 °C and Zeolite X (Na) 100 °C in figure 24 we conclude that crystalli- zation temperature influences the conductance of Zeolite X (Na) 100 °C modified bio- sensors compared to Zeolite X (Na) 80 °C, but the regression coefficient prefers the for- mer over the later. 12 Conductance at 100°C (µS/cm) ExpDecay2 Fit of Sheet1 B"Conductance at 100°C" Conductance at 80°C (µS/cm) ExpDecay2 Fit of Sheet1 D"Conductance at 80°C" 10 8 R 2=0.989 R2=0.999 6 4 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Concerntration (mM) Figure 25: An exponential decay fit (type 2) of the conductance curve as a function of urea concentration by Zeolite X (Na) 100°C and Zeolite X (Na) 80°C respectively. The exponential and fitted values are represented by the scatter plots and the solid lines respectively. The R-squared values computed is an indication of a highly steady decay profile of conductance measured with respect to urea concentration for both Zeolite X (Na) 100°C and Zeolite X (Na) 80°C. 52 conductance(µS/cm) University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE DISCUSSION 5.1 Discussion The aim of this study was to investigate the effect of crystallization temperature on the kaolinite-based synthesis of zeolite and urease immobilization for potential fabrication of electrochemical urea biosensor. The synthesis of Zeolite X (Na) from kaolin was car- ried out as a function of the crystallization temperature, based on established methodol- ogies from literature. The crystal phase identification of the individual materials was car- ried out by X-ray diffraction-methods and validated using a collection of simulated XRD powder patterns for zeolites. As shown in figure 16, the signature peaks labeled “K” and “Q” denote kaolinite and β-quartz peaks respectively, typical of kaolin XRD diffraction patterns. After calcining and undergoing fusion before hydrothermal (crystallization tem- perature) reaction from 60 °C to 100 °C, three kaolinite peaks at position 9, 12.5 and 25 completely disappeared, with new additional peaks formed as indicated by “x” (figure 16b–f). All of the peaks labeled “x” are confirmed signature peaks of Zeolite X (Na). The peak intensities of Zeolite X (Na) were rather low, and this might be due to the formation of a small fraction of a crystalline phase sitting on an amorphous material. Nonetheless, all the crystallization temperatures produced Zeolite X (Na). The SEM re- sults of Zeolite X (Na) indicate that there are significant amounts of the amorphous phase, which reflects the results obtained from the low-intensity XRD spectra. However, with regards to the morphological arrangement of particles, Zeolite X (Na) synthesized at 80 °C and 100 °C revealed well-defined crevices (figure 16c and 16e). 53 University of Ghana http://ugspace.ug.edu.gh The Zeolite X (Na) 60 °C, Zeolite X (Na) 70 °C and Zeolite X (Na) 90 °C have compa- rable surface areas of 64, 68 and 113 m2/g respectively, with an average pore size of 2 nm. The Zeolite X (Na) crystallised at 80 and 100 °C revealed a larger surface area, 295 and 389 m2/g, respectively, with an average pore size of 4.5 nm, indicating that Zeolite X (Na) 80 °C and Zeolite X (Na) 100 °C can accommodate more urease. There is no clear explanation to the inconsistent behaviour of Zeolite X (Na) 90 °C with reference to the BET results obtained. These results are consistent with studies from poly (vinyl alco- hol)-modified sol-gel materials, graphene, and gold nanoparticles, as well as zeolites for fabricating a urea biosensor, demonstrated by Doong’s and other groups. The EDX data in Table 2 revealed that the Si/Al ratio is fairly constant, which is obvious because no additional source of Si and Al were introduced into the material. The estimation of crys- tallite size using Shearer’s equation as well as other analytical tools also showed no sig- nificant differences in particle size as a function of the crystallization temperature and, therefore, the observed differences. Cyclic voltammetry (CV) is an important and easy-to-use analytical tool to monitor charge transfer on an electrode surface through the electron transfer process. Urease was chosen due to its stability and has been widely applied in the fabrication of biosensors. The functioning of the urease biosensor is based on the reaction shown in Equation (1), in which the cleavage of urea into ammonium (NH +4 ) ions is as a result of proton con- sumption leading to charge transfer. Different methods for zeolite attachment to the trans- ducer surface were used, and dip coating with baking at 200 °C turned out to be the most suitable method. The parameter-sets in Table 1 were used to measure CV signals as a result of the catalysis of the urea by the immobilized urease enzyme. The analytical con- trol responses were measured as the cyclic voltammogram profiles for buffer only, buffer 54 University of Ghana http://ugspace.ug.edu.gh and urease, buffer and urea, buffer, urease and 0.1 mM urea, and buffer, bio-zeolite and 0.1 mM urea as shown in figure 18. It was observed that the reaction rate of the bio- zeolite modified electrode was faster and enhanced compared to the urease on the surface of the gold electrode alone using the same amount of urea substrate. Again when we compared the cyclic voltammogram of the pure kaolin with Zeolite X (Na) 80 and 100 °C, there was a unique analytical response from the latter, indicating that the response was due to the synthesized zeolite rather than the amorphous phase. As a control, we compared the results with the free urease and found that the sensitivity of the immobilized enzyme was higher. It is important to note that the pure kaolin did not exhibit any ana- lytical response. As seen in figures 19d, 20d, 22d, there is a poor response of the biosensor at these crys- tallization temperatures, with the rate of response being erratic for Zeolite X (Na) syn- thesized at 60, 70 and 90 °C as compared Zeolite X (Na) 80 °C and Zeolite X (Na) 100 °C (figure 21d, 23d). The Zeolite X (Na) material synthesized at 80 and 100 °C showed a better response than those synthesized at 60, 70 and 90 °C. This was further confirmed by the extremely high R-squared valued computed from the exponential decay fit model of their conductance curves. These peaks could be attributed to the oxidation and reduction of the enzymati- cally produced NH +4 ions. However, we cannot explain why there was no unique cyclic voltammogram of Zeolite X (Na) at 60, 70 and 90 °C even though the XRD spectra con- firmed the formation Zeolite X (Na). It is, however, speculated that there must be well- 55 University of Ghana http://ugspace.ug.edu.gh defined pore structures as in other two Zeolites for the effective immobilization of the substrate. As shown in figure 24, the molar conductance of developed bio-zeolites at different crys- tallization temperatures was measured as a function of urea concentration (mM). The random response of the Zeolite X (Na) 60 °C, Zeolite X (Na) 70 °C, and Zeolite X (Na) 90 °C modified electrode with respect to urea concentration was attributed to the fact that the morphology of the material was not well defined; this resulted in the inefficient ad- sorption of the urease on the material as already speculated. The sensitivity of the urea biosensor at these temperatures was high but inconsequential due to the heterogeneous nature of the pore structures. An exponential regression analysis revealed R2 values of 0.193, 0.299, 0.999, 0.350 and 0.989 for Zeolite X (Na) 60 °C, Zeolite X (Na) 70 °C, Zeolite X (Na) 80 °C, Zeolite X (Na) 90 °C and Zeolite X (Na) 100 °C respectively. In the case of Zeolite X (Na) 80 °C and Zeolite X (Na) 100 °C, there was a decay correlation due to the creation of a homogenous active surface area as a result of the formation of well-defined surface properties. On the basis of the detection limit of 0.1– 0.8 and 0.1– 0.6 mM of the curves at 80 and 100 °C respectively as shown in figures 24, 25, we speculate that the material properties were influenced by parameters such as the crystallization temperature and pH variations. Even though the analytical response of Zeolite X (Na) 100°C showed high detection sen- sitivity as compared to Zeolite X (Na) 80°C, the decay analysis s the latter on the basis of the computed R2 values. The cyclic voltammetry revealed oxidation and reduction peaks that indicate the successful immobilization of urease within the pore network in 56 University of Ghana http://ugspace.ug.edu.gh Zeolite X (Na) 80 °C and Zeolite X (Na) 100 °C. To confirm this assertion, N2 adsorption and desorption measurements were undertaken at 77 K and the results displayed in Table 2. In this work, the only parameter that was adjusted was the crystallization temperature. Therefore, we precluded that this parameter was a critical determinant of the material properties which could influence enzyme immobilization. Certain temperatures would produce zeolites with good adsorbing characteristics with enhanced analytical responses, such as good detection limits and linearity in response, which had been key for develop- ing good biosensors. It is also worth noting that, all the experiments were conducted without electrolytic (charged electrolytes) additives; thus it is recommended for future studies that conductive electrolytes such as poly (3, 4-ethylene dioxythiophene), PEDOT, NaCl and KCl that could be used to modify the zeolites in order to optimize their conductance without the use of electrodes. 57 University of Ghana http://ugspace.ug.edu.gh CHAPTER SIX CONCLUSION AND RECOMMENTATION 6.1 CONCLUSION In this study, we have demonstrated that zeolites synthesised from kaolin deposits in Ghana at different crystallization temperature can macromolecule immobilisation on the electrode surface. This has implication in the development of biosensors for various biomedical applications. The findings in this study suggest that crystallization temperature influences the mor- phology of the zeolite, which consequently affects the adsorbed enzyme on the material surface. We did not perform our experiments with other commercial zeolites and also could not investigate the erratic behaviours of Zeolite X (Na) 60°C, Zeolite X (Na) 70°C as well as Zeolite X (Na) 90°C and suggest further studies in this regard. The test-of- concept approach is a promising endeavour to investigate further attributes of zeolites as immobilization agents and comparing with similarly produced commercially available zeolites. 6.2 RECOMMENDATION We recommend that the response of the current zeolites be compared to their commercial available analogies. Further studies must also be conducted to determine the dynamic range of the detection. We also look forward to the advancement of producing screen printed kaolinite-base synthetic zeolite electrodes. The challenge with this work was that we could not estimate the amount of zeolite that is deposited on the surface of the gold 58 University of Ghana http://ugspace.ug.edu.gh electrode, even though the same amount of zeolite paste was employed for the experi- ments that we carried out. 59 University of Ghana http://ugspace.ug.edu.gh REFERENCES [1] N Bhalla, P Jolly. N Formisan, P Estrela. Introduction to biosensors. Essays in bio- chemistry, 60 (2016), pp. 1-8. [2] L. M. Muresan, Zeolite-modified electrodes with analytical applications. Pure Appl. Chem., 83 (2011), pp. 325–343. [3] M. D. Gouda, M. A. Kumar, M. S. Thakur, N. G. Karanth, Enhancement of opera- tional stability of an enzyme biosensor for glucose and sucrose using protein-based sta- bilizing agents, Biosens. Bioelectron. 17 (2002), pp. 503–507. [4] D. Y. Kim, M. Kim, S. Shinde, J. S. Sung and G. Ghodake, Cytotoxicity and antibac- terial assessment of gallic acid capped gold nanoparticles. Colloids and surfaces. 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Malhotra, “Application of conducting polymers to biosensors,” biosensors and bioelectronics, 17 (2002), pp. 345–359. [82] C. Belviso, F. Cavalcante, A. Lettino, S. Fiore. A and X-type zeolites synthesised from Kaolinite at low temperature, Applied Clay Science, 80–81 (2013), pp. 162–168. 69 University of Ghana http://ugspace.ug.edu.gh APPENDIX A Cyclic Voltammogram of Base Material Kaolin 0.00008 0.00006 0.00004 0.00002 0.00000 -0.00002 0.1mM -0.00004 0.5mM -0.00006 0.8mM 1.0mM -0.00008 -0.00010 -1.0 -0.5 0.0 0.5 1.0 Potential Applied (V) Figure 26: Cyclic Voltammogram of Kaolinite with selected Concentrations (0.1, 0.5, 0.8, 1.0 mM) of Urea There is no observed anodic and cathodic peak with the voltammogram from the base material Kaolinite. The observed peaks are as a result of the set parameters for the cy- clic voltammetry as indicated in Table 1. 70 Current Response (A) University of Ghana http://ugspace.ug.edu.gh APPENDIX B Exponential Fitting Data Generated by Origin Pro 9 Software for Conductance of Zeolite X (Na) 60 °C. Parameters Value Standard Error Conductance at 60 Intercept 10.601 1.96989 °C B1 -13.34924 8.22709 B2 13.53788 7.28889 Statistics Conductance at 60 °C Number of Points 10 Degrees of Freedom 7 Residual Sum of Squares 19.63608 Adj. R-Square 0.19278 Summary Intercept B1 B2 Statis- tics Value Stand- Value Stand- Value Stand- Adj. R- ard Er- ard Er- ard Er- Square ror ror ror Conduct- 10.601 1.96989 - 8.2270 13.5378 7.28889 0.19278 ance at 60 13.34924 9 8 °C Anova Conduct- DF Sum of Mean F Value Prob>F ance at 60 Squares Square °C Model 2 11.63961 5.81981 2.07468 0.1961 Error 7 19.63608 2.80515 Total 9 31.27569 71 University of Ghana http://ugspace.ug.edu.gh APPENDIX C Exponential Fitting Data Generated by Origin Pro 9 Software for Conductance of Zeolite X (Na) 70 °C. Parameters Value Standard Error Conductance of Zeo- Intercept 14.56 1.87684 lite X (Na) 70 °C B1 -11.88788 7.83847 B2 7.33333 6.94459 Statistics Conductance at 70 °C Number of Points 10 Degrees of Freedom 7 Residual Sum of Squares 17.82481 Adj. R-Square 0.29938 Summary Inter- B1 B2 Statistics cept Value Standard Value Standard Value Standard Adj. R- Error Error Error Square Conduct- 14.56 1.87684 - 7.83847 7.33333 6.94459 0.29938 ance at 70 11.8878 °C Anova Conduct- DF Sum of Mean F Value Prob>F ance at 70 Squares Square °C Model 2 14.88584 7.44292 2.92292 0.11945 Error 7 17.82481 2.5464 Total 9 32.71065 72 University of Ghana http://ugspace.ug.edu.gh APPENDIX D Exponential Fitting Data Generated by Origin Pro 9 Software for Conductance of Zeolite X (Na) 80 °C. Parameters Value Standard Error Conductance at Intercept 9.326 0.22205 80 °C B1 -11.50879 0.92739 B2 6.19697 0.82163 Statistics Conductance at 80 °C Number of Points 10 Degrees of Freedom 7 Residual Sum of Squares 0.24951 Adj. R-Square 0.98431 Summary Intercept B1 B2 Statis- tics Value Stand- Value Stand- Value Stand- Adj. R- ard Er- ard Er- ard Er- Square ror ror ror Con- 9.326 0.22205 - 0.92739 6.19697 0.82163 0.98431 duct- 11.50879 ance at 80 °C Anova Conductance DF Sum of Mean F Value Prob>F at 80 °C Squares Square Model 2 20.19085 10.09542 283.22668 2.00955E- 7 Error 7 0.24951 0.03564 Total 9 20.44036 73 University of Ghana http://ugspace.ug.edu.gh APPENDIX E Exponential Fitting Data Generated by Origin Pro 9 Software for Conductance of Zeolite X (Na) 90 °C. Parameters Value Standard Error Conductance at 90 Intercept 12.5195 1.11448 °C B1 -4.93356 4.65453 B2 2.04924 4.12374 Statistics Conductance at 90 °C Number of Points 10 Degrees of Freedom 7 Residual Sum of Squares 6.28512 Adj. R-Square 0.34987 Summary Con- Intercept B1 B2 Statistics ductance Value Stand- Value Standard Value Stand- Adj. R- at 90 °C ard Er- Error ard Er- Square ror ror 12.51 1.11448 -4.93356 4.65453 2.0492 4.12374 0.34987 95 4 Anova Conduct- DF Sum of Mean F Value Prob>F ance at 90 Squares Square °C Model 2 6.14453 3.07226 3.42171 0.09194 Error 7 6.28512 0.89787 Total 9 12.42965 74 University of Ghana http://ugspace.ug.edu.gh APPENDIX F Exponential Fitting Data Generated by Origin Pro 9 Software for Conductance of Zeolite X (Na) 100 °C. Parameters Value Standard Error Conductance at 100 °C Intercept 12.71617 0.37504 B1 -16.93568 1.56632 B2 9.00379 1.3877 Statistics Conductance at 100 °C Number of Points 10 Degrees of Freedom 7 Residual Sum of Squares 0.71174 Adj. R-Square 0.98001 Summary Intercept B1 B2 Statistics Value Stand- Value Stand- Value Stand- Adj. R- ard Er- ard Er- ard Square ror ror Error Con- 12.71617 0.37504 - 1 6 . 9 3 56 1.56632 9.00379 1.3877 0.98001 duct- ance at 100 °C Anova Conductance DF Sum of Mean F Value Prob>F at 100 °C Squares Square Model 2 45.07022 22.53511 221.63403 4.68483E-7 Error 7 0.71174 0.10168 Total 9 45.78196 75 University of Ghana http://ugspace.ug.edu.gh APPENDIX G PLAGIARISM REPORT Turnitin Originality Report Processed on: 19-Jul-2017 15:33 GMT ID: 831822523 Word count: 12331 Plagiarism check for “INVESTIGATING THE INFLUENCE OF TEMPERA- TURE ON KAOLINITE-BASE SYNTHESIS OF ZEOLITE AND UREASE IM- MOBILIZATION FOR POTENTIAL FABRICATION OF ELECTROCHEMI- CAL UREA BIOSENSOR” by Anderson Ebo David Similarity index: 13% Similarity by source Internet source: 7% Publications: 10% Student Papers: 2% 76