PHAGE AT THE AIR ? LIQUID INTERFACE FOR THE FABRICATION OF BIOSENSORS Except where reference is made to the work of others, the work described in this dissertation is my own or was done in collaboration with my advisory committee. This dissertation does not include proprietary or classified information. ________________________________________ Viswaprakash Nanduri Certificate of Approval: _________________________ Valery A. Petrenko Professor Department of Pathology _________________________ Vitaly J. Vodyanoy, Chair Professor Anatomy, Physiology and Pharmacology _________________________ James M. Barbaree Professor Biological Sciences _________________________ Tatiana Samoylova Research Assistant Professor Scott Ritchey Research Centre _________________________ Stephen L. McFarland Dean Graduate School PHAGE AT THE AIR ? LIQUID INTERFACE FOR THE FABRICATION OF BIOSENSORS Viswaprakash Nanduri A Dissertation Submitted to The Graduate Faculty of Auburn University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Auburn, Alabama December 16, 2005 iii PHAGE AT THE AIR ? LIQUID INTERFACE FOR THE FABRICATION OF BIOSENSORS Viswaprakash Nanduri Permission is granted to Auburn University to ma ke copies of this dissertation at its discretion, upon request of individuals or institutions and at their expense. The author reserves all publication rights. ______________________________ Signature of Author ______________________________ Date of Graduation iv VITA Viswaprakash Nanduri, son of Shri Chinnam Raju Nanduri and Sheshamma Nanduri, was born on January 05, 1965. He attended University of Madras, India, and earned his Bachelor?s Degree in Zoology in May, 1986. Viswaprakash earn ed his Masters in Marine Biology and Oceanography from the Centre of Advanced Study in Marine Biology and Oceanography, Annamalai University, India in May 1990. After working as a Fisheries Science teacher in the Republic of Maldives till December 1995, Vi swaprakash migrated to New Zealand with his family. After working for one year as a teacher at De La Salle College, Mangere East, Auckland, he earned his diploma in Business Computing 1996 and was employed as a Manager and System Administrator for Visual Tech Systems till June 2001. Viswaprakash joined the Doctoral program in Biomedical Sciences, with an emphasis in Biophysics and Biosensor Research in the College of Veterinary Medicine, Auburn University in fall 2001. Viswaprakash was born with 2 other siblings and has 2 older brothers, Aravind and Krishnanand Nanduri. Viswaprakash married Nilmini and has two sons, Ajitan and Ajay. During his stay at Auburn University Viswaprakash was awarded the Presidential Scholarship 3 years in a row and also was awarded the Outstanding Graduate Student award for 2005. v DISSERTATION ABSTRACT PHAGE AT THE AIR ? LIQUID INTERFACE FOR THE FABRICATION OF BIOSENSORS Viswaprakash Nanduri Doctor of Philosophy, August 13, 2005 (M.S., Annamalai University, India, May 1990) (B.S. University of Madras, India, May 1986) 144 Typed pages Directed by Dr.Vitaly J. Vodyanoy Food borne diseases cause an estimated 76 million illnesses, accounting for 325,000 hospitalizations and more than 5000 deaths in the United States each year. Currently, there are more than 250 known food borne diseases caused by different pathogenic microorganisms, including viruses, bacteria, fungi. Conventional methods of detecting pathogens entail a minimum of 24-48 hours of investigation, only after which results can be obtained. Apart from the urgent need of detection of food?borne pathogens, there is an even urgent need for the development of biosensors for the specific, sensitive and rapid detection of probable bio -terror agents. The general working principles of molecular recognition using thickness shear mode (TSM) sensors have been studied by employing different techniques such as formation of monolayer, and self assembled monolayers (SAM ). But, the specific mechanisms of molecular interaction between the vi probe-analyte that provides the sensitivity and specificity to the biosensor have not been thoroughly investigated. As a part of a project for environmental monitoring of biothreat agents, this work was done to determine if filamentous phage could be used as a recognition molecule on a sensor. E.coli obtained from ?-galactosidase (?-gal) was used as a model threat agent. Binding of ??gal to the selected landscape phage was characterized by enzyme linked immunosorbent assay (ELISA), thickness shear mode (TSM) and a surface plasmon resonance (SPR-SPREETA ?) sensors and responses obtained were compared. The landscape phage was immobilized through physical adsorption. The characteristics of the gold surfaces of both the TSM and SPR sensors were investigated using an atomic force microscope (AFM). The orientation of phage on formvar, carbon coated copper grids was also studied using a transmission electron microscope (TEM). Results obtained from 52 independent experiments showed a dose dependency in a range of 0.013 to 210 nM. The results of this work provided evidence that phage can be used as a recognition element on biosensors instead of antibodies and achieve detection in nanomolar ranges. Dose response curves indicated a stronger binding on a biosensor than that seen in ELISA. The sensitivity and specificity of phage peptide binding to an analyte envisages future applications of phage for the detection of bio-threat agents in bio -sensors. The sensitivity of both SPR and QCM sensor show similarities. The binding valences were 3.1 and 1.4 for the TSM and SPR sensor respectively. The apparent dissociation constants (Kd) are not significantly different It was observed that apparent Kd of the phage/? -gal complex was 2.8 nM ? 1.1 (S.D.) in TSM quartz sensor. The affinity valences of 2.3 ? 0.8 (S.D.) were estimated. AFM studies were conducted using a The vii SPM -100? (Nanonics Imaging Ltd, Jerusalem Israel) NSOM & SPM System for studying the effect of the cleaning procedures used for both the TSM and SPR sensors. While the control set showed an Rq (average roughness) of 45.9 nm, the treated TSM samples showed an Rq of 31.2 nm. The values obtained from the SPR sensors on the other hand, showed a much smaller difference in Rq values. viii Style manual used: Biosensors and Bioelectronics Computer software used: Microsoft Word, Microsoft Excel, Microsoft Power Point, Microcal Origin 6.0, Adobe Photoshop, Macromedia Fireworks MX. ix ACKNOWLEDGMENTS First and foremost, I wish to thank my parents whose constant encouragement and belief in me coupled with the sacrifices they underwent to provide me with a sound education has enabled me to tread this of doctoral research. Thanks go to both my brothers for their presence through thick and thin. I owe my deepest gratitude to my wife Nilmini and my sons Ajitan and Ajay for their encouragement during my research years. Much of my successful work would have never been possible without the persistent and gentle encouragement and guidance of Dr. Vitaly Vodyanoy. His simplicity of approach to research and the capacity to inspire the thirst for knowledge in his students make him a true mentor. I feel that I have been blessed for having been given an opportunity to work under his guidance. I wish to express my heartfelt thanks to Dr. Valery Petrenko, Dr. James Barbaree and Dr. Tatiana Samoylova for their endless support and guidance during my research. Special mention goes out to Dr. Valery Petrenko ; Prof. Aleksandr Simonian for introducing me to optical biosensor research under his expert guidance. Special thanks go out to Dr. Alexander Samoylov for initial training, and assistance; Oleg Pustovyy, Dr Galina A Kouzmitcheva and Dr. Iryna B Sorokulova, Dr. Maria A. Toivio Kinnucan, Dr. Minseo Park and Dake Wang. Thanks to my in-laws, Mrs. Nagapooshany, Rajmanna and Thirumalini for their personal support during my research years. I dedicate this research to my parents, brothers, my wife and my sons. x TABLE OF CONTENTS LIST OF FIGURES ????????????????????????.. xv LIST OF TABLES ????????????????????????? xvii 1. INTRODUCTION ??????????????????????. 1 1.1 Biosensor-definition and principles ??????????????. 1 1.2 Bio -recognition layer???????????????????? 3 1.3 The physical transducer ??????????????????? 4 1.3.1 Quartz Crystal Microbalance ????????????? 4 1.3.2 Surface Plasmon Resonance ????????????? 7 1.3.2a Overview of SPR based on Kretschmann geometry? 7 1.4 Atomic Force Microscopy?????????????????? 10 1.5 Transmission Electron Microscopy ??????????????. 11 1.6 References ???????????????????????? 12 2 LITERATURE REVIEW ???????????????????.. 15 2.1 Immunosensors ?????????????????????? 15 2.1.1 Antibodies ????????????????????. 15 2.2 Binding forces ??????????????????????. 16 2.2.1. Kinetics of Binding ????????????????. 17 2.2.2. Ligand Immobilization ??????????????.. 18 2.2.3. Mass Transfer ??????????????????.. 18 xi 2.3 Classical Sensor Platform ?????????????????? 19 2.3.1 Electrochemical ??????????????????. 19 2.3.2 Piezoelectric Acoustic ???????????????... 21 2.3.3 Evanescent wave optical sensing devices ????????. 24 2.3.3.1 Surface Plasmon Resonance ?????????.. 26 2.4 References ????????.????????.???????. 28 3. OBJECTIVES AND CONTRIBUTIONS OF THIS STUDY TO THE EXISTING LITERATURE ????????.??????????... 37 3.1 Validity of selected probe ? -galactosidase????????? 38 3.2 Physical micro/macro -environment-TSM sensor ?????? 38 3.3 Surface plasmon resonance sensor ???????????. 38 3.4 Compare binding studies using three platforms (ELISA, TSM, AND SPR)??????????????????????. 38 3.5 Atomic force microscopy ???????????????. 39 3.6 Transmission electron microscope ???????????... 39 4. OPTICAL PHAGE BIOSENSOR BASED ON SURFACE PLASMON RESONANCE SPECTROSCOPY???????????????? 40 Abstract ??????????????????????????.. 40 1. Introduction ???????????????????????? 40 2. Materials and Methods ???????????????????... 42 2.1 Phage ???????????????????????... 42 2.2 ?-galactosidase ??????????????????? 43 2.3 Solutions, reagents and tubing ????????????? 43 2.4 Miniature two -channel SPR sensor???????????.. 43 xii 2.5 SPR sensor batch mode setup ?????????????.. 44 3. SPR sensor preparations ??????????????????? 45 3.1 ?-galactosidase binding measurements ?????????? 45 3.1.1 Flow through mode ?????????????... 46 3.1.2 Batch mode ????????????????????????. 46 3.2. Specificity of binding ????????????????.. 46 4. Results and discussion ???????????????????? 47 4.1 Binding studies ??????????????????? 47 4.2 Phage deposition and surface coverage ?????????.. 48 4.3 Specificity of Binding ????????????????. 50 5 Conclusions ????????????????????????. 50 6 Acknowledgments ?????????????????????.. 51 7 References 65 5. PHAGE AS A MOLECULAR RECOGNITION ELEMENT IN BIOSENSORS IMMOBILIZED BY PHYSICAL ADSORPTION???... 69 Abstract ????????????????.??????????. 69 1. Introduction ???????????????????????? 70 2. Materials and Methods ???????????????????... 71 2.1 Phage ??????????????????????? 71 2.2 ?-galactosidase ??????????????????? 72 2.3 Solutions, reagents ?????????????????... 72 2.4 Phage Sensor preparation ??????????????? 72 2.5 ?-galactosidase binding measurements ??????? 73 xiii 2.5.1 Acoustic wave device??????????. 73 2.5.2 Binding measurements ?????????... 73 2.5.3 Specificity of binding ?????????? 74 2.6 Enzyme Linked Immunosorbent Assay (ELISA) with ?-galactosidase ???????????????????... 74 2.7 Binding Equations??????????????????. 75 3. Results and Discussion ???????????????????... 78 3.1 Specificity and selectivity of ?-galactosidase binding ???? 78 4. References????????????????????????... 85 6. COMPARATIVE PHAGE BASED BIOSENSOR RESPONSES FROM ELISA, THICKNESS SHEAR MODE SENSOR AND A SURFACE PLASMON RESONACE SPREETA? SENSOR 89 Abstract ??????????????????????????.. 89 1. Introduction ???????????????????????? 90 2. Materials and Methods ???????????????????... 91 2.1 Phage???????????????????????. 91 2.2 ?-galactosidase???????????????????.. 92 2.3 Materials ?????????????.................................. 92 2.3a ELISA, TSM and SPR sensor??????????. 92 2.3b Atomic force microscopy ???????????? 93 2.3c Transmission electron microscopy ????????. 93 3. Phage immobilization on sensors???????????????? 93 3.1 TSM sensor preparation ???????????????... 93 3.2 SPR sensor preparation ????????????????. 94 xiv 4. ?-galactosidase binding measurements ?????????????.. 94 4.1 Enzyme-linked immunosorbent assay (ELISA)??????? 94 4.2 Acoustic wave device??????????.??????.. 95 4.2.1 Binding measurements ????????????... 95 4.3 Surface plasmon resonance (SPREETA?) sensor?????... 96 4.3.1 SPR binding measurements??????????? 96 4.4 Atomic force microscopy???????????????. 96 4.4.1 AFM Imaging???????????????? 96 4.4.2 Surface roughness calculation ?????????... 97 4.4.3 Preparations of samples for AFM imaging ????... 97 4.5 Transmission electron microscopy???????????? 97 4.5.1 Negative staining ??????????????.. 97 4.5.2 Phage loading procedures ???????????. 98 5. Results and discussion??????????.?????????.. 98 5.1 ELISA and TSM sensor ??????????.?????.. 98 5.2 SPR and TSM sensor??????????.??????... 99 5.3 Atomic force microscopy ???????????????. 99 5.4 Transmission electron microscopy ???????????.. 100 6. Conclusions??????????.??????????.???... 101 7. References??????????.??????????????.. 119 7. CONCLUSIONS??????????.????????????? 123 xv LIST OF FIGURES 1.1 Working principle of a biosensor????????????????.. 2 1.2 Schematic representation of the various components of the phage used in this study??????????.???????????????.. 4 1.3 Piezoelectricity??????????.?????????????. 5 1.4 A TSM sensor setup on an anti vibration chamber?????????... 6 1.5 Surface Plasmon Resonance-Principle??????????????. 7 1.6 Surface Plasmon Resonance-angle shift?????????????... 8 1.7 Schematic of the miniature SPREETA? sensor??????????... 9 1.8 Scanner head of the Atomic Force Microscope??????????? 10 4.1 Schematic of the flow through mode setup ????????????.. 52 4.2 The batch mode setup??????????.??????????.. 53 4.3.A.a A full range dose response curve for a SPR sensor?????????.. 54 4.3.A.b Hill plots of binding isotherms from SPR sensors?????????? 55 4.3.B.a A low range dose response curve.???????????????? 56 4.3.B.b Hill plots of binding isotherms from SPR sensors?????????? 57 4.3.C.a A high range dose response curve.???????????????... 58 4.3.C.b Hill plots of binding isotherms from SPR sensors?????????? 59 4.4 Graph shows a typical example of addition of 1G40 phage??????. 60 4.5 A A full range dose response curve????????????????.. 61 4.5 B Hill plots of binding from SPR sensors?????????????? 62 xvi 4.6 Typical binding mean responses from SPR sensors????????? 63 4.7 Specificity of phage using SPR sensor??????????????. 64 5.1 Dose Dependent binding of ?-galactosidase to the phage??????? 81 5.2A Dose dependency binding of ? -galactosidase to TSM sensor and ELISA... 82 5.2B Hill Plots from binding isotherms of a TSM sensor and ELISA????.. 83 5.3 Specificity of ?-galactosidase binding in TSM sensor????????. 84 6.1 Dose responses from ELISA and TSM sensor???????????. 102 6.2 Hill plots of binding isotherms for ELISA and TSM sensor?????? 103 6.3 Dose responses from SPR and TSM sensors???????????? 104 6.4 Hill plots of binding isotherms for SPR and TSM sensors??????.. 105 6.5a(I) Surface Topography of a TSM sensor surface before cleaning????? 106 6.5a(II) Three dimensional features of a TSM sensor surface before cleaning??. 107 6.5b(I) Surface Topography of a TSM sensor surface after cleaning?????... 108 6.5b(II) Three dimensional features of a TSM sensor surface after cleaning??? 109 6.6a(I) Surface Topography of a SPR sensor surface before cleaning?????. 110 6.6a(II) Three dimensional features of a SPR sensor surface before cleaning??.. 111 6.6b(I) Surface Topography of a SPR sensor surface after cleaning?????? 112 6.6b(II) Three dimensional features of a SPR sensor surface after cleaning??? 113 6.7a TEM image of phage on formvar, carbon coated grid????????.. 115 6.7b TEM image of phage on formvar, without using a wetting agent???? 116 6. 8 TEM image of phage on gold gilded grids, without using a wetting agent.. 117 6.9 TEM image of phage diluted in Millipore water??????????.. 118 xvii LIST OF TABLES 6 T. 1 Mean surface roughness of sensor surface samples?????????.. 114 6 T.2 Comparative EC50 and effective Kd values of an SPR and a TSM sensor? 114 1 1. INTRODUCTION 1.1 Biosensors -definition and principles A biosensor is a device that integrates a biological sensing element with a physical transducer to provide us a signal for a specific target analyte. Biosensors are nowadays used in a wide range of fields from the detection of pollutants in the environment to the detection of pathogens in the food industry. More recently, the use of biosensors for the detectio n of bio-terrorist agents has seen an increased wave of research in this field. Classifications of biosensors are based upon either the type of biomolecules employed or on the type of physical transducer that is coupled with the biological element. Biomolecules ranging from enzymes, antibodies, receptors, tissues, whole cells, DNA and phage have been employed. Further classification of biosensors is based upon the type of action that is involved viz., biocatalytic sensors use enzymes, microorganisms, and tissue elements that are involved in the catalytic activity of a specific biological reaction; and bioaffinity sensors that revolve on molecular recognition by antibodies, receptors, binding proteins and phage. Biosensors are also classified based upon the physical transducer that is involved such as acoustic wave sensors (Quartz crystal microbalance/ Thickness Shear Mode (TSM), surface acoustic wave device) and optical sensors (surface plasmon resonance, fibreoptic and waveguides). A successful biosensor is one which fulfills high selectivity and sensitivity. Selectivity of the sensor is a function 2 of the sensing element and its ability to interact with its target analyte. Efficient detection of the interaction between the sensing element and the target analyte results in a highly sensitive biosensor. High specificity of a biosensor is defined by the degree of interaction between the sensing element and the target analyte, even in the presence of interferents. Many biosensors directly detect the presence of the target analyte doing away with the cumbersome process of addition of different reagents and thus adding cost and time to the detection process. The prevalent use of biosensors for the detection of bacteria[1-5], African swine virus[6] and even screening of phage libraries[7] has proved the versatility of biosensors in a wide arena of detection . Diagnostic kits for the detection of small SIGNAL Bio-recognition Specific analyte Fig 1.1 : Working principle of a biosensor NO SIGNAL Bio-recognition Layer Bio-recognition Non specific analyte TRANSDUCER 3 amounts of drugs are assisting both doctors and law enforcement officers in assessing drug abuse. 1.2 Bio -recognition layer For the development of an ideal biosensor, it is essential that both the essential components of the biosensor, the bio -recognition layer and the physical transducer be selected appropriately. In this study, we use filamentous phage as the bio-recognition layer. Phages are viruses that infect bacterial cells . Many phages are vectors used in recombinant DNA research and the standard recombinant DNA host is E. coli. Filamentous phage M13, f1 and fd are thread-shaped bacterial viruses. By inserting random peptides into their major coat protein (PVIII), a landscape library with billions of variations on the outer coat peptides is constructed. The library has been exploited for selection of phage that binds to the target analyte, ?-galactosidase (? -gal). Fig 1.2 shows a schematic of both the wild type (no modification to the outer coat) filamentous phage and a modified phage. The various major and minor coat proteins are shown. The phages selected in this study are flexible rods about 1.3? m long and 10nm in diameter and composed mainly of a tube of helically arranged molecules of the major coat protein pVIII. There is a single-stranded viral DNA inside the tube. Five copies each of the 4 minor coat proteins-pIII, pVI, pVII, pIX close off the ends of the sheath. 4 1.3 The physical transducer 1.3.1 Quartz Crystal Microbalance/Thickness Shear Mode (TSM) sensor The direct piezoelectric effect was discovered by the Curie brothers in 1880. When a weight was placed on a quartz crystal, charges appeared on the surface of the crystal that was proportional to the weight. When a voltage is applied to the crystal, deformation occurs due to lattice strains. This is reverse piezoelectricity and this effect was demonstrated in 1881. Piezoelectric crystals lack a center of symmetry. When a force deforms the lattice, the centers of gravity of the positive and negative charges in the crystal can be separated so as to produce surface charges . When a crystal has a center of Fig1.2: Schematic representation of the various components of the phage used in this study 5 + + + + + + + + + + + + + + + + + + + + + _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ + + + + + + + + + + + + + + + + + + + + + _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ + + + + + + + + + + + + + + + + + + + + + _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ + + + + + + + + + + + + + + + + + + + + + _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ symmetry, i.e., when the properties of the crystal are the same in both directions along any line in the crystal, no p iezoelectric effect can occur. The quartz crystal microbalance (QCM) also popularly called the thickness shear mode (TSM) sensor uses reverse piezoelectricity viz., when voltage is applied to the quartz substrate, it induces motion in thickness-shear mode. When mass on the crystal is Piezoelectricity Voltage Fig 1.3: Piezoelectricity The figure shows one example of the effect in quartz. Each silicon atom is represented by the blue spheres, and each oxygen atom by the red spheres . When a strain is applied so as to elongate the crystal along the Y-axis, there are net movements of negative charges to the left and positive charges to the right (along the X-axis). (Adapted from: R. A. Heising; Quartz Crystals for Electrical Circuits - Their Design and Manufacture, D. Van Nostrand Co., New York, pp. 16-20. 1946.) ?g ?g ?g 6 increased, due to binding of the probe to the analyte of interest, there is a decrease in frequency which is corresponds to an increase in output voltage. The TSM sensor?s characteristics of being small; rugged and entailing low costs offer significant advantages over other detection technologies. When TSM sensors are used with antibodies specific for the antigen under study, their sensitivity compares with techniques such as ELISA[8, 9] an d the use of TSM sensors for the detection of different pathogens have been well documented [10]. 1.3.2 Surface Plasmon Resonance Surface Plasmon Resonance (SPR) has become a well established tool for the characterization of biorecognition. Rapid, real time monitoring of the association and dissociation processes without the added chore of complex preparation of the sample is Fig 1.4: A TSM sensor setup on an anti vibration platform. 7 possible using the SPR technique. Recent years have seen the use of SPR for the detection and characterization of various molecular reactions. Through this platform, the sample to be investigated can be studied without any labeling; provide continuous real time monitoring; regeneration of the sensor?s surface using low pH wash and can be used for the detection of both chemical and biological warfare agents [11]. 1.3.2a Overview of SPR based on Kretschmann geometry Surface plasmon resonance (SPR) is a phenomenon that occurs at metal surfaces (usually gold or silver) when an incident p-polarized light beam strikes the surface at a particular angle, greater than the total internal reflection (TIR) angle. In our experiments, we use the Kretschmann geometry, where the metal (gold) surface is exposed to light transmitted via a transparent prism. As molecules bind to immobilized targets on the gold surface, the optical properties of the medium closest to the surface change (fig 1.6 a). This causes a proportionate shift in the SPR angle, (fig 1.6 b) which provides a quantitative measurement of the amount of mass binding . This in turn, can be used to determine a bio-molecular interaction in real time. In our experiments we used Free Plasmons l Fig 1.5: Surface Plasmon Resonance-Principle 8 SPREETA? sensors produced by Texas Instruments. This inexpensive dual channel, miniature sensors are very suitable for fundamental investigations. The SPREETA? is a highly integrated SPR sensor that uses the Kretschmann geometry and detects binding at the gold surface using the principle of angle interrogation. Fig1.7 depicts a schematic of the typical components of the device. SPREETA? mainly consists of light emitting diode, polarizer, temperature sensor, 2 photodiode arrays, reflecting mirror and an optical plastic substrate. An AlGaAs light emitting diode (LED) with a wavelength of 830nm is enclosed within an absorbing apertured box[12] . The LED light, after passing through a polarizer illuminates the gold coated glass slide with a wide range of angles. Reflecting mirror Flow Cell Dual channel Rubber gasket b Fig 1.6: Surface Plasmon Resonance-angle shift b a 9 Fig 1.7: Schematic of the miniature SPREETA? sensor. 1.7 a shows the frontal view of the two channels, showing the gasket that makes it possible. 1.7 b shows the various components of the sensor and the detachable flow cell. The polarizer filters and helps in the emission of only the transverse magnetic (TM) component as the transverse electric component cannot produce surface plasmon oscillations. The glass slide is positioned to get an appropriate SPR signal. The light from the gold surface strikes the mirror on top of the sensor and reaches the photodiode arrays. Protective encapsulation of all the components is provided by the optical substrate[12]. Moreover, this encapsulation also does away with the chore of optical alignment[13]. 1.4 Atomic Force Microscope AFM studies were conducted using a The SPM-100? (Nanonics Imaging Ltd, Jerusalem Israel) NSOM & SPM System. The system essentially consists of a) The NSOM/AFM 100? scan head which contains (i) a 9 pin outlet controlling the stepper motor that enables the scanning;(ii) a 15 pin outlet to a position sensitive detector and laser (iii) and lastly a 9 pin outlet that is connected to the scanner controller. (fig. 1.8) b) The Topaz controller which controls the scan and feedback mechanism. 10 c) The DT box which interfaces the computer to the controller through a 50 pin flat band cable d) A NSOM Topaz interface box that interfaces the controller to the scan head. e) And a personal computer with Quartz and data translation software that aids in data acquisition and image processing. Fig 1.8 shows the scan head with the different components The system uses a piezoelectric flat scanner (thickness 7 mm) with a scan range of 70 ?m Z-range and 70 ?m XY-range. Fiberglass cantilevered probes of 20 nm tips size were used for all our experiments, which were conducted at room temperature. The average surface roughness, Rq of the samples was calculated using the Quartz software (provided by the manufacturer) and is derived from the equation ? ??= 2)( 1 ZZNRq n Fig 1.8: The scanner head of the AFM with the different components Cantilever probe a (i) a (iii) a (ii) 11 Where N is the number of points in the defined area; zn, is the z values within the scanned area and z, the current z value. 1.5 Transmission Electron Microscope Transmission electron microscopy studies were carried out using the Philips 301 Transmission Electron Microscope (TEM) [FEI Company Hillsboro, Oregon]. This study was conducted as a prelude to understanding the orientation of phage. Formvar carbon coated /copper grids/formvar carbon gold guilder grids were incubated on 20 ?l drops of 3.18 ?10 11 vir/mL of phage solution for 20 minutes, membrane side down. The grids were then rinsed in a drop of 2% PTA so as to aid in the removal of excess non-adhered material and then placed in a second drop of the same stain preparation for 2 minutes. The grids were dried before examination under a Philips 301 TEM at 60 Kv. Representative fields were photographed at an original magnification of 71,000, magnified 2.75 times giving us a final magnification of 195,250. 12 1.6 References: 1. Carter, R.M., Mekalanos, J.J., Jacobs, M.B., Lubrano, G.J. & Guilbault, G.G., Quartz crystal microbalance detection of Vibrio cholerae O139 serotype. Journal of Immunological Methods, 1995. 187: p. 121-125. 2. Pathirana, S. T.,Barbaree, J.,Chin, B. A.,Hartell, M. G.,Neely, W. C.,Vodyanoy, V. Rapid and sensitive biosensor for Salmonella. Biosensors and Bioelectronics, 2000. 15(3-4): p. 135-141. 3. He, Fengjiao.,Zhang, Liude.,Zhao, Jianwen.,Hu, Biaolong.,Lei, Jingtian., A TSM immunosensor for detection of M. tuberculosis with a new membrane material. Sensors and Actuators B: Chemical, 2002. 85(3): p. 284-290. 4. Naimushin., Alexei N.Soelberg., Scott D.Nguyen., Di K.Dunlap., Lucinda Bartholomew., Dwight Elkind., Jerry Melendez., Jose and Furlong, Clement E. Detection of Staphylococcus aureus enterotoxin B at femtomolar levels with a miniature integrated two-channel surface plasmon resonance (SPR) sensor. Biosensors and Bioelectronics, 2002. 17(6-7): p. 573-584. 5. Olsen, E. V.,Pathirana, S. T.,Samoylov, A. M.,Barbaree, J. M.,Chin, B. A.,Neely, W. C.,Vodyanoy, V. et al., Specific and selective biosensor for Salmonella and its detection in the environment. Journal of Microbiological Methods, 2003. 53(2): p. 273-285. 6. Abad, J. M.,Pariente, F.,Hernandez, L.,Lorenzo, E. A quartz crystal microbalance assay for detection of antibodies against the recombinant African swine fever 13 virus attachment protein p12 in swine serum. Analytica Chimica Acta, 1998. 368(3): p. 183-189. 7. Hengerer, Arne.,Decker, Jochen.,Prohaska, Elke.,Hauck, Sabine.,Ko[ss]linger, Conrad.,Wolf, Han s Quartz crystal microbalance (QCM) as a device for the screening of phage libraries. Biosensors and Bioelectronics, 1999. 14(2): p. 139- 144. 8. Park I.S., Kim N., Thiolated Salmonella antibody immobilization onto the gold surface of piezoelectric quartz crystal. Biosensors and Bioelectronics, 1998. 13(10): p. 1091-1097. 9. Park, I.-S. and N. Kim, Thiolated Salmonella antibody immobilization onto the gold surface of piezoelectric quartz crystal. Biosensors and Bioelectronics, 1998. 13(10): p. 1091-1097. 10. Pyun J.C., B.H., Meyer J.U., Ruf H.H., Development of a biosensor for E. coli based on a flexural plate wave (FPW) transducer. Biosensors and Bioelectronics, 1998. 13(7): p. 839-845. 11. Alexei N. Naimushin., Charles B. Spinelli., Scott D. Soelberg., Tobias Mann., Richard C. Stevens., Timothy Chinowsky., Peter Kauffman., Sinclair Yee.,Clement E. Furlong. Airborne analyte detection with an aircraft-adapted surface plasmon resonance sensor system. Sensors and Actuators B: Chemical. 12. Naimushin., Alexei N.Soelberg., Scott D.Nguyen., Di K.Dunlap., Lucinda Bartholomew., Dwight Elkind., Jerry Melendez., Jose and Furlong, Clement E., Detection of Staphylococcus aureus enterotoxin B at femtomolar levels with a 14 miniature integrated two-channel surface plasmon resonance (SPR) sensor. Biosensors and Bioelectronics, 2002. 17(6-7): p. 573-584. 13. Kari Kukanskis., J.E., Jose Melendez., Tiffany Murphy., Gregory Miller., Harold Garner., Detection of DNA Hybridization Using the TISPR-1 Surface Plasmon Resonance Biosensor. Analytical Biochemistry, 1999. 274,: p. 7?17. 15 2. REVIEW OF LITERATURE 2.1 Immunosensors Immunosensors are biosensors that use antibodies as the recognition element. Spurred by the multi-million dollar industry of immunodiagnostics, interest in immunosensors has increased by leaps and bounds. Modern immunosensors are able to provide precise measurements of myriad analytes in complex mixtures. The convenience of not having to accurately pipette various reagents in a multitude of steps, rapidity of testing, portability, and simultaneous multi-analyte measurement are some of the distinct advantages of modern day immunosensors over the conventional methods. 2.1.1 Antibodies Immunosensors work on the principle of highly selective molecular recognition systems in order to determine the presence/absence and the amount of antigen. An antigen is any molecular species that is seen and identified by the body as foreign and triggers an immune response. The different classes of immunoglobulins (IgG, IgA, IgM, IgD, and IgE) are structurally related glycoproteins that differ in size, charge, amino acid composition, and carbohydrate content. Although antibodies are often chosen as the biological recognition element, this study uses filamen tous phage designed for specific binding to the model antigen, ?-galactosidase (? -gal). While molecular recognition 16 between antibody and the antigen is through epitope interaction, the specific method of molecular interaction between phage and ?-galactosidase is not clearly understood. Upon antigen challenge, a variety of antibodies are generated that, although they respond to the same antigen, bind to different sites on the antigen and have different affinities for that antigen. They belong to different subclasses and have differences in epitope specificity. 2.2 Binding Forces A number of forces that are present in the biomolecular reaction are responsible for the stabilization of the interaction between the antibody and antigen. These are forces such as electrostatic, Van der Walls, and hydrophobic interactions. Hydrogen bonding also plays a major part and along with the other forces make up for the affinity interactions between antibody and antigen[1]. Electrostatic interactions can be of two types: i) Repulsive or attractive forces between charged molecules ii) Diole-dipole interactions between highly polar molecules. Hydrogen bonds are considered as a type of electrostatic interactions. Dipoles that are weaker than those seen in electrostatic interactions exhibit Van der Waals forces . The temporary dipoles that are responsible for these forces are a direct result of the electric fields of nearby molecules. Although when taken singly, each of the forces is weak, the collective force from the several interactions can contribute up to 50% of the total binding strength [2]. Repulsive forces such as those seen between nonpolar molecules and water are called hydrophobic interaction. Existence of nonpolar regions at reaction sites, mainly as a result of entropy driven water exclusion and attainment of low favorable energy levels leads to intermolecular stabilization and increased binding strength[1]. 17 Other forces beside electrostatic interactions hydrogen bonding are also responsible for intermolecular stabilization and these other forces add on to the attractive forces in the interaction [1]. Repulsions between interpenetrating electron clouds of non-bonded atoms are a result of steric hindrances. The effect of these repulsive forces become minimal as the complement between the reactants increases [3]. 2.2.1 Kinetics of Binding Based on the basic thermodynamic principle governing antibody/phage-antigen/?-gal interactions in solution can be expressed by: gPhkkgPh d a bb ?+ (2.1) Where, Ph represents free phage, and ?g represents free ?-gal, Ph?g is the Phage-?-gal complex, and ka and kd are the association and dissociation rate constants, respectively. The equilibrium constant, or the affinity, is given by: [ ][ ] [ ]gPh gPhkkK d a b b == (2.2) Both the as sociation and dissociation are relatively quicker in solution and while the former is mostly affected by the diffusion of the reactants, the latter is mainly determined by the strength of the phage-?-gal bond. Whatever maybe the immobilization technique employed, immobilization can alter the properties of the antibody (or antigen), thus affects the binding kinetics [1]. 18 2.2.2 Ligand Immobilization The physical and chemical environment of the antibody-antigen complex is crucial for determining the sensitivity of the biosensor, be it TSM or SPR. Factors such the position of the antigen (?-gal) capturing areas of the antibody (Phage), after the latter has been immobilized on the surface of the sensor plays a vital role in understanding the conformational freedom of the immobilized phage. This is largely dependant on the immobilization techniques that have been employed such as Langmuir-Blodgett (LB) method [4],[5],[6] and molecular self-assembling of phage layer using biotin/streptavidin [7] This will in turn, determine the stability of the complex. Attachment of affinity ligands to the hydrogel matrix is also accomplished through well-known methods [8]. The most elementary method of ligand immobilization is nonspecific adsorption. This method has been employed successfully for the detection of African swine fever virus protein [9], IgG [10], anti-vibro cholera [11] and recombinant protein fragments of HIV specific antibodies [12] 2.2.3 Mass Transfer Transport of the target through the bulk solution such as that occurs in the Surface Plasmon Resonance (SPR) experiments, is governed by active transport and the kinetics of binding that govern ligand-target interactions. The bulk flow rate will affect the macroscopic transport through the system to the sensor surface[13]. Secondly, diffusion through the non-stirred boundary layer depends on bulk flow rate, geometry of the flow cell, and the diffusion coefficient of the target in solution[13]. Interactions between antibody and antigen in solution have been well understood. The binding of an antibody in solution to antigen immobilized on a surface has been described as a two -step 19 process[14]. Lateral interactions between macromolecules are thought to stabilize the adsorbed protein and antigen -antibody complexes on the surface, leading to an increase rate of binding and an increase in the antibody concentration near the surface[14]. 2.3. Classical Sensor Platforms Based on the measuring principle that is used, immunosensors can be classified as electrochemical, piezoelectric/acoustic and thermometric. Furthermore, all types can be categorized as either direct or indirect. Direct sensors are designed so that formation of the probe-analyte complex induces physical changes such as changes in frequency, mass electrode potential, membrane potential or the optical properties allowing for target measurement[15]. Materials such as electrodes, membranes, piezoelectric material, or optically active material surfaces are used to construct direct immunosensors. Indirect sensors rely on labels conjugated to either the antibody or antigen to visualize the binding event. Increased sensitivity can be achieved by the inclusion of enzyme s, catalysts, fluorophores, electrochemically active molecules, and liposomes as labels [15]. The final step must include incorporation of a label, which is then determined by optical, potentiometric or amperometric, measurements. The principles of the classical sensing platforms, including electrochemical, piezoelectric/acoustic, and optical immunosensors based on evanescent wave phenomenon will be discussed. 2.3 .1 Electrochemical Potentiometric and amperometric are the two basic electrochemical sensors. The changes in potential at an ion selective electrode are measured in a potentiometric sensor. These changes are with reference to the reference electrode. The electrodes are either submerged into a sample or separated from the sample by a membrane and placed into a 20 defined electrolyte solution. The measured potential difference taken with respect to the reference electrode is dependent on all potential differences that appear at the various phase boundaries, including that of the reference electrode and differences between electrolytes[16]. The most common potentiometric devices are pH electrodes and other ion -selective electrodes. The chief drawback of this system is that changes in potential due to antibody-antigen binding are very small (1-5 mV) and, consequently, limitations on the reliability and sensitivity due to background effects is present [17]. Amperometric devices function by measuring the current produced by the oxidation/reduction of an electro active compound at an electrode while a constant potential is applied to this electrode with respect to the second electrode. The glucose biosensor, which makes use of the electrochemical detection of the species produced (hydrogen peroxide) or consumed (oxygen) by the enzyme glucose oxidase, which is immobilized on an electrode surface is a typical example. Results from potentiometric immunosensors for syphilis and blood typing have been reported by[15, 18, 19], human chorionic gonadotropin (hCG) in solution by coating the electrode surface with anti-hCG[15]. Another type of potentiometric immunosensor is the ion -selective field effect transistor (ISFET) immunosensor. The ISFET is based on the field effect transistor (FET) used in electronics to detect voltage variations with minimal current drain. Detection of Heparin in the range of 0.3 to 2.0 units/mL by coating the sensor with a protamine (an affinity ligand) immobilized membrane has been reported [20]. The FET devices have practical problems associated with membrane performance [21]. Furthermore, FET drift, lack of selectivity and difficulty in making a stable, miniaturized reference electrode has made commercial development of these sensors difficult[20]. These potentiometric 21 immunosensors demonstrate insufficient sensitivity. A low charge density compared with background interferences such as ions of most biological molecules is responsible for the low signal-to-noise ratios. They also show a marked dependence of signal response on sample conditions such as pH and ionic strength [21]. 2.3 .2 Piezoelectric/ Acoustic devices Piezoelectric biosensors such as Quartz Crystal Microbalance (QCM) or Thickness Shear Mode (TSM) resonators have found a wide range of biosensing applications. Metal transducers (e.g. gold) on the surface of the crystal send acoustic waves into the material at ultrasonic frequencies. The potential of QCM/TSM devices in sensor applications was made possible after the derivation of the frequency to mass relationship by Sauerbrey [22] A mff ? ??? =? 26103.2 (2.3) where ?f is the change in fundamental frequency of the coated crystal in Hz, f is the fundamental frequency of the crystal(Hz), A is the resonator active area in cm2 and ? m is the mass deposited on the crystal in grams. The crystal orientation, thickness of the piezoelectric material, and geometry of the metal transducer determine the type of acoustic wave generated and the resonance frequency[23]. A change in weight on the crystal can be determined by measuring the shift in resonating frequency, wave velocity, or amplitude. The frequency shift of the piezoelectric crystal is proportional to mass change. Changes in acoustic wave propagation are then correlated to the amount of analyte captured on the crystal surface. TSM sensors have been used for the detection of immunoglobulins[24], antibodies for African swine virus[25], and S. typhimurium [5]. 22 The characteristics of the TSM sensor being small, rugged and entailing low costs offer significant advantages over other detection technologies. When TSM sensors are used with antibodies specific for the antigen in study, their sensitivity compares with techniques such as ELISA[26] and the use of TSM sensors for the detection of different pathogens have been well documented [27]. A bulk wave sensor was used to observe antibody in liquid phase[28]. The sensor surface was coated with goat anti-human immunoglobulin (IgG) either by attachment to a polyacrylamide gel with glutaraldehyde or by silylation onto the surface, then exposed to human IgG in solution. The advantage of the indirect method over the direct is that for a given amount of analyte bound, the mass of precipitate is much greater than that of the original bound analyte, hence sensor response is amplified. Variations of the acoustic wave sensor include the use of bulk acoustic waves, surface acoustic waves, and acoustic plate waves [15]. The influence of compressional wave generation on a TSM response in a fluid was investigated and it was shown that it does not affect the frequency shift[29]. By analyzing the frequency sifts and bandwidths of quartz coated resonators, a method to calculate the viscoelastic coefficients was derived [30]. A functional relationship between the frequency shift and the density and viscosity of the solution using a QCM was shown[31]. It was also shown that the changes in the oscillation frequency of a QCM in contact with a fluid is dependent on the material parameters of the fluid and quartz[32]. Use of QCM for the detection of gases[33-35], herbicide[36], polar and non polar halogenated organic chemicals [37], cell adhesion [38], endothelial cell adhesion [39], detection of microtubule alteration in living cells at nM nocodazole concentrations[40, 41], detection of M13 phages in liquids[42] and genetically modified organisms [43] have been reported. 23 The physical and chemical environment of the probe-analyte complex is crucial for determining the sensitivity of the biosensor[44], be it TSM or SPR. Factors such the position of the analyte (?-gal) capturing areas of the probe (Phage), after the latter has been immobilized on the surface of the sensor plays a vital role in understanding the conformational freedom of the immobilized phage. This will in turn, determine the stability of the complex. The stability, consistence and sensitivity of detection using the TSM sensor is limited by the type of immobilization that is being used. A myriad of immobilization methods have been tried and the optimal method one can employ relies mainly on the nature of the biological compound that needs to be immobilized. Immobilization techniques that have been employed are the Langmuir-Blodgett (LB) method[4-6], molecular self-assembling of phage layer using biotin/streptavidin [7], functionalized self assembled monolayers [45], surface modifications using Protein A[46- 48], Protein G[49] and enzymatic immobilization[50]. In our study, we propose to use a significantly simpler method of immobilization viz., physical adsorption. The strict relationship between the frequency change and the mass of BSA adsorbed was determined[51]. Physical adsorption technique has been employed successfully for the detection of African swine fever virus protein [9], IgG [10], anti-vibro cholera [11] and recombinant protein fragments of HIV specific antibodies [12]. The major criteria for an ideal active surface is that it should be chemically stable, contain a high surface coverage of the active sites of the immobilized material the coating should be as thin and uniform as possible[52]. All the above traits determine the sensitivity of the biosensor, as higher sensitivity and stable signals can be obtained by active, thin and rigid layers[53].The 24 importance of uniform coating of the immobilized layer in order to obtain accurate measurements using the QCM was also shown[54]. 2.3.3 Evanescent Wave Optical Sensing Devices The improvement of optoelectronics, availability of better fabrication materials and improved methods of signal generation and detection[15] has led to the rapid growth of optical immunosensors. Several types of optical transducers that are currently popular are Surface Plasmon Resonance (SPR) sensors, planar waveguides or fiber optic sensors. Detection of the probe-analyte binding by the optical immunosensors is achieved through changes in absorption, rotation, bio/chemi -luminescence, fluorescence or refractive index. Optical immunosensors can also be classified as direct, which depend solely on the binding of the probe-analyte binding to change the signal whereas the indirect optical immunosensors use labels to detect the binding events. Immunosensors that use evanescent waves detect target binding by measuring parameters such as absorbance, fluorescence, or refractive index. Surface plasmon resonance (SPR) is a phenomenon resulting from the presence of evanescent waves. Optical biosensors based on the evanescent wave (EW) use the principle of attenuated total reflection (ATR) spectroscopy and surface plasmon resonance (SPR) to measure real-time interaction between biomolecules. The basis of ATR is the reflection of light inside the core of a waveguide when the angle of incidence is less than the critical angle. Waveguides can be slab guides, planar integrated optics or optical fibers. Light waves are propagated along fibers by the law of total internal reflection (TIR). This law states that incident light striking nearly parallel to the interface between two media of differing refractive indices, 25 entering through the media of higher refractive index will be reflected or refracted according to Snell?s Law: 221 1 sin sin ?=? nn (2.4) where n1 is the higher refractive index (core), ? 1 is the incident ray angle through the core, n2 is the lower refractive index (cladding), and ? 2 is the angle of either internal reflection back into the core or refraction into the cladding. TIR occurs when the angle of incidence is greater than the critical angle. The critical angle is defined as: 1 21sin n n c ?=? (2.5) Although TIR occurs, the intensity does not suddenly fall to zero at the interface. The intensity exponentially decays with distance, starting at the interface and extending into the medium of lower refractive index. The EW is the electromagnetic field created in the second medium, which is characterized by the penetration depth. The penetration depth is defined as the distance from the interface at which it decays to 1/e of its value at the interface[55]. The wavelength of light, ratio of the refractive indices, and angle of the light at the interface determine the penetration depth [56]. The penetration depth (dp) is related to these factors by: 2/1 2 21 2 1 2 )sin(2 nn dp ??= p l (2.6) 26 where T1 represents the incident ray angle with the normal to the n1/n2 (core/cladding) interface, and ? represents the wavelength of light[57]. Typical penetration depths range from 50 to 1000 nm for visible light (dp