Surface plasmon resonance (SPR) biosensor for rapid detection of Salmonella and Salmonella infections [Elektronische Ressource] / vorgelegt von Saikat Datta Mazumdar

Dattamaz

Sujets

• Gesundheit

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 36
• Langue: Deutsch
• Poids de l'ouvrage: 9 Mo

Extrait

Surface plasmon resonance (SPR)
biosensor for rapid detection of Salmonella
and Salmonella infections

Dissertation
zur
Erlangung des Doktorgrades
der Naturwissenschaften
(Dr. rer. nat.)

dem
Fachbereich Pharmazie, der Philipps-Universität Marburg

vorgelegt von
Saikat Datta Mazumdar
aus Shillong, Indien

Marburg/Lahn 2008Die Untersuchungen zur vorliegenden Arbeit wurden im Zeitraum von Juli 2005 bis
September 2008 am Fachbereich Pharmazie, Institut für Pharmazeutische Chemie
der Philipps-Universität Marburg unter der Leitung von Herrn Prof. Dr. Michael
Keusgen durchgeführt.





















Vom Fachbereich Pharmazie der Philipps-Universität Marburg als Dissertation
angenommen am: 19.09.08

Erstgutachter: Prof. Dr. Michael Keusgen
Zweitgutachter: Prof. Dr. Maike Petersen
Tag der mündlichen Prüfung am: 22.09.2008 This work is dedicated to my Parents and Elders
and inspired by my wife and our son Shyamanjan
(Riddhi) Table of Contents
Acknowledgements .................................................................................................... 1
List of Abbreviations ................................................................................................. 2
1 Introduction........................................................................................................ 3
1.1 Biosensors.................................................................................................... 3
1.1.1 Classification of Biosensors..................................................................... 3
1.1.1.1 Electrochemical sensors................................................................... 3
1.1.1.2 Nanomechanical sensors.................................................................. 6
1.1.1.3 Optical sensors................................................................................. 7
1.1.2 Surface Plasmon Resonance (SPR) ......................................................... 9
1.1.2.1 Fundamentals of SPR....................................................................... 9
1.1.2.2 Detection formats in SPR .............................................................. 14
1.1.2.3 Biorecognition elements in surface plasmon resonance ................ 16
1.1.2.4 Antibodies...................................................................................... 19
1.1.2.5 Antibody binding proteins ............................................................. 21
1.1.2.6 Biotin-avidin and biotin-streptavidin interactions ......................... 23
1.1.2.7 SPR–based biosensors for detection of bacteria ............................ 25
1.2 Salmonella ................................................................................................. 28
1.2.1 Taxonomy and nomenclature............................................................. 28
1.2.1.1 Kauffmann-White-Le Minor scheme (formerly Kauffmann-White
scheme) .............................................................................................. 30
1.2.1.2 Designation of antigenic formula of Salmonella serotypes ........... 33
1.2.1.3 Serotyping of Salmonella............................................................... 35
1.2.2 Salmonella Lipopolysaccharide......................................................... 36
1.2.2.1 Gram-negative and Gram-positive bacteria................................... 36
1.2.2.2 Structure of LPS............................................................................. 38
1.2.3 Salmonellosis..................................................................................... 41
1.2.3.1 Salmonellosis in humans ............................................................... 42
1.2.3.2 Salmonellosis in pigs ..................................................................... 42
1.2.3.3 Pathogenesis of Salmonella infection............................................ 43
1.2.3.4 Immunity to Salmonella in animals............................................... 44
1.2.4 Detection of Salmonella and Salmonella infections.......................... 45
1.2.4.1 Microbiological detection.............................................................. 45
1.2.4.2 Biochemical identification............................................................. 49
1.2.4.3 Serological detection...................................................................... 50
1.2.4.4 Polymerase chain reaction (PCR).................................................. 51
2 Aims and objectives ......................................................................................... 53
3 Materials and Methods.................................................................................... 55
3.1 Materials .................................................................................................... 55
3.1.1 Chemicals........................................................................................... 55
3.1.2 Buffers ............................................................................................... 57
3.1.3 Samples.............................................................................................. 58
3.1.4 Salmonella serovars ........................................................................... 59
3.1.5 Culture media..................................................................................... 59
3.1.6 Antibodies.......................................................................................... 60
3.1.7 Equipments ........................................................................................ 61
3.1.8 Kits and consumables ........................................................................ 62
3.2 Methods ..................................................................................................... 62
3.2.1 Culture and preparation of Salmonella antigens................................ 62
3.2.2 ELISA for detection of Salmonella infection .................................... 63
3.2.3 Modification of antibodies................................................................. 65
3.2.3.1 EDC/sulfo-NHS activation of carboxyl functional groups............ 65
3.2.3.2 Oxidation of the carbohydrate side chain of antibody................... 67
3.2.3.3 Antibody biotinylation................................................................... 68
3.2.4 Surface modification (functionalisation) of SPR chips ..................... 69
3.2.4.1 Cleaning and activation of the gold surface................................... 69
3.2.4.2 Amine coating of SPR prisms........................................................ 70
3.2.4.3 Biotin coating of SPR prisms......................................................... 70
3.2.4.4 Alkylation of SPR prisms (Hydrophobic or C coating) .............. 71 18
3.2.4.5 Carboxyl coating of SPR prisms.................................................... 72
3.2.4.6 Cobalt-coated SPR prisms ............................................................. 74
3.2.4.7 LPS-coated SPR prisms................................................................. 75
3.2.4.8 Contact angle (hydrophobicity) measurement of SPR chips......... 75
®3.2.5 Plasmonic SPR device ..................................................................... 76
3.2.5.1 Setup .............................................................................................. 76
3.2.5.2 Optics............................................................................................. 78
3.2.5.3 Calibration of the SPR device........................................................ 80
®3.2.6 SPR-based assays on Plasmonic ...................................................... 81
®3.2.6.1 Detection modes on Plasmonic SPR device ................................ 81
3.2.6.2 Assay setup of the SPR-based assay for detection of Salmonella . 82
3.2.6.3 SPR-based serogrouping protocol ................................................. 83
3.2.6.4 Serotyping protocol for SPR-based serotyping of S. Enteritidis ... 83
3.2.6.5 Assay setup of the SPR based assays for detection of Salmonella
infection ............................................................................................. 84
3.2.7 Determination of antibody concentration .......................................... 85
3.2.8 Acid hydrolysis of LPS to remove Lipid A ....................................... 85
3.2.9 Kinetic analysis.................................................................................. 86
3.2.9.1 Theory of kinetic analysis.............................................................. 86
3.2.9.2 Kinetic analysis using “initial rate analysis” model of Edwards and
Leatherbarrow................................................................................................ 87
4 Results............................................................................................................... 89
4.1 Detection of Salmonella............................................................................. 89
4.1.1 Capture antibody immobilisation....................................................... 89
4.1.1.1 Immobilisation on a carboxyl functionalised gold surface............ 89
4.1.1.2 Immob