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One-Step Gold Nanoparticle Size-Shift Assay Using Synthetic Binding Proteins and Dynamic Light Scattering

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One-Step Gold Nanoparticle Size-Shift Assay Using Synthetic Binding Proteins and Dynamic Light Scattering One-step gold nanoparticle size-shift assay using synthetic binding proteins and dynamic light scattering By Thanisorn Mahatnirunkul Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds School of Biomedical Sciences, Faculty of Biological Sciences September 2017 I confirm that the work submitted is my own and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. Acknowledgement I would like to express my deepest gratitude to my supervisor, Prof. Paul Millner, for his invaluable guidance, support, advice, jokes and for being a great supervisor throughout my PhD time. I also would like to thank all members in the Millner group in the past and present for academic discussion in a very friendly manner especially Asif, Kaniz, Por and Shazana for all the food, cakes and coffee to comfort me after a long day of experiment. Special thanks go to Dr. Carolyn Jackson for being the best lab manager. Also, Dr. Jack Goode and Dr. Lewis McKenzie for your kind editorial assistance, I am really appreciated. I would like to thank the BSTG for Affimer production support especially my co-supervisors, Prof. Michael McPherson and Dr. Darren Tomlinson. As well as Anna, Cristian and Tom, thank you for being very patient with my limited molecular biology knowledge. I would also like to thank Prof. John Colyer for his guidance throughout this project. Additionally, I would like to thank all people who have facilitated my project in terms of equipment: Particle CIC in Faculty of Engineering for DLS, Martin Fuller for TEM, Dr. James Robinson for SPR, Rachel Gasior for ICP-MS and Dr. James Ault and his FBS mass spectrometry unit for all the mass spectra. Thank you for your trainings and special tips when using all the equipment. I would not have this PhD opportunity without my sponsor. Thank you the Royal Thai Government and National Nanotechnology Center of Thailand (NANOTEC) for financial support. Another group of person that I would like to thank is all my friends in Leeds for poker lessons, drinks, parties, late night talks and all the cherished memories we have been sharing during my stay in the UK. Especially the original Leeds 2012, poker gangster, Sukhothai crews and all MSc Bionanotechnology friends (especially Jo, Stella and Iril) without you guys my life here would not be completed. My endless thanks go to Aleena who always makes me laugh and keeps me sane along the journey. As well as all my Thai friends back home, thank you for always there for me whenever I need you guys. To my family, Daddy, Mommy and my sister Pearl aka Purzy, a very big thanks to you for the unmeasurable love and support you have given me all my life. I would not have come this far in life without you guys. I am really sorry for being far away for so long. I specially dedicate this work to all of you and I cannot wait to go home. i Abstract Gold nanoparticles (AuNPs) have attracted significant interest for biosensing applications because of their distinctive optical properties including light scattering. Dynamic light scattering (DLS) is an analytical tool used routinely for measuring the hydrodynamic size of colloids and nanoparticles in liquid environment. By combining the light scattering properties of AuNPs with DLS, a label-free, facile and sensitive assay has been developed. There have been several reports showing that NP- coupled DLS size shift assays are capable of quantitative analysis for target analytes ranging from metal ions to proteins as well as being a tool for biomolecular interaction studies. The principle of the assay developed is to immobilise bioreceptors (antibodies, oligonucleotides or synthetic binding proteins) specific to the target analyte onto AuNPs to produce nanobiosensors. When the analyte is added to the system, binding of the target protein to the immobilised bioreceptors leads to a size increase of the functionalised AuNPs. The hydrodynamic diameter (DH) can then be measured by DLS for complete quantitation. However, the ability to use synthetic binding proteins (Affimers) in optical sensing has not been investigated. Here, anti- myoglobin (Mb) Affimers were selected by biopanning of a phage display library and subcloned into a bacterial plasmid for expression in a prokaryotic system. These Affimers were then expressed and characterised before being used as bioreceptors in the NP-coupled DLS size shift assay. The Affimer functionalised AuNPs were compared to those using polyclonal antibodies (IgG) as bioreceptors. The Affimer nanobiosensors could selectively detect Mb with a limit of detection of 554 fM when multiple Affimer clones were immobilized onto the AuNPs, which was comparable to IgG based nanobiosensors (LOD = 148 fM). These findings suggest that in general a polyclonal reagent is optimum for the assay. In addition, other factors, such as AuNP size and concentration, related to the assay were investigated. The detection range of the size shift assay could be tailored to each analyte by selecting the appropriate AuNP size and concentration. This fundamental data will serve as a base for future studies of using Affimers in DLS based sensing applications. ii Abbreviations 10Fn3 10th domain of fibronectin type 3 AAS Atomic absorption spectroscopy Ab Antibody AF Aflatoxin Affimer-AuNP Affimer conjugated gold nanoparticle AFM Atomic force microscopy AFP Alpha-fetoprotein Ag@Au CSNP Silver-core gold-shell nanoparticle AgNP Silver nanoparticle AuNP Gold nanoparticle AuNR Gold nanorod Biotin HPDP N-[6-(biotinamido)hexyl]-3’-(2’-pyridyldithio)propionamide Biotin NHS Biotin N-hydroxysuccinimide BLI Bio-layer interferometry BPH Benign prostate hyperplasia BSTG The Leeds BioScreening Technology Group cal Calprotectin CDK Cyclin-dependent kinases cDNA Complementary DNA CDR Complementarity determining region CEA Carcinoembryonic antigen CFCA Calibration-free concentration analysis CM Carboxymethylated dextran Con A Concanavalin A CTAB Cetrimonium bromide Cys Cysteine Cyt c Horse heart cytochrome c 퐷 Translational diffusion coefficient DCS Differential centrifugal sedimentation DH Hydrodynamic diameter DHLA Dihydrolipoic acid iii DLS Dynamic light scattering DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid dNTP Deoxynucleotide DSC Differential scanning calorimeter dsDNA Double stranded DNA DTSSP 3,3’-dithiobis(sulfosuccinimidyl propionate) DTT Dithiothreitol ECL Enhanced chemiluminescence E.coli Escherichia Coli EDA 1,2-ethylenediamine EDC 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide EDTA Ethylenediaminetetracetic acid ELISA Enzyme-linked immunosorbent assay FRET Fluorescence resonance energy transfer GADPH Glyceraldehyde 3-phosphate dehydrogenase GC-MS Gas chromatography-mass spectrometry GOx Glucose oxidase GSH Glutathione HBsAg Hepatitis B antigen hCG Human chorionic gonadotropin HER2 Human epidermal growth factor receptor type 2 HIV Human immunodeficiency virus HPLC High-performance liquid chromatography HPV Human papilloma virus HRP Horseradish peroxidase ICP-MS Inductively coupled plasma mass spectrometry IgG Immunoglobulin G IgG-AuNPs IgG conjugated gold nanoparticle IO Iron oxide IPTG Isopropyl β-D-1-thiogalactopyronoside Ka Association rate constant iv Kb Dissociation rate constant KD Dissociation constant kcps Kilo counts per second LB Luria-Bertani LOD Limit of detection LRR Leucine-rich repeat modules LSPR Localised surface plasmon resonance mAb Monoclonal antibody Mb Myoglobin Mb-AuNP Myoglobin conjugated gold nanoparticle MC-LR Microcystin-LR Met Methionine MFP Mean free path MI Myocardial infarction miRNA Micro RNA MMP Micro magnetic nanoparticle MMMQ Melamine monomer’s migratory quantity Mr Molecular weight MRSA Methicillin-resistant Staphylococcus aureus MUA 11-mercaptoundecanoic acid MWCO Molecular weight cut-off Ni2+-NTA Nickel-nitrilotriacetic acid NNLS Non-negative least squares NOS Nopaline synthase NP Nanoparticle NTA Nanoparticle tracking analyis OD Optical density pAb Polyclonal antibody pagA Protective antigen precursor PAP Prostatic acid phosphatase p-ATP Para-aminothiophenol PBS Phosphate buffered saline v PCR Polymerase chain reaction PCS Photon correlation spectroscopy PDB Protein data bank PDI Protein disulphide isomerase PEG Polyethylene glycol pI Isoelectric point PNA Peptide nucleic acid PP Pancreatic polypeptide ppb Parts per billion ppm Parts per million ppt Parts per trillion PSA Prostate specific antigen QCM Quartz crystal microbalance QCM-D Quartz crystal microbalance with dissipation monitoring QD Quantum dot RB Rhodamine B RIA Radioimmunoassay RNA Ribonucleic acid RTags Raman tags SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis SELEX Systematic evolution of ligands by exponential enrichment SERS Surface-enhanced Raman spectroscopy SLS Static light scattering SOB Super optimum broth SOC SOB with catabolite repression SPR Surface plasmon resonance ssDNA Single stranded DNA ssRNA Single stranded RNA strep-AuNP Streptavidin coated gold nanoparticle sulfo-NHS N-hydroxysulfosuccinimide TCEP Tris(2-carboxyethyl)phosphine hydrochloride TEM Transmission electron microscopy vi TMB 3,3’,5,5’-tetramethylbenzidine TNF-α Tumour necrosis factor alpha TNT 2,4,6-trinitrotoluene TrxA Thioredoxin TxB Clostridium difficile toxin B VOC Volatile organic compound WHO World Health Organization vii Table of Contents Acknowledgement ……………….……………….……………….……………….……………….………….….i
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