Browsing by Author "Taykoz, Damla"

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Development of a plasmonic biosensor for detection of exosomes
(Izmir Institute of Technology, 2020-07) Taykoz, Damla; Bulmuş Zareie, Esma Volga; Tekin, Hüseyin Cumhur
The aim of this work was to develop Localized Surface Plasmon Resonance (LSPR) surfaces for quantitative detection of exosomes from different sources. For this aim, gold nanorods (AuNRs) with a mean diameter of 40 nm with an aspect ratio of 2.9 were first synthesized and characterized. The self-assembly of AuNRs on glass wafers were optimized through several experiments. In parallel, PEGylation of cetrimonium bromide (CTAB) stabilized AuNRs was investigated using PEGs with three different molecular weights via LSPR, zeta potential and XPS techniques. PEGylated AuNRs were further self-assembled on silanized microscope slides as confirmed. Surface functionalization of AuNR patterned slides was performed using alkane thiol molecules having carboxylic acid and hydroxyl functional groups and confirmed via XPS, FTIR and zeta potential. Specific antibodies (Ab) were conjugated to the surface following two different methods, i.e. click and NHS/EDC chemistry. To perform click chemistry strategy, ImmuneLink® molecules were conjugated with Abs and the final conjugate was used to functionalize surfaces prepared beforehand using azide bearing molecules. The functionalization procedure was confirmed via XPS FTIR and LSPR spectroscopy. The orientation of the antibodies on the AuNRs patterned surfaces was investigated with LSPR in comparison with conventional EDC/NHS chemistry. The click-chemistry strategy proved to provide conjugation of antibodies through their Fc regions exposing Fab regions better for antigen recognition. Finally, surfaces functionalized with a variety of antibodies were used to detect first a pregnancy-associated protein, PLAP, and then exosomes obtained from human semen samples with pre-determined exosome concentrations. The LoD of the biosensor surfaces was found to be between 103-104 exosomes/mL and 5 ng/mL (0.3 pM) PLAP. Human breast cancer cell culture samples having an unknown concentration of exosomes were further analyzed using the newly developed LSPR biochips and the exosome concentration was determined as 108 exosomes/mL for MCF-7 cell line and 107 exosomes/mL for MDA-MB-231 cell line.
Development of arginine-containing well-defined polymers
(Izmir Institute of Technology, 2014-07) Taykoz, Damla; Bulmuş Zareie, Esma Volga
The aim of this work is to synthesize arginine-containing well-defined polymers via reversible addition-fragmentation chain transfer (RAFT) polymerization and perform preliminary investigation on the use of these polymers in nucleic acid complexation for potential gene therapy applications. Pentafluorophenyl methacrylate (PFMA) was chosen as an active ester monomer to produce polymers having functional groups available for further modification. RAFT polymerization of PFMA was performed varying polymerization conditions such as feed composition and polymerization temperature. Polymers (PPFMA) were characterized using nuclear magnetic resonance (NMR) spectroscopy and gel permeation chromatography. Linear increase in ln[M]0/[M] with polymerization time, and number average molecular weight (Mn) with monomer conversion indicated RAFT controlled polymerization of PFMA under the conditions tested. Furthermore, block copolymers of PFMA with poly(ethylene glycol) methacrylate (PEGMA) as a biocompatible component were prepared. Copolymerization was studied using both P(PFMA) and P(PEGMA) as macro RAFT agent. Copolymerization kinetic studies indicated that chain extension block copolymerizations were successfully performed using both macroRAFT agents. P(PFMA) was reacted with arginine methylester (AME) in the presence of triethylamine (TEA). 100% of P(PFMA) active ester groups could be modified with AME at a polymer/AME/TEA mole ratio of 1/1/3, as determined by 1H-NMR spectroscopy. The AME modified polymers were complexed with a 681-bp DNA fragment through electrostatic interactions at varying nitrogen/phosphate (N/P) ratios. Gel electrophoresis experiments revealed that AME-modified P(PFMA) was able to complex with DNA at a N/P ratio of 200. Furthermore, the hydrodynamic diameter (Dh) of polymer/DNA complexes in phosphate buffer saline was found to be 58 nm, while the free DNA displayed a Dh of 109 nm, indicating the complexation of DNA by AME-modified P(PFMA).