The aims of this research were to develop novel surface modification strategies that can be
used on a range of solid supports including polymeric and metallic matrices, as these could
have a significant impact on the performance of bio/sensors employing these surfaces. These
approaches were used to develop methods for immobilizing biomolecular recognition
elements, such as antibodies, on modified matrices, and to exploit these approaches for the
generation of high sensitivity bio-assays. Human fetuin A, mouse immunoglobulin G, and
horseradish peroxidase were employed as model analytes.
A silane-based surface modification strategy was designed and optimized for planar or flat
surfaces such as polymeric sheets, chips or microtitre plates. These polymeric surfaces were
activated prior to silane-functionalization using potassium hydroxide (aq.)-mediated mild
oxidation (wet method) and oxygen-plasma etching. This novel surface activation strategy
was further optimized in combination with surface functionalization and covalent
immobilization of antibodies, for enhancing immunoassay sensitivities. Sensitivities
obtained for immunoassays using antibodies immobilized with the developed and
adsorption-based conventional strategies were 39 and 625 pg/mL, respectively, for human
fetuin A. The strategy was demonstrated to be generic in nature and could be employed to
activate a wide range of polymeric and metallic surfaces. In addition, highly sensitive
detection of human fetuin A was achieved with antibodies captured in an oriented manner
on covalently immobilized protein A (EC50 3.7 ng/mL) in comparison to randomly captured
antibodies (EC50 5.8 ng/mL).
High-brightness NIR664 dye-doped silica nanoparticles were employed to probe various
activation states of platelets. These NPs were functionalized with silanes (viz. amine and
carboxy-terminal) followed by conjugation to a platelet surface biomarker-specific antibody
(anti-CD41) and successfully employed for probing platelet activation. The antibody-NP
conjugates were found to be highly sensitive (>95%) and specific (≈100%). In addition,
aggregation of NPs was minimized by controlling their surrounding chemical environment
and their stability after antibody conjugation.
Metadata
Item Type:
Thesis (PhD)
Date of Award:
November 2012
Refereed:
No
Supervisor(s):
McDonagh, Colette, O'Kennedy, Richard and MacCraith, Brian
Uncontrolled Keywords:
surface modification strategies; biomolecular recognition
elements