Aptamers as biosensors

Aptamers are short, single strands of artificial nucleic acids which bind to specific targets. This binding is similar to the binding exhibited by antibodies but aptamers offer improved properties and consequently are replacing antibodies in some applications. This first part of this project attempted to generate an aptamer which would bind to the molecules amphetamine and methamphetamine, with high selectivity and affinity. The process for generating an aptamer is called Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and is carried out in vitro. The strength of an aptamer / target binding interaction can be assessed using a variety of different techniques and in this work an Enzyme Linked Immunosorbent Assay (ELISA) was investigated. Other methods including surface plasmon resonance (SPR) and circular dichroism were attempted. The phenomenon of naturally occurring RNA molecules with catalytic properties (ribozymes) were the inspiration for the development of catalytic aptamers. Consequently, a novel SELEX system was deigned to select aptamers capable of catalysing a Diels Alder reaction between cyclohexadiene modified DNA and maleimide. A number of potential sequences were isolated and when one such sequence was incorporated into a test reaction a cycloadduct resulting from a successful Diels-Alder reaction was confirmed using MALDI - MS. In addition to generating novel aptamers, this thesis details work on the application of existing aptamers with the aim of creating novel assays and expanding the potential use of aptamers. Two protein targets, namely Protein kinase C (PKC) and thrombin were chosen and their respective aptamers investigated for use in metallic nanoparticle conjugates. The combination of aptamers and nanoparticles (in particular metallic nanoparticles) has grown in prominence in the literature and is regarded as an exciting development due to their desirable optical properties. Consequently, novel assays based on UV-visible spectroscopy were developed using the two specified DNA aptamers conjugated to gold and / or silver nanoparticles. In addition, the possibility of incorporating Surface enhanced resonance Raman scattering (SERRS) analysis was investigated.

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral-pg

Grantor dc:publisher.institution

University of Strathclyde

Year dc:date.issued

2010

Author dc:creator

• Dalton, Colette

DOI

10.48730/evsb-xc33

Identifier

T12785

OAI identifier oai:identifier

oai:strathclyde:9w0323064