Aptamer biosensors

Abstract

Aptamers are single stranded nucleic acids, typically composed of between twenty to eighty nucleotides in length, capable of binding selectively to non-nucleic acid ligands. Aptamers are selected through a combinatorial chemistry process called Systematic Evolution of Ligands by Exponential enrichment (SELEX), which is composed of successive cycles of selection based on target affinity, followed by amplification. This results in the Darwinian evolution of the nucleic acid library resulting in increasing library homogeneity and target affinity over time. Aptamers have been extensively investigated for potential application as sensing molecules, with roles similar to those traditionally occupied by antibodies. Aptamers and monoclonal antibodies have similar sensitivity in the pico to micro molar range. However aptamers have a number of advantages over protein antibodies, such as greater thermal stability, ease of chemical amplification, and amenability to modification, especially at the 5’ and 3’ prime ends. The work performed in this Thesis is divided into three categories. The first section describes the development of voltammetric Kanamycin and Tetracycline biosensors based on electrode immobilized, redox label bioconjugated nucleotide molecular beacons. These sensors relies on the target-aptamer binding induced spatial displacement of the redox label towards or away from the electrode surface as a means of signal generation. Further study was conducted to test the feasibility of this sensor design under likely field operation environments such as in soil sample analysis for microbial product discovery and in agricultural effluence for regulatory purposes. The biosensor was also enhanced by gel encapsulation for defense against nuclease degradation. Negative control was performed against structurally similar antibiotics of the same family in order to prove the specificity of the biosensor. Lastly, the sensor was moved onto an automated platform in a multichannel format in order to improve the utility of the sensor. The second section describes the development of a voltammetric biosensor based on Enzyme-Linked Oligonucleotide Assay (ELONA) technology. Two sub-types of ELONA-like biosensors were originally envisioned, based respectively on direct and indirect ELONA. Both sub-types depend on the mass of redox label rich Gold Nanoparticles (GNP) at the electrode surface as a means of signal generation. Negative controls was performed against globular proteins Bovine Serum Albumin and Lysozyme, the former since it is the most ubiquitous protein component of serum (the most likely biosensor operational environment), the latter as a worst case scenario for non-specific false positive results due to its positive charge. The last section describes an attempt to develop an automated SELEX device based on mesofluidic flow channels. It was hoped that by using flow channels of a millimeter scale it would be possible to retain both the advantages of the conventional auto sampler based SELEX protocols (large library and sequence variation), while also gaining the primary advantages of microfluidic SELEX (reduced contamination risk, low initial cost and maintenance). Essential components of the SELEX device, such as thermal cycler, liquid handling, electronics infrastructure, and software control were designed, tested and integrated. Lastly an attempt was made to perform automated SELEX against Lysozyme targets using the device, though no nucleic acid with high affinity to target had yet been successfully isolated by the end of this study.