Characterization and application of morpholino monolayers in nucleic acid diagnostics.

摘要

This thesis describes the formation and characterization of layers of morpholino molecules on gold supports and further expands on the implementation of these layers in the context of DNA diagnostics, where the hybridization of surface-bound morpholino 'probes' to solution-borne DNA 'targets' is sensed in a label-free manner through changes in interfacial capacitance by the use of electrochemical impedance. Morpholino molecules are attached onto a gold electrode surface through a thiol end-terminus after which the surface is passivated by the self-assembly of mercaptopropanol (MCP) in order to undermine non-specific interactions between morpholinos and the metal and make them more accessible for hybridization. The desired conformation of morpholino probes at the surface is characterized through the use of Fourier Transform Infrared Spectroscopy (FTIR) by tracking changes in C=O bands of thymine bases. AC impedance is then used to detect modulations in surface capacitance as a function of electrode bias potential that result from the hybridization event, a procedure that does not make use of labels. The experimental results are emulated by the use of a Poisson-Boltzmann model from which it is deduced that the changes in capacitance observed are the combination of changes in local ionic and dielectric properties. As a means to validate the above-described label-free assay, changes in capacitance are correlated to the amount of DNA targets captured. This is done by labeling morpholino and DNA molecules with an electroactive ferrocene moiety and measuring the amount of charge passed at the electrode in a cyclic voltammetric measurement. The results show that the label-free assay is at least as sensitive as other label-free techniques such as surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) with a limit-of-quantification(l QT) of 2.9x1010 targets cm -2 under 0.2 mol L-1 sodium phosphate buffer. The effects of variations in surface probe density and ionic strength on the sensitivity of the label-free method are then explored. The results reveal that ionic strength has a greater effect on assay sensitivity than does probe coverage, where decreasing ionic strength increases sensitivity. Our label-free method shows potential to reach lQT values of &sim2x108 targets cm under 0.001 mol L-1 buffer however these low values are obscured by experimental noise.