Molecular recognition for rapid bioanalysis: Applications using antibody and aptamer systems

Abstract

The main focus of this dissertation is to apply biomolecular recognition to carry out rapid, sensitive protein detection using a polyclonal antibody and a DNA aptamer. Rapid immunosorption kinetics using reduced diffusion distances in microreactors were demonstrated at reduced diffusion distances using a model microchannel (40 x 100 μm dimension) architecture, based on a photoablated poly ethylene terephthalate (PET) surface. The detection of a potential warfare agent staphylococcal enterotoxin B (SEB) was implemented. Characterization of the Rb α SEB antibody adsorption on the photoablated channel surface revealed a maximum antibody coverage of 19.4 pmol.cm −2 on the channel surfaces and 5.5 pmol.cm−2 on the sealing laminate surfaces. Characterization of the photoablated surface using scanning electron microscopy (SEM) and attenuated total reflection—Fourier Transform infrared spectroscopy (ATR-FTIR) reveals the presence of charged functional groups on the surface along with an overall increase in the surface hydrophobicity. The bioactivity of the adsorbed antibody was found to be close to 30%. The kinetics of the immunosorption reaction was found to be rapid and the reaction was completed within a minute inside the microchannel. The modeling of such reactions using Fick's law predicts a diffusion rather then kinetically-controlled process.

Biorecognition by a DNA aptamer was implemented in a homogeneous approach based on the fluorescence polarization (FP) detection of immunoglobulin E (IgE). The method is based on the anisotropy changes that occur during the binding event. End-labeled IgE aptamer using fluorescein and Texas Red as the fluorophores has been used for the successful detection of IgE. A LOD of 350 pM has been observed, under unoptimized conditions, for the IgE detection. The optimization of the sensitivity of the FP signal was a key objective of the current work. Factors found to be important in this regard include the choice of fluorophore, dye tether length, temperature and ionic composition of the binding buffer. A detailed understanding of the anisotropy results has been obtained using the time correlated single photon counting (TCSPC) spectroscopy. The importance of understanding the dye-DNA interactions and rotational dynamics of the labeled aptamer has proved to be crucial for the FP approach.