Development of Impedimetric Biosensors, Based on Phage-Modified Microarrays, for the Direct and Specific Detection of Bacteria.

摘要

Bacteriophages (or phages) are viruses that replicate only by entering specific host bacteria. This property has facilitated their application in specific pathogen detection. This thesis will address these issues with regard to the development of phage-based methods for the detection of bacteria.;The bacteriophages were attached to electrochemically functionalized screen-printed carbon electrode (SPE) microarrays using two different methods.;In the first method, T4 phage which specifically recognizes E. coli K12, was immobilized onto SPE networks that were electrochemically functionalized using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) in acidic media. In the second method, Gamma phage that specifically recognizes B. anthracis Sterne was immobilized onto SPE networks that were functionalized in two steps: by electrochemically generating phenyl-amino groups at the SPE surface, followed by reaction with glutaraldehyde to act as a linker. SPE surface functionalization and phage immobilization were confirmed using XPS and TOF-SIMS analysis. The phage-modified SPEs were then used to specifically detect target bacteria. Impedance measurements in the form of Nyquist plots (imaginary impedance (Zi) versus real impedance (Zr )) show shifts due to binding of the bacteria to the phage. No significant change in impedance was observed due to binding of non-target bacteria strains. The presence of surface bound bacteria was verified by scanning electron and fluorescence microscopies. Based on these results, the feasibility of using these microarrays for the direct and specific impedimetric detection of bacteria has been demonstrated.;We present a novel approach for the specific detection of E. coli K12 and B. anthracis Sterne bacteria, using bacteriophages as probes. We have also adapted this system to allow for separation of specific bacteria in more complex (real) samples using phage-coated magnetic beads and a simple magnetic manipulation system.