Real-Time and Selective Detection of Single Nucleotide DNA Mutations Using Surface Engineered Microtoroids.

Abstract

Mictoroids, as optical biosensors, can provide beneficial biosensing platforms to understand DNA alterations. These alterations could have significant clinical importance, such as the case of Pseudomonas aeruginosa, which is a commonly found pathogen in Cystic Fibrosis (CF) patients-causing poor prognosis by undergoing mutations during disease steps, gaining virulence and drug resistance. To provide a preliminary diagnosis platform for early-stage bacterial mutations, biosensing with a selective microtoroid surface was suggested. For this purpose, microtoroids with high quality factors were fabricated. The microtoroid surfaces were coated with (3-aminopropyl) triethoxysilane (APTES)/trimethylmethoxysilane (TMMS) mixed silane solution followed by EDC/NHS chemistry for covalent conjugation of DNA probes. Ethanolamine capping was applied to avoid unspecific interactions. The confocal studies confirmed homogeneous functionalization of the microtoroid surface. The DNA hybridization was demonstrated to be affected from the probe length. The optical biosensors showed a significant response (∼22 pm) to the complementary strand of the mutated type P. aeruginosa DNA, while showing substantially low and late response (∼5 pm) to the point mismatch strand. The limit of detection (LOD) for the complementary strand was calculated as 2.32 nM. No significant response was obtained for the noncomplementary strand. The results showed the microtoroids possessed selective surfaces in terms of distinguishing DNA alterations.