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

Area of Science:

• Biotechnology
• Nanotechnology
• Medical Diagnostics

Background:

• Pseudomonas aeruginosa mutations are critical in Cystic Fibrosis (CF) progression, leading to increased virulence and drug resistance.
• Early detection of these bacterial DNA alterations is crucial for improved patient prognosis and treatment strategies.
• Optical biosensors offer potential for sensitive and selective detection of microbial genetic changes.

Purpose of the Study:

• To develop and evaluate microtoroid-based optical biosensors for the selective detection of Pseudomonas aeruginosa DNA mutations.
• To establish a preliminary diagnostic platform for early-stage bacterial genetic alterations relevant to CF.

Main Methods:

• Fabrication of high-quality factor microtoroids.
• Surface functionalization using silane chemistry (APTES/TMMS) and EDC/NHS for DNA probe immobilization.
• Ethanolamine capping to minimize non-specific binding.
• Optical detection of DNA hybridization events using microtoroid resonance shifts.

Main Results:

• Homogeneous functionalization of microtoroid surfaces was confirmed.
• DNA hybridization sensitivity was influenced by probe length.
• Biosensors exhibited a significant response (∼22 pm) to complementary P. aeruginosa DNA, with a low response (∼5 pm) to mismatch strands.
• Limit of detection (LOD) for the complementary strand was determined to be 2.32 nM.
• High selectivity was demonstrated, with no significant response to non-complementary DNA.

Conclusions:

• Microtoroids serve as effective optical biosensors for distinguishing specific DNA alterations.
• The developed platform shows potential for selective and sensitive detection of mutated P. aeruginosa DNA.
• This technology could facilitate early diagnosis of bacterial mutations in CF patients.