Evaluation of microfluidic biosensor development using microscopic analysis of molecular beacon hybridization kinetics

Abstract. Molecular beacons, oligonucleotide probes that fluoresce upon hybridization to a target nucleic acid, can be used in microflu-idic devices to detect and quantify nucleic acids in solution as well as inside bacterial cells. Three essential steps towards the development of such devices as integrated microfluidic biosensors using molecular beacons were investigated in the present study. First, experiments using real-time confocal microscopy indicated that diffusion of DNA molecular beacons across a 100-µm diameter microfluidic channel took less than one minute after the flow of reagents was stopped. Sec-ond, experiments to evaluate hybridization kinetics of DNA molec-ular beacons with target nucleic acids in solution showed that DNA molecular beacons can be used to characterize hybridization kinetics in real time in microfluidic channels and that hybridization signals approached their maximum in approximately three minutes. Finally, it was demonstrated that peptide nucleic acid molecular beacons can be used to detect bacterial cells in microfluidic devices. These results suggest that the use of microfluidic devices to detect nucleic acids in solution and in bacterial cells is promising and that further develop-ment of an integrated microfluidic biosensor for bacterial detection based on this concept is warranted