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The development of a generic semi-disposable microfluidic biosensor for the highly sensitive detection of pathogens via their nucleic acid sequences is presented in this paper. Disposable microchannels with defined areas for capture and detection of target pathogen RNA sequence were created in polydimethylsiloxane (PDMS) and mounted onto a reusable polymethylmethacrylate (PMMA) stand. Two different DNA probes complementary to unique sequences on the target pathogen RNA serve as the biorecognition elements. For signal generation and amplification, one probe is coupled to dye encapsulated liposomes while the second probe is coupled to superparamagnetic beads for target immobilization. The probes hybridize to target RNA and the liposome-target-bead complex is subsequently captured on a magnet. The amount of liposomes captured correlates directly to the concentration of target sequence and is quantified using a fluorescence microscope. Dengue fever virus serotype 3 sequences and probes were used as a model analyte system to test the sensor. Probe binding and target capture conditions were optimized for sensitivity resulting in a detection limit of as little as 10 amol microL(-1) (10 pmol L(-1)). Future biosensors will be designed to incorporate a mixer and substitute the fluorescence detection with an electrochemical detection technique to provide a truly portable microbiosensor system.

Members of a panel of stress-responsive biosensors have been used to study the effect of megahertz frequency ultrasound onEscherichia coli. Insonification causes acoustic cavitation, the collapse of oscillating microbubbles in solution, which can damage bacterial cells. A focused 1-MHz ultrasound transducer, capable of generating a spatial peak pulse average intensity of 500 W/cm2, was used to treat liquid bacterial cultures. Stress-responsive promoters fused to luxCDABE allowed the continuous measurement of light produced as a result of protein damage, DNA damage, oxidative stress, and membrane perturbation. A promoter responsive to ammonia limitation was not transcriptionally activated under test conditions. In contrast to bacteria in exponentially growing cultures, those in stationary-phase cultures were more resistant to the effects of ultrasound treatment. Quantification of the degree of acoustic cavitation due to symmetric bubble collapse was measured by a 20-MHz passive transducer, the output of which appears to be only partially correlated with cellular damage and survival. The methods and results summarized here provide the basis for further investigation into applications, including the purification of water samples.

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The interaction of a complex acoustic cavitation field and suspended cells in vitro has been explored by many previous investigators. Of greatest interest is the effect of collapsing cavitation bubbles on the membrane structure and DNA of cells. By employing E. coli bacteria that have been genetically engineered to emit visible light when they are stressed in proscribed ways, the factors influencing bubble-cell interactions can be better explored. These factors include insonation frequency (1–5 MHz), duty cycle, PRF, initial bubble size distribution, and fluid viscosity. Results from this ongoing study will be presented. [Work supported by NSF.]