Design, Fabrication, and Testing of a Microsystem for Monitoring Bacterial Quorum Sensing
Koev, Stephan Todorov
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Most pathogenic bacteria communicate with each other using signaling molecules. Their coordinated behavior, known as quorum sensing (QS), enables them to infect host organisms collectively and form drug-resistant biofilms. The study of bacterial signaling pathways may lead to discovery of new antimicrobials. Lab-on-a-chip technology can significantly accelerate the screening of candidate drugs that inhibit QS. This dissertation develops for the first time miniaturized sensors embedded in microfluidic channels to monitor the activity of an enzymatic pathway that produces signaling molecules. These devices can be used as building blocks of future high-throughput systems for drug discovery. The sensors presented here are gold-coated microcantilevers, and they detect the aminoacid homocysteine, a byproduct of the bacterial signaling pathway. It binds to the gold surface, causing stress and cantilever displacement that is measured optically. Samples are synthesized using bacterial enzymes and tested with the sensors. The minimal detected concentration of homocysteine is 1uM. It is demonstrated that deactivation of the enzymes causes a change in the sensor response; this effect can be used for finding drugs that inhibit the enzyme. The traditional method for measuring cantilever displacement requires an elaborate optical setup, and it can only test one device at a time. Two new methods are developed here to overcome these limitations. The first one uses a transparent cantilever which is also an optical waveguide. Light is coupled from the cantilever to a fixed output waveguide and measured with a photodetector. The cantilever displacement is determined from the change in output power. The change is approximately 0.7% per nanometer displacement. The minimal detectable displacement and surface stress are 6nm and 1.3 mN/m respectively. The second measurement method uses a transparent cantilever that is close to a reflective substrate. When the device is imaged with an optical microscope, an interference pattern forms. The cantilever displacement is calculated from the lateral shift of the interference fringes. This shift is determined from images of the device using custom software. The response of multiple cantilevers is captured by translating the microscope stage. The minimal detectable displacement and surface stress are 1nm and 340 uN/m respectively.