Transport, kinetic and thermodynamic insight for the isolation, amplification and identification of viral RNA

When engineering integrated approaches for extracting medical diagnostic information from test samples, the main scientific problem is translating a biomolecular mixture into quantitative data. How does the secondary structure of RNA modify the hybridization thermodynamics of an identifying probe? How can conformation dependent biomolecular binding and reaction be integrated into transport models? How can the kinetic rate of in-vitro transcription by RNA polymerase be optimized in a continuous flow microreactor? These are some of the basic scientific and application development problems, which present hurdles for the biomedical field. This thesis addresses these challenges through focused investigation of microscale reactions applied to disease diagnosis. This dissertation presents the development of an integrated viral diagnostic microdevice for isolation, amplification and identification of the influenza subtype. Rapid viral classification enables a proper public health response in combating emerging infections. This compact device is designed for portable field use using non-PCR amplification methods. Microfluidic devices were designed and fabricated to study fundamental properties of proteins, RNA and DNA in diagnostic applications. New methods for measuring protein conformation, studying enzyme kinetics, amplifying RNA and sequence detection were integrated in a microfluidic platform. New parameters were defined to identify regimes for mass transport limited or reaction rate limited operation for determining reaction kinetics on immobilized templates in a continuous flow microfluidic device. New termination sequences were discovered that strongly regulate full-length transcription of pathogenic human H5 RNA. Lastly, the dependency of thermodynamic responses of dual labeled fluorescent probes to RNA and DNA targets on secondary structure of targeted RNA, temperature and salt conditions were determined. These new insights can be applied to diagnostics of other RNA or DNA sequences.