The objective of this proposal is to develop a platform to assess mRNA expression using a novel quantum dot FRET probe within a microfluidic environment. Characterization of the molecular probe and the microfluidic systems are proposed followed by a demonstration of the integrated system using a gene expression model. The education goals include BioMEMS course development, introducing a seminar series on Bio/Nano topics, undergraduate mentoring and an outreach component focused on involving high school students.
The research project aims to develop and implement novel molecular analysis technologies that enable accurate and high throughput measurements of genetic contents on a single cell basis. The education plan is to promote outreach and undergraduate education in the emerging field of nano-biotechnology. In the past years, we developed a highly sensitive DNA/RNA sensor based on semiconductor nanocrystals (also called quantum dots) that induce fluorescence signal when linking to nucleic acid targets. We further developed a circulating microfluidic channel that is integrated with micro pumps for measurements of biomolecular targets in a continuous flow manner. This device has been integrated with the quantum dot probes and molecular beacons for single-cell RNA measurements. In order to enhance quantification efficiency, we developed a microfluidic coupling device to deliver and concentrate targets to nanoliter-sized single-molecule detection (SMD) chambers from otherwise undetectably low concentrations of sample nucleic acid targets. Although in principle SMD can be highly quantitative, its current implementations limit its accuracy, throughput, and practical applicability. The minute size of the SMD observation volume enables high signal-to-noise ratio detection of even single fluorescent molecules due to highly suppressed background levels. However, the diffraction-limited observation volume that enables SMD also significantly hampers its application in quantification and burst parameter determination. We reported a new implementation of single molecule detection called cylindrical illumination confocal spectroscopy (CICS) that can be generically incorporated into any microfluidic system and allows highly quantitative and accurate analysis of single fluorescent molecules. Besides, we discovered a new phenomenon of altered fast relaxation process associated an organic fluorophore, Cy5 interacting with nucleic acids. Basing on these encouraging preliminary studies, we have investigated on new design of probes utilizing this unique property of Cy5 for DNA/RNA quantification. During the project period, one new undergraduate course, Micro / Nanoscience and Biotechnology, has been offered in fall 2006 at Johns Hopkins University. The developments of using nonmaterials as transducers from transferring biochemical signals to detectable physical signals (e.g. photons) have been incorporated into the course. In addition, this project provides graduate, undergraduate and high school students the training on the use of the state-of-the-art confocal spectroscopic techniques to measure bimolecular interactions at the most fundamental single molecule level.