Single-molecule detection and microfluidics: generating systems for the in vitro diagnostics of stroke

There is currently no available molecular diagnostic test for stroke; the common modality for diagnosis consists of computed tomography or magnetic resonance imaging. Unfortunately, the use of these diagnostic regimens can delay proper therapeutic treatment, which requires administration within the first 3 h of a stroke event. We are developing a molecular assay that can report, in near real time and at the point-of-care, the presence or absence of biomarkers specifically targeted for the diagnosis of ischemic or hemorrhagic stroke. The proposed strategy uses blood-borne mRNAs that are either under-expressed or over-expressed as a result of tissue damage within the brain. The ability to report on these diagnostic markers is enabled through the use of a fluidic bio-processor fabricated in polymers via micro-replication to provide autonomous sample processing. This bio-processor comprises a fluidic motherboard that possesses task-specific modules for the selection of white blood cells from a blood sample, cell lysis and solid-phase extraction of the mRNA markers, ligase detection reaction to identify the mRNA markers and an optical module for multiplexed detection. The sample-processing pipeline was streamlined to generate a rapid assay turn-around-time by employing single-molecule detection. The output of the clinical sample processing hardware are molecular beacons undergoing single pair Fluorescence Resonance Energy Transfer (spFRET) that are digitally counted to provide exquisite analytical sensitivity for the expression profiling of the relevant mRNA markers. The presentation will discuss the use of spFRET for mRNA expression profile with comparisons made to quantitative real-time PCR