Single-molecule detection of molecular beacons generated from LDR on thermoplastic microfluidic device for bioanalysis

Current clinical techniques for nucleic acid detection and analysis often involve PCR, lacking adequate specificity or sensitivity to meet the stringent requirements in certain applications. This research aims to develop an innovative molecular assay and the associated hardware to rapidly signal the presence of certain targets using reporter sequence found in their genome without requiring PCR. This assay coupled the sensitivity of single-pair fluorescence resonance energy transfer (spFRET) with the specificity of ligase detection reaction (LDR) to provide near real-time readout of target biomarkers. Heightened concerns on potential bioterrorism threats, such as rapid dissemination of pathogenic bacteria or viruses into water and/or food supplies, demand fast detection strategies. In this work, a pair of strain-specific primers was designed based on the 16S rRNA gene and were end-labeled with a donor (Cy5) and acceptor (Cy5.5) dyes. In the presence of the target bacterium, the primers were joined using LDR to form a reverse molecular beacon (rMB), thus bringing Cy5 and Cy5.5 into close proximity to allow FRET to occur. These rMBs were analyzed using single-molecule detection of the FRET pairs (spFRET). The LDR was performed in a Cyclic Olefin Copolymer (COC) microfluidic device equipped with 2 or 20 thermal cycles in a continuous flow format. Single-molecule photon bursts from the resulting rMBs were detected on-chip and registered using a laser-induced fluorescence (LIF) instrument. The presence of target pathogens could be reported in as little as 2.6 min using spFRET. In another development, a similar assay format was utilized to quantify mRNA expression levels of MMP-7 gene, which is highly relevant to invasion, metastasis and progression of a variety of tumors. HT-29 cells were found to express the highest levels of MMP-7 transcripts among the studied cell lines using LDR primers specific to MMP-7 gene. This observation is consistent with the results obtained with RT-qPCR. The LDR-spFRET assay was also used for stroke subtyping by designing primers specific to AMPH gene and using a microfluidic chip with tapered detection window to improve sampling efficiency. The detection could be completed in 15 min with extended readout time to glean low copy number transcripts