Single molecule detection of double-stranded DNA in poly(methylmethacrylate) and polycarbonate microfluidic devices

Single photon burst techniques were used to detect double-stranded DNA mole cules in poly(methylmethacrylate) (PMMA) and polycarbonate (PC) microfluidi c devices. A confocal epi-illumination detection system was constructed to monitor the fluorescence signature from single DNA molecules that were mult iply labeled with the mono-intercalating dye, TOPRO-5, which possessed an a bsorption maximum at 765 nm allowing excitation with a solid-state diode la ser and fluorescence monitoring in the near-infrared (IR). Near-IR excitati on minimized autofluorescence produced from the polymer substrate, which wa s found to be significantly greater when excitation was provided in the vis ible range (488 nm). A solution containing lambda -DNA (48.5 kbp) was elect rokinetically transported through the microfluidic devices at different app lied voltages and solution pH values to investigate the effects of polymer substrate on the transport rate and detection efficiency of single molecula r events. By applying an autocorrelation analysis to the data, we were able to obtain the molecular transit time of the individual molecules as they p assed through the 7 mum laser beam. It was observed that the applied voltag e for both devices affected the transport rate. However, solution pH did no t alter the transit time for PMMA-based devices since the electroosmotic fl ow of PMMA was independent of solution pH, In addition, efforts were direct ed toward optimizing the sampling efficiency (number of molecules passing t hrough the probe volume) by using either hydrodynamically focused flows fro m a sheath generated by electrokinetic pumping from side channels or reduci ng the channel width of the microfluidic device. Due to the low electroosmo tic flows generated by both PMMA and PC, tight focusing of the sample strea m was not possible. However, in PMMA devices, flow gating was observed by a pplying field strengths > -120 V/cm to the sheath flow channels. By narrowi ng the microchannel width, the number of molecular events detected per unit time was found to be four times higher in channels with 10 mum widths comp ared to those of 50 mum, indicating improved sampling efficiency for the na rrower channels without significantly deteriorating detection efficiency. A ttempts were made to do single molecule sizing of lambda -DNA, M13 (7.2 kbp ) and pUC19 (2.7 kbp) using photon burst detection. While the average numbe r of photons for each DNA type were different, the standard deviations were large due to the Gaussian intensity profile of the excitation beam. To dem onstrate the sensitivity of single molecule analysis in the near-IR using p olymer microfluidic devices, the near-IR chromophore, NN382, was analyzed u sing our confocal imager. A detection efficiency of similar to 94% for sing le NN382 molecules was observed in the PC devices.