Multiplexed target detection using DNA-binding dye chemistry in droplet digital PCR.

Two years ago, we described the first droplet digital PCR (ddPCR) system aimed at empowering all researchers with a tool that removes the substantial uncertainties associated with using the analogue standard, quantitative real-time PCR (qPCR). This system enabled TaqMan hydrolysis probe-based assays for the absolute quantification of nucleic acids. Due to significant advancements in droplet chemistry and buoyed by the multiple benefits associated with dye-based target detection, we have created a "second generation" ddPCR system compatible with both TaqMan-probe and DNA-binding dye detection chemistries. Herein, we describe the operating characteristics of DNA-binding dye based ddPCR and offer a side-by-side comparison to TaqMan probe detection. By partitioning each sample prior to thermal cycling, we demonstrate that it is now possible to use a DNA-binding dye for the quantification of multiple target species from a single reaction. The increased resolution associated with partitioning also made it possible to visualize and account for signals arising from nonspecific amplification products. We expect that the ability to combine the precision of ddPCR with both DNA-binding dye and TaqMan probe detection chemistries will further enable the research community to answer complex and diverse genetic questions.

Investigating the activity of quinine analogues versus chloroquine resistant Plasmodium falciparum.

Plasmodium falciparum, the deadliest malarial parasite species, has developed resistance against nearly all man-made antimalarial drugs within the past century. However, quinine (QN), the first antimalarial drug, remains efficacious worldwide. Some chloroquine resistant (CQR) P. falciparum strains or isolates show mild cross resistance to QN, but many do not. Further optimization of QN may provide a well-tolerated therapy with improved activity versus CQR malaria. Thus, using the Heck reaction, we have pursued a structure-activity relationship study, including vinyl group modifications of QN. Certain derivatives show good antiplasmodial activity in QN-resistant and QN-sensitive strains, with lower IC(50) values relative to QN.

Use of photopatterned porous polymer monoliths as passive micromixers to enhance mixing efficiency for on-chip labeling reactions.

We introduce a passive micromixer with novel architecture using photopatterned porous polymer monoliths (PPM) and demonstrate an improvement in mixing efficiency by monitoring the fluorescence of an on-chip labeling reaction. UV light was used to photopattern a periodic arrangement of PPM structures directly within the channel of a plastic microfluidic chip. By optimizing the composition of the polymerization solution and irradiation time we demonstrate the ability to photopattern PPM in regularly repeating 100 microm segments at the tee-junction of the disposable device. To evaluate the efficiency of this dual functional mixer-reactor fluorescamine and lysine were introduced in separate channels upstream of the tee-junction and the intensity of laser-induced fluorescence resulting from the fluorogenic labeling reaction was monitored. The fluorescence level after the photopatterned periodic monolith configuration was 22% greater than both an equivalent 1 cm continuous segment of PPM and an open channel. Results indicate that this periodic arrangement of PPM, with regularly spaced open areas between 100 microm plugs of PPM, is directly responsible for enhancing the mixing and overall rate of chemical reaction in the system. In addition to facilitating preparation of a dual functional mixer-reactor, the ability to accurately photopattern PPM is an enabling technology for seamlessly integrating multiple monoliths into a single device. This technology will be particularly important to proteomic applications requiring preconcentration, enzymatic digestion and two-dimensional separations.