Continuous flow real-time PCR device using multi-channel fluorescence excitation and detection.

High throughput automation is greatly enhanced using techniques that employ conveyor belt strategies with un-interrupted streams of flow. We have developed a 'conveyor belt' analog for high throughput real-time quantitative Polymerase Chain Reaction (qPCR) using droplet emulsion technology. We developed a low power, portable device that employs LED and fiber optic fluorescence excitation in conjunction with a continuous flow thermal cycler to achieve multi-channel fluorescence detection for real-time fluorescence measurements. Continuously streaming fluid plugs or droplets pass through tubing wrapped around a two-temperature zone thermal block with each wrap of tubing fluorescently coupled to a 64-channel multi-anode PMT. This work demonstrates real-time qPCR of 0.1-10 µL droplets or fluid plugs over a range of 7 orders of magnitude concentration from 1 × 10(1) to 1 × 10(7). The real-time qPCR analysis allows dynamic range quantification as high as 1 × 10(7) copies per 10 µL reaction, with PCR efficiencies within the range of 90-110% based on serial dilution assays and a limit of detection of 10 copies per rxn. The combined functionality of continuous flow, low power thermal cycling, high throughput sample processing, and real-time qPCR improves the rates at which biological or environmental samples can be continuously sampled and analyzed.

Passive droplet sorting using viscoelastic flow focusing.

We present a study of passive hydrodynamic droplet sorting in microfluidic channels based on intrinsic viscoelastic fluid properties. Sorting is achieved by tuning the droplets' intrinsic viscous and viscoelastic properties relative to the continuous oil phase to achieve a positive or negative lateral migration toward high or low shear gradients in the channel. In the presence of weakly viscoelastic fluid behavior, droplets with a viscosity ratio, κ, between 0.5-10 were found to migrate toward a high shear gradient near the channel walls. For all other κ-values, or Newtonian fluids, droplets would migrate toward a low shear gradient at the channel centerline. It was also found that for strongly viscoelastic fluids with low interfacial tension, droplets would migrate toward the edge even with κ-values lower than 0.5. The resulting bi-directional lateral droplet migration between different droplets allows size-independent sorting. Still, their sorting efficiencies are dependent on droplet size, intrinsic fluid elasticity, viscosity, droplet deformability, and overall fluid shear rates. Based on these findings, we demonstrate >200 Hz passive droplet sorting frequencies and achieve >100 fold enrichment factors without the need to actively sense and/or control active mechanisms. Using a low viscosity oil phase of 6.25 cPs, we demonstrate sorting discrimination of 1 cPs and 5 cPs aqueous droplets with κ-values of 0.2 and 0.8 respectively.

1-Million droplet array with wide-field fluorescence imaging for digital PCR.

Digital droplet reactors are useful as chemical and biological containers to discretize reagents into picolitre or nanolitre volumes for analysis of single cells, organisms, or molecules. However, most DNA based assays require processing of samples on the order of tens of microlitres and contain as few as one to as many as millions of fragments to be detected. Presented in this work is a droplet microfluidic platform and fluorescence imaging setup designed to better meet the needs of the high-throughput and high-dynamic-range by integrating multiple high-throughput droplet processing schemes on the chip. The design is capable of generating over 1-million, monodisperse, 50 picolitre droplets in 2-7 minutes that then self-assemble into high density 3-dimensional sphere packing configurations in a large viewing chamber for visualization and analysis. This device then undergoes on-chip polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the sample's nucleic acid contents. Wide-field fluorescence images are captured using a low cost 21-megapixel digital camera and macro-lens with an 8-12 cm(2) field-of-view at 1× to 0.85× magnification, respectively. We demonstrate both end-point and real-time imaging ability to perform on-chip quantitative digital PCR analysis of the entire droplet array. Compared to previous work, this highly integrated design yields a 100-fold increase in the number of on-chip digitized reactors with simultaneous fluorescence imaging for digital PCR based assays.

Tunable 3D droplet self-assembly for ultra-high-density digital micro-reactor arrays.

We present a tunable three-dimensional (3D) self-assembled droplet packing method to achieve high-density micro-reactor arrays for greater imaging efficiency and higher-throughput chemical and biological assays. We demonstrate the capability of this platform's high-density imaging method by performing single molecule quantification using digital polymerase chain reaction, or digital PCR, in multiple self-assembled colloid-like crystal lattice configurations. By controlling chamber height to droplet diameter ratios we predictively control three-dimensional packing configurations with varying degrees of droplet overlap to increase droplet density and imaging sensor area coverage efficiency. Fluorescence imaging of the densely packed 3D reactor arrays, up to three layers high, demonstrates high throughput quantitative analysis of single-molecule reactions. Now a greater number of microreactors can be observed and studied in a single picture frame without the need for confocal imaging, slide scanners, or complicated image processing techniques. Compared to 2D designs, tunable 3D reactor arrays yield up to a threefold increase in density and use 100% of the sensor's imaging area to enable simultaneous imaging a larger number of reactions without sacrificing digital quantification performance. This novel approach provides an important advancement for ultra-high-density reactor arrays.