Development of a disposable centrifugal droplet generation platform for digital nucleic acid analysis

Y. Zhu, X. Huang, X. Lin, J. Li, M.R. Hoffmann
California Institute of Technology,
United States

Keywords: centrifugal droplet generation, disposable, digital LAMP


Monodispersed water-in-oil droplets enable nanoliter-sized compartmentalization, which has wide applications in organic synthesis, microreactors, and digital nucleic acid analysis. However, microfluidic-based droplet generation methods generally require special fabrication capacity to fabricate sub-100 µm channels and may involve complicated operation. Representative designs, such as T-junction on PDMS or SlipChip fabricated by hot embossing, may take thousands of dollars just in personnel training and capital cost [1]. Here we report a simple centrifugal droplet generation device that can be made from off-the-shelf components (less than $0.5/unit), run in a common lab centrifuge, and potentially developed into a robust integrated system. The droplet generation performance was optimized and the application in digital loop-mediated isothermal amplification (LAMP) was demonstrated. The device generates droplets with a bent commercial Luer-lock needle in a centrifuge tube. The schematic and the photo of the design are shown in Figure 1A-B. The aqueous phase was added into the Luer-Lock of the commercial needle, with the oil phase at the bottom of the tube. With centrifugal acceleration, the aqueous phase is forced into the oil phase by elevated aqueous pressure difference between the reservoir surface and the narrow inlet, and then pinched off by the interfacial tension. With the oil phase denser, the droplet is transported away by buoyancy force. Droplet generation was optimized with the oil phase containing 5% FluoroSurfactant in HFE 7500 oil and the aqueous phase containing LAMP buffer with calcein. Droplets were extracted into a viewing chamber, imaged by fluorescent microscope, and analyzed in MATLAB. Droplet sizes were measured with varying needle sizes (30, 32 and 34 Gauge needles corresponding to internal diameter of 160, 110 and 80 µ, oil volume, and centrifugal acceleration. For digital LAMP experiments, Salmonella Typhi DNAs were extracted using commercial kit and serial diluted for real-time PCR and digital LAMP analysis. LAMP primers targeting Salmonella spp. were employed [2]. DNA concentrations were estimated by Poisson distribution from the fraction of dark droplets. Our device was capable of producing size-tunable droplets. The smallest droplets generated were 98.5±12.2 µm in diameter, and the best monodispersed ones were 175.3±9.8 µm. Figure 2A-C compares the mean and standard deviation of droplet size under varying conditions. The smaller needles generated smaller sized droplets. Droplets can be tuned smaller by increasing centrifugation speed or increasing bottom oil volume, which provide greater pressure difference forcing the aqueous inflow. Larger oil volume reduced size deviation likely by reducing oil phase height variation and limiting the amount of aqueous phase inlet during acceleration. Digital LAMP was demonstrated under the condition produced 175.3±9.8 µm droplets. Examples of positive and negative fluorescent pictures are shown in Figure 3A-B. Estimated DNA concentration was linearly correlated with real-time PCR cycle number (Figure 3C), indicating that DNA copies were partitioned following Poisson distribution. Absolution quantification capacity will be evaluated with digital PCR. We are working on incorporating 3D-printed processing units linked to the Luer-Lock to develop the device into a fully integrated platform with wider applications.