Elliptical Pipette Generated Large Microdroplets for POC Visual ddPCR Quantification of Low Viral Load

Abstract

Rapid point-of-care (POC) quantification of low virus RNA load would significantly reduce the turn-around time for the PCR test and help contain a fast-spreading epidemic. Herein, we report a droplet digital PCR (ddPCR) platform that can achieve this sensitivity and rapidity without bulky lab-bound equipment. The key technology is a flattened pipette tip with an elliptical cross-section, which extends a high aspect-ratio microfluidic chip design to pipette scale, for rapid (<5 min) generation of several thousand monodispersed droplets ∼150 to 350 μm in size with a CV of ∼2.3%. A block copolymer surfactant (polyoxyalkylene F127) is used to stabilize these large droplets in oil during thermal cycling. At this droplet size and number, positive droplets can be counted by eye or imaged by a smartphone with appropriate illumination/filtering to accurately quantify up to 100 target copies. We demonstrate with 2019 nCoV-PCR assay LODs of 3.8 copies per 20 μL of sample and a dynamic range of 4–100 copies. The ddPCR platform is shown to be inhibitor resistant with spiked saliva samples, suggesting RNA extraction may not be necessary. It represents a rapid 1.5-h POC quantitative PCR test that requires just a pipette equipped with elliptical pipette tip, a commercial portable thermal cycler, a smartphone, and a portable trans-illuminator, without bulky and expensive micropumps and optical detectors that prevent POC application.

Methods of Respiratory Virus Detection: Advances towards Point-of-Care for Early Intervention

“…In addition to the new technologies listed above, there are many promising ones with the potential to be adopted as future POC diagnosis tools for detection of respiratory viruses, such as digital PCR, surface-enhanced Raman scattering (SERS), 3D plasmonicbased chips. For example, rapid detection of low virus (SARS-CoV-2) RNA load was achieved by using a droplet digital PCR method [60]. In this study, a flat pipette head with an elliptical cross section was utilized to prepare monodisperse droplets.…”

Interfacial tension driven open droplet microfluidics

“…Our method enables the researcher to deliver the aqueous phase, followed by the carrier phase, each in a single pipetting step, to initiate the generation of droplets; there is no need for continuous pipetting or monitoring as in prior systems. 18,19 Our SCF-driven droplet generation method eliminates the need for external flow generators, expanding the accessibility of droplet microfluidics to labs without flow generators and opening up possibilities for novel applications that are not feasible in devices requiring electricity.…”

“…Other methods to generate droplets using pipettes include simple agitation of a biphasic solution either with or without cavity-containing microparticles. 18,34…”

“…Other methods to generate droplets using pipettes include simple agitation of a biphasic solution either with or without cavity-containing microparticles. 18,34 Herein we describe a system that achieves autonomous droplet generation of up to hundreds of droplets in an open microfluidic channel. We made this possible with only the open channel, pipettes, and commercially available carrier fluid; tubing, electronics, or pumps are not required, making droplet technology feasible for research and biology labs without external flow generators.…”

“…( 1, 24–27 ) Other methods to generate droplets using pipettes include simple agitation of a biphasic solution either with or without cavity-containing microparticles to create an emulsion of droplets in an immiscible phase. ( 1, 32, 33 ) Additionally, there are methods of autonomous droplet generation utilizing a closed channel but they have limited downstream analysis and manipulation capabilities. ( 34, 35 )…”