Droplet microfluidics: amphiphilic nanoparticles as droplet stabilizers for high-fidelity and ultrahigh-throughput droplet assays

S.K.Y. Tang
Stanford University,
United States

Keywords: droplet microfluidics, amphiphilic silica nanoparticles

Summary:

Droplet microfluidics, in which nanoliter- to picoliter-sized drops are used to encapsulate and compartmentalize molecules or cells, has enabled a wide range of biochemical applications. Examples include digital PCR and directed evolution of enzymes. Initial research on this technology demonstrated 100–1000x increase in throughput and up to 10^6x reduction in cost compared with state-of-the-art methods using robots and microtiter plates. Aspects of the technology are not yet fully optimized, however. Specifically, cross-contamination of droplet content and droplet instability during serial interrogation process compromise assay accuracy and limit the scalability of the technology. The first part of the talk will focus on the use of amphiphilic silica nanoparticles for the stabilization of aqueous drops in fluorinated oils to mitigate droplet cross-contamination. The success of droplet microfluidics has thus far relied on one type of surfactant (PFPE−PEG). However, these surfactants cause inter-drop transport of small, hydrophobic molecules, leading to the cross-contamination of droplet contents. The formation of reverse micelles has been found to be the predominant pathway for such cross-contamination. We show that replacing surfactants with nanoparticles (NPs) as stabilizers solves this problem. NPs are known to be adsorbed at the liquid-liquid interface irreversibly. As a result, the formation of reverse-micelles in NPs system is eliminated. The use of NPs, therefore, successfully mitigates the undesirable inter-drop transport. The second part of the talk will describe a high-throughput optofluidic droplet interrogation device capable of counting fluorescent drops at a throughput of 254,000 drops per second. In many biochemical assays, fluorescence is used as a read-out for the reactions occurring inside the drops, and can indicate the presence of cells or molecules of interest. The optical detection of fluorescence signal is commonly performed in a serial manner, where drops are injected into a funnel-shaped microchannel consisting of a narrow constriction which forces the drops to arrange in a single file, and to ensure that drops enter the detection region one at a time. We show that the throughput of the serial interrogation process is limited by the rate at which droplets become unstable and undergo undesirable break-up as they flow through the constriction. The key challenge in performing optical interrogation in a largely parallel manner is the trade-off between light collection efficiency and the field of view which determines the number of drops that can be imaged at a time. We describe a novel approach by integrating the microfluidic channel directly on an image sensor. Fluorescence signals emitted from the drops are collected by the sensor that forms the bottom of the channel. The proximity of the drops to the sensor facilitates efficient collection of fluorescence emission from the drops, and overcomes the trade-off between light collection efficiency and field of view in conventional microscopy. The interrogation rate of the device is currently limited by the acquisition speed of CMOS sensor, and is expected to increase further as high-speed sensors become increasingly available. More information can be found at http://web.stanford.edu/group/tanglab/