Formation of uniform reaction volumes using concentric amphiphilic microparticles

Abstract

Reactions performed in uniform microscale volumes have enabled numerous applications in the analysis of rare entities (e.g. cells and molecules), however, sophisticated instruments are usually required to form large numbers of uniform compartments. Here, uniform aqueous droplets are formed by simply mixing microscale multi-material particles, consisting of concentric hydrophobic outer and hydrophilic inner layers, with oil and water. The particles are manufactured in batch using a 3D printed device to co-flow four concentric streams of polymer precursors which are polymerized with UV light. The size of the particles is readily controlled by adjusting the fluid flow rate ratios and mask design; whereas the cross-sectional shapes are altered by microfluidic nozzle design in the 3D printed device. Once a particle encapsulates an aqueous volume, each "dropicle" provides uniform compartmentalization and customizable shape-coding for each sample volume to enable multiplexing of uniform reactions in a scalable manner. We implement an enzymatically-amplified affinity assay using the dropicle system, yielding a detection limit of <1 pM with a dynamic range of at least 3 orders of magnitude. Moreover, multiplexing using two types of shape-coded particles was demonstrated without cross talk, laying a foundation for democratized single-entity assays.

Monodisperse drops templated by 3D-structured microparticles

“…This manufacturing cycle is automated using LabVIEW to fabricate large batches of particles. We also confirm the reproducibility of particle shape across a population of the particles (28). After fabrication, all particles are collected in a 50-ml centrifuge tube and rinsed with a volume of ethanol more than 1000 times the sample volume to eliminate the effect of noncross-linked reagents.…”

“…These users can then perform assays using monodisperse nanoliter-scale drops through simple shaking and agitation using widely available laboratory equipment. A number of emerging approaches to fabricate shaped microparticles could also be applied to functionalize DCPs ( 26 , 27 ) and scale their size to be compatible with analysis by flow cytometers ( 28 , 29 ). We expect that a number of assays previously demonstrated using lab-on-a-chip infrastructure could be implemented in this “lab-on-a-particle” format in the future, providing greater access to the deployment of powerful biological assays.…”

Counting of Enzymatically Amplified Affinity Reactions in Hydrogel Particle-Templated Drops

“…Hydrogel particles are utilized here both as a solid support to immobilize protein molecules, and as a template for emulsification around the hydrogel matrix. Previously, we and others have shown that spherical particles 31,32 or particles with tailored surface chemistry 33,34 can be used to template drop formation to perform measurements of DNA and protein biomarkers. In this work, we performed digital counting of β-gal enzyme on spherical polyethylene glycol (PEG) hydrogel particles templating drops of ~20 pL volume and achieved femtomolar detection limit.…”

A platform for multiplexed colorimetric microRNA detection using shape-encoded hydrogel particles

“…The latter labelling technique necessitates containment of the fluorescent product and hence has found most utility in dropletbased or oil-encapsulated systems, where the soluble fluorescent product can be contained for sufficient signal generation and multiplexing. [27][28][29][30][31][32] Thus, a limited amount of substrate is accessible for the reaction. Moreover, specialized equipment such as fluorescent microscopes, fluorescent plate readers or fluorescent scanners are necessary to detect the fluorescent signal.…”

“…Moreover, specialized equipment such as fluorescent microscopes, fluorescent plate readers or fluorescent scanners are necessary to detect the fluorescent signal. 19,24,29,32 Efforts to develop a colorimetric readout for hydrogels have used gold nanoparticle labelling combined with dark-field microscopy. While the sensitivity of this method was comparable to fluorescent labelling, it still requires the use of a microscope for detection using dark-field imaging.…”