Microparticle parking and isolation for highly sensitive microRNA detection

Abstract

Isolating small objects, such as particles, cells, and molecules, in individual aqueous droplets is useful for chemical and biological assays. We have developed a simple microfluidic platform to immobilize (park) microparticles at defined locations, and isolate particles in monodisperse droplets surrounded by immiscible oil. While conventional methods can only achieve stochastic encapsulation of objects within larger droplets, our in situ method ensures that a single particle is entrapped in a similar-sized droplet, with ~95% yield for parking and isolation. This enables time-lapse studies of reactions in confined volumes and can be used to perform enzymatic amplification of a desired signal to improve the sensitivity of diagnostic assays. To demonstrate the utility of our technique, we perform highly sensitive, multiplexed microRNA detection by isolating encoded, functional hydrogel microparticles in small aqueous droplets. Non-fouling hydrogel microparticles are attractive for microRNA detection due to favorable capture kinetics. By encapsulating these particles in droplets and employing a generalizable enzyme amplification scheme, we demonstrate an order of magnitude improvement in detection sensitivity compared to a non-amplified assay.

Quantitative and multiplex microRNA assays from unprocessed cells in isolated nanoliter well arrays

“…As expected from our theoretical model, our LLOD is >100× lower than previously reported for hydrogel particle-based assays done without signal amplification. 45 , 48 While the PEGDA hydrogel-based miRNA hybridization assay's sensitivity can be further increased using signal amplification (not target amplification) after miRNA hybridization, 44 , 47 , 66 in this work we aimed to keep the assay protocol simple. Our current results demonstrate the ability to perform sensitive and quantitative miRNA assays across a large dynamic range.…”

“…Hydrogel posts were treated with 500 μM potassium permanganate (Sigma) to oxidize hydrophobic, non-reacted acrylate groups to reduce non-specific binding, as specified. 44 , 66 …”

“…Aqueous droplets in oil are another approach to isolate nanoliter-volume reactors for biological analysis. 44 , 66 , 75 – 77 While droplet platforms generally have advantages in throughput, their need for pumps, precise fluidic controls, and careful engineering design considerations has limited their broad adoption. 23 Reactor isolation can also be done via valving in microfluidic devices, 78 – 80 but because of their need for dedicated equipment and limited density, these devices are better suited for applications with less parallelization and more complex workflows.…”

Microfluidic platform for selective microparticle parking and paired particle isolation in droplet arrays

“…35 To enable selective parking of microparticles based on particle physical characteristics, we designed a microfluidic device with an array of hydrodynamic traps, based on design principles from prior work. 13,31 Figure 1(a) shows the overall device design: the inlet contains a dust filter to prevent debris from entering the main channel and posts along the channel direct particles to streamlines at the edge of the channel to facilitate parking into traps. There are a total of 39 traps, with one trap in each row of the serpentine channel.…”

“…There are 5 pores along the bottom of each trap, each $10 lm in width. The narrow width of each pore ensures that highly deformable particles do not pass through the bottom of the trap and prevents trespassing of the two-phase interface during the isolation process, 13 while multiple pores ensure sufficient fluid flow through the trap to guide and park particles. Figures 1(d) and 1(e) show that different critical pressures are required to park particles depending on particle diameter, trap entrance width, and particle composition.…”