(570b) Population-Based Detection of Cell Penetrating Peptide Uptake in a Microfluidic Droplet Trapping Array

Authors: 
Melvin, A., Louisiana State University
Safabakhsh, N., Louisiana State University
Charles, S., Louisiana State University
Vaithiyanathan, M., Louisiana State University
Elkhanoufi, R., Louisiana State University
Droplet-based microfluidic devices have emerged as a highly efficient technique for performing high-throughput, single cell analysis of heterogeneous samples such as tumor biopsies. The underlying principle involves compartmentalizing single cells in picoliter-sized monodisperse aqueous droplets in an inert continuous oil phase. Traditionally, this technology has required the use of off-chip incubation of samples coupled with high speed cameras for static measurements of cells or extracellular products. Incorporation of a trapping array downstream of the flow focusing junctions allows for the dynamic measurement of cellular behavior or intracellular enzyme activity. In this study, a microfluidic droplet trapping array was fabricated using polydimethylsiloxzne (PDMS) by established soft lithography and PDMS replication techniques, in order to encapsulate and trap single cells in aqueous droplets and assess the single cell uptake of cell penetrating peptides (CPPs) across an entire population. This technology is capable of delivering a more quantitative and complete understanding of the dynamic permeability efficiency of select CPPs. The continuous phase was fluorinated oil (Novec 7500, 3M) with a 0.2% w/w 008-fluorosurfactant (RAN Biotechnologies). Oil and water flow rates were optimized to maximize single cell encapsulation and droplet trapping efficiency in the array. Cell viability was determined in entrapped droplets to verify that encapsulation and extended trapping did not affect cellular viability. CPP permeability efficiency was determined across 767 droplets using both commercially available and novel CPPs. The microfluidic droplet trapping array consisted of a flow-focusing junction upstream of a droplet trapping array to allow for on-chip incubation and visualization of the encapsulated cells. The geometry was designed to produce droplets with diameters ranging from 60-140 microns by adjusting the flowrates of the inlet oil and water streams. The droplet trapping array consisted of 70 µm circles imprinted 40 µm into the PDMS directly above the downstream trapping channel. It was observed that cell viability was not affected in the trapped droplets for the duration of the experiment (~3-6 hours). Dynamic analysis of CPP uptake was easily visualized across the entire population of three different types of cancer cells (HeLa, OPM2, and HCT116). This study demonstrated a novel application of microfluidic droplet trapping array specifically relevant to cancer detection and diagnostics. Single cancer cells were encapsulated in aqueous droplets that were subsequently trapped and analyzed in a time-dependent manner. While the CPP studies are proof of concept, they demonstrate the capabilities of the technology to quantify the intracellular fluorescence in single cells across a heterogeneous sample.