On-Chip Analysis of Protein Secretion from Single Cells Using Microbead Biosensors

Abstract

The profiling of the effector functions of single immune cellsincluding cytokine secretioncan lead to a deeper understanding of how the immune system operates and to potential diagnostics and therapeutical applications. Here, we report a microfluidic device that pairs single cells and antibody-functionalized microbeads in hydrodynamic traps to quantitate cytokine secretion. The device contains 1008 microchambers, each with a volume of ∼500 pL, divided into six different sections individually addressed to deliver an equal number of chemical stimuli. Integrating microvalves allowed us to isolate cell/bead pairs, preventing cross-contamination with factors secreted by adjacent cells. We implemented a fluorescence sandwich immunoassay on the biosensing microbeads with a limit of detection of 9 pg/mL and were able to detect interleukin-8 (IL-8) secreted by single blood-derived human monocytes in response to different concentrations of LPS. Finally, our platform allowed us to observe a significant decrease in the number of IL-8-secreting monocytes when paracrine signaling becomes disrupted. Overall, our platform could have a variety of applications for which the analysis of cellular function heterogeneity is necessary, such as cancer research, antibody discovery, or rare cell screening.

Integrated Microfluidics for Single‐Cell Separation and On‐Chip Analysis: Novel Applications and Recent Advances

“…• Combination with microvalves to prevent‐cross‐talk between chambers and enable targeted solution exchange [ 195 ]…”

“…• Label-free separation • High versatility and compatibility with miniaturized bioassays for enhanced separation and analysis capabilities [202] • Well-controlled cell positions allow long-term single-cell analysis • Facilitates single-cell-bead pairings and singlecell co-cultures [195,203] • Motile cells might leave the compartments [263] • Limited control in ratios and distances between cells in single-cell co-culture [202] • Potential cross-talk between wells or traps [127] • Targeted delivery challenging, as reagents are distributed throughout the array [127] • Limited dynamic range in sorting cells of different sizes or types…”

“…• Stackable microwells for controlled single-cell co-cultures [115,116] • Combination with microvalves to prevent-cross-talk between chambers and enable targeted solution exchange [195] Droplet-based microfluidics…”

“…Cytokine secretion analysis under chemical stimulation [195] Human monocytes were purified from peripheral blood and suspended in cell culture medium -Microtraps (microvalve assisted)…”

“…Furthermore, they can be generated (e.g., dendritic cell generation and maturation from peripheral blood mononuclear cells [PBMCs]), or mimicked (e.g., establishing antibody secretion via transfection of cell lines). [195][196][197][198] In contrast to CTCs, WBCs (i.e., leukocytes, such as neutrophils, lymphocytes, monocytes) are relatively abundant in blood (estimated 4.5-11.0 Â 10 6 mL À1 ). This facilitates their direct capture and isolation from whole blood samples for revealing insights into the pathogenesis of various diseases and the identification of new prognostic biomarkers.…”