Ramesh Ramji

Jetting microfluidics with size-sorting capability for single-cell protease detection.

Activated proteases such as matrix metalloproteinases (MMPs) secreted from cancer cells can degrade the extracellular matrix (ECM) and contribute to tumour formation and metastasis. Measuring MMP activity in individual cancer cells can provide important insights on cancer cell heterogeneity and disease progression. Here, we present a microfluidic platform combining a droplet jetting generator and a deterministic lateral displacement (DLD) size-sorting channel that is capable of encapsulating individual cancer cells inside picoliter droplets effectively. Droplet jetting with cell-triggered Rayleigh-Plateau instability was employed which produced large droplets capable of cell encapsulation (diameter, ~25µm) and small empty droplets (diameter, ~14µm), which were then size-separated using a DLD size-sorting channel to enrich the single-cell encapsulated droplets (~78%), regardless of the cell density of input sample solutions. The droplets containing encapsulated cancer cells were collected in an observation chamber to determine the kinetic profiles of MMP secretion and the inhibitory response in the presence of the drug doxycycline at the single-cell level to reveal their heterogeneous MMPs secretion activities.

Single cell kinase signaling assay using pinched flow coupled droplet microfluidics

Droplet-based microfluidics has shown potential in high throughput single cell assays by encapsulating individual cells in water-in-oil emulsions. Ordering cells in a micro-channel is necessary to encapsulate individual cells into droplets further enhancing the assay efficiency. This is typically limited due to the difficulty of preparing high-density cell solutions and maintaining them without cell aggregation in long channels (>5 cm). In this study, we developed a short pinched flow channel (5 mm) to separate cell aggregates and to form a uniform cell distribution in a droplet-generating platform that encapsulated single cells with >55% encapsulation efficiency beating Poisson encapsulation statistics. Using this platform and commercially available Sox substrates (8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline), we have demonstrated a high throughput dynamic single cell signaling assay to measure the activity of receptor tyrosine kinases (RTKs) in lung cancer cells triggered by cell surface ligand binding. The phosphorylation of the substrates resulted in fluorescent emission, showing a sigmoidal increase over a 12 h period. The result exhibited a heterogeneous signaling rate in individual cells and showed various levels of drug resistance when treated with the tyrosine kinase inhibitor, gefitinib.

Rapid Quantification of Live Cell Receptors Using Bioluminescence in a Flow‐Based Microfluidic Device

The number of receptors expressed by cells plays an important role in controlling cell signaling events, thus determining its behaviour, state and fate. Current methods of quantifying receptors on cells are either laborious or do not maintain the cells in their native form. Here, a method integrating highly sensitive bioluminescence, high precision microfluidics and small footprint of lensfree optics is developed to quantify cell surface receptors. This method is safe to use, less laborious, and faster than the conventional radiolabelling and near field scanning methods. It is also more sensitive than fluorescence based assays and is ideal for high throughput screening. In quantifying β(1) adrenergic receptors expressed on the surface of H9c2 cardiomyocytes, this method yields receptor numbers from 3.12 × 10(5) to 9.36 × 10(5) receptors/cell which are comparable with current methods. This can serve as a very good platform for rapid quantification of receptor numbers in ligand/drug binding and receptor characterization studies, which is an important part of pharmaceutical and biological research.

Ligand Binding Kinetics of Cell Surface Receptors by Microfluidic Displacement

Cell Surface binding kinetics of bio-molecular interaction is of fundamental importance in advancing our understanding of numerous biological processes and developing bioengineered systems. We have adopted a displacement technique, wherein a ligand is displaced from the binding site, by an excess of a ligand analog perfused through the microchannel. The theoretical model describes transient convection and diffusion in the microchannel volume following dissociation of the ligand from the cell surface receptors. To incorporate living cell processes, the model includes cell surface receptor trafficking. The decay of eluting ligand concentration follows a mono-exponential curve for one receptor sub-type or kinetic dissociation rate constant. A numerical solution is obtained using the method of finite differences and verified with an analytical solution for the case of negligible dispersion. Results illustrate how the fluid velocity and receptor internalization rate influence the ligand concentration at the microchannel outlet. This modeling effort is expected to allow better experimental design and subsequently more accurate measurement of kinetic rate constants.

Fold-change detection of NF-κB at target genes with different transcript outputs

The transcription factor NF-κB promotes inflammatory and stress-responsive gene transcription across a range of cell types in response to the cytokine tumor necrosis factor-α (TNF). Although NF-κB signaling exhibits significant variability across single cells, some target genes exhibit fold-change detection of NF-κB, which may buffer against stochastic variation in signaling molecules. However, this observation was made at target genes supporting high levels of TNF-inducible transcription. It is unknown if fold-change detection is maintained at NF-κB target genes with low levels of TNF-inducible transcription, for which stochastic promoter events may be more pronounced. Here we used a microfluidic cell-trapping device to measure how TNF-induced activation of NF-κB controls transcription in single Jurkat T cells at the promoters of integrated HIV and the endogenous cytokine gene IL6, which produce only a few transcripts per cell. We tracked TNF-stimulated NF-κB RelA nuclear translocation by live-cell imaging and then quantified transcript number by RNA FISH in the same cell. We found that TNF-induced transcription correlates with fold change in nuclear NF-κB with similar strength at low versus high abundance target genes. A computational model of TNF-NF-κB signaling, which implements fold-change detection from competition for binding to κB motifs, was sufficient to reproduce fold-change detection across the experimentally measured range of transcript outputs. Nevertheless, we found that gene-specific trends in transcriptional noise and levels of promoter-bound NF-κB predicted by the model were inconsistent with our experimental observations at low abundance gene targets. Our results reveal a gap in our understanding of RelA-mediated transcription for low abundance transcripts and suggest that cells use additional biological mechanisms to maintain robustness of NF-κB fold-change detection while tuning transcriptional output.

Cell surface receptors: rapid quantification of live cell receptors using bioluminescence in a flow-based microfluidic device (small 8/2015).

C. T. Lim and co-workers describe a rapid and sensitive bioluminescence-based microfluidic method for quantifying receptor numbers on live cells. On page 943, this integrated, lens-free optical platform allows the determination of signals from the cell surface with high sensitivity. Compared to conventional approaches, the combined use of bioluminescence and microfluidics makes it safe to use, reduces background noise, improves sensitivity, requires smaller sample volumes, and allows high-throughput sampling over thousands of cells.