Single Cell Analysis of Leukocyte Protease Activity Using Integrated Continuous-Flow Microfluidics.

Leukocytes are the essential cells of the immune system that protect the human body against bacteria, viruses, and other foreign invaders. Secretory products of individual leukocytes, such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase (ADAMs), are critical for regulating the inflammatory response and mediating host defense. Conventional single cell analytical methods, such as flow cytometry for cellular surface biomarker studies, are insufficient for performing functional assays of the protease activity of individual leukocytes.

Liquid biopsy and therapeutic response: Circulating tumor cell cultures for evaluation of anticancer treatment.

The lack of a robust anticancer drug screening system to monitor patients during treatment delays realization of personalized treatment. We demonstrate an efficient approach to evaluate drug response using patient-derived circulating tumor cell (CTC) cultures obtained from liquid biopsy. Custom microfabricated tapered microwells were integrated with microfluidics to allow robust formation of CTC clusters without pre-enrichment and subsequent drug screening in situ.

Single cell multiplexed assay for proteolytic activity using droplet microfluidics.

Cellular enzymes interact in a post-translationally regulated fashion to govern individual cell behaviors, yet current platform technologies are limited in their ability to measure multiple enzyme activities simultaneously in single cells. Here, we developed multi-color Förster resonance energy transfer (FRET)-based enzymatic substrates and use them in a microfluidics platform to simultaneously measure multiple specific protease activities from water-in-oil droplets that contain single cells. By integrating the microfluidic platform with a computational analytical method, Proteolytic Activity Matrix Analysis (PrAMA), we are able to infer six different protease activity signals from individual cells in a high throughput manner (~100 cells/experimental run).

Microfluidics for research and applications in oncology.

Cancer is currently one of the top non-communicable human diseases, and continual research and developmental efforts are being made to better understand and manage this disease. More recently, with the improved understanding in cancer biology as well as the advancements made in microtechnology and rapid prototyping, microfluidics is increasingly being explored and even validated for use in the detection, diagnosis and treatment of cancer. With inherent advantages such as small sample volume, high sensitivity and fast processing time, microfluidics is well-positioned to serve as a promising platform for applications in oncology.

Low-volume multiplexed proteolytic activity assay and inhibitor analysis through a pico-injector array.

Secreted active proteases, from families of enzymes such as matrix metalloproteinases (MMPs) and ADAMs (a disintegrin and metalloproteinases), participate in diverse pathological processes. To simultaneously measure multiple specific protease activities, a series of parallel enzyme reactions combined with a series of inhibitor analyses for proteolytic activity matrix analysis (PrAMA) are essential but limited due to the sample quantity requirements and the complexity of performing multiple reactions. To address these issues, we developed a pico-injector array to generate 72 different reactions in picoliter-volume droplets by controlling the sequence of combinational injections, which allowed simultaneous recording of a wide range of multiple enzyme reactions and measurement of inhibitor effects using small sample volumes (~10 μL).

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.