Hui Min Tay

Rapid and label-free microfluidic neutrophil purification and phenotyping in diabetes mellitus.

Advanced management of dysmetabolic syndromes such as diabetes will benefit from a timely mechanistic insight enabling personalized medicine approaches. Herein, we present a rapid microfluidic neutrophil sorting and functional phenotyping strategy for type 2 diabetes mellitus (T2DM) patients using small blood volumes (fingerprick ~100 μL). The developed inertial microfluidics technology enables single-step neutrophil isolation (>90% purity) without immuno-labeling and sorted neutrophils are used to characterize their rolling behavior on E-selectin, a critical step in leukocyte recruitment during inflammation. The integrated microfluidics testing methodology facilitates high throughput single-cell quantification of neutrophil rolling to detect subtle differences in speed distribution. Higher rolling speed was observed in T2DM patients (P < 0.01) which strongly correlated with neutrophil activation, rolling ligand P-selectin glycoprotein ligand 1 (PSGL-1) expression, as well as established cardiovascular risk factors (cholesterol, high-sensitive C-reactive protein (CRP) and HbA1c). Rolling phenotype can be modulated by common disease risk modifiers (metformin and pravastatin). Receiver operating characteristics (ROC) and principal component analysis (PCA) revealed neutrophil rolling as an important functional phenotype in T2DM diagnostics. These results suggest a new point-of-care testing methodology, and neutrophil rolling speed as a functional biomarker for rapid profiling of dysmetabolic subjects in clinical and patient-oriented settings.

A tunable microfluidic 3D stenosis model to study leukocyte-endothelial interactions in atherosclerosis

Atherosclerosis, a chronic inflammatory disorder characterized by endothelial dysfunction and blood vessel narrowing, is the leading cause of cardiovascular diseases including heart attack and stroke. Herein, we present a novel tunable microfluidic atherosclerosis model to study vascular inflammation and leukocyte-endothelial interactions in 3D vessel stenosis. Flow and shear stress profiles were characterized in pneumatic-controlled stenosis conditions (0%, 50% and 80% constriction) using fluid simulation and experimental beads perfusion. Due to non-uniform fluid flow at the 3D stenosis, distinct monocyte (THP-1) adhesion patterns on inflamed [tumor necrosis factor-α (TNF-α) treated] endothelium were observed, and there was a differential endothelial expression of intercellular adhesion molecule-1 (ICAM-1) at the constriction region. Whole blood perfusion studies also showed increased leukocyte interactions (cell rolling and adherence) at the stenosis of healthy and inflamed endothelium, clearly highlighting the importance of vascular inflammation, flow disturbance, and vessel geometry in recapitulating atherogenic microenvironment. To demonstrate inflammatory risk assessment using leukocytes as functional biomarkers, we perfused whole blood samples into the developed microdevices (80% constriction) and observed significant dose-dependent effects of leukocyte adhesion in healthy and inflamed (TNF-α treated) blood samples. Taken together, the 3D stenosis chip facilitates quantitative study of hemodynamics and leukocyte-endothelial interactions, and can be further developed into a point-of-care blood profiling device for atherosclerosis and other vascular diseases.

Label-free leukocyte sorting and impedance-based profiling for diabetes testing

Circulating leukocytes comprise of approximately 1% of all blood cells and efficient enrichment of these cells from whole blood is critical for understanding cellular heterogeneity and biological significance in health and diseases. In this work, we report a novel microfluidic strategy for rapid (< 1 h) label-free leukocyte sorting and impedance-based profiling to determine cell activation in type 2 diabetes mellitus (T2DM) using whole blood. Leukocytes were first size-fractionated into different subtypes (neutrophils, monocytes, lymphocytes) using an inertial spiral sorter prior to single-cell impedance measurement in a microfluidic device with coplanar electrode design. Significant changes in membrane dielectric properties (size and opacity) were detected between healthy and activated leukocytes (TNF-α/LPS stimulated), during monocyte differentiation and among different monocyte subsets (classical, intermediate, non-classical). As proof-of-concept for diabetes testing, neutrophil/monocyte dielectric properties in T2DM subjects (n = 8) were quantified which were associated with cardiovascular risk factors including lipid levels, C-reactive protein (CRP) and vascular functions (LnRHI) (P < 0.05) were observed. Overall, these results clearly showed that T2DM subjects have pro-inflammatory leukocyte phenotypes and suggest leukocyte impedance signature as a novel surrogate biomarker for inflammation.