Integrated microfluidics for label-free leukocyte sorting and electrical phenotyping

Circulating leukocytes (white blood cells) in blood are known to orchestrate various biological processes and become activated during host defence (e.g. infection) or in pathogenesis of major diseases such as cancer, type 2 diabetes mellitus (T2DM) or cardiovascular diseases. Conventionally, fluorescence- and magnetic-activated cell sorting (FACS, MACS) techniques widely used in leukocyte studies require antibodies labelling which is expensive and time consuming. Leukocytes are also prone to activation during sample preparation which advocates the need to develop novel labelfree leukocyte sorting and analysis approaches. Microfluidic impedance cytometry is an established single-cell analysis tool based on intrinsic cellular dielectric properties. It is widely used for leukocyte enumeration and differential counting, but its application in leukocyte activation profiling remains unexplored. In this dissertation, two novel microfluidic technologies for label-free leukocyte sorting and electrical profiling towards rapid immune health profiling in type 2 diabetes mellitus are developed. In the first part of this project, a combinatorial microfluidic strategy for leukocyte phenotyping by enriching target leukocyte subtypes (neutrophils and monocytes) by Dean Flow Fractionation (DFF) prior impedance measurement is proposed. This increases the detection selectivity which is demonstrated for various applications namely monocyte activation, monocyte differentiation and monocyte subtype characterization. We also showed for the first time, that leukocyte impedance characteristics were associated with cardiovascular risk factors (lipid levels and Creactive protein (CRP)) in patients with T2DM, thus suggesting leukocyte impedance signature as novel surrogate biomarkers for diabetes testing. In the second part of the project, both cell sorting module and impedance detection module are integrated on a single chip. The integrated “sample in-answer out” platform provides several key advantages including high leukocyte separation efficiency, minimal sample manual handling, and rapid analysis (~5-15 mins). This platform was developed for direct neutrophil isolation and impedance characterization of neutrophil extracellular trap formation (NETosis), a recently discovered key defense mechanism of neutrophils. Our results showed distinct differences in impedance profiles of neutrophils undergoing NETosis and can be further developed to study neutrophil dysfunction in T2DM. Finally, by changing the dimensions of microfluidic design, it is demonstrated that it is possible to perform cancer cell sorting and electrical phenotyping for assessment of metastatic potential.Doctor of Philosoph

Label-free virtual staining of neutrophil extracellular traps (NETs) in microfluidics

Neutrophils are the most abundant circulating white blood cells and one of their critical functions to eliminate pathogenic threats includes the release of extracellular DNA, also known as neutrophil extracellular traps (NETs), which is dysregulated in many diseases including cancer, type 2 diabetes mellitus and infectious diseases. Currently, conventional methods to quantify the NET formation (NETosis) rely on fluorescence antibody-based NET labelling or circulating NET-associated protein detection by ELISA, which are expensive, laborious, and time-consuming. In this work, we employed a novel "virtual staining" using deep convolutional neural networks (CNNs) to facilitate label-free quantification of NETs trapped in a micropillar array in a microfluidic device. Virtual staining is constructed to establish relations between morphological features in phase contrast images and fluorescence features in Sytox-green (DNA dye) images. We first investigated the effect of different learning rates on model training and optimized the learning rate to achieve the best model which can provide outputs close to Sytox green staining based on various reconstruction metrics (e.g., structural similarity (SSIM) and pixel-wise error (MAE, MSE)). The virtual staining of different NET concentrations was investigated which showed a linear correlation with fluorescent staining. As a proof of concept for clinical testing, the model was used to characterize purified neutrophils treated with NETosis inducers, including lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), and calcium ionophore (CaI), and successfully detected different NET profiles for different treatments. Collectively, these results demonstrated the potential of using deep learning for enhanced label-free image analysis of NETs for clinical research, drug discovery and point-of-care testing of diseases.Ministry of Education (MOE)Submitted/Accepted versionH. W. H. would like to acknowledge the financial support from MOE AcRF Tier 2 (MOE-T2EP30120-0004), the Lee Kong Chian School of Medicine (LKCMedicine) Vascular Research Initiative and Dompé farmaceutici S.p.A

Advances in Single Cell Impedance Cytometry for Biomedical Applications

Microfluidics impedance cytometry is an emerging research tool for high throughput analysis of dielectric properties of cells and internal cellular components. This label-free method can be used in different biological assays including particle sizing and enumeration, cell phenotyping and disease diagnostics. Herein, we review recent developments in single cell impedance cytometer platforms, their biomedical and clinical applications, and discuss the future directions and challenges in this field.NRF (Natl Research Foundation, S’pore)MOE (Min. of Education, S’pore)NMRC (Natl Medical Research Council, S’pore)MOH (Min. of Health, S’pore)Published versio

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.NRF (Natl Research Foundation, S’pore)MOE (Min. of Education, S’pore)NMRC (Natl Medical Research Council, S’pore)MOH (Min. of Health, S’pore)Accepted versio

Microfluidic impedance-deformability cytometry for label-free single neutrophil mechanophenotyping

The intrinsic biophysical states of neutrophils are associated with immune dysfunctions in diseases. While advanced image-based biophysical flow cytometers can probe cell deformability at high throughput, it is nontrivial to couple different sensing modalities (e.g., electrical) to measure other critical cell attributes including cell viability and membrane integrity. Herein, an "optics-free" impedance-deformability cytometer for multiparametric single cell mechanophenotyping is reported. The microfluidic platform integrates hydrodynamic cell pinching, and multifrequency impedance quantification of cell size, deformability, and membrane impedance (indicative of cell viability and activation). A newly-defined "electrical deformability index" is validated by numerical simulations, and shows strong correlations with the optical cell deformability index of HL-60 experimentally. Human neutrophils treated with various biochemical stimul are further profiled, and distinct differences in multimodal impedance signatures and UMAP analysis are observed. Overall, the integrated cytometer enables label-free cell profiling at throughput of >1000 cells min-1 without any antibodies labeling to facilitate clinical diagnostics.Ministry of Education (MOE)Nanyang Technological UniversitySubmitted/Accepted versionThis research was supported by Singapore Ministry of Education Academic Research Fund (MOE ACRF) Tier 1 (RG53/18), MOE AcRF Tier 2 (MOE-T2EP30120-0004), and A. Menarini Biomarkers Pte Ltd. C.P. acknowledged support for the NTU-RSB Postdoctoral Fellowship

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.MOE (Min. of Education, S’pore)Published versio

Rapid screening of urinary tract infection using microfluidic inertial-impedance cytometry

Urinary tract infection (UTI) diagnosis based on urine culture for bacteriuria analysis is time-consuming and often leads to wastage of hospital resources due to false-positive UTI cases. Direct cellular phenotyping (e.g., RBCs, neutrophils, epithelial cells) of urine samples remains a technical challenge as low cell concentrations, and urine characteristics (conductivities, pH, microbes) can affect the accuracy of cell measurements. In this work, we report a microfluidic inertial-impedance cytometry technique for label-free rapid (<5 min) neutrophil sorting and impedance profiling from urine directly. Based on size-based inertial focusing effects, neutrophils are isolated, concentrated, and resuspended in saline (buffer exchange) to improve consistency in impedance-based single-cell analysis. We first observed that both urine pH and the presence of bacteria can affect neutrophil high-frequency impedance measurements possibly due to changes in nucleus morphology as neutrophils undergo NETosis and phagocytosis, respectively. As a proof-of-concept for clinical testing, we report for the first time, rapid UTI testing based on multiparametric impedance profiling of putative neutrophils (electrical size, membrane properties, and distribution) in urine samples from non-UTI (n = 20) and UTI patients (n = 20). A significant increase in cell count was observed in UTI samples, and biophysical parameters were used to develop a UTI classifier with an area under the receiver operating characteristic curve of 0.84. Overall, the developed platform facilitates rapid culture-free urine screening which can be further developed to assess disease severity in UTI and other urologic diseases based on neutrophil electrical signatures.Ministry of Education (MOE)Nanyang Technological UniversitySubmitted/Accepted versionThis research is supported by Singapore MOE AcRF Tier 1(RG53/18), HealthTech NTU-LKCMedicine-NHG POCT (ID POCT/17003) and MOE AcRF Tier2 (MOE-T2EP30120-0004). C.P acknowledged support for the NTU-RSB Postdoctoral Fellowship