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Blood tests are considered as standard clinical procedures to screen for markers of diseases and health conditions. However, the complex cellular background (>99.9% RBCs) and biomolecular composition often pose significant technical challenges for accurate blood analysis. An emerging approach for point-of-care blood diagnostics is utilizing "label-free" microfluidic technologies that rely on intrinsic cell properties for blood fractionation and disease detection without any antibody binding. A growing body of clinical evidence has also reported that cellular dysfunction and their biophysical phenotypes are complementary to standard hematoanalyzer analysis (complete blood count) and can provide a more comprehensive health profiling. In this review, we will summarize recent advances in microfluidic label-free separation of different blood cell components including circulating tumor cells, leukocytes, platelets and nanoscale extracellular vesicles. Label-free single cell analysis of intrinsic cell morphology, spectrochemical properties, dielectric parameters and biophysical characteristics as novel blood-based biomarkers will also be presented. Next, we will highlight research efforts that combine label-free microfluidics with machine learning approaches to enhance detection sensitivity and specificity in clinical studies, as well as innovative microfluidic solutions which are capable of fully integrated and label-free blood cell sorting and analysis. Lastly, we will envisage the current challenges and future outlook of label-free microfluidics platforms for high throughput multi-dimensional blood cell analysis to identify non-traditional circulating biomarkers for clinical diagnostics. Ministry of Education (MOE) Published version The authors would like to acknowledge support from Singapore Ministry of Education Academic Research Fund (MOE AcRF) Tier 2 (MOE-T2EP30120-0004) and under the RIE2020 Industry Alignment Fund – Industry Collaboration Projects (IAF-ICP) Funding Initiative, as well as cash and in kind contributions from the industry partner, HP Inc., through the HP-NTU Digital Manufacturing Corporate Lab.

Mandelic acid and its derivatives are an excellent source of chirality for the construction of chiral coordination polymers (CPs) and metal-organic frameworks (MOFs) for enantioselective applications. To date, limited reports exist for the incorporation of mandelic acid derivatives with halogen substituents such as fluorine and chlorine despite the extra intermolecular interactions possible. The extra sites for hydrogen and halogen bonding in these mandelic acid derivatives may afford interesting modes of coordination and packing in 3 D space. Six CPs containing mandelic acid and its halogenated analogues, (R)-2-chloromandelic acid, 3,5-difluoromandelic acid, 4-trifluoromethylmandelic acid, together with linear dipyridyl co-ligands are reported. These structures consist of three 2 D sql sheets and three 1 D chains with a range of packing motifs influenced by the differing intermolecular interactions present.

A fluorescent, chiral coordination polymer (CP) with a novel topology has been synthesised using a dipyridyl ligand derived from 1,1'-bi-2-naphthol (BINOL). Enantioselectivity ratios up to 2.61 were obtained in fluorescence sensing studies with chiral analytes.

Three MOFs with metalloporphyrin lined, large square 1D channels were used as colorimetric sensors for electron donors. Exposure to amine vapours caused a redshift of the Soret absorption bands of the metalloporphyrin.

Construction of supramolecular assemblies using hydrogen and halogen bonding between anions and the 3,5-bis(triazolyl)pyridinium motif was investigated.

The first examples of halogen bonding (XB) heteroditopic homo[2]catenanes were prepared by discrete Na+ template-directed assembly of oligo(ethylene glycol) units derived from XB donor-containing macrocycles and acyclic bis-azide precursors, followed by a Cu(I) mediated azide-alkyne cycloaddition macrocyclisation reaction. Extensive 1H NMR spectroscopic studies show that the [2]catenane hosts exhibit positive cooperative ion-pair recognition behaviour, wherein XB-mediated halide recognition is enhanced by alkali metal cation pre-complexation. Notably, subtle changes in the catenanes' oligo(ethylene glycol) chain length dramatically alters ion binding affinity, stoichiometry, complexation mode and conformational dynamics. Solution phase and single crystal X-ray diffraction studies provide evidence for competing host-separated and direct contact ion-pair binding modes. We further demonstrate the [2]catenanes are capable of extracting solid alkali metal halide salts into organic media.

Vascular stenosis caused by atherosclerosis instigates activation and aggregation of platelets, eventually resulting in thrombus formation. Although antiplatelet drugs are commonly used to inhibit platelet activation and aggregation, they unfortunately cannot prevent recurrent thrombotic events in patients with atherosclerosis. This is partially due to the limited understanding of the efficacy of antiplatelet drugs in the complex hemodynamic environment of vascular stenosis. Conventional methods for evaluating the efficacy of antiplatelet drugs under stenosis either fail to simulate the hemodynamic environment of vascular stenosis characterized by high shear stress and recirculatory flow or lack spatial resolution in their analytical techniques to statistically identify and characterize platelet aggregates. Here we propose and experimentally demonstrate a method comprising an

Exceptionally strong halogen bonding (XB) donor-chloride interactions are exploited for the chloride anion template synthesis of neutral XB [2]rotaxane host systems which contain perfluoroaryl-functionalised axle components, including a remarkably potent novel 4,6-dinitro-1,3-bis-iodotriazole motif. Halide anion recognition properties in aqueous-organic media, determined via extensive

Chiral coordination polymers constructed from mandelic acid have shown promise in enantioselective recognition and sensing, whereas its methoxy-protected derivative, α-methoxyphenyl acetic acid, is rarely used as a ligand in coordination polymers. In this study, mandelic acid, 3,5-difluoromandelic acid, and α-methoxyphenyl acetic acid were combined with divalent transition metals and bridging dipyridyl co-ligands to obtain a series of six 1D coordination polymers. The coordination polymers of mandelate and 3,5-difluoromandelate possessed densely packed structures stabilised by hydrogen bonding. In contrast, the use of (R)-α-methoxyphenyl acetic acid gave rise to zig-zag chains with significant solvent-accessible void space.

Adenosine is a metabolite that suppresses antitumor immune response of T and NK cells via extracellular binding to the two subtypes of adenosine-2 receptors, AWe report the synthesis and functional evaluation of five potent AWe provide data for six novel small molecules: five AOur results demonstrate that A

Delayed wound healing is commonly associated with diabetes. It may lead to amputation and death if not treated in a timely fashion. Limited treatments are available partially due to the poor understanding of the complex disease pathophysiology. Here, we investigated the role of leucine-rich α-2-glycoprotein 1 (LRG1) in normal and diabetic wound healing. First, our data showed that LRG1 was significantly increased at the inflammation stage of murine wound healing, and bone marrow–derived cells served as a major source of LRG1. LRG1 deletion causes impaired immune cell infiltration, reepithelialization, and angiogenesis. As a consequence, there is a significant delay in wound closure. On the other hand, LRG1 was markedly induced in diabetic wounds in both humans and mice. LRG1-deficient mice were resistant to diabetes-induced delay in wound repair. We further demonstrated that this could be explained by the mitigation of increased neutrophil extracellular traps (NETs) in diabetic wounds. Mechanistically, LRG1 mediates NETosis in an Akt-dependent manner through TGFβ type I receptor kinase ALK5. Taken together, our studies demonstrated that LRG1 derived from bone marrow cells is required for normal wound healing, revealing a physiological role for this glycoprotein, but that excess LRG1 expression in diabetes is pathogenic and contributes to chronic wound formation.

Homochiral coordination polymers have emerged as promising candidates for a range of chirotechnological applications, including asymmetric catalysis and enantioselective separation. The use of liga...

Vitamin D deficiency is a major public health problem worldwide, linked to several chronic diseases including cardiovascular diseases. While immunomodulatory effects of vitamin D on monocytes have been reported in cardiovascular and metabolic diseases, there is limited understanding on monocyte phenotype in healthy individuals with suboptimal vitamin D levels and without any clinical diseases. In this work, we performed label-free, microfluidic isolation of monocytes, and characterized their functional phenotype using flow cytometry and in vitro vascular models in healthy subjects with (n = 7) and without vitamin D deficiency (n = 16). Vitamin D deficient (VitD-Def) subjects (25(OH)D3 level

A semi-rigid chiral ligand (1R, 2R)-4,4′-(trans-cyclohexane-1,2-diyl)bis(azanediyl)bis(carbonyl) dibenzoic acid (H2cbba) was combined with various Co(II) salts and dipyridyl co-ligands to obtain four 2D homochiral coordination polymers of composition [Co(cbba)(dipyridyl)]·solvate (dipyridyl = 4,4′-bipyridyl (bipy), bis(4-pyridyl)ethylene (bpe), 1,4-bis(4-pyridyl)benzene (1,4-bpb), 2,5-bis(4-pyridyl)thiazolo [5,4-d]thiazole (2,5-bptztz)) (1–4). The topology of the frameworks is mediated by the length of the co-ligands, with short dipyridyl co-ligands (4,4′-bipy and bpe) resulting in the formation of (413·62) networks (1–2), while long co-ligands (1,4-bpb and 2,5-bptztz) led instead to (44·62) (sql) networks that undergo 2D → 2D parallel interpenetration to form a rare example of a homochiral polyrotaxane (3–4). The length of the dipyridyl co-ligand was also found to have an effect on the conformation of the flexible cbba2− ligands and the crystal packing of the networks.

Chirality is a key aspect of amino acids and is essential for life. Here, a chiral metal–organic framework, S-Mg2dobpdc, is used to determine the chirality of three BOC protected amino acids (alanine, valine and proline) by 13C solid-state NMR with chemical shift differences of up to 1.3 ppm observed between enantiomers. The chiral sensitivity persists upon in situ deprotection of the amino acids by thermolysis of the BOC group.

We attempted to use hydrogen, iodo and seleno 3,5-bis(triazolyl)pyridinium (btp) motifs to form 3D supramolecular frameworks assembled by hydrogen, halogen or chalcogen bonding to anions. Initially, we prepared flexible ditopic and tetratopic receptors where the pyridinium nitrogen atom was functionalized with a benzylic group but were unable to crystallise extended frameworks. We subsequently developed the use of Zincke methodology to prepare rigid ditopic and tetratopic hydrogen and halogen bonding tectons, and were able to crystallize the sulfate derivative of a tetratopic receptor, although unfortunately this did not have the desired open framework. Several crystal structures were obtained with the receptors and monovalent anions, including one containing an unusually short iodotriazole∙∙∙Cl– halogen bond. Generally, the btp triazole groups show a tendency to rotate away from the desired conformation and it appears that this may contribute to difficulties in obtaining 3D frameworks even when the receptors have a rigid core.

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 University Submitted/Accepted version This 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.

BackgroundAdenosine is a metabolite that suppresses antitumor immune response of T and NK cells via extracellular binding to the two subtypes of adenosine-2 receptors, A2ARs. While blockade of the A2AARs subtype effectively rescues lymphocyte activity, with four A2AAR antagonists currently in anticancer clinical trials, less is known for the therapeutic potential of the other A2BAR blockade within cancer immunotherapy. Recent studies suggest the formation of A2AAR/A2BAR dimers in tissues that coexpress the two receptor subtypes, where the A2BAR plays a dominant role, suggesting it as a promising target for cancer immunotherapy.MethodsWe report the synthesis and functional evaluation of five potent A2BAR antagonists and a dual A2AAR/A2BAR antagonist. The compounds were designed using previous pharmacological data assisted by modeling studies. Synthesis was developed using multicomponent approaches. Flow cytometry was used to evaluate the phenotype of T and NK cells on A2BAR antagonist treatment. Functional activity of T and NK cells was tested in patient-derived tumor spheroid models.ResultsWe provide data for six novel small molecules: five A2BAR selective antagonists and a dual A2AAR/A2BAR antagonist. The growth of patient-derived breast cancer spheroids is prevented when treated with A2BAR antagonists. To elucidate if this depends on increased lymphocyte activity, immune cells proliferation, and cytokine production, lymphocyte infiltration was evaluated and compared with the potent A2AAR antagonist AZD-4635. We find that A2BAR antagonists rescue T and NK cell proliferation, IFNγ and perforin production, and increase tumor infiltrating lymphocytes infiltration into tumor spheroids without altering the expression of adhesion molecules.ConclusionsOur results demonstrate that A2BAR is a promising target in immunotherapy, identifying ISAM-R56A as the most potent candidate for A2BAR blockade. Inhibition of A2BAR signaling restores T cell function and proliferation. Furthermore, A2BAR and dual A2AAR/A2BAR antagonists showed similar or better results than A2AAR antagonist AZD-4635 reinforcing the idea of dominant role of the A2BAR in the regulation of the immune system.

Extracellular vesicles (EVs) are recognized as next generation diagnostic bio-markers due to their disease-specific biomolecular cargoes and importance in cell–cell communications. A major bottleneck in EV sample preparation is the inefficient and laborious isolation of nanoscale EVs (≈50–200 nm) from endogenous proteins in biological samples. Herein, a unique microfluidic platform is reported for EV-protein fractionation based on the principle of size exclusion chromatography (SEC). Using a novel rapid (≈20 min) replica molding technique, a fritless microfluidic SEC device (μSEC) is fabricated using thiol-ene polymer (UV glue NOA81, Young’s modulus ≈1 GPa) for high pressure (up to 6 bar) sample processing. Controlled on-chip nano-liter sample plug injection (600 nL) using a modified T-junction injector is first demonstrated with rapid flow switching response time (

BackgroundPolypoidal choroidal vasculopathy (PCV), a subtype of age-related macular degeneration (AMD), is characterized by polyp-like dilatation of blood vessels and turbulent blood flow in the choroid of the eye. Gold standard anti-vascular endothelial growth factor (anti-VEGF) therapy often fails to regress polypoidal lesions in patients. Current animal models have also been hampered by their inability to recapitulate such vascular lesions. These underscore the need to identify VEGF-independent pathways in PCV pathogenesis.ResultsWe cultivated blood outgrowth endothelial cells (BOECs) from PCV patients and normal controls to serve as our experimental disease models. When BOECs were exposed to heterogeneous flow, single-cell transcriptomic analysis revealed that PCV BOECs preferentially adopted migratory-angiogenic cell state, while normal BOECs undertook proinflammatory cell state. PCV BOECs also had a repressed protective response to flow stress by demonstrating lower mitochondrial functions. We uncovered that elevated hyaluronidase-1 in PCV BOECs led to increased degradation of hyaluronan, a major component of glycocalyx that interfaces between flow stress and vascular endothelium. Notably, knockdown of hyaluronidase-1 in PCV BOEC improved mechanosensitivity through activation of Krüppel-like factor 2, a flow-responsive transcription factor, which in turn modulated PCV BOEC migration. Barrier permeability due to glycocalyx impairment in PCV BOECs was also reversed by hyaluronidase-1 knockdown. Correspondingly, hyaluronidase-1 was detected in PCV patient vitreous humor and plasma samples.ConclusionsHyaluronidase-1 inhibition could be a potential therapeutic modality in preserving glycocalyx integrity and endothelial stability in ocular diseases with vascular origin.

Background Polypoidal choroidal vasculopathy (PCV), a subtype of age-related macular degeneration (AMD), is a global leading cause of vision loss in older populations. Distinct from typical AMD, PCV is characterized by polyp-like dilatation of blood vessels and turbulent blood flow in the choroid of the eye. Gold standard anti-vascular endothelial growth factor (anti-VEGF) therapy often fails to regress polypoidal lesions in patients. Current animal models have also been hampered by their inability to recapitulate such vascular lesions. These underscore the need to identify VEGF-independent pathways in PCV pathogenesis. Results We cultivated blood outgrowth endothelial cells (BOECs) from PCV patients and normal controls to serve as our experimental disease models. When BOECs were exposed to heterogeneous flow, single-cell transcriptomic analysis revealed that PCV BOECs preferentially adopted migratory-angiogenic cell state, while normal BOECs undertook proinflammatory cell state. PCV BOECs also had a repressed protective response to flow stress by demonstrating lower mitochondrial functions. We uncovered that elevated hyaluronidase-1 in PCV BOECs led to increased degradation of hyaluronan, a major component of glycocalyx that interfaces between flow stress and vascular endothelium. Notably, knockdown of hyaluronidase-1 in PCV BOEC improved mechanosensitivity, as demonstrated by a significant 1.5-fold upregulation of Krüppel-like factor 2 (KLF2) expression, a flow-responsive transcription factor. Activation of KLF2 might in turn modulate PCV BOEC migration. Barrier permeability due to glycocalyx impairment in PCV BOECs was also reversed by hyaluronidase-1 knockdown. Correspondingly, hyaluronidase-1 was detected in PCV patient vitreous humor and plasma samples. Conclusions Hyaluronidase-1 inhibition could be a potential therapeutic modality in preserving glycocalyx integrity and endothelial stability in ocular diseases with vascular origin.

The ubiquity of anions in biological and environmental systems has motivated the development of many novel anion receptors and sensors over the past two decades. Optical anion sensors, which undergo a spectral change in response to anion binding, are particularly desirable due to the technical simplicity and fast response time of such systems. A myriad of macrocyclic host molecules have been shown to be effective anion receptors and present a promising platform for elaboration into optical anion sensors by incorporation of an appropriate fluorogenic or chromogenic group. The enhanced anion binding properties of the three-dimensional binding cavities in mechanically interlocked host molecules have also been exploited to design anion sensors that exhibit remarkable selectivity and sensitivity. This review summarises recent progress in the development of optical anion sensors based on macrocyclic and interlocked hosts. The major classes of macrocyclic receptors possessing neutral, cationic and metal-based binding motifs are examined, followed by a survey of optically-responsive interlocked anion hosts.

Extracellular vesicles (EVs) are key mediators of communication among cells, and clinical utilities of EVs-based biomarkers remain limited due to difficulties in isolating EVs from whole blood reliably. We report a novel inertial-based microfluidic platform for direct isolation of nanoscale EVs (exosomes, 50 to 200 nm) and medium-sized EVs (microvesicles, 200 nm to 1 μm) from blood with high efficiency (three-fold increase in EV yield compared to ultracentrifugation). In a pilot clinical study of healthy (n = 5) and type 2 diabetes mellitus (T2DM, n = 9) subjects, we detected higher EV levels in T2DM patients (P < 0.05), and identified a subset of "high-risk" T2DM subjects with abnormally high (∼10-fold to 50-fold) amounts of platelet (CD41a+) or leukocyte-derived (CD45+) EVs. Our in vitro endothelial cell assay further revealed that EVs from "high-risk" T2DM subjects induced significantly higher vascular inflammation (ICAM-1 expression) (P < 0.05) as compared to healthy and non-"high-risk" T2DM subjects, reflecting a pro-inflammatory phenotype. Overall, the EV isolation tool is scalable, and requires less manual labour, cost and processing time. This enables further development of EV-based diagnostics, whereby a combined immunological and functional phenotyping strategy can potentially be used for rapid vascular risk stratification in T2DM. Ministry of Education (MOE) Nanyang Technological University Singapore-MIT Alliance for Research and Technology (SMART) H. W. H. would like to acknowledge the kind financial support from SMART Innovation Centre (ING-000539 BIO IGN and ING-001058 BIO IGN), Singapore Ministry of Education (MOE) Academic Research Fund Tier 1 (RG53/18), as well as A. Menarini Biomarkers Singapore Pte Ltd. S. Y. L would like to acknowledge support from the NTU Research Scholarship.

Circulating lymphocytes are integral components of our adaptive immunity with emerging clinical applications in immune status monitoring in infectious diseases and cell-mediated cancer immunotherapies. Herein we present a novel impedance-based microfluidic assay for label-free lymphocyte activation profiling based on native or antigen-specific T-lymphocyte biophysical responses. Single cell impedance profiling of T-lymphocytes first revealed distinct biophysical differences in cell size and membrane electrical impedance of healthy, activated (CD3/CD28) and dead lymphocyte populations. Impedance characterization of peripheral blood mononuclear cells (PBMCs) stimulated with mitogen phytohemagglutinin (PHA) or Tuberculin Purified Protein Derivative antigen (PPD) after 24 h also showed an increase in lymphocyte cell size (∼8 to 10 μm) which corresponded to activated lymphocytes (CD69+CD137+). We next developed a spiral inertial microfluidics cell sorter integrated with coplanar electrodes for direct impedance quantification of activated lymphocytes. By removing non-activated smaller lymphocytes (< 8 μm) and employing hydrodynamic-based single stream particle focusing, we demonstrated significant enrichment of activated lymphocytes (∼11.7-fold) to electrically detect low levels of lymphocyte activation (< 5%). Finally, the developed biochip is coupled with magnetic activated cell sorting (MACS) to quantify CD4+ T-lymphocytes response in PBMCs stimulated with PPD. A differential impedance cell count ratio (stimulated/unstimulated) was defined to distinguish activated T-lymphocytes, which showed better sensitivity as compared to immunophenotyping by flow cytometry. Taken together, the integrated impedance biosensor can be further developed as a rapid multiplexed screening assay to detect antigen-specific T-lymphocyte responses to characterize host immunity and diagnosis of infectious diseases (e.g tuberculosis, dengue and COVID-19). Ministry of Education (MOE) National Research Foundation (NRF) This research is supported by Singapore Ministry of Education Academic Research Fund Tier 1 (RG53/18) and A. Menarini Biomarkers Pte Ltd, as well as the National Research Foundation, Prime Minister’s Office, Singapore under its Campus for Research Excellence and Technological Enterprise (CREATE) programme, through Singapore MIT Alliance for Research and Technology (SMART): Critical Analytics for Manufacturing Personalised-Medicine (CAMP) Inter-Disciplinary Research Group.

Delayed wound healing is commonly associated with diabetes. It may lead to amputation and death if not treated timely. Limited treatments are available partially due to the poor understanding of the complex disease pathophysiology. Here, we investigated the role of Leucine-rich alpha-2-glycoprotein1 (LRG1) in normal and diabetic wound healing. Firstly, our data showed that LRG1 was significantly increased at the inflammation stage of murine wound healing, and bone marrow-derived cells served as a major source of LRG1. LRG1 deletion causes impaired immune cell infiltration, re-epithelialization and angiogenesis. As a consequence, there is a significant delay in wound closure. On the other hand, LRG1 was markedly induced in diabetic wounds in both humans and mice. LRG1-deficient mice were resistant to diabetes-induced delay in wound repair. We further demonstrated that this could be explained by the mitigation of increased neutrophil extracellular traps (NETs) in diabetic wounds. Mechanistically, LRG1 mediates NETosis in an Akt-dependent manner through TGF-beta type I receptor kinase ALK5. Taken together, our studies demonstrated that LRG1 derived from bone marrow cells is required for normal wound healing, revealing a physiological role for this glycoprotein, but that excess LRG1 expression in diabetes is pathogenic and contributes to chronic wound formation.

Blood vessel narrowing and arterial occlusion are pathological hallmarks of atherosclerosis, which involves a complex interplay of perturbed hemodynamics, endothelial dysfunction and inflammatory cascade. Herein, we report a novel circular microfluidic stenosis model that recapitulates atherogenic flow-mediated endothelial dysfunction and blood-endothelial cell (EC) interactions in vitro. 2D and 3D stenosis microchannels with different constriction geometries were fabricated using 3D printing to study flow disturbances under varying severity of occlusion and wall shear stresses (100 to 2000 dynecm-2). Experimental and fluid simulation results confirmed the presence of pathological shear stresses in the stenosis region, and recirculation flow post stenosis. The resultant pathological flow profile induced pro-inflammatory and pro-thrombotic EC state as demonstrated by orthogonal EC alignment, enhanced platelet adhesion at the stenosis, and aberrant leukocyte-EC interactions post stenosis. Clinical utility of the vascular model was further investigated by testing anti-thrombotic and immunomodulatory efficacy of aspirin and metformin, respectively. Overall, the platform enables multi-factorial analysis of critical atherogenic events including endothelial dysfunction, platelets and leukocyte adhesion, and can be further developed into a liquid biopsy tool for cardiovascular risk stratification.

Circulating leukocytes are indispensable components of the immune system, and rapid analysis of their native state or functionalities can help to unravel their pathophysiological roles and identify novel prognostic biomarkers in health and diseases. Herein we report a novel high throughput “sample-in-answer-out” integrated platform for continuous leukocyte sorting and single-cell electrical profiling in a label-free manner. The multi-staged platform enables isolation of neutrophils and monocytes from diluted or lysed blood samples directly within minutes based on Dean flow fractionation (DFF) (stage 1). Next DFF-purified leukocytes are inertially focused in serpentine channels into a single stream (stage 2) prior to impedance detection (stage 3). As a proof-of-concept for neutrophil functional characterization towards diabetes testing, we characterized the formation of neutrophil extracellular traps (NETosis) of healthy and glucose-treated neutrophils and observed significant changes in dielectric properties (size and opacity) between both groups. Interestingly, the NETosis profiles induced by calcium ionophore (CaI) and phorbol 12-myristate 13-acetate (PMA) were also electrically different, which could be attributed to the differential rates of cell enlargement and attenuated membrane permeability. Taken together, these results clearly demonstrated the potential of the developed platform for rapid (∼mins) and label-free leukocyte profiling and the use of impedance signatures as novel functional biomarkers for point-of-care testing in diabetes. MOE (Min. of Education, S’pore) NMRC (Natl Medical Research Council, S’pore) Accepted version

The emergence of omics studies and single cell analysis in biomedicine has advocated a critical need to develop novel cell sorting technologies to process complex and heterogenous biological samples prior analysis. Spiral inertial microfluidics is an enabling membrane-free cell separation technique developed almost a decade ago for high throughput biophysical cell separation, and has since been widely exploited for different biomedical applications. In this chapter, we will provide a comprehensive review on spiral inertial microfluidics including (1) conventional and microfluidic cell sorting techniques, (2) introduction to inertial microfluidics and Dean-coupled inertial focusing, (3) classification of major spiral devices, (4) summary of different biomedical applications, (5) recent advances in next generation spiral cell sorters, and (6) highlight key challenges for future research. With increasing advancement in microfabrication and computational simulation, we envision that spiral inertial microfluidics will play a leading role in driving research and commercialization in clinical diagnostics, as well as other research areas in chemistry and material sciences.

Chiral coordination polymers and porous chiral MOFs have great potential as agents for enantioselective separations, sensing, and catalysis due to their well-structured internal chiral cavities. They also have physical properties that may be exploited, such as non-linear optical activity, ferroelectrics, magnetochiral dichroism, and use as chemical shift reagents. There are many ways in which chiral coordination polymers can be synthesised and the file has grown tremendously over the past two decades. In this review, we will discuss some of the main synthetic approaches that have been taken towards this fascinating class of materials. The main focus will be placed on synthetic approaches using enantiopure ligands, the route that is most likely to yield homochiral network solids. We will highlight the applications of many examples, noting how the design of the frameworks influences their application. Whilst this is a mature field, there still lies huge potential for functional chiral materials, and we aim to demonstrate areas that are ripe for exploration.

Monocytes and platelets play key roles in atherosclerosis and thrombosis, and circulating monocyte-platelet aggregates (MPA) in blood have been widely proposed as surrogate biomarkers for cardiovascular risk stratification and monitoring antiplatelet therapies. However, conventional MPA characterization is based on whole-blood fixation and flow cytometry analysis which adversely affect cell viability and detection accuracy due to significant leukocyte and platelet contaminations. Herein, we introduce a rapid and label-free microfluidic approach for complete size-based fractionation of peripheral blood mononuclear cells (PBMCs) into monocytes, lymphocytes, and platelets. The developed biochip enables gentle sorting of intact MPA in the enriched monocytes with efficient depletion of lymphocytes and platelets for accurate MPA quantification. We first compared the developed microfluidic technology (dean flow fractionation, DFF) with standard magnetic negative isolation (MACS) and observed that DFF-sorted monocytes had similar viability, purity, and key immune functions (phagocytosis, macrophage differentiation) as MACS-sorted monocytes. As proof of concept for diabetes testing, we isolated and characterized monocytes/MPA from a cohort of healthy ( n = 5) and type 2 diabetes mellitus (T2DM) subjects ( n = 8) in PBMCs and DFF-sorted monocytes. High-speed imaging, immunofluorescence, and flow cytometry analysis clearly indicated higher levels of MPA in T2DM patients ( P0.05) with enhanced MPA detection sensitivity in DFF-sorted monocytes ( P0.05). Taken together, the developed DFF technology greatly facilitates high-throughput (∼130 μL min

Monocytes represent a highly heterogeneous leukocyte population with the ability to differentiate into macrophages, a major cell type involved in the pathogenesis of atherosclerotic plaque in cardiovascular diseases [1,2]. Label-free analysis of their native cellular phenotypes and functions not only reduces assay cost and time, but is also essential for understanding disease progression and development of novel therapeutic strategies. Impedance cytometry is an emerging technology for high throughput cell phenotyping based on intrinsic electrical properties without the use of antibodies [3,4]. While parallel electrodes offer higher detection sensitivity as compared to co-planar electrodes [3,4], it is limited by laborious microfabrication. Herein, we present the development of an efficient microfluidics impedance cytometer using coplanar electrodes for rapid monocyte identification and phenotyping based on differentiation status. The microfluidics impedance cytometer consists of a two-inlet, two-outlet polydimethylsiloxane (PDMS) microchannel (30 μm (width) × 20 μm (height)) bonded on patterned coplanar electrodes (20 μm in width with 20 μm separation gap). Sample and sheath fluid are injected into the device to hydrodynamically focus the cells at the channel center prior electrical detection. As the cell moves through the detection region, it disrupts the electric field generated by coplanar electrodes, thereby causing a change in electrical impedance. Using our setup (Fig. 1), the change in electrical impedance was quantified based on current change, and various cellular information were extracted at different frequencies of the excitation signal. We measured two impedance parameters namely the 1) opacity (ratio of impedance magnitude at 0.3MHz (|ZLF|) to impedance magnitude at 1.7MHz (|ZHF|)) which reflects cell membrane capacitance, and 2) |ZLF| which characterize cell size. For identification of different blood cell types, human monocytes and lymphocytes purified using Dean Flow Fractionation (DFF) [5], and diluted red blood cells (RBCs) samples were separately injected into the microdevice. As shown in figure 2, different cell types were clearly differentiated based on |ZLF| due to distinct cell size differences (monocytes: 10 – 12 µm, lymphocytes: 7 – 8 µm, and red blood cells: 6 – 8 µm). Characterization of primary monocytes and leukemic THP-1 monocytic cell line also showed distinct differences in size and opacity (Fig. 3). Lastly, we compared the impedance profile of THP-1 and differentiated macrophages (PMA stimulated), and found that macrophages were more heterogeneous in size with significantly lower opacity than THP-1, demonstrating the feasibility of real-time assessment of monocyte differentiation using impedance-based sensing (Fig. 4). In conclusion, the developed microdevice enables continuous label-free monocyte phenotyping using coplanar electrodes configuration with sufficient sensitivity. With the design simplicity and low cost fabrication, we envision our method will greatly facilitate immunology research and point-of-care monocyte profiling in patients with cardiovascular diseases.

Herein, we introduce a novel Dean migration phenomenon in spiral microchannel, termed High-resolution Dean Flow Fractionation (HiDFF), for separation of small microparticles based on differential Dean migration profiles. Inertial microfluidics is an emerging class of passive size-based sorting technique for cell separation [1,2]. However, inertial focusing of small microparticles (particle size, ap 10 µm) focus near inner wall for separation. However, it cannot further size-fractionate small particles (1 µm vs. 2 µm) as they would recirculate continuously due to Dean vortices. Using HiDFF, we were able to demonstrate efficient separation of 1–3 µm microbeads mixture, and then apply the technology to size-fractionate different small biological components including bacteria and nanoparticles. The 2-inlet, 2-outlet spiral microdevice (60×300 µm (H×W)) is fabricated using polydimethylsiloxane (PDMS) (Fig. 1A). Small particles (ap/ Dh

Atherosclerosis, the leading cause of cardiovascular diseases [1–3], is a chronic inflammatory disorder characterized by deposition of cholesterol-containing low-density lipoproteins (LDL) in arterial walls, resulting in blood vessel narrowing and impaired perfusion, and increased leukocyte recruitment [4,5]. Current atherosclerosis in vitro models are 2-dimensional (2D), and fail to reproduce important features of atherogenesis including blood flow-induced shear stress and leukocyte-endothelial interactions [6–7]. Herein, we report a novel biomimetic blood vessel model to study the hemodynamics and leukocyte-endothelial interactions using a tunable, 3-dimensional (3D) endothelial barrier to mimic stenotic plaque. We first characterized the effects of THP-1 monocyte adhesion on activated endothelial monolayer, followed by whole blood perfusion at different channel constrictions to study leukocyte rolling phenotype. A schematic of the polydimethylsiloxane (PDMS) microfluidic device is shown in Figure 1A. It consists of 3 layers, with a top cell culture chamber (800×100 µm (H×W)) for endothelial (HUVECs) cell culture, and a bottom pneumatic channel (1000×100 µm (H×W)), separated by a thin PDMS membrane (10 µm thick). By varying air flow into the pneumatic channel, the membrane is deflected upwards, thereby creating tunable constrictions in the cell culture chamber (Figure 1B). Endothelial inflammation is mimicked by growing HUVECs to confluency and treating them with tumor necrosis factor-α (TNF-α) to study leukocyte-endothelial interactions in cell culture media (RPMI) and whole blood flow. We first performed confocal imaging to visualize channel constriction using FITC dye and 3D endothelial “stenotic plaque” (Figure 1B, C). The laminar flow profile over the 3D stenosis was studied from the rolling velocities of 10 µm beads, which varied significantly in the stenosis region (Figure 1D). As observed from Figure 2A, HUVECs expressed higher ICAM-1 due to TNF-α-treatment which facilitated binding of THP-1 cells. Interestingly, distinct adherence patterns of THP-1 for 50% and 80% constrictions was observed under flow (1 dyne/cm2) with increased adherence at the top of the constriction (Figure 2B, C). Upon increasing flow rate to 10 dyne/cm2, THP-1 adhesion was reduced in 80% constriction while completely eliminated in 50% constriction. (Figure 2D, E). Finally, whole blood was perfused through the device to study blood flow under stenosis conditions (Figure 3). As expected, significant leukocyte (mostly neutrophils) rolling and adhesion were observed, and average leukocyte rolling velocity was lowest with an 80% constriction (Figure 3D), possibly due to low shear at the top of the stenotic “plaque”. On-going studies are performed to gain better insights into the stenosis-induced hemodynamics effects on leukocyte-endothelial interactions. In conclusion, the developed atherosclerosis-on-a-chip mimics a physiologically-relevant 3D stenotic blood vessel which enables long-term perfusion cell culture and on-chip visualization of leukocyte-endothelial interactions (rolling, adhesion). This model can also be further developed to study thrombus formation and other endothelial-related dysfunctions in cardiovascular diseases.

Host defence peptides (HDPs) derived from the C-terminus of thrombin are proteolytically generated by enzymes released during inflammation and wounding. In this work, we studied the effects of the prototypic peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), on neutrophil functions. In vitro, GKY25 was shown to decrease LPS-induced neutrophil activation. In addition, the peptide induced CD62L shedding on neutrophils without inducing their activation. Correspondingly, GKY25-treated neutrophils showed reduced attachment and rolling behaviour on surfaces coated with the CD62L ligand E-selectin. The GKY25-treated neutrophils also displayed a dampened chemotactic response against the chemokine IL-8. Furthermore, in vivo, mice treated with GKY25 exhibited a reduced local ROS response against LPS. Taken together, our results show that GKY25 can modulate neutrophil functions in vitro and in vivo.

Neutrophil dysfunction is strongly linked to type 2 diabetes mellitus (T2DM) pathophysiology, but the prognostic potential of neutrophil biomarkers remains largely unexplored due to arduous leukocyte isolation methods. Herein, a novel integrated microdevice is reported for single-step neutrophil sorting and phenotyping (chemotaxis and formation of neutrophil extracellular traps (NETosis)) using small blood volumes (fingerprick). Untouched neutrophils are purified on-chip from whole blood directly using biomimetic cell margination and affinity-based capture, and are exposed to preloaded chemoattractant or NETosis stimulant to initiate chemotaxis or NETosis, respectively. Device performance is first characterized using healthy and in vitro inflamed blood samples (tumor necrosis factor alpha, high glucose), followed by clinical risk stratification in a cohort of subjects with T2DM. Interestingly, "high-risk" T2DM patients characterized by severe chemotaxis impairment reveal significantly higher C-reactive protein levels and poor lipid metabolism characteristics as compared to "low-risk" subjects, and their neutrophil chemotaxis responses can be mitigated after in vitro metformin treatment. Overall, this unique and user-friendly microfluidics immune health profiling strategy can significantly aid the quantification of chemotaxis and NETosis in clinical settings, and be further translated into a tool for risk stratification and precision medicine methods in subjects with metabolic diseases such as T2DM. MOE (Min. of Education, S’pore) NMRC (Natl Medical Research Council, S’pore) Accepted version

Vessel geometries in microengineered in vitro vascular models are important to recapitulate a pathophysiological microenvironment for the study of flow-induced endothelial dysfunction and inflammation in cardiovascular diseases. Herein, we present a simple and novel extracellular matrix (ECM) hydrogel patterning method to create perfusable vascularized microchannels of different geometries based on the concept of capillary burst valve (CBV). No surface modification is necessary and the method is suitable for different ECM types including collagen, matrigel and fibrin. We first created collagen-patterned, endothelialized microchannels to study barrier permeability and neutrophil transendothelial migration, followed by the development of a biomimetic 3D endothelial-smooth muscle cell (EC-SMC) vascular model. We observed a significant decrease in barrier permeability in the co-culture model during inflammation, which indicates the importance of perivascular cells in ECM remodeling. Finally, we engineered collagen-patterned constricted vascular microchannels to mimic stenosis in atherosclerosis. Whole blood was perfused (1-10 dyne cm-2) into the microdevices and distinct platelet and leukocyte adherence patterns were observed due to increased shear stresses at the constriction, and an additional convective flow through the collagen. Taken together, the developed hydrogel patterning technique enables the formation of unique pathophysiological architectures in organ-on-chip microsystems for real-time study of hemodynamics and cellular interactions in cardiovascular diseases. MOE (Min. of Education, S’pore) MOH (Min. of Health, S’pore) Accepted version

A series of lanthanide chloranilate frameworks containing a (4,4)-net with LaIII, CeIII, NdIII, SmIII, and EuIII have been synthesised and structurally characterised. Two structure types of square grids were obtained for these frameworks. Type 1 consists of the formula (Et4N)[Ln(can)2(H2O)] (Ln=LaIII, CeIII, NdIII; H2can=chloranilic acid) and crystallised in the tetragonal space group I4/m, featuring a nine-coordinate lanthanide ion with a coordinated water molecule and four chloranilate ligands. Type 2, (Et4N)[Ln(can)2] (SmIII and EuIII) crystallised in the I4/mcm space group, and contains an eight-coordinate lanthanide ion without a coordinated water molecule. A single-crystal-to-single-crystal transformation was carried out for (Et4N)[Nd(can)2(H2O)] on removal of the coordinated aqua ligand.