| Summary: | The body’s defence against pathogens and foreign invaders is mediated by white blood cells (WBCs), one of the most important blood constituents. In blood, there is only one WBC for every 1000 red blood cells (RBCs), which makes WBC isolation a difficult task. Conventional isolation methods such as differential centrifugation, or selective lysis of RBCs could result in undesirable activation of WBCs and require a relatively large volume of blood. In this work, we present a microfluidic cross-flow trapping chip capable of selectively capturing WBCs in a dense array from whole blood without the prerequisite of RBCs lysis. This chip exploits the size and deformability difference between blood cells, and consists of a two-layer trapping system fabricated utilising standard photolithography and soft lithography. This array enables high-resolution imaging of individual live WBCs, obtained by taking a ~50 μl blood sample by fingerprick, and does not require cell drying or cell fixation as in a blood smear. In this work, a wide range of two-layered microfluidic devices has been fabricated and investigated in terms of blood cell arraying and imaging performance. Selective WBC capturing from a whole blood sample was achieved, for the first time, and the optimal geometry of the microfluidic channels for efficient WBC arraying was identified and related to hydrodynamic trapping theory. The developed WBC arrays were then used for the identification and enumeration of WBC subtypes on the basis of cell size and cell nucleus morphology, visualized by a combination of off-chip and on-chip dye staining, and also by labelling with antibodies against specific cell surface receptors. Furthermore, cells of nanoparticle-incubated blood samples were arrayed on the device and a preliminary analysis was performed of the association of these silica nanospheres with the different blood cell species, revealing significant differences in nanoparticle association. Moreover, sub-cellular imaging suggested a lysosomal nanosphere location in monocytes, implying nanoparticle uptake by endocytosis. Such studies have not previously been performed with arrayed WBCs, exemplifying that the WBC array developed in this work is a promising platform for single-cell (nano) biomedical studies with, for example, diagnostic imaging and drug discovery applications.
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