We report the development of a novel quartz nanopillar (QNP) array cell separation system capable of selectively capturing and isolating a single cell population including primary CD4(+) T lymphocytes from the whole pool of splenocytes. Integrated with a photolithographically patterned hemocytometer structure, the streptavidin (STR)-functionalized-QNP (STR-QNP) arrays allow for direct quantitation of captured cells using high content imaging. This technology exhibits an excellent separation yield (efficiency) of ~95.3 ± 1.1% for the CD4(+) T lymphocytes from the mouse splenocyte suspensions and good linear response for quantitating captured CD4(+) T-lymphoblasts, which is comparable to flow cytometry and outperforms any non-nanostructured surface capture techniques, i.e. cell panning. This nanopillar hemocytometer represents a simple, yet efficient cell capture and counting technology and may find immediate applications for diagnosis and immune monitoring in the point-of-care setting.

With scanning electron microscopy analysis, we investigated the role of nanoscale topography on cellular activities; e.g. cell adhesion and spreading by culturing A549 cells (human lung carcinoma cell line cells) for 1-48 h on three sets of nanostructures; quartz nanopillars (QNPs), silicon nanopillars and silicon nanowire (SiNW) arrays, along with planar glass substrates. We found that cells on QNP arrays developed a longer shape than those on SiNW arrays. In addition, we studied how cell morphologies influence the cell-capture yield on the three sets of nanostructures. This research showed that the filopodial formations were directing the cell-capture yield on nanostructured substrates. This finding implies the possibility of using nanoscale topography features to control the filopodial formation including extension and migration from the cells. Using streptavidin-functionalized SiNW substrate, we further demonstrated a substantially higher yield (~91.8 ± 5.9%) than the planar glass wafers (~24.1 ± 7.5%) in the range of 200-3000 cells.

Direct CD4þ T lymphocytes were separated from whole mouse splenocytes using 1-dimensional ferromagnetic nickel silicide nanowires (NiSi NWs). NiSi NWs were prepared by silver-assisted wet chemical etching of silicon and subsequent deposition and annealing of Ni. This method exhibits a separation efficiency of $93.5%, which is comparable to that of the state-of-the-art superparamagnetic bead-based cell capture ($96.8%). Furthermore, this research shows potential for separation of other lymphocytes, B, natural killer and natural killer T cells, and even rare tumor cells simply by changing the biotin-conjugated antibodies. Silicides are widely used as metal contact materials in modern silicon microelectronics because of their low contact resistivity and the excellent junction interface on silicon (Si).1,2 With the recent improvement in nanoscale Si devices, metal silicides have received significant attention because of their unique properties and potential applications in electronic and photonic devices.2-4 Recently, several research groups have studied nickel silicide nanowires (NiSi NWs).3,5-8 They confirmed that NiSi NWs show lower resistivity when formed at lower processing temperature with less reaction phase at interface compared to other metallic silicide materials. As a result, NiSi NWs are considered a promising material for use as gate and source/drain electrodes in current complementary metal oxide semiconductor devices.

Nanostructured surfaces emerge as a new class of material for capture and separation of cell populations including primary immune cells and disseminating rare tumor cells, but the underlying mechanism remains elusive. Although it has been speculated that nanoscale topological structures on cell surface are involved in the cell capture process, there are no studies that systematically analyze the relation between cell surface structures and the capture efficiency. Here we report on the first mechanistic study by quantifying the morphological parameters of cell surface nanoprotrusions, including filopodia, lamellipodia, and microvilli in the early stage of cell capture (< 20 min) in correlation to the efficiency of separating primary T lymphocytes. This was conducted by using a set of nanohole arrays (NHAs) with varying hole and pitch sizes. Our results showed that the formation of filopodia (e.g., width of filopodia and the average number of the filopodial filaments per cell) depends on the feature size of the nanostructures and the cell separation efficiency is strongly correlated to the number of filopodial fibers, suggesting a possible role of early stage mechanosensing and cell spreading in determining the efficiency of cell capture. In contrast, the length of filopodial filaments was less significantly correlated to the cell capture efficiency and the nanostructure dimensions of the NHAs. This is the first mechanistic study on nanostructure-based immune cell capture and provides new insights to not only the biology of cell-nanomaterial interaction but also the design of new rare cell capture technologies with improved efficiency and specificity.