A quartz nanopillar hemocytometer for high-yield separation and counting of CD4+ T lymphocytes

Abstract

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.

Morphology of CD4+T Lymphocytes Bound on Nano-Patterened Substrates for Sensing the Size of Nanoholes

“…The QNHA samples are ready to collect incoming cell suspension containing primary mouse CD4 and CD8 T-lymphocytes, natural killer (NK), natural killer T (NKT), and B cells. We used this surface functionalized method to separate targeting specific cells (in our case CD4 + T-lymphocytes) among different kinds of cells via novel STR-biotin conjugation technique to capture the incoming targeting cells in PBS solution as we reported previously [8,9]. More detailed information can be found in previous reports [8,9].…”

“…The T-cells were first fixed with 4% GA in the refrigerator for 2 hr, followed by a post-fix process using 1% osmium tetroxide for 2 hr. The captured T-lymphocytes were then dehydrated through a series of ethanol concentrations (25, 50, 75, 95, and 100%) and slowly dried at vacuum-connected desiccators for 24 hr [7,9]. Once the samples were dry in the desiccators, the surface-bound T-lymphocytes were sputter-coated with platinum before performing the FE-SEM measurement.…”

“…To fabricate the QNHA structures on quartz substrates, colloidal PS suspension containing different sizes of PS nanoparticles NPs) (200, 300, 430, and 750 nm in diameter) was first carefully deposited on the QNHA substrate using a modified self-assembly technique we improved previously (Fig. 1a) [9]. To create space between the spin-coated PS NPs, their sizes were first reduced by O 2 plasma etching for 5 s (O 2 /Ar=35/10 sccm, RF power of 100 W and bias power of 50 W).…”

“…Furthermore, nanostructures have been increasingly used for studies on cell interaction with solid nanostructures because unique properties of nanostructured surfaces enable a variety of novel functions of immobilized cells on the nanostructured surfaces [4,5]. To date, these nanostructures were prepared by various methods including the polymeric templating method [6], vaporliquid-solid (VLS) growth [7], dry-etching process using nanosphere lithography technique [8,9]. Recently, we have also demonstrated a novel platform for separating CD4 + T lymphocytes from mouse splenocytes using streptavidin (STR)-functionalized and vapor-liquid-solid (VLS)-grown silicon nanowire (SiNW) [7] as well as transparent quartz nanopillar (QNP) arrays [8,9] having a higher separation efficiency of 93-95.3%.…”

“…To date, these nanostructures were prepared by various methods including the polymeric templating method [6], vaporliquid-solid (VLS) growth [7], dry-etching process using nanosphere lithography technique [8,9]. Recently, we have also demonstrated a novel platform for separating CD4 + T lymphocytes from mouse splenocytes using streptavidin (STR)-functionalized and vapor-liquid-solid (VLS)-grown silicon nanowire (SiNW) [7] as well as transparent quartz nanopillar (QNP) arrays [8,9] having a higher separation efficiency of 93-95.3%. However, there is no study on how the various size and shape-matched nanostructures and nanomaterials interact in the primary cells on the nanostructured substrates.…”