Schematic of the overall assay for transplant rejection monitoring envisioned on EWOD device. (a) Droplets containing the sample and MB-Abs are merged and (b) mixed so as to bind the target cells to the MBs. (c) The MBs and MB-bound cells are collected with a magnet. (d) Droplet is split to collect the MB-bound cells (in collected droplet), while removing some of the nontarget (CD8−) cells (in depleted droplet). (e) In the future, the steps of (b)–(d) can be repeated to improve the purity of cells, and (f) the collected cells can then be lysed chemically or electrically (not shown) before the mRNA or proteins in the lysate can be detected.
Schematic cross section of the EWOD device. EWOD actuation electrodes are patterned from the ITO layer of the “Active” chip. Contact pads are formed with Cr/Au (outside the given field of view and not shown). Electrodes are coated with silicon nitride as dielectric and Cytop® as hydrophobic coating. Thinner silicon nitride and Cytop® layers are deposited on the ITO-coated “Reference” chip, connected to the ground. The two chips are separated by a double-sided adhesive spacer.
Confirmation of cell binding to MBs, performed at macroscale (not using microfluidics) under EWOD-like conditions. Presented are flow-cytometry data obtained for lymphocyte distribution before and after magnetic separation using antibodies (for CD8 and CD3 membrane proteins) labeled with fluorescent dyes (APC and PerCP, respectively). In each figure, dots on the upper right quadrants, which have high APC as well as PerCP intensity, indicate T-lymphocytes, while those on the lower right quadrants, having high PerCP but low APC intensity, indicate CD8− T-lymphocytes. (a) Before separation. (b) After magnetic separation for MB-cell binding done at as per protocol, (c) After magnetic separation for MB-cell binding at room temperature in serum-free buffer containing Tween 20, as used during EWOD experiments. In both (b) and (c), most of the cells collected are T-lymphocytes, indicating that the collection efficiency under the EWOD-like conditions is similar to that under the protocol.
Schematic representation comparing the binding of cells [: (a)–(e); CD8−: (f)–(j)] to MBs conjugated with anti-CD8 antibodies (MB-Abs), followed by magnetic collection on the EWOD device. Droplets containing the MB-Abs and cells [: (a) and (b); CD8−: (f) and (g)] are merged and mixed. The circulating flow inside the droplet leads to high interaction between the cells and the MB-Abs specific to them (c). However, despite the high interaction between the CD8− cells and the MB-Abs, there is little binding (d). After the droplet is in position [: (c); CD8−: (h)], a magnet is introduced, collecting the MBs and the cells bound to them to the left edge of the droplet [: (d); CD8−: (i)]. The droplet is subsequently cut, collecting the MBs and the MB-bound cells in the left (collected) droplet, and leaving the right (depleted) droplet with only unbound cells [: (e); CD8−: (j)]. Bright-field image sequence [(k)–(o)] showing that the corresponding steps look virtually identical for the two cases.
Comparison between binding of (fluorescently stained) cells and (fluorescently stained) CD8− cells to the magnetic beads conjugated with anti-CD8 antibodies on the EWOD device. [(a)–(c)] cells: (a) the initial sample contained cells. After MB-cell binding and magnetic collection, (b) collected droplet contained cells, while (c) depleted droplet contained only cells. Moreover, the fluorescence pattern appears over the MB-Abs in (b) the collected droplet, suggesting binding of the cells to the MBs. [(d)–(f)] CD8− cells: (d) the initial sample contained cells. After MB-cell binding and magnetic collection, (e) collected droplet contained cells, while (f) depleted droplet contained cells. Very little fluorescence is seen over the MBs in (e), suggesting little binding between the CD8− cells and the MB-Abs.
Schematic representation [(a), (c), (e), and (g)] and superposed image sequence [(b), (d), (f), (h), and (i)] showing the separation of MB-bound and fluorescently labeled cells from the unlabeled CD8− cells. The sample is placed on the EWOD device [(a) and (b)], and a magnet is introduced [(c) and (d)] to collect the MB-bound fluorescent cells to the left [(d), inset)]. With the magnet in place, EWOD microfluidic operations are used to stretch the droplet to the left [(e) and (f)]. Droplet is split by EWOD so as to magnetically collect the fluorescent cells in the collected droplet [(g), (h), and (i)]. (Each pair of inset shows zoomed-in bright-field and fluorescence images of certain regions of the droplets.) As indicated by the fluorescence concentrated over the MBs at the left edge [(h), lower insets], most (conservatively ) of the fluorescently stained cells are in the collected droplet. Unstained CD8− cells are divided between the collected [(h), upper insets] and depleted droplets [(i), insets], similar to Figs. 5(d)–5(f). On the other hand, the lack of fluorescence [(i), insets] indicates that few cells are in the depleted droplet.
Summary of the results comparing the binding of and CD8− T lymphocytes to anti-CD8 MB-Abs on EWOD.
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