(a) Micrograph of the electrode array viewed from the top of the channel (with a CHO cell exiting the analysis volume, flowing left to right). Each electrode in the array is 25 μm wide, but the spacing between them varies: between the two central actuation electrodes, the gap is 15 μm; between the electrodes of the detection sets D1 and D2, it is 25 μm; finally, the spacing between the actuation set A and the two detection sets D1 and D2 is 35 μm on each side. (b) Zoomed-out (and rotated by 90°) view of the microelectrode array from the channel bottom shows the configuration of the electrode pads and the way in which the electrodes are connected and energized. Outlined by the rectangle is the approximate area depicted in the previous micrograph. (c) Schematic representation of a side view of the channel displays a trajectory of a small sphere flowing through the microfluidic channel and actuated by an nDEP force using a megahertz signal applied to the central electrodes in the actuation region. Corresponding electronic signature, S, shown in the background and obtained by capacitive detection at 1.29 GHz by electrodes D1 and D2, is an experimental signature produced by a PSS actuated by a DEP force at 0.1 MHz. Peaks P1 and P2 are produced when the sphere is situated directly above the gap centers at D1 and D2, which are 210 μm apart.
(a) Simulated detection signatures for 1 μm nDEP-induced increments in altitude. Figure indicates the expected signature profiles for different types of actuation. (b) Change of peak amplitude with altitude. Points obtained from simulation are connected with a spline interpolation curve.
(a) Trajectories for nDEP actuated 10 μm-diameter PSS for equally spaced steps in CMF in the interval [−0.5, 0]; assumed ; (b) trajectories simulated for 13 μm-diameter CHO cells for CMF in the interval [−0.3, 0.3]; assumed . Note the difference in vertical scale on the two plots. Inset bars drawn next to the trajectories on both plots represent the relative sizes of the smallest change in altitude observable with our apparatus; in both cases, it is about 0.25 μm. A slight change in altitude, expected as the cell exits the region of strong actuation and begins to return to equilibrium position, is not large enough to be observable by our apparatus.
Mean force index values for PSS populations actuated at 1 MHz by signals of amplitudes 0 to , in steps of . Inset shows an example of the force index histogram for with the mean force index and standard deviation as indicated. Examples of individual signatures produced by PSS entering the analysis volume at about the same altitude are provided for different actuation voltages, UA (values of 0, 4.5 and , as indicated by arrows). The response has a linear characteristic for signal amplitudes between about 2 and ; the change in voltage of about results in an observable change in force index of about 0.025. This allows us to use the force index to estimate the actuation and the corresponding expected change in polarizability.
Experimental signatures of CHO cells. (a) Signatures produced by cells entering the actuation region at about the same altitudes and with similar velocities, but actuated in a different way depending on the frequency of the applied signal. Examples were deliberately chosen from measurements performed at lower flow rates to allow characteristic features to be clearly observable. This allows, for example, the cell actuated by the pDEP to approach the electrodes close enough for the double peak to be resolvable at the second detection site; this feature disappears at higher flow rates as hydrodynamic focusing forces the flow to higher altitudes. Note also that evenly matched entrance peak amplitudes of these signatures become distinctly modified due to the DEP actuation in between detections. (b) Sample signatures produced by unactuated CHO cells (no DEP applied) at flow rates of 5 nl/s and cell density 0.5 × 106 cells/ml; (c) Signatures for a similar flow rate produced by actuated CHO cells. Both nDEP and pDEP signatures can be observed.
Detection of early stage apoptosis. (a) After the culture was maintained for 96 h through growth and stable phase, histograms of the force index values obtained from a sample population of CHO cells suspended in a medium of conductivity and actuated using a 6 MHz signal with an amplitude of show an incipient subpopulation of early apoptotic cells. Within the next 12 h, more than half of all cells show changes in dielectric properties indicating early apoptosis. After another 12 h, changes are evident in almost entire population. (b) The figure shows viability estimates of different assays plotted against the viability estimates provided by DEP cytometry, and indicates clearly the strong correlation between the DEP cytometer and the Annexin V assay. Note: ViaCount assay uses a proprietary mix of two DNA binding dyes to detect viable, apoptotic and dead cells.
Article metrics loading...
Full text loading...