DEP experiments were conducted using a prototype 3DEP DEP-Well electrode chip and reader system (DEPtech, East Sussex, UK), variations of which are detailed in prior publications [58-60]. Each DEP experiment using this system approximated DEP force (as defined by equation 3 in Section 1.3.1) by quantifying cell motion in response to application of a non-uniform AC electric field, by measuring change in light intensity as a function of both tim e and distance (Well radius) for each frequency applied to the cell solution (cells from the sample being tested suspended in DEP testing medium). The DEP-Well chip (DEPtech, East Sussex, UK) used in this study and shown in Figure 2.3, hosted several identical Well electrodes, each with an aperture diam eter of 0.75m m and a W ell depth of 1.54mm, sealed on the inferior surface by a glass cover-slip to allow both light transmission but filling of only the Well of interest. Each Well, only one of which was selected for use during experiments, was composed of seven insulator layers of 150 pm-thick FR4 (fibreglass reinforced epoxy)
sandwiched between eight conductor layers of gold-plated copper (top and bottom layers were 35 pm in thickness, six remaining layers were 70 pm in thickness).
Figure 2.3: Photographie close-up of a cluster of three DEP-Well electrode areas and how this cluster appeared on visual inspection of a DEP-Well chip.
Five frequencies per decade were used over a range of 4 kHz to 20 MHz to test each OBB sample and the DEP-Well electrode chip was energised by a 10 V peak-to-peak sinusoidal signal from a Digimess FG 100 function generator (Digimess, Reading, UK), whose output was monitored using a ISO-Tech IDS710 digital oscilloscope. For each frequency tested, approximately 5 pi of cell solution was injected (using a pipette with a bent tip to permit more precise Well filling) into the area bounded by the We!! e!ectrode on the DEP-Wel! chip, which was mounted above the light source of a Nikon Eclipse 50i upright microscope (Nikon, Surrey, UK) and viewed at 4x magnification. This filling continued to the point at which there was an excess of cell solution over the Well aperture area. A glass cover-slip (large enough to cover the area of the Well aperture) was then laid slowly at an angle to this excess (to reduce the possibility of trapping air in the Well). An AVT Dolphin F145B digital interface camera (Allied Vision Technologies, Germany), affixed to the microscope and connected to a PC, captured images of the cell solution into the area bounded by the Well electrode using
SmartView for W DM software version 0.1.3.3 (supplied with the aforementioned camera). This experiment setup is shown in labelled photograph in Figure 2.4.
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Figure 2.4: Photograph illustrating the main pieces of equipment comprising the DEP testing setup. A close-up of the DEP- Well chip can be seen in Figure 2.3.
The "Preview" button (Figure 2.5) was then selected to view the contents of Well in real-tim e prior to experiment commencement, to ensure that the whole of the Well area was in view and that no bubbles were present within the Well. Next, the frequency to be applied to the contents bounded by the Well electrode was entered into the GUI (Figure 2.5), in kilohertz.
Selecting the "Start experiment" button (Figure 2.5) commenced the application of AC signals at the selected frequency to the contents bounded by the Well electrode. Images were captured immediately prior to signal application ("zero seconds", to allow normalisation of the change in light intensity to this point) and every three seconds thereafter, for a period of 60 seconds. A MATLAB (The MathWorks Inc, Nantick, MA, USA) script was then used to assess the change in light intensity over the period that the electric current was applied and as a function of Well radius. A change in light intensity was observed when either positive or negative DEP occurred. Given that the electrodes were located around the perimeter of the Well aperture, and that the Well electrode chip was placed above the upright microscope light source, but below the camera capturing cell movement (recording images every three seconds over the course of each 60 second run, for each frequency tested), positive DEP was seen to occur when the cells in solution moved towards the Well aperture perimeter, thus allowing more light from the upright microscope to pass up through the centre of the
Well and be received by the camera unit above the Well. Conversely, negative DEP was seen to occur when the cells in solution were repelled by th e electrodes round the perim eter of the W ell, towards the centre of the Well aperture, thus impeding the amount of light from the upright microscope which could be received the the camera unit. The short video in Appendix 2 shows both positive and negative DEP occurring (not in real time-sped up), for tw o separate frequencies, for one OBB sample. Positive DEP shows the oral cells in solution moving towards the edges of the grey-coloured Well aperture area and during negative DEP, the cells appear to move towards the centre of the Well aperture area, creating a shadow in this region and decreasing the amount o f light able to transmit through the Well.
3 DEP_well_centre [s3 - Data capture- Save images on Set Path Filename Time [sec] Interval [sec] 60 r -D a ta analysis Start Experiment Bands 10 1 j Preview j j Intensay line {
[— Camera Settings Brightness Contrast Resolution: 100 50 640 X 480 No Display -Signal Generator- Frequency [kHz] 1 Voltage [Vpp] 10 -W aveform — Sine O Square
O
Triangel Offset Voltage [V] Port0 C0M1
g Oscilloscope Port
Vpp last run: NA COM3
Run Signal Generator
Threshold, 20 Min Rad 10 -S etup Chip ID Well used [ Diameter [um f -Sample information- Cell Type Concentration Conductivity medium Growth condition Treatment Remarks Operator
Path to Save: C:1DEP_well\data
Figure 2.5: The MATLAB graphic user interface (GUI) used in this study, named "DEP_well_centre", to control the DEP test settings applied for each frequency run, over the course of each DEP experiment.
Images were captured immediately prior to signal application ("zero seconds") and every three seconds thereafter, for a period of 60 seconds. A MATLAB (The MathW orks Inc, Nantick, M A, USA) script was then used to assess the change in light intensity over the period that the electric current was applied and the change in light intensity was normalised to the image captured at tim e zero seconds.
At the end of each frequency run, the glass cover-slip was removed, the Well was flushed through with fresh DEP testing medium and then dabbed dry. These steps ensured the results produced reflected the intrinsic frequency response of the cells, rather than the effects of prolonged cellular exposure to electric current.
This outlined protocol was repeated for each frequency tested.