Single-cell bioelectrical impedance platform for monitoring cellular response to drug treatment

Abstract

The response of cells to a chemical or biological agent in terms of their impedance changes in real-time is a useful mechanism that can be utilized for a wide variety of biomedical and environmental applications. The use of a single-cell-based analytical platform could be an effective approach to acquiring more sensitive cell impedance measurements, particularly in applications where only diminutive changes in impedance are expected. Here, we report the development of an on-chip cell impedance biosensor with two types of electrodes that host individual cells and cell populations, respectively, to study its efficacy in detecting cellular response. Human glioblastoma (U87MG) cells were patterned on single- and multi-cell electrodes through ligand-mediated natural cell adhesion. We comparatively investigated how these cancer cells on both types of electrodes respond to an ion channel inhibitor, chlorotoxin (CTX), in terms of their shape alternations and impedance changes to exploit the fine detectability of the single-cell-based system. The detecting electrodes hosting single cells exhibited a significant reduction in the real impedance signal, while electrodes hosting confluent monolayer of cells showed little to no impedance change. When single-cell electrodes were treated with CTX of different doses, a dose-dependent impedance change was observed. This enables us to identify the effective dose needed for this particular treatment. Our study demonstrated that this single-cell impedance system may potentially serve as a useful analytical tool for biomedical applications such as environmental toxin detection and drug evaluation.

Figure 1

Illustration of the cell impedance monitoring system. Cells to be monitored are first patterned on IMA chips prior to loading in the chip carrier, which is attachable/detachable from the circuit board, and kept in a live-cell incubator chamber for real-time optical imaging and impedance measurements. Data acquisition is controlled in real-time by computer interface with the lock-in amplifier and MUX/Preamplifier circuit board through LabVIEW and processed post-time using MATLAB.

Figure 2

Schematic of the microfabrication processes of IMA chips. (1) Photolithography patterning, Ti and Au electron-beam deposition, and lift-off. (2) Plasma enhanced chemical vapor deposition of SiO₂ and O₂ passivation/annealing. (3) Photolithography patterning with reactive ion etching, mask removal, and O₂ plasma cleaning. (4) Photolithography patterning for 2^nd/final Au layer by electron-beam deposition.

Figure 3

Frequency spectroscopy plot of impedance magnitude |Z| at a frequency range from 500 to 20 kHz when cell-free electrodes of 30 μm and 250 μm diameters are seeded with U87MG cells (n = 18 per condition).

Figure 4

(a) Brightfield and fluorescence images of U87MG single-cell adhered to 30 μm detecting electrodes of an IMA chip after continuous impedance monitoring for 24 hours using 100 nA current (top row) and 100 μA current (bottom row). From left to right: Brightfield, Hoechst 33342 staining on UV fluorescence channel, EtD-III staining on TRITC fluorescence channel, and the fluorescent overlay images. The scale bar is 30 μm. (b) Viability of U87MG cells, as determined by presence of Hoechst 33342 and absence of EtD-III signal and normalized to the total count.

Figure 5

ECM of the single-cell electrode heterostructure.

Figure 6

Influence of initial cell coverage (r_c,o/r_e) on normalized real impedance (Re[Z]) as a result of reduced coverage and retraction of cell morphology on the electrode (r_c/r_c,o).

Figure 7

(a) Schematic of IMA chip featuring optical epi-DIC micrographs of four single live U87MG cells on 30 μm detecting electrodes (right panel) and one 250 μm detecting electrode hosting a confluent monolayer of live U87MG cells (left panel) before and 16 hours after 5 μM CTX treatment. Both scale bars represent 30 μm. (b)–(e) Normalized real impedance waveforms of the four U87MG single cells exposed to 5 μM CTX over a period of 16 hours. Real impedance was normalized to the point just prior to CTX inoculation.

Figure 8

Average normalized real impedance waveform of multiple U87MG cells (blue) and single U87MG cells (green) in response to 5 μM CTX, as well as to single cells receiving no CTX treatment (brown). Real impedance was normalized to the point just prior to CTX inoculation.

Figure 9

Average normalized real impedance waveforms of U87MG single cells in response to CTX at concentrations of 500 nM (dark yellow), 1.0 μM (light blue), 5.0 μM (green), and 10.0 μM (blue), respectively, over a period of 16 hours. Real impedance was normalized to the point just prior to CTX inoculation.