Detection of drug-induced cellular changes using confocal Raman spectroscopy on patterned single-cell biosensors

Abstract

We report on a cell-based biosensor application that utilizes patterned single-cell arrays combined with confocal Raman spectroscopy to observe the time-dependent drug response of individual cells in real time. The patterned single-cell platform enables individual cells to be easily located and continuously addressable for Raman spectroscopy characterization of biochemical compositional changes in a non-destructive, quantitative manner so that discrete cellular behavior and cell-to-cell variations are preserved. In this study, human medulloblastoma (DAOY) cells were exposed to the common chemotherapeutic agent etoposide, and Raman spectra from patterned cells were recorded over 48 hours. It was found that 87.5% of the cells monitored exhibited a sharp decrease in DNA and protein associated peaks 48 hours after drug exposure, corresponding to cell death. The remaining 12.5% of the cells showed little to no reduction in key Raman biomarkers, indicating their drug resistance. Furthermore, the patterned cell population showed a very similar response to etoposide as confluent cell cultures, as confirmed by flow cytometry. Finally, patterned cells were assessed with TUNEL assay for apoptosis due to DNA fragmentation after etoposide exposure. The results agree well with those from the Raman spectroscopy analysis. This combined biosensor-Raman platform provides a quick, simple way to assess cell responses to chemical and biological agents with high throughput and can be potentially used for a wide variety of biomedical applications such as pharmaceutical drug discovery, toxin tests, and biothreat detection.

Figure 1

a) Schematic of the final surface modification of gold patterned silicon oxide substrate for subsequent single-cell adhesion. b) Optical DIC image of patterned DAOY cells on the surface-modified 20 μm × 20 μm gold squares with 40 μm spacing between squares.

Figure 2

Raman spectra for two representative cells acquired prior to (0 hr) and every 12 hours after etoposide exposure. a) Raman spectra of cell 1, suggesting that the cell died after 36 hours. b) Raman spectra of cell 5, indicating that the cell was viable over the course of the experiment.

Figure 3

Changes in intensity of selected Raman peaks of sampled patterned cells before and every 12 hours for 48 hours after etoposide exposure. Peaks are displayed as a ratio to the initial intensities (0 hr) for each individual cell.

Figure 4

a) Flow cytometry PI fluorescence intensity histograms of DAOY cultured cells at 12, 24, 36, and 48 hr after etoposide exposure. b) 1342 cm⁻¹ Raman peak (DNA A,G, protien C–H deformation) intensity histograms of n = 24 single patterned cells exposed to etoposide. c) Normalized viability of cultured DAOY cells determined by PI intensity (squares) and patterned DAOY cells determined by the Raman 1342 cm⁻¹ peak intensity (circles).

Figure 5

Results of TUNEL staining of patterned DAOY cells. a) DAPI (blue) and TUNEL (green) fluorescence images of typical control and etoposide exposed cells. b) Histogram of TUNEL intensities for control and etoposide exposed cells for n = 24 cells examined.