A living cell quartz crystal microbalance biosensor for continuous monitoring of cytotoxic responses of macrophages to single-walled carbon nanotubes

作者：Wang Gang; Dewilde Abiche H; Zhang Jianping; Pal Anoop; Vashist Malavika; Bello Dhimiter; Marx Kenneth A; Braunhut Susan J^*; Therrien Joel M

来源：Particle and Fibre Toxicology, 2011, 8(1): 4.

DOI：10.1186/1743-8977-8-4

Background: Numerous engineered nanomaterials (ENMs) exist and new ENMs are being developed. A challenge to nanotoxicology and environmental health and safety is evaluating toxicity of ENMs before they become widely utilized. Cellular assays remain the predominant test platform yet these methods are limited by using discrete time endpoints and reliance on organic dyes, vulnerable to interference from ENMs. Label-free, continuous, rapid response systems with biologically meaningful endpoints are needed. We have developed a device to detect and monitor in real time responses of living cells to ENMs. The device, a living cell quartz crystal microbalance biosensor (QCMB), uses macrophages adherent to a quartz crystal. The communal response of macrophages to treatments is monitored continuously as changes in crystal oscillation frequency (Delta f). We report the ability of this QCMB to distinguish benign from toxic exposures and reveal unique kinetic information about cellular responses to varying doses of single-walled carbon nanotubes (SWCNTs).
Results: We analyzed macrophage responses to additions of Zymosan A, polystyrene beads (PBs) (benign substances) or SWCNT (3-150 mu g/ml) in the QCMB over 18 hrs. In parallel, toxicity was monitored over 24/48 hrs using conventional viability assays and histological stains to detect apoptosis. In the QCMB, a stable unchanging oscillation frequency occurred when cells alone, Zymosan A alone, PBs alone or SWCNTs without cells at the highest dose alone were used. With living cells in the QCMB, when Zymosan A, PBs or SWCNTs were added, a significant decrease in frequency occurred from 1-6 hrs. For SWCNTs, this Delta f was dose-dependent. From 6-18 hrs, benign substances or low dose SWCNT (3-30 mu g/ml) treatments showed a reversal of the decrease of oscillation frequency, returning to or exceeding pre-treatment levels. Cell recovery was confirmed in conventional assays. The lag time to see the Delta f reversal in QCMB plots was linearly SWCNT-dose dependent. Lastly, the frequency never reversed at high dose SWCNT (100-150 mu g/ml), and apoptosis/necrosis was documented in conventional 24 and 48 hr-assays.
Conclusion: These data suggest that the new QCMB detects and provides unique information about peak, sublethal and toxic exposures of living cells to ENMs before they are detected using conventional cell assays.

• 出版日期2011-1-25