Abstract
BackgroundNumerous 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 (Δf). 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).ResultsWe analyzed macrophage responses to additions of Zymosan A, polystyrene beads (PBs) (benign substances) or SWCNT (3-150 μg/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 Δf was dose-dependent. From 6-18 hrs, benign substances or low dose SWCNT (3-30 μg/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 Δf reversal in QCMB plots was linearly SWCNT-dose dependent. Lastly, the frequency never reversed at high dose SWCNT (100-150 μg/ml), and apoptosis/necrosis was documented in conventional 24 and 48 hr-assays.ConclusionThese data suggest that the new QCMB detects and provides unique information about peak, sub-lethal and toxic exposures of living cells to ENMs before they are detected using conventional cell assays.
Highlights
Numerous engineered nanomaterials (ENMs) exist and new Engineered nanomaterial (ENM) are being developed
Cellular testing relies heavily on organic dyes, labels and spectrophotometric techniques. Such protocols have several technical drawbacks, including: i) interference of the label by ENMs via absorption of detection dyes onto the large surface area of ENMs [5,6,7,8]; and ii) light scattering and/or quenching of fluorescence used in detection in the assay by the ENMs
We describe experiments that demonstrate the ability of live cell based biosensors to detect and provide unique temporal response data following living cell exposures to single-walled carbon nanotubes (SWCNTs)
Summary
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 testing relies heavily on organic dyes, labels and spectrophotometric techniques (ultraviolet or fluorescence measurements) Such protocols have several technical drawbacks, including: i) interference of the label by ENMs via absorption of detection dyes onto the large surface area of ENMs [5,6,7,8]; and ii) light scattering and/or quenching of fluorescence used in detection in the assay by the ENMs. Such protocols have several technical drawbacks, including: i) interference of the label by ENMs via absorption of detection dyes onto the large surface area of ENMs [5,6,7,8]; and ii) light scattering and/or quenching of fluorescence used in detection in the assay by the ENMs In addition these types of protocols may be inadequate in detecting the mechanism of ENM toxicity because: i) discrete monitoring of toxic endpoints at particular time intervals may miss the recovery behavior of the community of cells between those endpoints following initial perturbations; ii) these assays do not reveal kinetics of such perturbations; iii) the monitored toxic endpoints may not include all mechanistic pathways leading to cellular toxicity; and iv) repeated ENMs dosimetry on cells is rarely done, missing a critical aspect of human exposure. Development of test systems capable of monitoring continuously and in real time communal behavior of cells over hrs to days using biologically-relevant global, label-free endpoints would alleviate several limitations of current cellular testing protocols
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