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We present here a perspective detailing the current state-of-the-art technologies for the characterisation of nanoparticles (NPs) in liquid suspension. We detail the technologies involved and assess their applications in the determination of NP size and concentration. We also investigate the parameters that can influence the results and put forward a cause and effect analysis of the principle factors influencing the measurement of NP size and concentration by NP tracking analysis and dynamic light scattering, to identify areas where uncertainties in the measurement can arise. Also included are technologies capable of characterising NPs in solution, whose measurements are not based on light scattering. It is hoped that the manuscript, with its detailed description of the methodologies involved, will assist scientists in selecting the appropriate technology for characterising their materials and enabling them to comply with regulatory agencies’ demands for accurate and reliable NP size and concentration data.

An important consideration in developing standards and regulations that govern the production and use of commercial nanoscale materials is the development of robust and reliable measurements to monitor the potential adverse biological effects of such products. These measurements typically require cell-based and other biological assays that provide an assessment of the risks associated with the nanomaterial of interest. In this perspective, we describe the use of cause-and-effect (C&E) analysis to design robust, high quality cell-based assays to test nanoparticle-related cytotoxicity. C&E analysis of an assay system identifies the sources of variability that influence the test result. These sources can then be used to design control experiments that aid in establishing the validity of a test result. We demonstrate the application of C&E analysis to the commonly used 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell-viability assay. This is the first time to our knowledge that C&E analysis has been used to characterize a cell-based toxicity assay. We propose the use of a 96-well plate layout which incorporates a range of control experiments to assess multiple factors such as nanomaterial interference, pipetting accuracy, cell seeding density, and instrument performance, and demonstrate the performance of the assay using the plate layout in a case study. While the plate layout was formulated specifically for the MTS assay, it is applicable to other cytotoxicity, ecotoxicity (i.e., bacteria toxicity), and nanotoxicity assays after assay-specific modifications.

SummaryDevelopment of reliable cell-based nanotoxicology assays is important for evaluation of potentially hazardous engineered nanomaterials. Challenges to producing a reliable assay protocol include working with nanoparticle dispersions and living cell lines, and the potential for nano-related interference effects. Here we demonstrate the use of a 96-well plate design with several measurement controls and an interlaboratory comparison study involving five laboratories to characterize the robustness of a nanocytotoxicity MTS cell viability assay based on the A549 cell line. The consensus EC 50 values were 22.1 mg/L (95% confidence intervals 16.9 mg/L to 27.2 mg/L) and 52.6 mg/L (44.1 mg/L to 62.6 mg/L) for positively charged polystyrene nanoparticles for the serum-free and serum conditions, respectively, and 49.7 µmol/L (47.5 µmol/L to 51.5 µmol/L) and 77.0 µmol/L (54.3 µmol/L to 99.4 µmol/L) for positive chemical control cadmium sulfate for the serum-free and serum conditions, respectively. Results from the measurement controls can be used to evaluate the sources of variability and their relative magnitudes within and between laboratories. This information revealed steps of the protocol that may need to be modified to improve the overall robustness and precision. The results suggest that protocol details such as cell line ID, media exchange, cell handling, and nanoparticle dispersion are critical to ensure protocol robustness and comparability of nanocytotoxicity assay results. The combination of system control measurements and interlaboratory comparison data yielded insights that would not have been available by either approach by itself.

The infrared spectra of the band of the 2Π–2Π asymmetric hydrogen stretching vibration in the three isotopic acetylene ions C2H2+ (ν3), 13C2H2+ (ν3), and DCCH+ (ν1) have been observed and analyzed. The high resolution infrared spectra were recorded using a difference-frequency laser spectrometer as the tunable coherent infrared source probing an ac glow discharge. Velocity modulation, noise subtraction, and unidirectional multipassing of the infrared beam through the discharge cell provided high sensitivity. C2H2+ was produced in a gas mixture of H2, He, and either CH4 or C2H2, with a total pressure of ≊7 Torr in multiple-inlet–outlet air-, water-, and liquid-nitrogen-cooled discharge tubes; C2H2 freezing precluded its use in liquid-N2-cooled discharges. Complicated by a strong perturbation whose maximum occurred at N′=15 for F1 and N′=14 for F2, the assignment of the spectrum of normal C2H2+ was made possible by (1) fortuitous discharge conditions which provided unambiguous discrimination of C2H2+ lines from among concurrent CH3+ and C2H3+ lines, and (2) fitting the ground state combination differences. Sufficiently high N transitions were observed where Λ doubling was evident. The average bond lengths rz(CH)=1.077 (5) Å and rz(CC)=1.257 (8) Å were calculated from the spectroscopic constants determined from nonlinear least-squares fitting. Vibration–rotation interactions, the Renner–Teller interaction of perturbing states, plasma chemistry, and the relevance of the work in astrophysics are discussed.

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