Accurate and precise nucleic-acid quantification is crucial for clinical and diagnostic decisions, as overestimation or underestimation can lead to misguided treatment of a disease or incorrect labelling of the products. Digital PCR is one of the best tools for absolute nucleic-acid copy-number determination. However, digital PCR needs to be well characterised in terms of accuracy and sources of uncertainty. With droplet digital PCR, discrepancies between the droplet volume assigned by the manufacturer and measured by independent laboratories have already been shown in previous studies. In the present study, we report on the results of an inter-laboratory comparison of different methods for droplet volume determination that is based on optical microscopy imaging and is traceable to the International System of Units. This comparison was conducted on the same DNA material, with the examination of the influence of parameters such as droplet generators, supermixes, operators, inter-cartridge and intra-cartridge variability, and droplet measuring protocol. The mean droplet volume was measured using a QX200™ AutoDG™ Droplet Digital™ PCR system and two QX100™ Droplet Digital™ PCR systems. The data show significant volume differences between these two systems, as well as significant differences in volume when different supermixes are used. We also show that both of these droplet generator systems produce droplets with significantly lower droplet volumes (13.1%, 15.9%, respectively) than stated by the manufacturer and previously measured by other laboratories. This indicates that to ensure precise quantification, the droplet volumes should be assessed for each system.Electronic supplementary materialThe online version of this article (10.1007/s00216-017-0625-y) contains supplementary material, which is available to authorized users.
Effective interaction and accumulation of nanoparticles (NPs) within tumor cells is crucial for NP-assisted diagnostic and therapeutic biomedical applications. In this context, the shape and size features of NPs can severely influence the strength of adhesion between NPs and cell and the NP internalization mechanisms. This study proved the ability of the PT45 and A549 tumor cells to uptake and retain magnetic Au-coated Ni 80 Fe 20 nanodisks (NDs) prepared by means of a bottom-up self-assembling nanolithography technique assisted by polystyrene nanospheres. The chosen geometrical parameters, i.e., diameter (≈650 nm) and thickness (≈30 nm), give rise to magnetic domain patterns arranged in vortex state at the magnetic remanence. PT45 and A549 cell lines were cultured in the presence of different concentrations of Au-coated Ni 80 Fe 20 nanodisks, and their biocompatibility was evaluated by viability and proliferation tests. Electron microscopy techniques and a combined CARS (Coherent Anti-stokes Raman Scattering) and TPL (two-photon photoluminescence) microscopy allow localizing and distinguishing the NDs within or attached to the tumor cells, without any labeling. A quantitative measurement of ND amount retained within tumor cells as a function of ND concentrations was performed by the Instrumental Neutron Activation Analysis (INAA) characterization technique.
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