(14) Still, light-scattering measurements for NPs with <300 nm are challenging and were only performed so far resorting to custom-made, not broadly available nFC setups, (14,15) while fluorescence detection requires NPs labeling and purification before analysis. In nFC, single-particle counting is performed by light scattering (label free) and/or fluorescence measurements after NPs irradiation by a laser beam. Considering these limitations, other techniques, such as nano flow cytometry (nFC), multiangle light scattering (MALS), and differential centrifugal sedimentation (DCS), have been also used for measuring NPs concentration, and/or to provide a means for results validation. (9,12) Nevertheless, TRPS requires NPs dilution in electrolytes, analysis at different pressure points, calibration with particle standards prior to each sample analysis and for each pressure point, and experienced operators (9,12) capable of optimizing measurement parameters, which renders the technique not straightforward for routine applications at industrial or medical facilities. (9,13) TRPS is a more robust technique, in which NPs concentration is assessed while they pass through a porous membrane, driven by voltage application. (5,13) Also, results depend on the parameters set for video acquisition and processing, which are operator-dependent, thus requiring trained personnel and often suffering from variability if undertaken by different operators. However, PTA analysis requires extensive sample dilution, which can induce NPs aggregation, causing inaccurate measurements. (9,12) PTA measurements result from light-scattering-based tracking of individual NPs in a given illumination volume. (10,11) Therefore, for organic NPs, concentration measurements are mainly performed by single-particle analysis techniques, such as particle tracking analysis (PTA) and tunable resistive pulse sensing (TRPS). Still, measuring NPs concentration resorting to the currently available techniques poses several challenges, (5,9) namely, for organic NPs, whose mass cannot be quantified by inductively coupled plasma mass spectrometry, (10) as occurs for inorganic NPs. Moreover, concentration measurements for PEG–PLGA NPs loaded with an anti-inflammatory drug, methotrexate (MTX), after their incubation in simulated gastric and intestinal fluids were successfully achieved (recovery values of 102–115%, as confirmed by PTA), showing the suitability of the proposed method to support the development of polymeric NPs targeting intestinal delivery. NPs size and concentration were maintained during analysis, as verified for NPs eluted from the LOV by particle tracking analysis (PTA). Determinations for polystyrene NPs (of 100, 200, and 500 nm) and for NPs made of PEGylated poly- d, l-lactide- co-glycolide (PEG–PLGA, a biocompatible FDA-approved polymer) were accomplished within 10 8–10 12 particles mL –1 range, depending on the NPs size and composition. Measurements were performed on polymeric NPs, as these represent one of the major classes of NPs under development for drug-delivery aims. Each analysis was accomplished in 2 min, rendering a determination throughput of 30 h –1 (6 samples h –1 for n = 5) and only requiring 30 μL (≈0.03 g) of NPs suspension. NPs concentration measurements were based on the decrease in the light transmitted to the detector due to the light scattered by NPs when passing through the optical path. Automatic NPs sampling and delivery to the LOV detection unit were set by flow programming. Herein, a miniaturized automated ensemble method to measure NPs concentration was established under the lab-on-valve (LOV) mesofluidic platform. Still, faster and simpler procedures, dismissing skilled operators and post-analysis conversions are needed to quantify NPs for research and quality control operations, and to support result validation. ![]() The evaluation of this parameter is required during NPs developmental and quality control stages, for setting dose–response correlations and for evaluating the reproducibility of the manufacturing process. Nanoparticles (NPs) concentration directly impacts the dose delivered to target tissues by nanocarriers.
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