Single-particle (digital) immunoassays offer significantly lower limits of detection (LODs) than traditional immunoassays, making them suitable for the detection of low-abundance biomarkers. The most common approach for digital detection is based on counting individual labels. Here, we introduce a novel dot-blot particle-linked immunosorbent assay (PLISA) with digital readout utilizing laser ablation (LA) of photon upconversion nanoparticle (UCNP) labels from the nitrocellulose substrate. Compared to conventional LA, our approach allows desorption of intact nanoparticles and their precise counting by single-particle inductively coupled plasma mass spectrometry (SP ICP MS), thus counting individual UCNP-labeled immunocomplexes. Digital signal processing filters instrument noise and nanoparticle aggregates, minimizing potential errors. The immunoassay and LA SP ICP MS readout were optimized using human serum albumin, a kidney damage biomarker, as a model analyte, obtaining LODs of 0.18 and 0.12 ng/mL for the reference upconversion luminescence (UCL) and LA SP ICP MS readout, respectively. Building upon these optimized conditions, we developed PLISA for prostate-specific antigen, the key prostate cancer biomarker, with LODs of 2.4, 1.4, and 0.3 pg/mL for the UCL, analog, and digital LA SP ICP MS readout, respectively. The LOD in the sub-pg/mL range highlighted the advantage of particle counting and its ability to detect low-abundance biomarkers, as superior performance was achieved compared to the UCL and analog LA ICP MS readout. Finally, clinical serum samples of patients tested for prostate cancer were analyzed, and a strong correlation with the reference electrochemiluminescence method confirmed the potential of LA SP ICP MS for clinical diagnostics.

• MeSH
• biologické markery analýza   MeSH
• hmotnostní spektrometrie * metody   MeSH
• imunoanalýza metody   MeSH
• laserová terapie * MeSH
• lasery MeSH
• lidé MeSH
• lidský sérový albumin * analýza   MeSH
• limita detekce MeSH
• nádorové biomarkery * krev   analýza   MeSH
• nanočástice chemie   MeSH
• prostatický specifický antigen * krev   analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biologické markery MeSH
• lidský sérový albumin * MeSH
• nádorové biomarkery * MeSH
• prostatický specifický antigen * MeSH

We present a simplistic and absolute method for estimating the number concentration of nanoparticles. Macroscopic volumes of a nanoparticle dispersion (several μL) are dropped on a glass surface and the solvent is evaporated. The optical microscope scans the entire surface of the dried droplet (several mm2), micrographs are stitched together (several tens), and all nanoparticles are counted (several thousand per droplet) by using an artificial neural network. We call this method evaporated volume analysis (EVA) because nanoparticles are counted after droplet volume evaporation. As a model, the concentration of ∼60 nm Tm3+-doped photon-upconversion nanoparticles coated in carboxylated silica shells is estimated with a combined relative standard uncertainty of 2.7%. Two reference methods provided comparable concentration values. A wider applicability is tested by imaging ∼80 nm Nile red-doped polystyrene and ∼90 nm silver nanoparticles. Theoretical limits of EVA such as the limit of detection, limit of quantification, and optimal working range are discussed.

• Publikační typ
• časopisecké články MeSH

Silver nanoparticles (AgNPs), widely used in various fields of technology as an antimicrobial agent, represent a new type of environmental pollutant. Through various routes, AgNPs might penetrate into agricultural crops and foodstuffs. It is important to know if AgNPs contained in food persist in digested food and are therefore available for entering the inner organs of the consumer's body. Using the technique of single-particle ICP-MS, we analysed the changes in the number and size distribution of AgNPs added to a sample of bread submitted to in vitro simulated gastrointestinal digestion. The majority of silver, in terms of mass, was transformed from the state of particles to the dissolved state during bread digestion, but the number of particles was reduced by 25% only. The most abundant particle size was reduced from 60 nm to 49 nm. Hence, a substantial part of transformed nanoparticles is still present in food digestate. This means that AgNPs consumed together with food can theoretically enter the inner cells of human body.

• Klíčová slova
• bread, food contamination, food safety, silver nanoparticles, simulated digestion, sp-ICP-MS,
• Publikační typ
• časopisecké články MeSH

With the growing numbers of nanomaterials (NMs), there is a great demand for rapid and reliable ways of testing NM safety-preferably using in vitro approaches, to avoid the ethical dilemmas associated with animal research. Data are needed for developing intelligent testing strategies for risk assessment of NMs, based on grouping and read-across approaches. The adoption of high throughput screening (HTS) and high content analysis (HCA) for NM toxicity testing allows the testing of numerous materials at different concentrations and on different types of cells, reduces the effect of inter-experimental variation, and makes substantial savings in time and cost. HTS/HCA approaches facilitate the classification of key biological indicators of NM-cell interactions. Validation of in vitro HTS tests is required, taking account of relevance to in vivo results. HTS/HCA approaches are needed to assess dose- and time-dependent toxicity, allowing prediction of in vivo adverse effects. Several HTS/HCA methods are being validated and applied for NM testing in the FP7 project NANoREG, including Label-free cellular screening of NM uptake, HCA, High throughput flow cytometry, Impedance-based monitoring, Multiplex analysis of secreted products, and genotoxicity methods-namely High throughput comet assay, High throughput in vitro micronucleus assay, and γH2AX assay. There are several technical challenges with HTS/HCA for NM testing, as toxicity screening needs to be coupled with characterization of NMs in exposure medium prior to the test; possible interference of NMs with HTS/HCA techniques is another concern. Advantages and challenges of HTS/HCA approaches in NM safety are discussed. WIREs Nanomed Nanobiotechnol 2017, 9:e1413. doi: 10.1002/wnan.1413 For further resources related to this article, please visit the WIREs website.

• MeSH
• buněčné linie MeSH
• cytologické techniky MeSH
• intracelulární prostor chemie   metabolismus   MeSH
• lidé MeSH
• myši MeSH
• nanostruktury toxicita   MeSH
• rychlé screeningové testy metody   MeSH
• testy toxicity metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH