Early-stage diagnosis of prostatic carcinoma is essential for successful treatment and, thus, significant prognosis improvement. In laboratory practice, the standard non-invasive diagnostic approach is the immunochemical detection of the associated biomarker, prostate-specific antigen (PSA). Ultrasensitive detection of PSA is essential for both diagnostic and recurrence monitoring purposes. To achieve exceptional sensitivity, we have developed a microfluidic device with a flow-through cell for single-molecule analysis using photon-upconversion nanoparticles (UCNPs) as a detection label. For this purpose, magnetic microparticles (MBs) were first optimized for the capture and preconcentration of PSA and then used to implement a bead-based upconversion-linked immunoassay (ULISA) in the microfluidic device. The digital readout based on counting single nanoparticle-labeled PSA molecules on MBs enabled a detection limit of 1.04 pg mL-1 (36 fM) in 50% fetal bovine serum, which is an 11-fold improvement over the respective analog MB-based ULISA. The microfluidic technique conferred several other advantages, such as easy implementation and the potential for achieving high-throughput analysis. Finally, it was proven that the microfluidic setup is suitable for clinical sample analysis, showing a good correlation with a reference electrochemiluminescence assay (recovery rates between 97% and 105%).
- MeSH
- imunoanalýza přístrojové vybavení metody MeSH
- lidé MeSH
- limita detekce MeSH
- mikrofluidní analytické techniky přístrojové vybavení MeSH
- nádory prostaty diagnóza krev MeSH
- nanočástice chemie MeSH
- prostatický specifický antigen * analýza krev MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Co to je...
Vydání 1. 23 stran : barevné ilustrace ; 21 cm
Brožura se zaměřuje na výzkumnou činnost Ústavu analytické chemie AV ČR. Určeno odborné veřejnosti.; Přehled vybraných výzkumných směrů na Ústavu analytické chemie AV ČR. Každé z pěti vědeckých oddělení ústavu představuje vybrané téma ze své činnosti.
- Klíčová slova
- Praha (Česko),
- MeSH
- akademie a ústavy MeSH
- analytická chemie MeSH
- chemie dějiny MeSH
- dějiny 20. století MeSH
- dějiny 21. století MeSH
- věda MeSH
- výzkum MeSH
- Check Tag
- dějiny 20. století MeSH
- dějiny 21. století MeSH
- Geografické názvy
- Česká republika MeSH
- Konspekt
- Analytická chemie
- NLK Obory
- chemie, klinická chemie
- věda a výzkum
- NLK Publikační typ
- brožury
Conventional immunochemical methods used in clinical analysis are often not sensitive enough for early-stage diagnosis, resulting in the need for novel assay formats. Here, we provide a detailed comparison of the effect of different labels and solid supports on the performance of heterogeneous immunoassays. When comparing three types of streptavidin-modified labels─horseradish peroxidase, carboxyfluorescein, and photon-upconversion nanoparticles (UCNPs)─UCNPs led to the most sensitive and robust detection of the cancer biomarker prostate-specific antigen. Additionally, we compared the immunoassay formats based on conventional microtiter plates and magnetic microbeads (MBs). In both cases, the highest signal-to-background ratios and the lowest limits of detection (LODs) were obtained by using the UCNP labels. The MB-based upconversion-linked immunosorbent assay carried out with a preconcentration step provided the lowest LOD of 0.46 pg/mL in serum. The results demonstrate that the use of UCNPs and MBs can significantly improve the sensitivity and working range of heterogeneous immunoassays for biomarker detection.
- MeSH
- imunoanalýza metody MeSH
- imunosorbenty * MeSH
- lidé MeSH
- limita detekce MeSH
- magnetismus MeSH
- nanočástice * MeSH
- streptavidin MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The detection of cancer biomarkers in histological samples and blood is of paramount importance for clinical diagnosis. Current methods are limited in terms of sensitivity, hindering early detection of disease. We have overcome the shortcomings of currently available staining and fluorescence labeling methods by taking an integrative approach to establish photon-upconversion nanoparticles (UCNP) as a powerful platform for cancer detection. These nanoparticles are readily synthesized in different sizes to yield efficient and tunable short-wavelength light emission under near-infrared excitation, which eliminates optical background interference of the specimen. Here we present a protocol for the synthesis of UCNPs by high-temperature co-precipitation or seed-mediated growth by thermal decomposition, surface modification by silica or poly(ethylene glycol) that renders the particles resistant to nonspecific binding, and the conjugation of streptavidin or antibodies for biological detection. To detect blood-based biomarkers, we present an upconversion-linked immunosorbent assay for the analog and digital detection of the cancer marker prostate-specific antigen. When applied to immunocytochemistry analysis, UCNPs enable the detection of the breast cancer marker human epidermal growth factor receptor 2 with a signal-to-background ratio 50-fold higher than conventional fluorescent labels. UCNP synthesis takes 4.5 d, the preparation of the antibody-silica-UCNP conjugate takes 3 d, the streptavidin-poly(ethylene glycol)-UCNP conjugate takes 2-3 weeks, upconversion-linked immunosorbent assay takes 2-4 d and immunocytochemistry takes 8-10 h. The procedures can be performed after standard laboratory training in nanomaterials research.
- MeSH
- imunosorbenty MeSH
- lidé MeSH
- nádorové biomarkery MeSH
- nádory * diagnóza MeSH
- nanočástice * chemie MeSH
- oxid křemičitý chemie MeSH
- polyethylenglykoly chemie MeSH
- streptavidin MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Sensitive immunoassays are required for troponin, a low-abundance cardiac biomarker in blood. In contrast to conventional (analog) assays that measure the integrated signal of thousands of molecules, digital assays are based on counting individual biomarker molecules. Photon-upconversion nanoparticles (UCNP) are an excellent nanomaterial for labeling and detecting single biomarker molecules because their unique anti-Stokes emission avoids optical interference, and single nanoparticles can be reliably distinguished from the background signal. Here, the effect of the surface architecture and size of UCNP labels on the performance of upconversion-linked immunosorbent assays (ULISA) is critically assessed. The size, brightness, and surface architecture of UCNP labels are more important for measuring low troponin concentrations in human plasma than changing from an analog to a digital detection mode. Both detection modes result approximately in the same assay sensitivity, reaching a limit of detection (LOD) of 10 pg mL-1 in plasma, which is in the range of troponin concentrations found in the blood of healthy individuals.
- MeSH
- fotony MeSH
- imunoanalýza MeSH
- lidé MeSH
- nanočástice * MeSH
- troponin MeSH
- velikost částic MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Due to increasing food safety standards, the analysis of mycotoxins has become essential in the food industry. In this work, we have developed a competitive upconversion-linked immunosorbent assay (ULISA) for the analysis of zearalenone (ZEA), one of the most frequently encountered mycotoxins in food worldwide. Instead of a toxin-conjugate conventionally used in competitive immunoassays, we designed a ZEA mimicking peptide extended by a biotin-linker and confirmed its excellent suitability to mimic ZEA by nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) analysis. Upconversion nanoparticles (UCNP, type NaYF4:Yb,Tm) served as background-free optical label for the detection of the peptide mimetic in the competitive ULISA. Streptavidin-conjugated UCNPs were prepared by click reaction using an alkyne-PEG-neridronate linker. The UCNP conjugate clearly outperformed conventional labels such as enzymes or fluorescent dyes. With a limit of detection of 20 pg mL-1 (63 pM), the competitive ULISA is well applicable to the detection of ZEA at the levels set by the European legislation. Moreover, the ULISA is specific for ZEA and its metabolites (α- and β-zearalenol) without significant cross-reactivity with other related mycotoxins. We detected ZEA in spiked and naturally contaminated maize samples using liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) as a reference method to demonstrate food analysis in real samples.
In this study, radish (Raphanus sativus L.) and common duckweed (Lemna minor L.) were treated with an aqueous dispersion of carboxylated silica-coated photon-upconversion nanoparticles containing rare-earth elements (Y, Yb, and Er). The total concentration of rare earths and their bioaccumulation factors were determined in root, hypocotyl, and leaves of R. sativus after 72 h, and in L. minor fronds after 168 h. In R. sativus, translocation factors were determined as the ratio of rare earths concentration in hypocotyl versus root and in leaves versus hypocotyl. The lengths of the root and hypocotyl in R. sativus, as well as the frond area in L. minor, were monitored as toxicity endpoints. To distinguish rare earth bioaccumulation patterns, two-dimensional maps of elemental distribution in the whole R. sativus plant and L. minor fronds were obtained by laser-induced breakdown spectroscopy with a lateral resolution of 100 μm. Moreover, the bioaccumulation was inspected using a photon-upconversion laser microscanner. The results revealed that the tested nanoparticles became adsorbed onto L. minor fronds and R. sativus roots, as well as transferred from roots through the hypocotyl and into leaves of R. sativus. The bioaccumulation patterns and spatial distribution of rare earths in nanoparticle-treated plants therefore differed from those of the positive control. Overall, carboxylated silica-coated photon-upconversion nanoparticles are stable, can easily translocate from roots to leaves, and are expected to become adsorbed onto the plant surface. They are also significantly toxic to the tested plants at nominal concentrations of 100 and 1000 μg/mL.
- MeSH
- fotony MeSH
- nanočástice chemie MeSH
- Raphanus účinky léků MeSH
- rostlinné extrakty chemie MeSH
- spektrální analýza metody MeSH
- Publikační typ
- časopisecké články MeSH
Upconversion nanoparticles (UCNPs) are an emerging class of optical materials with high potential in bioimaging due to practically no background signal and high penetration depth. Their excellent optical properties and easy surface functionalization make them perfect for conjugation with targeting ligands. In this work, capillary electrophoretic (CE) method with laser-induced fluorescence detection was used to investigate the behavior of carboxyl-silica-coated UCNPs. Folic acid, targeting folate receptor overexpressed by wide variety of cancer cells, was used for illustrative purposes and assessed by CE under optimized conditions. Peptide-mediated bioconjugation of antibodies to UCNPs was also investigated. Despite the numerous advantages of CE, this is the first time that CE was employed for characterization of UCNPs and their bioconjugates. The separation conditions were optimized including the background electrolyte concentration and pH. The optimized electrolyte was 20 mM borate buffer with pH 8.
- MeSH
- elektroforéza kapilární metody MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční spektrometrie metody MeSH
- kyselina listová chemie MeSH
- limita detekce MeSH
- lineární modely MeSH
- nanokonjugáty chemie MeSH
- protilátky chemie MeSH
- reprodukovatelnost výsledků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Metody pro studium jednotlivých biomolekul pomáhají objasnit jejich vlastnosti jinak skryté při měřeních v celém objemu vzorku. Velmi významnou metodou pro výzkum chování jednotlivých biomolekul je luminiscenční mikroskopie. Luminiscenční značky, které vykazují fotonovou up‑konverzi, přináší nové možnosti v této oblasti. Předpokládá se, že zjednoduší a přinesou větší rozšíření metod pro vizualizaci jednotlivých biomolekul, např. membránových receptorů, studium jejich interakcí v reálném čase a rovněž nové možnosti pro detekci jednotlivých biomolekul.
Single molecule methods allow us to explain properties of biomolecules on single molecule level, which are not apparent from bulk experiments. Luminescence microscopy is a very important method for single molecule experiments. Novel luminescent labels, which exhibit photon up‑conversion, revealed new possibilities in this field. It is considered that photon up‑conversion nanoparticles will allow for next development of single molecule methods and facilitate for visualization of individual biomolecules, e.g., membrane receptors, studies of their interactions in real time and also provide new possibilities for the detection of individual biomolecules.
