The ability to detect low concentrations of analytes and in particular low-abundance biomarkers is of fundamental importance, e.g., for early-stage disease diagnosis. The prospect of reaching the ultimate limit of detection has driven the development of single-molecule bioaffinity assays. While many review articles have highlighted the potentials of single-molecule technologies for analytical and diagnostic applications, these technologies are not as widespread in real-world applications as one should expect. This Review provides a theoretical background on single-molecule-or better digital-assays to critically assess their potential compared to traditional analog assays. Selected examples from the literature include bioaffinity assays for the detection of biomolecules such as proteins, nucleic acids, and viruses. The structure of the Review highlights the versatility of optical single-molecule labeling techniques, including enzymatic amplification, molecular labels, and innovative nanomaterials.

• Klíčová slova
• digital assays, immunoassays, optical detection, signal background, single-molecule detection,
• MeSH
• biologické markery analýza   MeSH
• ELISA MeSH
• fluorescenční barviva chemie   MeSH
• limita detekce MeSH
• nanostruktury chemie   MeSH
• nukleové kyseliny analýza   MeSH
• polymerázová řetězová reakce metody   MeSH
• poměr signál - šum MeSH
• proteiny analýza   MeSH
• vazebná místa MeSH
• viry izolace a purifikace   MeSH
• zobrazení jednotlivé molekuly metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• biologické markery MeSH
• fluorescenční barviva MeSH
• nukleové kyseliny MeSH
• proteiny MeSH

Here a novel digital bioassay readout concept is reported that does not rely on enzymatic amplification nor compartmenting of an analyzed liquid sample. Rather, it is based on counting individual affinity-captured target biomolecules via the use of a tethered catalytic hairpin assembly (tCHA) deployed on a solid sensor surface with spatial confinement utilized by a flexible polymer linker (FPL). Wide-field plasmon-enhanced fluorescence (PEF) imaging is employed for optical real-time probing of the reaction kinetics, where affinity-captured target molecules are manifested as spatially distinct bright fluorescent spots. The effect of the length of the FPLs is investigated, and the analytical performance of the dual amplification tCHA-PEF concept is tested by using a model short single-stranded DNA analyte. When applied in a sandwich immunoassay, the detection of target proteins at sub-femtomolar concentrations is demonstrated. The reported experiments are supported by diffusion-limited mass transfer models and document the potential of tCHA-PEF as a new class of generic enzyme-free bioanalytical tools enabling the ultrasensitive analysis of trace amounts of protein and nucleic acid analytes, making it attractive for future molecular diagnostics and research applications.

• Klíčová slova
• catalytic hairpin assembly, flexible DNA linker, plasmon‐enhanced fluorescence, sandwich immunoassay, single molecule detection,
• MeSH
• biosenzitivní techniky * metody   MeSH
• fluorescence MeSH
• imunoanalýza metody   MeSH
• jednovláknová DNA chemie   analýza   MeSH
• katalýza MeSH
• povrchová plasmonová rezonance * metody   MeSH
• zobrazení jednotlivé molekuly * metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• jednovláknová DNA MeSH

We report on the tailoring of rolling circle amplification (RCA) for affinity biosensors relying on the optical probing of their surface with confined surface plasmon field. Affinity capture of the target analyte at the metallic sensor surface (e.g., by using immunoassays) is followed by the RCA step for subsequent readout based on increased refractive index (surface plasmon resonance, SPR) or RCA-incorporated high number of fluorophores (in surface plasmon-enhanced fluorescence, PEF). By combining SPR and PEF methods, this work investigates the impact of the conformation of long RCA-generated single-stranded DNA (ssDNA) chains to the plasmonic sensor response enhancement. In order to confine the RCA reaction within the evanescent surface plasmon field and hence maximize the sensor response, an interface carrying analyte-capturing molecules and additional guiding ssDNA strands (complementary to the repeating segments of RCA-generated chains) is developed. When using the circular padlock probe as a model target analyte, the PEF readout shows that the reported RCA implementation improves the limit of detection (LOD) from 13 pM to high femtomolar concentration when compared to direct labeling. The respective enhancement factor is of about 2 orders of magnitude, which agrees with the maximum number of fluorophore emitters attached to the RCA chain that is folded in the evanescent surface plasmon field by the developed biointerface. Moreover, the RCA allows facile visualizing of individual binding events by fluorescence microscopy, which enables direct counting of captured molecules. This approach offers a versatile route toward a fast digital readout format of single-molecule detection with further reduced LOD.

• Klíčová slova
• biosensor, immunoassays, rolling circle amplification, single molecule, surface plasmon resonance, surface plasmon-enhanced fluorescence,
• MeSH
• biosenzitivní techniky * metody   MeSH
• jednovláknová DNA MeSH
• limita detekce MeSH
• povrchová plasmonová rezonance metody   MeSH
• techniky amplifikace nukleových kyselin * metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• jednovláknová DNA MeSH

Quantitative genomic mapping of DNA damage may provide insights into the underlying mechanisms of damage and repair. Sequencing based approaches are bound to the limitations of PCR amplification bias and read length which hamper both the accurate quantitation of damage events and the ability to map them to structurally complex genomic regions. Optical Genome mapping in arrays of parallel nanochannels allows physical extension and genetic profiling of millions of long genomic DNA fragments, and has matured to clinical utility for characterization of complex structural aberrations in cancer genomes. Here we present a new mapping modality, Repair-Assisted Damage Detection - Optical Genome Mapping (RADD-OGM), a method for single-molecule level mapping of DNA damage on a genome-wide scale. Leveraging ultra-long reads to assemble the complex structure of a sarcoma cell-line genome, we mapped the genomic distribution of oxidative DNA damage, identifying regions more susceptible to DNA oxidation. We also investigated DNA repair by allowing cells to repair chemically induced DNA damage, pinpointing locations of concentrated repair activity, and highlighting variations in repair efficiency. Our results showcase the potential of the method for toxicogenomic studies, mapping the effect of DNA damaging agents such as drugs and radiation, as well as following specific DNA repair pathways by selective induction of DNA damage. The facile integration with optical genome mapping enables performing such analyses even in highly rearranged genomes such as those common in many cancers, a challenging task for sequencing-based approaches.

• Klíčová slova
• CNV, Cancer genomes, Cytogenetics, DNA damage, DNA repair, Long-reads, Nanochannels, OGM, Osteosarcoma, Oxidative damage, RADD, S.V., Single molecule, Toxicogenomics,
• MeSH
• bromičnany toxicita   MeSH
• lidé MeSH
• mapování chromozomů * přístrojové vybavení   metody   MeSH
• mikrofluidní analytické techniky * přístrojové vybavení   metody   MeSH
• nádorové buněčné linie MeSH
• nanotechnologie * přístrojové vybavení   metody   MeSH
• oprava DNA genetika   MeSH
• oxidační stres účinky léků   genetika   MeSH
• poškození DNA * genetika   MeSH
• regulace genové exprese MeSH
• stanovení celkové genové exprese MeSH
• toxikogenetika * přístrojové vybavení   metody   MeSH
• variabilita počtu kopií segmentů DNA MeSH
• zobrazení jednotlivé molekuly * přístrojové vybavení   metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bromičnany MeSH
• potassium bromate MeSH Prohlížeč

Single-molecule (digital) immunoassays provide the ability to detect much lower protein concentrations than conventional immunoassays. As photon-upconversion nanoparticles (UCNPs) can be detected without optical background interference, they are excellent labels for so-called single-molecule upconversion-linked immunosorbent assays (ULISAs). We have introduced a UCNP label design based on streptavidin-PEG-neridronate and a two-step detection scheme involving a biotinylated antibody that efficiently reduces nonspecific binding on microtiter plates. In a microtiter plate immunoassay, individual sandwich immune complexes of the cancer marker prostate-specific antigen (PSA) are detected and counted by wide-field epiluminescence microscopy (digital readout). The digital detection is 16× more sensitive than the respective analogue readout and thus expands the limit of detection to the sub-femtomolar concentration range (LOD: 23 fg mL-1, 800 aM). The single molecule ULISA shows excellent correlation with an electrochemiluminescence reference method. Although the analogue readout can routinely measure PSA concentrations in human serum samples, very low concentrations have to be monitored after radical prostatectomy. Combining the digital and analogue readout covers a dynamic range of more than 3 orders of magnitude in a single experiment.

• MeSH
• bisfosfonáty MeSH
• dermatoskopie metody   MeSH
• fotony MeSH
• imunoanalýza metody   MeSH
• imunosorpční techniky * MeSH
• lidé MeSH
• nanočástice chemie   MeSH
• polyethylenglykoly MeSH
• prostatický specifický antigen krev   MeSH
• streptavidin MeSH
• zobrazení jednotlivé molekuly metody   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 6-amino-1-hydroxyhexane-1,1-diphosphonate MeSH Prohlížeč
• bisfosfonáty MeSH
• polyethylenglykoly MeSH
• prostatický specifický antigen MeSH
• streptavidin MeSH

DNA double stranded breaks (DSBs) are the most serious type of lesions introduced into chromatin by ionizing radiation. During DSB repair, cells recruit different proteins to the damaged sites in a manner dependent on local chromatin structure, DSB location in the nucleus, and the repair pathway entered. 53BP1 is one of the important players participating in repair pathway decision of the cell. Although many molecular biology details have been investigated, the architecture of 53BP1 repair foci and its development during the post-irradiation time, especially the period of protein recruitment, remains to be elucidated. Super-resolution light microscopy is a powerful new tool to approach such studies in 3D-conserved cell nuclei. Recently, we demonstrated the applicability of single molecule localization microscopy (SMLM) as one of these highly resolving methods for analyses of dynamic repair protein distribution and repair focus internal nano-architecture in intact cell nuclei. In the present study, we focused our investigation on 53BP1 foci in differently radio-resistant cell types, moderately radio-resistant neonatal human dermal fibroblasts (NHDF) and highly radio-resistant U87 glioblastoma cells, exposed to high-LET 15N-ion radiation. At given time points up to 24 h post irradiation with doses of 1.3 Gy and 4.0 Gy, the coordinates and spatial distribution of fluorescently tagged 53BP1 molecules was quantitatively evaluated at the resolution of 10⁻20 nm. Clusters of these tags were determined as sub-units of repair foci according to SMLM parameters. The formation and relaxation of such clusters was studied. The higher dose generated sufficient numbers of DNA breaks to compare the post-irradiation dynamics of 53BP1 during DSB processing for the cell types studied. A perpendicular (90°) irradiation scheme was used with the 4.0 Gy dose to achieve better separation of a relatively high number of particle tracks typically crossing each nucleus. For analyses along ion-tracks, the dose was reduced to 1.3 Gy and applied in combination with a sharp angle irradiation (10° relative to the cell plane). The results reveal a higher ratio of 53BP1 proteins recruited into SMLM defined clusters in fibroblasts as compared to U87 cells. Moreover, the speed of foci and thus cluster formation and relaxation also differed for the cell types. In both NHDF and U87 cells, a certain number of the detected and functionally relevant clusters remained persistent even 24 h post irradiation; however, the number of these clusters again varied for the cell types. Altogether, our findings indicate that repair cluster formation as determined by SMLM and the relaxation (i.e., the remaining 53BP1 tags no longer fulfill the cluster definition) is cell type dependent and may be functionally explained and correlated to cell specific radio-sensitivity. The present study demonstrates that SMLM is a highly appropriate method for investigations of spatiotemporal protein organization in cell nuclei and how it influences the cell decision for a particular repair pathway at a given DSB site.

• Klíčová slova
• 15N ion irradiation, repair cluster formation, repair cluster persistence, repair foci nano-architecture, single molecule localization microscopy (SMLM),
• MeSH
• 53BP1 metabolismus   MeSH
• konfokální mikroskopie metody   MeSH
• kultivované buňky MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• rekombinační oprava DNA * MeSH
• transport proteinů MeSH
• zobrazení jednotlivé molekuly metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 53BP1 MeSH

Fluorescence microscopy using single molecule imaging and localization (PALM, STORM, and similar approaches) has quickly been adopted as a convenient method for obtaining multicolor, 3D superresolution images of biological samples. Using an approach based on extensive Monte Carlo simulations, we examined the performance of various noise reducing filters required for the detection of candidate molecules. We determined a suitable noise reduction method and derived an optimal, nonlinear threshold which minimizes detection errors introduced by conventional algorithms. We also present a new technique for visualization of single molecule localization microscopy data based on adaptively jittered 2D histograms. We have used our new methods to image both Atto565-phalloidin labeled actin in fibroblast cells, and mCitrine-erbB3 expressed in A431 cells. The enhanced methods developed here were crucial in processing the data we obtained from these samples, as the overall signal to noise ratio was quite low.

• MeSH
• aktiny metabolismus   MeSH
• algoritmy * MeSH
• faloidin metabolismus   MeSH
• lidé MeSH
• metoda Monte Carlo MeSH
• mikroskopie metody   MeSH
• nádorové buněčné linie MeSH
• nelineární dynamika MeSH
• počítačová simulace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny MeSH
• faloidin MeSH

The ability to detect disease markers at the single molecule level promises the ultimate sensitivity in clinical diagnosis. Fluorescence-based single-molecule analysis, however, is limited by matrix interference and can only probe a very small detection volume, which is typically not suitable for real world analytical applications. We have developed a microtiter plate immunoassay for counting single molecules of the cancer marker prostate specific antigen (PSA) using photon-upconversion nanoparticles (UCNPs) as labels that can be detected without background fluorescence. Individual sandwich immunocomplexes consisting of (1) an anti-PSA antibody immobilized to the surface of a microtiter well, (2) PSA, and (3) an anti-PSA antibody-UCNP conjugate were counted under a wide-field epifluorescence microscope equipped with a 980 nm laser excitation source. The single-molecule (digital) upconversion-linked immunosorbent assay (ULISA) reaches a limit of detection of 1.2 pg mL-1 (42 fM) PSA in 25% blood serum, which is about ten times more sensitive than commercial ELISAs, and covers a dynamic range of three orders of magnitude. This upconversion detection mode has the potential to pave the way for a new generation of digital immunoassays.

• MeSH
• biologické markery analýza   MeSH
• imunoanalýza metody   MeSH
• imunosorbenty chemie   imunologie   MeSH
• limita detekce * MeSH
• luminiscence MeSH
• nanočástice chemie   MeSH
• prostatický specifický antigen analýza   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery MeSH
• imunosorbenty MeSH
• prostatický specifický antigen MeSH

During oocyte growth the cell accumulates RNAs to contribute to oocyte and embryo development which progresses with ceased transcription. To investigate the subcellular distribution of specific RNAs and their translation we developed a technique revealing several instances of localized translation with distinctive regulatory implications. We analyzed the localization and expression of candidate non-coding and mRNAs in the mouse oocyte and embryo. Furthermore, we established simultaneous visualization of mRNA and in situ translation events validated with polysomal occupancy. We discovered that translationally dormant and abundant mRNAs CyclinB1 and Mos are localized in the cytoplasm of the fully grown GV oocyte forming cloud-like structures with consequent abundant translation at the center of the MII oocyte. Coupling detection of the localization of specific single mRNA molecules with their translation at the subcellular context is a valuable tool to quantitatively study temporal and spatial translation of specific target mRNAs to understand molecular processes in the developing cell.

• Klíčová slova
• imaging, localization, mRNA, subcellular, translation,
• MeSH
• cyklin B1 genetika   MeSH
• cytoplazma genetika   MeSH
• embryo savčí chemie   MeSH
• hybridizace in situ fluorescenční MeSH
• messenger RNA genetika   MeSH
• myši MeSH
• nekódující RNA genetika   MeSH
• oocyty chemie   růst a vývoj   MeSH
• polyribozomy genetika   MeSH
• proteosyntéza MeSH
• protoonkogenní proteiny c-mos genetika   MeSH
• vývojová regulace genové exprese MeSH
• zobrazení jednotlivé molekuly metody   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Ccnb1 protein, mouse MeSH Prohlížeč
• cyklin B1 MeSH
• messenger RNA MeSH
• nekódující RNA MeSH
• protoonkogenní proteiny c-mos MeSH

The analysis of low-abundance protein molecules in human serum is reported based on counting of the individual affinity-captured analyte on a solid sensor surface, yielding a readout format similar to digital assays. In this approach, a sandwich immunoassay with rolling circle amplification (RCA) is used for single molecule detection (SMD) through associating the target analyte with spatially distinct bright spots observed by fluorescence microscopy. The unspecific interaction of the target analyte and other immunoassay constituents with the sensor surface is of particular interest in this work, as it ultimately limits the performance of this assay. It is minimized by the design of the respective biointerface and thiol self-assembled monolayer with oligoethylene (OEG) head groups, and a poly[oligo(ethylene glycol) methacrylate] (pHOEGMA) antifouling polymer brush was used for the immobilization of the capture antibody (cAb) on the sensor surface. The assay relying on fluorescent postlabeling of long single-stranded DNA that are grafted from the detection antibody (dAb) by RCA was established with the help of combined surface plasmon resonance and surface plasmon-enhanced fluorescence monitoring of reaction kinetics. These techniques were employed for in situ measurements of conjugating of cAb to the sensor surface, tagging of short single-stranded DNA to dAb, affinity capture of the target analyte from the analyzed liquid sample, and the fluorescence readout of the RCA product. Through mitigation of adsorption of nontarget molecules on the sensor surface by tailoring of the antifouling biointerface, optimizing conjugation chemistry, and by implementing weak Coulombic repelling between dAb and the sensor surface, the limit of detection (LOD) of the assay was substantially improved. For the chosen interleukin-6 biomarker, SMD assay with LOD at a concentration of 4.3 fM was achieved for model (spiked) samples, and validation of the ability of detection of standard human serum samples is demonstrated.

• Klíčová slova
• antifouling biointerface, biomarker, digital readout of assay, rolling circle amplification, single molecule detection, surface plasmon resonance, surface plasmon-enhanced fluorescence,
• MeSH
• jednovláknová DNA * MeSH
• lidé MeSH
• povrchová plasmonová rezonance * metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• jednovláknová DNA * MeSH