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

The growth of surface-attached single-stranded deoxyribonucleic acid (ssDNA) chains is monitored in situ using an evanescent wave optical biosensor that combines surface plasmon resonance (SPR) and optical waveguide spectroscopy (OWS). The "grafting-from" growth of ssDNA chains is facilitated by rolling circle amplification (RCA), and the gradual prolongation of ssDNA chains anchored to a gold sensor surface is optically tracked in time. At a sufficient density of the polymer chains, the ssDNA takes on a brush architecture with a thickness exceeding 10 μm, supporting a spectrum of guided optical waves traveling along the metallic sensor surface. The simultaneous probing of this interface with the confined optical field of surface plasmons and additional more delocalized dielectric optical waveguide modes enables accurate in situ measurement of the ssDNA brush thickness, polymer volume content, and density gradients. We report for the first time on the utilization of the SPR/OWS technique for the measurement of the RCA speed on a solid surface that can be compared to that in bulk solutions. In addition, the control of ssDNA brush properties by changing the grafting density and ionic strength and post-modification via affinity reaction with complementary short ssDNA staples is discussed. These observations may provide important leads for tailoring RCA toward sensitive and rapid assays in affinity-based biosensors.

• Klíčová slova
• DNA, biointerfaces, optical waveguide spectroscopy, polyelectrolyte brushes, rolling circle amplification, surface plasmon resonance, surface plasmon-enhanced fluorescence,
• MeSH
• biosenzitivní techniky MeSH
• časové faktory MeSH
• jednovláknová DNA genetika   MeSH
• optické jevy * MeSH
• povrchová plasmonová rezonance metody   MeSH
• spektrální analýza * MeSH
• techniky amplifikace nukleových kyselin * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• jednovláknová DNA MeSH

We report a highly sensitive and selective multiplex assay by empowering an electrochemical DNA sensor with isothermal rolling circle amplification. The assay could simultaneously detect and discriminate three common entero-pathogens in a single reaction, with femtomolar sensitivity. It is useful for field- or resource-limited settings.

• MeSH
• biosenzitivní techniky * MeSH
• DNA genetika   MeSH
• elektrochemické techniky * MeSH
• Salmonella typhi izolace a purifikace   MeSH
• Shigella flexneri izolace a purifikace   MeSH
• techniky amplifikace nukleových kyselin * MeSH
• Vibrio cholerae izolace a purifikace   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA MeSH

Integrating isothermal nucleic acid amplification strategies into immunoassays can significantly decrease analytical limits of detection (LODs). On the other hand, an amplification step adds time, complication, reagents, and costs to the assay format. To evaluate the pros and cons in the context of heterogeneous multistep immunoassays, we quantified prostate-specific antigen (PSA) with and without rolling circle amplification (RCA). In addition, we compared time-gated (TG) with continuous-wave (CW) photoluminescence (PL) detection using a terbium complex and a fluorescein dye, respectively. For both direct (non-amplified) and amplified assays, TG PL detection provided circa four- to eightfold lower LODs, illustrating the importance of autofluorescence background suppression even for multi-wash assay formats. Amplified assays required an approximately 2.4 h longer assay time but led to almost 100-fold lower LODs down to 1.3 pg/mL of PSA. Implementation of TG-FRET (using a Tb-Cy5.5 donor-acceptor pair) into the RCA immunoassay resulted in a slightly higher LOD (3.0 pg/mL), but the ratiometric detection format provided important benefits, such as higher reproducibility, lower standard deviations, and multiplexing capability. Overall, our direct comparison demonstrated the importance of biological background suppression even in heterogeneous assays and the potential of using isothermal RCA for strongly decreasing analytical LODs, making such assays viable alternatives to conventional enzyme-linked immunosorbent assays (ELISAs).

• Klíčová slova
• Diagnostics, ELISA, Fluorescence, PSA, TR-FRET, Terbium,
• MeSH
• imunoanalýza metody   MeSH
• lidé MeSH
• limita detekce * MeSH
• prostatický specifický antigen * krev   analýza   MeSH
• rezonanční přenos fluorescenční energie metody   MeSH
• techniky amplifikace nukleových kyselin * metody   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• prostatický specifický antigen * MeSH

The KRAS mutation is a crucial biomarker for determining targeted cancer therapies, making its accurate and cost-effective detection vital for precision oncology. However, current methodologies, such as next-generation sequencing (NGS) or PCR-based methods, are often expensive and technically complex, limiting their accessibility. Here, we present a novel bioassay for KRAS G12V mutation analysis that combines rolling circle amplification (RCA) with locked nucleic acid (LNA)-modified magnetic beads, electrochemical detection using carbon electrode chips, and AI-assisted analysis via a logistic regression classifier. Our platform demonstrated exceptional selectivity in distinguishing the KRAS G12V mutation from wild-type (wt) sequences, enabling analysis <1 % of mutated DNA in a wt sample. We validated the bioassay on 7 cancer cell lines and 11 patient-derived samples, achieving results that perfectly correlated with NGS data. This innovative approach simplifies the workflow, reduces costs, and offers high sensitivity and specificity, making it a promising tool for clinical diagnostics and personalized cancer treatment strategies.

• Klíčová slova
• DNA point mutation, Electrochemistry, KRAS gene, Locked nucleic acid, Rolling circle amplification,
• MeSH
• biotest * metody   MeSH
• bodová mutace * MeSH
• elektrochemické techniky * metody   MeSH
• lidé MeSH
• mutační analýza DNA metody   MeSH
• nádorové buněčné linie MeSH
• oligonukleotidy * chemie   genetika   MeSH
• protoonkogenní proteiny p21(ras) * genetika   MeSH
• techniky amplifikace nukleových kyselin * metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• KRAS protein, human MeSH Prohlížeč
• locked nucleic acid MeSH Prohlížeč
• oligonukleotidy * MeSH
• protoonkogenní proteiny p21(ras) * MeSH

Rodents are distributed throughout the world and interact with humans in many ways. They provide vital ecosystem services, some species are useful models in biomedical research and some are held as pet animals. However, many rodent species can have adverse effects such as damage to crops and stored produce, and they are of health concern because of the transmission of pathogens to humans and livestock. The first rodent viruses were discovered by isolation approaches and resulted in break-through knowledge in immunology, molecular and cell biology, and cancer research. In addition to rodent-specific viruses, rodent-borne viruses are causing a large number of zoonotic diseases. Most prominent examples are reemerging outbreaks of human hemorrhagic fever disease cases caused by arena- and hantaviruses. In addition, rodents are reservoirs for vector-borne pathogens, such as tick-borne encephalitis virus and Borrelia spp., and may carry human pathogenic agents, but likely are not involved in their transmission to human. In our days, next-generation sequencing or high-throughput sequencing (HTS) is revolutionizing the speed of the discovery of novel viruses, but other molecular approaches, such as generic RT-PCR/PCR and rolling circle amplification techniques, contribute significantly to the rapidly ongoing process. However, the current knowledge still represents only the tip of the iceberg, when comparing the known human viruses to those known for rodents, the mammalian taxon with the largest species number. The diagnostic potential of HTS-based metagenomic approaches is illustrated by their use in the discovery and complete genome determination of novel borna- and adenoviruses as causative disease agents in squirrels. In conclusion, HTS, in combination with conventional RT-PCR/PCR-based approaches, resulted in a drastically increased knowledge of the diversity of rodent viruses. Future improvements of the used workflows, including bioinformatics analysis, will further enhance our knowledge and preparedness in case of the emergence of novel viruses. Classical virological and additional molecular approaches are needed for genome annotation and functional characterization of novel viruses, discovered by these technologies, and evaluation of their zoonotic potential.

• Klíčová slova
• Diagnostics, High-throughput sequencing, Microarray, PCR, Pathogen, RT-PCR, Rodent, Rolling circle amplification, Workflow, Zoonosis,
• MeSH
• diagnostické techniky molekulární metody   MeSH
• hlodavci virologie   MeSH
• metagenomika metody   MeSH
• techniky amplifikace nukleových kyselin metody   MeSH
• virové nemoci epidemiologie   veterinární   virologie   MeSH
• viry klasifikace   genetika   izolace a purifikace   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• zoonózy epidemiologie   virologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Molecular diagnostics may provide tailored and cost efficient treatment for infectious disease and cancer. Rolling circle amplification (RCA) of padlock probes guarantees high specificity to identify nucleic acid targets down to single nucleotide resolution in a multiplex fashion. This makes the assay suitable for molecular analysis of various diseases, and interesting to integrate into automated devices for point-of-care analysis. A critical prerequisite for many molecular assays is (i) target-specific isolation from complex clinical samples and (ii) removal of reagents, inhibitors and contaminants between reaction steps. Efficient solid supports are therefore essential to enable multi-step, multi-analyte protocols. Superparamagnetic micro- and nanoparticles, with large surface area and rapid liquid-phase kinetics, are attractive for multi-step protocols. Recently, streptavidin-modified magnetic monodispersed poly(2-hydroxyethyl methacrylate) (STV-mag.PHEMA) microspheres were developed by multiple swelling polymerization. They are easily separated by a magnet and exhibit low non-specific protein sorption. In this study, the performance and the binding efficiency of STV-mag.PHEMA was addressed by circle-to-circle amplification (C2CA). A lower number of RCA products were detected as compared to the gold standard Dynabeads. Nevertheless, this study was the first to successfully adapt STV-mag.PHEMA microspheres as solid support in a DNA-based protocol, which is an important finding. The STV-mag.PHEMA microspheres were larger with about 16 times less surface area as compared to the Dynabeads, which might partly explain the lower rolling circle product (RCP) count obtained. Further research is currently ongoing comparing particles of similar sizes and optimizing reaction conditions to establish their full utility in the field. Ultimately, low cost and versatile particles are a great resource to facilitate future clinical molecular diagnostics.

• Klíčová slova
• DNA, Magnetic microspheres, Poly(2-hydroxyethyl methacrylate), Rolling circle amplification,
• MeSH
• DNA chemie   metabolismus   MeSH
• imobilizované proteiny chemie   metabolismus   MeSH
• magnetismus MeSH
• mikrosféry * MeSH
• mikroskopie elektronová rastrovací MeSH
• polyhydroxyethylmethakrylát chemie   MeSH
• spektroskopie infračervená s Fourierovou transformací MeSH
• streptavidin chemie   metabolismus   MeSH
• techniky amplifikace nukleových kyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• imobilizované proteiny MeSH
• polyhydroxyethylmethakrylát MeSH
• streptavidin 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

To understand cellular processes and events responsible for their perturbations, proteomic analyses are needed in bio-medical research and clinical diagnostics. Several techniques based on specifically binding reagents (antibodies) or recombinant proteins (GFP fusion protein, methods of fluorescence/ bio-luminescence resonance energy transfer) are generally used to study protein location and activity resulting from secondary modifications and interactions. The in situ proximity ligation assay represents a novel technique of in situ protein imaging using DNA as a reporter molecule and DNA amplification processes. This method enables direct visualization of single molecules, their levels, modifications and pattern of interactions in individual fixed cells and tissues. Proximity probes consist of specific antibody with attached oligonucleotides that are used as reporter molecules for identification of such events. Proximity probes guide the formation of a circular DNA strand when bound in close proximity. The DNA circle after that serves as a template for rolling circle amplification allowing the interaction to be visualized. Compared to available proteomic techniques benefiting from genetic engineering, in situ PLA enables study of endogenous proteins in their natural environment and thus can be used for clinical specimens. The areas of applicability where proximity ligation procedure can be used include any research field where protein interaction measurements are important, such as signal-ing pathway studies, monitoring of pharmacological treatment targets and oncological diagnostics.

• MeSH
• kruhová DNA MeSH
• lidé MeSH
• mapování interakce mezi proteiny metody   MeSH
• proteiny analýza   MeSH
• proteomika MeSH
• signální transdukce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kruhová DNA MeSH
• proteiny MeSH

Inverted repeats (IRs) can facilitate structural variation as crucibles of genomic rearrangement. Complex duplication-inverted triplication-duplication (DUP-TRP/INV-DUP) rearrangements that contain breakpoint junctions within IRs have been recently associated with both MECP2 duplication syndrome (MIM#300260) and Pelizaeus-Merzbacher disease (PMD, MIM#312080). We investigated 17 unrelated PMD subjects with copy number gains at the PLP1 locus including triplication and quadruplication of specific genomic intervals-16/17 were found to have a DUP-TRP/INV-DUP rearrangement product. An IR distal to PLP1 facilitates DUP-TRP/INV-DUP formation as well as an inversion structural variation found frequently amongst normal individuals. We show that a homology-or homeology-driven replicative mechanism of DNA repair can apparently mediate template switches within stretches of microhomology. Moreover, we provide evidence that quadruplication and potentially higher order amplification of a genomic interval can occur in a manner consistent with rolling circle amplification as predicted by the microhomology-mediated break induced replication (MMBIR) model.

• MeSH
• body zlomu chromozomu MeSH
• chromozomální inverze MeSH
• duplikace genu * MeSH
• genová dávka MeSH
• lidé MeSH
• myelinový proteolipidový protein genetika   MeSH
• Pelizaeusova-Merzbacherova nemoc genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• myelinový proteolipidový protein MeSH
• PLP1 protein, human MeSH Prohlížeč