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Sandwich immuno-RCA assay with single molecule counting readout: the importance of biointerface design

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    SYSNO ASEP0585186
    Document TypeJ - Journal Article
    R&D Document TypeJournal Article
    Subsidiary JČlánek ve WOS
    TitleSandwich immuno-RCA assay with single molecule counting readout: the importance of biointerface design
    Author(s) Schmidt, K. (AT)
    Riedel, Tomáš (UMCH-V) RID, ORCID
    de los Santos Pereira, Andres (UMCH-V) RID, ORCID
    Lynn Jr., Nicholas Scott (FZU-D) ORCID
    Dorado Daza, Diego Fernando (UMCH-V)
    Dostálek, Jakub (FZU-D) ORCID, RID
    Source TitleACS Applied Materials and Interfaces. - : American Chemical Society - ISSN 1944-8244
    Roč. 16, č. 14 (2024), s. 17109-17119
    Number of pages11 s.
    Languageeng - English
    CountryUS - United States
    Keywordsrolling circle amplification ; biomarker ; surface plasmon resonance
    Subject RIVCB - Analytical Chemistry, Separation
    OECD categoryAnalytical chemistry
    Subject RIV - cooperationInstitute of Physics - Biophysics
    R&D ProjectsGF21-16729K GA ČR - Czech Science Foundation (CSF)
    Method of publishingOpen access
    Institutional supportUMCH-V - RVO:61389013 ; FZU-D - RVO:68378271
    UT WOS001191166700001
    EID SCOPUS85189013649
    DOI https://doi.org/10.1021/acsami.3c18304
    AnnotationThe 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.
    WorkplaceInstitute of Macromolecular Chemistry
    ContactEva Čechová, cechova@imc.cas.cz ; Tel.: 296 809 358
    Year of Publishing2025
    Electronic addresshttps://pubs.acs.org/doi/10.1021/acsami.3c18304
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