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Sandwich immuno-RCA assay with single molecule counting readout: the importance of biointerface design
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SYSNO ASEP 0585186 Druh ASEP J - Článek v odborném periodiku Zařazení RIV J - Článek v odborném periodiku Poddruh J Článek ve WOS Název Sandwich immuno-RCA assay with single molecule counting readout: the importance of biointerface design Tvůrce(i) 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) ORCID
Dostálek, Jakub (FZU-D) ORCID, RIDZdroj.dok. ACS Applied Materials and Interfaces. - : American Chemical Society - ISSN 1944-8244
Roč. 16, č. 14 (2024), s. 17109-17119Poč.str. 11 s. Jazyk dok. eng - angličtina Země vyd. US - Spojené státy americké Klíč. slova rolling circle amplification ; biomarker ; surface plasmon resonance Vědní obor RIV CB - Analytická chemie, separace Obor OECD Analytical chemistry Vědní obor RIV – spolupráce Fyzikální ústav - Biofyzika CEP GF21-16729K GA ČR - Grantová agentura ČR Způsob publikování Open access Institucionální podpora UMCH-V - RVO:61389013 ; FZU-D - RVO:68378271 UT WOS 001191166700001 EID SCOPUS 85189013649 DOI https://doi.org/10.1021/acsami.3c18304 Anotace 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. Pracoviště Ústav makromolekulární chemie Kontakt Eva Čechová, cechova@imc.cas.cz ; Tel.: 296 809 358 Rok sběru 2025 Elektronická adresa https://pubs.acs.org/doi/10.1021/acsami.3c18304
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