Sandwich immuno-RCA assay with single molecule counting readout: the importance of biointerface design
The result's identifiers
Result code in IS VaVaI
<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F68378271%3A_____%2F24%3A00585186" target="_blank" >RIV/68378271:_____/24:00585186 - isvavai.cz</a>
Alternative codes found
RIV/61389013:_____/24:00585186
Result on the web
<a href="https://pubs.acs.org/doi/10.1021/acsami.3c18304" target="_blank" >https://pubs.acs.org/doi/10.1021/acsami.3c18304</a>
DOI - Digital Object Identifier
<a href="http://dx.doi.org/10.1021/acsami.3c18304" target="_blank" >10.1021/acsami.3c18304</a>
Alternative languages
Result language
angličtina
Original language name
Sandwich immuno-RCA assay with single molecule counting readout: the importance of biointerface design
Original language description
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.
Czech name
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Czech description
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Classification
Type
J<sub>imp</sub> - Article in a specialist periodical, which is included in the Web of Science database
CEP classification
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OECD FORD branch
10610 - Biophysics
Result continuities
Project
<a href="/en/project/GF21-16729K" target="_blank" >GF21-16729K: Digital plasmonic biosensor (DIPLAB)</a><br>
Continuities
I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace
Others
Publication year
2024
Confidentiality
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů
Data specific for result type
Name of the periodical
ACS Applied Materials and Interfaces
ISSN
1944-8244
e-ISSN
1944-8252
Volume of the periodical
16
Issue of the periodical within the volume
14
Country of publishing house
US - UNITED STATES
Number of pages
11
Pages from-to
17109-17119
UT code for WoS article
001191166700001
EID of the result in the Scopus database
2-s2.0-85189013649