Microbead-Based Heterodimer Assay for Amplification-Free Detection of Single-Stranded DNA
- 1LIMMS/CNRS-Institute of Industrial Science
Abstract
Direct detection of rare nucleic-acid targets without molecular amplification requires specific recognition events to be made observable while remaining distinguishable from nonspecific interactions. Here, we introduce a microbead-based heterodimer assay (MBHA) that converts two-site recognition of single-stranded DNA into spatially resolved, two-color bead-pairing events for direct nucleic acid detection. Two spectrally distinct microbead populations are functionalized with partially double-stranded DNA probes exposing target-binding domains complementary to separate regions of the target. Target-mediated bridging produces heterotypic bead pairs that are classified by fluorescence identity and an interparticle-distance criterion, while residual background is evaluated relative to blank measurements. Under the tested conditions, 166 fM was the lowest target concentration exceeding the blank-derived detection threshold. A sequence-scrambled synthetic non-target DNA remained below threshold across the tested concentration range, and detection at 166 fM was retained in the presence of 1 uM non-target DNA, corresponding to an approximately six-million-fold molar excess. Probe redesign further extended the assay to a second 60-nucleotide synthetic DNA target derived from the SARS-CoV-2 ORF1ab sequence. This proof-of-concept study establishes heterodimer counting as an amplification-free measurement strategy in which rare molecular-recognition events are made directly observable and individually classifiable without increasing target copy number.
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BibTeX
@misc{roy2026microbeadbased,
title = {{Microbead-Based Heterodimer Assay for Amplification-Free Detection of Single-Stranded DNA}},
author = {S. R. Roy and N. Lobato-Dauzier and J. Mateos and Y. Tauran and A. Brioude and A. Baccouche and T. Fujii and A. J. Genot and S. H. Kim},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.28.762408},
url = {https://www.biorxiv.org/content/10.64898/2026.10.28.762408v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Microbead-Based Heterodimer Assay for Amplification-Free Detection of Single-Stranded DNA
AU - S. R. Roy
AU - N. Lobato-Dauzier
AU - J. Mateos
AU - Y. Tauran
AU - A. Brioude
AU - A. Baccouche
AU - T. Fujii
AU - A. J. Genot
AU - S. H. Kim
PY - 2026
DA - 2026/10/28
PB - bioRxiv
DO - 10.64898/2026.10.28.762408
UR - https://www.biorxiv.org/content/10.64898/2026.10.28.762408v1
AB - Direct detection of rare nucleic-acid targets without molecular amplification requires specific recognition events to be made observable while remaining distinguishable from nonspecific interactions. Here, we introduce a microbead-based heterodimer assay (MBHA) that converts two-site recognition of single-stranded DNA into spatially resolved, two-color bead-pairing events for direct nucleic acid detection. Two spectrally distinct microbead populations are functionalized with partially double-stranded DNA probes exposing target-binding domains complementary to separate regions of the target. Target-mediated bridging produces heterotypic bead pairs that are classified by fluorescence identity and an interparticle-distance criterion, while residual background is evaluated relative to blank measurements. Under the tested conditions, 166 fM was the lowest target concentration exceeding the blank-derived detection threshold. A sequence-scrambled synthetic non-target DNA remained below threshold across the tested concentration range, and detection at 166 fM was retained in the presence of 1 uM non-target DNA, corresponding to an approximately six-million-fold molar excess. Probe redesign further extended the assay to a second 60-nucleotide synthetic DNA target derived from the SARS-CoV-2 ORF1ab sequence. This proof-of-concept study establishes heterodimer counting as an amplification-free measurement strategy in which rare molecular-recognition events are made directly observable and individually classifiable without increasing target copy number.
ER -