Microbead-Based Heterodimer Assay for Amplification-Free Detection of Single-Stranded DNA

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.