In Silico Pre-Screening as a Design-Assessment Tool for Gold Nanoparticle-Amplified DNA Displacement Assays: Identifying Sequence-Level Design Limitations Across Electrode and Magnetic-Bead Configurations

The growing demand for decentralised and point-of-care (POC) nucleic acid testing has increased interest in label-free, sequence-specific DNA biosensors and computational approaches that can guide their design before extensive experimental optimisation. Here, an in silico workflow was first employed to evaluate a gold nanoparticle (AuNP)-amplified, target-induced DNA displacement assay. Nearest-neighbour duplex thermodynamics, DNA secondary-structure analysis, and High Ambiguity Driven biomolecular DOCKing (HADDOCK) were used to assess sequence complementarity and displacement behaviour. The analyses confirmed Watson–Crick complementarity among the electrode capture, reporter, and target sequences and identified an unintended mismatch in the magnetic bead-capture probe. HADDOCK analyses using default RNA and explicit DNA settings consistently favoured target over non-target displacement, although control poses were more setting-sensitive. Experimental testing subsequently provided supporting evidence for the computational assessment. Electrochemical measurements showed the anticipated signal hierarchy of target > non-target > buffer, although differences were not statistically significant at the replicate numbers tested. The magnetic bead platform produced weak, non-differential responses, consistent with the sequence-level design limitation identified computationally. Together, these findings demonstrate the utility of computational pre-screening for interrogating displacement mechanisms, identifying correctable design errors, and guiding rational experimental optimisation of nucleic acid biosensors. No statistically significant target discrimination was achieved at the replicate numbers tested, and the proposed corrected bead-capture sequence has not been experimentally validated.