Tuneable one-step PEG-silane functionalisation strategy for controlled DNA probe immobilisation and enhanced...

Nucleic acid biomarkers are central to modern healthcare, supporting early diagnosis and personalised treatment decisions. At the same time, advances in silicon-based microtechnologies are enabling mass-manufacturable photonic sensors that are compact, multiplexed and suitable for point-of-care applications. A major challenge remains the reproducible control of DNA probe density on silicon, which critically influences hybridisation efficiency and overall biosensor performance. Many conventional silanisation routes are sensitive to reaction conditions and often require multi-step, time-intensive protocols, making it difficult to create functional DNA surfaces with minimal crowding and steric hindrance effects. Here, we introduce a tuneable, one-step PEG-silanisation strategy that offers a practical route to forming functional monolayers with controllable functional-group density on silicon substrates. By adjusting the ratio of functional to non-functional PEG-silanes, we achieve robust and reproducible modulation of DNA probe grafting density, enhancing probe immobilisation and improving target hybridisation performance. Surface composition and molecular arrangement were characterised using standard and angle-resolved X-ray photoelectron spectroscopy, atomic force microscopy and ellipsometry. Efficiency in DNA probe immobilisation and target hybridisation was verified using fluorescence-labelled biomolecules, revealing a strong dependence on molecular crowding at both the DNA probe immobilisation and target levels, yielding up to ten-fold enhancement in hybridisation efficiency. The method is fully compatible with silicon photonic platforms, as demonstrated using asymmetric Mach-Zehnder interferometers. Overall, this approach provides a standardisable, practical framework for engineering DNA-functionalised surfaces on silicon, suitable for label-free biosensing applications.