Design and\nValidation of Specific Oligonucleotide\nProbes on Planar Magnetic Biosensors

Planar DNA biosensors employ surface-tethered oligonucleotide\nprobes\nto capture target molecules for diagnostic applications. To improve\nthe sensitivity and specificity of biosensing, hybridization affinities\nshould be enhanced, and cross-hybridization with off-targets must\nbe minimized. To this end, assays can be designed using the thermodynamic\nproperties of hybridization between probes and on-targets or off-targets\nbased on Gibbs free energies and melting temperatures. However, the\nnature of heterogeneous hybridization between the probes on the surface\nand the targets in a solution imposes challenges in predicting precise\nhybridization affinities and the degree of cross-hybridization due\nto indeterminable thermodynamic penalties induced by the solid surface\nand its status. Herein, we suggest practical and convenient guidelines\nfor designing oligonucleotide probes based on data obtained from planar\nmagnetic biosensors and thermodynamic properties calculated by using\neasily accessible solution-phase prediction. The suggested requirements\ncomprised Gibbs free energy ≥ −7.5 kcal mol<sup>–1</sup> and melting temperature ≤10 °C below the hybridization\ntemperature, and we validated for the absence of cross-hybridization.\nAdditionally, the effects of secondary structures such as hairpins\nand homodimers were investigated for better oligonucleotide probe\ndesigns. We believe that these practical guidelines will assist researchers\nin developing planar magnetic biosensors with high sensitivity and\nspecificity for the detection of new targets.