Design and Validation of Specific Oligonucleotide Probes on Planar Magnetic Biosensors

Planar DNA biosensors employ surface-tethered oligonucleotide probes to capture target molecules for diagnostic applications. To improve the sensitivity and specificity of biosensing, hybridization affinities should be enhanced, and cross-hybridization with off-targets must be minimized. To this end, assays can be designed using the thermodynamic properties of hybridization between probes and on-targets or off-targets based on Gibbs free energies and melting temperatures. However, the nature of heterogeneous hybridization between the probes on the surface and the targets in a solution imposes challenges in predicting precise hybridization affinities and the degree of cross-hybridization due to indeterminable thermodynamic penalties induced by the solid surface and its status. Herein, we suggest practical and convenient guidelines for designing oligonucleotide probes based on data obtained from planar magnetic biosensors and thermodynamic properties calculated by using easily accessible solution-phase prediction. The suggested requirements comprised Gibbs free energy ≥ −7.5 kcal mol^–1 and melting temperature ≤10 °C below the hybridization temperature, and we validated for the absence of cross-hybridization. Additionally, the effects of secondary structures such as hairpins and homodimers were investigated for better oligonucleotide probe designs. We believe that these practical guidelines will assist researchers in developing planar magnetic biosensors with high sensitivity and specificity for the detection of new targets.