Application of central composite design for DNA hybridization onto magnetic microparticles

Central composite face-centered (CCF) design and response surface methodologies were used to investigate the effect of probe and target concentration and particle number in immobilization and hybridization on a microparticle-based DNA/DNA hybridization assay. The factors under study were combined according to the CCF design matrix, and the intensity of the hybridization signal was quantified by flow cytometry. A second-order polynomial was fitted to data and validated by analysis of variance. The results showed a complex relationship between variables and response given that all factors as well as some interactions were significant, yet it could explain 95% of the data. Probe and target concentration had the strongest impact on hybridization signal intensity. Increments in initial probe concentration in solution positively affected the hybridization signal until a negative influence of a compact probe layer emerged. This trend was attributed to probe–probe interactions. By manipulating particle number on both immobilization and hybridization, enhancements on the assay sensitivity could be obtained. Under optimized conditions, the limit of detection (LOD) at the 95% confidence level was determined to be 2.3 nM of target solution concentration.