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.
Institute for Biotechnology and Bioengineering (IBB), Centre for Biological and Chemical Engineering, Instituto Superior Técnico, 1049-001 Lisboa, Portugal.
S A M
SA
Martins
D M F
DM
Prazeres
L P
LP
Fonseca
G A
GA
Monteiro
D004247:DNA / Q000032:analysis*; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D008280:Magnetics; D008956:Models, Chemical; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry
D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D004247:DNA