Optical study of DNA surface hybridization reveals DNA surface density as a key parameter for microarray hybridization kinetics.
We investigate the kinetics of DNA hybridization reactions on glass substrates, where one 22 mer strand (bound-DNA) is immobilized via phenylene-diisothiocyanate linker molecule on the substrate, the dye-labeled (Cy3) complementary strand (free-DNA) is in solution in a reaction chamber. We use total internal reflection fluorescence for surface detection of hybridization. As a new feature we perform a simultaneous real-time measurement of the change of free-DNA concentration in bulk parallel to the total internal reflection fluorescence measurement. We observe that the free-DNA concentration decreases considerably during hybridization. We show how the standard Langmuir kinetics needs to be extended to take into account the change in bulk concentration and explain our experimental results. Connecting both measurements we can estimate the surface density of accessible, immobilized bound-DNA. We discuss the implications with respect to DNA microarray detection.
Michel
Wolfgang
W
University of Bayreuth, Bayreuth, Germany. woife@ep1.uni-bayreuth.de
Mai
Timo
T
Naiser
Thomas
T
Ott
Albrecht
A
D003198:Computer Simulation; D004247:DNA / Q000737:chemistry*; D015342:DNA Probes / Q000737:chemistry*; D017403:In Situ Hybridization / Q000379:methods*; D007700:Kinetics; D008956:Models, Chemical; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013499:Surface Properties
D015342:DNA Probes; D004247:DNA