Michel Wolfgang, Mai Timo, Naiser Thomas, Ott Albrecht
University of Bayreuth, Bayreuth, Germany.
Biophys J. 2007 Feb 1;92(3):999-1004. doi: 10.1529/biophysj.106.092064. Epub 2006 Nov 3.
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.
我们研究了玻璃基板上DNA杂交反应的动力学，其中一条22聚体链（固定化DNA）通过亚苯基二异硫氰酸酯连接分子固定在基板上，染料标记（Cy3）的互补链（游离DNA）在反应室的溶液中。我们使用全内反射荧光进行杂交的表面检测。作为一个新特点，我们在进行全内反射荧光测量的同时，对本体中游离DNA浓度的变化进行实时测量。我们观察到杂交过程中游离DNA浓度显著降低。我们展示了如何扩展标准的朗缪尔动力学以考虑本体浓度的变化，并解释了我们的实验结果。通过将这两种测量方法结合起来，我们可以估计可及的固定化结合DNA的表面密度。我们讨论了其对DNA微阵列检测的意义。
