Lehr H P, Reimann M, Brandenburg A, Sulz G, Klapproth H
Fraunhofer-Institute of Physical Measurement Techniques IPM, Heidenhofstrasse 8, D-79110 Freiburg, Germany.
Anal Chem. 2003 May 15;75(10):2414-20. doi: 10.1021/ac0206519.
This paper describes the development of an optical readout system for the real-time analysis of fluorescent-labeled DNA microarrays is described. The system is targeted toward research applications in genomics, agriculture, and life sciences, where the end-point detection of state-of-the-art readout systems does not provide sufficient information on the hybridization process. The hybridization progress of molecules from the liquid phase in a flow cell to immobilized oligonucleotides on a transducer surface can be observed. The excitation of fluorochromes is realized by a semiconductor laser, and the fluorescence emission is collected by a cooled CCD camera. Quantitative data can be extracted from the images for analysis of the microarray. For the signal transduction, the principle of total internal reflection is used. With a multiple internal reflection arrangement, the sensor chip was adapted to the standard microscope slide format and a homogeneous evanescent illumination of the active area of the sensor surface was achieved. An application measurement was carried out with this readout system. The hybridization of Cy5-labeled 30-mer single-stranded oligonucleotides to fully complementary immobilized strands was observed in real time. A kinetic analysis was demonstrated with the recorded data. Melting curves of a 140-mer PCR product from a hemochromatosis patient sample hybridized to immobilized wild-type mutant 15- and 17-mer oligonucleotides were recorded and single-point mutations could be detected.
本文描述了一种用于实时分析荧光标记DNA微阵列的光学读出系统的开发。该系统针对基因组学、农业和生命科学中的研究应用，在这些领域中，最先进读出系统的终点检测无法提供关于杂交过程的足够信息。可以观察到分子从流动池中的液相到换能器表面固定寡核苷酸的杂交过程。荧光团的激发由半导体激光器实现，荧光发射由冷却的电荷耦合器件（CCD）相机收集。可以从图像中提取定量数据以分析微阵列。对于信号转导，使用全内反射原理。通过多次内反射装置，将传感器芯片适配到标准显微镜载玻片格式，并实现了传感器表面活性区域的均匀倏逝照明。使用该读出系统进行了应用测量。实时观察到Cy5标记的30聚体单链寡核苷酸与完全互补的固定链的杂交。用记录的数据进行了动力学分析。记录了来自血色素沉着症患者样本的140聚体聚合酶链反应（PCR）产物与固定的野生型突变15聚体和17聚体寡核苷酸杂交的熔解曲线，并检测到单点突变。
