Real-Time Detection of Nucleic Acid Interactions by Total Internal Reflection Fluorescence

• Published in: Analytical chemistry (Washington) Vol. 75; no. 10; pp. 2414 - 2420
• Main Authors: Lehr, H.-P, Reimann, M, Brandenburg, A, Sulz, G, Klapproth, H
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15.05.2003
• Subjects: Acids; Analysis; Analytical chemistry; Chemistry; DNA microarrays; Exact sciences and technology; Hemochromatosis - genetics; Humans; Kinetics; Nucleic Acid Hybridization; Nucleic Acids - analysis; Nucleic Acids - genetics; Nucleic Acids - metabolism; Oligonucleotide Array Sequence Analysis - methods; Oligonucleotides - analysis; Oligonucleotides - chemistry; Optics and Photonics; Point Mutation; Spectrometric and optical methods; Spectrometry, Fluorescence - instrumentation; Spectrometry, Fluorescence - methods; Fluorescence; Total reflection; Detection; Real time; Nucleic acid; Fluorescence spectrometry
• ISSN: 0003-2700; 1520-6882
• 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.