Anal. Chem. 2003, 75, 2414-2420
Real-Time Detection of Nucleic Acid Interactions by Total Internal Reflection Fluorescence H.-P. Lehr,*,† M. Reimann,‡ A. Brandenburg,† G. Sulz,† and H. Klapproth§
Fraunhofer-Institute of Physical Measurement Techniques IPM, Heidenhofstrasse 8, D-79110 Freiburg, Germany, Gene Scan Europe AG, Engesserstrasse 4B, D-79108 Freiburg, Germany, and Institute for Microsystem Technology, Georges-Ko¨hler Allee 101-103, D-79110 Freiburg, Germany
This paper describes the development of an optical readout system for the real-time analysis of fluorescentlabeled 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 singlestranded 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 wildtype mutant 15- and 17-mer oligonucleotides were recorded and single-point mutations could be detected. Microarrays. The Human Genom Project was a driving force for the development of DNA microarray technology with the large amount of sequence information from the genetic code, which had to be decoded.1 The study of hybridization reactions plays an important role in genomic research and is exploited for analytical procedures, for example, in the PCR technique. A relatively new approach is microarray technology, which offers a massively parallel analysis platform for hybridization reactions.1,2 A part of the genetic analysis is already performed * Corresponding author. E-mail: HP.Lehr@ t-online.de. † Fraunhofer-Institute of Physical Measurement Techniques IPM. ‡ Gene Scan Europe AG. § Institute for Microsystem Technology. (1) Niemeyer, C. M.; Blohm, D. Angew. Chem. 1999, 111, 19, 3039-3043. (2) Schena, M. Microarray Biochip Technology; Eaton Publishing: Natick, MA, 2000.
2414 Analytical Chemistry, Vol. 75, No. 10, May 15, 2003
on microarrays. Hybridization assays on microarrays will play a key role in the future. A microarray is an ordered two-dimensional spatial arrangement of small structures on a solid support. The structures are oligonucleotide probes, which are either synthesized on the solid support by a photolithographic process or dispensed as small volumes (pL-nL) of oligonucleotide sequences. If the complementary sample sequence is labeled with a fluorophore prior to application onto the microarray, binding events can be detected by fluorescence imaging techniques. Microarray Readout System for R&D. Activities have been accompanied by the development of readout systems for microarrays. Due to the dominance of fluorescent microarrays, most of the existing systems are optical readout systems called microarray scanner or microarray reader from which a number of systems has been commercialized.3 The readout systems can be divided into two main classes, scanning systems4-6 and imaging systems.7,8 Most of the readout systems, which have been developed in research laboratories and which are commercially available, were designed to read out microarrays where the hybridization reaction on the chip has already been made with an external device. For research applications, more sophisticated readout instrumentation is required to gain more information about the progression of the hybridization, such as the association rate, the dissociation rate, and the affinity constant. This is achieved by integration of the hybridization process, fluid handling, temperature control, and optical readout into a single instrument. Permanent contact of the DNA sample with the sensor during the readout process allows one to monitor the continuous binding of molecules from the sample medium onto the sensor surface. Furthermore, repeated cycles of hybridization and denaturation are possible with a single sensor chip. This speeds up the development of DNA chip assays, where a large number of (3) http://www.gene-chips.com. (4) Alexay, C.; Kain, R. C.; Hanzel, D. K. Proc. SPIE-Int. Soc. Opt. Eng. 1996, 2705, 63-72. (5) Montagu, J.; Honkanen, P. Proc. SPIE-Int. Soc. Opt. Eng. 1999, 3779, 284292. (6) Trulson, M. O.; Stern, D. Proc. SPIE-Int. Soc. Opt. Eng. 1997, 2980, 145148. (7) Bogdanov, V.; Rogers, Y. H.; Lan, G.; Boyce-Jacino, M. Proc. SPIE-Int. Soc. Opt. Eng. 1998, 3259, 156-163. (8) Karcheter, S. Aufbau und Charakterisierung eines fluorometrischen Detektionssystems zur ortsaufgelo¨sten Analyse von Biochips. Diplomarbeit, Fraunhofer Institut Physikalische Messtechnik, 1999. 10.1021/ac0206519 CCC: $25.00
© 2003 American Chemical Society Published on Web 04/12/2003
hybridization experiments with different target and probe sequences and buffer solutions under various temperature conditions are required. Optical evanescent field methods have been used extensively in the past and present as biosensors to study real-time binding events on surfaces.9-11 They have in common that a electromagnetic wave, termed an evanescent field, propagates only by a wavelength distance from the sensor surface in the lower refractive index sample medium. Thus, the binding of ligands from the bulk medium onto the surface generates a sensor signal while the ligands in bulk medium do not interfere. No washing steps are required. Free dye-labeled oligonucleotides in close proximity to the sensor surface inside the domain of the evanescent field (∼100 nm) are also excited by the laser illumination. They contribute to the fluorescence signal as a weak background signal with uniform intensity over the sensing area. The majority of free dye-labeled oligonucleotides are not excited by the laser illumination because these molecules are located outside the domain of the evanescent field between a distance of 100 and 1000 nm (depth of the flow cell) from the sensor surface. The evanescent field method, which can detect fluorescently labeled molecules, is based on the principle of total internal reflection fluorescence (TIRF).12-14 The application of the TIRF principle for biosensing has been described extensively in the literature. Planar waveguides with a single sensing pad for the monitoring of antibody-antigen reactions have been used.15-17 The fluorescent emission from the sensing pad was detected with a photomultiplier tube. Fiber-optic TIRF biosensors with a single sensing pad have been used.18,19,20 Later multichannel systems having a few sensing pads (
