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Technical Notes
Chip-Based Optical Detection of DNA Hybridization by Means of Nanobead Labeling Jo 1 rg Reichert, Andrea Csa´ki, J. Michael Ko 1 hler, and Wolfgang Fritzsche
Institute of Physical High Technology, P.O. Box 100 239, D-07702 Jena, Germany
A new scheme for the detection of molecular interactions based on optical readout of nanoparticle labels has been developed. Capture DNA probes were arrayed on a glass chip and incubated with nanoparticle-labeled target DNA probes, containing a complementary sequence. Binding events were monitored by optical means, using reflected and transmitted light for the detection of surface-bound nanoparticles. Control experiments exclude significant influence of nonspecific binding on the observed contrast. Scanning force microscopy revealed the distribution of nanoparticles on the chip surface. Highly paralleled detection of DNA hybridization using microarrays shows tremendous promise for medical, pharmaceutical, forensic, and other applications.1,2 Microarrays are 2D patterns consisting of surface-immobilized molecules of defined molecular species. For detection of specific molecular binding, the arrays are incubated with a solution containing binding partners of the immobilized molecules. Specific binding is visualized by use of tagged molecules. The pattern of tags on the array is compared with the known 2D arrangement of the surface-immobilized molecules, which results in the identification of molecules bound by specific interactions. Besides radioactive labeling with its inherent safety problems, fluorescence labels are broadly used in standard applications of microarrays. Although hampered by the need for sophisticated fluorescence microscopes/scanners as well as strongly environment-depending quantum yields, no other scheme for readout could supersede fluorescence detection for standard use. However, quantitative measurements remain challenging because of low fluorescence intensities, low dye stability, and changing environmental conditions. An interesting new detection approach is the use of colloidal gold as a color label for detection of DNA hybridization in solution.3 Colloidal gold particles were modified with attached DNA-oligonucleotides with sequences complementary to either * Corresponding author: (e-mail)
[email protected]; (phone) xx493641-206304; (fax) xx49-3641-206399. (1) Southern, E.; Mir, K.; Shchepinov, M. Nat. Genet. Suppl. 1999, 21, 5-9. (2) Eggers, M. D.; Hogan, M. E.; Reich, R. K.; Tamture, J. B.; Beattie, K. L.; Hollis, M. A.; Ehrlich, D. J.; Kosicki, B. B.; Shumaker, J. M.; Varma, R. S.; Burke, B. E.; Murphy, A.; Rathman, D. D. Adv. DNA Sequencing Technol. 1993, 1891, 113-126. (3) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078-1080. 10.1021/ac000567y CCC: $19.00 Published on Web 11/14/2000
© 2000 American Chemical Society
Figure 1. Oligonucleotides used in the study. Surface-immobilized capture probe X1; Target probe X2 complementary to X1 labeled with gold nanoparticle.
ends of the target DNA. In the presence of the target DNA, the colloids become bridged, resulting in a color change of the solution. Such a signal can be easily detected, without sophisticated and expensive fluorescence equipment. By using a silver enhancement technique, combinatorial DNA arrays were monitored by a conventional flatbed scanner.4 On the other hand, this technique cannot deliver the high parallelization needed for highthroughput DNA analysis. In this paper, we demonstrate a new scheme for the detection of specific molecular binding, which is based on optical visualization of nanobead-labeled molecules on a microstructured chip surface. This combination of an easily detectable signal with chip technology has the potential for high-throughput applications. MATERIALS AND METHODS DNA Arrays on Patterned Glass Substrate. A glass substrate was passivated by a perfluoroalkyl.5 Standard photolithographic techniques were applied to open 60-µm quadratic windows on the substrate surface. The windows were modified with a layer of glycidoxypropyltrimethoxysilane,6 which was activated by incubation with ethylene glycol prior to solid-phase synthesis (onchip) of the DNA oligonucleotide X1 (Figure 1) according to.7 Before hybridization, the chips were washed for 30 min with 0.1 M NaCl/10 mM sodium phosphate buffer at pH 7.0. Preparation of Gold-Labeled Oligonucleotides. 3′-Alkylthiolated oligonucleotides (X2, BioTez, Berlin, Germany) were preincubated with 30-nm gold nanoparticle solution (Plano, (4) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 17571760. (5) Parikh, A. N.; Allara, D. L.; Azouz, I. B.; Rondelez, F. J. Phys. Chem. 1994, 98, 7577-7590. (6) Henke, L.; Pinno, P. A. E.; McClure, A. C.; Krull, U. J. Anal. Chem. Acta 1997, 334, 201-213. (7) Opitz, A.; Kirschstein, O.; Reichert, J.; Birch-Hirschfeld, E.; Kittler, L.; Ko ¨hler, J. M., submitted.
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Figure 2. Optical micrograph of DNA chips after hybridization with the 30-nm gold-labeled target probe X2: (A, B) reflection; (C) transmission; (D) reflection. To illustrate the signal-to-noise ratio, the averaged intensity of the central region of (B) and (C) (as marked by the arrows) was plotted in the respective insets. (A) Overview, revealing the pattern of 60-µm squares. (B) Higher magnification, showing individual spots. (C) The same spot as in (B) observed in transmitted light. (D) The potential of the method is demonstrated by imaging a field of microstructured squares (4 × 4 µm) with high speed (exposure time in the millisecond range).
Figure 3. SFM of the gold-labeled chip shown in Figure 2 revealing the surface topography. The height is brightness-coded; higher regions appear brighter. The scale bars in the cross sections (insets in C and D) are 30 (height) and 100 nm (lateral), respectively. (A) Scheme of the chip surface; the squares represent the areas where the capture probe DNA X1 was immobilized on the surface. (B) Corner of a square after incubation with the 30-nm gold-labeled complementary target DNA X2. (C) Zoom into the gold-covered square. (D) Zoom into the background region.
Wetzlar, Germany) for 16 h at room temperature using concentrations of 0.33 (gold) and 200 nM (DNA). After the solution was adjusted to 0.1 M NaCl/10 mM sodium phosphate buffer at pH 7.0, it was incubated for 40 h at room temperature. The DNAnanoparticle complexes were washed with buffer and redispersed in 0.3 M NaCl/10 mM sodium phosphate buffer at pH 7.0,resulting in a final concentration of 2.0 OD regarding the gold colloids. Hybridization. The glass substrates (3 × 6 mm) were incubated in the prepared solution of gold-labeled oligonucleotides using a modified protocol from the literature.8 The solution were heated to 65 °C for 10 min and then allowed to cool to room temperature (∼12 h). Concentrations of 0.5, 1, and 2 OD were used for the concentration-response curve. (8) Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mikron, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 1959-1964.
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Microscopy. For microscopy, the samples were washed in di-ionized water and air-dried. An optical microscope Axiotech (Carl Zeiss Jena, Germany) equipped with a CCD camera Sensicam (PCO Computer Optics, Kehlheim, Germany) was used in transmission and reflection mode. For quantification of the optical signals, microstructured samples were imaged using the CCD. A linear dependency of the measured intensity from the exposure time was found for the CCD in the limits of an error of 10%. The intensity of signal regions as well as of the background was determined by averaging over 10 regions of 5 × 5 µm each on different parts of the substrate surface using the program NIH Image 1.61. The reference sample for a 100% reflectivity was a gold-sputtered surface. To exclude distributions by unspecific interactions between gold-labeled DNA and
Figure 4. Concentration dependency of the particle density (SFM, A-C) and the optical transmissive signal (D-F). Glass chips with DNA immobilized in microstructured pattern were incubated with three different concentrations of gold particles modified with the complementary DNA: 0.5 (A, E), 1.0 (B, F), and 2.0 OD (C, G).
the substrates, a glass substrate incubated with gold-labeled oligonucleotides with a sequence noncomplementary to the surface-immobilized DNA was used as reference value for 0%. Scanning force microscopy (SFM, also known as atomic force microscopy, AFM) was conducted with a Dimension 3100 (Digital Instruments, Santa Barbara, CA) in tapping mode in air. RESULTS AND DISCUSSION Hybridization of Gold-Labeled DNA to an Oligonucleotide Array. One species of DNA-oligonucleotides (X1, cf. Figure 1) was arrayed in microstructured squares (60 µm) on glass substrates and used as capture probescomparable to the setup in typical fluorescence-based experiments. However, the labels of the target species were gold nanobeads with an average diameter of ∼30 nm. These nanobeads were modified with the target oligonucleotide X2 containing a sequence complementary to the surface-immobilized capture probe X1. The chip was now incubated with the target solution, washed, and dried. A checkerboard pattern became visible on the surface of the chip. Optical Characterization. An optical micrograph of this pattern in reflecting light is shown in Figure 2A. An array of squares is clearly visible. It reproduces the pattern predefined by
surface-synthesized DNA, thereby demonstrating the preferential binding of the labeled DNA to the surface-immobilized DNA. The squares appear as bright areas on a darker background. This contrast could be explained by light scattering on the adsorbed gold particles, pointing to a successful specific binding of the labels. A zoom of a chip structure in reflected light reveals a reddish color of the sample (Figure 2B), comparable to the color of the solution of colloidal gold. Observation of the same location in transmitted light (Figure 2C) reverses the contrast; the squares are now visible as dark structures on a bright background. The color changes to brown, and the contrast is slightly decreased. The insets in Figure 2B and C show horizontal brightness profiles for the region between the two arrows. In the case of reflected light (Figure 2B), the signal appears as well-defined bright regions (arrows), and the noise level is low. The profile for the transmission measurement (Figure 2C) is inverted compared to the reflection profile; now the adsorbed gold particles result in a lower brightness (due to decreased transmission). Although the overall pattern is comparable, the signal is less defined and the noise is more pronounced. These apparent advantages of reflection measurements were confirmed by shortAnalytical Chemistry, Vol. 72, No. 24, December 15, 2000
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time exposure experiments (Figure 2D), which showed that reflective measurements of the same sample need less (∼1 order of magnitude) exposure time compared to the transmissive mode.9 Unspecific Binding. How sequence-specific is the binding of DNA-modified particles to immobilized DNA? Gold nanoparticles bridged by DNA allow the discrimination down to a single basepair mismatch.10 In the case of chip-immobilized DNA, we could show that fluorescently labeled DNA strands with the same lengths but different sequence yielded no detectable signal.11 A low unspecific binding was also observed for nanoparticle-labeled oligonucleotides bound to DNA immobilized on a gold substrate.12 Detection of Individual Label Molecules. The optical analysis of the gold-labeled DNA chips yielded a high signal-tonoise ratio. After gold labeling, the squared surface regions with immobilized capture DNA were clearly visible compared to the background. An evaluation of an optical background signal is usually based on integral absorption measurements, because the individual dye molecules are hardly accessible by microscopical techniques. In the case of the presented experiments, the signal is due to a layer of colloidal gold particles, which are easily resolvable by SFM. So the surface density of particles can be determined, and related to optical measurements, as described below. SFM Reveals Surface Distribution of Particles. The chip (which was studied in the optical contrast in Figure 2) was investigated by SFM. Figure 3B shows a scanning force micrograph of a corner of one of the squares, revealing the gold beads as round particles. A high surface coverage with 50-100 particles/ µm2 is apparent in the upper right corner, which represents a part of a square. A zoom into this region is shown in (C), revealing aggregates of particles. A cross section (inset) demonstrates that the beads adsorb only in one layer. The surface coverage is incomplete. The background (the area between the squares) shows also adsorption of gold beads (lower part of Figure 3A). However, the density of the particles in the background region is significantly lower compared to inside the squares. Concentration-Response Curve. The influence of the solution concentration of DNA-modified particles on the surface density of adsorbed particles was investigated. Particle concentrations of 0.5, 1.0, and 2.0 OD were applied to complementary DNA arrayed on a chip. With increasing solution concentration, a bright pattern becomes visible in the SFM images (Figure 4A-C). This effect is based on colloidal particles, which bind in increasing number on microstructured squares of immobilized DNA. So the signal is represented by the particle density, which is plotted in Figure 5A. The background relates to particles unspecifically adsorbed between the DNA spots (cf. dark regions in Figure 4C). Although both signal and background increase with the concentration (Figure 5A), the effect is much more pronounced for the signal, resulting in an enhanced signal-to-background ratio for larger concentrations. (9) Ko ¨hler, J. M.; Csa´ki, A.; Reichert, J. r.; Mo ¨ller, R.; Straube, W.; Fritzsche, W. Sens. Actuators B, in press. (10) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078-1080. (11) Mo ¨ller, R.; Csa´ki, A.; Ko ¨hler, J. M.; Fritzsche, W. Nucleic Acids Res. 2000, 28, e91. (12) Csa´ki, A.; Mo ¨ller, R.; Straube, W.; Ko ¨hler, J. M.; Fritzsche, W., submitted.
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Figure 5. Concentration dependency of the particle density (A) and the intensity of reflected light (B) of the samples shown in Figure 4. (A) The particle density was extracted from SFM images. (B) The optical measurements were related to a gold-sputtered surface (100%) and a substrate with immobilized DNA after incubation with particles modified with noncomplementary DNA (0%).
Similar samples were studied in optical contrast (Figure 4DF). Although the general phenomenon of both signal and background increase with higher concentration is confirmed in the case of reflectivity (Figure 5B), the signal-to-background ratio is decreased. Further experiments will show the significance of the signal for concentrations at 1.0 OD and lower. Advantages of Optical Detection. What are the advantages of the presented technique? The detection is significantly simplified by relying on optical transmission or reflection, compared to fluorescence techniques. The stability of the samples is greatly enhanced, so that high light intensities can be applied for readout. This is in contrast to the sensitive fluorescence dyes and their bleaching problem and could result in a significantly shorter detection time (one high-intensity shot of the whole chip area compared to the scanning approach used in most fluorescence techniques, Figure 2D). The increased stability should apply also to the long term, providing the means for archiving as well as reanalyzing of samples after months or years. The influence of chemical and physical environment on the signal intensity is significantly reduced, resulting in a robust measurement. So the
reproducibility is enhanced, and comparability between different assays and chips is provided. An interesting point is the potential of miniaturization. The high signal-to-noise ratio in the case of the particle labels points to the possibility of spot sizes approaching the refraction limit in the submicrometer range, compared to the medium micrometer range used in fluorescence chips. So the packing density of spots as an extremely important aspect for parallelization could be enhanced by orders of magnitude. Our results show that colloidal gold particles can be used for the chip-based detection of specific biomolecular binding. This approach enables the application of optical readout without the
need of fluorescence equipment, thereby providing the means for a fully new class of chip detection systems. ACKNOWLEDGMENT Supported by VCI/BMBF and DFG grants (Fr1348/3-1, -2). We thank H. Stu¨rmer for help with the optical micrographs and E. Birch-Hirschfeld for on-chip DNA synthesis.
Received for review May 17, 2000. Accepted October 7, 2000. AC000567Y
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