Parallel Multispot Detection of Target Hybridization to Surface-Bound

Dec 7, 2002 - We introduce surface-plasmon field-enhanced fluorescence microscopy for the on-line determination of hybridization reactions between tar...
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Langmuir 2003, 19, 1567-1572

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Parallel Multispot Detection of Target Hybridization to Surface-Bound Probe Oligonucleotides of Different Base Mismatch by Surface-Plasmon Field-Enhanced Fluorescence Microscopy† Thorsten Liebermann and Wolfgang Knoll* Max-Planck-Institut fu¨ r Polymerforschung, Ackermannweg 10, 55128 Mainz, Germany Received July 18, 2002. In Final Form: October 9, 2002 We introduce surface-plasmon field-enhanced fluorescence microscopy for the on-line determination of hybridization reactions between targets from solution to complementary probe oligonucleotides which are surface-bound to a sensor substrate in a matrix format. This way, the parallel and real-time detection of the association and dissociation kinetics (rate constants) of hybrids of different mismatch situations can be quantitatively analyzed.

1. Introduction The use of fluorescence detection schemes in combination with the resonant excitation of surface plasmons has been shown to increase the sensitivity for bioanalyte monitoring considerably.1 The obtainable enormous enhancement of the optical field at and near the metal/ dielectric interface2 which can amount to more than 2 orders of magnitude for Ag (at λ ) 633 nm)3 and still reaches a factor of 16 for Au2 can be directly used to improve the signal-to-noise level in the quantitative analysis of biorecognition and interfacial binding events in sensor formats. We have recently demonstrated that this principle for bioanalyte detection can be used to monitor the association and dissociation reactions between surface-attached probe oligonucleotides and their complementary strands from solution.4 Rate constants, that is, kon and koff rates, and affinity constants, Ka, could be determined quantitatively,5 for example, as a function of the mismatch situation between probe and target.4 Various detection schemes have been tested.3 They were based (1) on having either the target fluorescently labeled or (2) the probe strand, (3) on using a donor/acceptor energy transfer principle to monitor hybridization reactions, or (4) on the competition between single strand binding proteins and the complementary oligonucleotide.6 But all examples given so far were based on counting photons with the help of a photomultiplier that in one way or another indicated details of the hybridization in a spectroscopic mode. † Part of the Langmuir special issue entitled The Biomolecular Interface.

(1) Attridge, J. P.; Daniels, P. B.; Deakon, J. K.; Robinson, G. A.; Davidson, G. P. Biosens. Bioelectron. 1991, 6, 201-214. (2) Liebermann, T.; Knoll, W. Surface-plasmon field-enhanced fluorescence spectroscopy. Colloids Surf., A 2000, 171 115-130. (3) Neumann, T.; Johansson, M.-L.; Kambhampati, D.; Knoll, W. Surface-Plasmon Fluorescence Spectroscopy. Adv. Mater., in press. (4) Liebermann, T.; Knoll, W.; Sluka, P.; Herrmann, R. Complement hybridization from solution to surface-attached probe-oligonucleotides observed by surface-plasmon field-enhanced fluorescence spectroscopy. Colloids Surf., A 2000, 169, 337-350. (5) Kambhampati, D.; Nielsen, P. E.; Knoll, W. Investigating the kinetics of DNA-DNA and PNA-DNA interactions using surface-plasmon resonance-enhanced fluorescence spectroscopy. Biosens. Bioelectron. 2001, 16, 1109-1118. (6) Neumann, T.; Knoll, W. Mismatch discrimination in oligonucleotide hybridization reactions using single strand binding protein. A surface-plasmon fluorescence study. Isr. J. Chem. 2001, 41, 69-78.

Here, we want to extend this technique toward the parallel detection of hybridization reactions to a whole array of individual sensor spots by using a highly sensitive CCD camera as the recording element. The camera takes time-dependent images of the fluorescence intensities emitted from each sensor element arranged on the chip surface in a matrix format using an ink-jet preparation protocol.7 This way, a large number of individually functionalized spots can be monitored simultaneously. For simplicity, we will limit our studies to an array of 3 × 3 spots of three different probe oligonucleotide sequences. 2. Experimental Procedures The presented detection scheme is a direct extension of our earlier work on surface-plasmon microscopy7-10 combined with the principle of detecting fluorescence light from chromophores located near the metal/dielectric interface, excited by the resonantly coupled surface-plasmon modes propagating along this interface.2 A simplified schematic diagram showing the combination setup that allows for recording by both techniques, that is, surfaceplasmon microscopy and surface-plasmon field-enhanced fluorescence microscopy, is given in Figure 1a. A light beam from a HeNe laser (Uniphase 5 mW, λ ) 632.8 nm) is controlled in its intensity and polarization by two polarizers and then passes a beam-expanding unit (spatial filter) with a pinhole (25 µm) for spectral cleaning of the wave fronts. The light is coupled via a high-index prism (LaSFN9) in this Kretschmann configuration to the (Au) metal-coated substrate which is index-matched to the prism (cf. Figure 1b) in contact with the flow cell (volume V ) 150 µL) (cf. Figure 1c) used for on-line recordings of hybridization reactions. The reflected light is imaged via an objective lense (Rodenstock, f ) 50 mm) onto a CCD camera (Hamamatsu, C 5405-01).10 Sample cell and camera are mounted to a two-stage goniometer such that θ-2θ angular scans can be performed in the normal reflection mode of surface-plasmon microscopy.9 The video signal of the camera is digitized by a framegrabber unit (Stemmer, ICP-AM-VS) and further analyzed by a PC. For the fluorescence microscopy, a particularly sensitive CCD camera (Photometrics, PVCAM) is mounted to that part of the (7) Zizlsperger, M.; Knoll, W. Multispot parallel on-line monitoring of interfacial binding reactions by surface plasmon microscopy. Prog. Colloid Polym. Sci. 1998, 109, 244-253. (8) Rothenha¨usler, B.; Knoll, W. Surface-plasmon microscopy. Nature 1988, 332, 615-617. (9) Hickel, W.; Kamp, D.; Knoll, W. Surface-plasmon microscopy. Nature 1989, 339, 186-190. (10) Hickel, W.; Knoll, W. Surface plasmon microscopy of lipid layers. Thin Solid Films 1990, 187, 349-356.

10.1021/la026263j CCC: $25.00 © 2003 American Chemical Society Published on Web 12/07/2002

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Liebermann and Knoll Table 1. Comparison of Some Thickness and Intensity Data of Interfacial Architectures Built with Streptavidin as a Matrix (Coupled to a Mixed Biotinylated Thiol SAM) and with Directly Chemisorbed Thiolated Streptavidin, Respectively layer thickness thickness of DNA probe layer fluorescence intensity (MM0) unspecific dye adsorption

Figure 1. (a) Schematic experimental setup for surfaceplasmon and surface-plasmon field-enhanced fluorescence microscopy in the Kretschmann configuration. (b) Schematic of the flow cell attachment to the prism/slide/Au-layer surfaceplasmon coupling device. (c) Cross section of the flow cell with inlet and outlet for solution rinsing.

Figure 2. Schematic setup used for the fabrication of an array of sensor spots on an Au-coated surface-plasmon substrate by the ink-jet principle. A piezo pump ejects nanoliter droplets of solutions provided in a 96 well plate by means of a computercontrolled nanoplotter unit. goniometer that rotates the sample cell (θ) thus ensuring that the camera always looks at a fixed angle (typically normal) to the substrate surface. A software package (V for Windows, Photometrics) allows for the recording of images either as the sample rotates for angular scans or in a kinetic mode as a function of time at a fixed angle of incidence (and observation). This camera is operated at an internal temperature of T ) -35 °C and with an integration time of ∆t ) 30 s. For the preparation of the substrate chip with an array of sensor spots, the drop-on-demand unit shown in Figure 2 was used.7 The high-index glass slide coated with Au and SHstreptavidin (cf. below) is placed on an XYZ positioning module (Isel, Typ EP 1090/4, positioning accuracy of 10 µm) together with a microtiter plate that contains all the sample solutions. A Si piezo pump (GeSiM, Typ Mikropipette SPIP) is used to apply droplets of sample solution from the supply plate to the sensor surface with drop volumes in the range of 1-2 nL. A dilutor unit

streptavidin

streptavidin-SH

4.0 nm 1.6 nm 1 × 106 cps 1 × 104 cps

5.3 nm 2.3 nm 4 × 105 cps 5 × 104 cps

(GeSiM) is used for the fully automated application of the various sample solutions and the rinsing of the pump. The preparation of a 3 × 3 array of nine spots takes about 5 min. If the application of the droplets is performed in a (nearly) saturated humid atmosphere, the individually deposited droplets can be kept stable for ca. 3 h, allowing, for example, a self-assembly of thiol molecules to proceed to (near) completion.7 In principle, the patterning of the substrate for the array fabrication can be done at different levels of the chip preparation: One could evaporate the Au layer through a mask confining the application of, for example, a thiol solution to the individual Au spots. Suitable masks, however, with a sufficiently high integration density of spots are not easy to fabricate and use. In many of our earlier studies, we used a streptavidin monolayer coupled to a biotinylated thiol self-assembled monolayer (SAM)11 as a generic binding matrix for other biotinylated functional units.12 For the ink-jet approach, however, it turned out that this streptavidin surface is too hydrophilic to allow for a precise placement of aqueous droplets of oligonucleotide solutions in an array format: the wetting of that protein surface by the water drops leads to a total loss of control over the position and the size of the individual sensor spot. (The same would happen if one were to spot the aqueous solution of streptavidin onto the OH/biotin-mixed SAM surface!) To overcome this difficulty, we choose SH-functionalized streptavidin as the spotted agent. An aqueous solution of this analogue (streptavidin-SH) can be placed onto a slightly aged (t > 30 min in air after evaporation) and hence sufficiently hydrophobic Au substrate rather precisely because the relatively high contact angle of the corresponding drops on that precontaminated Au surface leads to a contact area of ca. 300 µm with only little jitter in the position of the center of the droplets. The highly reactive SH-groups at the surface of this streptavidin derivative (ca. 8 SH-groups per streptavidin molecule), however, guarantee a stable chemisorption of the protein monolayer to the Au substrate. To make sure that this streptavidin analogue has otherwise similar binding properties for biotinylated species, we compared the layer parameters of streptavidin and streptavidin-SH directly by surface-plasmon spectroscopy and by surface-plasmon fluorescence spectroscopy. The results are summarized in Table 1. The obtainable surface coverage of streptavidin-SH (assuming in the data analysis the same refractive index of n ) 1.45 for both proteins) seems to be slightly higher with a correspondingly higher average layer thickness (i.e., surface coverage) of the biotinylated probe DNA matrix (cf. the chemical structure of the probes and targets given in Figure 3c). However, contrary to this higher probe density the fluorescence intensity seen after hybridization of the chromophore-labeled target oligonucleotide strands is considerably lower. Although differences in the hybridization efficiency to probes coupled to the two different streptavidin layers might contribute to this observation, we do favor the interpretation that this is caused by an enhanced quenching of excitation energy from the target chromophore to the Au substrate. As shown in Figure 3a, the protein is assumed to be in close contact to the surface, chemisorbed to the metal. (11) Spinke, J.; Liley, M.; Schmitt, F. J.; Guder, H. J.; Angermaier, L.; Knoll, W. Molecular Recognition at Self-assembled monolayers: optimization of surface functionalization. J. Chem. Phys. 1993, 99, 70127019. (12) Knoll, W.; Zizlsperger, M.; Liebermann, T.; Arnold, S.; Badia, A.; Liley, M.; Piscevic, D.; Schmitt, F.-J.; Spinke, J. Streptavidin arrays as supramolecular architectures in surface-plasmon-optical sensor formats. Colloids Surf., A 2000, 161, 115-137.

Multispot Detection of Target Hybridization

Langmuir, Vol. 19, No. 5, 2003 1569 The corresponding target strands, T1, T2, and T3, were of full complementarity with respect to their probe strands and exhibited a mismatch 1 or 2 situation upon cross-hybridization; for example, P1/T3 is a mismatch 1 (MM1) hybrid, whereas P2/T3 is a MM2, and so forth. Earlier studies had shown that the nature of the mismatch situation controls the kinetics of the association and dissociation processes and results in a substantially different stability of the hybrid, that is, determines the affinity constant.4,5 Each of the target strands carries a chromophore, MR121 (structure formula given also in Figure 3c), that could be excited by the red HeNe laser line (λ ) 632.8 nm) and emitted fluorescence photons recorded (imaged) at λ ) 656 nm.

3. Results

Figure 3. Schematic cross section of a monolayer of streptavidin-SH chemisorbed to an Au substrate and loaded with a monolayer of biotinylated catcher oligonucleotide probes able to hybridize to target strands from solution. (b) Arrangement of the 3 × 3 probe spots on the streptavidin-SH matrix. Each vertical row consists of 3 spots of identical probe sequences (cf. (c)). (c) Probe sequences, P1, P2, and P3, used in this study: 15mers of thymine nucleotides acting as spacers are functionalized by biotin at their 5′-ends and carry the specific 15mer probes with slightly different base sequences. The corresponding target sequences, T1, T2, and T3, respectively, allow for hybridization studies with mismatch 0 (MM0), 1 (MM1), and 2 (MM2) situations. All targets carry at their 5′-ends a fluorophore, MR121, the chemical structure of which is also shown. As a consequence, the chromophores at the end of the hybridized targets are slightly closer to the quenching metal than the ones in the case of normal streptavidin which is separated from the metal by an additional 1.5 nm of the thiol SAM. Since the energy transfer shows a very strong distance, d, dependence between chromophore and acceptor metal surface (∝[1 + (d/d0)4]-1, with d0 ∼ 5-7 nm, the Fo¨rster radius),13 this slight difference in separation could account for the observed loss in fluorescence intensity.14 For the following investigations, however, this difference and its interpretation are not relevant. When tested for nonspecific adsorption of dye molecules, the streptavidin-SH layer seems to offer more active sites; however, this background contribution still is in the range of only 10% of the signal from hybrids. With this approach, a 3 × 3 spot array was then prepared as is schematically depicted in Figure 3b: 3 rows of 3 spots each with a functionality given by the three different sequences P1, P2, and P3, respectively, which are shown in (c). The specific 15mer base sequences were separated from the biotin anchor group by 15 thymine nucleotides acting as spacers. (13) Kuhn, H.; Mo¨bius, D.; Bu¨cher, H. In Physical Methods of Chemistry; Weissberger, A., Rossiter, B. W., Eds.; Wiley-Interscience: New York, 1972; Part IIIB, Chapter 7. (14) Neumann, T. Ph.D. Thesis, Johannes Gutenberg-Universita¨t, Mainz, Germany, 2001.

3.1. Surface-Plasmon Microscopy (SPM) of Oligonucleotide Arrays. A first idea of the matrix arrangement of the sensor spots can be obtained from imaging the 3 × 3 matrix by regular surface-plasmon microscopy. Two of those images, taken at two different angles of excitation, θ ) 56.75° and θ ) 57.50°, showing the typical angle-dependent contrast (inversion),7 are given in parts a and b of Figure 4, respectively. Several features are noteworthy: the individual spots with an average diameter of ca. 300 µm appear as ellipses due to the oblique angle at which the surface is imaged and an additional distortion by refraction at the prism/air interface (cf. Figure 1a,b). The average spot-to-spot separation is relatively narrowly distributed (cf. the discussion above) and amounts to ca. 600 µm. By reducing both the spot size and the relative distance, one could enhance the array density substantially, but that was outside the scope of this work. The individual images show a relatively weak contrast between Au/streptavidin/probe oligonucleotide-coated spot areas and the mere Au/streptavidin background, respectively, and are further disturbed by optical patterns originating from dust particles and an overlaid interference pattern. Moreover, the spots appear to be homogeneous in their central parts but exhibit a thickness (or refractive index) profile toward their periphery. As has been discussed before,7 all these “optical impurities” do not disturb (too much) the quantitative analysis of the local resonance behavior of each spot by image analysis computer routines. Defining for each sensor spot a suitable pixel frame on the camera image, taking all the individual gray value histograms constructed from all the pixels within each of these frames, and reducing these data to a single average gray value obtained from these histograms, one then obtains for each spot a sequence of average gray values which, if plotted as a function of the angle of incidence at which the image was taken, represents a local surface-plasmon resonance curve. This can be quantified in the usual way by fitting these data to a Fresnel simulation. One example is shown in Figure 4c for one of these spots, before and after binding of the biotinylated probes from the small droplet of probe solution that was applied via the ink-jet preparation step (cf. above). Both series of measurements were taken with the chip in contact with an aqueous phase, that is, the hybridization buffer. For the spot image taken after the catcher probe binding to the streptavidin-SH matrix, several pixel frames were selected at this spot in order to gain a better statistical average. Already from the raw data one can clearly see the shift of the resonance curve that was caused by the binding of the biotinylated probes to the streptavidin-SH matrix which is equivalent to an increase of the optical thickness of the surface coating. Given in Figure 4c are also Fresnel fit curves to both the Au/streptavidin-SH background

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Figure 4. Two surface-plasmon optical images of the 3 × 3 array of oligonucleotide probe spots prepared by the ink-jet setup shown in Figure 2 on top of the streptavidin-SH matrix: (a) was taken at θ ) 56.75° at the low-angle side of the surface-plasmon resonance (cf. (c)), and (b) was recorded with the incidence angle θ ) 57.5°, i.e., at an angle larger than the minimum. Note the contrast inversion between the two images. The round spots appear as ellipses because of the oblique angle of observation (relative to the plane of the sample). Both bars correspond to ca. 300 µm. (c) Quantitative analysis of images such as those shown in (a) and (b), taken at many different angles near the resonances of the two areas (spots and background). Plotted are the reflected intensities obtained (by an image analysis computer routine) from pixel fields corresponding to the Au/streptavidin-SH background (∆) and to the DNA probe spots (O), respectively. The full curves are Fresnel fits to the data.

and the data obtained from one of the probe-loaded sensor spots. Assuming an average refractive index of the probe layer of n ) 1.375,15 we obtain a probe layer thickness of d ) (2.1 ( 0.3) nm which is in good agreement with the determination of this layer thickness by surface-plasmon spectroscopy! 3.2. Surface-Plasmon Fluorescence Microscopy (SPFM). As we have seen for many experiments of (15) Zizlsperger, M. Ph.D. Thesis, Johannes Gutenberg-Universita¨t, Mainz, Germany, 1999.

oligonucleotide hybridization with surface-plasmon spectroscopy (SPS), the binding of relatively short target oligonucleotides (15mers) to a probe matrix of very low catcher density (ca. 1 probe strand per 40 nm2) results in a final effective thickness increase which is too low to be detected and reliably quantified by label-free surface-plasmon optics, even at 100% efficiency. Hence, no change in the surface-plasmon microscopic images upon injection of target solution into the flow cell could be observed.

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Figure 6. Vertical fluorescence intensity (pixel) line scans of the reference, i.e., the streptavidin-SH background, and across the 3 DNA probe spots, P1, taken during the T3 desorption period (cf. Figure 5b) after ca. 5 min dissociation. For details, see the text.

Figure 5. Series of time-lapse surface-plasmon fluorescence microscopy images taken during hybridization (a,c,e) and dissociation (b,d,f) of different targets, T1-T3, to the surfaceattached probes, P1-P3, on the chip. For details, see the text.

However, since in our experiments here all the targets carry a fluorophore we can use the resonantly coupled surface-plasmon waves as the light source that excites those chromophores that are within the evanescent tail of the propagating SPS mode and, in particular, are bound to the interface by hybridization to the surface-attached catcher probes. Since we operate at a very low bulk target concentration of c0 ) 10-6 M, the constant, that is, hybridization-independent, fluorescence background of free targets in the evanescent tail of the surface-plasmon mode2 does not give a significant contribution to the total signal and we are mostly concerned with the enrichment of chromophores at the interface by the association event. What was found for the surface-plasmon fluorescence spectroscopic experiments is also true for the microscopic mode of operation. In Figure 5, we present a series of SPFM images taken from a 3 × 3 catcher matrix during the sequential association and dissociation of the target molecules T1, T2, and T3, respectively, to the 3 rows of catcher probes P1, P2, and P3 (cf. also Figure 3b). The images in the first row (Figure 5a) were taken with a 30 s integration time per image over a time period of ca. 20 min, following the injection of a 1 µM solution of target T3. One can observe that both the P3 (MM0, middle column of spots) and the P1 (MM1, left column) spots are “decorated” by the hybridizing targets and thus gain fluorescence intensity although not to the same full level of saturation intensity. The right column of sensor spots

on the chip functionalized with the probe P2 with a base sequence that shows two mismatches relative to the injected target T3 remains totally dark. This is fully in line with earlier observations.4 When the cell is rinsed with pure buffer (second row of images, Figure 5b), the middle column in each image (MM0) remains fluorescent while the left column (MM1) gradually loses its intensity as a consequence of the efficient dissociation for this hybrid.16 As a consequence, at the end of the rinsing period only the MM0 column still is fluorescently labeled. If, in a next step of the experiment, the T1 target sequence is injected into the flow cell (Figure 5c) again the respective MM0 probe (P1, left column) and the MM1 probe (P2, right column) are decorated and strongly emit fluorescence light. Rinsing with pure buffer (Figure 5d) leads to a complete dissociation of the hybrid and hence loss of fluorescence emitted from this column of spots whereas the MM0 hybrid (left column) again is stable. And finally, the injection of the T2 targets (Figure 5e) leads to the decoration of the P2 probe spots (right column) which are then also stable against rinsing (Figure 5f) (at the time scale of these experiments). Similar to the case of the SPM data analysis, the quantification of these SPFM images has a spatial and a time-dependent component and is also based on the averaging of the pixel intensities of the respective spot image on the CCD camera. A vertical line scan of pixel intensities scaled in their spatial coordinate to the real sample dimensions is given in Figure 6. Here, we compare the intensities of a single vertical line of pixels taken from one of the images displayed in Figure 5b, crossing the 3 sensor spots of the left column (P1/T3, cf. Figure 5) (“DNA-Probes” in Figure 6), with that of the bare streptavidin-SH background intensity (“Reference” in Figure 6) taken between the left and the middle column of sensor spots (Figure 5b). Each sensor spot is clearly seen as an increase of the fluorescence intensity above the background. Between the individual spots, the fluorescence returns to the background which indicates that nonspecific adsorption of targets to the streptavidin-SH matrix can be neglected in the data analysis. (16) Liebermann, T. Ph.D. Thesis, Johannes Gutenberg-Universita¨t, Mainz, Germany, 1999.

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Figure 7. Kinetic scans of the association (a) and dissociation (b) of T3 targets to P1 (MM1) and P2 (MM2) spots, respectively. Also shown is the time-dependent fluorescence intensity measured from bare streptavidin-SH areas (reference).

The single line scan gives pixel intensities which are relatively noisy. In addition, the signal to background is relatively low because this line scan was taken ca. 5 min after the desorption of the T3 targets from these P1 probe spots was initiated by rinsing buffer through the flow cell (cf. Figure 7). Nevertheless, if averaged over the full area of the individual sensor spot the resulting pixel fluorescence value histogram gives rather precise averaged fluorescence intensities which can be monitored and evaluated as a function of time, for example, after injection of target solution (i.e., during the hybridization) or after replacement of the target solution by bare buffer (i.e., during the dissociation step). This then gives kinetic information on the reaction rate constants for all imaged spots simultaneously. Two examples for the obtained kinetic scans are given in Figure 7. We choose the P1/T3 (i.e., MM1) and the P2/ T3 (i.e., MM2) situations. The data displayed in Figure 7, hence, were taken from the images displayed in Figure 5a during the association of targets T3 and from images in Figure 5b during the desorption, by averaging the fluorescence intensities of the left (P1) and of the right vertical rows (P2), respectively. Moreover, fluorescence intensities collected from the background are also displayed as a reference. As shown before by surface-plasmon field-enhanced fluorescence spectroscopy,16 the target T3 hybridizes rapidly and efficiently to the probe spots P1 (MM1)

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whereas the lack of fluorescence intensity increase at the P2 spots indicates the dramatic loss of affinity upon the introduction of a second mismatched base in the probe sequence (cf. Figure 7a). Hence, no binding at all is seen at this concentration of target T3 (c0 ) 10-6 M). The moderate intensity rise within the first several tens of seconds after injection of the target solution indicates the gradual mixing within the cuvette which leads to the replacement of buffer by target solution with the corresponding fluorescence increase from chromophores excited within the evanescent tail of the surface-plasmon mode. This increase is identical to the one found in front of the background areas (cf. “reference” in Figure 7a) because no surface-bound targets give any additional fluorescence intensity. Upon exchange of target solution by pure buffer, this background fluorescence intensity decreases (again identical for the P2/T3, i.e., MM2, spots and for the background reference) within a few tens of seconds (cf. Figure 7b). The surface-bound targets at the P1 spots dissociate, and within a few hundred seconds the fluorescence intensity decays completely to finally reach the background level (cf. Figure 7b). The quantitative analysis (within a Langmuir adsorption model) of the hybridization and dissociation kinetic curves gives rate constants, kon and koff, that are roughly similar to the ones determined by spectroscopy: we find kon ) 1.2 × 104 M-1 s-1 and koff ) 3.3 × 10-3 s-1. The corresponding rate constants found in the spectroscopic experiments were16 kon ) 2 × 104 M-1 s-1 and koff ) 0.13 × 10-3 s-1. We believe that the apparently faster koff rate constant in the microscopic mode is an artifact due to the relatively long integration time needed for the recording of the images (ca. 30 s). The limited photostability of the MR121 fluorescence dye results in a substantial loss of the fluorescence due to photobleaching of chromophores. This is not so evident in the kon process because here the continuous exchange of targets T3 bound to the surfaceattached probes P1 with those from solution constantly replaces bleached chromophores by new ones. However, once the target solution is replaced by pure buffer and the dissociation of targets from their probes leads to a decrease of the measured fluorescence intensity as a function of time, this bleaching of the still-bound chromophores appears in the data as an additional decay channel resulting in an increased effective koff rate constant. This problem can be solved by the use of chromophores with increased photostability, for example, luminescent quantum dots,17 or imaging devices with a higher sensitivity and, hence, reduced integration time. Acknowledgment. We thank G. Batz, R. Herrmann, P. Sluka, and M. Zizlsperger for stimulating discussions. Financial support from Boehringer Mannheim (now Roche Diagnostics) and by the European Community (Project QLK1-2000-01658, “DNA-Track”) is gratefully acknowledged. The MR121-labeled oligonucleotides and the thiolated streptavidin were kindly provided by Boehringer Mannheim. LA026263J (17) Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Semiconductor nanocrystals as fluorescent biological labels. Science 1998, 281, 2013-2016.