Characterization and Optimization of a Real-Time, Parallel, Label-Free

We describe in this paper a methodology to quantify multispot parallel DNA ..... Figure 8 shows the differential reflectivity as a function of time of...
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Anal. Chem. 2000, 72, 6003-6009

Characterization and Optimization of a Real-Time, Parallel, Label-Free, Polypyrrole-Based DNA Sensor by Surface Plasmon Resonance Imaging Philippe Guedon,† Thierry Livache,‡ Franc¸ oise Martin,† Fre´de´ric Lesbre,‡ Andre´ Roget,‡ Ge´rard Bidan,‡ and Yves Levy,*,†

Laboratoire Charles Fabry de l’Institut d’Optique The´ orique et Applique´ e, UMR8501, Bat. 503, BP147, 91403 Orsay Cedex, France, and De´ partement de Recherche sur la Matie` re Condense´ e, LEMSI, UMR 5819 (CEA, CNRS, Universite´ J. Fourier), CEA-Grenoble 17 Rue des martyrs, 38054 Grenoble Cedex 09, France

We describe in this paper a methodology to quantify multispot parallel DNA hybridizations and denaturations on gold surfaces by using, on one hand, a polypyrrolebased surface functionalization based on an electrospotting process and, on the other hand, surface plasmon resonance imaging allowing real-time measurements on several DNA spots at a time. Two characterization steps were performed in order to optimize the immobilization of oligonucleotide probes and, thus, to increase the signalto-noise ratio of monitored hybridization signals: the first step consisted of characterizing the signal dependence upon the density of immobilized 15-mer probes, and, the second step, in analyzing the hybridization response versus spot thickness. We further demonstrated that a surface density of polypyrrole/DNA probes of ∼130 fmol/ mm2 (590 pg/mm2) optimizes the hybridization signal that can be detected directly. Optimal thickness of the spot was found to be close to 11 nm. Specificity and regeneration steps on each spot have also been demonstrated successfully, showing this method to be very competitive and convenient in use. The identification of biological samples through their nucleic acid sequences is one of the fastest growing fields at this time.1 Basically, most of the systems rely on hybridization between chemically immobilized single-stranded DNA probes and a nucleic acid sequence. There are two ways usually adopted to detect optically duplex creations when working on several spots at a time. The more sensitive is detection by fluorescence,2 where hybridization is not directly detected but is revealed by fluorescent labels. Although this method is quite sensitive, it needs a twostep protocol and quantification is not that easy. An alternative way is based on surface plasmon resonance (SPR) imaging that allows real-time monitoring and quantification.3-5 * To whom correspondence should be addressed: (phone) 33-1 69 35 87 95; (fax) 33-1 69 35 87 00; (e-mail) [email protected]. † Laboratoire Charles Fabry de l’Institut d’Optique The ´ orique et Applique´e. ‡ CEA, CNRS, Universite ´ J. Fourier. (1) Gerhold, D.; Rushmore, T.; Caskey, T. Trends Biotechnol. 1999, 24, 168-173. (2) Graves, D. J. Trends Biotechnol. 1999, 17, 127-134. (3) Bier, F.; Kleinjung, F.; Scheller, F. Sens. Actuators B 1997, 38-39, 78-82. 10.1021/ac000122+ CCC: $19.00 Published on Web 11/14/2000

© 2000 American Chemical Society

The SPR technique has been known for years as a means for detecting biomolecular interactions as antibody-antigen, proteinprotein.6-8 A surface plasmon is a TM electromagnetic wave that propagates along a metal/dielectric interface. The SPR system is built up in the Kretschmann configuration; i.e., a gold layer is evaporated on the top of a 60° prism. Then, when p-polarized light illuminates the interface through the prism under conditions of total internal reflection, at a certain incident angle, incident light can be coupled into surface plasmon mode, noticed as a decrease in the reflectivity. This angle is called the resonance angle and largely depends on the refractive index of the medium close to the gold surface. Because of the total internal reflection, the light reflected at the interface creates an evanescent wave that penetrates into the dielectric medium as a probe and that is sensitive to any change in optical thickness detected by a shift of the resonance angle. At a fixed angle, this shift is monitored as a variation of the intensity of the reflected beam leading to the quantification of the optical thickness changes. Several SPR configuration schemes have been investigated, as for instance, when illuminated with a collimated beam, the incident angle can be scanned by rotating the sample, and the reflectivity can be monitored in real time by a simple photodiode. This configuration will be called the regular SPR device in the following text. The SPR imaging configuration is closely based on the same configuration, except the metal surface is imaged on a CCD camera via an imaging lens. With SPR imaging, a new advantage appears: sensing on several areas of the gold surface at the same time.9,10 For a few years, it has also been explored as a DNA sensor. This technique combines many advantages for biosensing such as realtime, label-free recognition, fast quantification, and measurements (4) Marraza, G.; Chiannella, I.; Mascini, M. Biosens. Bioelectron. 1999, 14, 43-51. (5) Nilsson, P.; Persson, B.; Larsson, A.; Uhlen, M.; Nygren, P. A. J. Mol. Recognit. 1997, 10, 7-17. (6) Lo ¨fas, S.; Malmqvist, M.; Ro ¨nneberg, I.; Stenberg, E.; Liedberg, B.; Lundstro ¨m, I. Sens. Actuators B 1991, 5, 79-84. (7) Liedberg, B.; Lundstro ¨m, I.; Stenberg, E. Sens. Actuators B 1993, 11, 63-72. (8) Ha¨ussling, L.; Ringsdorf, H.; Schmitt, F.; Knoll, W. Langmuir 1991, 7, 18371840. (9) Thiel, A. J.; Frutos, A. G.; Jordan, C.; Corn, R.; Smith L. Anal. Chem. 1997, 69, 4948-4956. (10) Zizlsperger, M.; Knoll, W. Prog. Colloid Polym. Sci. 1998, 109, 244-253.

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of affinity constants. SPR is sensitive to small changes in optical thickness at the interface between a thin gold layer and a bulk solution.11,12 Furthermore, imaging a gold surface with SPR can lead to large-scale sensing only limited by a spatial resolution of a few micrometers.13 Parallel DNA sensing offers two main applications as clinical diagnostics and sequencing by hybridization. In both cases, the main difficulty is to build up addressable DNA arrays14-16si.e. to order the deposition of different DNA sequences on a chipsand to reuse the chip by denaturing the duplex after hybridization. Different chemical functionalization schemes have been investigated to bind the oligonucleotide (ODN) probes to the surface. For instance, surface linkage can be ensured either by selfassembled monolayers such as thiolate17 or silane18 or by polymers such as dextran19 or polypyrrole.20 This latter process involves the use of pyrrole/pyrrole-ODN copolymerizations carried out on a silicon substrate bearing individually addressed microelectrodes. Hybridization detection on these biochips is performed by fluorescence microscopy. To use the polypyrrole chemistry in conjunction with a SPR detection process on a nonpatterned homogeneous gold layer, we have developed a new electrodirected system involving the use of a pipet tip as an electrochemical cell allowing a localized one-step synthesis of a polypyrrole film including covalently linked ODN. This technology offers the following advantages: (i) no need of a bifunctional linker between gold and polymer; (ii) the in situ copolymerization of each ODN allows a good precision in localization and, thus, a control of the probe density at the spot surface based on the ratio between pyrrole monomers and oligonucleotides, plus a good precision on thickness owing to copolymerization time control; and (iii) the film synthesis is very fast since it takes 0.5 s to spot an 11-nmthick film. The main purpose of this work is to optimize the surface chemistry parameters for SPR imaging in order to obtain the highest hybridization signal by optimizing the probe density for each spot. A previously reported oligonucleotide/pyrrole-based functionalization scheme used with fluorescent detection of hybridization reactions consisted of 20-nm-thick spots with a 1:29000 oligonucleotide/pyrroles ratio and could detect less than 1 nM 15-mer complementary oligonucleotide.20 Nevertheless, these parameters are not directly applicable to SPR measurements, because on one hand, polypyrrole is highly absorbant in the visible spectrum (n ) 1.7 - j0.3 at a wavelength λ ) 633 nm) and a thickness of 20 nm would severely weaken the SPR sensitivity, and on the other hand, a 1:29000 ratio of immobilized probes would lead to quasi-undetectable optical thickness changes. (11) Burstein, E.; Chen, W.; Chen, Y.; Harstein, A. J. Vac. Sci. Technol. 1974, 11, 1004-1019. (12) Kretschmann, E.; Raether, H. Naturforsch: Astrophys, Phys., Phys. Chem. 1968, 23, 2135-2136. (13) Hickel, W.; Knoll, W. J. Appl. Phys. 1990, 67, 3572-3575. (14) Fidanza, J.; McGall, G. Nucleosides Nucleotides 1999, 18, 1293-1295. (15) Southern, E.; Mir, K.; Schepinov, M. Nat. Genet. Suppl. 1999, 21, 5-9. (16) Baier, M.; Hoheisel, J. Nucleic Acids Res. 1999, 27, 1970-1977. (17) Spinke, J.; Lliley, M.; Schmitt, F.; Guder, H.; Angermaier, L.; Knoll, W. J. Chem. Phys. 1993, 99, 7012-7019. (18) Jing, J.; Reed, J.; Huang, J.; Hu, X.; Clarke, V.; Edington, J.; Housman, D.; Anantharaman, T.; Huff, E.; Mishra, B.; Porter, B.; Shenker, A.; Wolfson, E.; Hiort, C.; Kantor, R.; Aston, C.; Schwartz, D. Proc. Natl. Acad. U.S.A. 1998, 95, 8046-8051. (19) Lo ¨fas, S. Pure Appl. Chem. 1995, 67, 829-834. (20) Livache, T.; Fouque, B.; Roget, A.; Marchand, J.; Bidan, G.; Teoule, R.; Mathis, G. Anal. Biochem. 1998, 255, 188-194.

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We report herein the hybridization experiments of biotinylated oligonucleotides to electrochemically immobilized ODN carried out on a four-spot chip. Two sets of experiments were performed, one with a regular SPR device leading to the optimal thickness of the chemical linkage and the other with SPR imaging leading to the optimal density of probes per spot. Once the immobilization step is optimized, there is no need to label the complementary sequence anymore. Hence, we used a quantification process relying on Fresnel equations applied to SPR imaging with a sensitivity of a few picograms per millimeter squared (assuming a detection limit of 0.1 nm). EXPERIMENTAL SECTION Oligonucleotides. Oligonucleotides were synthesized on an Applied Biosystems 381A DNA synthesizer. Probes were labeled at their 5′ end with a pyrrole residue according to a previously described procedure using a pyrrole-phosphoramidite building block.21 5′ Biotinylated oligonucleotides were used for the hybridization experiments. All the oligonucleotides were purified by HPLC on a C18 reversed-phase column with a gradient of acetonitrile (5-50%) in 25 mM triethylammonium acetate pH 7.0. Preparation of the Gold Slides. Gold thin films (thickness between 45 and 50 nm, and permittivity  ) -11.9 + j1.3) were vacuum-evaporated at a pressure below 10-7 mbar onto clean glass substrates (n ) 1.717 at λ ) 633 nm). Before being used, the gold-coated glass substrates were irradiated with UV rays to clear the gold slides from any pollution and then kept clean under filtered air conditions. SPR Chip Preparation. The electrospotting methodology developed herein is an adaptation of a chemical process used for the construction of biochips on individually addressable microelectrode arrays.20 This new methodology allows a one-step preparation of a biosensing layer on a gold substrate. It is based on an electrocopolymerization of a solution containing pyrrole and pyrrole linked to an ODN leading to the synthesis of a solid polypyrrole film including the ODN. The basic setup is outlined in Figure 1: a pipet tip including a platinium wire used as the counter electrode is filled with the polymerizable solution containing the pyrrole-ODN and moved to a precise location on the gold layer used as the working electrode. Then, after depositing a droplet of solution, an electrochemical pulse allows the synthesis of the polypyrrole film. Successive copolymerizations with different ODNs lead to the straightforward construction of a submillimeter (diameter is close to 500 µm) spot ODN array. Systematic fluorescence quality control is carried out by hybridization to check each spot in terms of fluorescence intensity and homogeneity. Copolymerization of the Pyrrole-Oligonucleotide Probes on the Gold Layer. The electrochemical directed copolymerization was carried out on the gold layer through the use of a 200µL pipet tip as the electrochemical cell. Electrical contact was established at the tip by inserting a platinum wire used as a counter electrode. The tip was filled with 20 µL of a solution containing 20 mM pyrrole (Tokyo Kasei), 0.1 M LiClO4 (Fluka), and 0.7 µM ODN (unless specified) bearing a pyrrole group. The tip was then applied to a precise location on the gold layer used (21) Livache, T.; Roget, A.; Dejean, E.; Barthet, C.; Bidan, G. ; Teoule, R. Nucleic Acids Res. 1994, 22, 2915-2921.

Figure 1. General scheme of the polypyrrole electrospotting methodology: The different tubes A-D containing different pyrrole-ODN and pyrrole monomer solutions are on the left. The spotting is carried out on the gold surface via the plastic tip containing the solution to be copolymerized.

as the working electrode. This electrochemical system was connected to an EGG Princeton Applied Research model 283 potentiostat and to an 8300 Schlumberger X/Y recorder. Prior to use, this simplified electrochemical cell (two electrodes) was calibrated with a saturated calomel reference electrode. The polypyrrole film was synthesized by electrocopolymerization on the gold layer (working electrode) by a 2-V electrochemical pulse for 0.5 s (unless specified). During the pulse, the synthesis charge (C) was recorded. Following the electrosynthesis, the tip was emptied and rinsed with water. The successive polymerizations were carried out by the same process with the different ODNs to be copolymerized on spatially defined areas of the gold slide. When all the ODN spots were synthesized, the slide was disconnected, rinsed with water, and stored at 4 °C. Fluorescence Quality Control of the ODN Spots. Quality control of the ODN spots was carried out with biotinylated complementary probes according to the procedure described for the DNA chips.20 Briefly, the hybridization was carried out at 45 °C for 15 min in a 20-µL volume containing 0.1 pmol of biotinylated oligonucleotide diluted in a hybridization buffer: PBS (Sigma) containing 0.5 M NaCl, 100 µg/mL herring sperm DNA (Sigma), and 10 mM EDTA. Following a washing in PBS/0.5M NaCl/0.5% Tween (washing buffer) at room temperature, the slide was incubated for 10 min in a solution of 5% streptavidin-R-phycoerythrin (Molecular Probes) diluted in the washing buffer. The fluorescence was recorded for 1 s with an epifluorescence microscope (BX 60, Olympus) equipped with a Peltier cooled CCD camera (Hamamatsu) and image analysis software (Imagepro plus, Media Cybernetics). Regeneration of the spots was carried out by a 10-s denaturation step in 0.1 M NaOH followed by a water rinse. SPR Instrumentation. Instead of a laser source as used in the regular SPR device, the SPR imaging used a light-emitting diode (wavelength centered at 660 nm, half-width 30 nm) as the light source. To obtain a uniform intensity distribution of the beam, we used two microscope objectives ×10 and ×20 (Leitz, Germany) and a small aperture placed between the two lenses. A polarizer was set in the p-polarized position during all the experiments. Changes in the optical thickness on the gold surface were interpreted by the 8-bit CCD camera as gray-level contrasts in the plasmon reflected beam. During hybridization experiments,

images were recorded at fixed intervals of time (20 s). All the images captured during an experiment were digitized by a framegrabber card (LG3, Scion) and then recorded and analyzed on a computer as usual image files. Data were analyzed with Igor Pro software (Wavemetrics). Oligonucleotide Materials: Hybridization and Denaturation. The biorecognition experiments were carried out in a 110-nL Teflon hybridization cell connected to a peristaltic pump (Gilson). The flow of running solutions within the cell was 10 µL/min. Hybridization of the Biotinylated Oligonucleotide to the Immobilized Probe. Complementary ODN, noted M5* (5′-Biotin-ACGCCA-GCA-GCT-CCA-3′), or noncomplementary ODN, noted CP (5′-biotin-TGG-AGC-TGC-TGG-CGT-3′), was injected on the surface functionalized with oligonucleotide probes, noted M5 (5′pyrrole-T10TGG-AGC-TGC-TGG-CGT-3′), in a PBS buffer (Sigma, pH 7.4) in the flow cell for 20 min at room temperature (20 °C) at a concentration of 20 µM (unless specified). After the oligonucleotide injection, the sensor surface was rinsed with PBS to remove unbound molecules. Duplex Denaturation. The regeneration step was performed by rinsing the chip with water for a few seconds and then injecting 20 mM NaOH for 10 s to open the double helix. The chip was rinsed again with water and then with running buffer to start another cycle. RESULTS AND DISCUSSION Measurements with the SPR Imaging Device. As can be seen in Figure 2, the polypyrrole/gold layer deposited on glass is fixed to the prism (with index matching oil) for the surface plasmon resonance to be excited through the illuminating lightemitting diode (LED). We preferred to use a simple LED source rather than a laser in order to suppress interference patterns and speckle due to laser coherence, generating much noise on the pictures. The example taken in Figure 2 shows how an optical thickness variation (index versus geometrical thickness) is converted into a gray-level variation on the CCD camera. Plotting the reflectivity of each point of the surface versus the angle of incidence is possible with SPR Imaging and leads to Fresnel-like curves. Measurements on the Spots. As optimal sensitivity occurs on the right slope area of the reflectivity dip, we determined the Analytical Chemistry, Vol. 72, No. 24, December 15, 2000

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Figure 2. Schematic representation of the SPR imaging device. A collimated, p-polarized LED beam illuminates the sensor surface via a coupling prism (n ) 1.7170 ( 5 × 10-4 at λ ) 633 nm). Reflected light, which contains all of the SPR response information is imaged on a CCD camera. The example shows on the monitor, at a fixed angle, a great contrast due to plasmonic enhancement. The angle of the resonance is adjusted for the bare gold layer giving a dark field, whereas at the same angle, the reflectivity of the dielectric coating has reached a higher gray-level depending on the optical thickness of the film.

Figure 3. Pictures of the four-element sensor: (a) at the angle of resonance for the four spots, giving the minimum of reflectivity and (b) at the angle at which the kinetics are monitored. Gold gray level appears in the background of the pictures. Dark rings are revealed on the periphery of each spot.

angular position corresponding to this point through the graylevel value averaged on the center (on a ∼100 × 100 µm2 square) of the functionalized spots. The measurements are always performed on the same area during an experiment. We show on the Figure 3 two pictures taken at different angles. In the part a, the angle of incidence corrresponds to the resonance angle for the four spots. The quasi-elliptic shape of the spot is due to the nonparallelism between the sensitive surface and the CCD camera. Measurements at the angle of resonance on the center area (see white square) of the four spots leaded to an averaged value of 33 ( 3. In the part b, the angles have been adjusted to optimize the sensitivity of the response. A contrasted ring appears around the spots which is due to optical thickness variation during the 6006 Analytical Chemistry, Vol. 72, No. 24, December 15, 2000

Figure 4. Attempts performed with the SPR imaging device to show specificity of the DNA sensor on a 11-nm-thick spot with a 1:700 ratio. Here are three kinetics proving the specificity of the sensor: (a) negative control to ensure specific recognition; (b) positive control. Rinsing with PBS is symbolized by dashed arrows and injections by full arrows.

copolymerization process. The angle that optimizes the sensitivity corresponds to the resonance angle for the ring. Obviously, these areas are not taken into account for the measurements. Inside the rings, the spots are homogeneous enough (variations are less than 1%) to be significant. Measurements on the four spots at the angle that optimizes the sensitivity leaded to an averaged value of 85 ( 15. The standard deviation is characteristic of the small difference of the thickness of each spot ((1.5 nm) but does not interfere in the experiments, as we measure variations and not absolute values. A contrario, the inner surfaces are more inhomogeneous for thin spots (close to 5 nm) because of the lack of copolymer matter (experiments not shown performed with fluorescence microscopy). Spots are homogeneous as soon as the thickness reaches a value close to 7-8 nm. Specificity of the Hybridization Reaction. The specificity of the biorecognition was studied on the slide by the SPR imaging system. Figure 4 shows a sensorgram including a noncomplementary ODN control and then a hybridization kinetic with the complementary sequence (positive control) on a 11-nm-thick sensor element, covered with a film made from a 1:700 oligonucleotide/pyrrole ratio. Unspecific interactions always occur and have to be minimized, and the most interesting information yielded by Figure 4 is the signal-to-noise level between negative and positive controls, ensuring the method to be reliable (positive over negative control, 33). Optimization of the Electrochemical Surface Functionalization for SPR Imaging. As described above, surface functionalization consists of the copolymerization of pyrrole monomers with oligonucleotides coupled to a pyrrole moiety. This electrodirected process allows easy monitoring of both the film thickness through the charge control and the grafting density through the copolymerization ratio. These parameters can be optimized in view of the SPR detection. Optimization of the Spot Thickness. Because of the high imaginary part of the refractive index, of the polypyrrole (n ) 1.7 - j0.3),22 the sensitivity of the SPR response may be weakened because of the decrease in reflectivity right dip slope value. A (22) Kim, Y.-T.; Collins, R. W.; Vedam, K.; Allara, D. L. J. Electrochem. Soc. 1991, 138, 3266-3274.

Figure 5. Reflectivity curves versus incident angle performed with the regular SPR device for different polypyrrole thicknesses. Solid lines are the results of the Fresnel calculations; the points are for experimental data. Parameters used are gold permittivity, -11.9 + j1.3; gold thickness, 47.5 ( 0.2 nm; polypyrrole index 1.7 - j0.3, at λ ) 633 nm.

Figure 6. Reflectivity variation monitored for the hybridization signal versus spot thickness. Hybridization kinetics were monitored on the regular SPR device.

special slide was prepared with four spots of different thickness (Figure 5). The slopes are 8%/deg for a 20-nm-thick spot, instead of 60%/deg for bare gold. Indeed, the thicker the polypyrroleODN film is, the smaller the dynamic range of the reflectivity will be, mainly due to the broadening of the resonance curve and to the higher value of the minimum of reflectivity. Conversely, if the functionalized spot is too thin, hybridization signals decrease as shown by Livache et al.21 with fluorescent detection, probably due to inhomogeneous surface distribution. Six different thickness ODN spots were made on the same slide by performing the syntheses for 0.25-1 s leading to 9-14-nm-thick films, respectively. As the spot thickness is not the same for all the spots, parallel experiments cannot be attempted in this section, because resonance angles are not the same for the six spots. As a consequence, hybridizations were performed one spot after another with a regular SPR device. The parameters derived from the fitting procedure are the refractive index, thickness of the gold, and thickness dlayer of the biological layer. We first fitted the experimental data of the bare gold reflectivity to determine the initial conditions (thickness and permittivity of gold). After hybridization, we fitted the reflectivities curves by varying the thickness of the biological layer by setting the gold parameters constant. The optimal surface coverage value was reached with a 11-nm-thick spot (Figure 6). As expected, this thickness value lies between the sensitivity-limiting values and the values leading to inhomogeneous spots. Optimization of the Probe Density on a Sensor Surface. To obtain the best sensitivity, the probe density has to be optimized in order to provide a legible hybridization signal. But, this density has to

Figure 7. Kinetics of hybridization reactions performed on a fourchannel sensor with the SPR imaging device. Each channel bears a different oligonucleotide/pyrrole ratio on its surface. Surface coverages given respectively in regard to the volume ratio: (a) Γ ) 740 pg/mm2 (1:300), (b) Γ ) 660 pg/mm2 (1:700), (c) Γ ) 340 pg/mm2 (1:1500), and (d) Γ ) 160 pg/mm2 (1:3000).

be properly determined to avoid steric hindrance, which occurs when the surface density is so high that complements cannot reach the probes, as described by Piscevic and al.23 In fluorescence detection experiments, the ratio of oligonucleotides with respect to pyrrole monomers, is taken to 1:29000. This ratio has been optimized for the fluorescence method and is due, on one hand, to the great sensitivity of the fluorescence that does not need more probes on the surface and, on the other hand, to steric hindrance considerations due to the size of the fluorescent label (R-phycoerythrin, MW 240 000) usually coupled to an avidin molecule. Our purpose is then to determine the optimal ratio of oligonucleotide per pyrrole monomers for SPR detection. This ratio should be obviously higher than 1:29000 and lower than the ratio involving steric hindrance limitation. We synthesized on a bare gold surface, four different polypyrrole spots, each made from a different oligonucleotide/pyrrole ratio. The ratios were chosen on a 2D geometric model leading to the following values: 1:3000, 1:1500, 1:700, and 1:300. The model assumes that the probes have a contact surface with the polymer corresponding to their diameter and allows us to estimate the surface density from the volumic ratio. As a matter of fact, the experiments were performed on 11-nm-thick spots. Figure 7 shows related hybridization kinetics, monitored with the SPR imaging device, and gives the surface coverage Γ, expressed in mass unit per surface unit, for each spot. Surface coverages (Γ ) [nlayer - ncover][dn/dc]-1dlayer), derived from the hybridized amount on these spots, were calculated with a value of dn/dc taken at 0.14 mL/g,24 a refractive index of the biological layer nlayer taken fixed to 1.41,25 an index of the cover medium ncover measured equal to 1.3340 ( 5 × 10-4 at λ ) 633 nm, and a thickness of the biological layer derived from Fresnel coefficients. The biphasic behavior of the kinetics derives from a two-step interaction: first, most of targets hybridize with the probes, and then, in the second phase, interactions are diffusion limited. The 1:300 ratio is very close to the ratio giving the best hybridization signal, and a stronger ratio would probably set the chip in a steric (23) Piscevic, D.; Lawall, R.; Veith, M.; Liley, M.; Okahata, Y.; Knoll, W. Appl. Surf. Sci. 1995, 90, 425-436. (24) Peterlinz, K.; Georgiadis, R.; Herne, T.; Tarlov, M. J. Am. Chem. Soc. 1997, 119, 3401-3402. (25) Persson, B.; Stenhag, K.; Nilsson, P.; Larsson, A.; Uhlen, M.; Nygren, P. Anal. Biochem. 1997, 246, 34-44.

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Figure 8. Sensorgrams performed on the four-channel sensor with the SPR imaging device at the same time. The oligonucleotide/pyrrole ratios are 1:300, 1:700, 1:1500, and 1:3000. Four cycles of oligonucleotide M5* (M5 complementary sequence) hybridization/denaturation have been performed between the kinetics indexed by a, b, and c. A negative control with M5 sequence (c) kinetics have also been performed on the chip.

Table 1. Surface Coverage for Every Ratio for Second to Fourth Cycles, Including Negative Controlsa Γ (pg/mm2)b

hybridization 1 hybridization 2 negative control hybridization 3 hybridization 4

1:300

1:700

1:1500

1:3000

740 650 10 300 270

660 590 15 260 210

340 340 20 230 200

160 130 25 130 100

a The accuracy reaches (20 pg/mm2 for all the values. b Ratio step number.

hindrance-limited configuration. At this ratio and assuming a 2D and brush ODN structure (the ODN length ∼10 nm is in the same range of the film thickness) and a perfect copolymerization yield, the calculated distance between each single-stranded ODN molecule is 3 nm (taking 0.4 nm for the pyrrole monomer). The diameter of the double helix resulting from the DNA hybridization is ∼2 nm, so it is quiet conceivable that steric hindrance occurs for this ratio if the brush structure is not perfect. Moreover, the calculated amount of hybridized ODN on such a support is 750 pg/mm2, which is in the same range of the highest value practically found (740 ( 20 pg/mm2, Table 1). These data show that the ratio of pyrrole/pyrrole-ODN in the film is roughly the same as the ratio from the feed solution; this was also checked on thin film by hydridization with radiolabeled ODN whereas hybridization on 3D thick film (200 or 600 nm) leads to different results.21 Using this optimized 1:300 ratio support, we performed a study of the hybridization signal versus the concentration (not pre6008 Analytical Chemistry, Vol. 72, No. 24, December 15, 2000

sented). This led us to measure a hybridization with a concentration of 15 nM complementary ODN (Γ ) 90 pg/mm2), which is of the same order as can be seen elsewhere.25,26 Repeatability of the SPR Multichannel Detection Regeneration of the Chip. Chip regeneration is really useful to test several sequences with the same initial immobilized probe level, for instance, for sequencing by hybridization or mutations screening. Furthermore, it leads to a smaller cost. We then performed four cycles on the four sensor elements at a time, i.e., complementary sequence hybridization followed by a duplex denaturation. Figure 8 shows the differential reflectivity as a function of time of these four cycles. Between the second and the third cycles, negative control with probe sequence kinetics was attempted in order to check the specificity of the method. Table 1 shows the surface coverage values derived from these experiments. As can be seen in this table, the baseline decreases cycle after cycle, but relative responses are still the same among the different ratio sensor elements, though we observed a loss of the hybridization rate. CONCLUSION The electrospotting process described allows an easy and rapid preparation of ODN matrixes directly onto a gold substrate without the need of thiolated reagents or multistep synthesis. These supports can be well qualified (thickness, coupling ratio) and can be easily coupled to SPR imaging monitoring of DNA hybridization. Specificity of the ODN/ODN interaction has been demonstrated, and measurements performed with SPR imaging were in (26) Feriotto, G.; Lucci, M.; Bianchi, N.; Mischiati, C.; Gambari, R. Hum. Mutat. 1999, 13, 390-400.

good agreement with those performed with a regular SPR method. The detection method, once the optimized parameters were found, no longer requires labeled DNA targets. We demonstrated the reliability of the SPR imaging, allowing real-time monitoring of parallel, label-free, hybridization experiments. Besides, surface functionalization was stable enough for the sensor chip to be reused several times. In future work, SPR imaging with oligonucleotide/pyrroles chemical functionalization will be used to study DNA minisequencing and mutation screening assays. Moreover, it has been shown that this kind of chemistry can also be applied to peptide immobilization,27 opening the field of real-time protein interaction studies.

ACKNOWLEDGMENT We thank Sandrine Villette for her technical support about picture acquisitions.

Received for review September 13, 2000.

February

3,

2000.

Accepted

AC000122+ (27) Livache, T.; Bazin, H.; Caillat, P.; Roget, A. Biosen. Bioelectron. 1998, 13, 629-634.

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