Planar Waveguides as High-Performance Sensing Platforms for

A major challenge for the construction of multiplexed biosensor platforms is the ... The single-channel ink-jet printer used in the present study allo...
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Anal. Chem. 1999, 71, 3347-3355

Planar Waveguides as High-Performance Sensing Platforms for Fluorescence-Based Multiplexed Oligonucleotide Hybridization Assays Wolfgang Budach,* Andreas P. Abel, Alfredo E. Bruno, and Dieter Neuscha1 fer

Novartis Pharma AG, DMPK, 4002 Basel, Switzerland

16-Mer and 22-mer oligonucleotide capture probes with an amino function at the 5′-end were covalently immobilized on (3-glycidoxypropyl)trimethoxysilane (GOPTS) silanized planar waveguides in “checkerboard”-style 2-dimensional arrays by means of ink-jet printing in order to demonstrate the potential of multiplexed planar waveguide biosensing. The laser-induced fluorescence was collected by a CCD camera, providing quantitative information regarding the intensity of individual sensing pads. In model hybridization assays with Cy5-labeled complementary oligonucleotides, sample concentrations down to 50 fM were successfully detected within 12 min. The overall spot-to-spot variability of the ink-jet immobilization in combination with amino functionalized oligonucleotides and GOPTS silanized waveguides was found to be about 12% and almost independent of the formation of an equilibrium between the labeled analyte and capture probes. Both pipet-made and ink-jet-made oligonucleotide spots showed comparable assay performances.

A variety of optical biosensors have been developed in the past few years for rapid detection of biospecific interactions between molecules. Many research activities increasingly concentrate on miniaturized sensor platforms for drug discovery, food processing, genomics, proteomics, and point-of-care diagnostics, since features such as small sample volume, high sensitivity, reduced consumption of working solutions, and short time-to-result are demanded for the realization of novel concepts in research and development. However, the technical and scientific hurdles grow with increasing degrees of miniaturization, especially if samples containing blood, cell extracts, or serum are to be analyzed. As a contribution to overcome the complex technical requirements of miniaturized biosensors,1,2 we report in the present paper on miniaturized multiplexed biosensors based on planar waveguide technology. Among the various types of evanescent waveguide transducers, including fibers,3 tapered fibers,4 hollow cylinders,5 and channel * Corresponding author. E-mail: [email protected]. (1) Turner, A. P. F Ann. Chim. 1997, 87, 225-260. (2) Go ¨pel, W. Sens. Actuators, A 1996, 56, 83-102. (3) Kao, P.; Yang, N.; Schoeniger, J. S. J. Opt. Soc. Am. 1998, A15, 21632171. (4) Pilevar, S.; Davis, C. C.; Portugal, F. Anal. Chem. 1998, 70, 2031-2037. (5) Wolfbeis, O. F. Trends Anal. Chem. 1996, 15, 225-232. 10.1021/ac990092e CCC: $18.00 Published on Web 07/02/1999

© 1999 American Chemical Society

waveguides,6 planar waveguides appear to offer the broadest range of applications with enhanced sensitivity. Two different classes of evanescent field transducers can be distinguished: waveguide sensors based on changes in the refractive index (∆n) in the propagation media of the evanescent field due to specific molecular adsorption at the sensor surface7-9 and luminescence-based planar waveguide sensors, which offer superior sensitivity.10,11 For biosensor applications, the luminescence-based sensor platforms have shown impressive detection limits and selectivity for both immunoand oligonucleotide hybridization assays, as well as for assays with functional membrane fragments.12,13 In contrast to luminescence detection principles based on confocal microscopy,14-16 where the light source is focused to a defined volume element leading to a strong local electrical field (i.e., epifluorescence), the evanescent field excitation provides an enhanced excitation probability/efficiency of the surface-bound fluorophores along the entire planar waveguiding surface. In the present study, 150 nm thin dielectric metal oxide layers (Ta2O5) were used as single-mode waveguides.17,18 The electrical fields of the waveguides decay exponentially in the probe media and vanish within a distance corresponding to the wavelength λ of the guided modes.19 As a consequence, labeled analyte molecules bound to recognition elements (antibodies, antigens, oligonucleotides, bioreceptors, etc.) at the sensor surface are strongly excited. This (6) Eldada, L.; Xu, C.; Stengel, K. M. T.; Shacklette, L. W.; Yardley, J. T. J. Lightwave Technol. 1996, 14, 1704-1713. (7) Berger, C. E. H.; Beumer, T. A. M.; Kooyman, R. P. H.; Greve, J. Anal. Chem. 1998, 70, 703-706. (8) Lukosz, W. Sens. Actuators, B 1995, 29, 37-50. (9) Du ¨ bendorfer, J.; Kunz, R. E.; Schu ¨ rmann, E.; Duveneck, G. L.; Ehrat, M. J. Biomed. Opt. 1997, 2, 391-400. (10) Neuscha¨fer, D.; Budach, W.; Ba¨r, E.; Pawlak, M.; Duveneck, G. L. Proc. SPIE-Int. Soc. Opt. Eng. 1996, 2836, 221-234. (11) Duveneck, G. L.; Pawlak, M.; Neuscha¨fer, D.; Ba¨r, E.; Budach, W.; Pieles, U.; Ehrat, M. Sens. Actuators, B 1997, 38-39, 88-95. (12) Abel, A. P.; Weller, M. G.; Duveneck, G. L.; Ehrat, M.; Widmer, H. M. Anal. Chem. 1996, 68, 2905-2912. (13) Pawlak, M.; Grell, E.; Schick, E.; Ansellmetti, D.; Ehrat, M. Faraday Discuss., submitted for publication. (14) Eigen, M.; Rigler, R. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5740-5747. (15) Chiu, D. T.; Zare, R. N. Chem.sEur. J. 1997, 3, 335-339. (16) Fister, J. C.; Jacobson, St. C.; Davis, L. M.; Ramsey, J. M. Anal. Chem. 1998, 70, 431-437. (17) Srivastava, R.; Bao, C.; Go´mez-Reino, C. Sens. Actuators, A 1996, 165171. (18) Kogelnik, H. In Guided-Wave Optoelectronics, 2nd ed.; Tamir, Th., Ed.; Springer-Verlag: New York, 1990; Chapter 2. (19) Yeh, P. Optical Waves in Layered Media; John Wiley & Sons: New York, 1988.

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contrasts with impurities or unbound fluorophores which remain outside the evanescent field in the electronic ground state. This inherent feature of the planar waveguides allows effective discrimination between bound and unbound labeled affinity partners and also allows the analysis of either scattering or absorbing samples which might contain blood, serum, or cell extracts. A major challenge for the construction of multiplexed biosensor platforms is the realization of structured immobilization of multiple recognition elements. A variety of methods, including photoimmobilization20 (i.e., on-chip synthesis of oligonucleotides via photodeprotection), gel pad immobilization,21 dip-and-spot techniques,22 microcontact printing23 (µCP), ink-jet printing,24 and laser ablation25) have been developed. However, all these methods differ widely in their performance, e.g. morphology of the sensing pad, number of active recognition elements per area, surface contact of the printing device, and carryover of recognition elements. Thus, the decision for a specific immobilization method has a strong influence on both the format and performance of the envisioned sensor platform. The single-channel ink-jet printer used in the present study allowed precise deposition of minute amounts of recognition elements (approximately 500 pL per droplet) in combination with a well-defined circular shape and flexible architecture of the sensing pads. However, the handling of multiple capture probes is complicated since a fast parallel printing approach requires individual printing heads for each compound when compared to a simpler sequential approach that would be relatively time consuming. Printing heads with multiple jets to generate an equivalent number of recognition elements are crucial in terms of cross-contamination, and the immobilization remains relatively time consuming as long as the pitches of jets and sensing pad spacings do not match. The surface functionalization of the transducers is an important prerequisite for the immobilization in order to attach the capture probes covalently at the sensor surface. This was accomplished by silanization of planar waveguides with (3-glycidoxypropyl)trimethoxysilane (GOPTS), which leads to epoxy-functionalized waveguide surfaces.26 For the patterned immobilization, oligonucleotides were used with an amino function at the 5′-end, assuming covalent coupling of the capture probes to the modified waveguide surface. In the present paper, results are reported that were obtained with ink-jet immobilization of oligonucleotide capture probes on planar waveguides in order to gain information on ink-jet performance, feasibility of covalent immobilization of oligonucleotides, and, finally, on the detection limits resulting from checkerboardlike immobilized oligonucleotide capture probes on fluorescencebased planar waveguide biosensors. (20) Fodor, St. P. A. Science 1997, 277, 393-395. (21) Guschin, D.; Yershov, G.; Zaslavsky, A.; Gemmell, A.; Shick, V.; Proudnikov, D.; Arenkov, P.; Mirzabekov, A. Anal. Biochem. 1997, 250, 203-211. (22) Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. Science 1995, 467470. (23) Bernard, A.; Delamarche, E.; Schmid, H.; Michel, B.; Bosshard, H. R.; Biebuyck, H. Langmuir 1998, 24, 2226-2229. (24) Baldeschwieler, J. D.; Gamble, R. C.; Theriault, T. P. World Patent Application, WO 95/25116, 1995. (25) Schwarz, A.; Rossier, J. S.; Roulet, E.; Mermod, N.; Roberts, M. A.; Girault, H. H. Langmuir 1998, 14, 5526-5531. (26) Maskos, U.; Southern, E. M. Nucleic Acids Res. 1992, 20, 1679-1684.

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EXPERIMENTAL SECTION Chemicals. Chloroform (puriss. p.a., >99.8% (GC)), acetonitrile (for HPLC, >99.8% (GC)), and o-xylene (puriss. p.a., >99% (GC)) were used as solvents (Fluka). The silanization reagents (3-glycidoxypropyl)trimethoxysilane (purum, >97% (GC)) and N-ethyldiisopropylamine (purum, >98% (GC)) were purchased from Fluka. The complementary 16-mer and 22-mer oligonucleotide pairs, purchased from Microsynth (Balgach, Switzerland; HPLC-purified), were labeled at the 5′-end with either an amino group or a Cy5 fluorophor (addition of a linker phosphoramidite on a DNA synthesizer, followed by coupling to a Cy5-carboxylic acid NHS ester, according to the Amersham labeling kit). The following abbreviations were used for the oligonucleotides: 16*NH2, H2N-5′-CAC AAT TCC ACA CAA C-3′; 16*cCy5, complementary 16-mer oligonucleotide with a Cy5 label at the 5′-end; 22*NH2, H2N-5′-CAC TTC ACT TTC TTT CCA AGA G-3′; 22*cCy5, complimentary 16-mer oligonucleotide with a Cy5 label at the 5′end. Stock solutions (10 nM) of both Cy5-labeled oligonucleotides were prepared in hybridization buffers (the concentrations were determined via UV absorption at 260 nm with a Perkin-Elmer Lambda 2 spectrometer). Subsequently, the fluorescence intensities of the stock solutions at 650 nm emission wavelength were determined with a Perkin-Elmer LS50B luminescence spectrometer (excitation at 630 nm). The fluorescence intensity of the 16mer stock solution was found to be about 10% lower than the fluorescence of the 22-mer stock solution. The amino-labeled capture oligonucleotides used for the inkjet printing were dissolved in 50 mM bicarbonate buffer, pH 8.5 (0.21 g of NaHCO3, Fluka, 71627, MicroSelect >99.5%, in 50 mL of deionized water). The concentration of the capture probes was approximately 0.7 mM, and the tracer solutions were prepared by diluting the Cy5-labeled oligonucleotides in the hybridization buffer. A solution containing 75 mL of 70 mM phosphate buffer (pH 7; containing 0.04 M Na2HPO4 and 0.03 M KH2PO4), 6.5 g of KCl, 0.02 g of EDTA‚2H2O, 0.25 g of NaN3, 0.5 g of poly(acrylic acid) 5100 sodium salt, and 0.5 g of Tween 20 was used as the hybridization buffer. The pH was adjusted to pH 7.7 with 1 M NaOH (filled to 1 L with deionized water). The regeneration solution was prepared from 500 g of urea (Fluka, 51456, MicroSelect for molecular biology >99.5%) in 500 mL of deionized water. Poly(dimethoxysilane) (PDMS), the polymer used for the construction of the flow cells, was purchased from Dow Corning (Sylgard 184, Silicone Elastomer Kit). Chip Silanization Protocol. The planar waveguide chips were mounted into chip racks, placed into a glass beaker, covered completely with CHCl3, and then sonicated for 30 min at room temperature. The solvent was decanted, and the chips were sonicated once more for 30 min in fresh CHCl3 and then, to avoid hydration, transferred directly into a solution of 10.0 mL (2% v/v) of 3-(glycidoxypropyl)trimethoxysilane and 1.0 mL (0.2% v/v) of N-ethyldiisopropylamine in 500 mL o-xylene freshly prepared at 52 °C. The crystallizing dish reaction vessel was closed with a watch glass and the reaction mixture stirred at 56-58 °C and 800 rpm. After 7 h, the chips were removed from the reaction mixture, immersed in CH3CN, and sonicated for 5 min. The CH3CN was discarded, and the chips were washed twice with fresh CH3CN,

Figure 1. Experimental setup used for the excitation and detection of fluorophores at the planar waveguide surface: (1) helium-neon laser, 1.3 mW, vertically polarized; (2) goniometer for the adjustment of the coupling angle; (3) diffractive optical element (DOE) or cylindrical lens (f ) 5 mm, convex) for the expansion of the laser beam; (4) convex lens (f ) 200 mm) to collimate the eight diffracted beams retaining a pitch of 1 mm; (5) prism; (6) grating and (7) Ta2O5 waveguide on the glass substrate; (8) PDMS cast with the integrated flow cell; (9) inlets, and outlets. The helium-neon laser, the diffractive optical element, and the 200 mm lens are jointly mounted onto the goniometer stage.

each with 5 min of sonication. The CH3CN was drained off, and the chips were dried for 20 min in a desiccator under vacuum. Ink-Jet Immobilization. A single-channel ink-jet printer (Microdrop GmbH, Norderstedt, Germany) was used for the patterned immobilization of the capture probes. The ink-jet nozzle was coupled to a 1 mL reservoir for the working solution. The ink-jet nozzle and reservoir were washed with 2-propanol and deionized water prior to filling with the working solution. The printer was installed in a chamber with a saturated water vapor atmosphere to reduce the drying of the printed droplets. The reservoir was typically filled with 50 µL of the respective capture oligonucleotide solution to be printed. The reservoir was connected to a pump system that controlled the pressure above the solution. Pressure within the system was reduced by 10 mbar in relation to the ambient pressure to prevent leakage of the working solution. The printing system was mounted to an XYZ translation stage which allowed deposition of single or multiple droplets onto substrates with 10 µm precision. The dispensing frequency was adjusted to 100 Hz and the pulse length to 30 µs (the droplet formation was controlled with a stoboscope/camera assembly). After the printing, the chip was kept overnight in a saturated water vapor atmosphere, then flushed with water, and dried with N2. The smallest achievable sensing pad diameter of 0.1 mm corresponds to 1 droplet/position. Due to the limited optical resolution of the detection system used, spot diameters of 0.8 mm (200 droplets/position) and 0.3 mm (20 droplets/position) were chosen to demonstrate the potential of miniaturization for multiplexed planar waveguide sensors. For comparison of the assay performance, three rows of oligonucleotide spots (200 droplets/position corresponding to a total volume of 0.1 µL/spot, pitch 1 mm) were printed with the ink-jet, each in the upper and lower parts of the chip (cf. Figure 5a). Along the central axis of the chip, 1 µL droplets were applied manually using an Eppendorf pipet. The resulting diameters were 0.8 mm (0.5 mm2 area) for the ink-jet spots at the top and the bottom of the chip and 2 mm (3 mm2 area) for the central spots prepared with the pipet. Calculated values for the dispensed

volumes per spot areas were 0.2 µL/mm2 for the ink-jet and 0.3 µL/mm2 for the manually prepared spots. Immobilization was carried out as described above. Planar Waveguides. Single-mode planar waveguides (dimensions 48 × 16 mm2) were manufactured by Balzers AG (Balzers, Liechtenstein) according to specifications described elsewhere11 [glass substrates were coated with a Ta2O5 layer of 150 nm thickness as high-refractive-index waveguiding material (n ) 2.25), and waveguides had integrated optical gratings (period 320 nm) to launch light into the waveguiding layer]. Dammann Grating Based Multiplexing. The multiplexing concept is based on a diffractive optical element (DOE) known as Dammann grating.27 A DOE is a wave front processor capable of transforming a monochromatic coherent beam into an arbitrary and relatively complex output. The desired output for the present purpose was a fan-out element that splits the incoming beam into a 1-dimensional array of eight beams, each one corresponding to a diffraction order of equal intensity and profile. The Dammann grating used in this study consisted of a binary structure etched in fused silica according to exact specifications (i.e., beam intensities within 5%, relief depth maximized for λ ) 633 nm, and pitch ) 1 mm with a f ) 200 mm lens). Design and fabrication were done by Weible OpTech (Neuchaˆtel, Switzerland). This type of DOE was similarly used by Bruno et al.28 (see Figure 1 in ref 2), where the n diffracted beams propagate parallel and have an internal pitch of 1 mm. This was achieved using a single lens of focal number f placed exactly at a distance f from the DOE. In this way, the lens both collimates the n diffracted beams and focuses them at a distance f; i.e., the beam waist will be located at a distance 2f from the DOE. Excitation and Detection Setup. The experimental setup is shown schematically in Figure 1. A HeNe laser (632.8 nm, 1.3 mW; Melles Griot, Zevenaar, The Netherlands), mounted onto a (27) Dammann, H.; Go¨rtler, K. Opt. Commun. 1971, 3, 312-315. (28) Bruno, A. E.; Baer, E.; Vo¨lkel, R.; Effenhauser, C. S. Micro Total Analysis Systems ‘98; Kluwer Academic Publishers: Boston, MA, 1998; pp 281-285.

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goniometer (MM3000 controller, UBG120 goniometer, Newport Corp., Irvine, CA), was used as the excitation light source. The laser beam was either multiplexed by use of a diffractive optical element as described above or expanded by a cylindrical lens (f ) 5 mm). The alternative cylindrical lens elongated the laser beam into a 1 × 20 mm2 line. In both cases, the beam(s) was (were) adjusted in relation to the incoupling grating by means of a small prism. To achieve a maximum coupling efficiency, both the coupling angle and the position of the laser beam on the coupling grating were optimized. The incoupling grating for launching light into the metal oxide layer lies virtually in the center of rotation of the goniometer, allowing adjustment of the optimum coupling angle without changing the position of the laser on the planar waveguide during alignment. In addition, the flow cell/ transducer unit was shifted along the longitudinal axis of the chip for the optimum position of the incident laser beam in relation to the coupling grating by means of a translation stage (Newport MM3000 controller, 850F linear actuator). The coupling conditions were adjusted prior to each experiment. The coupling angle (≈4° with respect to the surface normal) depends on the refractive indices of substrate, superstrate, and waveguide, thickness of the waveguiding Ta2O5 layer, period of the incoupling grating (320 nm), and wavelength of the HeNe laser (632.8 nm). Propagation losses (scattering, absorption) of the guided modes were 3 dB/ cm for those waveguides used. An Astrocam back-illuminated CCD slow-scan camera was used as the photodetector (LSR Astrocam, Cambridge, U.K.). The camera head carried a thinned EEV 30-11 chip with a 16 bit dynamic range operated at a temperature of -50 °C (Peltier cooling). A Nikon Noct 58 mm/1.2 lens was used as the collection optics (Nikon Corp., Tokyo). Fluorescence was separated from excitation light by means of an Omega interference filter (model 680RDF40, Omega, Brattleboro, VT) centered at the maximum of the Cy5 emission at 680 nm with a 40 nm half-width value. The exposure time was 1 s per image for all measurements, and the laser beam was automatically blocked by a shutter in the dead time between the image acquisitions to reduce photobleaching. Fluidics. The fluidic components consisted of a flow cell coupled to a Cavro valve (six-port), a sample loop29 (sequential injection analysis, SIA), a stepper motor driven syringe (CAVRO Scientific Instruments, Sunnyvale, CA), and a Gilson autosampler (model 221, Gilson, Villier Le Bel, France). The fluidic system allowed automated prewashing with hybridization buffer, incubation with analyte, postwashing to remove unbound material, and regeneration of the sensor chip with a urea solution. Fluidic handling (volumes and flow rates of the respective solutions) and image acquisition (series of fluorescence images) were synchronized by means of a computer. The flow cells were designed inhouse and cast from PMMA masters with poly(dimethoxysilane) (PDMS). A small PDMS flow cell with the dimensions 12 × 7 × 0.2 mm3 (volume 20 µL; schematically shown in Figure 1; flow direction perpendicular to the mode propagation) and a larger one with the dimensions 15 × 8 × 0.2 mm3 (volume 25 µL; not shown; flow direction parallel to the mode propagation) were used as described in the following paragraphs. (29) Ruzicka, J.; Marshall, G. D. Anal. Chem. 1990, 62, 1861-1872.

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RESULTS AND DISCUSSION Multiplexed Model Assays. The objective of this part of the study was to prove the feasibility of ink-jet immobilization of different capture probes on planar waveguide chips. Checkerboardlike patterns with alternating positions of the two capture probes were prepared on the silanized planar waveguides by sequential printing of the two 16-mer and 22-mer capture probes with an amino function at the 5′-end. The transducer was mounted into the flow cell of the experimental setup that was equipped with a cylindrical lens for the laser beam expansion. A total of six consecutive assay cycles were measured at three different analyte concentrations (0.5, 5, and 50 pM; increasing concentration) for both the 22-mer and 16-mer analyte solutions. For each assay cycle, the chip was automatically exposed to the following sequence of solutions: prewash with 0.5 mL of hybridization buffer (1 min), incubation with 0.2 mL of analyte (3.5 min, flow injection), postwash I with 0.2 mL of hybridization buffer (1 min), regeneration with 1 mL of urea solution (2 min), and postwash II with 1 mL of hybridization buffer (1 min). Each individual assay required 12.5 min and included breaks for filling/washing of the sample loop/port. Some 25 fluorescence images were taken from the chip with each assay with a 30 s delay between each image acquisition. Examples of the fluorescence images obtained during the assays are shown in Figure 2. Each of the six assay cycles per chip (three concentrations and two different labeled analytes; see numbering; mode propagation from right to left and flow vertical in the images) is represented by a pair of images in the figure. The patterns with the large-diameter spots are seen in the upper half of each of the six pairs of images. All images were scaled logarithmically (1000-50 000 counts/s) to show the wide dynamic range of the experiment. The left image of each pair represents the background image of the respective assay captured at the start of the assay cycle, whereas the right image corresponds to the maximum fluorescence signal that was obtained after 6 min of incubation for each analyte. The checkerboard-like patterns of the 16-mer (bottom series in Figure 2) and 22-mer (upper series in Figure 2) capture probes on the chip are clearly visible even at the lowest analyte concentration of 0.5 pM, denoted as 1 and 2 in Figure 2. At the beginning of each assay cycle, the chip is almost completely regenerated. The remaining fluorescence signal after the regeneration is below the detection limit of the system. At the highest concentration of 50 pM, denoted as 5 and 6 in Figure 2, the background intensity is slightly increased and is attributed to the scattering of guided fluorescence photons that have been coupled into the high refractive planar waveguide by optical near-field effects.30 At 5 and 50 pM analyte concentrations, the spots appear to be slightly out of focus due to aberrations of the optical system. In addition, the streaky texture of the images is probably caused by small contamination particles at the chip surface acting as scattering centers. In Figure 3, the mean intensities calculated from small oligonucleotide spots of the complete series of fluorescence images are plotted against time and the intensities obtained for the large spots (not shown) are similar. In principle, the intensity (30) Apel, O.; Neuscha¨fer, D.; Pawlak, M.; Roders, O.; Ehrat, M.; Marowsky, G. Submitted for publication in Appl. Opt.

Figure 2. Fluorescence images obtained with six different analyte solutions in oligonucleotide model assays (16-mer and 22-mer labeled oligonucleotides, each in three different concentrations). Each pair of images represents one assay cycle. The left image of each pair corresponds to the background measured at the beginning of the assay; the right image represents the maximum fluorescence signal obtained after 6 min of incubation with the corresponding analyte. All images are scaled logarithmicaly (1000-50 000 counts/s). Dimensions: diameter of large spots, 0.8 mm; diameter of small spots, 0.3 mm. Mode propagation is from right to left in the images.

variations of the individual spots are mainly affected by the Gaussian profile of the laser beam and by the parabolic flow profile of the analyte in the flow cell. As a result of the parabolic flow profile (flow vertical in Figure 2), the amount of analyte molecules applied to the recognition elements at the edges of the flow channel is expected to be reduced. This effect depends on the dimensions of the flow cell, diffusion coefficient of the analyte molecules, and the flow rate.31 Such local differences in analyte delivery can be reduced by injection of the analyte into the flow cell (stopped flow) in combination with an incubation time that allows diffusion of the analyte molecules to all regions of the sensor surface. In addition, a flat intensity profile of the laser beam can be achieved with special beam homogenizers or DOEs. However, the optical design of such elements for planar waveguides requires the maintenance of the nondivergent characteristics of the laser beam. Remaining intensity variations could then be attributed solely to the flow profile of the analyte in the flow cell. In the data calculated from the fluorescence images, the sequential hybridization and dehybridization cycles for three different analyte concentrations are seen for both the 16-mer and the 22-mer oligonucleotides. The 16-mer analytes showed slightly lower fluorescence intensities compared to the 22-mer analytes, most likely due to their lower content of fluorescence-labeled oligonucleotides and the lower stability of the hybridized 16-mer oligonucleotide complexes compared to the 22-mer complexes at room temper(31) Ruzika, J.; Hansen, E. H. Flow Injection Analysis; John Wiley & Sons: New York, 1988.

ature. Thermally induced variations of the laser pointing stability have been identified as the reason for the periodic drifts in the observed baseline. The background intensity of the chip was found to increase by about 2% of the net fluorescence signal (Figure 3) which can be attributed to scattering of guided fluorescence photons. This effect is most clearly visible for the 50 pM concentrations of the analytes: the signal for the 16-mer oligonucleotide spots is increased by about 1000 counts/s while the incubation with the 22-mer analyte gives a net signal of about 5 × 104 counts/s (and vice versa). In a negative control experiment, a chip with immobilized 22-mer capture oligonucleotide spots showed no significant increase of the background intensity (10 count/s) upon exposure to a 50 pM solution of a noncomplementary labeled oligonucleotide (see inset Figure 3). Bare regions of the chip and regions where the capture oligonucleotides were immobilized showed similar background characteristics. To determine the limit of the detection (LOD), the “checkerboard” chip was exposed to a 50 fM solution of 22*cCy5 as the analyte. The signal development was analyzed by extracting horizontal signal intensity profiles from the fluorescence images as indicated in Figure 4, and the profiles corresponded to a cross section through eight small capture spots: four of the 22-mer and four of the 16-mer capture probes. Intensity profiles obtained at the start of the assay (chip in contact with hybridization buffer) and after incubation with the 50 fM analyte, denoted as “baseline” and “22*cCy5”, are plotted in Figure 4. Upon incubation with the 22* analyte (200 µL), the fluorescence signals of the four 22-mer oligonucleotide sensing pads Analytical Chemistry, Vol. 71, No. 16, August 15, 1999

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Figure 3. Signal development as a function of assay time calculated from small oligonucleotide spots of a series of fluorescence images that were recorded during the course of model assays with two analytes each in three concentrations (six assay cycles). The CCD camera has an offset of 1000 counts/s. Insert: negative control with 50 pM noncomplementary labeled oligonucleotide as the analyte.

Figure 4. Determination of the level of detection (LOD). Intensity profiles obtained at the start of the assay and after incubation with 50 fM 22-mer analyte are denoted as “baseline” and “22*cCy5”.

exceeded the value for the sum of the baseline and 3 × standard deviation (SD), thus demonstrating a detection limit of about 50 fM, while the intensities of the four 16-mer oligonucleotide sensing pads remained almost unchanged. 3352 Analytical Chemistry, Vol. 71, No. 16, August 15, 1999

To determine the stability and regenerability of the immobilized capture probes, repetitive assay cycles were measured with constant analyte concentration. The coefficient of variation for the net signal was found to be 2%, and a loss of binding capacity

Figure 5. (a) Fluorescence image of manually deposited (by means of pipet, large spots, in the middle of the chip) and ink-jet-deposited (small spots, in the upper and lower parts of the chip) oligonucleotide sensing pads after incubation with labeled analyte. The squares and circles indicate the areas of interest which have been analyzed to compare the assay performances. Mode propagation is from right to left in the images. (b) Comparison of the mean fluorescence intensities calculated from various regions of interest as indicated in Figure 5a.

of the capture probes was not detectable (data not shown). Ink-jet technology has been shown to be valuable for the preparation of oligonucleotide sensing pads. The results reported in the present paper do not make use of the full miniaturization potential since the diameter of the sensing pads was 0.3 mm and larger (multiple droplets per position). A further miniaturization could be achieved by printing arrays of single droplets resulting in sensing pad diameters of 100 µm. Dimensions could be further reduced by the use of even smaller jets. The time requirement for the exchange of a large number of solutions and the lack of controls for jet functioning (droplet formation is very sensitive regarding impurities and air bubbles/spots) are technical hurdles which have to be overcome for arraying multiple capture elements routinely with such instruments. Comparison of Assay Performance. To compare the assay performances between ink-jet- and pipet-immobilized oligonucleotides, a 16-mer 5′-amino oligonucleotide was immobilized by means of ink-jet printing, as well as by manual application using a pipet, onto a planar waveguide. For this experiment, the planar waveguide setup was equipped with a Dammann grating to divide the laser beam into eight parallel excitation beams of equal intensity (pitch 1 mm). The chip and the laser beams were aligned to center the ink-jet spots (cf. Figure 5a). Stopped-flow conditions were used to avoid a parabolic flow profile and thus reduce the fluidic induced inhomogeneities in the fluorescence pattern; i.e., after a 3 min prewash with the hybridization buffer, the flow cell volume (25 µL) was completely exchanged with 10 pM labeled analyte. Fluorescence images were automatically taken each 50 s. A fluorescence image captured 4 min after incubation of the analyte (central area of the chip) is shown in Figure 5a.

In the upper and the lower parts of the image, all six rows of the ink-jet-prepared spots can be monitored. Because the dimension of the laser beams is smaller than the diameter of the oligonucleotide spots, the Gaussian profile of each of the eight laser beams appears in the fluorescence pattern. Since the modes propagate from right to left in Figure 5a, the fluorescence intensity also slightly decreases from right to left due to losses of the guided mode. In addition, the intensity profile of the laser beam has to be taken into account for the quantitative comparison of the fluorescence intensities of the various spots, since the mean intensity depends on the geometrical definition of the analyzed image regions. This effect is seen in Figure 5b. The mean values computed from the large circular regions (O ink-jet in Figure 5) are lower than the mean values calculated from small square regions (0 ink-jet in Figure 5) that were centered at the maximum intensity of the laser beam. To compensate for such differences in the sensing pad geometry and excitation, small squares centered in the maximum of the respective spots were chosen to compare the mean net signals of ink-jet- and pipet-prepared spots (0 ink-jet and 0 pipet in Figure 5). The manually prepared spots systematically showed a 30% higher net signal in comparison to the ink-jet-prepared spots. All fluorescing spots displayed higher intensities at the edges in comparison to the center of the respective spots, indicating inhomogeneities within the capture spots. These spot inhomogeneities were probably due to processes that occurred during the immobilization procedure, since it has been reported that drying solution droplets at surfaces show a material transport from the center to the edges of the droplet resulting in crust formation at the edges.32 Besides the variations in the spot morphology, the Analytical Chemistry, Vol. 71, No. 16, August 15, 1999

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This can be attributed to the photobleaching of the fluorophores in the evanescent field and/or desorption due to photoinduced warming of the surface. A variety of reasons contribute to the spot-to-spot signal variations and comprise inhomogeneous analyte distribution within the flow cell and differences in the local light intensity caused by mode jumping or damping of the mode or by energy dissipation due to scattering centers at the chip surface as well as inhomogeneous immobilization of the capture probes due to variations of the ink-jet printer itself. CONCLUSION

Figure 6. Mean fluorescence net signals and spot-to-spot variations of ink-jet-made oligonucleotide spots (Figure 5a) after injection of labeled analyte as a function of assay time.

higher net signal of the manually prepared spots can be explained by the different amount of oligonucleotide solution per area applied in the above immobilization procedure. A 10 pM analyte solution was used for the experiments (stopped flow). Therefore, the absolute number of analyte molecules in the flow cell (25 µL volume) was approximately 2 × 108. The entire area of all sensing pads on the chip was 60 mm2 (6 rows with 15 ink-jet spots having 0.5 mm2 area per spot and 5 pipet spots each having 3 mm2 area). The resulting net signals (mean) plotted in Figure 5b (0 ink-jet and 0 pipet) were larger than 5000 counts/s and exceeded the limit of detection (3 × SD of baseline ) 342 counts/s) by a factor >10. The measured signal intensities corresponded to about 3 fluorophores/µm2 of sensing pad area (under the assumption that all labeled oligonucleotides are bound to the chip surface) and thus demonstrate the high sensitivity of fluorescence-based evanescent field biosensors. Spot-to-Spot Reproducibility. The spot-to-spot signal variations of the ink-jet sensing pads in relation to the assay time were investigated in detail to reduce the assay duration. For this experiment, the analyte was applied under stopped-flow conditions as described in the previous section. The mean fluorescence net signal from six ink-jet spots and the resulting standard deviations calculated from the circular regions as indicated in Figure 5a as a function of assay time are shown in Figure 6. An increase in the mean fluorescence signal of the six ink-jet spots was seen immediately after injection of the analyte and reached the maximum 3 min after the injection. The standard deviations (error bars in Figure 6) as a function of assay time appeared to be almost independent of the formation of the chemical equilibrium between capture probes and analyte molecules and remained nearly constant after the increase of the signal. This feature allows the assay time to be shortened for applications where absolute precision, in terms of equilibrium and removal of nonspecifically bound labels, is not required but a short time to result is needed. About 4 min after the injection of the analyte, the signal intensity started to decrease with assay time. (32) Deegan, R. D.; Bakajin, O.; Dupont, T. F.; Huber, G.; Nagel, S. R.; Witten, T. A. Nature 1997, 398, 827-829.

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Planar waveguides enable efficient evanescent field excitation of surface-bound chromophores and thus sensitive fluorescencebased detection of 2-dimensional arrays of recognition elements in a nonscanning approach. This is of particular interest for the measurement of reaction kinetics and for applications where a short time to result is needed without compromising high sensitivity. In the present paper, the potential of planar waveguide transducers was demonstrated in multiplexed hybridization model assays with capture oligonucleotides, arranged in checkerboardlike arrays, using fluorescence-labeled oligonucleotides as the analytes. For the preparation of the oligonucleotide arrays, the surfaces of the planar waveguides were epoxy-functionalized with GOPTS and 5′-amino oligonucleotides were immobilized on the chip surfaces by means of ink-jet printing. The arrayed planar waveguide chips prepared in this way showed an improved assay performance (detection of 50 fM analyte), could be regenerated by urea solution, and were used many times without visible loss of activity. The spot-to-spot variations of the sensing pads were found to be almost independent of the equilibrium between capture probes and analyte molecules, and the observed regenerability and signal reproducibility of the oligonucleotide chips appear suitable for multiple-use applications. The ink-jet system allows the precise deposition of recognition elements, since the applied quantities and the resulting circular sensing pad shape are highly reproducible. Manually prepared and ink-jet prepared oligonucleotide spots have both shown similar assay performances, indicating that there was no significant degradation of the oligonucleotides due to physical processes, such as acceleration of solution or exposure to sound waves and shear forces occurring within the ink-jet. Fabrication of arrays with large numbers of recognition elements appears technical challenging and requires, depending on the application, specially constructed printing systems. Future work will focus on the realization of multiplexed biosensors based on the evanescent field excitation. ACKNOWLEDGMENT The authors thank Markus Ehrat and Gerhard Kresbach for related discussions and helpful suggestions, Gerolf Kraus for aid in the fluidic setup, Edgar Baer and Erwin Baeriswyl for help in the experimental setup, Andreas Helg for support in the silanization of the planar waveguides, Reinhard Vo¨lkel (IMT, Universiy of Neuchaˆtel) for suggestions regarding the optical setup, and

Paul Skelton-Stroud for reading and correcting the original manuscript. Abbreviations: 16*/22*, 16/22-mer oligonucleotide; CCD, charge-coupled device; CV, coefficient of variation; Cy5, Amersham cyanine dye; DOE, diffractive optical element; GOPTS, (3-glycidoxypropyl)trimethoxysilane; HeNe laser, helium-neon laser; LOD, limit of detection; µCP, microcontact printing; PMMA, poly-

(methyl methacrylate); PDMS, poly(dimethoxysilane); SD, standard deviation; SIA, sequential injection analysis.

Received for review January 28, 1999. Accepted May 10, 1999. AC990092E

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