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A Scanning Near-Field Optical Microscope Approach to Biomolecule Patterning Claude Philipona,† Yann Chevolot,‡ Didier Le´onard,‡,§ Hans Jo¨rg Mathieu,‡ Hans Sigrist,| and Fabienne Marquis-Weible*,† Institut d’Optique Applique´e, Ecole polytechnique Fe´de´rale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland, De´partement des Mate´riaux, Ecole polytechnique Fe´de´rale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland, and Centre Suisse d’Electronique et de Microtechnique, Jaquet-Droz 1, CH 2007 Neuchaˆtel, Switzerland. Received July 20, 2000; Revised Manuscript Received December 20, 2000
In view of future generations of biosensors and advanced biomaterials, photochemistry in the near field using scanning near-field optical microscopy is investigated. The potential of direct near-fieldinduced photoactivation is demonstrated on standard photoresist. Photoimmobilization of maleimidoaryldiazirine on silicon substrates and bovine serum albumin on glass substrates is achieved, opening the way to a controlled biopatterning of surfaces with submicrometer feature size. The obtained patterns are characterized using atomic force microscopy, time-of-flight secondary ion mass spectroscopy (ToF-SIMS), and near-field fluorescence microscopy.
INTRODUCTION
In view of future generations of biosensors and advanced biomaterials, the construction and integration of complex biological systems onto selected materials or transducers is of primary importance. Chemical treatment of a material surface through functionalization is the first step in engineering of biosensors and biomaterials. Immobilization of biomolecules on transducer surfaces can be achieved by different means: physical adsorption (physisorption), receptor-mediated adsorption (bioaffinity), or covalent binding. The first two processes are reversible, while the latter leads to the formation of irreversible chemical bonds (1). An elegant way toward covalent immobilization is the use of photoactivable reagents. Indeed, the interaction of light with the reagents induces reaction intermediates that can lead to covalent chemical bonds to the substrate surface. Among the photoactivable reagents available, aryldiazirine has been successfully used on a wide range of materials (from polymer to metal oxides) (2). Its activation wavelength is in the range of 350 nm, which does not impair biological activity. Meanwhile, topical addressability, which allows multicomponent domain-limited devices, has been achieved with a standard masking technique as it is commonly used in microtechnology. However, this technique is limited in spatial resolution by the diffraction of light. The use of deep-UV wavelengths to improve the resolution is not possible without destroying the biological properties of the attached molecules. Future * To whom correspondence should be addressed. Phone: ++41 21 693 7012. Fax ++41 21 693 3701. E-mail:
[email protected]. † Institut d’Optique Applique ´ e, Ecole polytechnique Fe´de´rale de Lausanne (EPFL). ‡ De ´ partement des Mate´riaux, Ecole polytechnique Fe´de´rale de Lausanne (EPFL). § Current address: GE Plastics, Analytical Technology, Plasticslaan 1, PO Box 117, 4600 AC Bergen op Zoom, The Netherlands. | Centre Suisse d’Electronique et de Microtechnique.
needs for photopatterning of biomolecules include high spatial localization of the biomolecules, and thus it involves the development of new techniques going beyond the diffraction limit. Near-field illumination potentially allows to overcome this limit. Scanning near-field optical microscopy (SNOM) is, in first use, a technique to optically investigate surfaces beyond the diffraction limit. An optical probe is scanned a few nanometers above the object to be investigated where it interacts with the sample via the near electromagnetic field either emitted or scattered by the probe or by the object. The resolution of SNOM is not limited by diffraction, but rather by the size of the optical probe and the distance between the probe and the sample. Although already proposed decades ago (3), the principle has been rediscovered and experimentally realized only in 1984 with the demonstration of optical resolution below diffraction limit (4), when technology allowed fabrication of sub-wavelength size probes and control of its distance to the sample with nanometer precision. This technique offers a wide area of applications for the study of biomaterials, allowing exploitation of most of the optical contrast mechanisms (fluorescence, phase, polarization, etc). SNOM has been successfully used in investigation of single molecules (5), cells (6), and in combination with fluorescence energy transfer (7). This work demonstrates that SNOM can be used not only as an observation tool, but also as a structuring tool, that exploits the high localization of the light in the probe’s near field, to very locally induce photochemical effects. Combining SNOM lateral resolution with photochemical immobilization of aryldiazirine on material surfaces, we perform near-field photochemistry to immobilize nanostructures on surfaces obtaining submicrometric domains of recognition. In this paper, we first illustrate the potential of direct near-field induced photoactivation on standard photoresist and show that subwavelength patterning is achievable. In a second step, immobilization of maleimidoaryldiazirine (MAD) on a
10.1021/bc0000841 CCC: $20.00 © 2001 American Chemical Society Published on Web 04/14/2001
Biomolecule Patterning by Near-Field Optical Microscopy
silicon substrate is obtained by near-field photoactivation, opening the way to a controlled biopatterning of surfaces. This configuration offers the advantage of a small molecule that forms a thin and homogeneous layer on the sample surface. Finally, the technique is applied to photoimmobilize a larger biomolecule (bovine serum albumin, BSA), obtaining patterns of proteins with submicrometer feature size on a glass substrate. EXPERIMENTAL SECTION
Near-Field Instrumentation. Near-field photoimmobilization and near-field sample observation are performed with a homemade scanning near-field optical microscope, mounted on a standard inverted optical microscope (Zeiss Axiovert 100). The SNOM head is composed of a near-field probe holder and a compact distance control module. The probe-to-sample distance control is based on interferometric detection of the lateral force induced when the tip is laterally vibrating over the sample. This technique, described in detail in ref 8, allows interaction forces between the probe and the sample to be kept constant. Assuming that the distance-to-sample is directly correlated to the interaction forces, topographical information of the sample is obtained. The near-field probes are obtained by chemical etching of monomode optical fibers and subsequent metallization with aluminum (9). Before using them for photoimmobilization, the optical probe quality is tested by a set of optical measurements with a characterization of transmission and light distribution at the apex. Scanning of a calibrated test object allows evaluation of the diameter of the optical probe. Tips used in this study show a diameter between 60 and 110 nm with transmission between 6 × 10-4 and 5 × 10-3. The samples are scanned under the tip with an x-y-z linear piezo scanner (Physik Instrument). The linearity and the absence of creep are ensured by a closed loop control of the scanner. Near-field photochemistry is induced by UV light emitted from an argon laser (Spectra Physics 2020) coupled into the optical fiber-based probe. Detection of fluorescein-labeled BSA is performed by near-field fluorescence, excited by 488 nm Argon laser light. Emitted fluorescence light is collected by a microscope objective (Zeiss 40x, N.A: 0.6) and, after filtration of the excitation light with a notch filter (OD6), it is finally analyzed with an avalanche photodiode (APD, EG&G, SPCMAQ131FC). Photoresist. Photoresists are radiation-sensitive materials commonly used in semiconductor technology for patterning. The resist is exposed to ultraviolet radiation through a mask containing the desired patterns. In positive photoresists, the exposed regions are made soluble, whereas in negative photoresists they remain insoluble and the nonilluminated resist is dissolved away. A diazoquinone-inhibited novolak positive photoresist S1813 available from Shipley is used for all experiments. The hydrophobic diazoquinone inhibitor strongly absorbs UV light (350-400 nm). The addition of the hydrophobic inhibitor to the hydrophilic novolak resin retards the dissolution of the mixture in alkaline developer. Upon photoexposure, the inhibitor is destroyed and rearranged into hydrophilic acid, which dissolves in the alkaline developer. The coating and the developing processes are made using Rite Track 88 series equipment. Four-inch diameter plain silicon wafers are coated with 1.5 µm photoresist. MF-319 is used for development of the exposed wafer. The obtained patterns are preliminary
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observed with a far-field optical microscope. Precise characterization of patterns is carried out with an atomic force microscope (Autoprobe CP, Thermomicroscopes). Photoreagents. N-(m-(3-(Trifluoromethyl)diazirine3-yl)phenyl)-4-maleimidobutyramide (MAD) is synthesized according to Collioud et al. (10). Silicon wafers with a naturally grown oxide layer are used as substrate. The 5 × 5 mm2 samples are cut from the wafer, washed with an ultrasonic treatment (2 × 5 min in ethanol Uvasol and then 2 × 5 min in hexane Uvasol), and dried for 2 h at 10-2 mbar. The MAD solution (0.5 mM in ethanol, 10 µL) is deposited as a droplet released by a micropipette. The samples are dried for 2 h at room temperature under vacuum of 50 mbar before irradiation. Irradiation is performed at 350 nm, keeping a constant irradiance of 3.8 mW/cm2 during 5 min. The samples are then washed according to the following process: 2 × 5 min ethanol Uvasol, 2 × 5 min hexane Uvasol. Samples are characterized and analyzed with time-of-flight secondary ion mass spectroscopy (ToF-SIMS). The ToF-SIMS system used is a mass spectrometer from PHI-EVANS & Associates (described in detail in (11)) equipped with a pulsed FEI Ga+ ion gun operated at 25 kV for high lateral resolution (unbunched beam). Sample surface is biased ( 3 kV with respect to the grounded extraction electrode for positive and negative mode SIMS, respectively. The 400 pA DC ion beam current is pulsed at 5 kHz repetition rate (8 ns pulse width, measured with the unbunched beam). The analyzed area is estimated to 126 × 126 µm2. The analysis is performed under so-called “static” conditions with an ion dose in the order of 9 × 1011 ions/cm2. F- is the most characteristic ion of the MAD molecule (12), and images of this ion are displayed to demonstrate the topical addressability with ToF-SIMS imaging. Bovine serum albumin (BSA) is functionalized with 3-(trifluoromethyl)-3-(m-isocyanophenyl)diazirine (TRIMID), a light activable agent that absorbs UV light at 350 nm to obtain T-BSA. The detailed protocol for the preparation of T-BSA is described in (13). After modification, T-BSA carries 7 to 9 mol aryldiazirines per mole of BSA. For subsequent fluorescence detection, BSA labeled with fluorescein (F-BSA) is mixed with T-BSA. Glass substrates are prepared according to the following protocol: overnight treatment in HCl 37%, 3 × 5 min sonification in Millipore quality water, and drying 2 h under vacuum/3 mbar at ambient temperature. A solution of proteins (4 µg of F-BSA, 4 µg of T-BSA, 10 µL of PBS 1:100 ) 0.5 mM sodium phosphate buffer pH 7.4 containing 1.5 mM NaCl) is deposited on the glass substrate as a droplet released from a micropipette and then dried under vacuum at 30 to 40 mbar. After photoimmobilization by UV argon laser light, the sample is washed to eliminate the physically adsorbed molecules according to the following process: 3 × 10 min in 3 M KSCN, overnight in PBS/0.05% Tween solution, 3 × 10 min in PBS/0.02% Tween, 2 × 5 min in PBS 1:100, 10 min in 10 mM HCl, 3 × 5 min in Millipore quality water. The obtained patterns are observed and characterized using near-field fluorescence optical microscopy and atomic force microscopy in contact and noncontact mode (Autoprobe CP, Thermomicroscopes). RESULTS
Cruciform patterns have been polymerized into photoresist, scanning the near-field probe above the deposited resist in two successive perpendicular lines. Figure 1 shows an AFM observation (contact mode) of the resulting pattern obtained after resist development. The
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Figure 1. AFM recording of a near-field-induced cruciform pattern in S1813 positive photoresist (AFM image by E. Dupas, IGA-EPFL).
Figure 2. Imaging of ToF-SIMS (registration of F-) of a MADcoated surface after mask-assisted photopatterning.
estimated size of the near-field probe used in this experiment is 120 nm (optical aperture), for a transmission of 9.2 × 10-4. This leads to an estimated illumination dose of 29.8 mJ/cm2. The line width measured at a typical line section is 223 nm FWMH, with a depth of 43 nm. This shows that sub-wavelength patterning is possible with the SNOM technique, even with a standard photoresist deposited as a 1.5 µm thick layer. The same technique applied to polymerize the photoresist is used to photobond a cruciform pattern of biomolecules. Two approaches are considered: In the first one, demonstration of near-field photoimmobilization is performed with a low molecular weight diazirine-based molecule (MAD), that can be used as a “building block” in the functionalization of surfaces. In the second approach, a biologically relevant molecule is directly photoimmobilized onto the surface. This is demonstrated using BSA functionalized with the photoactive diazirine and illuminated in the near-field of the probe. Figure 2 is a ToF-SIMS image of MAD photobonded onto a Si surface. The photobonding is obtained by illuminating a monolayer of MAD deposited onto a Si wafer, using a mask patterning technique with parallel beam far field illumination with a light irradiance of 8.5 mW/cm2. The light dose for optimal photobonding of MAD is measured in the range between 1.1 J/cm2 to 1.6 J/cm2. At lower doses, no immobilization occurs. The square
Philipona et al.
patterns of 40 × 40 µm2 show a transition width of 2 µm from immobilized to nonimmobilized zones. Using nearfield immobilization with a comparable light dose, it is possible to immobilize MAD with a finer spatial resolution. However, we have not succeeded in clearly demonstrating sub-wavelength patterns of photoimmobilized MAD. This is related to the difficulty to observe a deposited monolayer with submicrometer lateral feature size. Several analysis techniques have been applied to observe the near-field immobilized MAD pattern, such as ToF-SIMS and scanning microscopies (AFM, SEM, fluorescence SNOM). ToF-SIMS submicrometer imaging is in principle possible. However, it requires a small probed area that implies the use of an increased ion dose. This leads, in the case discussed here, to surface damage, which prohibits the acquisition of images on ultrathin submicrometer MAD structures. Further imaging techniques such as AFM, or SEM of OsO4-treated surfaces (OsO4 reacts with the maleimido group, thus enhancing contrast), do not lead to a sufficient contrast to allow quantitative characterization of the patterns. Even nearfield fluorescence microscopy with fluorescence-labeled MAD leads to inconclusive results when performed on immobilized monolayer. This is attributed to quenching of the fluorescence when the fluorescent marker is directly bound to the surface. In a second approach, biomolecules have been photoimmobilized using the same technique. This is performed onto a glass substrate using BSA, previously functionalized with the photoactivable diazirine and labeled with fluorescein isothiocyanate for subsequent detection. Immobilization in the near-field is obtained with a light dose of 2.1 J/cm2. The threshold for immobilization is sharp at the lower end. When the dose is increased up to 5 times above that threshold, no significant difference in the attachment is observed. The thickness of the adsorbed proteins is measured by AFM before immobilization, the average result being 165 nm (( 30 nm). Far-field fluorescence microscopy is used to localize where immobilization has taken place and to optimize the illumination dose. Working with small structures and low concentration requires an optimal washing process. This process, described in the previous paragraph, is 2.5 times more efficient to eliminate the adsorbed proteins compared to standard procedures without chaotropic agents. Figure 3 shows the topography (a) and the near-field fluorescence image (b) of the immobilized BSA pattern. Observation and immobilization are performed with the same tip. The cruciform pattern is clearly visible, both in topography and as near-field fluorescence contrast. Figure 4 shows the topographical and light profile of the smallest structure obtained. The width (FWHM) of the topographical profile is 470 nm. The immobilized proteins thickness is 40 nm ( 15 nm as confirmed by independent AFM observations. For comparison, the best resolution obtained on BSA by mask-assisted photoimmobilization is 3.8 µm applying mask-contact modes and 1.9 µm with the mask-projection procedure. DISCUSSION
The potentiality of SNOM for nanostructuring is clearly demonstrated using standard photoresist chemistry. Sub-wavelength feature size has been obtained with standard coating and development processes. Improvement in feature size can be obtained by using an ultrathin photoresist layer (
