Photothermal Laser Fabrication of Micro- and Nanostructured

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Photothermal Laser Fabrication of Micro- and Nanostructured Chemical Templates for Directed Protein Immobilization Anja Schröter, Steffen Franzka, and Nils Hartmann Langmuir, Just Accepted Manuscript • DOI: 10.1021/la503814n • Publication Date (Web): 14 Nov 2014 Downloaded from http://pubs.acs.org on November 26, 2014

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Photothermal Laser Fabrication of Micro- and Nanostructured Chemical Templates for Directed Protein Immobilization Anja Schröter, Steffen Franzka, Nils Hartmann* Fakultät für Chemie, Universität Duisburg-Essen, 45117 Essen, Germany; CENIDE – Center for Nanointegration Duisburg-Essen, 47048 Duisburg, Germany Photothermal patterning of polyethylene glycol terminated organic monolayers on surfaceoxidized silicon substrates is carried out using a microfocused beam of a CW laser operated a wavelength of 532 nm. Trichlorosilane and trimethoxysilane precursors are used for coating. Monolayers from trimethoxysilane precursors show negligible unspecific protein adsorption in the background, i. e. provide platforms of superior protein-repellency. Laser patterning results in decomposition of the monolayers and yields chemical templates for directed immobilization of proteins at predefined positions. Characterization is carried out via complementary analytical methods including fluorescence microscopy, atomic force microscopy and scanning electron microscopy. Appropriate labeling techniques (fluorescent markers and gold clusters) and substrates (native and thermally oxidized silicon substrates) are chosen in order to facilitate identification of protein adsorption and ensure high sensitivity and selectivity. Variation of the laser parameters at a 1/e2 spot diameter of 2.8 µm allows for fabrication of protein binding

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domains with diameters on the micrometer and nanometer length scale. Minimum domain sizes are about 300 nm. In addition to unspecific protein adsorption on as-patterned monolayers, biotin-streptavidin coupling chemistry is exploited for specific protein binding. This approach represents a novel facile laser-based means for fabrication of protein micro- and nanopatterns. The routine is readily applicable to femtosecond laser processing of glass substrates for the fabrication of transparent templates.

*To whom correspondence should be addressed: Tel: +49 203 379 8033. Email: [email protected]


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1. Introduction Patterning of self-assembled organic monolayers (SAMs) has evolved as a versatile approach towards well-defined micro- and nanostructured chemical templates for directed adsorption of biomolecules,1-4 e. g. targeting biochip fabrication and cell studies.5-7 A common strategy for directed immobilization of proteins considers patterning of polyethylene glycol (PEG) terminated monolayers.1-4 PEG-terminated monolayers are protein-repellent coatings.8-9 During patterning the monolayers are decomposed or modified. This allows for local protein adsorption in predefined domains via unspecific interactions.1-4 In addition, a variety of chemical strategies can be used for specific protein binding.10 Along this path distinct patterning techniques have been employed including photolithography, electron beam lithography and scanning probe techniques.11-13 More recently, also laser patterning have come into play.14-17

The general

interest in employing laser techniques originates from their peculiar set of technical features, which makes them a valuable tool in fundamental research as well as medical and technical applications ranging from implant fabrication to optical data storage.18 Generally, laser techniques offer fast processing speeds and a high degree of flexibility.14,18 Also, parallel processing of large-areas is feasible, e. g. using micro lens arrays or interference patterns.15-16,1819

In addition, direct patterning of glass – the preferred platform in many biomedical and

biotechnical applications – is feasible and processing can be carried out in ambient air or in liquid environments, i. e. in aqueous solutions.14, 18, 20 Micropatterning can be achieved following standard procedures.18 Optimization of the laser parameters allows one to maximize the size of the fabricated structures.21 When it comes to nanopatterning, however, the optical diffraction limit represents a significant challenge.18, 22-23 A means to extend the lateral resolution of laser patterning techniques from the micrometer to the nanometer range are near-field techniques.17-18,


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22-23

Leggett and coworkers used scanning near-field lithography for the fabrication of

nanostructured biomolecular templates using PEG-terminated monolayers.17 Domain sizes are in the range of 50-100 nm. An alternative for the fabrication of nanostructured biomolecular templates takes advantage of nonlinear effects, e. g. exploiting multiphoton absorption processes or photothermally induced reactions.18, 22-34 Because of their high thermal and chemical stability, silane-based SAMs offer particular prospects in nonlinear laser patterning.25,30-34 In previous studies, photothermal laser patterning of alkylsiloxane monolayers has been reported.26,31-34 Photothermal micro- and nanoprocessing can conveniently be carried out using microfocused CW lasers at wavelength in the visible range. Minimum structure sizes are below 100 nm.26,33 Here we expand this approach to PEG-terminated silane-based monolayers for fabrication of protein micro- and nanopatterns.

2. Experimental Section Commercial Si(100) wafers are cut into small pieces, about 10x10 mm2 in size, and used as substrates. For characterization via fluorescence microscopy silicon substrates with a surface oxide layer of dOx = 100 nm are used as preferred substrates.35-36 For this purpose, the silicon samples are thermally oxidized in air at 1200 °C for 6 h using a furnace.31 Native silicon substrates with a surface oxide layer of dOx = 1-2 nm are used for characterization via scanning electron microscopy (SEM). Prior to coating, all samples at first are cleaned with ethanol (p.a., VWR Prolabo) and hot piranha solution, a 3:1 mixture of 96% sulfuric acid (suprapur, Merck) and 30% hydrogen peroxide (AppliChem), for 30 min, thoroughly rinsed in ultrapure water (18.2 MΩ) from a Millipore system (Simplicity, Millipore) and dried in a stream of high-purity argon (5.0, Air Liquide). The samples then are coated with PEG-terminated monolayers, using: 2-


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[methoxy(polyetheneoxy)propyl]trichlorosilane (PEGCl, CH3O(CH2CH2O)6-9(CH2)3SiCl3, 90 %, ABCR) and 2-[methoxy(polyetheneoxy)propyl]trimethoxysilane (PEGMeO, CH3O(CH2CH2O)69(CH2)3Si(OCH3)3,

90 %, ABCR). For the preparation of PEGCl-monolayers, the substrates are

placed in a closed container and are covered with a droplet of PEGCl for 4 h. For the preparation of PEGMeO-monolayers PEGMeO is spin-coated onto the substrates at 2500 rpm. Because of the low reactivity of the methoxy groups the substrates are heated to 70 °C for 10 min. After coating both PEGCl- and PEGMeO-coated substrates are extensively washed with ethanol in order to remove excess precursor molecules and subsequently are dried in a stream of high purity argon. Contact angle measurements and measurement of the film thickness by atomic force microscopy (AFM) are carried out in order to characterize the SAMs. Prior to AFM measurements the coating is removed via laser patterning (Fig. 1). This allows one to determine the film thickness from height profiles. PEGCl-monolayers exhibit a thickness of about 1 nm and a static water contact angle of 38°. PEGMeO-monolayers exhibit a thickness of about 1 nm and a static water contact angle of 33°. These values are in good agreement with data reported in the literature.37-38 Photothermal laser processing is carried out at ambient conditions using a microfocused scanning CW-laser setup operating at a wavelength of 532 nm and a 1/e2 laser spot size of d1/e2 = 2.8 µm. Briefly, the laser beam of a diode-pumped solid state laser (Laser Quantum, Ventus) is focused onto the sample surface using an optical microscope objective with a numerical aperture of NA = 0.25. For focusing, the objective is mounted on a stepper motor stage (Micos, PLS-85). For positioning in the focal plane, the sample can be moved within an area of 26 x 26 mm2 using two additional stepper motor stages (Micos, PLS-85). An acousto-optical modulator (A.A. OptoElectronic, A.A.MTS.110/AS.VIS) allows one to adjust the laser power P and to switch the laser


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beam on and off and hence adjust the duration of the irradiation time τ. Generally, patterning can be carried out either in pulse-mode operation or in continuous-mode operation.29 In pulse-mode operation the laser is switched on at fixed positions for predefined irradiation times in order to create dot patterns. In continuous-mode operation the laser beam is turned on all time while the motor stage is moving in order to fabricate line patterns. For protein adsorption experiments, a droplet of protein solution in phosphate buffered saline (PBS) with a concentration of c = 50-500 µg/mL is placed onto the patterned sample for specific times in the range of t = 1-18 hours. A closed container is used for coating in order to prevent evaporation of water. The PBS-buffer solution (0.01 M total phosphate, 0.0027 M KCl, 0.137 NaCl in water) is prepared via dissolving PBS-tablets (Sigma Aldrich) in an appropriate amount of ultrapure water. As proteins bovine serum albumine (BSA) and streptavidin (STV) are used: AlexaFluor555-labeled BSA (3-6 mol AlexaFluor555 per mol BSA, Molecular Probes, c = 250 µg/mL, t = 1 h), AlexaFluor555-labeled STV (2-4 mol AlexaFluor555 per mol STV, Molecular Probes, c = 50 µg/mL, t = 4 h), Nanogold-labeled STV (Molecular Probes, c = 80 µg/mL, t = 4 h) and biotinylated-BSA (8-16 mol biotin per mol BSA, Sigma Aldrich, c = 500 µg/mL, t = 18 h). Complementary protein adsorption experiments have been carried after addition of Triton X-100 (10% aqueous solution, Sigma Aldrich). Both proteins, BSA and STV, exhibit a size of a few nanometers and can easily be detected via AFM.39-40 AlexaFluor555 shows maximum absorption at 555 nm and maximum emission at 565 nm. After protein adsorption the samples are extensively washed with PBS-buffer and ultrapure water and dried in a stream of high-purity argon. For characterization, contact angle goniometry (Surftens Universal, OEG), AFM (Autoprobe CP and NanoScope Multimode IIIa, Veeco), SEM (ESEM Quanta 400, FEI and JSM-7500F,


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JEOL) and fluorescence microscopy (BX41TS microscope, Olympus, with X-Cite 120 from ExFo and ColorViewII CCD camera from Olympus) is used. Oxide layer thicknesses are determined via ellipsometry (EL X-02C, DRE GmbH).31 AFM images are recorded in intermittent-contact mode with standard cantilevers. Fluorescence micrographs are recorded using Olympus MPLFLN and LMPLFLN Objectives (including: 50x, NA = 0.5; 100x, NA = 0.8, 100x, NA = 0.9), an excitation filter at 532-554 nm, a dichroic mirror at 562 nm and an emission filter at 570-613 nm at imaging times of t = 1 – 100 s. Domain diameters are determined using the ImageThreshold operation in combination with the Fuzzy-Mean-Gray method of IGOR Pro (Version 6.3, Wavemetrics). For graphical visualization the brightness and the contrast of the micrographs have been adjusted. Relative fluorescence intensities and S/N values shown in bar charts are extracted from the raw data without image processing. Thermokinetic analysis of data from laser patterning experiments has been carried out on the basis of an established approach taking into account the measured 1/e2 laser spot diameter and the experimental laser powers.32 All material specific parameters are chosen as detailed in reference 32. Given errors of the effective kinetic parameters represent statistical uncertainties. In addition, systematic uncertainties apply because of the limited accuracy of the temperature calculations and errors of the measured 1/e2 laser spot diameter and laser powers.32

3. Results and discussions Fabrication of micro- and nanostructured chemical templates for directed protein adsorption at predefined positions necessitates a proper choice of the underlying substrate and the applied surface chemistry as well as optimization of the patterning procedure.1-7 The general approach considered here is displayed in Scheme 1. At first, surface-oxidized silicon samples are coated


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with PEG-terminated SAMs in order to provide protein-repellent platforms. Subsequently, photothermal laser patterning is carried out. This induces monolayer decomposition and results in the formation of reactive domains for local protein adsorption. For characterization of protein adsorption in micro- and nanosized domains distinct analytical methods and labeling techniques are employed.

3.1. Protein micropatterns Fluorescence microscopy is the method of choice for characterization of protein adsorption in microsized domains. For this purpose, at first, the monolayer and the substrate are optimized in order to increase the sensitivity, i. e. the S/N ratio. The S/N ratio, here, represents the ratio of the fluorescence intensities in the laser-fabricated patterns (signal) and the surrounding PEGterminated areas in the background (noise). Fig. 2 displays respective fluorescence micrographs of laser-fabricated line patterns after adsorption of AlexaFluor555-labeled BSA. The best S/N ratio is obtained on PEGMeO-coated substrates with a thermally grown silicon oxide layer of 100 nm (Fig. 2a). In contrast, the S/N ratio is significantly lower on PEGCl-coated substrates (Fig. 2b). Again lower S/N values are observed on substrates with native silicon oxide layers exhibiting a thickness of 1-2 nm (Fig. 2c). Fig. 2d displays relative S/N ratios and fluorescence intensities measured on the line patterns and in the PEG-terminated areas in the background. Note, despite a longer exposure time t during imaging the relative fluorescence intensities in the domains are significantly reduced in Fig. 2b-c when compared with Fig. 2a. In addition, the relative fluorescence intensities in the PEG-terminated areas are significantly increased in Fig. 2b-c when compared with Fig. 2a.


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The higher S/N ratio on PEGMeO-coated substrates largely results because of an enhanced protein repellency of this coating. Fig. 3 displays respective fluorescence images, which have been recorded at a higher magnification in the PEG-terminated area on PEGMeO- and PEGClcoated substrates. Note, for better graphical visualization the contrast of the micrographs has been inverted. Hence, dark spots in these images indicate protein adsorption. The nearly uniform size and intensity of the dark spots suggest that these features represent single proteins. On this basis, an analysis of these images yields ≈ 2x104 proteins/mm2 on PEGMeO-coated substrates versus ≈ 9x105 proteins/mm2 on PEGCl-coated substrates. Note, the different protein resistance of PEGMeO- and PEGCl-based monolayers provides no clear information on the packing density and structural quality of these SAMs. In particular, both an increased and a decreased packing density may reduce the protein resistance.41 The lower protein repellency of PEGCl-coated substrates results in an increased background fluorescence intensity. Noteworthy, at the same time the fluorescence intensity in the proteincovered lines is significantly increased. This could point to incomplete decomposition or contamination of laser-fabricated lines on these substrates. These effects result in superior S/N values on PEGMeO-coated substrates when compared with PEGCl-coated substrates. The higher S/N ratio on thermally oxidized substrates with a silicon oxide layer of 100 nm, in turn, is attributed to an enhanced fluorescence intensity because of interference effects.35-36 Moreover, the thick oxide layer impedes quenching caused by the underlying silicon.42 In addition, on silicon substrates with native silicon oxide layers the background fluorescence intensity is significantly higher. Fluorescence microscopy is also used in order to analyze and compare the size of protein domains on PEGMeO- and PEGCl-coated substrates (Fig. 4). Note, thermally oxidized Si


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substrates are used in these experiments because of the high sensitivity of measurements on these platforms. Previous studies demonstrated that photothermal laser patterning of silane-based monolayers on surface-oxidized silicon substrates with distinct oxide layer thickness yields very similar structure sizes.31 Because of the lower reflectivity, though, the required laser power is significantly lower on thermally oxidized silicon substrates.31 Photothermal laser patterning of the substrates in these experiments is carried out in pulsemode operation at distinct laser parameters. For local protein adsorption, AlexaFluor-labeled BSA and STV are used (Fig. 4a-c). As displayed in Fig. 4d-e, an analysis of the domain diameters yields very similar results for both SAMs. Slight variations of the laser power are within the error of the laser patterning experiments. Generally, the structure size in photothermal laser patterning depends on the thermal stability of the coating and the absorption of the laser light and the heat conduction in the underlying substrate.26-27 Here, the same type of substrate is used. Also, during coating PEGMeO and PEGCl become hydrolyzed and couple to the surface via siloxane bonds. Hence, the chemical structure of the coupled molecular entities on the surface is expected to be very similar. Of course, the packing density and degree of order might be quite different (cf. above). The data in Fig. 4, however, suggest that these monolayers exhibit about the same stability. The lateral resolution of fluorescence microscopy, of course, is limited by optical diffraction. For microsized domains with diameters ≥ 1 µm, comparative AFM measurements yield very similar values. The analysis of domains with diameters

Photothermal Laser Fabrication of Micro- and Nanostructured

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