Controlled Modification of Protein-Repelling Self-Assembled

Article pubs.acs.org/JPCC

Controlled Modification of Protein-Repelling Self-Assembled Monolayers by Ultraviolet Light: The Effect of the Wavelength Y. L. Jeyachandran,† Andreas Terfort,‡ and Michael Zharnikov*,† †

Applied Physical Chemistry, Heidelberg University, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany Institute for Inorganic und Analytical Chemistry, Frankfurt University, Max-von-Laue-Straße 7, 60438 Frankfurt, Germany

‡

ABSTRACT: Exposure of protein-repelling oligo(ethylene glycol) (OEG) terminated alkanethiolate (AT) monolayers to ultraviolet (UV) light results in the damage of the OEG chains and photooxidation of the thiolate headgroups, which can be used for controlled tuning of protein-repelling properties within the so-called UV direct writing (UVDW) approach or for the preparation of mixed OEG-AT/specificreceptor films by so-called UV-promoted exchange reaction (UVPER). Using several model systems, we studied the effect of the wavelength (254−365 nm) on the course and efficiency of UVDW and UVPER applied to different OEG-AT matrices. The cross sections of the UV-induced damage were found to decrease significantly with increasing wavelength of UV light. In accordance with this behavior, the efficiencies of both UVDW and UVPER were maximal at a wavelength of 254 nm, somewhat lower at 313 nm, and lowest at 365 nm. Both UVDW and UVPER allowed a fine-tuning of protein affinity for nonspecific and specific adsorption, respectively, but UVDW did not occur below a certain, wavelength-dependent threshold dose. Performing UVPER below this dose enables us to suppress possible nonspecific adsorption of proteins even in the case of noncomplete exchange of the UV-damaged molecules of the primary OEGAT matrix by receptor-bearing moieties. The obtained results are of direct relevance for the preparation of high-quality mixed OEG-AT/specific-receptor films and the fabrication of complex protein patterns by UVDW and UVPER lithography.

1. INTRODUCTION Advanced lithographic techniques applied to monomolecular resists (so-called self-assembled monolayers, SAMs) enable the fabrication of well-defined patterns of functional biomolecules, above all proteins, and specific receptors, which are the key elements of biosensors, biofouling analysis assays, cell studies, and tissue engineering applications.1−6 An essential element of such patterns is a protein-repelling “background” surrounding the preselected sensing areas and preventing nonspecific adsorption of proteins beyond these regions. Suitable moieties to form such a background are oligo(ethylene glycol) (OEG) or poly(ethylene glycol) (PEG) based compounds, following the pioneering work by Whitesides and co-workers who, in particular, documented that certain OEG-terminated alkanethiolate (OEG-AT) SAMs on gold are completely resistant to protein adsorption under standard biological conditions.7−9 Further studies have demonstrated that protein-repelling properties depend on the length of the OEG part, packing density of the OEG-AT moieties, and the character of the terminal group (e.g., −OMe or −OH).8−14 Most frequently, the protein-repelling background is fabricated by a backfilling process, following the preparation of the specific sensing areas by a specific lithographic approach such as microcontact printing (μCP) or dip-pen (DPN) techniques.1,15 Alternatively, the background can be formed by an exchange reaction between the nonmodified areas of a primary aromatic SAM matrix and OEG-based moieties, © 2012 American Chemical Society

following the chemical modification and cross-linking of preselected areas in this matrix by deep UV or electron beam lithography. These modified areas can be subsequently used for the attachment of the protein receptors, becoming the sensing areas of a protein chip.16,17 The respective exchange reaction takes, however, quite long, which is unfavorable for the practical implementation of the above approach. This drawback and necessity to perform the backfilling procedure (μCP, DPN) can be, however, overcome by an alternative, single-step route which we have suggested recently.18 The major idea is to use a protein-repelling, OEG-based matrix from the very beginning and write the desired pattern directly in it by electron-beam lithography. Such a direct writing (DW) lithography relies on the very high sensitivity of the OEG moieties to electron irradiation, which allows us to destroy, in a controlled way, the OEG segments of the OEGbased SAM constituents without significantly affecting the other parts of the molecules. Since the OEG segments are responsible for the protein repulsion, one can easily adjust this property by selection of a suitable irradiation dose, which, among other possibilities, allows us to fabricate templates for complex protein patterns, e.g., gradient-like ones.18 The only disadvantage of this approach is that the irradiation-modified areas Received: January 13, 2012 Revised: April 4, 2012 Published: April 10, 2012 9019

dx.doi.org/10.1021/jp300436n | J. Phys. Chem. C 2012, 116, 9019−9028

The Journal of Physical Chemistry C

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2. EXPERIMENTAL SETUP Several OEG-AT compounds, differing by the number of the EG units, length of the alkyl part, and the terminal group, were used as precursors for the primary SAMs (see Figure 1).

adsorb proteins nonspecifically, relying, presumably, on a combination of physisorption and covalent binding. To transform such a nonspecific template to a specific one, one needs either to attach a protein bearing to a specific binding group for the target protein18 or to exchange the partly damaged OEG-functionalized moieties in the irradiated areas for molecules bearing a specific receptor for the target protein using the so-called irradiation-promoted exchange reaction (IPER).19−21 The rate and extent of IPER depend on the irradiation dose (i.e., defect concentration in the primary matrix),19,20 so that the content of the protein receptors can be precisely adjusted within a certain range.21 The advantages of the IPER approach, which is still under development and optimization, are high flexibility and ultimate lateral resolution provided by electron lithography (down to a few nanometers).22,23 A major disadvantage is the requirement to work under vacuum and, in the case of the writing with a focused electron beam, the necessity to use complex and expensive lithographic setups such as a scanning electron microscope (SEM) with a pattern generator system. These drawbacks can be potentially overcome by ultraviolet (UV) lithography, as demonstrated by Leggett and co-workers recently.22,24−26 Thus, the OEG part of an OEG-based monomolecular matrix or a PEG-based brush can be selectively damaged by UV light (≤250 nm), as in the case of electrons. Using an advanced setup with a powerful UV laser in combination with a scanning near-field optical microscope (SNOM), they achieved high-resolution patterning (down to 9 nm).22 They have also demonstrated large area patterning by using diffraction of the UV laser beam (interferometric lithography).27 The use of deep UV light (≤250 nm) generated by a quite expensive UV laser source bears several disadvantages. Apart from the aspect of high cost, a serious concern in using deep UV radiations is degradation of the optical surfaces limiting significantly the use of standard optical elements. A suitable solution of this problem could be UV patterning at longer wavelengths, which, consequently, might offer more versatile patterning strategies in combination with developing technologies, such as digital micromirror devices (DMD) or micro lens arrays (MLA).28,29 However, this option has not been tested so far with respect to OEG-terminated monolayers, although there is a significant body of work related to the patterning of photosensitive SAMs.30−32 Further, the possibility of UV-promoted exchange reaction (UVPER) has not been proven so far for the OEG-based matrix, even though it was principally demonstrated that UVPER is well applicable to SAMs in general.33 In this context, in the present work, we studied the effect of the wavelength (254−365 nm) on the course and efficiency of UVDW and UVPER applied to the OEG-AT matrix, taking several OEG-AT SAMs on Au(111) as representative test systems. We varied the lengths of both OEG and AT parts, in view of the fact that the rate and extent of IPER (and presumably those of UVPER) depend strongly on the length of the SAM constituents.20 The efficiency of UVDW was monitored using avidin as a test protein, whereas a biotinylated OEG-AT molecule was used as a substituent for UVPER to fabricate templates for specific avidin adsorption and patterning based on the specific biotin−avidin interaction.34,35 The results were directly implemented for the fabrication of protein patterns relying on the specific interaction between the target protein and the chemical template.

Figure 1. Chemical structures of the molecular constituents used for the preparation of primary OEG-AT monolayers (C2EG6OMe, C2EG8OMe, C11EG4OH, C11EG5OH, and C11EG6OH) and as substituent for UVPER (EG3-Bio). EG3-Bio bears the terminal group (biotin) which has a specific affinity to avidin.

Among them, the molecules with a general formula HS− (CH2)11(OCH2CH2)n−OH (C11EGnOH; n = 4−6) were purchased from ProChimia Surfaces, Poland. Further precursors, viz., HS−(CH2)2(OCH2CH2)n−OMe (C2EGnOMe, n = 6 and 8), were custom-synthesized in analogy to a previously described protocol.36 As a receptor-bearing substituent, we used biotin-terminated tri(ethylene glycol) hexadecanethiol (EG3Bio; C36H68N4O6S2; see Figure 1); it was purchased from Asemblon Inc. As the target proteins, we used avidin (A9275), bovine serum albumin (BSA) (A7638), and biotin-functionalized BSA (A8549; Bio-BSA) as purchased from Sigma-Aldrich. As substrates for the primary SAMs, we used gold. The substrates were prepared by thermal evaporation of 100 nm of gold (99.99% purity) onto polished single-crystal silicon (100) wafers (Silicon Sense) primed with a 5 nm titanium adhesion layer. The resulting gold films were polycrystalline, with the predominant (111) orientation of the individual grains and a grain size of 20−50 nm. The primary OEG-substituted SAMs were prepared by immersion of fresh gold substrates into 1 mmol solutions of the precursor compounds in absolute ethanol (Sigma-Aldrich) for 24 h at room temperature. After immersion, the samples were thoroughly rinsed with pure ethanol, blown dry with argon, and used immediately for characterization or further treatment. The UV irradiation treatment was performed under ambient conditions. UV light with three different wavelengths, viz., 254, 313, and 365 nm, was used; it was provided by short-wave (UVC), medium-wave (UV-B), or long-wave (UV-A) Hg vapor lamps (Benda Konrad Laborgeräte), respectively. Irradiation at 254 nm was performed without any filters because of the highest UV cross-section at this wavelength (see Section 3.3) 9020



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Figure 2. Schematic representation of the general experimental procedure. Step 1 is common for UVDW and UVPER, with the UV wavelength being varied. Alone, this step corresponds to UVDW; the resulting monomolecular templates can be used as substrates for nonspecific protein adsorption (step 2). Successive steps 1 and 3 correspond to UVPER; the resulting monomolecular templates can be used as substrates for specific protein adsorption (step 4). The primary monolayer should be protein repelling and, specifically for UVPER, nonprone to ZDER. This is schematically shown by the crossed red arrows.

and only a minor (∼5%) contribution of longer wavelengths in the case of the UV-C source. For irradiation at wavelengths of 313 and 365 nm, suitable band-pass filters (Optoprim GmbH) with an effective transmission window of ∼10 nm centered at ∼313 nm (XHQA313) and ∼365 nm (XHQA365), respectively, were used. The intensity of the UV light at a distance of 3 cm away from the lamp was ∼2.2 mW/cm2 for 254 nm (UVC lamp), ∼0.57 mW/cm2 for 313 nm (UV-B lamp with the filter), and ∼0.52 mW/cm2 for 365 nm (UV-A lamp with the filter) as measured using the respective UVX radiometer sensors (Ultraviolet Products Ltd.). The irradiation doses were estimated by multiplication of the exposure time with the light intensity. The primary SAMs were either irradiated homogeneously or patterned in proximity printing geometry using commercial (Plano) metal grids as masks. After irradiation, the samples were washed in ethanol and dried under argon flow prior to characterization or further use. The only exceptions were the S 2p X-ray photoelectron spectroscopy (XPS) characterization, to monitor the photooxidized species at the headgroup−substrate interface, and UVPER, because the SAMs were automatically “washed” in the course of this procedure. In these cases, the irradiated samples were not washed. The exchange reaction was performed by immersion of the pristine or UV-irradiated primary SAMs into the 0.1 mmol solution of EG3-Bio in ethanol for 5 min to 2 h at room temperature. After immersion, the samples were rinsed thoroughly with pure ethanol, dried under argon flow, and used immediately for characterization or further treatment. The protein adsorption experiments were performed on the pristine monolayers, testing protein repulsion/resistance; after

the UV treatment step (UVDW), testing nonspecific adsorption of proteins; and after the exchange reaction procedure (UVPER), testing specific adsorption of proteins. In all cases, the samples were immersed into a 0.1 mg/mL solution of the respective protein in PBS (P4417TAB, pH = 7.4 at 25 °C, Sigma-Aldrich) solution prepared using Millipore water. The incubation time was kept at 30 min. After protein adsorption, the samples were rinsed thoroughly in PBS solution, washed in Millipore water, and dried under argon flow. The characterization of the samples was performed by XPS, protein adsorption experiments (see above), and scanning electron microscopy (SEM). For the XPS characterization, a Leybold−Heraeus Max100 system equipped with a hemispherical LHS 11 analyzer was used. The measurements were performed using a Mg Kα X-ray source (λ = 1253.6 eV) operated at 260 W and positioned ∼1.5 cm away from the sample. The spectra were recorded in normal emission geometry with an energy resolution of ∼0.9 eV. The energy scale was referenced to the Au 4f7/2 peak of the gold substrate at a binding energy (BE) of 84.0 eV. Apart from the characterization of the primary OEG-AT SAMs, XPS was used to monitor protein adsorption, which was performed on the basis of the characteristic N 1s signal following the methodology of our previous publications.18,21 Note that this approach is quite common and frequently used for this purpose; it is sensitive enough and delivers as reliable data as such established (in this particular field) techniques as surface plasmon resonance or quartz crystal microbalance. The fabricated protein patterns were imaged using a Leo 1530 9021



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Gemini SEM device (Zeiss, Germany). The images were recorded at an acceleration voltage of 1 kV. The residual gas pressure was ≈5 × 10−6 mbar.

3. RESULTS AND DISCUSSION 3.1. General Procedure. The general experimental procedure is illustrated by Figure 2. The primary OEG-AT monolayers were first tested for their protein repulsion which is a prerequisite for the adjustment of protein affinity by both UVDW and UVPER. Then, the primary SAMs were checked for their stability against the so-called zero-dose exchange reaction (ZDER), i.e., the lack of molecular exchange (EG3-Bio as the substituent) for the pristine monolayers. Otherwise, in the case of UVPER lithography, the exchange reaction would occur at both the irradiated and nonirradiated areas of an UVpatterned primary OEG-AT SAM, which would result in the appearance of the protein receptors in both areas and, subsequently, as far as a protein pattern will be fabricated, in contrast to reduction or even disappearance. Further, when the primary monolayers exhibited protein repulsion and the lack of ZDER, they were irradiated by UV light and subsequently tested for nonspecific adsorption of proteins (efficiency of UVDW) or subjected to UVPER with EG3-Bio as the substituent. The efficiency of UVPER was monitored by adsorption of avidin that specifically binds to EG3-Bio. The adsorbed avidin was, in some cases, used for subsequent specific coupling of Bio-BSA. 3.2. Primary SAMs: Protein Repulsion and Zero-Dose Exchange. Protein repulsion is a crucial requirement to utilize the primary OEG-AT monolayers for both UVDW and UVPER lithography; otherwise, it will be impossible to provide a completely protein-repelling background for the pattern fabrication. Luckily, all the C11EGnOH and C2EGnOMe SAMs used in the present study exhibited protein-repelling behavior as demonstrated by the experiments on the adsorption of avidin and BSA onto the native SAMs. Note that these findings agree well with the literature data,10,11,14 especially for the C11EGnOH SAMs on gold, for which protein repulsion was reported at n > 3.13 Another crucial requirement, important for UVPER lithography only (see Section 3.1), is the stability of the primary OEG-AT SAMs against ZDER. It was found that these films are quite diverse in this regard. This is illustrated by Figure 3 which shows the estimated surface coverage of avidin adsorbed on the OEG-AT monolayers which were preliminarily exposed to EG3-Bio for different time, varied from 5 min to 2 h. Since the adsorption of avidin relies on the presence of the biotin receptor in the molecular film, avidin coverage represents a direct measure of the EG3-Bio content, at least at low and moderate portion of the EG3-Bio moieties in a mixed EG3Bio/OEG-AT monolayer (see Section 3.4),21,37,38 which can be expected for the nonirradiated films. In addition, this coverage could be monitored much easier than the EG3-Bio content itself since the N 1s XPS signal from the avidin is much stronger than that from biotin. Moreover, even very low surface concentration of biotin could mediate noticeable avidin adsorption,21,37,38 which increases the sensitivity of the EG3Bio content monitoring significantly. According to Figure 3, most of the primary OEG-AT SAMs are not stable against ZDER, even at a short exposure to EG3Bio. In particular, both C2EG6OMe and C2EG8OMe films exhibit very strong extent of ZDER, with faster exchange kinetics and higher extent of exchange in the former case, which

Figure 3. Surface coverage of avidin on the pristine (i.e., nonirradiated) OEG-AT monolayers which were subjected to ZDER, i.e., immersed in EG3-Bio solution for different time. The avidin coverage values are referenced to the value for the single-component EG3-Bio monolayer. The solid lines are guides to the eye.

is likely related to the shorter molecular chain of C2EG6OMe. Thus, both these films are not suitable for the UVPER lithography and can only be used for the direct writing approach. As for the C11EGnOH system, the respective monolayers exhibit continuous variation of the stability against ZDER with increasing n, with very fast exchange kinetics and considerable extent of ZDER in the case of C11EG4OH, stability for 5 min and abrupt start of ZDER in the case of C11EG5OH, and stability at moderate (up to 30 min) exposure to EG3-Bio in the case of C11EG6OH. This behavior correlates nicely with our previous results for the EG3OH and EG7OH monolayer, with the former film exhibiting a significant extent of ZDER and the latter one being stable against ZDER for 2 h exposure.21 Summarizing, the above results show that a suitable OEG-AT monolayer for UVPER lithography could be prepared using molecules with a sufficiently long alkyl chain (11 methylene units in the given case) and at least five EG units in the OEG part. On the basis of these results, only the relatively stable C11EG6OH monolayer was used for further UV damage (also C11EG5OH to some extent), UVDW and UVPER studies. Considering the course of ZDER for this particular system, the exchange reaction was always performed for a fixed time of 5 min which is far below the onset of ZDER after ca. 30 min. Note that the high values of the avidin coverage observed for some of the investigated films as compared to the singlecomponent EG3-Bio monolayer can be explained by the orientational disorder in the latter system resulting in the spreading of the biotin tail groups throughout the entire monolayer.38 Consequently, the exposure of the biotin moieties to the SAM−ambience interface occurs to a limited extent only, so that their content there is comparably low.37,38 Obviously, the mixed OEG-AT/EG3-Bio films, produced by ZDER, do not suffer from this problem and exhibit a better orientational order. 3.3. Effect of UV Irradiation. Following the findings described in the previous section, the C11EG6OH monolayer, which is both protein repelling and resistant to ZDER, was subjected to UV light of different wavelengths. The most prominent UV-induced process in AT SAMs, as reported, 9022



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GJL2006,33 is photooxidation of the thiolate headgroups at the SAM−substrate interface to sulfonates (SOx) and, to a much lesser extent, damage of the alkyl matrix. In contrast, as has been shown by the example of the C11EG3OH monolayers exposed to 250 nm UV light,22 the most prominent and rapid UV-induced process in OEG-AT SAMs is the damage of their OEG part, which includes cleavage of the C−O and C−C bond, desorption of the released fragments, and appearance of carbonyl or carboxylate species. Photooxidation of the thiolate headgroups occurs in parallel but with a somewhat lower rate. This is exactly what we observe for the C11EG6OH monolayer in the present work, analyzing the C 1s and S 2p XPS spectra of this film in the course of UV treatment. The spectra, for the case of 254 nm, are depicted in Figure 4,

for irradiation with wavelengths of 254, 313, and 365 nm, respectively. The photooxidation of the thiolate headgroups occurs in parallel to the damage of the OEG segments as demonstrated by the S 2p XPS spectra in Figure 4b; these spectra were acquired for the samples immediately after UV irradiation without washing in ethanol. The spectrum of the pristine sample exhibits a single S 2p3/2,1/2 doublet at a BE ∼ 162.0 eV (S 2p3/2) corresponding to the intact Au−thiolate bonds in a well-defined OEG-AT SAM.39−41 The spectra of the irradiated samples feature an additional doublet at a BE ∼ 168.2 eV assigned to the photooxidized thiolate species;22,33 ethanol washing resulted in the disappearance of this doublet. The latter doublet becomes perceptible in the spectra at doses above 7.9 J/cm2 at 254 nm irradiation and at doses greater than 100 J/ cm2 at 313 and 365 nm irradiation. Intensity of this doublet increases at the initial stages of the UV treatment but decreases with the progressive irradiation following a partial desorption of the SOx bonded species. Along with the qualitative considerations, the data presented in Figure 4 can be analyzed in a quantitative way. For this purpose, the intensity ratio for the C−O and C−C emissions in the C 1s spectra and the intensity ratio for the thiolate and SOx related doublets in the S 2p spectra of the C11EG6OH monolayer as functions of irradiation dose are presented in Figure 5a (for all three wavelengths) and Figure 5b (for 254

Figure 4. C 1s (a) and S 2p (b) XPS spectra of the pristine and UV irradiated (254 nm) C11EG6OH monolayers; the character of the UV-induced changes is representative for the other wavelengths as well. The C 1s spectra are decomposed into the peaks related to the OEG and alkyl parts of the SAM constituents and photooxidation species. The S 2p spectra exhibit doublets related to the thiolate and photooxidized sulfur species. The irradiation doses are given at the respective curves. The vertical lines are guides to the eyes.

representative of the other wavelengths as well. The C 1s spectrum of the pristine monolayer in Figure 4a exhibited two characteristic emissions at BEs of 284.6 and 286.4 eV associated with the alkyl (C−C) and OEG (C−O) parts of the SAM constituents, respectively.18 With progressive UV irradiation, the intensity of the C−O emission decreased rapidly, whereas that of the C−C emission changed much more slowly or remained unchanged, suggesting preferential decomposition of the OEG part of the SAM constituents. In addition, two new, weak emissions at BEs of 287.9 and 289.4 eV appeared. In accordance with the previous work,22,24 they can be tentatively assigned to photooxidation products of the ether carbon atoms, such as carbonyl (presumably aldehyde) and carboxylate moieties. The rate of the decomposition of the OEG chain depends strongly on the wavelength of the UV light. Whereas this process is very rapid at 254 nm, it is much slower at 313 nm and very slow at 365 nm (see below). Also, the onset of photooxidation of the OEG chain, i.e., the appearance of the emissions related to carbonyl and carboxylate, depends strongly on the wavelength. The respective features become perceptible in the spectra starting from doses of ≈0.5, ≈ 12, and ≈28 J/cm2

Figure 5. Intensity ratio for the C 1s emissions related to the OEG and alkyl parts of the SAM constituents (a,c) and intensity ratio for the thiolate and SOx related doublets (b,d) for the C11EG6OH (a,b) and C11EG5OH (c,d) SAMs as functions of UV dose. The values for wavelengths of 254, 313, and 365 nm are shown in (a) by black squares, gray circles, and light gray triangles, respectively. (b), (c), and (d) present the values for a wavelength of 254 nm (black squares). The solid lines are the first-order exponential decay function fits to the experimental points.

nm), respectively. For comparison, analogous data for the C11EG5OH film are depicted in Figures 5c and 5d (for 254 nm). All of the observed dependencies can be fitted by a firstorder exponential decay function (first-order kinetics) following the formalism of ref 42 9023



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avidin, relying, as mentioned in Sections 1 and 2, on the wellknown and documented biotin−avidin affinity. The surface coverage of avidin was estimated with respect to its coverage on the one-component EG3-Bio SAM.21 Parallel to the specific adsorption, the nonspecific adsorption was probed, using BSA and Bio-BSA as test moieties. Figure 6 shows the N 1s XPS spectra of the C11EG6OHmonolayer-based systems measured after UVPER and sub-

I = Isat + (Iprist − Isat) × exp( −σ ΦUV )

where I is the characteristic parameter value as a function of the UV exposure, ΦUV (in J/cm2); Iprist and Isat are the parameter values for the pristine and strongly irradiated (a leveling off behavior) OEG-AT films, respectively; and the cross-section σ (expressed in cm2) is a measure of a rate at which a process occurs and the saturation behavior is achieved. The crosssection values are presented in Table 1. Taking the 254 nm data Table 1. Cross Sections of the Major UV Irradiation Induced Processes for the C11EG6OH and C11EG5OH Monolayers cross section, σ × 10−20 cm2 C11EG6OH irradiation induced process damage of the OEG chain damage of the SAM/Au interface

254 nm 33.9 ± 2 6.8 ± 0.1

C11EG5OH

313 nm

365 nm

5.1 ± 0.5

1.7 ± 0.1

-

-

254 nm 37.0 ± 4 7.9 ± 0.3

and comparing the cross sections for the decomposition of the OEG segments in both C11EG6OH and C11EG5OH monolayers with those for the photooxidation of the thiolate groups in these films, we see that the former values are noticeably higher. This means that the UV-induced decomposition of the OEG chains progresses much more rapidly than the photooxidation of the thiolate−substrate interface. Further, taking the data for C11EG6OH and comparing the cross sections for the UV-induced decomposition of the OEG chains at the different wavelengths, we can conclude that the rate of the above process depends drastically on the wavelength, being very high at 254 nm and progressively lower at 313 and 365 nm. Finally, comparing the data for C11EG6OH and C11EG5OH (at 254 nm only), we see that the cross sections for both major UV-induced processes, viz., the decomposition of the OEG chains and the photooxidation of the thiolate− substrate interface, are higher for C11EG5OH as compared to C11EG6OH. This means that the efficiency of the UV-induced damage tends to increase for OEG-AT monolayers comprised of shorter molecules (as can be expected). However, as mentioned in Section 3.2, the use of the monolayers comprised of C11EG5OH or shorter molecules for UVPER applications is limited because they are not stable with respect to ZDER. At the same time, SAMs built of shorter OEG-AT molecules can be of interest for direct writing (UVDW) applications, considering the higher efficiency of UV irradiation in these systems. 3.4. Exchange Reaction and Protein Adsorption. Following the UV irradiation experiments described in the previous section, we tested the possibility to imbed the molecules bearing a specific protein receptor into an OEG-AT template by UVPER. As such a molecule, we used EG3-Bio and as a primary template an C11EG6OH monolayer. This film was subjected to a sufficient dose (15 J/cm2) of 254 nm UV light to achieve a high extent of the exchange reaction. The EG3-Bio exchange and protein adsorption were monitored using the characteristic N 1s XPS signal (see Section 2). The surface concentration of the EG3-Bio molecules that were exchanged into the primary C11EG6OH matrix was calculated relative to the density of these moieties in the one-component EG3-Bio SAM. As a test protein for the specific adsorption, we used

Figure 6. Panel A: N 1s spectra of strongly irradiated (254 nm; 15 J/ cm2) C11EG6OH monolayer after exchange reaction with EG3-Bio (a) and subsequent exposure to BSA (b), Bio-BSA (c), or avidin (d). Panel B presents difference spectra calculated on the basis of the spectra in panel A.

sequent exposure to the different proteins (panel A) as well as the respective difference spectra (panel B). The spectrum measured after the EG3-Bio exchange reaction (a) exhibits a pronounced N 1s emission, manifesting the formation of a mixed C11EG6OH/EG3-Bio monolayer. This spectrum and the ones measured after the subsequent exposure of the mixed film to BSA (b) or Bio-BSA (c) exhibit similar intensities of the N 1s signal, which is additionally evidenced by the lack of intensity gain in the resultant difference spectra (b − a) and (c − a). This behavior suggests an almost complete blocking of nonspecific adsorption of BSA or Bio-BSA by the mixed monolayer. In contrast, the spectrum taken after the exposure of the C11EG6OH/EG3-Bio monolayer to avidin (d) exhibits a significant gain in the N 1s signal and a characteristic peak in the (d − a) difference spectrum, manifesting the specific adsorption of avidin. Note that the exposure of the resulting bilayer to BSA produces no gain in the N 1s intensity (not shown), manifesting its inertness to the nonspecific adsorption. In contrast, the exposure of the bilayer to Bio-BSA causes an increase in the N 1s intensity (not shown), suggesting that the bilayer can be used for specific adsorption of a secondary protein. Note also that the adsorption of Bio-BSA on a nonspecific C11EG6OH template prepared by UVDW will presumably transform it into a specific one (with respect to avidin), similar to the case of electron irradiation.18 The complete blocking of nonspecific protein adsorption after UVPER in the given case has several important implications. Generally, any essential defect produced in the primary protein-resistant OEG-AT matrix by UV irradiation can facilitate nonspecific protein adsorption as far as it still persists after UVPER.22 Accordingly, a complete blocking of such an adsorption after UVPER suggests that all such defects were eliminated by the exchange of the damaged SAM 9024



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dose of 70 J/cm2 (presumably due to the low intensity of the respective N 1s signal), their portion increased rapidly with increasing UV dose after 313 nm UVPER and even more rapidly after 254 nm UVPER, achieving fractions of ∼40% (313 nm; 57.5 J/cm2) and ∼80% (254 nm; 74.5 J/cm2). The surface coverage of avidin on the respective C11EG6OH/EG3-Bio substrates depends, as expected, on the EG3-Bio portion. For 254 nm, this coverage exhibits characteristic nonlinear behavior observed previously for the mixed OEG-AT/EG3-Bio monolayers,21,37 viz., a steep increase of the protein coverage with increasing EG3-Bio fraction in the SAM until a maximum coverage was reached, followed by a gradual decrease. Such a nonlinear dependence is related to the variable degree of the orientational order in the mixed OEG-AT/EG3-Bio films. This degree is highest at low content of EG3-Bio, resulting in the exposure of the biotin moieties to the SAM−ambience interface, but progressively decreases with increasing EG3-Bio content, resulting in the spreading of the biotin tail groups throughout the film, so that their exposure to the SAM− ambience interface occurs to a limited extent only.37,38 Accordingly, the optimal adsorption of avidin occurs not at the highest but at a moderate EG3-Bio portion. The highest coverage was ∼220%, suggesting, in view of such a comparably high value,21 a good intermixture of the C11EG6OH and EG3Bio species in the mixed film (as expected); it occurred at an EG3-Bio portion of ∼40%. The high coverage value as compared to the single-component EG3-Bio monolayer is explained by the orientational disorder and the following low content of biotin at the SAM−ambience interface of this monolayer (see also Section 3.2).38 In view of the lower fractions of EG3-Bio, the avidin coverage curve for 313 nm UVPER reflects only the initial part of the general curve observed at 254 nm, exhibiting a steep increase with increasing EG3-Bio fraction in the SAM and leveling-off behavior at higher fractions. A similar highest coverage value of ∼220% is achieved at a similar fraction of EG3-Bio (∼40%). As for the 365 nm UVPER, an immediate and progressive increase in the avidin coverage with increasing UV dose is observed, even though the EG3-Bio could not be directly detected. The latter is presumably related to a limited sensitivity of XPS. Obviously, the UVPER occurred to some extent and in a progressive way following the UV exposure, but its result could only be visualized by the avidin adsorption. The outcome of UVPER relying on the specific adsorption of avidin can be compared to the outcome of UVDW relying on nonspecific adsorption. For this purpose, adsorption of avidin on the irradiated C11EG6OH substrate (before the exchange reaction) was monitored. The results are also presented in Figure 7. Similar to UVPER, the kinetics and extent of UVDW depend strongly on the wavelength of the UV light, with UVDW being more efficient at 254 nm, less efficient at 313 nm, and least efficient at 365 nm. Apart from this general trend, the dependence of the avidin coverage on UV dose is distinctly different as compared to UVPER. In the case of UVDW, avidin does not adsorb below a certain threshold dose, and then its coverage increases slowly and further rapidly with increasing dose and, finally, exhibits a leveling-off behavior at higher doses. The maximum coverage by UVDW (320%) is higher than that for UVPER, which is understandable in view of the fact that nonspecific adsorption relies on the variety of different interactions and is not affected by any orientational effects. In contrast, the specific adsorption of avidin onto the singlecomponent EG3-Bio monolayer relies on the specific biotin−

constituents with the EG3-Bio molecules. Such an efficient exchange suggests that the damage occurs individually to each molecule, with no or negligible cross-linking between them (cross-linking can significantly hinder the exchange reaction). However, in view of the strong effect of the molecular composition and length on the efficiency of the exchange reaction, the above conclusion is only strictly valid to the systems of our study. Generally, a noncomplete exchange of the strongly damaged molecules by receptor-bearing species can occur in the course of UVPER, resulting in the proneness of the OEG-AT/receptor film to nonspecific adsorption. 3.5. UVPER versus UVDW. The experiments described in the previous section were performed at a certain UV dose and a wavelength. In further experiments, we varied the latter parameter and monitored both the composition of the mixed C11EG6OH/EG3-Bio film and nonspecific and specific protein adsorption after UVDW and UVPER as functions of UV dose. The results are presented in Figure 7. All the experimental curves exhibit a strong effect of the wavelength. In particular, both rate and extent of UVPER increase significantly with decreasing wavelength. Whereas the EG3-Bio species were not directly perceptible after 365 nm UVPER up to a maximum

Figure 7. Surface coverage of avidin adsorbed on the UV-irradiated C11EG6OH matrix before (UVDW) and after (UVPER) the exchange reaction with EG3-Bio as well as a portion of EG3-Bio in the mixed C11EG6OH/EG3-Bio films as functions of UV dose. The vertical dotted lines in the graphs indicate the threshold dose for UVDW. The solid lines are guides to the eye. The portion of EG3-Bio is referred to the single-component EG3-Bio SAM. The avidin coverage is related to the coverage on the single-component EG3-Bio SAM. 9025



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one EG unit shortening of the OEG chain (compare the curves for C11EG6OH and C11EG5OH; compare the curves for C11EG5OH and C11EG4OH). The latter assumption correlates with the results of the protein-repulsion test for the pristine monolayers (cf. Section 3.2)even the C11EG4OH monolayer, for which the OEG chain is noticeably shorter than that of C11EG6OH, is protein-repelling, i.e., nonprone to protein adsorption. Thus, the UV-induced shortening of the EG6 segments in the C11EG6OH monolayer by one or two EG units will not change its protein-repelling ability but promote the subsequent exchange reaction with EG3-Bio. This is presumably true as long as an irradiation dose is kept below the threshold. In contrast, at the doses above the threshold, more extensive damage of the OEG chains and photooxidation of the thiolate headgroups occur, resulting in a partial loss of the protein-repelling properties and a higher extent of UVPER as far as it is performed. 3.6. UVPER Lithography. The experimental curve in Figure 8 allows a course estimation of the threshold dose values at higher UV wavelengths, for example, at 375 and 390 nm. These wavelengths are significant in view of the availability of the compatible, commercial patterning technology that could be directly employed for both UVDW and UVPER. For the moment, to demonstrate the possibility of UVPER lithography, we have fabricated simple mesh patterns of specifically adsorbed proteins in proximity printing geometry (see Section 2). The patterns were prepared on the C11EG6OH/EG3-Bio templates fabricated by UVPER lithography using 254, 313, and 365 nm UV light with the dose either below or above the threshold value for UVDW. The SEM images of these patterns are shown in Figure 10. A good contrast between the proteincovered (dark) and protein-repelling (light) areas is observed in all the cases. The patterns corresponding to the below-thethreshold and above-the-threshold conditions are quite similar. This can be expected since UVPER does not exhibit any threshold. The UV treatment below the threshold dose for UVDW is relevant and can be utilized to suppress possible nonspecific adsorption of proteins, resulting from noncomplete exchange of the UV-modified molecules as long as this will be a problem for a particular matrix−substituent system. Apart from the above considerations, the data presented in Figure 7 suggest that both the protein-repelling properties in the case of UVDW and the density of specific protein receptors in the case of UVPER can be varied in a controlled way by the selection of a proper UV dose with dynamic range depending on the wavelength and the necessity to suppress nonspecific adsorption events. In the case of UVPER, this can be used for the fabrication of high-quality mixed OEG-AT/receptor films (molecular level mixture). In the case of UVDW and UVPER lithography, this can be used for the fabrication of complex protein patterns, including gradient ones.

avidin interaction, requiring the presence of the biotin moieties at the SAM−ambience interface. The threshold doses for UVDW are ca. 1.6, 12, and 28 J/cm2 for wavelengths 254, 313, and 365 nm, respectively, following a second-order exponential dependence on the wavelength, as shown in Figure 8. In contrast, UVPER seems to occur without

Figure 8. Threshold dose for UWDW plotted as a function of the wavelength (circles). The solid gray line is an exponential fit to the data points, projected for the higher wavelengths.

any threshold under the conditions of our experiments. This means that UVPER is more sensitive to UV-induced defects as compared to UVDW in the case of OEG-AT templates. Further, this means that one can prevent a nonspecific adsorption, related to a possible, noncomplete exchange of UV-damaged SAM constituents, completely, as long as one stays below the threshold dose for UVDW. Of course, the exact values of this parameter given in the present article are characteristic of the C11EG6OH monolayer only, but the occurrence of the threshold and its behavior as a function of the wavelength should be generally applicable to the OEG-AT matrix. The proposed models of the OEG-AT monolayer modification at the doses below and above the threshold value for UVDW are presented in Figure 9. We propose that

4. CONCLUSIONS We studied the effect of the wavelength (254−365 nm) on the course and efficiency of UVDW and UVPER applied to the OEG-AT matrix, taking several different OEG-AT SAMs on Au(111) as representative test films and EG3-Bio as the UVPER substituent. Whereas all the SAMs exhibited proteinrepelling properties, which are a prerequisite for both UVDW and UVPER, only SAMs comprised of molecules with sufficiently long alkyl and OEG segments (C11EG5OH and C11EG6OH) were not prone to ZDER which is a prerequisite for UVPER lithography.

Figure 9. Proposed models of the UV irradiation induced processes in the primary OEG-AT matrix exposed to UV light with a dose below (a) and above (b) the threshold value for UVDW.

the irradiation-induced modification at doses below the threshold value is restricted to only a soft cleavage of the few outermost OEG units promoting the subsequent exchange reaction but allowing the monolayer to retain its proteinresistant characteristics (Figure 9a). The former assumption is in accord with Figure 2 which demonstrates that the rate and extent of the exchange reaction increase significantly at even 9026



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Figure 10. SEM images of protein patterns taken after the specific adsorption of avidin and subsequent specific adsorption of Bio-BSA on C11EG6OH/EG3-Bio templates prepared by UVPER exploiting UV light with wavelengths of 254, 313, and 365 nm. The dose was set either below (two columns at the left) or above (two columns at the right) the threshold dose for UVDW. Scale bar is 100 μm. Some patterns are digitally enhanced (contrast/brightness) for better visibility.

The detailed UV irradiation, UVDW, and UVPER experiments performed on the C11EG6OH film resulted in a variety of valuable findings. First, it was found that the major UVinduced processes in OEG-AT monolayers are damage of the OEG segments of the SAM constituents and photooxidation of the thiolate headgroups (in accordance with literature data), with the former process occurring with a significantly higher rate. Both processes exhibit first-order kinetics with the rate constantsdenoted as the cross sectionsdecreasing with increasing wavelength of UV light. In accordance with this behavior, the efficiency of both UVDW and UVPER was highest at a wavelength of 254 nm, somewhat lower at 313 nm, and lowest at 365 nm. Both UVDW and UVPER allowed a finetuning of protein affinity for nonspecific and specific adsorption, respectively. However, the exact dependences of the protein (avidin) coverage on the UV dose were distinctly different for UVDW and UVPER. In the case of UVPER relying on specific adsorption of proteins, we observed an immediate and steep increase of the avidin coverage with increasing UV dose until a maximum coverage was reached, followed by a gradual decrease. This behavior was correlated with the EG3Bio fraction in the mixed C11EG6OH/EG3-Bio monolayer. In the case of UVDW relying on nonspecific adsorption of proteins, avidin does not adsorb below a certain threshold dose, and then its coverage increases slowly and further rapidly with increasing dose and, finally, exhibits a leveling off behavior at higher doses. In view of this difference, the UV treatment below the threshold dose for UVDW can be utilized in the case of UVPER to suppress possible nonspecific adsorption of proteins, which can result from a noncomplete exchange of the UVmodified SAM constituents by receptor-bearing molecules. This was however not a problem for the C11EG6OH/EG3Bio system of the present study as was demonstrated by a series of experiments involving the exposure of the C11EG6OH/ EG3-Bio monolayers to specific and nonspecific proteins. Further, we demonstrated the principal possibility of UVPER lithography, fabricating several different specific protein patterns at variable dose and wavelength of UV light. The above results can be directly implemented for the fabrication of mixed OEG-AT/receptor films which exhibit a

better molecular intermixture as compared to the analogous films prepared by standard approaches such as coadsorption. Most significantly, these results can be utilized for UWDW and UVPER lithography, which can be especially attractive at longer wavelength of UV light and hopefully enable the use of comparably nonexpensive, commercial tools. Progressive and controlled character of UWDW and UVPER should make possible the fabrication of complex protein patterns such as gradient-like ones.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49-6221-54 4921. Fax: +49-6221-54 6199. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank M. Grunze for the support of this work. This work has been supported by DFG (ZH 63/9-3 and TE 247/9-2).

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REFERENCES

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Controlled Modification of Protein-Repelling Self-Assembled

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