Comprehensive Analysis of the Effect of Electron Irradiation on Oligo

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Comprehensive Analysis of the Effect of Electron Irradiation on Oligo(ethylene glycol) Terminated Self-Assembled Monolayers Applicable for Specific and Nonspecific Patterning of Proteins Yekkoni Lakshmanan Jeyachandran and Michael Zharnikov* Applied Physical Chemistry, University of Heidelberg, Im Neuenheimer Feld 253, D-69120 Heidelberg, Germany S Supporting Information *

ABSTRACT: Controlled modification of protein-repelling self-assembled monolayers (SAMs) of oligo(ethylene glycol) (OEG) terminated alkanethiols (AT) by electron irradiation was investigated, and the usability of these templates for the fabrication of proteins patterns by electron beam lithography was explored. The attachment of proteins was performed either directly after irradiation, based on nonspecific interaction with the modified SAM constituents, or after the successive exchange reaction with molecular substituents bearing special receptor group for protein adsorption, based on the respective specific interaction. Characteristic dose ranges for the above approaches, viz. direct writing (DW) and irradiation-promoted exchange reaction (IPER), were defined. On the basis of these findings, patterns of both specifically and nonspecifically bound proteins of various shapes and on different length scales were fabricated in the OEG-AT templates. In addition, a simple, one-step irradiation process was suggested to prepare multiprotein patterns. As an example, side-by-side patterning of bovine serum albumin and avidin is described.

1. INTRODUCTION Protein microarrays are a useful tool for proteomics research, toward the development of protein based diagnostics and therapeutics.1 Moreover, single or multicomponent protein patterns provide an important platform for cell culture analyses.2−9 Frequently, the techniques designed for the fabrication of such patterns rely on molecular templates provided by self-assembled monolayers (SAMs).10,11 Along with the protein-binding areas, these template comprise protein-repelling regions to prevent nonspecific adsorption of proteins beyond the sensing areas. Popular components to achieve the protein repelling property are SAMs of oligo(ethylene glycol) (OEG)-terminated alkanethiols (OEG-ATs) on gold, introduced in the pioneering work by Whitesides and Prime.12,13 Subsequently, significant efforts were made to establish the structure−property relationship in these systems and explore their potential for lithographic applications.14−16 A thorough survey of the OEG-AT monolayers of various molecular compositions, including the variation of the OEG and/or alkyl chain lengths and the use of different terminal groups, suggested that a typical combination of the structural and chemical characteristics, such as helical conformation of the OEG chain, relaxed lateral package density, internal hydration and terminal hydrophilicity should be required in the monolayers to exhibit a protein repelling property.14,16 Accordingly, it was suggested that the OEG-AT monolayers of molecules with three or more EG units could exhibit protein © 2012 American Chemical Society

repelling characteristics regardless of their alkyl chain length or the character of the tail group (e.g., −OMe or −OH).14−17 Using OEG-AT monolayers, as the basic protein-repelling material, different approaches have been developed to fabricate protein patterns. The most popular technique is the backfilling method. Within this approach, the protein adsorbing molecules are first patterned employing, e.g., microcontact printing or dippin lithography, and then, the OEG-AT molecules are introduced around the patterned spots.18−20 In some cases, backfilling occurs over an exchange reaction with the primary SAM used as template for the patterning.21,22 A promising alternative to the approaches involving backfilling is the use of an OEG-AT SAM as a primary template and electrons or UV light as primary patterning tools. The patterning can then be performed in different ways. The most straightforward technique is the so-called direct writing (DW) method relying on the high sensitivity of the OEG backbone to electron irradiation or UV light.23−25 Accordingly, the irradiation of an OEG-AT film by electrons or its exposure to UV light result in preferable degradation of the OEG part of the SAM constituents, leading, in its turn, to a continuous change in the protein-repelling ability with progressive irradiation. The UV/electron induced defects act as proteinadsorbing sites in the monolayer so that the required protein Received: April 19, 2012 Revised: June 13, 2012 Published: June 25, 2012 14950

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process were systematically investigated. Similar to ref 36, we tried the primary OEG-AT SAMs with different OEG chain lengths but put emphasis on only one system that delivered an optimal performance in the IPER framework. We relied again on the well-known biotin−avidin interaction37,38 using a biotinterminated OEG-AT molecule as the substituent. In addition to the spectroscopic and protein-adsorption experiments, we performed protein patterning using both DW-EBL and IPEREBL, including multicomponent protein patterns.

adsorbing patterns can be written in proximity printing geometry or using a focused electron beam or UV light.23−25 Since the density of the defects depends on the irradiation dose, protein-adsorbing ability of the exposed areas can be precisely adjusted by the selection of a proper dose, so that not only simple, dot-like or stripe-like but also complex, gradient-like protein patterns can be fabricated.24,25 The DW method is a comparably simple and flexible approach, which can be applied not only to OEG-AT monolayers but to OEG-based polymer films as well.24 However, the molecular templates fabricated by DW do not exhibit special affinity to a certain type of proteins but are the subjects of an arbitrary, nonspecific adsorption, at least, in the first adsorbed layer of proteins. In some cases, such a layer can serve as a specific template for a secondary protein but an additional passivation of nonspecific surface vacancies remaining after the adsorption of the first protein layer is frequently required,26−30 which increases the complexity of the system.31 A further complication is possible denaturation of the nonspecifically adsorbed proteins due to a complex and poorly controlled character of individual interactions, which mediate the adsorption. A way to avoid the above complications is so-called irradiation-promoted exchange reaction (IPER).32 Within this approach, an irradiated or irradiation-patterned SAM is immersed into a solution of a potential molecular substituent. Under these conditions, the rate and extent of the exchange reaction between the molecules in the primary SAM and the substituents in the solution depend on the density of irradiation-induced defects in the SAM, which can be precisely controlled by the selection of a proper dose.32−36 Usually IPER occurs at a much faster rate compared to the normal displacement reaction, and also, the dose level required to induce a sufficient defect density is quite small.24,34−36 However, there are also certain limitations in the IPER process, such as, its low efficiency in regard to the primary monolayers composed of long molecules and limited dose range associated with the onset of cross-linking in the primary monolayer at high irradiation doses.33,35,36 Employing DW and IPER methods in combination with electron beam lithography (EBL), we have previously succeeded to fabricate protein-adsorbing and protein-repelling patterns, including gradient ones.24,35 Protein-adsorbing patterns were produced by DW using the primary OEG-AT matrix, while the protein-repelling patterns were fabricated by IPER using protein-adsorbing primary matrix comprising nonsubstituted alkanethiols (ATs) and OEG-AT molecules as substituents. Furthermore, by using OEG-AT molecules with different length of OEG chain (3 or 7 EG units) as the primary monolayers and biotin-terminated OEG-AT molecules as the substituents, we have analyzed the formation of mixed OEGAT/receptor SAMs by IPER process with respect to electron irradiation dose and explored the possibility of specific protein patterning based on biotin−avidin coupling.36 This study provided basic knowledge about the formation of mixed SAMs and their response to the adsorption of specific and nonspecific proteins. However, no attempt to implement the above results for IPER-EBL was made, which was partly related to the complex character of the underlying processes requiring a more detailed investigation and analysis. These shortcomings were overcome in the present work in which the electron irradiation induced defects in the OEG-AT monolayers and their influence on the efficiency of the IPER

2. EXPERIMENTAL DETAILS 2.1. Substrate and Materials. The Au substrates used in the present study were prepared by thermal evaporation deposition of 100 nm Au (99.99% purity) film onto polished single-crystal silicon (100) wafers primed with a 5 nm titanium adhesion layer. The prepared Au films are polycrystalline in nature with the grain size in a range of 20−50 nm and exhibiting predominant (111) orientation.39 The SAM precursors with a general formula HS− (CH2)11(OCH2CH2)n−OH (EGn; n = 3, 5, and 6) and substituent (EG3-bio) molecules used in the present study are shown in Figure 1. The EG3 and EG3-bio substances were

Figure 1. Chemical structures of the molecular constituents used for the preparation of primary OEG-AT monolayers (EG3, EG5, and EG6) and as substituent for IPER (EG3-bio). EG3-bio bears the terminal group (biotin) that has a specific affinity to avidin.

purchased from Asemblon Inc., while EG5 and EG6 were obtained from ProChimia Surfaces. Absolute ethanol, phosphate buffered saline (PBS) tablets, and the proteins, viz. avidin (A9275), bovine serum albumin (BSA, A7638), and biotinylated-BSA (bio-BSA, A8549) were purchased from SigmaAldrich. Distilled water that was additionally purified in a Milli Q plus system (Millipore) was used to prepare all aqueous solutions required for the experiments. 2.2. Monolayer Preparation. The primary OEG-AT SAMs were prepared by immersion of the freshly prepared Au substrates into 1 mM solutions of the SAM precursors (EG3, EG5, and EG6) in ethanol 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 experiments. 14951

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resolution of ∼0.9 eV. The binding energy (BE) scale was referenced to the Au 4f7/2 emission of alkanethiol-coated gold at a BE of 84.0 eV. SEM and AFM were used to image the fabricated patterns. AFM measurements were performed with a Digital Instruments DI3100 microscope equipped with a Nanoscope IIIa controller (Veeco instruments). The images were acquired in tapping mode using standard cantilevers in the repulsive force regime. The SEM images were obtained using the Leo 1530 system at an accelerating voltage of 1 kV.

2.3. Electron Irradiation and EBL. The primary OEG-AT monolayers were either exposed to homogeneous electron irradiation or patterned by focused electron beam (EBL approach). The homogeneous electron irradiation was performed using a flood gun delivering electrons with a kinetic energy of 10 eV in normal incidence geometry. The vacuum in the chamber during irradiation was around 10−8 mbar. The samples were placed at a distance of ∼11 cm to the electron gun to ensure uniform irradiation. The irradiation dose was estimated by multiplication of the exposure time with the current density (∼2 μA/cm2). EBL was performed with a Leo 1530 (Gemini, Zeiss) scanning electron microscope (SEM) equipped with Raith Elphy plus pattern generator system (REPGS) software. The kinetic energy of the electron beam was set to 1 keV. The beam current as measured was around 0.25 nA. The base pressure during the patterning was around 2 × 10−6 mbar. The electron irradiated (or patterned) samples were washed in pure ethanol and dried under argon flow before any measurements or further experiments. 2.4. Exchange Reaction and IPER. Exchange reaction was performed for 5 or 30 min at room temperature by immersion of the pristine or electron irradiated primary OEG-AT monolayers into 0.1 mM solution of the EG3-bio in ethanol. After exchange reaction, the samples were washed thoroughly using pure ethanol to remove the physically adsorbed EG3-bio molecules. Then, the samples were dried under argon flow and used immediately for the characterization or protein adsorption experiments. 2.5. Protein Adsorption. Pristine, irradiated, or IPERprocessed OEG-AT monolayers were subjected to specific and nonspecific protein adsorption. The solutions of avidin, bioBSA and BSA were prepared at a concentration of 0.1 mg/mL using PBS (pH 7.4 at 25 °C) buffer. Protein adsorption experiments were performed by immersion of the monolayer samples in the respective protein solution for an incubation period of 30 min at room temperature. In the experiments involving adsorption of a second protein, the samples bearing the first protein layer were washed in PBS solution and thoroughly rinsed in Millipore water prior to immersion into the solution of the second protein. This procedure is usually performed to remove any weakly adsorbed first protein molecules to avoid their interference with the second protein adsorption. After protein adsorption experiments, the samples were rinsed thoroughly using PBS, washed in Millipore water, and dried under argon flow before the characterization. 2.6. Sample Characterization. X-ray photoelectron spectroscopy (XPS), atomic force microscope (AFM), and SEM were used to characterize the samples. The quality of the primary OEG-AT SAMs, damage caused by electron irradiation, as well as outcomes of the exchange reaction, IPER and protein-adsorption experiments were studied step-bystep using XPS. In particular, the monitoring of the protein adsorption was performed on the basis of the characteristic N 1s signal following the methodology of our previous publications.24,25,36 Such an approach is quite common and frequently used for this purpose; it is sensitive enough and delivers as reliable data as such alternative techniques as surface plasmon resonance or quartz crystal microbalance. A LeyboldHeraeus Max100 XPS system equipped with a hemispherical LHS 11 analyzer was used. The measurements were performed using a Mg Kα X-ray source (E = 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

3. RESULTS AND DISCUSSION 3.1. Primary SAMs: Suitability for DW and IPER. The suitability of the primary monolayers for DW-EBL and IPEREBL had to be proven. The foremost property, crucial for both types of lithography, is the protein-repelling ability. A further requirement, relevant for IPER-EBL only is the inertness of the nonirradiated, primary EGn monolayers toward exchange reaction with the EG3-bio substituent; we will name this specific case as the zero-dose exchange reaction (ZDER). If the primary templates are not inert to ZDER, protein receptors will be imbedded into both irradiated and nonirradiated areas of an EGn template upon IPER, so that the contrast in the resulting protein pattern will be deteriorated. To test the protein-repelling ability, pristine EGn monolayers were exposed to avidin solution and subsequently characterized by XPS, looking for a protein-specific N 1s emission at a BE around 400.1 eV.24,25,36 The respective N 1s XPS spectra are shown in Figure 2. No N 1s peak could be detected for all EGn SAMs of this study, which evidence that they all are protein repelling.

Figure 2. N 1s XPS spectra of pristine (solid lines) and ZDERprocessed (open circles) EGn SAMs after incubation in avidin solution.

To test the inertness to ZDER, primary EGn monolayers were exposed to EG3-bio solution for 30 min (the same conditions as for IPER), subsequently immersed in avidin solution, and characterized by XPS, looking for the proteinspecific N 1s emission (see above). As far as the EGn SAMs are prone to ZDER, the first step of the above procedure resulted in the formation of the mixed EGn/EG3-bio film, which mediated the specific adsorption of avidin upon the immersion of this film into the respective solution. Note that the direct detection of the EG3-bio exchange on the basis of the N1s XPS spectra was inconclusive, presumably because of the low content of N in the EG3-bio molecules and low concentration of these molecules in the mixed EGn/EG3-bio monolayer, which can be expected for the nonirradiated films. However, even such low concentration of EG3-bio could mediate 14952

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noticeable coverage of avidin36,40,41 due to the multivalent nature of the biotin−avidin interaction. Moreover, the N1s XPS signal from the avidin is much stronger than that from biotin, which improves the sensitivity of the ZDER tracking. The N1s XPS spectra of the EGn films acquired after ZDER and immersion into avidin solution are shown in Figure 2. As can be seen in this figure on the basis of the intensity of the characteristic N1s emission, the stability of the monolayers against ZDER continuously improves with the increasing length of the OEG chain. EG3 and EG5 are not stable, but the intensity of the N1s peak for EG5 is lower suggesting a smaller extent of ZDER. In contrast, no signal is observed for EG6, which implies that this film is completely stable against ZDER, at least at the conditions of our experiments (30 min exposure to EG3-bio solution). This behavior correlates well with our previous findings for the EG3 and EG7 monolayers, with the former exhibiting poor stability against ZDER and the latter one being stable for at least 2 h of exposure to EG3-bio solution.36 Combining, the above results shows that the OEGAT molecular layers should contain at least six EG units to exhibit stability against ZDER required for application in IPEREBL. The above findings provide a basis for the use of EGn SAMs in DW-EBL and IPER-EBL. On the basis of these results, further experiments, described in the following sections, were focused mainly on EG6 monolayers that fulfill all the requirements for the lithography, with some additional experiments on other monolayers when necessary for comparison or more detailed analysis. 3.2. Electron Irradiation of EGn SAMs. Modification of aliphatic SAMs by electron irradiation has been investigated extensively.42,43 The primary processes are decomposition of alkyl chain and cleavage of Au−thiolate bonds.42,43 The closely related secondary processes are the loss of conformational and orientational order, partial dehydrogenation of the film, trapping and desorption of the released fragments, and crosslinking between residual moieties.42 In this section, we present a brief description of the irradiation induced modification of OEG-AT SAMs with particular emphasis on EG6 monolayers. The irradiation-induced processes in EGn SAMs were monitored by XPS on the basis of the C 1s and S 2p spectra as shown in Figure 3 for EG6 monolayers. The C 1s spectrum of the pristine monolayers exhibits two emissions at 284.5 and 286.3 eV associated with the OEG (C−O) and alkyl (C−C) parts of the EG6 molecules, respectively.23,24 Upon irradiation, the C−O component decreases in intensity, which is related to progressive decomposition of the OEG chain.24 In contrast, the C−C component remains mostly unchanged, which means, in accordance with literature, 24 that the OEG chain is preferentially damaged. The S 2p spectra of the pristine monolayers exhibits a characteristic doublet at ∼162.0 eV (S 2p3/2) related to the thiolate moiety bonded to the gold surface.42,43 Upon irradiation, an additional doublet (S*) at ∼163.6 eV appears and increases in intensity. This doublet is assigned to irradiation-induced dialkylsulfide species trapped in the monolayer matrix.44,45 The kinetics of the above processes was studied in detail by evaluating the XPS spectra. The intensity ratio of the C−O and C−C components (ICO/ICC) representing the preferential damage of the OEG chain and the relative intensity of the thiolate signal (Ithiolate) as a measure of the damage at the SAM−substrate interface are plotted against the irradiation dose for EGn monolayers in Figure 4. The observed dependencies could be fitted by a first order

Figure 3. C 1s (left panel) and S 2p (right panel) XPS spectra of the pristine and electron irradiated EG6 monolayers. The C 1s spectra are decomposed into two peaks related to the OEG (C−O) and alkyl (C− C) parts of EG6, respectively. The S 2p spectra are decomposed into two doublets associated with the thiolate (Au−S) and dialkylsulfide (S*) components. The irradiation doses (in mC/cm2) are given at the respective spectra. The vertical dashed lines are guides to the eye.

Figure 4. Intensity ratio of the C−O and C−C components in the C 1s XPS spectra of EG3 (black circles), EG5 (dark gray squares), and EG6 (gray triangles) monolayers as well as the relative intensity of the thiolate-related doublet in the S 2p spectra of EG5 (dark gray squares) and EG6 (gray triangles) monolayers as functions of irradiated dose. The solid lines are the first order exponential decay function fits to the experimental data. These fitting curves were used to calculate the cross-sections of the irradiation induced processes. The respective cross-sections for EG3, EG5, and EG6 monolayers are given in the inset tables.

exponential decay function (first-order kinetics) following the formalism of refs 42 and 46 I = Isat + (Iprist − Isat) × exp( −σQ /eSirrad)

where I is the value of a characteristic film parameter in the course of irradiation, Iprist and Isat are the parameters for the pristine and strongly irradiated (a leveling off behavior) film, respectively, Q is the cumulative charge delivered to the surface in Coulombs, e is the electron charge, Sirrad is the area irradiated by the electron beam, and the cross-section σ (expressed in cm2) is a measure of a rate at which the saturation behavior is achieved. The calculated cross-section values for the irradiationinduced processes are given as inserts in Figure 4. These crosssections were found to be higher for EG6 monolayer as compared to the EG5 and EG3 cases. Interestingly, the ICO/ICC curves look similar for all the SAMs in terms of the leveling off behaviors, whereas the Ithiolate curves for EG6 and EG5 films are different in this regard. This implies that the decomposition mechanism of the OEG chain is similar in all EGn monolayers, 14953

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monolayers are likely to be restricted only to a partial cleavage and damage of the EG units due to the high sensitivity of these groups to electron irradiation.24 Under these conditions, the irradiated monolayer could still exhibit protein resistance as far as at least three intact EG units remain (see section 3.1).14,17,49 In contrast and in agreement with literature data,36 even such subtle defects can be successfully utilized for IPER. As shown in Figure 5, specific adsorption of avidin, following the IPER procedure, could be detected at all dose conditions. Although EG3-bio exchange could not be detected directly at lower doses, probably due to a low concentration, the observed specific adsorption of avidin reveals its presence. Interestingly, the avidin coverage increases progressively with increasing dose in the given dose range reaching ∼250% at 2 mC/cm2. This behavior is typical for low coverage of EG3bio25,36,40 correlating with our values for the EG3-bio coverage in Figure 5. Note that, typically, the amount of avidin adsorbed on a mixed EGn/EG3-bio film increases initially with increasing portion of EG3-bio, achieves a maximum at 20−30% EG3-bio coverage, and decreases at a higher EG3-bio portion.36,40 The maximum avidin coverage observed in the present work is somewhat higher than the previously recorded values of around 220%,25,36 which could be due to an additional nonspecific adsorption process mediated by the residual, damaged EG6 species in the EGn/EG3-bio monolayer. Such species can remain after IPER at high irradiation doses because of the cross-linking between the SAM constituents. 3.4. Efficiency of the IPER Process. As discussed in the previous section, the efficiency of the IPER process depends generally on the nature of the irradiation-induced defects in the monolayers. Whereas most of the subtle defects produced at low irradiation doses favor the exchange reaction, some of the more severe defects generated as a result of cross-linking or trapping at higher doses can hinder this reaction. Such defects are usually difficult to characterize directly, at least in the case of aliphatic SAMs.33,35,36,42 In this section, we describe an indirect method using protein adsorption to assess the extent of irradiation-induced defects at higher doses and their effect on the IPER process. The EG6/EG3-bio template prepared by the IPER process is supposed to adsorb avidin specifically (as described in the previous section). Such a specificity can only be achieved with a high efficiency exchange reaction, i.e., a complete exchange of the SAM constituents with severe irradiation-induced defects, which otherwise could lead to nonspecific adsorption of other proteins. Therefore, by the adsorption of nonspecific proteins such as BSA on the EGn/EG3-bio templates, the efficiency of the exchange reaction and hence that of the IPER process could be assessed. Differences between the N 1s XPS spectra of EGn/ EG3-bio films (n = 6, 5, and 3) acquired after and before their exposure to BSA are shown in Figure 6; the N 1s signal from EG3-bio is eliminated by the subtraction procedure. The EG6/ EG3-bio film prepared at low irradiation doses (e.g., at 0.3 mC/ cm2) showed no adsorption of nonspecific BSA, which suggests a complete exchange of the severe irradiation-induced defects by EG3-bio molecules (100% efficiency of IPER). In contrast, at irradiation doses of 0.6 mC/cm2 and above, nonspecific adsorption of BSA was detected and found to increase with increasing dose. These observations imply that the damaged SAM constituents in EG6 monolayers irradiated at higher doses were not completely exchanged by the EG3-bio species and capable to mediate the nonspecific adsorption of BSA. The increase in nonspecific adsorption coverage of BSA with

whereas the stability of the SAM−substrate interface seems to increase to some extent with increasing OEG chain length. This can be tentatively explained by a higher extent of irradiationinduced cross-linking in the SAMs with long OEG chain. Similar to the situation in aromatic SAMs,47 such a cross-linking binds the residual species to each other preventing, at least to some extent, breaking of the S−Au bonds. Note, however, that in contrast to the aromatic SAM, modification of which, including the chemical transformation of the attached tail groups, requires doses in the range of 40 mC/cm2,47,48 much lower (by two orders of the magnitude) doses are sufficient for the modification of the OEG-AT monolayers. This is a significant advantage since it allows to reduce the time necessary for the pattern writing considerably. 3.3. IPER and Protein Adsorption. The EG6 monolayers, which were found to be suitable for both DW-EBL and IPEREBL (see section 3.1), were studied in detail with respect to the basic processes behind the above lithographic approaches. In the framework of DW, EG6 SAMs were homogeneously irradiated, immersed in protein solution, and characterized by XPS to estimate the protein coverage. In the framework of IPER, EG6 SAMs were homogeneously irradiated, immersed in EG3-bio solution for exchange reaction, exposed to protein solution, and characterized by XPS. In both cases, we used avidin as a test protein, which corresponded to nonspecific and specific adsorption conditions in the cases of DW and IPER, respectively. The EG3-bio exchange reaction time was fixed at 5 min in all the experiments involving IPER described here and hereafter to ensure a complete absence of ZDER. The surface coverage of avidin adsorbed on the irradiated EG6 monolayer before (DW) and after (IPER) the exchange reaction with EG3-bio as well as surface concentration of the exchanged EG3-bio in the mixed EG6/EG3-bio films (IPER) are presented in Figure 5 as functions of irradiation dose. The

Figure 5. Surface coverage of avidin adsorbed on the irradiated EG6 monolayer before (DW) and after (IPER) the exchange reaction with EG3-bio as well as surface concentration of exchanged EG3-bio in the mixed EG6/EG3-bio films as functions of irradiation dose. The solid lines are guides to the eye. The surface concentration of EG3-bio is referenced to the single-component EG3-bio SAM. The avidin coverage is referenced to the coverage on the single-component EG3-bio SAM.

respective N 1s XPS spectra can be found in Supporting Information (Figure S1). In the case of DW, nonspecific adsorption of avidin was not detected until the irradiation dose of 0.6 mC/cm2 above which a gradual increase in the coverage was observed. This adsorption behavior is directly related to the nature of the irradiation-induced defects in the EG6OH monolayer. At low dose, the defects produced in the 14954

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cm2 predicted for the onset of cross-linking in dodecanethiol monolayers on gold. The practical significance of the above results is the exact definition of the electron irradiation dose range suitable for IPER with the resulting mixed EGn/EG3-bio films exhibiting only specific adsorption of proteins. For EG6 monolayers, the IPER process could be employed efficiently in a dose range of 0 to 0.6 mC/cm2 above which the mixed EG6/EG3-bio film will be partly prone to nonspecific adsorption. Similarly, for EG5 and EG3 monolayers, the efficient dose ranges for IPER process are 0−1.1 and 0−1.5 mC/cm2, respectively. Interestingly, the dose of 0.6 mC/cm2 for EG6 monolayers coincides practically with the onset of nonspecific protein adsorption in the case of DW (Figure 5). This shows that the underlying processes are quite similar, which is understandable since DW relies upon the extensive damage of the OEG chain, which can involve subsequent cross-linking of the residual species. 3.5. Specificity of EG6/EG3-Bio/Avidin Template. In a variety of practical cases, first protein layer serves as a template for the adsorption of a second protein, relying on specific interaction between these two proteins. In such cases, the activity in the first adsorbed protein should be retained to enable specific adsorption of the second protein. Otherwise, denaturation or structural changes of the first protein could cause a decline in the efficiency and specificity of the system.50−52 Both denaturation and structural changes are rather unlikely if the first protein adsorbs by specific interaction only, whereas such changes can occur in the case of nonspecific adsorption due to multiple and nonstrictly defined interaction with the primary substrate. Since the nonspecific adsorption of proteins was found to occur on EGn/EG3-bio films fabricated by IPER above a certain onset dose as discussed in the previous section, it was important to test the specificity of the EGn/ EG3-bio/protein templates toward specific or nonspecific adsorption of the second proteins. For this purpose, EG6/EG3-bio films were prepared by IPER at a dose value of 1.1 mC/cm2 and exposed to avidin, resulting in EG6/EG3-bio/avidin templates. Note that, at this dose value, according to Figure 6, EG6/EG3-bio films are prone to both specific and nonspecific adsorption, so that a part of the avidin moieties was adsorbed nonspecifically. Figure 8 shows the N1s XPS spectra of EG6/EG3-bio/avidin template and those recorded after the subsequent exposure of the template to BSA (nonspecific adsorption) or to bio-BSA (specific adsorption). The spectrum of the template (Figure 8A, curve a) and that obtained after the exposure to BSA (Figure 8A, curve b) were found to be similar without any loss or gain (Figure 8B, curve b−a) in the N 1s intensity. However, the exposure to bio-BSA (Figure 8A, curve c) produced a significant gain (Figure 8B, curve c−a) in the N 1s intensity. The lack of the nonspecific adsorption of BSA shows that the templates remain stable when exposed to biological conditions and that the avidin moieties adsorbed by both specific and nonspecific interactions retain their bioactivity. The detection of the specific adsorption of bio-BSA, facilitated by the avidin− biotin interaction, further proves the bioactive nature of the adsorbed avidin layer. While the specifically adsorbed avidin is expected to be bioactive, it was interesting to observe that the nonspecifically adsorbed avidin also exhibits bioactive behavior to some extent by remaining passive to nonspecific adsorption of BSA. This ability can be of potential importance for biosensors and immunoassays.

Figure 6. Difference between the N 1s XPS spectra of EGn/EG3-bio films acquired after and before their exposure to BSA. The irradiation doses (in mC/cm2) are given at the right side of the respective spectra, while the BSA coverage values are given at their left side. The latter are calculated with respect to the BSA coverage on a reference dodecanethiol SAM.

increasing dose gives a rough estimate for the density of such nonexchanged, damaged molecules at higher doses. As seen in Figure 6, EG5 and EG3 based films exhibit similar behavior as EG6 based ones, but the onset of the nonspecific adsorption of BSA occurs at the higher dose values as compared to the EG6/EG3-bio monolayers. Moreover, this onset shifts to higher doses with a decrease of the OEG chain length (compare the spectra for EG5/EG3-bio and EG3/EG3-bio). This behavior can be explained by the assumption that the efficiency of IPER increases with decreasing molecular length in the primary film as suggested in our earlier studies.33,36 Moreover, the observed strong correlation between the onset dose and the OEG chain length suggests that the cross-linking or related processes occur predominantly between the decomposed OEG residues in the monolayers. Unfortunately, the shorter chain OEG-ATs cannot be used as straightforward as EG6 monolayers for IPER based lithographic patterning due to the limitation imposed by the ZDER as described in section 3.1. The dependence of the onset dose for the formation of crosslinking-like defects on the number of the EG units is presented in Figure 7. This dependence could be fitted with an exponential function that exhibits the leveling off behavior at ∼1.65 mC/cm2. The latter value can be considered as the onset of the IPER hindering cross-linking in nonsubstituted alkanethioate SAMs. In accordance with our previous results,33,35 this value is within the dose range of 1 to 2 mC/

Figure 7. Onset dose for the formation of cross-linking-like structures as a function of the number of the EG units in the SAM constituents. The dark gray line is an exponential fit to the experimental data extrapolated to the lower number of EG units. The black dashed line represents the saturation dose of IPER. 14955

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of the OEG chain could occur even at a low dose so that the nonspecific protein adsorption in step 1a could be possible. The schematic summary in Figure 9 could be used to identify the appropriate pathways for the different types of protein patterning. By following the steps 1b and 1d or 1b, 1d, and 1e, completely specific protein patterns can be prepared. However, irradiation with a high dose (step 2) will make the OEG-AT monolayers prone to nonspecific protein adsorption. Performed directly after irradiation (step 2a) such an adsorption is described as DW as mentioned several times above. At the same time, by employing the IPER process at these conditions (step 2b), patterning by specific binding, at least at the secondary adsorption level (bio-BSA), is possible as shown in step 2e following step 2d. As demonstrated in the previous section, EGn monolayers are not selective for the first adsorbing protein after irradiation with a high dose; however, the selectivity could be attained for the adsorption of a suitable second protein. Finally, a combination of the DW and IPER processes is possible (step 2c), which enables the fabrication of multiprotein patterns as represented in step 2f. The nonspecific and specific protein patterns fabricated by EBL according to the procedures shown in Figure 9 are described below. The images of the EBL patterned EG6 monolayer and the respective patterns obtained after protein adsorption are shown in Figure 10. The image 10a is an SEM image obtained after the EBL writing in the primary monolayer; the irradiation dose was continuously varied along each stripe from 0 to a certain value given in the image. The images 10b and 10c shows avidin patterns produced by DW (step 2a in Figure 9) and IPER processes (step 1d in Figure 9), respectively. The pattern prepared by IPER shows certain avidin coverage at all doses from 0 to 3 mC/cm2, while that fabricated by DW exhibits the adsorbed avidin starting only

Figure 8. Panel A: N 1s XPS spectra of irradiated (1.1 mC/cm2) EG6 monolayer after exchange reaction with EG3-bio and avidin adsorption (a) and subsequent exposure to BSA (b) or bio-BSA (c). Panel B presents difference spectra calculated from the spectra in panel A.

3.6. Protein Patterning. The above results provide a reliable basis to fabricate specific or nonspecific protein patterns in protein-repelling OEG-AT templates by EBL. A summary of possible procedures is schematically presented in Figure 9. The schemes best represent EG6 monolayers for which the low (1) and high (2) dose regimes are those within the IPER dose range (0−0.6 mC/cm2) and above, respectively. In the cases of EG5 and EG3, the scheme after low dose irradiation (1) could be somewhat different; in particular, a complete decomposition

Figure 9. Schematic representation of the processes adopted for the fabrication of specific (boxed) and nonspecific (NS; gray shaded) protein patterns in primary OEG-AT monolayers. Irradiation at low doses (1) is suitable for IPER (1b) through which only specific adsorption of proteins is possible (1d and 1e). Throughout the IPER process, nonspecific adsorption of proteins does not occur (1a or 1c). Irradiation with high dose (2) results in the monolayers that are prone (DW, 2a) or partly prone (after exchange reaction, 2c and 2d) to nonspecific adsorption of proteins. The specific adsorption of proteins was then only possible at the secondary level as depicted in step 2e with an example of bio-BSA on avidin. By combining the IPER and DW processes, multiprotein adsorption is possible as represented by step 2f. 14956

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Figure 10. SEM image of EBL-generated, gradient-like stripe patterns in EG6 monolayers (a) as well as AFM images of the protein patterns prepared on such templates for the cases of direct adsorption of avidin (b), avidin adsorption after exchange reaction with EG3-bio (c), BSA adsorption after exchange reaction with EG3-bio (d), and subsequent adsorption of BSA and avidin after exchange reaction with EG3-bio (e). The length of each stripe is 20 μm; the dose along the stripes was varied from 0 to the values shown in panel a. The scale bar is 10 μm. The height profiles along the white lines in the AFM images are presented below the respective images.

Figure 11. SEM image of EBL-generated pattern in EG6 monolayers (a) as well as AFM images of this pattern obtained after exchange reaction with EG3-bio and subsequent BSA (b) and avidin adsorption (c). Different doses were used to write the squares and rectangles in the EBL-generated pattern; they are given in the SEM image. Areas covered by BSA and avidin are marked in panels b and c. Scale bar is 10 μm.

from a dose of ∼0.25 mC/cm2. This behavior agrees well with the results of the avidin adsorption experiments presented in Figure 5. The difference in the onset dose for DW as compared to the value in Figure 5 (0.5−0.6 mC/cm2) is related to the different kinetic energies of electrons used for the homogeneous irradiation (10 eV) and EBL patterning (1 keV).48 Similarly, the BSA pattern prepared on EG6/EG3-bio templates fabricated by IPER exhibits the adsorbed BSA only above a dose of ∼0.25 mC/cm2. As mentioned in section 3.4, this adsorption relies on the nonspecific interaction of BSA with the severely damaged but not exchanged EG6 molecules in the primary templates. The complete exchange does not occur at high irradiation doses because of the cross-linking between the SAM constituents. The above dose of ∼0.25 mC/cm 2 correlates well with the analogous value of ∼0.6 mC/cm2 obtained by the spectroscopic experiments (section 3.4) as far as one considers the different impacts of the 1 keV (EBL) and 10 eV (spectroscopy) electrons.48 The BSA pattern shown in Figure 10d was exposed to avidin (step 2f in Figure 9) resulting in the pattern shown in Figure 10e. As seen in the latter figure, avidin adsorbed on all irradiated areas that were not covered by BSA. This adsorption relies upon the specific interaction between the avidin moieties and biotin tail groups of EG3-bio. Thus, the pattern presented

in Figure 10e is an example of a multiprotein pattern comprising of both avidin and BSA, which, however, cannot be clearly distinguished from each other. In order to clearly distinguish BSA and avidin as well as to address properly the multiprotein patterning challenge, the patterns as shown in Figure 11 were fabricated. For this purpose, an array of squares and rectangles was written by EBL in EG6 template at dose values of 0.15 and 2 mC/cm2, respectively, and the resulting pattern was subjected to the exchange reaction with EG3-bio (IPER). The former dose lies in the exclusive IPER range (0−0.25 mC/cm2), so that only specific adsorption of avidin was possible on the square areas. The latter dose lies far above this range, so that predominately nonspecific adsorption was possible on the rectangular areas. Accordingly, after the exposure of the pattern to BSA, its adsorption was observed only over the rectangular spots as demonstrated by the AFM image in Figure 11b. Subsequently, after this pattern was exposed to avidin, its adsorption occurred over the square areas as demonstrated by the AFM image in Figure 11c. This method is an easy way to fabricate multiprotein patterns, at least in a two protein system; however, the success of this method relies on the selection of a suitable receptor and, most importantly, on the correct sequence of protein adsorption. Note that, in the above system, 14957

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Figure 12. AFM images (3D) of complex, gradient-like protein patterns fabricated by EBL in EG6 matrix. Panels I and II show avidin patterns produced by DW-EBL and IPER-EBL, respectively, and the images g and h in panel III correspond to additional bio-BSA adsorption on the patterns e and f, respectively. The irradiation doses used in all the patterns are in a range of 0−2 mC/cm2. In patterns a and d, the dose was increased in spiral fashion along the feature; in patterns b, e, and g, the dose was increased from an edge of the triangle to the opposite boundary (in a stripe-like fashion for g); and in patterns c, f, and h, the dose was increased from the center of the square toward its boundaries in all directions. The gradients are, however, poorly visible in some cases since the dose range is too broad. Electron energy was 1 keV. Scale bar is 1 μm. The height profiles along the white lines in the AFM images b, e, and g are shown in panels i, j, and k, respectively.

the reverse sequence as first avidin and then BSA would not have worked because avidin will cover both squares and rectangles completely already in the first step similar to the situation shown in Figure 10c. Such patterns can, however, be useful to compare the activity of specifically and nonspecifically adsorbed proteins. Note that, parallel to the relatively simple (in terms of form) stripe-like or array-like patterns that we used to investigate the target phenomena and to demonstrate the reliability of the approach, complex patterns can be prepared as demonstrated in Figure 12. EBL does not have any limitations in terms of the form of the patterns or the dose distribution within the written features, which allows the fabrication of gradient-like patterns. The respective dynamical ranges are given by the curves in Figure 5. As far as the dose is varied within these ranges, the surface density of specifically or nonspecifically adsorbed proteins can be precisely controlled.

possible. At higher doses, which could induce cross-linking between the SAM constituents so that they cannot be easily exchanged by IPER, nonspecific adsorption of proteins was always present. Within the above dose ranges, the outcomes of both DW and IPER could be well-controlled in terms of the coverage of nonspecifically and specifically adsorbed proteins. Accordingly, specific and nonspecific protein patterns of various shapes (including gradient-like) were fabricated by EBL. Along with the one-component patterns, multiprotein ones were prepared by a single-step irradiation procedure, applying doses within both characteristic dose ranges of IPER to the different features in the patterns. In view of the high flexibility of EBL, there are no limitations regarding the form and length scale of the fabricated features. The extremely low doses required for the modification of the OEG-AT SAMs enable a rapid and efficient patterning even if it performed in a successive mode, by a focused electron beam.

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4. CONCLUSIONS We investigated modification of OEG-AT monolayers by electron irradiation to explore the conditions suitable for the fabrication of protein patterns by specific and nonspecific interactions. In the former case, the IPER process was employed, based, as a test, on the interaction between the imbedded biotin-bearing receptors and avidin proteins, whereas DW approach was followed in the case of nonspecific adsorption. All tested OEG-AT SAMs with at least three EG units in the OEG chain were found to be suitable for the DW process, while, for the lithographic applications of IPER, at least six EG units are required to possess the necessary stability against ZDER. DW was found to be possible starting from a certain onset dose, below which no nonspecific adsorption of proteins was observed after irradiation. For IPER, two different dose regimes were identified. At low doses, at which the modification in the monolayers was only restricted to subtle defects and all damaged SAM constituents were exchanged by IPER, protein patterning exclusively by specific adsorption was

ASSOCIATED CONTENT

S Supporting Information *

N 1s XPS spectra related to Figure 5 and more examples of protein patterns of different size and shapes, including gradients, fabricated on EG6 monolayers by DW and IPER processes. This material is available free of charge via the Internet at http://pubs.acs.org.

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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. This work has been financially supported by DFG (ZH 63/9-3). 14958

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