Article pubs.acs.org/Langmuir
Biofunctional Surface Patterns Retaining Activity after Exposure to Whole Blood Ryosuke Ogaki* and Morten Foss Interdisciplinary Nanoscience Center (iNANO), Faculty of Science and Technology, Aarhus University, Aarhus, Denmark S Supporting Information *
ABSTRACT: Biofunctional surface patterns capable of resisting nonspecific bioadsorption while retaining bioactivity play crucial roles in the advancement of life science and biomedical technologies. The currently available functional surface coatings suffer from a high level of nonspecific surface adsorption of proteins under biologically challenging conditions, leading to a loss of activity in functional moieties over time. In this study, the recently discovered facile method of temperature-induced polyelectrolyte (TIP) grafting has been used to graft two biofunctional variants (biotin and nitrilotriacetic acid, NTA) of poly(L-lysine)-grafted PEG (PLL-g-PEG) onto a titanium surface. A significant increase in the polymer adsorption was observed from the TIPgrafted surfaces assembled at 80 °C, compared to the polymer surfaces assembled at ambient temperature (20 °C). These functional PLL-g-PEG surfaces were subsequently incubated in whole human blood continuously for up to 7 days, and the TIP-grafted surfaces achieved close-to-zero nonspecific protein adsorption, as confirmed by ultrasensitive time-of-flight secondary ion mass spectrometry (ToF-SIMS). To test the maintenance of the bioactivity of the biotin and NTA moieties, submicrometer-scale mono- (biotin) and bi- (biotin/NTA) functional surface chemical patterns were fabricated via two-step TIP grafting using colloidal lithography (CL), preincubated in blood for up to 7 days and sequentially exposed to streptavidin and Ni2+-histidine-tagged calmodulin. The fluorescence microscopy studies revealed that the PLL-g-PEG-NTA and -biotin surfaces grafted from the TIP method were still capable of recognizing the corresponding affinity proteins for up to 1 and 7 days of preincubation in blood, respectively. These results highlight the bioresistant robustness realized by the facile TIP grafting method, which in turn preserves the activities of biofunctional moieties over a prolonged period in whole blood.
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INTRODUCTION Functional surface patterns capable of recognizing multiple specific chemical and biological entities while minimizing nonspecific bioadsorption from biologically stringent conditions are vital for numerous biomedical applications, including biosensing, drug screening, functional implants, medical diagnostics, and fundamental cell studies.1−4 In general, biofunctional surface patterns contain protein-coupling and protein-resisting regions.1,3,5 The specific proteins of interest are immobilized on the protein-coupling regions via covalentor affinity-based coupling methods to retain the activity and orientation of the proteins, while nonfouling polymers such as poly(ethylene glycol) (PEG)-based polymers are used to create protein-resisting regions to minimize the nonspecific adsorption of proteins and cells from the biological milieu.5−10 The strategy becomes complicated for generating multiple biological surface patterns, for instance, in protein pattering, where multiple proteins must be individually and precisely positioned on a surface. In this case, several nonfouling polymers with functional groups are used, and each polymer must be capable of coupling protein specifically, while resisting all other proteins.11 Despite the successful surface patterning of single or multiple12−14 proteins using the top-down and bottom-up methodologies reported so far, a low level of nonspecific © 2014 American Chemical Society
bioadsorption under challenging conditions remains a challenge. Furthermore, the currently available fabrication procedures require several conjugation steps, which introduce a number of issues such as the coupling efficiency, presence of unconjugated reactive groups, and orientation of biomolecules. The recent promising nonfouling functional materials include zwitterionic polymers such as polycarboxybetaine (pCB),15−19 and poly(oligo(ethylene glycol) methacrylate (pOEGMA).20,21 In particular, pCB is capable of overcoming the issue associated with the unconjugated reactive groups, where COO-N-hydroxyl succimide (NHS) groups can be hydrolyzed back to the nonfouling state of COO−.16,17 However, the reactivity of the carboxylate moiety after exposure to biological fluid is yet to be shown. Furthermore, despite the successful detection of multiple analytes demonstrated from rabbit blood using pOEGMA22 and undiluted human plasma using pCB,15,17−19 the nonspecific adsorption studies were conducted for several minutes to a few hours at most. Thus, there is an unmet need in numerous applications that require long-term robust device performance under stringent conditions, for instance, bloodReceived: February 24, 2014 Revised: April 28, 2014 Published: May 27, 2014 7014
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Figure 1. Overall concept of the presented study. The effect of incubation temperature (20 and 80 °C) on the surface adsorption and bioresistance of PLL-g-PEG and its functional derivatives of PLL-g-PEG biotin and PLL-g-PEG NTA (a) were first studied on flat surfaces using XPS and ToFSIMS (b). The bioreconition tests were conducted subsequently by first fabricating the chemical patterns consisting of monofunctionality (biotin) and bifunctionality (biotin and NTA) via colloidal lithography and physical vapor deposition (c, i−iii), second, incubating in whole blood for up to 7 days (iv), and finally, incubating the blood-exposed patterns to Ni2+, streptavidin, and His-tagged calmodulin (v). The success of the biorecognitions were confirmed by incubating the patterns sequentially in mouse IgG anticalmodulin primary antibody, atto-565-labeled biotin, and atto-488-labeled goat IgG antimouse secondary antibody and visualized by fluorescence microscopy (vi).
contacting device applications such as long-term implantable biosensors.23−25 Furthermore, the antibody patterns made in the aforementioned studies were fabricated using array printers, which provided pattern sizes of several hundred micrometers, but this is yet to be demonstrated for significantly smaller patterns. The minituarization of the pattern features gives rise
to an issue in achieving sufficiently discriminating signal-tonoise (S/N) performance, as the number of antibodies per patterned area inevitably decreases with decreasing pattern size.8 Thus, ideally, the surface-patterning strategy for biological applications should be (1) straightforward (e.g., by using a “grafting-to” approach for the patterning of functional 7015
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found to be advantageous due to the wide commercial availablilty of His-tagged proteins, and the reaction can be rapid and reversible.35 The overall concept of the study is given in Figure 1. We first examined the level of adsorption of PLL-g-PEG, PLL-g-PEG biotin, and PLL-g-PEG NTA at 20 °C (conventional grafting) and 80 °C (TIP) on Ti surfaces by X-ray photoelectron spectroscopy (XPS). The chemical structures of the studied polymers are given in Figure 1a. Using XPS and ToF-SIMS, we further investigated the bioresistant capabilities of the functional PLL-g-PEG polymer surfaces against whole blood from 1 to 7 days (Figure 1b). Finally, monofunctional and bifunctional chemical patterns consisting of PLL-g-PEG biotin/PLL-g-PEG and PLL-g-PEG biotin/PLL-g-PEG NTA, respectively (Figure 1c), were fabricated via colloidal lithography (CL) and physical vapor deposition (PVD) to test the biorecognition capability of the TIP-grafted functional polymer surfaces. These patterns were exposed to whole blood for up to 7 days, and the recognition of streptavidin and Ni2+/His-tagged calmodulin by the patterned biotin/NTA functional groups was subsequently tested. To confirm the success of biorecognition, the patterned streptavidin and/or Ni2+-His-tagged calmodulin surfaces incubated in Atto-565-labeled biotin, mouse IgG anticalmodulin primary antibody, and Atto-488-labeled goat IgG antimouse secondary antibody were visualized by fluorescence microscopy. The nonspecific adsorption and biorecognition capabilities of PLL-g-PEG biotin36,37 and PLL-g-PEG NTA35 assembled at room temperature have been studied previously against serum and bacterial cytoplasmic fractions for a short duration of less than 1 h. However, the retention of biofunctionality was not investigated after biological fluid exposure. It is important to determine whether the bioactivity of these functional moieties can be maintained in a biological fluid over an extended period, as the functionality may be lost immediately from nonspecific bioadsorption from the surrounding environment.8
polymers); (2) able to exhibit robust bioresistant performance over a long period in a biologically demanding environment; and (3) capable of scaling down the feature size while maintaining a detectable S/N level (i.e., maintain a high biomolecular capturing capacity in the patterned area). We have previously shown26 that the temperature-induced polyelectrolyte (TIP) grafting of nonfouling poly(L-lysine)grafted poly(ethylene glycol) (PLL-g-PEG) permits extremely dense surface grafting of the polymer. The study investigated the sole effect of temperature (from 20 to 80 °C) on the adsorption of PLL-g-PEG onto titanium (Ti) surfaces where an almost 4 times higher grafting density of the polymer was achieved at the highest incubation temperature of 80 °C compared to that at 20 °C. The small-angle X-ray scattering (SAXS) data further indicated that there was a negligible change in the hydrodynamic volume of the polymer observed from 20 °C up to 90 °C in solution (reduction in the crosssectional radius of the polymer, R, of ∼14%), thus we concluded that the cloud-point effect27 (i.e., collapsing the polymer chains owing to the reverse solubility nature of the PEG with increasing temperature, leading to high surface grafting) may not be the main contributing effect for the increased polymer surface graft density observed with higher grafting temperatures. However, it was noted that the cloudpoint effect cannot be fully neglected, as the SAXS data also qualitatively showed a decrease in interpolymer repulsion with an increase in temperature. The systematic studies into the effect of varying ionic strength (NaCl varied from 0 to 2.4 M) during and after polymer assembly at 20 and 80 °C showed that the overall adsorbed polymer amount during the assembly and the stability of the assembled polymer under high ionic strength conditions was significantly higher for PLL-g-PEG prepared at 80 °C compared to that prepared at 20 °C. These results indicated that the temperature may also affect electrostatic as well as nonelectrostatic interactions between the polymer and the Ti surface, where some of the nonelectrostatic forces are expected to be temperature-dependent but are not strongly sensitive to the presence of salt.28 The importance of having a high surface graft density for achieving robust and long-term bioresistance was demonstrated against undiluted serum and three types of mammalian cells for up to 36 days, as well as against whole human blood for up to 1 day. A vanishingly low nonspecific protein adsorption was achieved as confirmed by time-of-flight secondary ion mass spectrometry (ToF-SIMS), which can provide a typical detection limit of attomolar29 or 0.1 ng/cm2.30 From these findings, we hypothesized that the TIP grafting method may also be applicable to functional PLL-g-PEG derivatives containing bioreactive groups (PLL-g-PEG-biotin and -nitrilotriacetic acid, NTA) for obtaining robust bioresistance while retaining the activity of the functional moieties in blood. We selected biotin and NTA as model systems since these affinity ligands are widely used in numerous biomedical applications including immunoassays, chromatographic protein purification, and diagnostics.31 Biotin is a vitamin which can strongly interact with tetrameric streptavidin by forming multiple noncovalent interactions without altering its structure and remains stable over a wide range of pH and temperature.32,33 NTA is a quadridentate chelate former which can bind strongly via four positions in octahedral metal coordination spheres such as Ni2+, and the remaining two free ligand positions in the sphere can bind with oligo-histidine tags (also known as His-tags).34 This chelator-based strategy is
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EXPERIMENTAL SECTION
Materials. Polystyrene (PS) particles with a diameter of 1.77 μm (10% w/v) were purchased from Microparticles GmbH (Berlin, Germany). PLL-g-PEG (PLL (20 kDa)-g-[4.0]-PEG (5 kDa)), PLL-gPEG biotin (PLL (20 kDa)-g-[3.4]-PEG (2 kDa)/PEG (3.4 kDa) biotin (51%)), and PLL-g-PEG NTA (PLL (20 kDa)-g-[3.5]-PEG (3.4 kDa) NTA (96%)) were purchased from SuSoS AG (Dübendorf, Switzerland). His-tag calmodulin and anticalmodulin IgG monoclonal primary antibody were purchased from Merck Millipore KGaA (Darmstadt, Germany). Nickel(II) chloride hexahydrate, streptavidin, atto-565-labeled biotin, and atto-488-labeled antimouse IgG monoclonal secondary antibody from goat were purchased from SigmaAldrich (Copenhagen, Denmark). PLL-g-PEG Adsorption. Sputter-coated Ti substrates (100 nm) were sonicated in ethanol and MQ water for 20 min and UV/ozone treated for 20 min. The substrates were dried in N2 and immediately immersed in PLL-g-PEG and its derivatives in 10 mM HEPES buffer at concentrations of 50 (PLL-g-PEG biotin and NTA) and 100 (PLL-gPEG) μg/mL and incubated for 24 h at either 20 or 80 °C. The surfaces were subsequently rinsed copiously in MQ water and dried in N2. Mono- and Bifunctional Surface Patterning. The pattern was fabricated as follows. PLL-g-PEG biotin was first assembled at 80 °C onto a Ti-sputtered SiO2 substrate. The polystyrene (PS) particles (diameter 1.77 μm) were deposited onto the PLL-g-PEG biotin surface by transferring a preassembled PS particle layer formed at an air/water interface, as previously described by our group.38 The PSparticle-covered surface was subsequently sputtered with Ti again using the PS particle layer as a mask. Thus, the PLL-g-PEG biotin surfaces were overcoated only at the exposed area in the interstitial 7016
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spaces between the particles. The high mobility and less-directional nature of the Ti atoms from the sputtering process38 resulted in partially depositing the Ti under the PS-shadowed region, which led to the retention of the PLL-g-PEG biotin region of ∼1 μm. The PS particles were subsequently removed, revealing the areas of PLL-gPEG biotin uncoated by the second Ti sputtering, surrounded by a newly sputtered Ti surface. Finally, the surface was incubated in PLL-gPEG or PLL-g-PEG NTA to generate mono- and bifunctional patterned surfaces, respectively, and dried with N2. Bioadsorption Testing from Whole Blood for XPS and ToFSIMS. The PLL-g-PEG surfaces prepared at 20 and 80 °C as well as mono- and bifunctional patterns were incubated separately in human whole blood (venous blood was drawn from apparently healthy donors into blood bags containing citrate−phosphate−dextrose (CPD) solution and used within 24 h; blood was collected at Skejby Hospital Blood Bank, Denmark) and exposed to the surfaces for 1, 3, and 7 days at 37 °C. After the incubation, all surfaces were washed copiously in PBS buffer and MQ water and analyzed using XPS and ToF-SIMS. XPS. XPS data acquisitions were performed using a Kratos Axis UltraDLD instrument (Kratos Analytical Ltd., Telford, U.K.) supplied with a monochromated Al Kα X-ray source (hν = 1486.6 eV) operating at 10 kV and 15 mA (150 W). Survey spectra (binding energy (BE) range of 0−1100 eV with a pass energy of 160 eV) and high-resolution spectra (with a pass energy of 20 eV) of C 1s, O 1s, and N 1s were obtained to determine the chemical state information. All data acquisitions were performed over three areas per sample, repeated at least once and at the electron take-off angle of 0° (defined here as the angle between analyzer lens axis and the substrate normal). The acquired data were converted to the VAMAS format and analyzed using CASAXPS software (CASA XPS Ltd., U.K.). The BE scales for the high-resolution spectra were calibrated by setting the BE of the O 1s Ti−O−Ti component to 530.0 eV.37 ToF-SIMS. ToF-SIMS data acquisition spectra were acquired using a ToF-SIMS V time-of-flight secondary ion mass spectrometer (IONTOF GmbH, Muenster, Germany) in high mass resolution mode (high current bunched mode). The data was acquired using 15 keV Bi1+ ions rastered in a 128 × 128 (x, y) line format over a 150 μm × 150 μm area. The ion current (Bi1+) was below 1 pA with a cycle time of 150 μs, which provides a m/z range of 0 to 2000. Mass resolution (m/Δm) was measured on the surface of the clean silicon wafer, and the m/Δm in the positive mode at m/z 29 was found to be above 9000 with an H pulse width of 0.56 ns. All data acquisition was performed over three areas per sample, repeated at least once, and acquired in static mode not exceeding 1013 primary ions/cm2. All of the acquired SIMS data were analyzed using Surface Lab 6 software (IONTOF GmbH, Germany). Mass calibration of the positive spectra was performed by selecting CH3+ (m/z 15), C2H5+ (m/z 29), C3H7+ (m/z 43), and C7H7+ (m/z 91). High spatial resolution ToF-SIMS images were acquired in the high spatial resolution mode (burst alignment mode), and the positive ion images were acquired using Bi1+ ions rastered in a 256 × 256 (x, y) line format over a 20 μm × 20 μm area, providing a spatial resolutions of 78.1 nm per pixel, respectively. The beam current measured in the dc mode was ∼0.4 nA. All of the acquired SIMS data were analyzed using Surface Lab 6 software (IONTOF GmbH, Muenster, Germany). Biorecognition of Single and Binary Chemical Patterns. For protein patterning and visualization, mono- and bifunctional PLL-gPEG patterns were prepared at 20 and 80 °C in triplicate per surface type, and all surfaces were sequentially incubated in the following order: nickel(II) hexahydrate (200 mg/mL, 20 min), streptavidin (5 μg/mL, 1 h), His-tag calmodulin (5 μg/mL, 1 h), mouse IgG anticalmodulin primary antibody (5 μg/mL, 1 h), atto-565 labeled biotin (10 μg/mL, 1 h), and atto-488 labeled goat IgG antimouse secondary antibody (10 μg/mL, 1 h). The fluorescent images were captured using a fluorescence microscope (Leica DM6000B) and Leica Qwin software at ×100.8 magnification.
Article
RESULTS AND DISCUSSION Representative high-resolution XPS O 1s spectra of PLL-gPEG, PLL-g-PEG biotin and PLL-g-PEG NTA assembled at 20 and 80 °C are shown in Figure 2. As the polymer assembly
Figure 2. XPS high-resolution O 1s spectra of PLL-g-PEG derivatives at 20 °C (solid line) and 80 °C (dashed line). The spectra are calibrated to Ti−O−Ti at BE = 530 eV. Note that only three components have been fitted (20 °C surfaces) and the BE positions of the additional components from biotin and NTA (OC−N and C O, respectively) are also highlighted. A significant increase in the intensities of C−O/CO component and a decrease in the Ti−O−Ti component were observed for the polymers assembled at 80 °C as compared to those assembled at 20 °C.
temperature was increased from 20 to 80 °C, a decrease in the substrate (Ti−O−Ti) component at the binding energy (BE) of 530.0 eV and an increase in the ether (C−O) and amide (OC−N) components from PLL-g-PEG at ∼533.5 eV and ∼531.8 eV were observed, respectively. To quantify the difference in the level of polymer adsorption between the two assembly temperatures, the polymer-to-substrate ratio (C− O/Ti−O−Ti) was calculated from the high-resolution O 1s spectra. The C−O/Ti−O−Ti ratio increased from 0.3 to 2.0 for PLL-g-PEG, 0.3 to 2.8 for PLL-g-PEG biotin, and 0.5 to 1.9 for PLL-g-PEG NTA from 20 to 80 °C. These surfaces were subsequently exposed to whole human blood for 1, 3, and 7 days and analyzed by XPS. Figure 3a,b represents the XPS highresolution C 1s and N 1s spectra of the polymers assembled at 20 and 80 °C on flat Ti surfaces. The level of nonspecific protein adsorption from blood was monitored by the change in the spectral peak intensities of N 1s at BE ≈ 400 eV, as well as 7017
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Figure 3. XPS high-resolution C 1s (a) and N 1s (b) spectra of PLL-g-PEG (PEG), PLL-g-PEG biotin (Biotin), and PLL-g-PEG NTA (NTA) surfaces assembled at 20 and 80 °C before and after blood incubation for 1, 3, and 7 days. The nonspecific protein adsorption from blood was monitored by the change in the intensity of the amide (OC−N) functionality at BE ≈ 288.2 eV in the C 1s spectra (dashed line) and the N 1s peak intensity at BE ≈ 400 eV.
the amide (OC−N) functionality at BE ≈ 288.2 eV in the C 1s spectra. Note that the spectral intensities were normalized to the total counts in order to compare between the samples. For bare Ti surfaces, a significant increase in the intensities of amide functionality and the N 1s peak from day 0 can be seen at day 1, and the intensity continues to increase up to day 7 of blood incubation, highlighting the considerable amount of nonspecific protein adsorption. A similar trend was observed for the three polymer types assembled at 20 °C, but lower relative increases in the intensities of N 1s and the amide component in C 1s were observed, suggesting that some level of bioresistance was achieved from the polymer surfaces assembled at 20 °C. In contrast, a significant improvement in the bioresistance was observed for the polymers assembled at 80 °C, where negligible changes in the intensities of the amide and N 1s peaks were observed at all time points. Gradual desorption of the polymers was observed for the surfaces grafted at 80 °C with an increase in the incubation time, highlighted by the increase in the Ti 2p signal from the substrate (relative surface atomic percentages are given in SI Figure S1) and the decrease in the ether C 1s signals indicative of the PEG side chains (C−O, BE ≈ 286.5 eV). To probe the level of nonspecific adsorption further, we employed ToF-SIMS and monitored the change in the secondary ion intensities (normalized by the total ion counts) of 13 amino acid fragments (Figure 4 and Table 1). The fragments selected were solely derived from nonspecifically adsorbed proteins from blood and not from the PLL-g-PEG
derivatives or the substrate.26,39 The amino acid fragment of asparagine (Asn, C3H6NO2+, m/z 88.045), a typical SIMS mass fragment used for amino acid detection, was omitted from the analysis as the mass peak significantly overlaps with the C4H8O2+ (m/z 88.053), which is a polymer-related fragment and contributes significantly to the intensity of the Asn (overlay of ToF-SIMS spectra given in SI Figure S2). From the positive ToF-SIMS normalized intensities of amino acids, the proportional intensity changes from the unexposed surfaces were calculated and given in Figure S3. In accord with the XPS data, a considerable increase in the intensities were observed on the bare Ti surface for all amino acid fragments at day 1 of blood incubation, indicating a significant level of protein adsorption from blood. In particular, the normalized intensities of serine (m/z 60.04) and histidine (m/z 110.07) fragments showed a proportional increase of beyond 100 times the mass peak intensities observed at day 0. For all PLL-g-PEG types assembled at 20 °C, improved levels of bioresistance were observed but still showed an average proportional increase of up to 9 times at day 1 and further increased to up to 13 times at day 7. In contrast, excellent bioresistance were achieved for all of the polymers assembled at 80 °C, where the proportional increase in the intensities of up to 2, 4, and 7 times at day 7 were observed for PLL-g-PEG, PLL-g-PEG biotin, and PLL-gPEG NTA, respectively, which is considerably lower than for bare Ti or the polymers assembled at 20 °C. Evidence of superior bioresistance can be highlighted further by the negligible differences observed in the overall spectra (m/z 0 7018
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Figure 4. ToF-SIMS positive secondary ion intensities of 13 amino acid fragments from the polymer surfaces exposed to blood from 0 (unexposed) to 7 days. The intensites are normalized by the total ion counts.
∼100 times more sensitivity for protein detection compared to XPS.26,30 Finally, to investigate the site-specific biorecognition capability of the TIP grafted surfaces, mono- and bifunctional PLL-g-PEG micropatterns were fabricated as outlined in Figure 1c, exposed to whole blood for 0, 1, and 7 days, and subsequently incubated in streptavidin and histidine-tagged (his-tag) calmodulin. In order to fluorescently visualize and confirm the site-specific positioning of the proteins, the bloodexposed patterns were incubated in the following order (Figure 1c): (1) nickel(II) chloride hexahydrate for chelation of Ni2+ on PLL-g-PEG NTA, followed by His-tag calmodulin; (2)
to 150) before and after the exposure to blood (SI Figure S4). The PLL-g-PEG NTA surfaces were found to be most prone to nonspecific adsorption with long-term incubation in blood. This may be due to the differences in the molecular architecture, where a high proportion of the side chains are NTA-functionalized (96%) and do not contain any PEG side chains as in the case for PLL-g-PEG biotin. Interestingly, ToFSIMS has successfully tracked the change in the amino acid intensities with blood incubation time for the surfaces assembled at 80 °C which could not be unambiguously identified using XPS. This discrepancy is due to the difference in the sensitivity of the instrument with ToF-SIMS having 7019
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The control studies were also carried out on the following: (1) autofluorescence from the bifunctional polymer pattern containing PS particles (Figure S5a); (2) Ni2+-exposed binary patterns were incubated in atto-565 biotin only (Figure S5b), atto-488 IgG secondary antibody only (Figure S5c), and anticalmodulin and atto-488 IgG secondary antibody (Figure S5d). Autofluorescence was absent from the polymer and the PS particles, and no site-specific patterns of fluorescently labeled biotin and secondary antibody were observed without streptavidin and His-tag calmodulin. Some aggregates were observed on all surfaces from atto-488 IgG. The secondary antibody was not filtered prior to incubation, as a considerable amount of secondary antibody was lost in the filter. Successful site-specific recognitions of streptavidin and his-tagged calmodulin were confirmed for both types of patterns by fluorescently labeled biotin and the antimouse IgG secondary antibody before and after 1 day of blood incubation. However, antimouse IgG secondary antibody was not recognized on the bifunctional patterns after day 7 of blood incubation. Remarkably, the PLL-g-PEG biotin features from both single and binary patterns were still capable of coupling with streptavidin, even after 7 days in blood. This difference in the retention of bioactivity observed between the two moieties result from the difference in the affinity of protein/ligand
Table 1. List of Monitored Amino Acid Positive Secondary Ions mass (m/z)
amino acid
assignment
43.03 60.04 61.01 70.03 72.08 74.06 84.04 86.10 98.02 110.07 120.08 130.07 136.08
arginine (Arg)/phenylalanine (Phe) serine (Ser) methionine (Met) asparagine (Asn) valine (Val) threonine (Thr) glutamine (Gln)/glutamic acid (Glu) isoleucine (Ile)/leucine (Leu) asparagine (Asn) histidine (His) phenylalanine (Phe) tryptophan (Trp) tyrosine (Tyr)
CH3N2+ C2H6NO+ C2H5S+ C3H4NO+ C4H10N+ C3H8NO+ C4H6NO+ C5H12N+ C4H4NO2+ C5H8N3+ C8H10N+ C9H8N+ C8H10NO+
streptavidin on PLL-g-PEG biotin; (3) mouse monoclonal anticalmodulin IgG primary antibody, and finally (4) Atto-565labeled biotin (red) and Atto-488-labeled antimouse monoclonal IgG secondary antibody (green). The fluorescent images highlighting the single and binary protein pattern formation of the corresponding PLL-g-PEG patterns are shown in Figure 5.
Figure 5. Site-specific single (streptavidin) and binary (streptavidin and histidine-tagged calmodulin) affinity protein recognition of functional PLLg-PEG patterns after incubation in whole human blood for 0, 1, and 7 days. The protein patterns were visualized by fluorescently labeled biotin (red, b) and the antimouse secondary antibody from anticalmodulin mouse primary antibody (green, c). The details of protein immobilization are given in the main text. The DIC (a) and merged overlay (d) of red and green fluorescent images are also shown. The cross-sectional profiles of bifunctional patterns (e, each cross-sectional region highlighted by a white dashed line) provide evidence that PLL-g-PEG NTA and PLL-g-PEG biotin prepared from TIP grafting retain their functionality for 1 and 7 days, respectively, in whole blood. Scale bar = 10 μm. 7020
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Figure 6. XPS high-resolution C 1s (a) and N 1s (b) spectra of mono and bifunctional chemical patterns assembled at 80 °C before and after continuous blood incubation at 1 and 7 days. The nonspecific protein adsorption from blood was monitored by the change in the intensity of the amide (OC−N) functionality at BE ≈ 288.2 eV in the C 1s spectra (dashed line) and the N 1s peak intensity at BE ≈ 400 eV.
Figure 7. Normalized ToF-SIMS positive secondary ion intensities of 13 amino acid fragments from the mono- and bifunctional surface patterns exposed to blood from 0 (unexposed) to 7 days.
and analyzed in the same manner as for the flat surfaces. The XPS C 1s and N 1s spectra are given in Figure 6a,b, respectively (relative surface atomic % of the surfaces are given in Figure S6). A similar trend was observed for the flat surfaces assembled at 80 °C, where negligible differences in the intensities of the amide functionality in the C 1s as well as N 1s peak before and after the blood incubation were observed. The results again highlighted that the level of nonspecific protein adsorption on the TIP grafted surfaces was below the detection limit of the XPS. Figure 7 and Figure S7 represent the ToF-SIMS normalized secondary ion intensities of 13 amino acid fragments for the mono and bifunctional patterns, and the calculated proportional intensity changes from the unexposed surfaces, respectively. The average proportional changes in the
interaction, where the biotin/streptavidin interaction possess a higher affinity per single ligand compared to the NTA-Ni2+/ oligohistidine interaction (KD ≈ 10−14 and 10−6 M for biotin/ stretptavidin40 and NTA-Ni2+/oligohistidine,41 respectively), and the difference in the molecular architecture between PLL-gPEG biotin and PLL-g-PEG NTA, where PLL-g-PEG biotin possesses a greater number of PEG side chains and fewer biotin groups compared to PLL-g-PEG NTA, where most of the PEG side chains are terminated with NTA groups (96%). The greater number of negatively charged NTA groups present on the surface leads to higher amount of nonspecific adsorption of positively charged proteins from blood.35 The level of nonspecific protein adsorption was also investigated for mono- and bifunctional patterns using XPS and ToF-SIMS 7021
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surfaces exposed to blood, and further analysis by ToF-SIMS indicated a very low level of nonspecifically adsorbed proteins. The results highlighted the critical importance in using highly surface sensitive techniques for accurately monitoring protein adsorption, as the protein detection capability of XPS was not sufficient to detect the adsorbed proteins on the TIP-grafted surfaces. The bioactivities of the TIP-grafted biotin and NTA moieties were tested on submicrometer-scale single and binary functional patterns fabricated from a combination of PLL-gPEG, PLL-g-PEG biotin, and PLL-g-PEG NTA using CL and PVD. The successful formation of ligand−protein complexes involving biotin−streptavidin and NTA- Ni2+-his-tagged calmodulin after blood incubation were confirmed via fluorescence microscopy. The biorecognition capabilities of NTA and biotin were maintained for at least 1 day and 7 days of preincubation in blood, respectively. The result from this study is an example of how carefully designed polymer architecture in combination with the TIP grafting method can be straightforwardly used to present any bioactive moieties on surfaces with excellent bioresistance and without a loss of activity. This is further highlighted by its extremely simple single-step grafting-to approach: it does not require any uncoupled moieties to be returned to the inert state, and it may be readily integrated into a wide range of existing fabrication strategies to provide longterm robust bioresistance while maintaining chemical reactivity in challenging biological environments.
amino acid-normalized intensities were found to be 1 and 2 for the mono and bifunctional patterns, respectively, which indicated excellent bioresistance against blood. Monofunctional patterns maintained excellent bioresistance even after 7 days, with only 2 times the average proportional increase in the intensities observed. However, a 6-fold increase in the average intensities was observed on the bifunctional surface due to the lack of robust bioresistance offered by PLL-g-PEG NTA compared to PLL-g-PEG and PLL-g-PEG biotin as already highlighted on the flat surfaces. The monofunctional patterns were also prepared at 20 °C, but the ∼1 μm pattern features could not be fluorescently visualized and resolved at day 0 (data not shown). This may be due to having a lower number of fluorescently tagged biotin binding to the streptavidin-PLL-gPEG biotin pattern assembled at 20 °C than at 80 °C, given that the chemical patterning of PLL-g-PEG biotin at both temperatures has been successful as confirmed by highresolution ToF-SIMS imaging (SI Figure S8a,b, respectively). The absolute difference in the normalized N 1s peak area before and after exposure to streptavidin was monitored to determine whether a greater amount of streptavidin can be captured on the PLL-g-PEG biotin surface prepared at 80 °C compared to those at 20 °C (Figure S9). A significantly higher amount of N, i.e., a greater number of streptavidin molecules, has been captured on the surfaces assembled at 80 °C, with ∼1.4 times the amount of streptavidin adsorbed compared to that for the surfaces prepared at 20 °C. Interestingly, the earlier study has previously showed nearly full streptavidin coverage (∼99% of a closely packed streptavidin monolayer) achieved by using the PLL-g-PEG biotin assembled under ambient conditions with similar molecular architecture (PLL (20 kDa)-g-[3.5]-PEG (2 kDa)/PEG (3.4 kDa) biotin (50%)).36 The results indicate that the higher coverage of streptavidin achieved for the surfaces assembled at 80 °C is due to the different orientation in the packing of the streptavidin, where a greater amount of streptavidin may be adsorbed in a tilted configuration. The higher amount of streptavidin captured on the surface explains the larger number of attached fluorescently tagged biotins. However, we cannot conclude exclusively as to whether this is the case from this study alone, as the subsequent coupling of fluorescently tagged biotin on streptavidin can be hindered sterically by the neighboring streptavidin and/or the nearby biotins from the polymer occupies more than two biotin binding sites via the tilted streptavidin. Furthermore, the initial PLL-g-PEG biotin surface concentration, streptavidin solute concentration, and adsorption time as well as the dimensions of the biotinylated molecule are thought to govern the architecture of the PLL-g-PEG−biotin−streptavidin−biotinylated molecule complex, and further studies are needed to precisely characterize such systems.36
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ASSOCIATED CONTENT
S Supporting Information *
XPS relative surface atomic concentrations (%) of flat and patterned PLL-g-PEG derivatives; ToF-SIMS positive ion spectral overlay of bare Ti exposed to blood and PLL-g-PEG assembled at 80 °C for 0 days (no exposure); plot of proportional changes in the ToF-SIMS normalized intensities of 13 amino acid fragments at various times of blood exposure; ToF-SIMS positive ion spectral overlays of bare Ti and the PLL-g-PEG and its functional derivatives in blood; fluorescent images of bifunctional PLL-g-PEG biotin and NTA patterns; XPS relative surface atomic concentrations of mono- and bifunctional surface patterns; plot of proportional changes in the ToF-SIMS normalized intensities of 13 amino acid fragments at various times of blood exposure; high-resolution ToF-SIMS images of the PLL-g-PEG biotin array pattern generated by CL and PVD with polymer incubation; highresolution XPS N 1s spectra of PLL-g-PEG biotin surfaces. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Notes
The authors declare no competing financial interest.
CONCLUSIONS This study has conclusively shown that the TIP grafting of nonfouling functional PLL-g-PEG provides excellent long-term bioresistance and retention of the bioactivity of functional moieties after prolonged exposure to whole blood. The XPS and ToF-SIMS techniques were employed to determine the level of nonspecific protein adsorption. The adsorbed proteins were readily detectable on blood-incubated bare Ti and the polymer surfaces assembled at ambient temperature, indicating that the level of nonspecifically adsorbed proteins was well within the detection limit of XPS. In contrast, the protein adsorption could not be detected from the TIP (80 °C)-grafted
ACKNOWLEDGMENTS R.O. is grateful for a Danish Research Council DFF individual postdoc grant for funding. We thank Jacques Chevallier for sputtering and David Kraft and Lisbeth Abildtrup for blood studies.
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REFERENCES
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