Selective and Reversible Binding of Thiol-Functionalized

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SELECTIVE AND REVERSIBLE BINDING OF THIOLFUNCTIONALIZED BIOMOLECULES ON POLYMERS PREPARED VIA CHEMICAL VAPOR DEPOSITION POLYMERIZATION Aftin Ross, Hakan Durmaz, Kenneth Cheng, Xiaopei Deng, Yuwei Liu, Jonathan Oh, Zhan Chen, and Joerg Lahann Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b00654 • Publication Date (Web): 14 Apr 2015 Downloaded from http://pubs.acs.org on April 18, 2015

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SELECTIVE AND REVERSIBLE BINDING OF THIOL-FUNCTIONALIZED BIOMOLECULES ON POLYMERS PREPARED VIA CHEMICAL VAPOR DEPOSITION POLYMERIZATION

Aftin Ross1, Hakan Durmaz2,3,4, Kenneth Cheng3,5, Xiaopei Deng3,6, Yuwei Liu7, Jonathan Oh4, Zhan Chen6,7, Joerg Lahann1,3,4,5,6 *

1

Institute of Functional Interfaces, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-

Platz 1, 76344 Eggenstein-Leopoldshafen, Germany 2

Department of Chemistry, Istanbul Technical University, Maslak, 34469 Istanbul, Turkey

3

Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA

4

Chemical Engineering, University of Michigan, Ann Arbor, MI 48109, USA

5

Materials Science and Engineering, University of Michigan, Ann Arbor, MI 48109, USA

6

Macromolecular Science and Engineering, University of Michigan, Ann Arbor, MI 48109, USA

7

Chemistry, University of Michigan, Ann Arbor, MI 48109, USA

*To whom correspondence should be addressed: [email protected]

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ABSTRACT We use chemical vapor deposition polymerization to prepare a novel dibromomaleimidefunctionalized polymer for selective and reversible binding of thiol-containing biomolecules on a broad range of substrates. We report the synthesis and CVD polymerization of 4-(3,4dibromomaleimide)[2.2]paracyclophane to yield nanometer-thick polymer coatings. Fourier transformed infrared spectroscopy and x-ray photoelectron spectroscopy confirmed the chemical composition of the polymer coating. The reactivity of the polymer coating towards thiolfunctionalized molecules was confirmed using fluorescent ligands. As a proof of concept, the binding and subsequent release of cysteine-modified peptides from the polymer coating was also demonstrated via sum frequency generation spectroscopy. This reactive polymer coating provides a flexible surface modification approach to selectively and reversibly bind biomolecules on a broad range of materials, which could open up new opportunities in many biomedical sensing and diagnostic applications where specific binding and release of target analytes are desired.

Keywords: Click chemistry, Surface modification, Chemical vapor deposition polymerization, Reversible reactions, Multifunctional surfaces, Patterning


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INTRODUCTION Bioconjugation of proteins and peptides to solid surfaces plays an important role in many applications, ranging from sensing and diagnostics to tissue engineering and food packaging.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12

An arsenal of different attachment strategies for proteins and peptides has

been developed, including physical entrapment, encapsulation, physical adsorption and covalent immobilization. 13 Among these attachment strategies, covalent immobilization is found to be the most appropriate, because the target biomolecules form rigid chemical bonds with the solid surface. Common chemical immobilization strategies exploit the reactivity of lysine and cysteine residues of proteins or peptides.14, 15 Lysine residues are commonly found in native proteins and can readily react with surfaces containing activated carboxyl groups, allowing for facile protein/peptide immobilization. However, proteins often contain multiple lysine residues, resulting in poor control over the orientation of the immobilized proteins/peptides.15 Cysteine residues offer access to thiol groups that react rapidly with maleimides, via Michael-addition, under neutral or physiological conditions.16,

17, 18

Unlike lysine, cysteine residues are not as

abundant in proteins. In some cases, the introduction of additional cysteine groups enables precise control of the immobilization site of the protein or peptide, thereby achieving uniform orientation-specific immobilization.15 To date, several thiol-reactive surface modification techniques have been developed.18,

19, 20, 21, 22

A reversible bioconjugation strategy can be

advantages to many biomedical applications, such as bio-sensing and therapeutic diagnostics, in which the biomolecular receptors can bind to the dibomomaleimide surface, capture analytes and subsequently release the analytes for further analysis. Dibromomaleimide not only covalently binds with thiol groups, but also allows for the reversal of the binding, when exposed to an excess amount of stronger nucleophiles, such as


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tris(2-chloroethyl) phosphate (TCEP) or 2-mercaptothanol following an addition-elimination reaction.23, 24 By developing a coating that provides access to dibromomaleimide groups on a surface, one can chemically immobilize proteins/peptides on the surface with the desired protein/peptide orientation, and subsequently release the immobilized protein/peptide ondemand. A polymer coating that could introduce dibromomaleimide groups to a broad range of solid surfaces would thus be desirable for a number of biomedical and biotechnological applications. A surface modification technique that is particularly versatile with respect to substrate choice is chemical vapor deposition (CVD) polymerization of substituted paracyclophanes.25, 26 The resulting reactive polymer coatings have xylylene backbone with tunable reactive side groups for straightforward surface modification. A range of different functionalized coatings including aldehyde27, active esters28, ketones, anhydrides29, alkynes30, activated alkynes31 have been reported. Reactive coatings can be applied conformally to virtually any solid surfaces of complex geometries.32, 33, 34, 35, 36 In this article, we demonstrate the use of CVD polymerization as a generic method to develop novel polymers featuring dibromomaleimide groups for controlled attachment and release of thiol-containing biomolecules. EXPERIMENTAL METHODS Synthesis of 4-(3,4-dibromomaleimide)[2.2]paracyclophane (1) The starting material, 4-(hydroxymethyl)[2.2]paracyclophane, was synthesized using a previously

described

synthetic

route.37

The

synthesis

of

4-(3,4-

dibromomaleimide)[2.2]paracyclophane 1 from the starting material is illustrated in Scheme 1a. Triphenylphosphine (1.05 g, 4.01 mmol) was dissolved in 100 mL of anhydrous THF and cooled to -78°C in a dry ice/acetone bath. The solution was stirred at this temperature for 30 min under 4 ACS Paragon Plus Environment

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nitrogen atmosphere. Diisopropyl azodicarboxylate (0.79 mL, 4.01 mmol) was added dropwise to the solution over 5 min and stirred for 30 min. 4-(hydroxymethyl)[2.2]paracyclophane (1.05 g, 4.41 mmol) was dissolved in 5 mL of THF and added dropwise to the mixture over 10 min. Finally, neopentyl alcohol (0.176 g, 2.0 mmol) and 3,4-dibromomaleimide (1.02 g, 4.01 mmol) were added to the mixture, respectively. The reaction mixture was stirred at -78 oC for 2 h and then overnight at room temperature. The solvent was removed in vacuo yielding yellow oil, which was purified by column chromatography over silica gel eluting with ethyl acetate/hexane (1:4). A yellow solid was placed in a beaker and diethyl ether (20 mL) was added and stirred at room temperature for 1 h, filtered, and washed several times with diethyl ether. A yellow powder was obtained after drying. Rf = 0.55 (1:4, ethyl acetate/hexane). 1H NMR and

13

C NMR are

provided in Figure S1 of the Supporting Information. Yield (0.7 g, 37%). 1H NMR (400 MHz, CDCl3) 6.8-6.2 (m, 7H, ArH), 4.71 (d, 1H, ArCH2N), 4.51 (d, 1H, ArCH2N), 3.65 (m, 1H, ArCH2CH2Ar), 3.2-2.8 (m, 7H, ArCH2CH2Ar).

13

C NMR (100 MHz, CDCl3, δ): 163.5, 140.3,

139.5, 139.2, 137.9, 135.3, 134.5, 133.7, 133.5, 133.1, 132.6, 132.2, 129.4, 129.2, 41.5, 35.2, 34.9, 34.6, 33.3. Chemical Vapor Deposition (CVD) Polymerization of Precursor 1 Precursor 1 was polymerized to yield poly[4-(3,4-dibromomaleimide)-p-xylylene-co-pxylylene] 2 by CVD polymerization using a custom-built CVD system that has been previously described34. As shown in Scheme 1b, precursor 1 was first sublimed at approximately 120°C. The precursor vapor then underwent pyrolysis at 750°C, and was deposited and polymerized simultaneously onto different substrate surfaces, including calcium fluoride (CaF2) prisms, gold, silicon, copper, or glass cover slips, at 15oC. The whole process occurred under a pressure of 0.1 mbar. The CVD polymerization of non-functionalized [2,2]paracyclophane into poly(p-xylyene) 5 ACS Paragon Plus Environment

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was performed using the same procedure described above but with a pyrolysis temperature of 660oC. To generate a microstructured surface as illustrated in Figure 2a, a two-step CVD process was utilized. The surface was first coated with a non-functionalized poly(p-xylylene). Subsequently, polymer 2 was deposited spatial-selectively onto the poly(p-xylylene)-coated surface via the vapor-assisted micro-patterning in replica structures (VAMPIR) technique which has been previously described.38 Briefly, a patterned PDMS shadow mask (preparation of this PDMS mask has been described elsewhere38) was placed in conformal contact with a poly(pxylylene)-coated substrate. The PDMS-masked sample then underwent the same CVD process to deposit polymer 2 onto the exposed regions of the substrate (the regions not covered by the PDMS mask). The PDMS mask was subsequently peeled off of the substrate and a microengineered surface with two spatial-selectively-deposited polymer coatings was obtained. Surface Characterization: FT-IR, XPS, Ellipsometry Fourier transformed infrared spectroscopy (FT-IR) was utilized to characterize polymer 2. In all instances, a Nicolet 6700 spectometer with the grazing angle accessory (Smart SAGA) at a grazing angle of 80° was utilized. A total of 128 scans were taken for each sample. X-ray photoelectron spectroscopy (XPS) was used to assess the composition on the polymer coating before and after surface modification. Specifically, data were recorded with an Axis Ultra X-ray photoelectron spectrometer (Kratos Analyticals, UK) outfitted with a monochromatized Al Ka X-ray source at a power of 150 kW. Both survey and high-resolution spectra were taken at 160 eV and 20 eV respectively. All spectra were calibrated with respect to the non-functionalized aliphatic carbon with a binding energy of 285 eV. Information on film thickness and visualization of surface patterning was provided by a multi-wavelength imaging null6 ACS Paragon Plus Environment

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ellipsometer (EP3 Nanofilm, Germany). Thickness was determined by fitting ellipsometric delta and psi with fixed values of the real and refractive index of the CVD coating (n=1.6 and k=0). Measurements were taken at an angle of incidence of 60° for gold substrates, 65° for copper substrates, and 70° for silicon substrates. A CCD camera incorporated into the ellipsometer was used to capture images and the field of view was 0.5 mm2. Surface modification and Visualization via Fluorescence Microscopy A fluorescent-labeled protein, streptavidin-TRITC, was incubated onto micro-engineered surfaces with the help of a thiol-terminated biotin linker (biotin-PEG-thiol) (Nanocs, New York, NY). The surfaces were first immersed in an aqueous solution containing 10mM biotin-PEGthiol for 12.5-14 hours. After subsequent rinsing, the surfaces were immersed in phosphate buffer solution (PBS) (pH7.4) containing 0.4 µg/mL streptavidin-TRITC, Tween 20 (0.01% w/v) and bovine serum albumin (BSA) (0.2% w/v) for 1 hour. Afterwards, the surfaces were rinsed 3 times with PBS and deionized water and were then dried prior to fluorescence imaging. Protein immobilization was visualized via fluorescence microscopy on a Nikon Eclipse 80i.

Evaluating the Reversible Binding of Thiol-Containing Ligands via XPS analysis A thiol-containing molecule, N-acetyl-L-cysteine-methyl-ester (NALCME) (Sigma Aldrich, St. Louis, MO), was immobilized onto the surface of polymer 2 by simply immersing the samples into an aqueous solution containing 1mM NALCME for 12.5-14 hours, followed by subsequent rinsing with deionized water. The coated surfaces were then analyzed via XPS. Subsequently, the same samples were incubated in a aqueous solution containing 300mM tris(2chloroethyl) phosphate (TCEP), and were analyzed again via XPS.


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Evaluating the Reversible Binding of Thiol-based Biomolecules via Sum Frequency Generation (SFG) Spectroscopy Sum frequency generation (SFG) spectroscopy was utilized to evaluate the reversible binding of cysteine-terminated cecropin P1(CP1c) (New England Peptide, USA) on polymer 2. In the SFG setup (EKSPLA, Lithuania), spectra were obtained by overlapping a fixed pulsed visible laser beam (532 nm) and a tunable pulsed infrared beam spatially and temporally at the sample/solution interface. Right-angle CaF2 prisms coated with polymer 2 were used as the substrates for this SFG study. Prior to the SFG spectral collection, a small volume of TCEP solution (1:1 equivalent ratio to CP1c) was added to the CP1c stock solution and incubated for 2 hours to avoid formation of the di-sulfide bonds between peptide molecules. SFG spectrum at 1655cm-1 (indicative of the amide I band) as a function of time was recorded to study the reversible binding of CP1c on polymer 2. The recording started when the polymer-coated prism was in contact with a 2mL reservoir containing 5mM (pH 7.2) phosphatebuffered saline (PBS) solution. A solution containing 2uM CP1c (from CP1c stock solution) was then added to the PBS solution for the immobilization to happen, in which the SFG signal at 1655cm-1 began to increase. When the SFG signal plateaued, the reservoir was replaced continuously with fresh PBS to rinse off any non-specific binding of the CP1c on the polymercoated prism. After the rinsing process, a PBS solution containing TCEP was injected into the reservoir to release any chemically immobilized CP1c from the polymer-coated prisms, followed by a final rinsing step. The recording stopped after the final rinsing step.

RESULTS AND DISCUSSION The three-step synthesis of precursor 1 and subsequent CVD polymerization of precursor 1 into polymer 2 are shown in Scheme 1. Both 1HNMR and

13

CNMR confirmed the chemical 8

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structure of precursor 1 (Figure S1). The overall yield of precursor 1 was 37%. For polymer 2, FT-IR revealed a strong band at 1723 cm-1 that is characteristic for the C=O group, as seen in Figure 1a. In addition, characteristic bands at 2857-3039 cm-1 indicate the presence of symmetric and asymmetrtic C-H stretching modes. Furthermore, the elemental composition of polymer 2 was in reasonably good accordance with the theoretically expected values, as determined by XPS (Figure 1b). We note that the amount of Br, obtained with XPS, is less than the predicted value, indicating that there could be some loss in Br during the pyrolysis stage of the CVD polymerization process. The high resolution C1s spectrum revealed five different signals: (1) aliphatic hydrocarbon (C—C/H) at a binding energy of 285.0 eV, (2) carbon-nitrogen (C—N) at 286.3 eV, (3) carbon-bromide (C—Br) at 286.8 eV, (4) carbonyl in immediate neighborhood to a nitrogen (N—C=O) at 289.0 eV, and (5) π—π* transitions at 291.5 eV that are characteristic for aromatic polymers (Figure 1b).39 Both FT-IR and XPS confirmed the chemical composition of polymer 2. The thicknesses of polymer 2 were determined, by means of imaging ellipsometry, to range between 30-50 nm depending on the reaction conditions.

Reactivity of Polymer 2 towards Thiol-Containing Molecules After validating the chemical composition of polymer 2, the reactivity of polymer 2 towards thiol-containing moieties was tested and visualized using thiol-terminated fluorescence ligands. Specifically, polymer 2-coated surfaces were incubated in an aqueous solution containing thiol-terminated biotin (biotin-PEG-thiol). After subsequent rinsing, the surface was immersed into a phosphate buffer solution containing streptavidin-TRITC. Strepavidin is known to strongly and selectively bind with biotin. If the binding of biotin-PEG-thiol onto polymer 2 were successful, the surface would contain free biotin groups that would bind preferentially to


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streptavidin, and the TRITC moieties on streptavidin-TRITC would be on the surface to be visualized via fluorescence microscopy. In order to better visualize the binding of the thiol-terminated biotin and strepavidinTRITC on polymer 2, we utilized the VAMPIR technique to spatial-selectively deposit polymer 2 onto surfaces previously coated with non-functionalized poly(p-xylylene), as illustrated in Figure 2a. These microstructured surfaces enable direct comparison between polymer 2 and a polymer coating with no functional groups under the same immobilization process. Prior to the immobilization process, the microstructured surfaces were analyzed via imaging ellipsometry (Figure 2b). Imaging ellipsometry confirmed that polymer 2 were conformally coated onto the exposed regions of poly(p-xylylene)-coated surfaces during the VAMPIR process and that the thickness of polymer 2 on top of poly(p-xylylene) is approximately 10-12 nm thick, confirming the successful preparation of the micropatterned surfaces. As shown in Figure 2c, the fluorescence signal on the regions with polymer 2 was much stronger than that on poly(pxylylene), indicating that both the thiol-terminated biotin and strepavidin-TRITC bound selectively to the surface of polymer 2. This experiment unambiguously confirmed the reactivity of polymer 2 towards thiol-containing molecules.

Reversible Binding of Thiol-Containing Molecules and Thiol-Based Biomolecules After the specific binding of thiol-containing molecules was validated, polymer 2 was further used to study reversible binding of thiol-containing molecules. This was done through a combination of XPS and sum frequency generation (SFG) spectroscopy. In the XPS study, Nacetyl-L-cysteine-methyl-ester (NALCME) was used as the probe molecule. Polymer 2 was first incubated in a solution containing NALCME and was subsequently analyzed by XPS (Figure 3).


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XPS detected a small amount of sulfur and a decrease in Br from 3.6 at% to 2.4 atom-%, indicating NALCMEs were immobilized onto polymer 2. A small amount of Br was still detected after the NALCME immobilization, because XPS detects the first 10 nm of the sample, while the chemical reaction occurred solely on the polymer surface. Therefore, it is reasonable to expect detection of some amount of Br from the bulk polymer, even after the reaction with the thiol. After XPS analysis, the same sample was then incubated in an solution containing excess amount of tris(2-carboxyethyl)phosphine (TCEP), which is a sufficiently strong nucleophile to cleave the thioether bond between thiol and dibromomaleimide. Subsequently, the sample was analyzed again using XPS. XPS detected no sulfur but a small amount of phosphorus, indicating that the immobilized NALCMEs were successfully cleaved and replaced by TCEP. To further investigate this finding, sum frequency generation (SFG) spectroscopy was utilized. SFG spectroscopy is a second-order nonlinear optical spectroscopic technique with submonolayer surface sensitivity that has been shown to be a powerful tool for studying the secondary structures and orientations of peptides/proteins immobilized on an interface. 40, 41, 42 In light of this, SFG spectroscopy is an ideal tool for investigating the immobilization and subsequent release of peptides/proteins on polymer 2. An antimicrobial peptide, cysteine-terminated cecropin P1 (CP1c), was used as a model peptide. CP1c exhibits α-helical structures, which showed a characteristic amide I band at 1655cm-1 in SFG when immobilized on a surface.40, 41 The cysteine end group on CP1c contains thiol groups that are known to chemical bind with dibromomaleimide. To study the binding and subsequent release of CP1c on polymer 2, time-dependent SFG measurements monitored at 1655cm-1 were performed. As shown in Figure 4, upon the addition of Cp1c, SFG signal at 1655 cm-1 increases, indicating the binding of CP1c on polymer 2. The SFG signal then reached a


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plateau, indicating saturation in binding of CP1c on polymer 2. Please note that the binding of Cp1c to polymer 2, at this stage, can be both physical adsorption and chemical immobilization. Therefore, to remove physical adsorption of CP1c from the polymer coating, the sample was subsequently rinsed with PBS. SFG signal at 1655 cm-1 dropped until a certain point, which represents only chemically immobilized CP1c. To justify that the chemically immobilized CP1c can be cleaved off with a stronger nucleophiles, an excess amount of TCEP was injected into the system. As shown in Figure 4, the SFG signal decreased to the baseline value after the injection of TCEP, and subsequent rinsing revealed no further decrease in SFG signal, signifying the successful release of the immobilized CP1c when exposed to TCEP. Both XPS and SFG unambiguously show the successful cleavage of thiol-containing moieties from the dibromomaleimide functionalized surface further enhancing the viability of this platform for use in biomolecular sensing arrays, or drug delivery. CONCLUSIONS We have successfully prepared a new dibromomaleimide-functionalized polymer coating via CVD polymerization that enables chemical immobilization and subsequent release of thiolcontaining biomolecules on and from the coated surfaces. The binding of dibromomaleimides and thiols occurs under physiological conditions without the use of any additives or catalysts. In addition, because the polymer coating is prepared via chemical vapor deposition polymerization, the coating can be applied to virtually any solid materials, allowing for provide a generic strategy for reversible binding of biomolecules on a broad wide of materials. This generic strategy could be crucial in biomedical sensing and diagnostic applications, in which specific binding and release of target analytes are desired.


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SUPPORTING INFORMATION 1

H-NMR and 13C-NMR spectra of 4-(3,4-dibromomaleimide)[2.2]paracyclophane. This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGEMENTS We acknowledge support from DTRA, under project HDTRA1-12-1-0039 and the Army Research Office (ARO) under Grant W911NF-11-1-0251. A.M.R would like to acknowledge funding support from the Whitaker International Scholars Program. H.D gratefully acknowledges support from the Scientific and Technical Research Council of Turkey (TUBITAK) for 2219International Postdoctoral Research Scholarship Programme.


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FIGURES

Scheme 1. (a) Synthesis of 4-(3,4-dibromomaleimide)[2.2]paracyclophane (1) and (b) chemical vapor deposition polymerization of precursor 1 into poly[4-(3,4-dibromomaleimide)-p-xylyleneco-p-xylylene] 2.


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Figure 1. (a) Fourier Transform Infrared (FT-IR) spectrum of polymer 2. (b) (Left) a table showing the chemical composition of polymer 2, in atomic percentage, determined experimentally by x-ray photoelectron spectroscopy (XPS). The theoretical (calculated) composition of polymer 2 is included for comparison. (Right) high-resolution C1s spectrum of polymer 2.


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Figure 2. a) Schematic diagram illustrating the immobilization of thiol-PEG biotin and streptavidin-TRITC on a patterned polymer film prepared via the vapor-assisted micro-patterning in replica structures (VAMPIR) technique. b) Thickness profile showing the thickness difference between the two spatial-selectively deposited polymer coatings on a substrate surface. c) A fluorescence micrograph of a patterned polymer film after the immobilization of thiol-PEGbiotin and strepavidin-TRITC on the patterned polymer film. Scale bar: 100µm.


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Figure 3. (a) Reaction scheme showing the immobilization of N-acetyl-L-cysteine-methyl-ester (NALCME) on polymer 2 and subsequent release of NALCME from polymer 2 upon exposure to a stronger nucleophile (Nuc), which is tris(2-chloroethyl) phosphate (TCEP) in this case. (b) A table showing the chemical composition of 2, 3 and 4 in at% determined by XPS.


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Figure 4. (a) Reaction scheme showing the immobilization of cysteine-terminated cecropin P1 (CP1c) on polymer 2 and subsequent release of CP1c from polymer 2 upon exposure to a stronger nucleophile (Nuc), which is tris(2-chloroethyl) phosphate (TCEP) in this case. (b) Timedependent sum frequency generation (SFG) measurement recording of the signal at 1655cm-1 i) the immobilization of CP1c, ii) subsequent rinsing with PBS, iii) injection of TCEP, and iv) final rinsing with PBS on polymer 2.


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REFERENCES (1)

Ratner, B. D.; Bryant, S. J. Biomaterials: Where we have been and where we are going.

Annu. Rev. Biomed. Eng. 2004, 6, 41-75. (2)

Lin, S.-P.; Chi, T.-Y.; Lai, T.-Y.; Liu, M.-C. Investigation into the Effect of Varied

Functional Biointerfaces on Silicon Nanowire MOSFETs. Sensors 2012, 12, 16867-16878. (3)

Frasconi, M.; Mazzei, F.; Ferri, T. Protein immobilization at gold-thiol surfaces and

potential for biosensing. Anal. Bioanal. Chem. 2010, 398, 1545-1564. (4)

North, S. H.; Lock, E. H.; Taitt, C. R.; Walton, S. G. Critical aspects of biointerface

design and their impact on biosensor development. Anal. Bioanal. Chem. 2010, 397, 925-933. (5)

Sun, K.; Liu, H. L.; Wang, S. T.; Jiang, L. Cytophilic/Cytophobic Design of

Nanomaterials at Biointerfaces. Small 2013, 9, 1444-1448. (6)

Wu, J. D.; Mao, Z. W.; Tan, H. P.; Han, L. L.; Ren, T. C.; Gao, C. Y. Gradient

biomaterials and their influences on cell migration. Interface Focus 2012, 2, 337-355. (7)

Hynes, R. O. Integrins - Versatility, Modulation, and Signaling in Cell-Adhesion. Cell

1992, 69, 11-25. (8)

Slowing, I.; Trewyn, B. G.; Lin, V. S. Y. Effect of surface functionalization of MCM-41-

type mesoporous silica nanoparticleson the endocytosis by human cancer cells. J. Am. Chem. Soc. 2006, 128, 14792-14793. (9)

Hoa, X. D.; Kirk, A. G.; Tabrizian, M. Towards integrated and sensitive surface plasmon

resonance biosensors: A review of recent progress. Biosens. Bioelectron. 2007, 23, 151-160. (10)

Bora, D. K.; Rozhkova, E. A.; Schrantz, K.; Wyss, P. P.; Braun, A.; Graule, T.;

Constable, E. C. Functionalization of Nanostructured Hematite Thin-Film Electrodes with the


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 24

Light-Harvesting Membrane Protein C-Phycocyanin Yields an Enhanced Photocurrent. Adv. Funct. Mater. 2012, 22, 490-502. (11)

Ihssen, J.; Braun, A.; Faccio, G.; Gajda-Schrantz, K.; Thony-Meyer, L. Light Harvesting

Proteins for Solar Fuel Generation in Bioengineered Photoelectrochemical Cells. Current Protein & Peptide Science 2014, 15, 374-384. (12)

Veluchamy, P.; Sivakumar, P. M.; Doble, M. Immobilization of Subtilisin on

Polycaprolactam for Antimicrobial Food Packaging Applications. J. Agric. Food Chem. 2011, 59, 10869-10878. (13)

Brady, D.; Jordaan, J. Advances in enzyme immobilisation. Biotechnol. Lett. 2009, 31,

1639-1650. (14)

Tischer, W.; Wedekind, F. Immobilized enzymes: Methods and applications.

Biocatalysis - from Discovery to Application 1999, 200, 95-126. (15)

Jonkheijm, P.; Weinrich, D.; Schroder, H.; Niemeyer, C. M.; Waldmann, H. Chemical

Strategies for Generating Protein Biochips. Angewandte Chemie-International Edition 2008, 47, 9618-9647. (16)

Kalia, J.; Raines, R. T. Catalysis of imido group hydrolysis in a maleimide conjugate.

Bioorg. Med. Chem. Lett. 2007, 17, 6286-6289. (17)

Gregory, J. D. The Stability of N-Ethylmaleimide and Its Reaction with Sulfhydryl

Groups. J. Am. Chem. Soc. 1955, 77, 3922-3923. (18)

Hermanson, G. T. Bioconjugate Techniques, 2nd Edition. Bioconjugate Techniques, 2nd

Edition 2008, 1-1202.


Page 21 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(19)

Subramani, C.; Cengiz, N.; Saha, K.; Gevrek, T. N.; Yu, X.; Jeong, Y.; Bajaj, A.; Sanyal,

A.; Rotello, V. M. Direct Fabrication of Functional and Biofunctional Nanostructures Through Reactive Imprinting. Advanced Materials 2011, 23, 3165-3169. (20)

Billiet, L.; Gok, O.; Dove, A. P.; Sanyal, A.; Nguyen, L. T. T.; Du Prez, F. E. Metal-Free

Functionalization of Linear Polyurethanes by Thiol-Maleimide Coupling Reactions. Macromolecules 2011, 44, 7874-7878. (21)

Tsai, M. Y.; Lin, C. Y.; Huang, C. H.; Gu, J. A.; Huang, S. T.; Yu, J. S.; Chen, H. Y.

Vapor-based synthesis of maleimide-functionalized coating for biointerface engineering. Chemical Communications 2012, 48, 10969-10971. (22)

Zhu, J.; Waengler, C.; Lennox, R. B.; Schirrmacher, R. Preparation of Water-Soluble

Maleimide-Functionalized 3 nm Gold Nanoparticles: A New Bioconjugation Template. Langmuir 2012, 28, 5508-5512. (23)

Smith, M. E. B.; Schumacher, F. F.; Ryan, C. P.; Tedaldi, L. M.; Papaioannou, D.;

Waksman, G.; Caddick, S.; Baker, J. R. Protein Modification, Bioconjugation, and Disulfide Bridging Using Bromomaleimides. J. Am. Chem. Soc. 2010, 132, 1960-1965. (24)

Tedaldi, L. M.; Smith, M. E. B.; Nathani, R. I.; Baker, J. R. Bromomaleimides: new

reagents for the selective and reversible modification of cysteine. Chemical Communications 2009, 6583-6585. (25)

Deng, X. P.; Lahann, J. Orthogonal Surface Functionalization Through Bioactive Vapor-

Based Polymer Coatings. J. Appl. Polym. Sci. 2014, DOI: 10.1002/app.40315. (26)

Chen, H. Y.; Lahann, J. Designable Biointerfaces Using Vapor-Based Reactive

Polymers. Langmuir 2011, 27, 34-48.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(27)

Page 22 of 24

Nandivada, H.; Chen, H. Y.; Lahann, J. Vapor-based synthesis of poly [(4-formyl-p-

xylylene)-co-(p-xylylene)] and its use for biomimetic surface modifications. Macromol. Rapid Commun. 2005, 26, 1794-1799. (28)

Lahann, J.; Choi, I. S.; Lee, J.; Jenson, K. F.; Langer, R. A new method toward

microengineered surfaces based on reactive coating. Angewandte Chemie-International Edition 2001, 40, 3166-3169. (29)

Lahann, J.; Klee, D.; Hocker, H. Chemical vapour deposition polymerization of

substituted [2.2]paracyclophanes. Macromol. Rapid Commun. 1998, 19, 441-444. (30)

Nandivada, H.; Chen, H. Y.; Bondarenko, L.; Lahann, J. Reactive polymer coatings that

"click"". Angewandte Chemie-International Edition 2006, 45, 3360-3363. (31)

Deng, X. P.; Friedmann, C.; Lahann, J. Bio-orthogonal "Double-Click" Chemistry Based

on Multifunctional Coatings. Angewandte Chemie-International Edition 2011, 50, 6522-6526. (32)

Tawfick, S.; Deng, X. P.; Hart, A. J.; Lahann, J. Nanocomposite microstructures with

tunable mechanical and chemical properties. Phys. Chem. Chem. Phys. 2010, 12, 4446-4451. (33)

Hu, W. W.; Elkasabi, Y.; Chen, H. Y.; Zhang, Y.; Lahann, J.; Hollister, S. J.; Krebsbach,

P. H. The use of reactive polymer coatings to facilitate gene delivery from poly (epsiloncaprolactone) scaffolds. Biomaterials 2009, 30, 5785-5792. (34)

Chen, H. Y.; Lahann, J. Surface patterning strategies for microfluidic applications based

on functionalized poly-p-xylylenes. Bioanalysis 2010, 2, 1717-1728. (35)

Zhang, Y.; Deng, X. P.; Scheller, E. L.; Kwon, T. G.; Lahann, J.; Franceschi, R. T.;

Krebsbach, P. H. The effects of Runx2 immobilization on poly (epsilon-caprolactone) on osteoblast differentiation of bone marrow stromal cells in vitro. Biomaterials 2010, 31, 32313236.


Page 23 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(36)

Seymour, J. P.; Elkasabi, Y. M.; Chen, H. Y.; Lahann, J.; Kipke, D. R. The insulation

performance of reactive parylene films in implantable electronic devices. Biomaterials 2009, 30, 6158-6167. (37)

Lahann, J.; Langer, R. Surface-initiated ring-opening polymerization of epsilon-

caprolactone from a patterned poly(hydroxymethyl-p-xylylene). Macromol. Rapid Commun. 2001, 22, 968-971. (38)

Chen, H. Y.; Lahann, J. Vapor-assisted micropatterning in replica structures: A

solventless approach towards topologically and chemically designable surfaces. Advanced Materials 2007, 19, 3801-3808. (39)

Gardella, J. A.; Ferguson, S. A.; Chin, R. L. Pi-Star[-Pi Shakeup Satellites for the

Analysis of Structure and Bonding in Aromatic Polymers by X-Ray Photoelectron-Spectroscopy. Appl. Spectrosc. 1986, 40, 224-232. (40)

Han, X. F.; Liu, Y. W.; Wu, F. G.; Jansensky, J.; Kim, T.; Wang, Z. L.; Brooks, C. L.;

Wu, J. F.; Xi, C. W.; Mello, C. M.; Chen, Z. Different Interfacial Behaviors of Peptides Chemically Immobilized on Surfaces with Different Linker Lengths and via Different Termini. J. Phys. Chem. B 2014, 118, 2904-2912. (41)

Han, X. F.; Uzarski, J. R.; Mello, C. M.; Chen, Z. Different Interfacial Behaviors of N-

and C-Terminus Cysteine-Modified Cecropin P1 Chemically Immobilized onto Polymer Surface. Langmuir 2013, 29, 11705-11712. (42)

Liu, Y. W.; Ogorzalek, T. L.; Yang, P.; Schroeder, M. M.; Marsh, E. N. G.; Chen, Z.

Molecular Orientation of Enzymes Attached to Surfaces through Defined Chemical Linkages at the Solid-Liquid Interface. J. Am. Chem. Soc. 2013, 135, 12660-12669.


Langmuir

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Selective and Reversible Binding of Thiol-Functionalized

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