Reactivity and Kinetics of Vinyl Sulfone-Functionalized Self

Mar 3, 2015 - Reactivity and Kinetics of Vinyl Sulfone-Functionalized Self-Assembled Monolayers for ... A new vinyl sulfone (VS) disulfide, 1,2-bis(11...
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Reactivity and Kinetics of Vinyl Sulfone-Functionalized SelfAssembled Monolayers for Bioactive Ligand Immobilization Hanqi Wang,†,‡ Fang Cheng,*,†,‡ Mingyang Li,†,‡ Wei Peng,§ and Jingping Qu†,‡ †

State Key Laboratory of Fine Chemicals, and ‡School of Pharmaceutical Science and Technology, Dalian University of Technology, Dalian, Liaoning 116024, People’s Republic of China § School of Physics and Optoelectronic Technology, Dalian University of Technology, Dalian, Liaoning 116023, People’s Republic of China S Supporting Information *

ABSTRACT: A new vinyl sulfone (VS) disulfide, 1,2-bis(11(vinyl sulfonyl)undecyl)disulfane, was synthesized to enable the preparation of VS-presenting self-assembled monolayers (VS SAMs) on Au substrates. The VS SAMs were used as a model system to assess the reaction kinetics of bioactive ligands, i.e., glutathione (GSH), N-(5-amino-1-carboxypentyl)iminodiacetic acid (ab-NTA), and mannose, toward the VS groups on the SAM surface. The VS SAMs and the ligand immobilization were characterized by X-ray photoelectron spectroscopy (XPS), contact angle goniometry, and proteinbinding experiments using a quartz crystal microbalance (QCM). Kinetic studies showed that the surface VS groups undergo pseudo-first-order reactions with various ligands, with the observed rate constant being 0.057 min−1 for GSH at pH 7.5, 0.011 min−1 for ab-NTA at pH 8.5, and 0.009 min−1 for mannose at pH 10.5. This work advanced our understanding of the reactivity of VS-bearing functional surfaces and further demonstrated the versatile potential of VS chemistry to prepare ligandimmobilized bioactive surfaces.

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solutions.19,20 Importantly, the VS group shows great stability in aqueous solution within a broad pH range at room temperature. For example, the half-life of the VS hydrolysis reaction at pH 8 is estimated to be more than 4 years.21 In contrast to the long half-life of VS chemistry, the half-lives of NHS and maleimide hydrolysis reactions at pH 8 are approximately 2 and 1 h, respectively.8,22 Although VS chemistry-based biomedical applications have been increasing, little is known on the reaction kinetics of a surface-anchored VS group with species containing mercapto, amino, or hydroxyl groups. To study reactions taking place at surfaces, various methods have been used, such as X-ray photoelectron spectroscopy (XPS),23 infrared (IR) spectroscopy,24 contact angle,25 and isotope exchange.26 Among these techniques, contact angle measurement is simple and straightforward.27,28 The occurrence of a surface reaction and its progression inevitably bring changes to the associated surface wettability, which has made contact angle measurement a convenient tool for surfacerelated research, with examples ranging from studies of surfacebased hydrolysis,29 polymerization,30 to catalyst screening.31

INTRODUCTION The development of reactive and specific chemical/biological molecule immobilization strategies for bioactive ligands has attracted broad attention because of their increasing applications in biosensing,1 disease detection,2 drug delivery,3 protein-receptor functions,4 bioactive materials,5 etc. The most commonly used immobilization strategy involves selective acylation of amino/thiol groups with activated esters, e.g., Nhydroxyl succinimide (NHS) for amines6 and maleimide for thiols.7 Unfortunately, both NHS and maleimide are highly prone to alkaline hydrolysis on a solid surface and in aqueous solution as well.8−10 Thus, it is desirable to seek alternative immobilization chemistries with high efficiency and improved resistance against hydrolysis. Vinyl sulfone (VS) chemistry has been used for protein immobilization in affinity chromatography since 1975.11 Because of its versatile reactivity toward mercapto, amino, and hydroxyl groups under ambient conditions, VS chemistry has been widely used in bioconjugation,12 drug delivery,13 biosensors,14 and affinity separation.15 Recently, VS surface chemistry was developed to present a variety of bioactive ligands on Au-,16 silicon-,14 and paper-based17 biosensors, on which biomolecule interactions (e.g., ligand−virus) were quantitatively examined. While the reaction of VS with mercapto groups occurs in neutral buffers,18 the reactions of VS with amino and hydroxyl groups typically proceed in basic © 2015 American Chemical Society

Received: October 16, 2014 Revised: February 14, 2015 Published: March 3, 2015 3413

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Supporting Information) δ: 139.2 (CH=CH2), 114.1 (CH=CH2), 45.2 (ClCH 2 ), 33.8 (CH 2 CHCH 2 ), 32.7 (ClCH 2 CH 2 ), 26.9 (ClCH2CH2CH2), 28.9−29.4 (other CH2). MS (EI+): m/z [M]+ 188.13. Synthesis of 2-((11-Chloroundecyl)thio)ethanol (3). Compound 2 (4.1 g, 19 mmol), mercapto ethanol (3 mL, 30 mmol), and azodiisobutyronitrile (1.8 g, 10.8 mmol) were added to 40 mL of petroleum ether, and the mixture was heated at 84 °C for 4 h. The solvent was then evaporated, and the resulting crude was purified with a silica gel column, affording compound 3 as a white solid (4.5 g, 88.2%). 1H NMR (400 MHz, CDCl3; see SI Figure 2a of the Supporting Information) δ: 3.72 (t, 2H, J = 6.0 Hz, CH2OH), 3.53 (t, 2H, J = 6.8 Hz, ClCH2), 2.73 (t, 2H, J = 6.0 Hz, SCH2CH2OH), 2.52 (t, 2H, J = 7.6 Hz, SCH2CH2CH2), 2.28 (s, 1H, CH2OH), 1.78 (m, 2H, ClCH2CH2), 1.58 (m, 2H, SCH2CH2CH2), 1.28−1.44 (m, 14H, other CH2). 13C NMR (100 MHz, CDCl3; see SI Figure 2b of the Supporting Information) δ: 60.2 (CH2OH), 45.2 (ClCH2), 35.3 (SCH2CH2OH), 32.6 (ClCH2CH2), 31.7 (SCH2CH2CH2), 26.8−29.7 (other CH2). MS (EI+): m/z [M]+ 266.15 Synthesis of 2-((11-Chloroundecyl)sulfonyl)ethanol (4). Compound 3 (2.7 g, 10 mmol) and 30% H2O2 (6 mL, 30 mmol) were added to 20 mL of acetic acid, and the mixture was heated at 77 °C for 3 h. The solvent was evaporated, and the residue was recrystallized with methanol, affording compound 4 as a white solid (2.6 g, 89.6%). 1H NMR (400 MHz, DMSO-d6; see SI Figure 3a of the Supporting Information) δ: 3.77 (t, 2H, J = 5.6 Hz, CH2OH), 3.61 (t, 2H, J = 6.4 Hz, ClCH2), 3.18 (t, 2H, J = 6.0 Hz, SO2CH2CH2OH), 3.09 (t, 2H, J = 8.0 Hz, SO2CH2CH2CH2), 1.68 (m, 4H, SO2CH2CH2CH2 and ClCH2CH2), 1.26−1.37 (m, 14H, other CH2). 13C NMR (100 MHz, CDCl3; see SI Figure 3b of the Supporting Information) δ: 56.3 (SO 2 CH 2 CH 2 OH), 54.9 (SO2CH2CH2CH2), 54.6 (SO2CH2CH2OH), 45.2 (ClCH2), 32.6 (ClCH2CH2), 26.8 (ClCH2CH2CH2), 28.4−29.3 (other CH2). MS (ESI+): m/z [M + H]+ 299.20, [M + NH4]+ 316.18, [M + Na]+ 321.19. Synthesis of 2,2′-(11,11′-Disulfanediylbis(undecane-11,1diylsulfonyl))diethanol (5). Compound 4 (1.8 g, 6 mmol) and sodium hydrosulfide (5.5 g, 60 mmol) were added to 50 mL of 70% ethanol, and the mixture was refluxed overnight. After cooling, hydrochloride acid was added to afford a white precipitate. The precipitate was filtered and dissolved in 30 mL of ethyl acetate. A total of 2 mL of H2O2 and 5% equivalent I2 were added, and the mixture was stirred for 3 h at room temperature. Compound 5 precipitated as a white solid (1.3 g, 73.4%). 1H NMR (400 MHz, DMSO-d6; see SI Figure 4a of the Supporting Information) δ: 3.78 (t, 4H, J = 6.8 Hz, CH2OH), 3.18 (t, 4H, J = 5.6 Hz, SO2CH2CH2OH), 3.09 (t, 4H, J = 8.0 Hz, SO2CH2CH2CH2), 2.69 (t, 4H, J = 6.8 Hz, SSCH2), 1.62−1.68 (m, 8H, SO2CH2CH2CH2 and SSCH2CH2), 1.26−1.36 (m, 28H, other CH2). 13C NMR (100 MHz, DMSO-d6; see SI Figure 4b of the Supporting Information) δ: 55.5 (SO 2 CH 2 CH 2 OH), 55.2 (SO2CH2CH2CH2), 54.0 (SO2CH2CH2OH), 38.3 (SCH2), 28.3− 29.4 (other CH2). MS (ESI+): m/z [M + K]+ 629.23. Synthesis of 1,2-Bis(11-(vinyl sulfonyl)undecyl)disulfane (1). Compound 5 (0.4 g, 0.6 mmol) and triethylamine (2 mL, 14 mmol) were added to 20 mL of dichloromethane. A solution of methanesulfonyl chloride (1.2 mL, 1.3 mmol) in dichloromethane (3 mL) was added dropwise to the mixture in an ice bath. The mixture was warmed to room temperature and stirred for 2 h. The solvent was removed, and the crude was purified with a silica gel column, affording compound 1 as a white solid (0.3 g, 88.6%). 1H NMR (400 MHz, CDCl3; see SI Figure 5a of the Supporting Information) δ: 6.63 (dd, 2H, J1 = 16.6 Hz, J2 = 10.0 Hz, SO2CH=CH2), 6.42 (d, 2H, J = 16.6 Hz, SO2CHCH2), 6.18 (d, 2H, J = 10.0 Hz, SO2CHCH2), 2.98 (t, 4H, J = 8.4 Hz, SO2CH2CH2), 2.68 (t, 4H, J = 7.6 Hz, SSCH2), 1.78 (m, 4H, SO2CH2CH2), 1.65 (m, 4H, SSCH2CH2), 1.27−1.43 (m, 28H, other CH2). 13C NMR (100 MHz, DCCl3; see SI Figure 5b of the Supporting Information) δ: 136.1 (SO2CH=CH2), 130.5 (SO 2 CH=CH 2 ), 54.3 (SO 2 CH 2 CH 2 ), 39.1 (SCH 2 ), 22.3 (SO2CH2CH2CH2), 28.4−29.4 (other CH2). MS (ESI+): m/z [M + H]+ 555.30, [M + NH4]+ 572.36, [M + Na]+ 577.39.

With the aid of self-assembly and subsequent surface modification, reactive groups can be readily introduced on solid surfaces, creating highly ordered two-dimensional thin films. Such thin films have been used as model systems for the understanding of surface reactions,32 protein resistance,33,34 and biosensors35 as well as serving as the basis for immobilization of molecules. While preparation of such thin films can be achieved in a number of ways, the most widely used approach is chemisorption of alkanethiols/disulfide on Au because of its convenience.36 Herein, we described the preparation of VS-presenting selfassembled monolayers (VS SAMs) on Au using a newly designed disulfide molecule with VS terminal groups. The VS SAMs were characterized by XPS and contact angle goniometry. The reactivity of the VS groups on the SAM surface toward mercapto, amino, and hydroxyl species is demonstrated using three affinity ligands, i.e., glutathione (GSH), N-(5-amino-1-carboxypentyl)iminodiacetic acid (abNTA), and mannose. Success of such reactions was confirmed via protein-binding experiments and XPS measurements. Furthermore, the effects of solution pH on these reactions as well as the reaction kinetics were quantitatively examined through water contact angle measurements.

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EXPERIMENTAL SECTION

Materials. 10-Undecen-1-ol was purchased from Alfa (Ward Hill, MA). Triphenyl phosphine, azodiisobutyronitrile (AIBN), mercapto ethanol, hydrogen peroxide, acetic acid, sodium hydrosulfide, methanesulfonyl chloride, triethylamine, and sodium chloride were purchased from Guangfu Fine Chemical Research Institute (Tianjin, China). Sodium phosphate monobasic, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and calcium chloride were purchased form J&K (Beijing, China). GSH and bovine serum albumin (BSA) were purchased from Solarbio (Beijing, China). AbNTA and glutathione S-transferase (GST) were purchased from Sigma (St. Louis, MO). Mannose was purchased from TCI (Tokyo, Japan). Concanavalin A (Con A) was purchased from Vector Laboratories (Burlingame, CA). All chemical reagents were used as received without further purification. pET21a-Streptavidin-Alive was obtained from Addgene (Addgene 20860). The expression and purification of Histagged streptavidin (SA) have been reported elsewhere.37,38 Oligo(ethylene glycol) thiol (OEG-thiol) was synthesized as previously described.39 Ultrapure Millipore deionized water with 18.2 MΩ cm resistivity was used in all experiments. Solution Preparation. GSH solution (2 mM, pH 5.5−11.5) was freshly prepared in 0.1 M phosphate-buffered saline (PBS) containing 5 mM ethylenediaminetetraacetic acid (EDTA). Ab-NTA solution (10 mM, pH 5.5−11.5) was prepared in 0.1 M PBS. Mannose solution (10%, w/v) was prepared in 0.1 M PBS (pH < 9.5) or 0.5 M Na2CO3/ NaHCO3 buffer (pH ≥ 9.5). OEG-thiol and mercapto ethanol solutions (1 mM, pH 8.5) were freshly prepared in 0.1 M PBS containing 5 mM EDTA. GST, His-tagged SA, and BSA solutions were all prepared at a final concentration of 0.1 mg/mL in 0.01 M PBS containing 150 mM NaCl with pH adjusted to 7.5. Con A solution (0.1 mg/mL, pH 8.5) was prepared in 0.01 M HEPES buffer containing 0.15 M NaCl and 0.1 mM CaCl2. Synthesis of 11-Chloro Undece-1-ne (2). A solution of 10undecan-1-ol (8 mL, 39.8 mmol) and triphenyl phosphine (10.7 g, 40 mmol) in 20 mL of CCl4 was refluxed for 3 h. After cooling, petroleum ether (60 mL) was added to afford a white precipitate. The precipitate was removed. The filtrate was concentrated and purified with a silica gel column, resulting in compound 2 as a colorless oil (7.1 g, 94.6%). 1 H NMR (400 MHz, CDCl3; see SI Figure 1a of the Supporting Information) δ: 5.80 (m, 1H, CH=CH2), 4.97 (dd, 2H, J1 = 16.8 Hz, J2 = 10.0 Hz, CHCH2), 3.53 (t, 2H, J = 6.8 Hz, ClCH2), 2.03 (m, 2H, CH2CHCH2), 1.77 (m, 2H, ClCH2CH2), 1.28−1.44 (m, 12H, other CH2). 13C NMR (100 MHz, CDCl3; see SI Figure 1b of the 3414

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Langmuir Scheme 1. Synthesis of 1,2-Bis(11-(vinyl sulfonyl)undecyl)disulfane (Compound 1)

9.5), or mannose (pH 9.5, 10.5, and 11.5) solutions at 25 °C for different reaction times. Contact angle measurements were then carried out immediately.

Preparation of Au Substrates. Silicon wafers were immersed in piranha solution (a 3:1 mixture of 97% sulfuric acid and 30% hydrogen peroxide) for 30 min. Caution: piranha solution reacts violently with most organic materials and must be handled with extreme care. Once the wafers were removed from piranha solution, they were rinsed with copious amount of water and ethanol and then dried under a stream of nitrogen. Titanium (5 nm) and gold (45 nm) were sequentially deposited onto the cleaned silicon wafers (10 × 10 mm) using a Turbo sputter coater K575XD (Kent, U.K.). Preparation of SAMs. The Au substrates were cleaned in an ultraviolet (UV)−ozone cleaner for 30 min and rinsed intensively with ethanol. Then, the cleaned substrates were immediately dipped into a 1.0 mM solution of compound 1 in dichloromethane/ethanol (1:1, v/ v) for 24 h at room temperature and rinsed with ethanol. Finally, the SAM samples were dried under a stream of nitrogen and stored in the dark at room temperature. XPS Analysis. XPS measurements were conducted using a Thermo Scientific ESCALAB 250Xi X-ray photoelectron spectrometer (Waltham, MA). All XPS data were acquired at a photoelectron takeoff angle of 90°. Thermo advances and XPS peak software were used to calculate elemental compositions and fit high-resolution spectra, respectively. Protein-Binding Experiments. Protein-binding experiments were conducted on a 5 MHz quartz crystal microbalance (QCM, Q-sense E1, Biolin Scientific, Sweden). The VS SAM-coated Au sensor chips were immersed in solutions of affinity ligands, i.e., GSH (pH 7.5) for 4 h, ab-NTA (pH 8.5) for 10 h, or mannose (pH 10.5) for 10 h, and rinsed intensively with water. The chips then were blocked with OEGthiol or mercapto ethanol (1 mM, pH 8.5) for 20 min and rinsed with water. The ab-NTA-treated chip was further immersed in a 1 mM NiCl2 solution for 5 min and rinsed with water before use. The affinity ligand-treated sensor chips were mounted in the module and equilibrated with buffer solutions. GST, His-tagged SA, Con A, and BSA were injected into the module separately, and the QCM frequency changes were monitored over time. Finally, the frequency responses were converted to binding mass according to the Sauerbrey equation (eq 1) Δm = −

C Δf n

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RESULT AND DISCUSSION Synthesis of VS-Functionalized Disulfide Compound. A new disulfide compound with VS terminal groups [1,2bis(11-(vinyl sulfonyl)undecyl)disulfane (1)] is designed and synthesized to prepare VS SAMs on Au substrates using the facile self-assembly process. The synthetic routine toward compound 1 is shown in Scheme 1. Briefly, 11-undecen-1-ol was selected as the starting material and underwent an Appel reaction by treatment with triphenyl phosphine in tetrachloromethane to generate compound 2. Next, the vinyl group in compound 2 reacted with mercapto ethanol through a radical addition reaction with by AIBN treatment to afford compound 3. Compound 3 was then converted into sulfone 4 by oxidization with H2O2 in acetic acid. Refluxing with sodium hydrosulfide resulted in the substitution of the chloro group in compound 4 by the mercapto group. The product was easily oxidized by H2O2 with I2 as the catalyst, generating disulfide 5. Finally, elimination of the β-hydroxyl group in compound 5 by treatment with methanesulfonyl chloride led to compound 1. It is worth noting that the generation of the VS group via elimination of the β-hydroxyl group with methanesulfonyl chloride41 offers several advantages over other previously reported VS-generating strategies. For example, the addition of divinyl sulfone to hydroxyl-bearing compounds can introduce the VS group. However, it requires violent basic conditions (e.g., NaOH or potassium tert-butoxide), and the conversion is low.42,43 Another method is elimination of the βhalogen in basic solutions44 to produce the VS group. Although this method has high efficiency and conversion, it requires additional chlorination and purification steps. In our study, elimination of the β-hydroxyl group using methanesulfonyl chloride not only affords the VS group in high yield but does so under mild reaction conditions with easy steps. Characterization of VS SAMs by XPS Analysis and Contact Angle Measurements. VS SAMs, featuring terminal vinyl sulfone groups (Figure 1a), were prepared by immersing Au substrates in a solution of compound 1. Hydroxylterminated SAMs (OH SAMs) with the same length hydrocarbon spacer were prepared to serve as a control (Figure 1b). The two SAMs on Au were characterized by XPS,and the corresponding elemental compositions of the two SAMs are summarized in Table 1. As we can see, the amount of Au detected was comparable between the two types of SAM surfaces, suggesting that the effective thickness of VS and OH SAMs is similar because of the hydrocarbon tail groups being the same length. In comparison to the OH SAMs, the packing density of VS SAMs would be slightly lower because of the

(1)

where Δm denotes the binding mass of proteins on the sensor chips, Δf denotes the QCM frequency response, n denotes the overtone number, and C = 17.7 ng Hz−1 cm−2 is a constant for the 5 MHz quartz crystal chips used in these experiments.40 Contact Angle Measurements. The water contact angle measurements were conducted on a contact angle goniometer DHHV1351UM (Beijing, China). To ensure reproducibility of the results, all of the measurements were repeated at least 3 times. pH Effect on VS Surface Reactions and Kinetics Study. The VS SAM samples were immersed in GSH, ab-NTA, or mannose solutions with pH ranging from 5.5 to 11.5 for 4 h at 25 °C. Control experiments were performed in parallel by immersing an additional set of VS SAM samples in corresponding plain buffer solutions. Following the reaction, the samples were rinsed with copious amounts of water, dried, and immediately characterized for water contact angles. To study the kinetics of VS surface reactions, the VS SAM samples were immersed in GSH (pH 6.5, 7.5, and 8.5), ab-NTA (pH 7.5, 8.5, and 3415

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chain45 and another peak around 286.3 eV that corresponds to the carbon atoms adjacent to the sulfone groups. The S 2p spectrum exhibited two distinct peaks, which can be fitted using two S 2p doublets with a 2:1 area ratio and a splitting of 1.2 eV.46 One is around 162.0 eV, corresponding to the thiolate species bonded directly to the Au substrate, while the other is around 168.0 eV, commonly assigned to the sulfone group according to a previous study.47 Besides XPS, contact angle measurements were carried out to confirm the success of VS SAM preparation from the new disulfide compound. The water contact angle on the unmodified Au substrates was 32° ± 1°. In contrast, after immersion in a solution of compound 1, the water contact angle increased to 67° ± 1°. This increase in the water contact angle suggests that the surface has become more hydrophobic, direct effect induced by the formation of SAMs based on the less polar compound 1. QCM Measurement of Protein Binding. Protein-binding experiments were conducted to demonstrate that (1) the VS groups on the SAM surface are reactive, and (2) ligands immobilized through these VS groups are bioactive. Three ligands have been selected for this study, i.e., GSH, ab-NTA, and mannose, with their structures shown in Figure 3. These ligands were chosen because each contained functional groups that are known to be reactive toward VS groups in solution, specifically, the functional groups of the mercapto group in GSH, the amino group in ab-NTA, and the hydroxyl group in mannose, respectively. Furthermore, all three reactants are affinity ligands for specific protein binding, i.e., GSH for GST,48 ab-NTA for His-tagged proteins,49 and mannose for Con A,16 which allows for direct bioactivity evaluation of the immobilized ligands after VS reactions. GST, His-tagged SA, Con A, and BSA were used to examine the presence of the ligands on VS SAMs. The four protein solutions were pumped through the QCM chip individually, and the frequency responses were monitored over time (see SI Figures 6−9 of the Supporting Information). The overall protein-binding mass for the four proteins on the untreated and ligand-treated VS SAMs is shown in Figure 4. In contrast to the large amount of non-specific adsorption on the as-prepared VS

Figure 1. Illustration of the structures of (a) VS SAMs and (b) OH SAMs on Au.

Table 1. Elemental Compositions of the VS and OH SAMs on Au Determined by XPS element

Au

C

S

O

VS SAMs OH SAMs

57.2 ± 0.9 58.1 ± 1.1

24.0 ± 1.3 32.6 ± 1.2

8.4 ± 1.2 3.5 ± 1.3

10.4 ± 1.1 5.9 ± 1.0

terminal sulfone groups being more sterically hindered than the hydroxyl groups. Both S/Au and O/Au ratios for VS SAMs roughly double those of OH SAMs, indicating the existence of extra S and O atoms in the VS SAMs. Although the theoretical ratio of sulfur/oxygen for both VS and OH SAMs is 1.0, the experimental ratio determined by XPS for both SAMs is less than 1. This discrepancy is due to the sulfur signal attenuation by the monolayers. In comparison to the OH SAMs, with the experimental S/O ratio being 0.59, a higher value of the S/O ratio (∼0.81) is obtained for the VS SAM. This finding confirms a fact that the terminal sulfone group is away from the Au substrate. The high-resolution spectra of C 1s and S 2p of VS SAMs were provided in Figure 2. The C 1s spectrum showed two peaks following peak fitting: a main peak at 285.0 eV that is characteristic of the repeating units of the tail oligomethylene

Figure 2. High-resolution XPS spectra of (a) C 1s and (b) S 2p obtained from the VS SAMs on Au. 3416

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Figure 3. Chemical structures of (a) GSH, (b) ab-NTA, and (c) mannose.

and after treatment with the ligands were summarized in SI Table 1 of the Supporting Information. For the VS SAMs treated with GSH and ab-NTA, nitrogen signals (see SI Figure 10a of the Supporting Information) were observed, originating from the amino groups in the ligands. For the VS SAMs treated with mannose, a slight increase in the oxygen signals (see SI Figure 10b of the Supporting Information) and O/S ratio (see SI Table 1 of the Supporting Information) was obtained, likely because of the multiple hydroxyl groups in the mannose molecule. Altogether, the protein-binding study and the XPS results indicate that the three ligands (GSH, ab-NTA, and mannose) are successfully immobilized on the VS SAMs after treated with the corresponding ligand solutions. pH Effect on VS Surface Reactions. The objective of this section is to examine the effect of solution pH on the VS surface reaction toward mercapto, amino, and hydroxyl groups. The water contact angles of the VS SAMs treated with the three ligands in buffer solutions are plotted versus the reactant solution pH (Figure 5). As mentioned earlier, the water contact angle of the as-prepared VS SAMs is around 67°. After incubation in any of the three ligand solutions at pH 5.5 for 4 h, the water contact angle of the VS SAMs remained around 67°, suggesting the lack of reaction at this pH. However, when the reaction solution pH increased, changes in the water contact angle were observed. Specifically, for the GSH ligand, a drastic decrease in the water contact angle was noted at pH 6.5 (Figure 5a). For the ab-NTA ligand, that critical solution pH for reaction was 8.5 (Figure 5b). As for mannose, a small decrease in the water contact angle was obtained for reaction at pH 9.5, while a more distinct change in the contact angle was observed for reactions at pH 10.5 or higher (Figure 5c). These results suggest that the reactivity of the three ligands toward the VS groups on SAMs ranks in the following order: GSH > ab-NTA > mannose. This finding is consistent with the reactivity difference among the respective functional groups present in

Figure 4. Protein-binding experiments on the VS SAMs before and after treated with GSH, ab-NTA, and mannose using QCM. Four different proteins are used, including GST (0.1 mg/mL in 0.01 M PBS at pH 7.5), His-tagged SA (0.1 mg/mL in 0.01 M PBS at pH 7.5), Con A (0.1 mg/mL in 0.01 M HEPES buffer containing 0.1 mM CaCl2 at pH 8.5), and BSA (0.1 mg/mL in 0.01 M PBS at pH 7.5).

SAMs for all four proteins, the VS SAMs treated with ligands show the expected binding patterns for these proteins, where the GSH-modified surface shows specific binding to GST, the ab-NTA-modified surface shows specific biding to His-tagged SA, and the mannose-modified surface shows specific binding to Con A. These results confirm that the VS SAMs are highly reactive toward the three ligands, and the immobilized ligands are bioactive. XPS measurements were also conducted to confirm the surface reactions of VS SAMs with GSH, ab-NTA, and mannose. The organic compositions of the VS SAMs before

Figure 5. Contact angle measurements of the VS SAMs treated with (a) GSH, (b) ab-NTA, and (c) mannose. All of the treatments are conducted in buffer solutions for 4 h at 25 °C. 3417

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Figure 6. Contact angle measurements of the VS SAMs after treated with (a) GSH, (b) ab-NTA, and (c) mannose. All of the treatments are conducted in buffer solutions at 25 °C.

Figure 7. Natural logarithm of VS coverage on the SAMs after treated with (a) GSH, (b) ab-NTA, and (c) mannose at 25 °C under various reaction pH values.

surface coverage was calculated from the contact angle data according to the Cassie equation (eq 2)51,52

these ligands, with mercapto groups being highly reactive, followed by amine groups being more reactive than hydroxyl groups.50 It is worth noting that the reaction of the VS SAMs with mannose occurred at pH 9.5 or higher (Figure 5c), whereas the reaction with glycol took place at pH 11.5 (see SI Figure 11 of the Supporting Information), indicating the high reactivity of the α-hydroxyl group in the mannose molecule.16 Two sets of control experiments were also carried out, where the VS SAMs were treated with either plain buffer solutions with pH ranging from 5.5 to 11.5 for 4 h or a strong basic buffer (pH 11.5) for 4−24 h. Both contact angle results (see SI Figures 12 and 13 of the Supporting Information) and the XPS analysis of Au % (an indicator of the effective film thickness; see SI Figure 14 of the Supporting Information) showed no significant changes between the buffer-treated and as-prepared VS SAMs, indicating the stability of the VS SAMs at various basic pH and with different times, which rule out the occurrence of either VS hydrolysis or SAM deterioration in aqueous solutions. In addition, the water contact angle on the VS SAMs showed no obvious decrease when treated with the oxidized form of GSH (glutathione disulfide) until pH > 8.5 (see SI Figure 15 of the Supporting Information), thus excluding the effect of chemisorption of GSH species onto Au on the decrease in the contact angle. Kinetics Study of the VS Reaction on the Surface. To examine the kinetics of the VS reactions toward the three ligands, we focused on the following pH range for each ligand: pH 6.5−8.5 for GSH, pH 7.5−9.5 for ab-NTA, and pH 9.5− 11.5 for mannose. The progress of the surface reaction was determined from the corresponding changes in the water contact angles as a function of time. As shown in Figure 6, the contact angles on the VS SAM surface decreased with the reaction time and reached plateaus at pH 7.5 or higher for GSH (Figure 6a), pH 8.5 or higher for ab-NTA (Figure 6b), and pH 10.5 or higher for mannose (Figure 6c). To evaluate the kinetics of these reactions, the VS

cos θexp = χVS cos θ VS + χreactant cos θreactant

(2)

where χVS and χreactant are the surface coverage of the two components, θVS = 67° ± 1° is the contact angle on the asprepared VS SAMs, and θGSH = 47° ± 1°, θab‑NTA = 45° ± 1°, and θmannose = 47° ± 1° are the plateau contact angle values on the VS SAMs treated with the three ligands. The natural logarithm of the corresponding VS coverage was plotted versus reaction time, as shown in Figure 7. For all reaction pH studied, we observed a linear dependence of the VS surface coverage on the reaction time before the plateaus, suggesting that the surface reactions can be described by a standard pseudo-first-order kinetics. The rate constant and halflife of the reaction are described by χ ln VS = −kobs(t − t0) χVS (3) 0

ln

1 = −kobst1/2 2

(4)

where χVS and χVS0 denote the measured and initial coverages of the VS group on the surface, respectively, t and t0 denote the measurement time and the initial time, respectively, kobs denotes the pseudo-first-order rate constant, and t1/2 denotes the half-life of the reaction. The rate constants of VS toward the three ligands are summarized in Table 2. As seen, for all three ligands, the surface reactions are pH-dependent. For example, the observed rate constant of GSH increases 10-fold as pH increases from 6.5 to 8.5, while the observed rate constant of mannose increases 5fold as pH increases from 9.5 to 11.5. The pseudo-first-order reactions of VS chemistry toward GSH have been examined previously using an aqueous solution of VS-conjugated polymer in aqueous solution.43 The reported 3418

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monolayers presenting VS groups well-suited for the preparation of a variety of bioactive surfaces. First, the facile immobilization versatility of ligands bearing mercapto, amino, or hydroxyl groups provides specific interactions with GST, His-tagged proteins, and lectins, respectively. Second, less hydrolysis in aqueous solutions at high pH ensures the high efficiency in ligand immobilization. Finally, the detailed reaction kinetics obtained in this study provides valuable information to guide bioactive surface preparation with ligand density varied in a controlled manner.

Table 2. Pseudo-First-Order Rate Constants and Half-Lives of the Reactions of the VS SAM Reaction with GSH, abNTA, and Mannosea ligand GSH

ab-NTA

mannose

a

pH

kobs (min−1)

t1/2 (min)

6.5 7.5 8.5 7.5 8.5 9.5 9.5 10.5 11.5

0.010 0.057 0.117

69 12 6

0.011 0.012 0.002 0.009 0.011

63 58 347 77 63

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ASSOCIATED CONTENT

* Supporting Information S 11

H and 13C NMR spectra for compounds 1−5, QCM records of protein-binding experiments, XPS composition of the VS SAMs treated with ligands, contact angle measurements of the VS SAMs treated with glycol, plain buffer, and the oxidized form of GSH, XPS assessments of the VS SAMs treated with plain buffer, chemical structures of blocking reagents, and QCM and XPS assessments of the blocking reagents. This material is available free of charge via the Internet at http:// pubs.acs.org.

Measurements were conducted at 25 °C.

rate constant in that study is 0.065 min−1 at pH 8.5, whereas the value obtained in our current work is 0.117 min−1 at pH 8.5. Such a faster reaction occurring for VS presented on surfaces compared to VS in solution is likely due to the easy access of VS groups of the SAMs to thiol reactants. The reaction of VS SAMs with GSH at pH 6.5−8.5 takes place with half-lives ranging from 69 to 6 min, considerably slower than the reaction of maleimide with GSH that typically completes within 1 min.53 However, it is worth noting that, unlike maleimide undergoing slow hydrolysis (half-life of 6.8 h at pH 7.5), VS chemistry exhibits infinite stability in aqueous solution.33 Such an intrinsic stability has also made VS chemistry advantageous over NHS chemistry. For example, the optimal pH value for NHS-able ab-NTA immobilization has been reported to be 8.5.54 Unfortunately, up to 70% of the NHS species is hydrolyzed under the same conditions.55 For ligand immobilization, it is often desirable to achieve high surface coverage. In Figure 7, the natural logarithm values of the corresponding VS coverage at the plateaus are −2.48 for GSH at pH 8.5, −3.69 for ab-NTA at pH 9.5, and −4.96 for mannose at pH 11.5. It suggests that a small percentage of the VS groups remains on the surface, i.e., 8.4% for the GSH treatment, 2.5% for ab-NTA, and 0.7% for mannose. The unreacted VS groups could be explained by the steric hindrance effect of the immobilized GSH or ab-NTA ligands, which subsequently prevent these VS groups from further reacting. To reduce any non-specific protein binding to the residual VS groups, blocking reagents, such as OEG-thiol and mercapto ethanol (chemical structures illustrated in SI Figure 16 of the Supporting Information), were used to consume the remaining free VS groups. It is worth noting that no significant difference of the blocking reagents or blocking batches was observed with respect to the protein-binding profile (see SI Figure 17 of the Supporting Information), the binding mass (see SI Figure 18 of the Supporting Information), and organic composition (see SI Figure 19 of the Supporting Information).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: ff[email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21104008, 21231003, and 61137005), the “111” Project of the Ministry of Education of China, and the Fundamental Research Funds for the Central Universities (DUT13LAB07). The authors also thank Dr. Daniel M. Ratner at University of Washington, Seattle, WA, for meaningful discussion.

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REFERENCES

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CONCLUSION In summary, we have successfully prepared VS-presenting SAMs on Au using a newly developed VS-terminated disulfide molecule 1. A detailed kinetic study of VS chemistry toward bioactive ligands, i.e., GSH, ab-NTA, and mannose, was carried out on the monolayers. The pseudo-first-order rate constants and half-lives of the VS surface chemistry in neutral and basic aqueous solutions were obtained. As expected, the reactivity of the three ligands ranks in the following order: GSH > ab-NTA > mannose. We demonstrated several features that make the 3419

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