pH-Switchable Interaction of a Carboxybetaine Ester-Based SAM with

Article pubs.acs.org/Langmuir

pH-Switchable Interaction of a Carboxybetaine Ester-Based SAM with DNA and Gold Nanoparticles Jaroslav Filip,†,‡ Anton Popelka,† Tomas Bertok,§ Alena Holazova,§ Josef Osicka,† Jozef Kollar,∥ Marketa Ilcikova,† Jan Tkac,§ and Peter Kasak*,† †

Center for Advanced Materials, Qatar University, P.O. Box 2713, Doha, Qatar Department of Environmental Protection Engineering, Faculty of Technology, Tomas Bata University in Zlin, Vavreckova 275, 76001 Zlín, Czech Republic § Department of Glycobiotechnology, Institute of Chemistry, Slovak Academy of Sciences, Dubravská cesta 9, 842 36 Bratislava, Slovak Republic ∥ Polymer Institute of the Slovak Academy of Sciences, Dúbravská cesta 9, 845 41 Bratislava, Slovak Republic ‡

S Supporting Information *

ABSTRACT: We describe a self-assembled monolayer (SAM) on a gold surface with a carboxybetaine ester functionality to control the interaction between DNA and gold nanoparticles via pH. The negatively charged phosphate backbone of DNA interacts with and adsorbs to the positively charged carboxybetaine esters on the SAM. DNA release can be achieved by the hydrolysis of carboxybetaine ester (CBE) to a zwitterionic carboxybetaine state. Furthermore, the adsorption of negatively charged citrate-capped gold nanoparticles to a SAM-modified plain gold surface can be controlled by the pH. The SAM based on carboxybetaine ester allows for the homogeneous adsorption of particles, whereas the SAM after hydrolysis at high pH repels AuNP adsorption. The antifouling surface properties of the surface modified with carboxybetaine were investigated with protein samples.

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INTRODUCTION In recent decades, interest in new platforms enabling the local delivery of DNA from implants or interventional devices has increased.1,2 Moreover, many applications in biomolecular and analytical fields such as genetic material diagnostics, biosensing, and biochips require robust, simple, and inexpensive approaches for immobilizing DNA on surfaces.3−7 The physical adsorption of DNA to surfaces is preferential for these applications compared to covalent immobilization because the DNA does not need to be modified and the adsorption process is simple and straightforward via immersion steps. In this regard, the key challenge in the fabrication of surfaces relies on the development of a surface to allow for the immobilization of DNA and, subsequently, its controlled release. These so-called smart surfaces undergo transition via an external input in the form of an electrochemical, thermal, mechanical, photochemical, or chemical trigger. The mechanism of DNA delivery or release by a pH-induced trigger relies on changing the positively charged protonated tertiary amine moieties that interact with the negatively charged phosphate backbone of DNA. Examples involve DNA immobilization and release from a layer-by-layer (LBL) architecture by the pH-induced deprotonation of amine groups,8−10 hydrolysis,11 or degradation12,13 of a polymer. These approaches are limited to © 2017 American Chemical Society

polymers and are accompanied by the release of a large quantity of the degradation product. Similar to DNA from a synthesis perspective, the controlled adsorption of gold nanoparticles (AuNPs) is an emerging topic of interest.14 AuNPs possess unique physical characteristics such as size and shape dependency, excellent biocompatibility, simple synthesis, easy bioconjugation, and so forth15 and are suitable candidates for use as materials for diagnosis, treatment, delivery, conjugation, implantation, or use as an imaging biologically active component.16,17 Self-assembled monolayers (SAMs)18 would be beneficial for particular applications such as implants or device coatings in biosensing because SAMs are easy to prepare and their functionality on a surface can be tailored to specific properties such as various architectures19 or the prevention of nonspecific interactions.20 Carboxybetaine ester derivatives contain a permanent quaternary ammonium group covalently linked to a carboxylate ester.21 They represent a suitable candidate for smart and switchable materials because they can switch from a cationic Received: February 19, 2017 Revised: June 16, 2017 Published: June 19, 2017 6657

DOI: 10.1021/acs.langmuir.7b00568 Langmuir 2017, 33, 6657−6666

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Langmuir

Scheme 1. Chemical Structure of CBE and Schematic Illustration of the Interaction of the SAM Derived from CBE with DNA and Au Nanoparticles (AuNPs) and the Change in the Interaction to Carboxybetaine Character after pH-Induced Hydrolysis

adsorption before and after hydrolysis. It should be noted that after hydrolysis and DNA release the surface possesses zwitterionic carboxybetaine character with antibiofouling properties, as examined by SPR measurements using model proteins.

carboxybetaine ester to a charge-balanced zwitterionic form.22−27 We previously showed that a polymer bearing a carboxybetaine ester with a photocleavable 2-nitrophenyl ester group can dramatically change the interaction between DNA and a surface. The polymer on the surface was able to immobilize DNA, and after irradiation at 365 nm, almost complete release was observed.28 Moreover, the hydrolysis of carboxybetaine polymers based on a hydrolyzable ester group and with the same distance between ammonium and carboxybetaine ester moieties as in solution was investigated.29 It was revealed that polymers are suitable gene vectors and that polymers with a methylene spacer after hydrolysis to the zwitterion form exhibited a range of amino acid buffering in endosomal environments. Furthermore, carboxybetaine-modified surfaces exhibited hemocompatible,30 nonbiofouling31,32 properties that allow for further functionalization by biomolecules.33,34 Thus, in this report, we designed and characterized a SAM formed from a carboxybetaine ester derivative of lipoic acid, namely, (R)-3-(5-(1,2-dithiolan-3-yl)pentanamido)-N-(2ethoxy-2-oxoethyl)-N,N-dimethylpropan-1-aminium chloride (CBE), whose structure is shown in Scheme 1. The 1,2dithiolane-based end group ensured anchoring of CBE to the gold surface, and the positively charged carboxybetaine ester moiety allowed for immobilization of the negatively charged phosphate backbone of DNA molecules or negatively charged particles. An investigation of the interactions between the gold surface modified with CBE and DNA and citrate-capped Au nanoparticles (AuNPs) was carried out as schematically depicted in Scheme 1. The immobilization and pH-controlled release of DNA from the modified surface and the transformation of the carboxybetaine ester moiety to carboxybetaine was examined. A set of characterization tools, including electrochemical, surface plasmon resonance (SPR), quartz crystal microbalance (QCM,) atomic force microscopy (AFM), and X-ray photoelectron spectrometry (XPS) analyses, were applied to prove the physical absorption of DNA to the modified gold surface and the subsequent release of DNA after pH-induced hydrolyses of the carboxybetaine esters. Moreover, interaction with AuNPs revealed a dramatic change in

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EXPERIMENTAL PROCEDURES

Materials. N,N-Dimethylpropane-1,3-diamine, ethyl chloroacetate, (R)-lipoic acid N-hydroxysuccinimide, dicyclohexylcarbodiimide (DCC), acetone, and CH2Cl2 were purchased from Aldrich and used as received. DNA sodium salt (70 kDa, double-stranded, approximately 110 bp) was supplied by Acros Organics and used as received. Human serum albumin (HSA) and immunoglobulin G (IgG) were purchased from Sigma-Aldrich (USA). Gold-coated silicon wafers were prepared according to the literature.33 Ultrapure deionized water (DW) was obtained from a Millipore system (Direct Q3, France). Phosphate buffer (PB; 10 mM, pH 7.4) and phosphate-buffered saline (PBS; PB with 0.9 wt % NaCl) were purchased from Sigma-Aldrich. The gold nanoparticles (AuNPs) stabilized by negatively charged sodium citrate were prepared according to the literature.35 A nanoparticle size of 24 nm and a zeta potential of −45.2 mV were measured with a Zetasizer Nano ZS (Malvern, USA). Synthesis of (R)-3-(5-(1,2-Dithiolan-3-yl)pentanamido)-N-(2ethoxy-2-oxoethyl)-N,N-dimethylpropan-1-aminium Chloride (CBE). Preparation of (R)-N-(3-(Dimethylamino)propyl)-5-(1,2-dithiolan-3-yl)pentanamide. Dissolved DCC (2.4 g, 11.6 mmol) in dry CH2Cl2 (4 mL) was added dropwise to a stirred solution of (R)-lipoic acid (2 g, 9.69 mmol) in CH2Cl2 (80 mL). The mixture was stirred at ambient temperature for 30 min. A solution of N-hydroxysuccinimide (NHS; 1.34 g, 11.6 mmol) in acetonitrile (2 mL) was added. After the reaction mixture was stirred for 30 min at ambient temperature, N,Ndimethylpropane-1,3-diamine (3.66 mL, 29 mmol) was added dropwise and stirred overnight. The reaction mixture was filtered and washed with 1 M NaOH (3 × 35 mL). CH2Cl2 was evaporated. The product was dissolved in 1 M HCl (80 mL) and stirred for 1 h. The suspension was filtered, and the solution was added to chloroform (100 mL). The solution was adjusted to pH 12−13 (2 M NaOH) and mixed for 30 min. The phases were separated, and the aqueous phase was extracted with CH2Cl2 (2 × 40 mL). The collected organic layers were dried with Na2SO4, filtered and evaporated, and then dried under high vacuum. The product was obtained in 57% yield as a yellowish oil. IR (ATR): 3315, 2933, 2848, 2764, 1639 (amide stretching CO), 1540, 1443, 1304, 1241, 1043, 879, 750, 639 cm−1. 1H NMR (400 6658


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Langmuir Scheme 2. Synthesis of the Carboxybetaine Ester Derivative, CBE

MHz, CDCl3) δ: 3.56 (m, 1H, H−C3), 3.31 (2t, J = 5.5 Hz, 2H, HN− CH2−CH2), 3.24−3.02 (m, 2H, CH2−5), 2.90−2.68 (m, 2H, CH2− CH2−NMe2), 2.56−2.34 (m, 3H, Ha-4 and CH2-2′), 2.22 (s, 6H, NMe2), 1.93 (3, 1H, Ha-4), 1.80−1.54 (m, 8H, CH2-3′ and CH2-5′), 1.54−1.34 (m, 2H, CH2-4′) ppm. Preparation of CBE. Tertiary amine from the previous reaction (0.66 g) was dissolved in THF (6 mL), and 0.485 mL of ethyl chloroacetate was added under a flow of argon. The reaction mixture was stirred at ambient temperature overnight in the dark under an argon atmosphere. The product was cleaned and dried by precipitation to diethyl ether, centrifuged, evaporated, and dried under high vacuum. The CBE product was obtained in 86% yield (0.7919 g) as a yellow solid. FTIR (FTIR): 3403, 3236, 2932, 2848, 1740 (ester, stretching C O), 1640 (amide stretching CO), 1538, 1448, 1397, 1217, 1185 (ester, stretching C−O), 1027, 848, 707, 600 cm−1. 1H NMR (400 MHz, D2O) δ: 4.25 (m, 2H, O−CH2), 3.70−3.50 (m, 4H, H-5 and NH−CH2−), 3.25 (s, 6H, N+Me2), 3.20 (m, 2H, N+−CH2), 3.10 (m, 1H, H-3), 2.80 (s, 2H, CO−N+−CH2) 2.45 (m, 1H, H-4), 2.25 (m, 2H, H-2′), 2.00−1.80 (m, 3H, CH2−CH2−N, H-4), 1.60−1.50 (m, 4H, H-3′ and H-5′), 1.35 (m, 2H, H-4′), 1.25 (m, 3H, CH3) ppm. 13C NMR (100 MHz, D2O) δ: 176.8 (N−CO), 175.5 (C−CO), 63.3 (CO−CH2), 61.2 (N−CH2), 56.5 (C-3), 52.1 (N−CH3), 51.9 (N− CH3), 48.8 (CH2), 40.2 (CH2, C-4), 38.0 (CH2, C-5), 35.7 (CH2), 33.7 (CH2), 27.9 (CH2), 24.9 (CH2), 22.5 (CH2), 13.1 (CH3) ppm. Nuclear Magnetic Resonance Spectroscopy (NMR). 1H NMR spectra were recorded using a Varian 400 MR spectrometer instrument at 298 K. Chemical shifts are reported in ppm downfield of the solvent standard. The solvent was used as a reference. The working frequencies were 400 and 100 MHz for 1H and 13C NMR, respectively. Coupling patterns were designated as s, singlet; d, doublet; t, triplet; q, quartet; and m, multiplet. The concentration of sample was approximately 10 mg of sample dissolved in 0.7 mL of solvent. Fourier Transfer Infrared (FTIR). FTIR spectra were recorded using a FTIR 760 infrared spectrophotometer (PerkinElmer, USA) with an attenuated total reflectance accessory. The spectra were recorded using 64 scans in the middle infrared region (400−4000 cm−1). The spectra were characterized in wavenumbers. SAM Formation. Preparation of a Bare Gold Surface. The goldcoated silicon wafer was first washed with acetone, ethanol, and DW, treated with UV, and dried in a stream of nitrogen. The freshly cleaned gold wafers were dried in vacuum for SAM formation. SAM Formation of CBE. Ten milliliters of an aqueous 4 mM solution of CBE was prepared in an argon atmosphere and was applied to 1 × 1 cm2 gold-coated silicon wafer samples for 12 h in the dark under an argon atmosphere at room temperature. The sample with the SAM was removed, washed with DW, dried in a stream of nitrogen, and used directly for further experiments or analysis. Electrode Pretreatment and SAM Formation on Electrodes. All electrochemical measurements were performed using a VSP potentiostat (Biologic, France) connected to an electrochemical cell. Three-electrode connections were employed with either a polycrystalline gold electrode (AuE; 1.6 mm, Bioanalytical Systems) or a goldcoated quartz crystal microbalance (QCM) chip and a platinum wire as working and auxiliary electrodes, respectively, whereas a Ag/AgCl/ sat KCl electrode was used as a reference electrode. Nyquist plots were evaluated by fitting with EC-Lab software (Biologic, France) using Circle Fit function. The AuE was cleaned according to a previously published protocol.36 Immediately after cleaning, the AuE was immersed in 300 μL (4 mM) of CBE. Reductive desorption of the thiol-modified AuE in Ar-bubbled NaOH (0.1 M) was performed to determine the number of thiol molecules attached to the gold surface.

The surface density of sulfur−gold bindings was calculated from the charge measured during the reduction. This value was obtained from the integration of the cathodic peak between −1275 and −870 mV (vs Ag/AgCl/saturated KCl reference electrode) of the first CV scan. QCM measurements were performed in an EQCM flow cell (ALS Co., Ltd., Japan) connected to an external peristaltic pump. An eQCM10 M oscillator (Gamry, USA) was connected to a chip mounted to the EQCM cell. In a typical experiment, an aqueous solution of CBE flowed through the cell with the fresh gold QCM chip mounted inside while the frequency change was recorded using Resonator software (Gamry). Afterward, this solution was replaced with PB, and after signal stabilization, the PB was changed to a solution of DNA in PB without an interruption of the signal recording. Surface Plasmon Resonance Studies. A surface plasmon resonance (SPR) tool (SR7000DC, Reichert, USA) was used for the determination of the self-assembly of CBE on gold-sputtered SPR chips (12 × 12 × 0.3 mm3, Xantec, Germany) and the subsequent adsorption and desorption of DNA. The flow rate was set to 10 μL· min−1. For protein adsorption measurements, CBE-modified goldsputtered SPR chips (12 × 12 × 0.3 mm3, Xantec, Germany) after hydrolysis to the zwitterionic state were examined with PBS as a blank and then with a solution of protein at a concentration of 1 mg·mL−1 in PBS and subsequently washed with PBS solution to determine the absorbed amount. Adsorption of AuNPs. Plain surfaces with the SAM of CBE before and after hydrolysis were incubated with aqueous solutions of citratecapped AuNPs (1 × 10−4 M) for 10 min under a nitrogen atmosphere. The surface was gently rinsed with DW, dried in stream of nitrogen, and further investigated by AFM and SEM analysis. Surface Characterization Methods. The SEM images were recorded using a Nova NanoSEM 450 (FEI, USA) microscope with an acceleration voltage. Good resolution of SEM images was ensured by a few nanometer sputter-coated Au layer on the samples. X-ray Photoelectron Spectroscopy. The XPS signals on squareshaped gold chips of 10 × 10 × 0.3 mm3 modified with the SAM were recorded using an AXIS ULTRA DLD (Kratos Analytical Ltd., U.K.) system equipped with an Al Kα X-ray source. The spectra were acquired in constant analyzer energy mode with a pass energy of 160 eV, 10 kV, and a 10 mA emission current for the survey. The individual scans were performed with a pass energy of 10 eV, 15 kV, and a 15 mA emission current. Vision Manager 2 software was used for digital acquisition and data processing. Spectral calibration was performed using the automated calibration routine and the internal C 1s standard. The surface compositions (in atom %) were determined by considering the integrated peak areas of detected atoms and the respective sensitivity factors. Atomic Force Microscopy. An atomic force microscope (MFP-3D Asylum Research, USA) equipped with a silicon probe (Al reflexcoated Veeco model OLTESPA, Olympus model AC160TS) was used. Analyses were carried out under ambient conditions using the standard topography of alternating current in air (ac or tapping mode). In this mode, the AFM tip is in contact with the sample surface for a short period of time, which dramatically decreases the tip deformation and dragging effects. The measurements were carried out at a 1 Hz scan rate and high resolution (512 lines and points) to obtain surface images of particles.

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RESULTS AND DISCUSSION The carboxybetaine ester derivative with a dithiolane terminal group CBE was applied as a single enantiomer for SAM formation. CBE was synthesized in two simple steps from (R)6659


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spectra also showed no detection of the hydrolysis of amide groups during hydrolysis with absorption at 1630 cm−1. By applying 0.1 M NaOH, the hydrolysis proceeded within 5 min. Complete hydrolysis and stability of the amide groups was also confirmed by 1H NMR spectra, as depicted in Figure S4, where the disappearance of signals corresponding to methyl groups at 1.2 ppm and methylene groups at 4.27 ppm from the ethyl ester moiety was observed. This is in accordance with the previously reported stability of amide groups during the hydrolysis of carboxybetaine ester derivatives.24 The formation of SAMs is a simple, fast, cost-effective approach to the modification of a variety of surfaces to control the chemical nature of a surface.18,38 In our case, submerging the Au surfaces in a 4 mM aqueous solution of CBE for 4 h formed a well-ordered SAM of CBE. Both qualitative and quantitative assessments of SAM from CBE on AuE (AuE-CBE) were performed using electrochemical reductive desorption. Reduction peaks typical for thiols on the Au surface were observed and were integrated using the B-spline baseline calculated with Origin software. This option was selected over the typically used linear baseline to reflect the nonlinearity of voltammograms in the given potential window, and it was found that the B-spline had corrected the integrated peak area by less than by 10% as compared to evaluation with a linear baseline. Reduction desorption revealed a thiol surface coverage of 0.84 ± 0.02 molecules per nm2 of geometric surface area, as shown in Figure 2a. Analogous results were obtained in experiments using the quartz crystal microbalance technique (QCM). In a typical experiment, an aqueous solution of CBE (1.5 mg mL−1) was first streamed through the QCM cell, resulting in a frequency change of 64.7 ± 2.7 Hz (Figure 2b). This corresponds to a surface coverage of 0.86 ± 0.04 CBE molecules per nm2, a value lower than that for a theoretical monolayer of aliphatic thiols39 (4.67 molecules per nm2) and lower than 3.7 molecules per nm2 as reported for linear thiols terminated with quaternary amines.40 Such a value reflects the steric hindrance of dithiolane units binding at position 3. Repulsive forces among individual molecular heads with positively charged ammonium moieties should also be taken into account. Because our value is close to the reported density of 1.7 molecules per nm2 of mercaptoundecanoic acid on a

lipoic acid according to Scheme 2. The reaction of (R)-lipoic acid with N,N-dimethylaminopropylamine in the presence of dicyclohexylcarbimide (DCC) and hydroxysuccine imide (NHS) yielded a tertiary amine derivative,37 which was subsequently quarternized with ethyl chloroacetate. The final product, CBE, was characterized by FTIR and NMR (Figures S1−S3, Supporting Information). It should be noted that several approaches to the synthesis of the linear thiol carboxybetaine derivative were tested. However, it was not possible to obtain a pure carboxybetaine ester derivative because of partial hydrolysis or an inseparable difficult reaction mixture during the course of the synthesis. The CBE derivative did not hydrolyze in solution over 2 days at pH 7.3. The hydrolysis of CBE was also carried out with 0.1 M NaOH aqueous solution or with solution at pH 9. Hydrolysis was evaluated by FTIR measurements where the absorbance peak at 1740 cm−1 associated with the ester CO stretching vibration was decreased and the peak at 1610 cm−1 due to carboxylate increased during the course of hydrolysis, as shown in Figure 1. Figure 1 shows the FTIR spectra at pH 9

Figure 1. FTIR spectra during the hydrolysis of CBE after 5 (solid line), 30 (dashed line), and 50 min (dotted line).

after 5, 30, and 50 min, indicating the progression of hydrolysis to completion. With the application of pH 9 TRIS buffer solution, complete hydrolysis occurred within 50 min. The

Figure 2. (a) Cathodic part of a representative CV from the electrochemical reductive desorption of the SAM of CBE obtained on AuE in argondeaerated NaOH (100 mM) (red line), with a scan rate 1 V s−1 and a calculated baseline (black line). (b) QCM recording of SAM formation in which the arrow indicates the addition of CBE solution. 6660


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Figure 3. (a) CVs of a gold electrode (AuE) modified with a SAM of CBE (AuE-CBE, black curve) and incubated for 10 min (red curve) or 1 h (green curve) in pH 7.4 phosphate buffer (PB) and then for 5 min in 100 mM NaOH (blue curve). (b) Nyquist plots (obtained from EIS investigation) of a CBE-modified AuE (AuE-CBE, black dots) and the same layer after interaction with DNA (AuE-CBE-DNA, red dots). All experiments were measured in 100 mM KCl + 5 mM ferricyanide/ferrocyanide.

Figure 4. (a) QCM recording of DNA adsorption on Au-coated QCM chips with an arrow marking the DNA addition. (b) Recording of SAM formation, DNA adsorption, and DNA desorption using SPR. In the sensogram, areas are marked with blue lines relevant to (I) the adsorption of CBE and washing with deionized water (DI) and PB, (II) DNA adsorption and PB washing, and (III) hydrolysis of CBE and DNA desorption after the pH of the running buffer was increased to 9 and washing with PB. The course of applied solutions is indicated by arrows and described above the plot. PB stands for washing the SPR chip with PB.

the SAM moieties and the conversion of the cationic character of the SAM to a zwitterionic state. Similarly, the presence of a functional monolayer on the QCM chip was further confirmed electrochemically using CV in PB containing ferricyanide; after the incubation of the CBE-modified QCM chip with 0.1 M NaOH, a large separation of CV peaks was observed as opposed to the same modified QCM chips without NaOH treatment (data not shown), in accordance with the experiments of the AuE. It should be noted that all CVs were performed at a 100 mV s−1 scan rate where the ferri-/ ferrocyanide redox pair is known to undergo a reversible redox transformation without a kinetic limitation.42,43 Hence, the limitations observed can be assigned only to surface modifications. In the next step, the electrochemical detection of DNA molecules adsorbed on AuE-CBE surfaces was performed. It was found that the ferricyanide peak separation increased by only 6 ± 1% after the incubation of AuE-CBE with the DNA solution. Electrochemical impedance spectroscopy was also applied to investigate the modification of electrode surfaces. After DNA adsorption on the AuE-CBE, AuE-CBE-DNA was formed and a 23.5 ± 10% increase in charge-transfer resistance

plain gold surface, it can be suggested that the carboxybetaine moiety is also responsible for the relatively low density of CBE. Furthermore, a surface coverage of 0.72 molecules per nm2 was reported for a spiropyran moiety attached to a lipoic acid similar to CBE,41 which justifies the conclusion that we achieved a dense monolayer. Therefore, a positively charged SAM should be formed on the AuE surface during the incubation time. To investigate the stability of SAM and DNA immobilization, electrochemical methods were employed using ferricyanide as a negatively charged electrochemical probe (Figure 3). It can be seen that even after long-term incubation in pH 7.4 phosphate buffer neither anodic nor cathodic ferricyanide peaks exhibited significant changes, suggesting the stability of the SAM. The stability is also retained at low pH, after 5 min of incubation in 100 mM H2SO4 (data not shown). Cyclic voltammograms of the SAM of CBE on gold electrodes (AuE-CBE) before and after 5 min of incubation in 0.1 M NaOH were performed, and the results are shown in Figure 3a. Conversely, a diminishing of ferricyanide CV peaks occurred upon incubation of AuE-CBE in NaOH solution, with a negative surface charge resulting from the hydrolysis of CBE in 6661


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Figure 5. High-resolution XPS spectra with binding energies for C, C 1s (a); N, N 1s, (b); P, P 2p (c); and S, S 2p (d).

(RCT) was observed, as is seen in Figure 3b. This finding is in accordance with the expectation that negatively charged DNA will undergo partial complexation with the positive charge of the SAM layer and impart to the surface a certain amount of negative charge, which will slow the ferricyanide redox transformation. It should be noted, however, that not the entire surface is covered by the DNA charge shield because even after the adsorption, the measured RCT was approximately 1400 Ω, suggesting that ferricyanide still undergoes the redox transformation depicted in Figure 3b. The same can be suggested from the obvious peaks of the CV voltammograms. On the other hand, positively charged thionine is known to interact well with DNA. It can be anticipated that this probe will be most likely repulsed by a SAM containing deprotonated quaternary ammonium moieties but can be complexed with DNA molecules attached to the surface. The Thi presence on the surface can alter the overall surface charge that, in turn, can be detected using a ferricyanide probe, unrestrained by the positively charged SAM of CBE. This system (Scheme S1) can thus be employed as a signal amplifier for the detection of DNA molecules scarcely spread on the otherwise positively charged surface. Indeed, a ferricyanide peak separation of 117.1 ± 3.3 mV was observed for the AuE-CBE-DNA electrodes incubated with thionine, which was lower than 158 ± 15 mV recorded at electrodes without DNA, where thionine will dock only onto flaws in the thiol SAM. The relative difference here (i.e., 35%) was even higher than the difference observed in the EIS study. Similarly, anodic peak heights decreased from 13.4 ± 0.5 mA detected for AuE-CBE-DNA-Thi to 9.6 ± 1.3 mA for AuECBE-Thi (27% decrease). Illustrative anodic parts of the CVs

for these electrodes are shown in Figure S5. These results have confirmed a hypothesis that a DNA-covered SAM-modified surface will gain its thionine surface coverage that, on the other side, could facilitate negative probe (ferricyanide) redox transformation. Moreover, XPS analysis of the SAM-modified gold surfaces showed evidence of CBE SAM formation and subsequent hydrolysis, as discussed below. To obtain quantitative information regarding DNA adsorption, the quartz crystal microbalance (QCM) method was employed and is depicted in Figure 4a. In a typical experiment, while a QCM cell with 20 μM DNA in 10 mM PB at pH 7.4 was being washed, a frequency change of 72.8 ± 14.9 Hz was observed, corresponding to 66.1 ± 13.5 ng of DNA per square cm of chip. This value is close to 70 and 80 ng cm−2 for ∼4000 and 2000 bp DNA, respectively, adsorbed on a zwitterionic lipid bilayer from a solution containing Na+ ions.44 Previously, 120 ng·cm−2 was observed for the adsorption of an RNA building block onto a cationic lipid bilayer,45 and approximately 700 ng·cm−2 was reported for 80-bp-long tRNA adsorption onto a liquid-crystalline, zwitterionic bilayer. Taking into account the geometric area of the chip and the average number of adsorbed CBE molecules, it can be estimated that 1 molecule of DNA adsorbs an average of 168 molecules of CBE. From the cited results, it is also obvious that, in the case of deposition on a planar surface, the smaller the nucleic acid fragments, the higher the surface density that can be achieved. It should be also noted that the DNA adsorption was a rather unexpectedly slow process and the mentioned values were obtained from the 6662


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Figure 6. SEM micrographs of a surface with a SAM of CBE after incubation with AuNPs (a) and after hydrolysis (b). The scale bars are 500 nm.

of DNA and the ammonium groups of CBE, the contact angle decreased to 58° ± 3 as a result of an increase in roughness and changes in the chemical composition of the surface because of adsorbed DNA. After hydrolysis and desorption, the contact angle changed to 53° ± 2. This was higher compared to the linear thiol derivative with a contact angle of 34°33 as a result of the lower surface density of CBE, as previously discussed. The measured value, however, is within the range of contact angles of 33−53°, as achieved for surfaces modified with poly(carboxybetaine methacrylamides).47,48 Furthermore, the formation of SAM from CBE, the modification of DNA, and subsequent hydrolysis were also confirmed by XPS measurements. XPS was performed on gold chips with SAMs formed by CBE after treatment with DNA and after hydrolysis with NaOH, and the results are shown in Figures S7−S9, indicating the expected elements. Highresolution spectra for C 1s, N 1s, P 2p, and S 2p are shown in Figure 5 to distinguish the chemical shifts due to the presence of functionalities. The expected elements for SAM formation were observed, and the C 1s spectrum (Figure 5a) indicates the presence of C−C, C−N, C−S, C−O, and CO based on the signals with binding energies of 284.6, 286.5, 286.5, 287.1, and 288.6 eV, respectively. In the N 1s spectrum, two peaks at 399.3 and 402.2 eV for amidic and quaternary ammonium functionalities, respectively, are present. After DNA modification, peaks in the C 1s spectrum related to CO and CN (288.6 eV) increased in intensity as a result of the chemical bonds in the DNA structure. More importantly, DNA immobilization was also demonstrated by a new peak in the N 1s spectrum (Figure 5b) for −NC− at 400.2 eV due to DNA bases and by the presence of a peak in the P 2p spectrum at 134.2 eV (Figure 5c) because DNA consists of only phosphoester groups from the deoxyribose backbone.49,50 After the hydrolysis of CBE, the C 1s spectrum confirmed the loss of the ethyl group due to the decreased signals for C− C and C−O compared to the CO signal at 288.6 eV. In all S 2p spectra (Figure 5d), typical double signals could be ascribed to the surface interaction of sulfur. No peak is present above 164 eV, suggesting that unbounded derivatives of CBE are not present in the SAMs.51 Hence, the XPS results confirmed the formation of the SAM from CBE, the successful immobilization of DNA molecules, and the pH-triggered transformation of CBE to zwitterionic carboxybetaine species.

sensograms only after stabilization of the signal, which typically took 30−40 min. To support the above-mentioned achievements, an SPR experiment was performed on a gold chip, and the course of SAM formation, the immobilization of DNA and its subsequent release, and hydrolysis after pH exchange are shown in Figure 4b. The experiment was divided into several stages based on the solution applied, and each stage consisted of applying a certain solution for association and a buffer solution for dissociation. The course of this process is shown in Figure 4b. In the first stage, the results suggested unambiguously that CBE formed a SAM monolayer from aqueous solution on the gold surface. The SAM was stable in PBS solution (stage I). Subsequently, in stage II, the adsorption of DNA molecules to the surface occurred. Many DNA molecules were observed to attach to the surface in the first 30 s, as was also shown in the QCM experiment, and further adsorption was restricted because of the bulkiness and rigidity of DNA chains. Furthermore, in the last stage, stage III, after an increase in the pH of the running buffer to 9, DNA molecules dissociated from the surface, as is the expected result of the hydrolysis of CBE esteric moieties and the formation of zwitterionic moieties with an overall neutral charge that repels the negatively charged DNA molecules. Hydrolysis was also verified by the decreased SPR signal after hydrolysis compared to the signal after SAM formation, which can be assigned to the release of ethyl ester groups and counterions. This type of switch from the cationic to zwitterionic form was previously observed for polymer materials with hydrolyzable or photolyzable moieties.26,28,34 It should be noted that even though the SPR signal is correlated to the mass of adsorbed molecules it is not correct to use such a correlation for the quantification of either CBE or DNA molecules because the known correlation is valid only for globular proteins. It can be concluded that SPR confirmed the results of our other experiments; however, only QCM can be used for the quantification of these steps. It should also be noted that DNA molecules are stable even in 0.1 M NaOH solution, which is used for the lysis of cells and the isolation of DNA,46 in addition to the esteric group hydrolysis. The contact angle of the modified gold surfaces was also studied, and representative images are shown in Figure S6. The bare gold surface changed after modification with CBE from 85 ± 3 to 64° ± 1, confirming the modification. After the adsorption of DNA to the surface through electrostatic attractions between the negatively charged phosphate backbone 6663


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Langmuir

Figure 7. Three-dimensional height AFM images (upper) and line profile image (lower) of a surface with a SAM of CBE before (a) and after (b) hydrolysis following incubation with AuNPs.

methodology offers a path for designing 3D systems with AuNP architecture on surfaces for controlled interactions in cellular arrangements,54 biorecognition devices,55 or other plasmonic structures.56 It is well known that zwitterionic monolayers57−60 or polyzwitterionic brushes32,61,62 are suitable candidates for antibiofouling materials. The overall neutral charge of zwitterions and their ability to form a hydration layer prevent nonspecific protein interactions on zwitterionic surfaces after the hydrolysis of CBE. To screen the adsorption properties of a monolayer from a zwitterionic derivative, SPR measurements were performed, and sensorgrams for HSA and IgG are depicted in Figure 8. Adsorption was performed with a 1 mg·

Furthermore, the tunable capability of the SAM derived from CBE to absorb AuNPs was investigated by SEM and AFM (Figures 6 and 7). As a model material, citrate-capped AuNPs were selected because they are hydrophilic with a negative zeta potential of −45.2 mV. In the SEM images, the bright spots represent AuNPs with sizes of approximately 24 nm, as depicted in Figure 6. A dramatic change in the distribution of these AuNPs was observed after the hydrolysis of the SAM of CBE. In the case of the CBE surface, the number of adsorbed AuNPs was calculated from SEM as 1200 ± 200 per μm2 with an overall distribution on the surface as shown in Figure 6a. The positive charge character of the CBE surface with quaternary ammonium moieties was favorable toward AuNP adsorption. AuNPs were distributed over the whole surface but in some areas were present in higher concentrations. This difference in the distribution of AuNPs can be attributed to the higher exposure of the positive charges on curvature on boundaries of Au crystalline domains,52 with better access of AuNP compared to that of flat areas where access can be partially shielded by the ethyl carboxymethyl group on the quaternary nitrogen atom of CBE. In contrast, in the case of the hydrolyzed surface with carboxybetaine moieties, the adsorption of AuNPs on the substrate dramatically weakened with 2 ± 1 particles per μm2 on the substrate, as shown in Figure 6b. AFM analysis also showed the same trend and height of absorbed AuNPs corresponding to the size of single AuNPs. A dense layer of AuNPs was formed on the CBE monolayer with a surface roughness of 6.1 nm (Figure 7a). In the case of the carboxybetaine surface (Figure 7b), i.e., after hydrolysis, almost no adhered AuNPs were observed, with a roughness similar to that of the plain gold surface. The overall neutral charge and zwitterionic character of the hydrolyzed surface prevented the adsorption of AuNPs as a result of repulsive interactions and did not allow the interaction of the negatively charged AuNPs with quaternary ammonium groups. This observation is in agreement with previously reported tailored AuNP adsorption based on counterion switchability of the linear quaternary ammonium groups on SAMs40 or based on CO2-responsive amidinium lipoic-derived SAMs.53 Switchable and controlled adsorption of AuNPs can be realized by the pH trigger, and this

Figure 8. SPR sensorgrams for the adsorption of HSA and IgG to the SAM in the zwitterionic state after hydrolysis. The course of applied solutions is indicated by the dashed line and described above the plot. The proteins were dissolved in PBS at a concentration of 1 mg/mL−1.

mL−1 protein solution, and the mass of adsorbed proteins was determined to be 39.26 and 36.32 ng·cm−2 for HSA and IgG, respectively. These results suggest a reduction of protein adsorption, even though, because of the lower density of zwitterion moieties on the surface, the values are slightly higher in comparison to those of previously reported zwitterionic 6664


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Langmuir linear derivatives58−60 or densely packed polyzwitterionic brushes.32,62

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SUMMARY AND CONCLUSIONS A system with a charged SAM containing a carboxybetaine ester moiety capable of switching to a zwitterionic carboxybetaine state by increasing the pH was studied. A carboxybetaine ester derivative based on lipoic acid was synthesized, and its hydrolysis to a carboxybetaine state was confirmed by FTIR and NMR analyses. The carboxybetaine ester-based SAM allowed for the immobilization of DNA, and the pH-induced and -controlled hydrolysis of esteric moieties led to the release of DNA and the formation of a neutrally charged zwitterionic surface. A dramatic change in the adsorption of AuNPs on the carboxybetaine ester surface before and after hydrolysis was observed, and hence the ability to modulate AuNP adsorption was demonstrated. This approach not only paves the way for the simple delivery of DNA vectors and DNA immobilization with controlled release and transformation to a nontoxic carboxybetaine coating for biomaterial applications but is also transferable to other types of nanoarchitectures, such as gold nanoparticles, quantum dots, and 2D materials. Moreover, this platform offers the opportunity to further develop surface systems for adjustable wettability and for controlled interactions in cellular arrangements,54 biosensing and recognition,55 and other plasmonic structures.56

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b00568. FTIR and NMR spectra of CBE, CV, schematic illustration of thionine experiments, photographs of water droplets on modified surfaces, and XPS surveys (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jan Tkac: 0000-0002-0765-7262 Peter Kasak: 0000-0003-4557-1408 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge Mr. Ahmed Suliman, Gas Processing Center, Qatar University, for carrying out the XPS analysis. This publication was made possible by NPRP grant no. NPRP-6-381-1-078 from the Qatar National Research Fund (a member of the Qatar Foundation). The statements made herein are solely the responsibility of the authors.

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REFERENCES

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pH-Switchable Interaction of a Carboxybetaine Ester-Based SAM with

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