One-Pot Synthesis of a Zwitterionic Small Molecule Bearing Disulfide

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Biological and Environmental Phenomena at the Interface

One-pot synthesis of a zwitterionic small molecule bearing disulfide moiety for anti-biofouling macro and nanoscale gold surfaces Seungjoo Yi, Won Kyu Lee, Ji-Ho Park, Jae-Seung Lee, and Ji-Hun Seo Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01532 • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 15, 2018

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One-pot synthesis of a zwitterionic small molecule bearing disulfide moiety for anti-biofouling macro and nanoscale gold surfaces Seungjoo Yi,†,⊥ Won Kyu Lee,†,⊥ Ji-Ho Park,‡ Jae-Seung Lee,*,† Ji-Hun Seo*,†

†

Department of Materials Science and Engineering, Korea University, 145 Anam-ro,

Seongbuk-gu, Seoul, 02841, Korea ‡

Department of Chemistry, Sogang University, Baekbeom-ro 35, Mapo-gu, Seoul 04107,

Korea

ABSTRACT

The goal of this study is to develop a simple one-pot method for the synthesis of a zwitterionic small molecule bearing disulfide moiety, which can effectively inhibit nonspecific protein adsorption on macroscopic and nanoscopic gold surfaces. To this end, the optimal molecular structure of a pyridine disulfide derivative was explored and a zwitterionic small molecule was successfully synthesized from the tertiary amine residue on the pyridine ring through a one-pot method. The coating conditions of the synthesized zwitterionic molecules on the gold surface were optimized through contact angle measurements, and the strong interactions between the gold surface and the disulfide moiety of the zwitterion small molecule were confirmed by surface plasmon resonance (SPR) analysis and X-ray

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photoelectron spectroscopy. The anti-biofouling properties of the coated gold surface were analyzed by fluorescence microscopic observations after contacting with FITC-labeled bovine serum albumin (BSA) and SPR sensor as contacting with BSA solution. In addition, the effect of zwitterion-coating on the salt stability of and protein adsorption on nanoscopic gold surfaces were examined through a NaCl stability test and BSA adsorption test, respectively. From the obtained results, it was confirmed that the simply synthesized zwitterionic small molecule was effective in inhibiting nonspecific protein adsorption on macroscopic and nanoscopic gold surfaces; further, it enhanced the salt stability of gold nanoparticle surfaces.

INTRODUCTION Gold is widely used in various advanced analytical devices and nanobiotechnology platforms because of its high thermal and electrical conductivity, corrosion resistance, and convenient features for the synthesis of nanoscale particles.1-3 For example, a gold substrate is used as the surface in advanced analytical devices, such as quartz crystal microbalancedissipation (QCM-D) and surface plasmon resonance (SPR) sensors, and gold nanoparticles synthesized in the form of nanoscale spheres, discs, and rods , etc., are widely used in nanotherapeutics, such as drug delivery systems and bio-imaging. However, in spite of the versatile uses of gold in many analytical and nano-therapeutic fields, when it is used in biological environments, the problem of biofouling, induced by nonspecific protein adsorption, is always encountered.4, 5 Nonspecific protein adsorption leads to the irreversible adsorption of a large amount of proteins on the surface by nonspecific ionic or hydrophobic interactions between proteins and the material surface in a biological environment. Surface proteins are the main cause of reduced signal-to-noise ratio in advanced analytical devices, resulting in decreased sensitivity.4 Moreover, nonspecific


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protein adsorption on gold nanoparticles can initiate particle aggregation, which is the main cause of coarsening and absorption wavelength changes.6 For this reason, many researchers have been trying to effectively impart anti-biofouling properties to gold surfaces, which is a very important issue in the field of advanced analysis and nanobiotechnology. Proteins have complex surface properties due to the presence of both negative and positive charges as well as hydrophobic and hydrophilic functional groups. In order to effectively repel the adsorption of proteins, the surface of a material should follow the Whiteside rule; it should be polar, electrically neutral, and not be a hydrogen bond donor but a hydrogen bond acceptor.7, 8 Poly(ethylene glycol) (PEG) and zwitterionic polymers are known to satisfy these requirements. For this reason, many functional coating materials containing PEG or zwitterionic polymers have been developed to impart anti-biofouling properties to gold surfaces. In order to effectively immobilize PEG on gold surfaces, thiolated terminal groups should be introduced into the PEG structure. However, in this case, complicated synthesis processes are required; furthermore, the commercially available materials are also very expensive because of the complicated synthesis and ease of oxidation of the thiolated groups.6, 9 Zwitterionic polymers are relatively free of these limitations. Because zwitterionic polymers can be synthesized with a variety of molecular structures with different chemical groups, it has been suggested that zwitterionic polymers can replace thiolated PEG as antibiofouling coating materials.10-13 A representative coating method is surface-initiated polymerization using atom transfer radical polymerization (ATRP) or reversible additionfragmentation chain transfer (RAFT) polymerization, in which a thiolated initiator or a chain transfer agent is pre-coated on a gold surface and a zwitterionic polymer is grown from these sites. Although this method shows an outstanding anti-biofouling performance, there are several disadvantages as well. The coating process, which includes polymerization and


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purification, is still very complicated and in situ coating is impossible in many applications.11, 12

Furthermore, in the case of gold nanoparticles whose physical properties are sensitive to

particle size, it is difficult to control the roughness and thickness of the grafted zwitterionic polymers.14 In order to overcome these problems, synthesizing zwitterionic small molecules which can be applied on both macroscopic and nanoscale gold surfaces seems to be an attractive proposition. For example, a sulfobetaine small molecule bearing a cyclic disulfide group, which can strongly interact with gold surfaces, was synthesized and it effectively inhibited the aggregation of gold nanoparticles in biological environments.15 In addition, thiolated zwitterionic small molecules, such as phosphorylcholine, carboxybetaine, and sulfobetaine, have been synthesized by various methods, including Michael type addition, and it has been reported that they exhibit notable anti-biofouling properties.16-19 Although these zwitterionic small molecules could successfully induce anti-biofouling properties on macroscopic or nanoscopic gold surfaces, they can only be synthesized in multi-step or harsh process conditions, which is an impediment to mass production and cost-effectiveness. Moreover, free thiol groups are easily oxidized and need to be protected before the introduction of the zwitterionic functional groups, which is the cause of the complicated synthetic process. In contrast, disulfide derivatives are relatively free from these problems even though they show similar affinity to gold surface with free thiol groups.20 Thus, there is no worry about the oxidation problem and disulfide derivatives can stably maintain their structure in various synthetic process. In this study, therefore, a simple one-pot method was developed for the synthesis of zwitterionic small molecules which can be easily immobilized on gold surfaces. The ultimate purpose of this study is to develop a versatile zwitterionic coating agent applicable not only on macroscopic but also on nanoscopic gold surfaces to induce anti-biofouling properties through a very simple and effective synthetic process.


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EXPERIMENTAL SECTION Materials. 4,4’-Dipyridyl disulfide (p-DPDS), 2,2’-dipyridyl disulfide (o-DPDS), and 1,3propanesultone (PS) were purchased from TCI (Tokyo, Japan). Dulbecco’s phosphatebuffered saline (DPBS, without calcium chloride and magnesium chloride) was purchased from WELGENE Inc. (Daegu, Korea). [2-(Methacryloyloxy)ethyl]dimethyl-(3sulfopropyl)ammonium hydroxide (SBMA), albumin-fluorescein isothiocyanate conjugate (FITC-BSA), gold(III) chloride trihydrate (99.9%, Cat. # 520918), sodium citrate tribasic dihydrate (99.0%, Cat. # S4641), Tween 20 (Cat. # P7949), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All the organic solvents were purchased from Samchun Chemical (Gyeonggi-Do, Korea). Synthesis of Zwitterionic Small Molecule (p-DPDSPS). PS was dissolved in 2 mL of acetonitrile to form a 2 M solution and p-DPDS, which was dissolved in 3 mL of acetonitrile (0.33 M), was slowly added through a dropping funnel to this solution. The reaction was allowed to proceed for 72 h until yellowish precipitates covered the whole wall of the reaction vial.21 The precipitate was washed with an excess of acetone to remove residual reactants. The structure of p-DPDSPS was characterized by 1H-nuclear magnetic resonance (NMR) spectroscopy (MERCURY 400 MHz, Varian, CA, USA) and ultra-high-performance liquid chromatography-quadrupole time-of-flight (UPLC-Q/TOF) mass spectrometry (SYNAPT G2-Si, Waters, MA, USA) (see Figure S1, S2 in the supporting information). The reaction yield was ~72%. 1H-NMR (D2O) δ = 8.7–8.8 (d, 2H, pyridyl), 8.1–8.2 (d, 2H, pyridyl), 4.7 (t, 2H, N+CH2CH2), 2.9–3.0 (t, 2H, CH2CH2CH2), 2.4 (m, 2H, CH2CH2S).21 UV-visible (UV-vis) Monitoring of the Reaction of PS with p-DPDS and o-DPDS. The reaction between o-DPDS and PS was performed under the same conditions as those used for the synthesis of p-DPDSPS. A UV-vis spectrophotometer (Agilent 8453, Agilent, CA, USA)


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was used to monitor the intensity of the absorption peaks of the unreacted pyridine rings of DPDS. Aliquots (100 µL) of the reaction mixture were extracted at pre-determined reaction times (0, 0.5, 1, 3, 12, 24, 48, and 72 h) and diluted with 0.9 mL of acetonitrile four times before analysis. The path length of the quartz cell was 10 mm. UV-vis spectra were obtained in the wavelength range of 200–1000 nm. Preparation of p-DPDSPS-Coated Gold Substrates. The gold substrates were prepared using an e-beam evaporator. Initially, a 10 nm-thick Ti layer was deposited on the glass substrate (10 mm × 10 mm) at a rate of 0.1 nm/s. Later, a 100-nm gold (4N) layer was deposited on the titanium layer at a rate of 0.3 nm/s. The obtained gold substrates were sequentially washed with excess ethanol, acetone, and deionized (DI) water and dried with argon gas. p-DPDSPS was dissolved in DI water at 0.05, 0.1, 0.5, and 1 mg/mL concentrations and gold substrates were immersed in these solutions for 5 min.22 In the case of the 0.1 mg/mL solution, the coating time was varied from 1 min to 10 min to find the optimal coating time. In addition, p-DPDS was also dissolved in ethanol at a concentration of 0.047 mg/mL and a gold substrate was immersed in the solution for 5 min. After the coating process, all the substrates were sequentially washed with ethanol, acetone, and DI water and dried with argon gas. Measurement of Water Contact Angle. Sessile drop contact angles were measured to optimize the coating conditions, i.e., the concentration of coating solution and coating time. Further, the hydrophilicity of bare Au and p-DPDS- and p-DPDSPS-coated substrates were compared. The volume of the sessile drop was 3 µL and contact angles at three different positions were measured for each sample. At least 3 samples were tested for each coating condition. The contact angles were measured by the tangential method using a goniometer (Phoenix 150; Surface Electro Optics, Gyeonggi-Do, Korea).


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X-ray Photoelectron Spectroscopy (XPS) Measurements. Three substrates were prepared – bare Au and p-DPDS- and p-DPDSPS-coated gold substrates. The presence of sulfur (S), nitrogen (N), and negatively charged sulfonyl groups (SO3–) on the surfaces of the gold substrates were confirmed by XPS (X-tool; ULVAC-PHI, Kanagawa, Japan) measurement. An X-ray detector was located at an angle of 45° and Al Kα radiation was used as the excitation source at 24.1 W. Synthesis of Gold Nanoparticles (AuNPs). In an Erlenmeyer flask, 49 mL of ultrapure water was heated up to its boiling point. While stirring, 1 mL of gold chloride solution (12.7 mM) was added to the boiling water and 0.94 mL of trisodium citrate solution (38.8 mM) was rapidly injected. After 5 min, the color of the solution changed from pale yellow to red, indicating that 15-nm AuNPs were synthesized. The AuNP solution was cooled to 25 °C with stirring and stored at 4 °C until use. Functionalization of Gold Nanoparticles (AuNPs) with p-DPDSPS. An aqueous p-DPDSPS solution was combined with pre-synthesized AuNPs (final [AuNPs] = 1 nM, [p-DPDSPS] = 0, 5, and 50 µM) and incubated at room temperature for 2 min for the conjugation of pDPDSPS on the AuNP surface. Unconjugated p-DPDSPS was removed by centrifugation (13000 rpm for 20 min). The supernatant was removed and p-DPDSPS-AuNPs were redispersed in an aqueous solution of Tween 20 (0.01%). The surface density of the immobilized zwitterionic molecules on the AuNP was calculated based on the method described in the literature.23 In brief, we combined the p-DPDSPSAuNPs with dithiothreitol (DTT) at a high concentration (20 mM), and incubated the mixture at 80 oC for 1 hour to displace p-DPDSPS on the AuNPs by DTT. The released p-DPDSPS was quantitatively analyzed using UV-vis spectroscopy by obtaining the maximum absorbance at 290 nm (extinction coefficient of p-DPDSPS at 290 nm is 1.7 × 104 cm-1 M-1 based on the spectrum in Figure 1A).


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NaCl Stability Test. The optical properties of p-DPDSPS-AuNPs were tested after injecting with NaCl (final [NaCl] = 0, 0.05, 0.10, 0.15, 0.20, and 0.25 M) using a UV-vis spectrophotometer (Agilent 8453, Agilent, CA, USA). Protein Adsorption Test FITC-BSA adsorption test. A bare gold substrate and substrates coated with p-DPDSPS (5 min, 0.1 mg/mL) and p-DPDS (5 min, 0.047 mg/mL) were prepared according to the protocol described for the preparation of p-DPDSPS-coated gold substrates. FITC-BSA was dissolved in DPBS at a concentration of 2 mg/mL. The substrates were then immersed in this solution and incubated for 30 min at 37 °C followed by 10 s of washing in 100 mL of DI water (stirred at 120 rpm). The adsorbed proteins were observed using a fluorescence microscope (Eclipse, Nikon, Tokyo, Japan). The mean intensity of the substrate surface was calculated by the NIS-Elements Basic Research (BR) software. Protein adsorption measurement using a surface plasmon resonance (SPR) sensor. Initially, the coating process of p-DPDSPS in DI water (0.1 mg/mL) was monitored in real time with a two-channeled SPR sensor (Reichert2SPR, Reichert Inc., NY, USA). The pDPDSPS solution flowed only into the left channel at a flow rate of 15 µL/min for 5 min. Later, DI water was allowed to flow into the left channel for a time period sufficient enough to wash out any unbound residual molecules. After washing, the water was replaced with DPBS. It was allowed to flow through the whole channel for a time period enough to stabilize the SPR sensor. When the sensor was stabilized, BSA in DPBS solution (1 mg/mL) flowed into the both channels at a flow rate of 20 µL/min for 10 min. Then, the solution was replaced with DPBS to wash out unbound BSA. SBMA aqueous solution (0.06 mg/mL) was used as the negative control to compare the degree of coating of p-DPDSPS on the gold substrates.


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BSA adsorption on p-DPDSPS-functionalized gold nanoparticles (p-DPDSPS-AuNP). AuNPs conjugated with p-DPDSPS or citrate anions were dispersed in BSA (final [AuNPs] = 1 nM, [p-DPDSPS] = 0 and 50 µM, and [BSA] = 15 nM) and analyzed by dynamic light scattering (DLS) and zeta potential measurement using a Zetasizer Nano ZS90 (Malvern Panalytical, Malvern, UK) instrument.

Scheme 1. Schematic explanation of the synthesis of p-DPDSPS.


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Figure 1. (A) UV-vis spectrum of p-DPDSPS, which cannot be observed in the spectrum of the reaction mixture due to natural precipitation. (B) UV-vis monitoring of the reaction between p-DPDS and PS. There is only one absorption band of p-DPDS (242 nm), which decreases with increasing reaction time. (C) Variation in the concentration of p-DPDS, calculated from the absorbance at 242 nm. (D) UV-vis monitoring of the reaction between oDPDS and PS. Absorption bands of o-DPDS alone (235 nm and 279 nm) could be observed; further, intensity changes at these wavelengths are negligible. (E) Changes in the concentration of o-DPDS, calculated based on the absorption at 279 nm.


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RESULTS AND DISCUSSION Optimizing the Synthetic Process of p-DPDSPS. The purpose of the present study is to develop a one-pot process for the synthesis of a zwitterionic small molecule capable of binding onto gold surfaces to provide anti-biofouling properties. To this end, pyridine derivatives containing disulfide moiety were chosen as the starting materials because the tertiary amine residue in pyridine derivatives can induce a nucleophilic ring-opening attack on 1,3-propanesultone to form zwitterionic sulfobetaine groups (Scheme 1).21 Moreover, the disulfide residue is known to be strongly bound to the gold surface, but does not take part in the ring-opening reaction as a nucleophile species.24 In order to find out which is the better molecular structure for pyridine disulfide derivatives, the reaction of PS with p-DPDS and oDPDS was monitored by measuring the consumption of pyridine disulfide derivatives by UVvis spectrophotometry. The characteristic absorption peaks of unreacted p-DPDS and oDPDS appeared at 242 nm and 279 nm, respectively, while the absorption peak of PS did not appear below 360 nm.25 In addition, pyridine disulfide derivatives were precipitated from the reaction mixture when zwitterionic sulfobetaine was synthesized (Figure 1A). Therefore, the degree of conversion of p-DPDS or o-DPDS to zwitterionic disulfide derivatives could be indirectly monitored by analyzing the changes in the heights of the characteristic peaks of pDPDS and o-DPDS. Figure 1B shows the UV-vis spectra of p-DPDS at different reaction times. It can be clearly seen that the characteristic absorption peak at 242 nm gradually decreased with an increase in the reaction time. The concentration of residual p-DPDS was calculated based on Beer-Lambert’s law and was plotted as a function of the reaction time. As shown in Figure 1C, the concentration of residual p-DPDS continuously decreased as the reaction proceeded and only 7% remained when the reaction time was over 72 h. In contrast, the UV-vis spectra and resulting residual concentrations of o-DPDS did not significantly


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change with variation in the reaction time, as shown in Figure 1D and 1E. This result indicates that the zwitterionization of pyridine disulfide derivatives proceeded very well from p-DPDS over a reaction period of 72 h at room temperature; however, the reaction was not initiated from o-DPDS. In many organic reactions, it is known that there exist differences in the reactivity of para- and ortho-isomer structures of aromatic hydrocarbons.26, 27 In most cases, this regioselectivity originates from steric hindrance or the stability of the resonance structures of the aromatic hydrocarbon. In this study, steric hindrance of the dimerized pyridine derivatives is thought to be the cause of the difference in reactivity. The tertiary amine group immediately adjacent to the disulfide linkage in o-DPDS could be hindered by the aromatic hydrocarbon from approaching the PS molecule for the ring-opening reaction. In contrast, the tertiary amine group in p-DPDS is completely open and can interact with the approaching PS molecules to initiate the ring-opening reaction. For this reason, we chose pDPDS as the reactant instead of o-DPDS and the reaction was carried out for 72 h to develop a high-yield synthesis process. The synthesized zwitterionic disulfide small molecule (pDPDSPS) was naturally precipitated from the reaction mixture and thus it could be easily collected without further purification processes. The molecular structure and exact mass of the synthesized p-DPDSPS were successfully confirmed by 1H-NMR, UV-vis spectrophotometry, and mass spectrometry. These results indicate that the synthesis process proposed in this study is a well optimized one-pot process for obtaining a highly pure zwitterionic small molecule bearing the disulfide group.


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Figure 2. Real-time coating process of p-DPDSPS (0.215 mM) and SBMA (0.215 mM). The coating solutions always flowed into the left (L) channel, while the blank solution flowed into the right (R) channel. The red/black graphs and brown/grey graphs were measured on a single gold substrate. SBMA, which does not have disulfide moiety, was used as the negative control.


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Figure 3. Contact angles of the gold substrates coated with (A) different concentrations of pDPDSPS solution for 5 min, (B) p-DPDSPS solution at a concentration of 0.1 mg/mL for different time periods, and (C) p-DPDSPS (0.215 mM) and p-DPDS (0.215 mM) for 5 min. Coating of the Zwitterionic Small Molecule on Gold Substrate. Disulfide derivatives are well known to contain chemical groups that can be used to coat gold surfaces.24 Therefore, it is anticipated that the p-DPDSPS molecules can be easily immobilized on the surface of gold by simply immersing a gold substrate in a p-DPDSPS solution. In order to confirm the strong interactions between the disulfide moiety and gold surface, the coating process was monitored by SPR measurement. The p-DPDSPS aqueous solution (0.1 mg/mL, 0.215 mM) was allowed to flow over the gold surface and the obtained SPR response was compared with the SPR responses obtained from different solutions, including pure water and an aqueous solution of the sulfobetaine monomer (SBMA) without disulfide groups. As shown in Figure 2, p-DPDSPS molecules were strongly bound to the gold surface; they exhibited an intense SPR signal and the signal remained intact even after washing with fresh water, which indicated that p-DPDSPS molecules were irreversibly adsorbed on the gold substrate. In contrast, the SBMA solution induced only a small amount of irreversible adsorption on the surface of the gold substrate. This result indicates that the disulfide group is responsible for the strong binding of p-DPDSPS molecules on gold surfaces rather than the sulfobetaine moiety. In order to optimize the coating conditions, the coating process was conducted at different immersion times and concentrations of the p-DPDSPS solution; changes in the water contact angle were monitored to evaluate the conditions at which the contact angle was minimum. Figure 3A shows the results of water contact angle measurement on surfaces coated with pDPDSPS solutions of different concentrations. The water contact angle was found to be


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minimum when the concentration was lower than 0.1 mg/mL. The reason for the increase in contact angles on gold surfaces coated with higher-concentration solutions is thought to be due to the increased electrostatic interaction between sulfobetaine zwitterionic molecules in the solution. As the amount of solute increases, the charged zwitterionic groups at the edges of the p-DPDSPS molecules begin to interact with opposite charges, which inhibits the pDPDSPS molecules from densely adsorbing on the gold surface.18 This might be the same phenomenon occurring in the case of SBMA, which is known to exhibit concentrationdependent hydrophilicity. If the concentration of the sulfobetaine moiety is high, its zwitterion groups are known to electrostatically interact with each other and the zwitterionic properties, for e.g., hydrophilicity, are reduced.28, 29 Therefore, more dilute coating solutions (0.1 and 0.05 mg/mL) might result in more homogeneous solute states and uniformly coat pDPDSPS on the gold substrate surface. Figure 3B shows the changes in the contact angles of gold surfaces coated for different time periods using 0.1 mg/mL p-DPDSPS. The contact angle of the coated surfaces was high when the coating time was only 1 min. However, when the coating time was 5 min or more, the contact angle reached and saturated at a minimum value of around 20°. In order to confirm whether the hydrophilicity of the p-DPDSPS-coated surface was induced by the zwitterionic sulfobetaine group or not, the surface was coated with p-DPDS over 5 min and its contact angle was measured (Figure 3C). There was no significant decrease in the contact angle of the p-DPDS-coated gold surface, which indicates that zwitterionic sulfobetaine is the cause of the hydrophilicity of p-DPDSPS-coated gold surfaces. The successful coating of p-DPDSPS on gold substrates was confirmed by XPS analysis. Figure 4 shows the XPS results of the three substrates: p-DPDS-coated, p-DPDSPS-coated, and bare Au substrates. Both p-DPDS- and p-DPDSPS-coated substrates showed N1s peaks around 398 eV, which originated from the nitrogen of the pyridine ring.30 The slight increase


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in the binding energy on the p-DPDSPS surface is due to the change of tertiary amine to quaternary amine by forming the zwitterionic sulfobetain group.31 In addition, strong S2p peaks were observed for both substrates at 161 eV, owing to the covalent bonding between sulfur and the Au substrate.30 Another characteristic S2p peak was observed in the spectrum of the p-DPDSPS-coated substrate at 167 eV owing to the presence of the zwitterionic sulfobetaine group.31 This result is a strong evidence of the presence of zwitterionic sulfobetaine groups on the p-DPDSPS-coated surface.

Figure 4. XPS analysis of N1s and S2p on three different substrates – bare Au and p-DPDS (0.215 mM)- and p-DPDSPS (0.215 mM)-coated gold substrates.


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Figure 5. Fluorescence, optical, and merged microscopy images of (A) bare Au, p-DPDS (0.215 mM)-coated, and p-DPDSPS (0.215 mM) coated gold substrates after contacting with the FITC-BSA solution. The scale bar is 100 µm. (B) Relative fluorescence intensity of each substrate, as calculated using the NIS-elements BR software.

Figure 6. BSA adsorption analysis using a SPR sensor. The values were measured right after coating with p-DPDSPS (0.215 mM), as mentioned in Figure 2. Protein Adsorption Resistance of the Zwitterionic Small Molecule-Coated Gold Substrates. The amount of protein adsorbed on the gold substrates was evaluated by immersing the substrates in the FITC-BSA solution. The intensity of fluorescence from the surface was


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measured by fluorescence microscopy and analyzed by the NIS-Elements BR software, as shown in Figure 5A and 5B. As shown in Figure 5A, the p-DPDS-coated substrate exhibited the highest fluorescence intensity. Because protein adsorption occurs mainly due to hydrophobic interactions, the increased hydrophobicity of the p-DPDS-coated surface is thought to induce high protein adsorption. According to previous studies, strong hydrophobic interactions between pyridine rings and proteins occur when the pH is around 7 to 8.32 In this study, a similar result was obtained on the p-DPDS-coated surface. In contrast, apparently small amounts of proteins were adsorbed on the p-DPDSPS-coated surface compared to the p-DPDS-coated and bare Au substrates. The quantitative results of the fluorescence intensity measurements (Figure 5B) show that the amount of protein adsorption on p-DPDSPS decreased almost to the background level. Because sulfobetaine is a zwitterionic chemical group with excellent anti-biofouling properties, this result proves the anti-biofouling performance of the single sulfobetaine molecule on the surface of the gold substrate.10, 33 In order to confirm the limitations of using p-DPDSPS molecule to prevent protein adsorption, its anti-biofouling property was analyzed at the molecular level by SPR measurement. Figure 6 shows the results of the BSA adsorption test. When 1 mg/mL BSA solution flowed over the bare gold surface, a large SPR signal was detected, which indicates that significant nonspecific protein adsorption occurred on the surface. In contrast, the SPR signal reduced to 51% when the p-DPDSPS solution was allowed to flow over the substrate before contacting with the BSA solution. Although an obvious decrease in protein adsorption was observed on the p-DPDSPS-coated surface, the amount of protein adsorbed did not significantly decreased during SPR measurements compared to the fluorescence data in Figure 5. There are two possible reasons for this result. The first possible reason is the low density of the p-DPDSPS molecules on the gold surface. In order to obtain outstanding anti-biofouling performance using small zwitterionic molecules, the formation of a self-assembled monolayer (SAM)


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structure is essential.16 Because the pyridine disulfide-induced zwitterionic molecule has a short aromatic ring under the outermost sulfobetaine group, inter-chain stabilization may not be strong enough as a long alkyl chain to develop a densely coated structure.34 The second possible reason is the nature of the sulfobetaine group of p-DPDSPS itself. Sulfobetaine groups are obviously effective in preventing non-specific protein adsorption on material surfaces. However, their performance is slightly lower than that of other types of zwitterionic molecules, such as carboxybetaine or phosphorylcholine groups, due to its relatively high degree of self-association.35-38 In this study, it was confirmed that the p-DPDSPS molecules are very effective in preventing non-specific protein adsorption at the visible level as confirmed by fluorescence labeling, while their performance is limited at the molecular level, as confirmed by SPR measurement. Stability and Anti-Biofouling Properties of p-DPDSPS Conjugated Gold Nanoparticles. Although the anti-biofouling effect of p-DPDSPS was clearly demonstrated on twodimensional surfaces, it is still a question if nanoparticles can be protected by p-DPDSPS from protein adsorption. While two dimensional flat surfaces and curved nanoparticle surfaces have the surface chemistry of gold in common, there are several issues to be addressed, including (1) nanoparticle stability after ligand conjugation,39 (2) ligand density on the nanoparticle surface,40, 41 and (3) the resultant anti-biofouling effect against proteins.42 We first examined how to control the density of p-DPDSPS on AuNP surfaces by adjusting the conditions under which p-DPDSPS and AuNPs were conjugated. In a typical conjugation reaction of nanoparticles and ligands, the surface density of the ligands is known to increase as the ligand concentration increases because chemical equilibrium shifts according to Le Chatelier’s principle.23 To prepare AuNPs with different surface coverage extents using pDPDSPS (p-DPDSPS-AuNPs), they were conjugated with p-DPDSPS at concentrations of 5 and 50 µM. In addition, as-synthesized AuNPs coated with citrate anions (Cit-AuNPs) were


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evaluated as the control. After conjugation with p-DPDSPS, we observed that the pDPDSPS-AuNPs were well-dispersed without aggregation as demonstrated by their UV-vis spectra (see Supporting Information, Figure S3), which indicates that p-DPDSPS can play a role in stabilizing AuNPs. We further increased the ionic strength of the solutions containing Cit-AuNPs or 5/50 µM p-DPDSPS-AuNPs and observed their stability by monitoring their extinction at 525 nm in the presence of 0 to 0.25 M NaCl (Figure 7). In the case of CitAuNPs, extinction at 525 nm decreased gradually in a linear manner as [NaCl] increased and became almost 0 when [NaCl] = 0.15 M. As citrate anions are weakly bound to AuNPs,43 their poor stability against NaCl was somewhat expected. On the other hand, p-DPDSPSAuNPs, regardless of the conjugation conditions, exhibited excellent stability even at high NaCl concentrations, up to 0.15 M. Moreover, AuNPs conjugated with p-DPDSPS at 50 µM exhibited higher extinction at 525 nm than p-DPDSPS-AuNPs prepared at 5 µM, which demonstrates that the higher p-DPDSPS concentration resulted in a higher surface coverage on the AuNP surface. We calculated the surface density of the immobilized zwitterionic molecules on the AuNP based on the UV-Vis spectroscopy taken after detaching the zwtterionic molecules from the AuNP. The final number of the p-DPDSPS molecules per AuNP was determined to be approximately 2001, and the footprint was calculated to be 0.35 nm2, which seems to be comparable with the literature values of thiolated small organic molecules on AuNPs.44-46 Eventually, at 0.25 M NaCl, the AuNPs completely precipitated owing to their irreversible assembly, regardless of the surface density and type of surface ligands. We further examined the protein anti-biofouling properties of AuNPs densely coated with p-DPDSPS using BSA as the model protein. To evaluate the effectiveness of p-DPDSPS, CitAuNPs were compared under the same experimental conditions.47 After the incubation of pDPDSPS-AuNPs and Cit-AuNPs with BSA for 30 min, their UV-vis spectra were collected;


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the spectra did not exhibit any noticeable changes (Figure 8A). This result indicates that BSA adsorption on the surface of AuNPs, if any, did not lead to irreversible aggregation based on non-specific protein binding. We more precisely monitored the adsorption of BSA on AuNPs as a function of adsorption time using DLS for a time period of 30 min (see Supporting Information, Figure S4). The initial hydrodynamic diameters of both p-DPDSPS-AuNPs and Cit-AuNPs were determined to be approximately 30 nm (Figure 8B). As adsorption proceeded, however, the hydrodynamic diameter of Cit-AuNPs increased up to 44 nm, clearly indicating the surface adsorption of BSA, particularly when the hydrodynamic diameter of BSA (8 nm) is considered.48 In contrast, the hydrodynamic diameter of pDPDSPS-AuNPs stopped increasing after 5 min at 34 nm. Even though zwitterionic pDPDSPS is designed to provide “anti-biofouling” surfaces, the protein adsorption, while very negligible, is still practically inevitable. In Figure 8B, the average increase in hydrodynamic diameter of the p-DPDSPS-AuNPs is less than 5 nm, indicating only one or no BSA molecule (8 nm in diameter) is adsorbed on the surface of the p-DPDSPS-AuNPs. Importantly, any additional protein adsorption after 5 min is strictly prohibited by the surface coating of pDPDSPS, indicating its highly reliable antifouling functions.

Figure 7. Salt-stability of AuNPs conjugated (coated/protected) with p-DPDSPS at different concentrations ([p-DPDSPS] = 0, 5, and 50 µM). The AuNPs are more densely conjugated


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(coated/protected) with p-DPDSPS at a higher concentration as demonstrated by the smaller changes in plasmonic properties, thus indicating their higher stability against NaCl.

Figure 8. (A) UV-vis spectra of AuNPs conjugated (coated/protected) with citrate or pDPDSPS (Cit-AuNP or p-DPDSPS-AuNP) in the presence or absence of BSA. The negligible changes in the spectral properties of the AuNPs indicate that the AuNPs are highly dispersed, regardless of the adsorbed BSA. (B) Changes in the diameters of Cit-AuNPs and p-DPDSPS-AuNPs in the presence of 15 nM BSA as functions of the incubation time. While p-DPDSPS-AuNPs exhibited only a slight increase in diameter, thus indicating the limited adsorption of BSA on p-DPDSPS-AuNPs, protein adsorption on Cit-AuNPs kept increasing under the experimental conditions. (C) Zeta potentials of Cit-AuNPs and p-DPDSPS-AuNPs


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in the presence or absence of BSA. In contrast to Cit-AuNPs, p-DPDSPS-AuNPs exhibited negligible changes in the presence of BSA, indicating the efficient antifouling properties of pDPDSPS on the AuNP surface. In addition, the adsorption of BSA on AuNP surfaces was investigated by measuring the changes in the zeta potential of the particles. Before the adsorption of BSA, Cit-AuNPs exhibited a fairly negative zeta potential (–29.6 mV), owing to the negative charges on the citrate anion. Interestingly, p-DPDSPS-AuNPs also exhibited a similar negative zeta potential (–29.2 mV). In fact, p-DPDSPS has an overall negative charge at pH 7, because most of the sulfonate groups are negatively charged (yet the alkylpyridinium groups are partially neutralized), as observed with other sulfobetaine zwitterions.49 After the incubation of nanoparticles with BSA, the zeta potential of Cit-AuNPs noticeably increased up to –19.6 mV, owing to the adsorbed BSA on the Cit-AuNP surface. On the other hand, p-DPDSPSAuNPs still maintained almost the same negative zeta potential (–31.2 mV), demonstrating that p-DPDSPS-AuNPs are almost intact after incubation with BSA. Both DLS and zeta potential results clearly prove the powerful anti-biofouling properties of p-DPDSPS in the context of curved nanoparticle surfaces, verifying its strong potential as a versatile and universal coating ligand, regardless of the surface texture.

CONCLUSIONS The synthesis of zwitterionic small molecules capable of adhering on gold surfaces generally requires multi-step processes or sophisticated purification processes. In this study, a one-pot process for the synthesis of zwitterionic sulfobetaine small molecules bearing a disulfide moiety without the need for complicated purification techniques is proposed. The synthesized zwitterionic molecule was very effective in inhibiting non-specific protein


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adsorption on the surface of a gold substrate at the macroscopic visible level or nanoscale particle level. Although future work is required to completely inhibit non-specific protein adsorption on gold surfaces at the molecular level, the proposed small molecule can be synthesized using a facile process and it is anticipated that it can be applied on macroscopic gold substrates or nanoscale gold particles to inhibit non-specific protein adsorption.

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Materials characterization with 1H-NMR and mass spectrometry (PDF) Salt stability of p-DPDSPS-AuNPs measured by UV-vis spectrophotometry (PDF) DLS diameter distributions in BSA adsorption test (PDF) AUTHOR INFORMATION Corresponding Author *[email protected] (J.-S. Lee); *[email protected] (J.-H. Seo) ORCID Jae-Seung Lee: 0000-0002-4077-2043 Ji-Hun Seo: 0000-0001-6193-4008 Author Contributions ⊥These

authors contributed equally. All authors have given approval to the final version of

the manuscript.


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Funding Sources This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF, 2015R1C1A1A01054022). Notes The authors declare no competing financial interest. ACKNOWLEDGMENT Mass spectrometry, liquid chromatography, and total ion chromatogram data were obtained using the UPLC-Q/TOF MS device at the Korea Basic Science Institute (KBSI; Seoul, Republic of Korea). SPR data were obtained using the facilities at the Daegu Gyeongbuk Institute of Science and Technology (DGIST). REFERENCES (1) Shan, J.; Tenhu, H. Recent Advances in Polymer Protected Gold Nanoparticles: Synthesis, Properties and Applications. Chem. Commun. 2007, 44, 4580–4598. (2) Sih, B. C.; Wolf, M. O.; Jarvis, D.; Young, J. F. Surface-Plasmon Resonance Sensing of Alcohol with Electrodeposited Polythiophene and Gold Nanoparticle-Oligothiophene Films. J. Appl. Phys. 2005, 98, 114314–114317. (3) Bearinger, J. P.; Terrettaz, S.; Michel, R.; Tirelli, N.; Vogel, H.; Textor, M.; Hubbell, J. A. Chemisorbed Poly(propylene sulphide)-based Copolymers Resist Biomolecular Interactions. Nat. Mater. 2003, 2, 259–264. (4) Jia, X.; Jiang, X.; Liu, R.; Yin, J. Ultrafast Generation of Thick Poly(ether amine) (PEA) Brush on a Gold Surface and its Protein Resistance. Chem. Commun. 2011, 47, 1276–1278. (5) Xiong, Z.; Wang, Y.; Zhu, J.; Li, X.; He, Y.; Qu, J.; Shen, M.; Xia, J.; Shi, X. Dendrimers meet Zwitterions: Development of a Unique Antifouling Nanoplatform for Enhanced Blood Pool, Lymph Node and Tumor CT Imaging. Nanoscale 2017, 9, 12295–12301. (6) Kim, D.; Park, S.; Lee, J. H.; Jeong, Y. Y.; Jon, S. Antibiofouling Polymer-Coated Gold Nanoparticles as a Contrast Agent for In Vivo X-ray Computed Tomography Imaging. J. Am. Chem. Soc. 2007, 129, 7661–7665. (7) Chapman, R. G.; Ostuni, E.; Takayama, S.; Holmlin, R. E.; Yan, L.; Whitesides, G. M. Surveying for Surfaces that Resist the Adsorption of Proteins. J. Am. Chem. Soc. 2000, 122, 8303–8304. (8) Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M. A Survey of Structure-Property Relationships of Surfaces that Resist the Adsorption of Protein. Langmuir 2001, 17, 5605–5620.


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One-Pot Synthesis of a Zwitterionic Small Molecule Bearing Disulfide

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