Mar 27, 2019 - Atomic-resolution Raman spectroscopy. By combining scanning probe microscopy, laser spectroscopy, and surface science, researchers have ...
Mar 27, 2019 - Our results also provide additional evidence of the critical role of DHI in controlling the thickness of PDA coatings, through competitive events ...
May 1, 1984 - Jo Anne K. Bonesteel, Babul Borah, Richard D. Bates Jr. J. Phys. Chem. , 1984, 88 (10), pp 2141â2144. DOI: 10.1021/j150654a040.
May 1, 1990 - Sialic acidresidues are required in cellular receptors for many different mammalian viruses. Sialic acid could have a direct role, being an ...
also expressed the wild-type MRP1 protein and some of the FLAG-tagged mutants in ... results demonstrate that the accessibility of the external FLAG epitope is ...
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Jul 11, 2002 - and to assess the role of the extracellular domains of this transporter, the FLAG epitope tag was introduced into different extracellular loops of ...
... of major drug transporters in the ATP-binding cassette family and solute carrier family .... American Journal of Physiology-Regulatory, Integrative and Comparative ... Portrait of multifaceted transporter, the multidrug resistance-associated ...
The structure of the alkylzincâtetramethylethyl-enediamine (TMEDA) cluster cation 3 has been determined in the gas phase by a combination of tandem mass spectrometry, infrared multiphoton dissociation (IRMPD) spectroscopy, and DFT calculations. Bot
Feb 14, 2011 - The structure of the alkylzincâtetramethylethyl-enediamine (TMEDA) cluster cation 3 has been determined in the gas phase by a combination of tandem mass spectrometry, infrared multiphoton dissociation (IRMPD) spectroscopy, and DFT ca
Article Cite This: Langmuir XXXX, XXX, XXX−XXX
Direct Evidence for the Critical Role of 5,6-Dihydroxyindole in Polydopamine Deposition and Aggregation Qinghua Lyu, Nathanael Hsueh, and Christina L. L. Chai* Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543
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ABSTRACT: The deﬁnitive role of the intermediate 5,6dihydroxyindole (DHI) in the formation of polydopamine (PDA) coatings from aqueous dopamine (DA) has not been clearly elucidated and remains highly controversial. Our foray into this debate as reported in this study agrees with some reported assertions that DHI-based coatings are not synonymous with PDA coatings. Our conclusion arises from a systematic comparison of the components and properties of DHI-based coatings and PDA coatings. In addition, through careful copolymerization studies of DA and DHI, our studies reported herein unequivocally suggest that both DA and DHI are partial building blocks for PDA formation. Our results also provide additional evidence of the critical role of DHI in controlling the thickness of PDA coatings, through competitive events between PDA aggregation in solutions and deposition onto substrates. These ﬁndings highlight the complex interplay between both DHI and uncyclized DA moieties in the formation of adhesive catechol/amine materials.
Dreyer et al. proposed that PDA consists of 5,6-dihydroxyindoline and its quinone forms held together by a combination of charge transfer and π−π stacking interactions (Figure 1E).11 A recent study by Alﬁeri et al. postulated that PDA coatings do not involve any DHI-based oligomers but are comprised of 7(3,4-dihydroxyphenethyl)pyrano[3,4,5-kl]acridine-6,9,10(7H)trione, derived from intermolecular quinone−amine conjugation (Figure 1F), as the use of DHI under alkaline conditions did not yield any coatings.12 Our recent study has also noted that the oxidative polymerization of DHI at pH 8.5 did not give signiﬁcant coatings, but under acidic and neutral conditions DHI coatings can be obtained.14 In this initial investigation, it was also noted that DHI can indeed be formed during the oxidative polymerization of DA under classical conditions (i.e., 10 mM DA, Tris buﬀer, pH 8.5), but the conversion from DA to DHI is a slow process.14 In view of the controversies that surround PDA formation and its structure, this work reports an investigation into the relationship between the putative intermediate DHI and PDA formation. To achieve this aim, the constituents of DHI-based coatings and PDA coatings were ﬁrst characterized using diﬀerent analytical techniques such as X-ray photoelectron spectroscopy (XPS) and matrix-assisted laser desorption/ ionization time-of-ﬂight (MALDI-TOF) mass spectroscopy. Copolymerization studies between DA and DHI were also Received: February 7, 2019 Revised: March 25, 2019 Published: March 27, 2019 A
Figure 1. Proposed PDA structures in reported studies. pixel of the image so that the lateral forces can be greatly minimized, making nondestructive imaging straightforward. All images were processed using the JPK data processing software (Version 6.1.22). X-ray Photoelectron Spectroscopy (XPS) Analysis. XPS measurements were performed on a Kratos AXIS Ultra HAS spectrometer with a monochromatized Al Kα X-ray source (1486.6 eV photons) at a constant dwelling time of 100 ms and pass energy of 40 eV. All binding energies were referenced to the C 1s hydrocarbon peak at 284.6 eV. ESI-MS Analysis. After polymerization for a predetermined time (0.5, 2, 4, 12, and 24 h), 1 mL aliquots of the above solution samples were acidiﬁed with 1 M HCl to pH 2−4 and ﬁltered using a syringe ﬁlter (0.2 μm, Millex-FG, Millipore Corporation, USA). ESI-mass analyses of the solution samples were performed on a LC/MS-2020, SHIMADZU Scientiﬁc instrument under ESI-MS (+) mode. At each time-point, three aliquots of each sample were prepared and analyzed. A blank Tris buﬀer sample was used for comparison. MALDI-TOF Analysis. Stainless steel substrates modiﬁed by the speciﬁed coating samples including PDA, DHI-based coatings, DA/ DHI, and DA/4,7-d2-DHI coatings were analyzed using a 4800 MALDI TOF analyzer from Applied Biosystems, Framingham, MA. The m/z data were manually acquired in the reﬂector mode by using the reﬂectron method (accelerating voltage: 20000 V; laser intensity: 3300−3600). For each coating sample, at least three diﬀerent areas of the substrate surfaces were examined for reproducibility. Synthesis of 5,6-Dihydroxyindole (DHI). DHI was prepared under a nitrogen atmosphere using the reported method.15 A mixture of K3[Fe(CN)6] (6.6 g, 20 mmol) and NaHCO3 (2.5 g, 30 mmol) in H2O (60 mL) was added dropwise over 5 min to a stirred solution of L-DOPA (0.99 g, 5 mmol) in 500 mL of H2O. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 3 h, and then 600 mg of Na2S2O4 was added. The solution was then adjusted to pH 4 with 3 M HCl(aq) and extracted with ethyl acetate (250 mL × 3). The combined organic phases were washed with saturated brine (100 mL × 3) and were dried over Na2SO4. Evaporation of ethyl acetate to 5 mL followed by the addition of hexane (50 mL) yielded a pale brown solid; the solids were then redissolved in ethyl acetate (5 mL) followed by recrystallization from 50 mL of hexane to give 287 mg (27% yield) of DHI as an oﬀ-white solid. The 1H and 13C NMR data were consistent with the literature.15 1 H NMR (CD3OD): δ (ppm) 6.209 (d, J = 3.08 Hz, 1H), 6.84 (s,
performed to examine the possible contribution of this pathway in PDA formation. These studies provide hitherto unreported insights into the role of DHI in the formation of PDA adhesive coatings.
Materials. All chemicals were purchased from Sigma-Aldrich or Alfa Aesar and were used without further puriﬁcation unless otherwise speciﬁed. Silicon (Si) wafers were obtained from Mitsubishi Silicon America, USA. Quartz microscope slides (fused, 76.2 mm × 25.4 mm × 1.0 mm) were purchased from Alfa Aesar. AISI type 304 stainless steel foils (0.05 mm thickness) were purchased from Goodfellow Ltd. of Cambridge, U.K. Poly(lysine) slides (Thermo Scientiﬁc, 25 mm × 75 mm × 1.0 mm) were purchased from Fisher Scientiﬁc. Si wafers were precleaned in a fresh H2O/NH4OH/H2O2 (6:1:1) solution at 100 °C for 10 min and then rinsed in deionized (DI) water and ethanol. Quartz slides, 304 stainless steel, and poly(lysine) were cleaned in an ultrasound water bath for 30 min, rinsed with ethanol, and then blown dry under nitrogen gas. NMR Analysis. NMR spectra were recorded on a Bruker Avance III 400 MHz spectrometer at 400 MHz for 1H and at 100 MHz for 13 C with methanol-d4 (CD3OD) as solvent. The chemical shifts are given in ppm, using the proton solvent residue signal (CD3OD: δ 3.31) as reference in the 1H NMR spectrum. The deuterium coupled signal of the solvent was used as reference in 13C NMR (CD3OD: δ 49.00). The following abbreviations were used to describe the signals: s = singlet, d = doublet, t = triplet, m = multiplet, q = quartet. ESImass spectra were recorded on a Shimadzu liquid chromatograph− mass spectrometer. AFM Analysis. AFM characterization was performed using a Nanowizard III instrument (JPK Instruments AG, Berlin, Germany) equipped with a NanoWizard head and controller. The triangularly shaped silicon nitride cantilevers (Nano World, PNP-TR) were used throughout the scanning, and the spring constant was calibrated using the thermal noise method in the range 0.07−0.09 N/m. The experiments were performed in air, letting the system equilibrate for 30−60 min. To carry out the measurement, the coated surfaces were gently scratched with a scalpel to estimate the thickness of the coating layer. The quantitative imaging mode (QITM) was performed for imaging. The QITM is a force spectroscopy based imaging mode that enables the user to have full control over the tip−sample force at each B
Langmuir 1H), 6.946 (s, 1H), 6.972 (d, J = 3.08 Hz, 1H). 13C NMR (CD3OD): δ (ppm) 98.0, 101.4, 101.5, 122.4, 123.7, 132.1, 141.2, 143.5. Synthesis of 4,7-d2-DHI.16,17 500 mg of nondeuterated DOPA was dissolved in 10 mL of 6 M DCl/D2O and then reﬂuxed at 90 °C under a nitrogen atmosphere for 6 h. The D2O solution of the reaction mixture was evaporated under vacuum and redissolved in cold MeOH. Evaporation of MeOH solvent gave the deuteriumlabeled L-DOPA(485 mg, >95% yield) as a white solid, which was used in the next step without puriﬁcation. 1H NMR (400 MHz, D2O) δ (ppm) 3.90 (dd, J = 7.4, 5.6 Hz, 1H), 2.80 (dd, J = 14.7, 5.6 Hz, 1H), 2.69 (dd, J = 14.7, 7.5 Hz, 1H). ESI-MS: m/z calcd for C9H8D3NO4 [M + 1]+ 201.09; found 201.10. To a solution of 480 mg (2.45 mmol) of deuterium-labeled LDOPA in 250 mL of water was added in 5 min a solution of 3.3 g (10 mmol) of K3[Fe(CN)6] and 1.3 g (15 mmol) of NaHCO3 in 30 mL of water. The wine-red solution was then kept at room temperature (rt) under a nitrogen atmosphere for 3 h and was extracted with ethyl acetate (300 mL × 3). The combined ethyl acetate extracts were washed successively with a saturated aqueous NaCl solution (100 mL) containing 0.10 g (0.5 mmol) of Na2S2O5 and then a saturated aqueous NaCl solution (50 mL × 2) and dried. Evaporation of the organic solvent gave a pale brown solid, which was recrystallized from 20 mL of hexane to aﬀord 107 mg (27% yield) of 4,7-d2-DHI as a colorless solid with 1H NMR data consistent with the literature.17 1H NMR (400 MHz, CD3OD) δ (ppm) 6.97 (d, J = 3.1 Hz, 1H), 6.19 (d, J = 3.1 Hz, 1H). Coating Procedure. The speciﬁed substrates were immersed in 20 mL of the buﬀer solutions as indicated with speciﬁed precursors (for PDA coatings: 10 mM DA, 10 mM Tris buﬀer, pH 8.5; for DHIbased coatings: 10 mM DHI, 10 mM Tris or phosphate buﬀer, pH 8.5 or 7; for DA/DHI coatings: 10 mM DA, 1 mM DHI, 10 mM Tris or phosphate buﬀer, pH 8.5; for DA/4,7-d2-DHI: 10 mM DA, 1 mM 4,7d2-DHI, 10 mM Tris buﬀer, pH 8.5). After polymerization for a speciﬁed time (30 min, 4 h, or 24 h), the substrates were rinsed with water and ethanol and dried with a stream of nitrogen gas. These coated substrates were subsequently characterized as described below. Investigation into the Eﬀect of PDA Particles on Coating Formation. PDA particles were collected from an aged DA-Tris solution (200 mg DA in 100 mL of Tris, pH 8.5, 24 h) under centrifugation using a HERMLE centrifuge Z300k (4000 rpm, 5 min). The obtained PDA solids were washed with 50 mL of deionized water twice and then dried under vacuum overnight. The obtained dry PDA solids (ca. 40 mg) were then added to a fresh DA solution (10 mM DA, 25 mL of Tris buﬀer, pH 8.5). Poly(lysine) slides were vertically dipped in this fresh solution. After 24 h, the slides were then rinsed with water and ethanol, followed by dryness under nitrogen gas and subjected to AFM characterization. Investigation into the Adsorption of DHI onto PDA Particles. To examine whether PDA particles sequester DHI and the oxidation eﬀects on this adsorption process, four DHI solutions, i.e. (i) 1 mM DHI in 10 mM Tris buﬀer (1 mL) at pH 7.4, (ii) 1 mM DHI in 10 mM Tris buﬀer (1 mL) at pH 7.4 in the presence of PDA particles (5 mg), (iii) 1 mM DHI in 10 mM degassed Tris buﬀer (1 mL) at pH 7.4, and (iv) 1 mM DHI in 10 mM degassed Tris buﬀer (1 mL) at pH 7.4 in the presence of PDA particles (5 mg) were prepared. After 10 min, the amounts of DHI present in both solutions were analyzed by HPLC. HPLC analysis was performed on a LC/MS2020, SHIMADZU Scientiﬁc apparatus equipped with a UV detector set at 280 nm using a Phenomenex column (5 μm, 4.6 × 150 mm2). Deionized water (eluant A) and acetonitrile (eluant B) gradients were used as follows: 0−5 min 10% eluant B, 5−10 min 10−50% eluant B, and 50−80 min 80% eluant B (ﬂow rate of 1 mL/min). Investigation into the Eﬀect of Poly(vinyl alcohol) on Coating Formation. To examine the eﬀect of PVA on the deposition/aggregation of PDA and DA/DHI coatings, silicon and poly(lysine) substrates were immersed in to fresh DA-Tris solution (10 mM DA, 20 mL of Tris buﬀer pH 8.5) containing 0.1 wt % PVA or DA/DHI solution (10 mM DA:1 mM DHI, 10 mL of Tris buﬀer, pH 8.5) containing 0.1 wt % PVA. After polymerization for 24 h, the substrates were subjected to the post-treatment procedure (rinsed
with water and ethanol and dried with a stream of nitrogen gas), followed by AFM characterization as indicated above.
RESULTS AND DISCUSSION Preparation of DHI-Based and PDA Coatings. The composition of DHI-based and PDA coatings was ﬁrst analyzed using XPS and MALDI-TOF MS. In these studies, phosphate buﬀer was used instead of the classical Tris buﬀer to avoid the interference of the amine-containing Tris molecules in the coating deposition. An authentic DHI compound was synthesized using the reported procedure,15 and DHI-based coatings were prepared at pH 7 following our previously reported procedure.14 PDA coatings were prepared under alkaline conditions, i.e., phosphate buﬀer at pH 8.5,1 while silicon wafer and stainless steel were used as the surface substrates for XPS and MALDI-TOS MS analyses, respectively. XPS Analysis of DHI-Based and PDA Coatings. The high-resolution XPS spectra of the nitrogen region for DHIbased and PDA coatings deposited from the respective solutions after 24 h is shown in Figure 2. The XPS of DHI-
Figure 2. High-resolution XPS spectra of N 1s region for (A) DHIbased and (B) PDA coatings deposited from the phosphate solution at pH 8.5 for 24 h.
based coatings (Figure 2A) shows a major peak at 400 eV (ca. 90%), which arises from secondary amine species derived from DHI-based oligomers. The small peak at 398 eV (ca. 3%) can be assigned to tertiary amines, associated with the tautomeric species of DHI and its quinone. The minor peak at 401−402 eV (ca. 7%) is normally assigned to primary amines in catecholamine based coatings and may be related to NH2containing impurities; however, this minor component could also derive from protonated pyrrole and other quaternary nitrogen species.18,19 In comparison, PDA coatings contained larger amounts of primary amines (ca. 15%) and tertiary amines (ca. 8%), with 77% of secondary amines (Figure 2B). This is in good agreement with the reported XPS data of PDA ﬁlms by Zangmeister (15% NH2-R; 10% = N-R; 74% R-NHR).20 The fraction of primary amines in PDA ﬁlms could be C
Figure 3. MALDI-TOF mass spectrum of DHI-based coatings on stainless steel after 24 h of polymerization.
ascribed to the presence of the uncyclized DA species while the secondary and tertiary amines in PDA could be related to the species derived from cyclized intermediates such as 5,6dihydroxyindoline/quinone and DHI/quinone. MALDI-TOF Analysis of DHI-Based and PDA Coatings. To gain a better understanding of the components of DHIbased and PDA coatings, MALDI-TOF mass spectroscopy was utilized to analyze the coatings samples on stainless steel substrates. Under MALDI-TOF MS analysis, DHI-based coatings (10 mM, phosphate buﬀer at pH 7, 24 h) revealed major peaks at m/z 294, 441, 522, and 550 (Figure 3). As compared with the major components detected in DHI solution (Figure S1), the observed major peaks at m/z 294 and 441 in the MALDI-TOF mass spectrum are very close to the values that correspond to the m/z peak of the DHI dimer (m/z 297) and trimer (m/z 444). The reasons underlying these small mass discrepancies could conceivably be explained by considering that the DHI dimer and trimer exist as oxidized forms in the coating sample. A common peak at m/z 577 was observed in both the solution and coating sample of DHI at pH 7. This was tentatively assigned as the ion derived from the degradation of DHI tetramer, as discussed in another report.6 The observed major peaks at m/z 522 and 550 in the MALDITOF mass spectrum of DHI-based coatings may also be derived from other degradation products derived from the proposed DHI tetramer. In contrast, the MALDI-TOF mass spectrum of the PDA coatings showed an intense peak at m/z 402 (Figure 4), which could originate from a trimeric oligomer derived from the oxidative polymerization of DA. In addition, some complex peaks with lower intensities (∼200 au) were also observed in the MALDI-TOF spectrum of PDA ﬁlms (Figure 4A, shaded area). Direct analysis of the aged DA−phosphate solution after 24 h using MALDI-TOF MS revealed a single peak at m/z 402 (Figure 4B) but did not show any complex peaks as observed in the spectrum of PDA coating ﬁlms (Figure 4A). Ding and Alﬁeri et al. also reported that a dominant peak at m/z 402 was detected in the PDA ﬁlms under time-of-ﬂight secondary ion mass spectrometry (Tof-SIMS) and MALDI-TOF MS analyses, but no other signiﬁcant peaks were observed,7,12 consistent with our observations (Figure 4B). It should be noted that the methods of sample preparation used in these reported studies were similar to that used in our analysis of PDA solution (dropping the DA-aged solution or the solution of PDA ﬁlm onto a MALDI target plate and allowing to air-dry for further analysis). In other words, our MALDI-TOF MS
Figure 4. MALDI-TOF mass spectra of (A) PDA coating sample on stainless steel and (B) solution sample after 24 h of polymerization.
analysis of authentic PDA ﬁlm grown on a substrate reveals the presence of a complex peak pattern in addition to a strong peak at m/z 402 (Figure 4A). Earlier literature analysis of aged solution of DA, or solubilized PDA ﬁlms dried on a MALDI steel plate, shows only a single peak at m/z 402.7,12 While present in lower intensities, the components corresponding to these minor peaks (Figure 4A, shaded area) could play important roles in adhesive PDA ﬁlm formation (discussed below). Based on our observations, the species at m/z 402 in PDA is not likely to be derived from the self-polymerization of DHI in PDA solution, as no intense peak at m/z 402 was observed in either the MALDI-TOF mass spectrum of DHI-based ﬁlms or the ESI mass spectrum of DHI-based coating solutions. Furthermore, prior work in the literature on DHI-derived oligomers/polymers did not show any signiﬁcant peak at m/z 402 via either Tof-SIMS or MALDI-MS.12,21−23 Coupled with the XPS data above, these results do not support the notion that DHI-based oligomers are the major components of PDA coatings.24−26 In addition, these data do not support Ding’s D
MALDI-TOF mass (m/z) ESI-MS analyses on solutionsb (m/z)
DA (10 mM, pH 8.5)
∼20 nm 0.5−1 μm disk-shaped broadband spectra from 300 to 800 nm strong bands at 1150 and 859 cm−1
−c −c −c
40−50 nm 0.5−2 μm granules broadband spectra from 300 to 800 nm
294, 441, 522, 550, and 577
bands with moderate intensity at 1500 and 1300 cm−1 402
298, 444, 577, and 588
Coating samples were obtained from the oxidative polymerization of DA or DHI for 24 h. bSolution samples were obtained from the oxidative polymerization of DA or DHI for 6 h. cNo signiﬁcant DHI-based coating ﬁlms were formed at pH 8.5 for further analyses. dThe data details were reported in our previous studies.14
Scheme 1. Possible Reaction Pathways of DA and DHI in PDA Formation
Figure 6. DA, DHI, and DA/DHI solutions after 30 min and poly(lysine) glass slides dip-coated in these solutions after 30 min.
proposal that the structure of the species at 402 m/z is (DHI)2/PCA, a degraded product of DHI trimer.7 UV−Vis Analysis of DHI-Based and PDA Coatings. To compare the optical property of DHI-based and PDA coatings, the absorption of both coatings on quartz substrates was measured using ultraviolet−visible (UV−vis) spectroscopy. As shown in Figure 5A, both coatings showed very similar broadband spectra from 300 to 800 nm, with the absorption increasing exponentially toward the higher energy ultraviolet end. These observations are consistent with the reported optical properties of PDA coating and naturally occurring eumelanin materials.27−30 The broadband UV−vis spectrum of PDA coatings is suggested to be associated with the chemical disorder in the primary structures of monomers/oligomers derived from DA and/or excitonic eﬀects in the aggregation of these monomers/oligomers.2 Micillo et al. investigated the UV−vis absorbance of the solution sample of the oxidative polymerization of DHI over 24 h reaction time and suggested that the broadband spectra could be also related to the delocalized π-electron systems of DHI-based oligomers (Figure 5B).31 Comparison of DHI-Based and PDA Coatings. The components and properties of DHI-based and PDA coatings are given in Table 1. Although both ﬁlms share similar optical properties, our data above suggest that PDA coatings are not synonymous with DHI coatings; i.e., PDA coatings are not solely composed of DHI or DHI oxidation intermediates/ products (Scheme 1, pathway A). However, the contribution of DHI as an intermediate in the formation of PDA ﬁlms cannot be completely excluded at this stage. Considering that there exists a large amount of unreacted DA precursor (and also other possible intermediates in PDA solution), the highly reactive DHI may copolymerize with these species resulting in PDA ﬁlm formation. Speciﬁcally, DHI-containing species could also be derived from other pathways, e.g., pathway B in Scheme 1, a postcyclization of linear dopamine oligomers, or pathway C, a postcyclization of DA/DHI dimer, contributing to PDA formation. Copolymerization of DA/DHI. To investigate the likelihood of the contributions of pathway C (Scheme 1) to the formation of PDA, DA and DHI were copolymerized. Our previous study demonstrated that the conversion of DA to DHI in DA-Tris solution (10 mM DA, 10 mM Tris buﬀer, pH 8.5) is a very slow process, and the real-time concentration of DHI was ca. 4 μM.14 To mimic the conditions of PDA
formation, a relatively small amount of DHI (1 mM) with DA (10 mM) under classical PDA conditions (i.e., Tris buﬀer at pH 8.5) was used for dip-coating on poly(lysine) slides and silicon wafers. As shown in Figure 6, the DA/DHI mixture rapidly gave visible dark coatings on the poly(lysine) surface after 30 min. Simultaneously, visible sheet-like aggregates in solution were also observed, suggesting that the copolymerization of DA and DHI precursors can give rise to adhesive oligomers/polymers (i.e., coating) and aggregates. These observations are in contrast to the observations for when DHI (1 mM) or DA (10 mM) are used alone under alkaline conditions, as neither experiments produced visible coatings on poly(lysine) slides at such short time points. In addition, no visible aggregates or particles were observed in the solution samples of both DA-Tris and DHI-Tris at these time points. The successful deposition of DA/DHI coatings on surfaces was further supported by AFM and XPS results. Under the optical microscope of the AFM instrument, it was observed that a black ﬁlm was successfully fabricated on the silicon surfaces (Figure 7A). Further AFM analysis revealed that a DA/DHI coating with ca. 10−15 nm was obtained (Figure 7B), and XPS data showed that signiﬁcant nitrogen content was detected on the silicon surface (Figure 7C). In contrast,
Figure 7. (A) Image of DA/DHI ﬁlm on silicon substrate under AFM optical microscope. (B) AFM and (C) XPS analyses of DA/DHI coatings on silicon surface after 30 min of polymerization F
Table 2. ESI (+)-MS Data of the Oxidative Polymerization Products of DA/DHI, DA, and DHI Precursors in the Early (t ≤ 2 h) and Later Stages (t ≥ 3 h) of Polymerization polymerization time
precursors and conditions DA/DHI (10 mM:1 mM) in Tris solution (pH DA (10 mM) in Tris solution (pH 8.5) 8.5) m/z 297 and 444; m/z 405, 458, 544, 597, 689, and major peaks at m/z 402 and 420 723 major peaks at m/z 402 and 420 major peaks at m/z 402 and 420; minor peaks at m/z 405, 458, 544, etc.
DHI (1 mM) in Tris solution (pH 8.5) m/z 297, 444, etc. complex peak patterns
Scheme 2. Possible Reaction Pathways Involved in the Copolymerization of DA/DHI
derived from DA/DHI copolymerization were analyzed and compared to those found in the respective solution and solid samples of PDA using ESI-MS and MALDI-TOF MS analyses. The ESI-MS spectra of solution samples of DA/DHI prepared at short time points (30 min, 1 h, and 2 h) showed complex peak patterns (Figure S3). Among the major species detected, some species were proposed to derive from the selfpolymerization of DHI. For example, the peaks at m/z 297 and 444 can be assigned to the ions of the DHI dimer and trimer. Other major peaks at m/z 405, 458, 544, 597, 689, and 723 were also observed in the spectra of DA/DHI copolymerization. However, none of these peaks were signiﬁcantly detected in the solution samples at these same time points when pure DA or DHI was used as monomer (Table 2; Figures S4 and S5), which suggests that these peaks could be related to the cross-linking of DA- and DHI-based species. ESI-MS monitoring of the copolymerization of DA and DHI reveals that the above-mentioned species (including the peaks assigned to both DHI-based oligomers and DA/DHI cooligomers) dramatically decreased with time and were no longer detectable after 3 h. Meanwhile, species at m/z 402 and 420 were signiﬁcantly detected in the samples of DA/DHI prepared after polymerization times of 3, 6, 12, and 24 h (Figure S6). The peak patterns were also identical to that observed in the spectra of the polymerization of DA alone measured between 5 min and 24 h (Figure S4). Thus, it would appear that the species observed at m/z 402 and 420 in the spectra of DA/DHI were derived from the degradation of species corresponding to DA/DHI co-oligomers detected in the early stages of polymerization (t ≤ 2 h). However, this notion cannot be supported by the observations in our mass spectral studies on the polymerization of DA alone. The abovementioned peaks corresponding to DA/DHI co-oligomers (m/ z 405, 458, 544, 597, 689, and 723) were not signiﬁcantly detected in the DA-Tris samples prepared at <12 h but were
the use of DA alone gave a ca. 3−5 nm thickness coating after 30 min of polymerization under alkaline conditions. The above results suggest that the addition of DHI to the aqueous DA solution under alkaline conditions can facilitate coating formation at short time points as compared to the oxidative polymerization of DA alone under similar conditions. As the use of DHI alone at pH 8.5 does not give signiﬁcant coatings,12,14 these observations suggest the complex interplay between DHI-based species and uncyclized DA species; i.e., amine functionalities play critical roles in the adhesive properties of DA/DHI coatings. To further verify the presence of primary amine functionalities in DA/DHI coatings, highresolution XPS analysis on DA/DHI coatings deposited from phosphate buﬀer at pH 8.5 after 4 h of polymerization was carried out. The presence of NH2-containing species was signiﬁcantly detected, as shown in Figure S2A. In addition, the relative amounts of diﬀerent amine species (ca. 18% of primary amines; 74% of secondary amines; 8% of tertiary amines) were much closer to that observed in PDA ﬁlms (Figure 2B). During the copolymerization of DA/DHI in Tris or phosphate buﬀers, we also noted that the growth of coatings plateaued after ca. 3−4 h, and an 15−20 nm thick coating can be obtained (Figure S2B) along with large amounts of aggregates in the solution. Prolonging the polymerization time to 24 h did not further increase the coating thickness. As compared to the coatings derived from the polymerization of DA at pH 8.5 for 24 h (ca. 40−50 nm), the thickness of DA/ DHI coatings obtained after 24 h is relatively thinner. This can be rationalized by the competitive events between deposition and aggregation as described in our the previous report: the addition of DHI in DA solution can lead to rapid coating growth in the early stages of polymerization, but the simultaneous formation of large aggregates and particles in solution also retards the subsequent coating growth.7,14 ESI-MS Analyses of DA/DHI Copolymerization. The components present in the solution and solid (coating) sample G
Langmuir Scheme 3. Polymerization of DA and 4,7-d2-DHI To Form Coatings
and 24 h) (Figure S10). These observations support the above hypothesis that the major species observed at m/z 402 and 420 in the spectra of DA/DHI samples at longer polymerization times (t ≥ 3 h) are not derived from the copolymerization pathway between DA and DHI but from other pathways during the self-polymerization of the remaining DA precursor. MALDI-TOF MS Analysis of DA/DHI Copolymerization. To gain a better understanding of the composition in the DA/DHI coatings, MALDI-TOF MS analysis on the steel substrates dipped in DA/DHI solution after 4 h of copolymerization was carried out. As shown in Figure 8, the
observable as minor species in the sample prepared after 24 h of reaction time. In contrast, the peaks at m/z 402 and 420 were consistently observed as the major species present in the DA-Tris sample measured between 5 min and 24 h. Therefore, a plausible explanation for these observations with DA/DHI copolymerization as well as the presence of diﬀerent components in the early (t ≤ 2 h) and later stages (t ≥ 3 h) of polymerization could be because the species at m/z 402 and the detected DA/DHI sample (t ≥ 3 h) do not arise from the degradation of DA/DHI co-oligomers but are derived from other pathways in the oxidative polymerization of DA. Based on the above, the copolymerization of DA/DHI could be described as shown in Scheme 2. At short time points (t ≤ 2 h), the presence of DHI in DA-Tris solution will result in large amounts of DA/DHI co-oligomers and DHI-based oligomers in solution, as DHI is a more reactive precursor than DA. These co-oligomers and oligomers were no longer detectable after ca. 3 h, presumably due to rapid aggregation and deposition. What was evident is the formation of large amounts of aggregates in solutions and a signiﬁcant amount of coatings onto the surfaces in the ﬁrst 2−3 h (Figures 6 and 7). Meanwhile, the DA-based species, e.g. quinones, could also be consumed in the copolymerization with DHI-based species. As a consequence, the self-polymerization of DA (pathway B, Scheme 2) is competitively suppressed due to the presence of reactive DHI, and thus no signiﬁcant species at m/z 402 and 420 were detected at this stage. At polymerization times >3 h, the DHI precursor has been consumed and the remaining DA precursor can undergo self-polymerization, leading to the formation of the species at m/z 402 and 420 (pathway A, Scheme 2). Copolymerization of DA and Deuterated DHI. To further support the above, a deuterium-labeled precursor, 4,7d2-DHI was synthesized following the procedures reported in the literature (Figure S7)16,17 and used in copolymerization studies with DA in Tris buﬀer at pH 8.5 (Scheme 3). ESI-MS studies on the solution samples of DA and 4,7-d2-DHI (10:1) in Tris buﬀer at pH 8.5 were carried out. An ESI-MS spectral study of the polymerization of 4,7-d2-DHI alone was also examined for comparison. The ESI-MS spectra of the solution samples of DA and 4,7d2-DHI in Tris buﬀer at pH 8.5 after 2 h of polymerization revealed major peaks at m/z 300, 331, 447−449, 462, and 584 (Figure S8). In comparison, none of these peaks were observed in the spectra of DA-Tris sample prepared at this time point (t ≤ 2 h) (Figure S3). As compared to the spectra of the polymerization of 4,7-d2-DHI in Tris buﬀer (pH 8.5) at these time points (t ≤ 2 h) (Figure S9), common peaks at m/z 300 and 447−449 were observed, which could be assigned to the ions of the dimer and trimer derived from 4,7-d2-DHI. Further ESI-MS studies showed that all the above peaks in the spectra of DA/4,7-d2-DHI were no longer detected after 4 h of reaction time, and the peaks at m/z 402 and 420 were signiﬁcantly detected in the sample prepared afterward (6, 12,
Figure 8. MALDI-TOF MS analyses of four diﬀerent surface spots of the DA/DHI coatings after 4 h of polymerization.
mass spectrum of DA/DHI coatings revealed complex peak patterns ranging from m/z 250−800 with relatively low intensities (∼200−300 au), suggesting a complex combination of heterogeneous mixtures. This complexity could be related to the complex pathways available in the copolymerization of DA/DHI. In addition, the instability of DHI-based species under alkaline conditions could be another factor resulting in these complicated mixtures. Previous reports have demonstrated the oxidative degradation of DHI-based species, leading to degraded components containing pyrrole-carboxylic acid.32−34 This view could be supported by the observation that the mass diﬀerences between the majority of the adjacent major peaks were 16−18 mass units (Figure 8), presumably due to the loss of hydroxyl group of the pyrrole-carboxylic acid moieties. Further MALDI-TOF MS analysis on the DA/4,7-d2DHI coatings also revealed a complex peak pattern (Figure S11), similar to that of DA/DHI coatings. While the DA/DHI ﬁlm obtained does not show a clear and signiﬁcant peak at m/z 402 characteristic of PDA, the DA/ DHI ﬁlm shows a complex peak pattern of lower intensity, similar to that observed in the MALDI-TOF spectrum of PDA ﬁlms (Figure 4A, shaded area). This suggests that while the DA/DHI coating is not completely identical with PDA coatings, they may share commonality, e.g., DA/DHI coH
Langmuir oligomers; nevertheless, the species corresponding to m/z 402 (characteristically found in PDA coatings) may represent a separate structural component. Our results do not rule out a role for the species corresponding to m/z 402 but suggest that the structural components derived from DA/DHI copolymerization play a signiﬁcant role in adhesive PDA ﬁlm growth. The importance of DHI in PDA formation may be related to its ability to couple rapidly with DA and other intermediates, giving rise to planar oligomeric species that can rapidly deposit onto surfaces via π−π stacking interactions,35−38 while uncyclized DA species could be associated with the protonated amine functionalities for cation−π interaction, which has been demonstrated as a crucial mechanism for PDA deposition.39,40 To our knowledge this is the ﬁrst report of the characterization of a dip-coated copolymer of DA and DHI via XPS and MALDI-TOF MS, which, while not identical to PDA, is highly similar to it. On the basis of the above, we propose that DHIrelated species, while produced in very low quantities during the oxidative polymerization of DA, can contribute significantly to PDA formation. Our conclusion stands in contrast to that reported by Alﬁeri et al., who suggested that DHI was unlikely to play a signiﬁcant role in PDA coating formation.12 However, our results are in agreement with several other studies.5,6,38 Hong et al. isolated a DHI/(DA)2 trimer complex from aqueous DA that had been left to oxidize and found that this isolated DHI/(DA)2 complex could further form insoluble particulates.5 Nevertheless, Hong et al. did not further characterize the precipitate obtained from the polymerization of DHI/(DA)2 or investigate whether coating can be formed from DA/DHI. More recently, Filip et al. demonstrated via 13 C/1H/2H solid state NMR that a covalent linkage between the aromatic core of DA and DHI-related species accounts for up to half of the phenyl ring positions.38 Roles of DHI in Deposition/Aggregation. To verify the competitive events proposed above and to investigate the roles of DHI in deposition/aggregation process, a series of experiments were designed. In an initial experiment, PDA particles (ca. 40 mg) were collected from the classic DA-Tris solution after 24 h and then added to a fresh DA solution (10 mM, 25 mL of Tris buﬀer 8.5, poly(lysine) glass sheets were vertically orientated) for dip-coating study (Figure 9A,B). The thickness of the coatings obtained after 24 h was measured to be ca. 7 nm. In contrast, when the fresh DA-Tris solution at pH 8.5 was used (in the absence of the added PDA particles), the coating thickness was around 40−50 nm after 24 h (Figure 9C,D), and a large amount of PDA suspensions was formed in solution. Interestingly, after ﬁltering oﬀ the suspended particles, an additional ca. 20 nm thick coating (leading to a total coating thickness of 60−70 nm) was obtained in this aged DA solution after 24 h. These observations suggest that PDA particles can indeed competitively retard coating growth via the sequestration of its key intermediate/s (e.g., DHI) in solution, so that at the late stages of the polymerization of DA there would not be enough reactive intermediates as feed for deposition onto coating surfaces. Assuming the key intermediate of PDA formation is DHI, PDA particles are proposed to sequester DHI. To conﬁrm this, PDA particles were added to a freshly prepared solution of 1 mM DHI (10 mM Tris buﬀer, pH 7.4). The amount of DHI present after 10 min as monitored by HPLC was reduced by 62% (Figure 10). In contrast, only a 16% reduction in the amount of DHI was observed in the absence of added PDA particles: the former presumably occurs due to the self-
Figure 9. Coating studies of DA-Tris with (A) and without (C) PDA particles and the AFM analyses (B, D) of the respective coatings.
Figure 10. HPLC analysis of the remaining DHI in the presence and absence of PDA particles.
polymerization of DHI. Under a nitrogen atmosphere, a rapid 53% loss of DHI was also noted in the presence of added PDA particles. In both of these experiments, we also noted that the color of DHI solution in the absence of added PDA particles darkened with time in the ﬁrst 10 min. In the presence of added PDA particles, the color of the DHI solution did not change signiﬁcantly at this time point (Figure S12). The latter could be due to the absorption of oxidized DHI species and the derived DHI-based oligomers onto PDA particles. These results support the hypothesis that PDA particles can eﬃciently sequester DHI. To further investigate the roles of DHI in the deposition/ aggregation processes, the eﬀects of a water-soluble poly(vinyl alcohol) (PVA) in PDA formation were examined, as PVA can eﬃciently bind to DHI species and was thus used to suppress the aggregation/polymerization of DHI oligomers.41,42 In this I
Langmuir study, 0.1 wt % PVA was added to DA-Tris solution (10 mM DA, Tris buﬀer pH 8.5, 24 h). It was noted that the deposition of PDA coating on the poly(lysine) slide was signiﬁcantly inhibited, and only coatings with a few nanometers of thickness were obtained. In addition, no PDA particles were observed (even after centrifugation) by the naked eye after 24 h (Figure S13). This suggests that the use of PVA can suppress both the deposition and aggregation of PDA. Similarly, in another separate experiment, it was also noted that PVA can dramatically suppress the deposition and aggregation of DA/ DHI copolymers. After copolymerization of DA/DHI for 24 h in the presence of PVA, only a few of nanoparticles deposited onto the surfaces but no coating ﬁlms were formed (Figure S14), and no signiﬁcant aggregates in the solution were observed (Figure S15). Based on these observations, DHI is likely to be an important precursor responsible for PDA deposition/aggregation. These phenomena can provide further insights into PDA ﬁlm assembly. Although PDA coating has been touted as a universal coating on virtually any type of surfaces, our ﬁndings shown here demonstrate that the deposition of PDA on some surfaces (e.g., glass, poly(lysine), and silicon) can be retarded in the presence of other “attractive” surfaces, e.g., PDA particles and PVA. Similar inhibitory eﬀects on PDA deposition in the presence of other polymers such as poly(N-vinylpyrrolidone) or surfactants such as sodium dodecyl sulfate (SDS) and hexadecyltrimethylammonium bromide (HTAB) have also been reported by others.43,44
Christina L. L. Chai: 0000-0002-9199-851X Notes
The authors declare no competing ﬁnancial interest.
ACKNOWLEDGMENTS This work was ﬁnancially supported by Ministry of Education, Singapore (No. R-148-000-252-114 and No. R-148-000-269114).
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CONCLUSION This work presents an investigation into the roles of DHI in PDA formation. On the basis of the comparative analyses of DHI-based and PDA coatings using diﬀerent analytical techniques, it can be concluded that PDA coatings are not synonymous with DHI coatings; i.e., PDA coatings are not solely composed of DHI or DHI oxidation intermediates/ products. The observations in the DA/DHI copolymerization studies strongly suggest that DHI may play a role in the crosslinking of DA molecules to form coatings, contributing to the formation of PDA. These ﬁndings suggest that both DA and DHI are partial building blocks in PDA formation and support the notion that DA provides a critical structural element, i.e., the free primary amine, in the formation of adhesive catechol/ amine materials.40,45,46 Our work here shows that the interactions between amine functionalities and DHI-based species are critical for PDA adhesion. In principle this knowledge could be further exploited in the design and synthesis of new melanin-like materials.
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.9b00392. Figures S1−S15 (PDF)
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