Gradient Coating of Polydopamine via CDR - ACS Publications

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Gradient Coating of Polydopamine via CDR Mei-Xia Zhao,*,†,‡ Junwei Li,† and Xiaohu Gao*,† †

Department of Bioengineering, University of Washington, Seattle, Washington 98195, United States Key Laboratory of Natural Medicine and Immune-Engineering of Henan Province, Henan University, Kaifeng 475004, China

‡

S Supporting Information *

ABSTRACT: Surfaces with gradient properties are of central importance for a number of chemical and biological processes. Here, we report rapid generation of a polydopamine (PDA) gradient on hydrophobic surfaces by a simple, low cost, and general technology, cyclic draining-replenishing (CDR). Due to the unique surface chemistry of PDA, it enables continuous and precise control of surface wettability and subsequent deposition of organic and inorganic compounds. Using kanamycin as a model compound, we show that the gradient PDA membrane potentially can be used to prepare minimum inhibitory concentration (MIC) test strips for quantifying resistance of antimicrobial agents from microorganisms. Because CDR is experimentally simple, scalable, fast, and does not require specialized reagents or instruments, we envision this platform can be easily adopted to create a variety of functional surfaces.

1. INTRODUCTION Creating gradients of physicochemical features on surface have found a broad spectrum of applications such as mimicking biological systems, screening functional drug combinations and catalysts, directing flow motions, and controlling cell migrations.1−7 Toward these uses, a bioinspired polymer coating material, PDA, recently has attracted considerable interest for surface functionalization and pattering because of its unique abilities of grafting onto a variety of surfaces and chemical conjugation.8−13 Indeed, the versatility of this special polymer has enabled applications in biotechnology, medicine, purification, sensor development, and photovoltaics.14 More interestingly, gradients of PDA on surface have recently been created by controlling the reactant concentrations and mixing process. For example, Shi et al. have explored the use of a microfluidic device to mix dopamine and its polymerization reaction buffer to create a gradient, whereas Yang and coworkers placed the membrane to be coated in dopamine solution with an oblique angle, taking advantage of the oxygen (oxidant for PDA formation) diffusion gradient in solution.15,16 These methods are clever in design but have drawbacks, including scalability and generalizability. Here, we report a general technology to deposit PDA onto surfaces into a gradient pattern. The design is built on a recent invention we made to bypass the diffusion limitation for physical adsorption (or binding events) and chemical reactions occurring at solid−liquid interfaces.17,18 When a compound is being deposited from a solution onto a submerged surface (regardless through physical interactions or chemical reactions), the coating compound near the surface will be captured first, creating a depletion layer. Addition compounds in the bulk solution slowly diffuse toward the surface to replenish the depletion layer for continuous coating. This diffusion limited process is typically slow, especially when the compounds are of large sizes (e.g., macrobiomolecules, nanoparticles, and © XXXX American Chemical Society

polymers) and low concentrations (e.g., to help reduce costs). Although mechanical mixing (stirring, shaking, and rocking) is effective in homogenizing bulk solutions, it often has little effect on surface because fluids near surface are largely stationary (diffusion limited zone). To address this fundamental problem, we recently reported a cyclic draining−replenishing (CDR) technology to bypass diffusion limitation.18 In contrast to mechanical mixing, CDR repeatedly pushes away the incubation solution (e.g., by flipping the incubation vessel upside down), thus, eliminating the depletion layer in the draining step. In the following replenishing step, it brings back the bulk incubation solution (e.g., returning to the original upright position), thus, restoring the coating compound concentration at the surface. When the rate of CDR cycling is much higher than that of compound deposition (formation of a depletion layer), the diffusion limitation can be completely eliminated or at least significantly reduced. In contrast, if the rate of CDR cycling is much slower than that of compound deposition, CDR will have minimal effect on improvement of the compound surface deposition rate. To create a gradient PDA coating on surface, here we operate the CDR process at an intermediate rate using a simple setup shown in Figure 1. The membrane strip to be coated is place at one end of the incubation chamber filled with dopamine-Tris solution (PDA polymerization buffer). When the chamber rocks to an angle, the incubation solution is drained from the membrane. When the chamber returns to the upright position, the entire strip is submerged in the PDA solution. As this process is repeated for many cycles, the near end of the membrane is incubated in the PDA solution for longer time Received: April 28, 2017 Revised: June 16, 2017

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DOI: 10.1021/acs.langmuir.7b01463 Langmuir XXXX, XXX, XXX−XXX

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2.6. Surface Characterization. The surface morphologies and chemical elements of the membranes were characterized with a SEM equipped with energy-dispersive X-ray spectroscopy (EDS, TCS SP5, Leica, German). Silver nanoparticle deposition was also analyzed with X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha).

3. RESULTS AND DISCUSSION A hydrophobic membrane, polyvinylidene fluoride (PVDF), was used to demonstrate CDR-aided PDA coating. As shown in Figure 2a, the coated PVDF membrane shows a gradient color

Figure 1. Schematic illustration of the simple CDR setup for gradient PDA coating onto a membrane. During the draining cycle, the container is tilted toward one side to remove the coating solution from the membrane; whereas during the replenishing cycle, the membrane is fully submerged in the solution. This process enables coating materials to effectively transport from solution to the surface.

than the distal end, and vice versa, which can create a PDA gradient on the membrane.

2. EXPERIMENTAL SECTION 2.1. Materials. Dopamine hydrochloride, tris(hydroxymethyl)aminomethane (Tris), silver nitrate, gold chloride, and the bacteriarelated reagents were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. Immobilon-FL transfer membrane (PVDF, mean pore size ∼0.45 μm) was obtained from Merck-Millipore (Billerica, MA). Tris buffer consisting of 10 mM Tris (pH 8.5) was used throughout the PDA coating experiments. 2.2. Fabrication of PDA Gradient Using CDR. PVDF membranes were cut into 2 × 5 cm2 strips and laid in a 100 × 20 mm culture vessel. The strips were prewetted with ethanol then immersed in dopamine-Tris solutions of 2 mg/mL (unless otherwise specified). The reaction vessel was placed on a common benchtop rocker (2 rpm otherwise stated) at room temperature, and the coated membranes were rinsed with deionize water and dried in an oven at 30 °C for 4 h. 2.3. Ag Deposition on PDA-Coated Gradient PVDF Membranes. The PDA-coated gradient PVDF membranes were immersed in AgNO3 solution (0.1 M) at room temperature for 6 h with gentle rocking, rinsed with deionized water, and dried in an oven at 30 °C for 4 h. 2.4. Antibacterial Assay of Ag-Coated PVDF Gradient Membranes. Single colony of the E. coli was inoculated in 5 mL of LB media overnight at 37 °C with shaking (150 rpm). A total of 1 mL of the bacterial suspension was inoculated in 50 mL of fresh LB media and incubated for 5 h with shaking (250 rpm) at 37 °C. The silver particle-decorated PVDF membranes (cut at different locations) were placed into 24-well culture plates and covered with 200 μL of the E. coli suspension in PBS (107 cfu/mL). After incubation at 37 °C for 3 h, 1.8 mL of fresh PBS was added into each well. The E. coli cells were ultrasonically detached and diluted by 1000-fold. Cell viability in the E. coli suspension (100 μL) was measured using agar plates. 2.5. Antibacterial Assay of Kanamycin-Coated PVDF Gradient Membranes. The PDA-coated PVDF gradient membranes were dipped in 50 μg/mL kanamycin solution for 2 min. The membranes were placed into agar plates with E. coli overnight at 37 °C.

Figure 2. Characterization of the water contact angles on a gradient PDA-coated PVDF membrane. (a) Photographs of the PDA-coated PVDF membrane. (b) Quantitative WCA measurements at representative locations of the gradient membrane.

from the original white to dark brown, the characteristic color of PDA. Due to the surface properties of PDA coating layer, the wettability of the hydrophobic PVDF is also changed gradually (Figure 2b). Along the membrane from the near end to the distal end, the water contact angel (WCA) of the PVDF gradually reduces from the original 132.5° to 88.4° at 0.5 cm and 9.5° at 4.5 cm positions. It is interesting and perhaps counterintuitive to observe that the PDA deposition at the distal end (shortest exposure to the PDA solution) of the membrane is more effective than the near end (longest incubation time). This can be attributed to two factors that are both resulted from the CDR process, although their individual contribution cannot be quantified due to the complicated PDA polymerization mechanism (still not entirely clear after decades of research).19 First, the dopamine solution in Tris buffer quickly turned black, indicating formation of PDA polymers and their aggregates in the solution which can attach to the membrane surface. When the CDR rocking speed is similar to the rate of depletion layer formation, the distal end from the PDA solution wavefront drains more effectively, thus better removes the depletion layer compared to the near end, which is still partially B

DOI: 10.1021/acs.langmuir.7b01463 Langmuir XXXX, XXX, XXX−XXX

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Ag deposition. In addition, the resulting Ag nanoparticles bind with PDA tightly due to a combination of forces between the quinone units in PDA and metal surfaces (such as electrostatic interaction and chelation).26,27 Indeed, as shown in the scanning electron microscopy (SEM) micrographs (Figure 3),

diffusion limited. This process renders more preformed PDA polymers and aggregates in solution to deposit at the distal end. Second, dopamine monomers and oligomers are added to the surface through polymerization, a reaction oxidized by oxygen in air. Again because of the efficient drainage at the distal end (longer exposure to oxygen in air), the reactive forms of dopamine are added to the surface faster. It has been shown previously that the cross-linking degree of catechol-tethered polysaccharide increases with incubation time when the solution is exposed to air,20 and dopamine-related catechol amines-containing polymeric solutions spontaneously forms free-standing films by the exposure to air.21,22 Thus, in our experiments, when the polydopamine solution was drained in this system, the dopamine on the surface layer might be more exposed to oxygen in the air, resulting in effective polymerization. In the current study, PDA coating benefits from the combination of these two separate effects resulted from CDR. Depending on the chemistry, we expect other coating materials can utilize one or both of these mechanisms to create a gradient on surface. As aforementioned, the rocking rate is important to produce the PDA coating gradients, because if the CDR cycling rate is much faster than PDA depletion at the interface, both the near end and the distal end will be coated efficiently; where as if the cycling rate is too slow, deposition at both ends will become diffusion limited. In both cases, CDR will not be able to create gradients effectively. Indeed, at the current dopamine concentration (2 mg/mL), a cycling rate of 2 rpm in the CDR process is the most efficient to create a gradient (Figure S1). We have also probed a number of other experimental conditions. For example, we show that increasing the dopamine concentration (e.g., to 5 mg/mL) requires a slower rocking rate to create a similar gradient (higher concentration of the coating material results in slower formation of the interfacial depletion layer), whereas decreasing the dopamine concentration to 1 mg/mL requires a faster CDR cycling rate (3 rpm) to achieve similar results (Figure S1). Furthermore, the CDR coating time also plays an important role. As shown in Figure S2, at the dopamine concentration of 2 mg/mL and CDR rocking rate of 2 rpm, 1 h deposition time leads to the best gradient. This is easy to understand because when the coating time is too short neither end is well coated by PDA (thus both hydrophobic), and when the coating time is too long, both ends are well coated (thus, both hydrophilic). To confirm the wettability change is indeed due to PDA coating, we quantitatively measured the PDA coating thickness using atomic force microscopy (AFM). Because AFM measurements require a flat surface, we replaced the porous PVDF membrane with a flat silicon wafer and conducted the same CDR-aided coating process. Although the PDA thickness is not uniform on the silicon surface, the trend of increasing average thickness from the near end to the distal end is clear (12.8 nm at 0.5 cm, 13.8 nm at 1.5 cm, 20.0 nm at 2.5 cm, 45.7 nm at 3.5 cm, and 45.9 nm at 4.5 cm), which helps explain the tunable wettability (Figure S3). Following the PDA coating and characterization, we proceeded to utilize the PVDF membrane coated with a gradient PDA layer as a general platform for immobilization of both organic and inorganic antibacterial compounds. To introduce an inorganic compound, the membrane was incubated in a silver salt solution. It is well established that PDA is an excellent reductant and chelator for synthesizing Ag nanoparticles.23−25 Thicker PDA coating should lead to more

Figure 3. SEM image of (a) blank PVDF, (b) PVDF@PDA, and (c− g) gradient PDA-coated PVDF after treatment with AgNO3. (h) XPS spectrum showing silver deposition at locations showing in c−g. From bottom to top, the black curve corresponds to a sample obtained at 0.5 cm from the near end, red at 1.5 cm, blue at 2.5 cm, pink at 3.5 cm, and green at 4.5 cm. Thicker PDA leads to formation of more silver particles. Scale bar: 2 μm.

the total amount of Ag deposition appears to directly correlate with PDA coating thickness on the PVDF membrane. X-ray photoelectron spectroscopy (XPS) results shown in Figure 3h also confirmed the formation of Ag nanoparticles (Ag0). Because XPS only surveys the very surface of the sample, for more quantitative elemental analysis, energy dispersive X-ray spectroscopy (EDS) was used. Figure S4 reveals that the Ag ion content keeps increasing from the near end to the distal end (e.g., wt%, 0.5% at 0.5 cm, and 2.5% at 4.5 cm), confirming the qualitative SEM observations. The gradient Ag nanoparticle deposition is also reflected by their antibacterial activities. Escherichia coli (E. coli) were used as a model bacterium models to investigate the antibacterial properties of the Ag gradient decorated PVDF membrane. Small bits of the membrane of the same size cut at various locations were cocultured with E. coli. As shown in Figure 4, compared with the control group (untreated PVDF), PDAcoated PVDF membrane has negligible effect on bacterial viability. In contrast, E. coli counts are gradually reduced along C

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4. CONCLUSIONS In conclusion, we have further developed the CDR technology, and by balancing the diffusion limitation and coating rate, we demonstrated its use in producing a gradient PDA layer on hydrophobic membranes. Compare to other gradient coating technologies, a unique feature of CDR is its simple setup, without the need of specialized equipment or additional reagents. We further show the use of the gradient PDA membrane in immobilizing antimicrobial agents such as silver nanoparticles and antibiotics, which potentially can become an economical approach to fabricate MIC test strips. We envision the simple, low-cost, green CDR technology can be broadly adopted to engineer other gradient surfaces for applications such as tissue engineering, sensing, and controlling fluid motion.

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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.7b01463. Figure S1 showing the combined effect of dopamine concentration and CDR rocking rate on gradient formation, Figure S2 showing the coating time effect, Figure S3 showing AFM measurement of coating thickness, and Figure S4 showing EDS elemental analysis (PDF).

Figure 4. Viability of E. coli incubated with the samples are shown, specifically: (a) blank PVDF, (b) PDA-coated PVDF, (c−g) Ag particle decorated gradient membrane. (h) Bar plot of E. coli colony counts of the samples shown in a−g.

the increasing thickness of PDA coating (and, consequently, the increasing amount of Ag nanoparticles) on the PVDF membrane (growth inhibition from 8% at 0.5 cm to 99% at 4.5 cm position). Next, we investigated an organic-molecule antibacterial agent, kanamycin, on the gradient strip. Depositing drug molecules with a gradient concentration is a useful assay for determining the minimal inhibition concentration (Figure 5a).

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Xiaohu Gao: 0000-0002-6054-0530 Author Contributions

M.X.Z., J.W.L., and X.H.G. designed the experiments, M.X.Z. performed majority of the experiments with help from J.W.L. M.X.Z., J.W.L., and X.H.G. analyzed the results and wrote the manuscript. Funding

The Howard Hughes Medical Institute, The Ministry of Education of China, Scholarship File Number 201408410251 Notes

The authors declare no competing financial interest.

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Figure 5. Antibacterial property of the gradient membranes coated with kanamycin. (a) Schematic of a MIC assay. (b) Experiment data showing the gradient antibacterial activity.

ACKNOWLEDGMENTS This work was supported in part by the Department of Bioengineering at the University of Washington. J.W.L. thanks the Howard Hughes Medical Institute (HHMI) for a student fellowship, and M.X.Z. thanks the Ministry of Education of China for a CSC scholarship, and the National Natural Science Foundation of China (21501044).

MIC is the lowest concentration of an antimicrobial drug that inhibits visible growth of microorganisms after overnight incubation. Measuring MIC can be useful for both clinical diagnostics and research evaluating the efficacy of antimicrobial compounds. Because the wettability gradient of the PDAcoated PVDF, kanamycin was loaded on the membrane through simple incubation of the antibiotics aqueous solution. Although the test strip with gradient kanamycin shown in Figure 5b by no means represents a quantitative MIC assay, it demonstrates the potential of the gradient PDA membrane in fabricating MIC test strips.

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ABBREVIATIONS PDA, polydopamine; CDR, cyclic draining−replenishing; PVDF, polyvinylidene fluoride; SEM, scanning electron microscopy; WCA, water contact angle; AFM, atomic force microscopy; MIC, minimum inhibitory concentration; EDS, energy-dispersive X-ray spectroscopy; XPS, X-ray photoelectron spectroscopy D

DOI: 10.1021/acs.langmuir.7b01463 Langmuir XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.langmuir.7b01463 Langmuir XXXX, XXX, XXX−XXX