Protein immobilization and fluorescence quenching on polydopamine thin films

Journal of Colloid and Interface Science 477 (2016) 123–130

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Protein immobilization and fluorescence quenching on polydopamine thin films Daqun Chen, Lei Zhao, Weihua Hu ⇑ Institute for Clean Energy & Advanced Materials, Faculty of Materials and Energy, Southwest University, Chongqing 400715, China

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a r t i c l e

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Article history: Received 14 April 2016 Revised 22 May 2016 Accepted 23 May 2016 Available online 24 May 2016 Keywords: Polydopamine Protein immobilization Surface plasmon resonance Protein microarray Fluorescence quenching

a b s t r a c t Mussel inspired polydopamine (PDA) film has attracted great interest as a versatile functional coating for biomolecule immobilization in various bio-related devices. However, the details regarding the interaction between a protein and PDA film remain unclear. Particularly, there is very limited knowledge regarding the protein immobilization on PDA film, even though it is of essential importance in various fields. The situation is even more complicated if considering the fact that quite a number of approaches (e.g., different oxidizing reagent, buffer pH, grown time, grown media, etc.) have been developed to grow PDA films. In this work, protein attachment on PDA film was systematically investigated by using the real-time and label-free surface plasmon resonance (SPR) technique. The kinetics of protein-PDA interaction was explored and the influence of buffer pH and deposition media on the protein attachment was studied. Fluorescent protein microarray was further printed on PDA-coated glass slides for quantitative investigations and together with SPR data, the interesting fluorescence quenching phenomenon of PDA film was revealed. This work may deepen our understanding on the PDA-protein interaction and offer a valuable guide for efficient protein attachment on PDA film in various bio-related applications. Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction Interaction of proteins with a surface is implicated in various important biological activities. Understanding, and in turn ⇑ Corresponding author. E-mail address: [email protected] (W. Hu). http://dx.doi.org/10.1016/j.jcis.2016.05.042 0021-9797/Ó 2016 Elsevier Inc. All rights reserved.

controlling this interaction is essentially important for both fundamental research and practical applications [1]. Inspired by the adhesive protein of marine mussels for attachment to wet surfaces, polydopamine (PDA) film has been explored as a versatile surface coating to modify eventually all types of solid surfaces [2,3]. Due to the presence of functional groups such as indole, catechol, quinone and indolic/catecholic p-system, the PDA film possesses

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excellent adhesion stability to surface and also provides a versatile platform for further conjugation with other interesting species for surface functionalization and modification [2–8]. Particularly interesting is its ability to covalently bind with nucleophiles such as amines and sulfhydryls via Schiff’s base formation or Michael addition, thus offering a universal strategy for protein attachment on solid surfaces [4,5,9]. Although PDA has been successfully used for protein attachment for years, the important details remain unclear regarding the interaction between protein and PDA film, for example, the conjugation density, interaction kinetics, immobilization stability, etc., which limits various practical applications. The situation is further complicated if considering the following three facts. (1) The oxidative polymerization mechanism of PDA is elusive at this time due to the complex redox processes involved and a series of intermediates generated during the reaction [10,11]. The exact chemical components of PDA are still under debate and therefore it is hard to figure out a clear picture about the protein-PDA interaction [12,13]; (2) A number of methods have been established to grow PDA films (different buffer, oxidant, polymerization duration, buffer pH, etc.); the film thickness, roughness, and chemical components may significantly vary with the synthetic conditions [3,14–17]; (3) the chemical components in the PDA film may also vary upon the environment change such as pH value due to the disturbed equilibriums of several reactive component pairs [11–13]. Therefore, it is particularly critical to investigate the protein-PDA interaction for fulfill the full potential of PDA film as a reactive while universal coating for protein attachment. Considering the long-standing ambiguity on protein immobilization on PDA, in this work, we systematically studied the interaction of proteins with PDA film by using surface plasmon resonance (SPR) combined to fluorescent protein microarray. SPR is a powerful optical tool for study of protein-surface interactions with the inherent advantages of being label-free, real time, quantitative and sensitive [18–20]. On the other side, fluorescence is one of indispensable approaches in biological research, and particularly it is widely used for quantitative high-throughput detection, such as in fluorescent protein microarray [21,22]. In this work, we investigated the kinetics of protein-PDA interaction and studied the influence of deposition pH and deposition media of PDA film on the protein attachment. We also unveiled the compelling fluorescence quenching property of PDA film, which greatly depends on the film thickness. This work may help clarify the questions on protein-PDA interaction, and offer a useful guide for PDA’s application involved biomolecules. 2. Experimental 2.1. Chemicals and materials Dopamine hydrochloride, Tris(hydroxymethyl)aminomethane (Tris), and 0.01 M phosphate buffered saline (PBS, pH 7.4) were obtained from Sigma-Aldrich. Other chemicals and reagents were purchased from Aladdin (China). All chemicals were used as received without further purification. All solutions are prepared with deionized (DI) water from a Millipore water purification system with a resistivity of 18.2 MX cm. Tris buffer and phosphate buffer (PB) were used in these experiments and their pH value was adjusted if necessary.

hydrochloride in Tris buffer or PB buffer (pH 6.4, 7.4 and 8.5, adjusted with HCl or NaOH). It is worth noting that for PDA deposition on SPR chip, the growing duration is 20 min for pH 8.5 and 20 min  3 (repeating three times) for pH 7.4 and 6.4. After the growth, the substrate was rinsed with DI water to remove the loosely absorbed PDA deposits and dried by gentle nitrogen flow for subsequent experiments. 2.3. SPR measurements SPR measurements were conducted using a BI-4000 instrument (Biosensing Instruments, Tempe, AZ, USA), which is based on Kretschmann configuration and coupled with a monochromatic ppolarized laser (670 nm) as the light resource. The SPR chip was separated as two channels, namely, sensing channel and reference channel and the SPR signals from these two channels were simultaneously collected at a frequency of 10 Hz. All reported SPR data were obtained by subtracting the reference (background) signal from the sensing signal to eliminate the possible disturbance from environment. A flow injection system based on BI-DirectFlowTM Technology was used to deliver the sample solution onto the chip surface with near-zero dispersion. During the detection, 0.01 M PBS was first flowed through the sensing surface at a constant rate of 3.0 lL min
1 to obtain a stable baseline, followed by the injection of protein solution (in 0.01 M PBS) via a sample loop to the sensing surface at the same flowing rate, followed by rinsing with 0.01 M PBS buffer. The surface density of adsorbed proteins was calculated by the increase in the SPR response (micro-degree, m°) measured in PBS solution before and after protein solution injection. 2.4. Printing of protein microarray Fluorescent protein microarrays were printed on PDA-coated glass slides by using Cy3-conjugated anti-Rabbit IgG was used as a fluorescent ink. The fluorescent protein was diluted with a printing buffer (0.01 M PBS with 10% glycerol and 0.005% Triton X-100) to a final concentration of 100 lg mL
1 and then printed with a Personal ArrayerTM16 system (CapitalBio Corporation, Beijing, China) under contacting mode [21–23]. The surfactant Triton X-100 in the ink is able to suppress the coffee-ring effect for uniform protein attachment and its presence does not influence the stability of protein at the concentration used according to previous work [23–25]. After printing, the slides were kept in dark for overnight incubation and washed gently with TBST (0.05 M Tris, 0.138 M NaCl, Ò 0.0027 M KCl and 0.05% Tween 20, pH 8.0) to remove unbound proteins. After drying, the slides were immediately imaged with LuxScanTM 10K-A Microarray Scanner (CapitalBio Corporation, Beijing, China) and the fluorescent images were obtained at 532 nm excitation. The obtained images were analysed to obtain the statistical fluorescent intensities. 2.5. Characterizations X-ray photoelectron spectra (XPS) of PDA films deposited on silicon were collected by using an ESCALAB 250Xi system from Thermo Scientific. Atomic force microscopy (AFM) images were collected using a Nanoman AFM (Veeco metrology group, USA) in tapping mode.

2.2. Deposition of PDA films

3. Results and discussion

PDA film was grown on SPR gold chip or standard glass slides by immersing the substrate into a freshly prepared PDA growing solution at ambient atmosphere for certain time. The PDA growing solution was prepared by dissolving 2.0 mg mL
1 dopamine

3.1. Thickness and roughness of PDA films Various methods have been explored for growing PDA films on a wide variety of substrates and the film growth kinetics has also

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been investigated by different groups [16,26]. In this work we particularly concern the PDA thickness because (1) the measurable SPR angle range of our instrument is from 70 to 76 degree, and thick PDA film results in high SPR angle and beyond this range; (2) SPR is intrinsically sensitive only to the local refractive index change near the solid/liquid interface due to the limited penetration depth of surface plasmon wave (SPW) [19,20]. If a thick PDA film was grown, it will block the SPW and the SPR will lose its sensitivity to sense the RI change occurring on the PDA surface. In order to ensure that the PDA film is thin enough to allow for sensitive SPR measurements, SPR simulation based on Fresnel equation was first carried out to determine the highest thickness of PDA film [19,20]. The simulated SPR plots (reflectivity vs. incident angle) for a four-layer system (prism/gold film/PDA film/aqueous solution, with the refractive index of 1.514, 0.1372 + 3.7852i, 1.5, and 1.333 @ 670 nm, respectively) with varying PDA film thicknesses were shown in Fig. 1, from which it is observed that the SPR angle (angle with minimal reflectivity) linearly increases from 70.0545 to 85.4838° when the PDA thickness increases from 0 to 60 nm. Meanwhile, for the thickness of 40 nm or higher, both the minimal reflectivity (resonance depth) and curve shape (resonance width) distinctly increase as well, suggesting the decrease of SPR signal-to-noise ratio toward the local RI change [19,20]. Therefore, to ensure reliable SPR study, the thickness of PDA film was controlled under 20 nm by using the short-immersion method to deposit PDA film on SPR chip [15]. In details, the deposition duration was controlled at 20 min at pH 8.5, which produces PDA film with a thickness of around 5 nm according to previous report [15]. For film deposited at lower pH, the immersion step was repeated for three times (denoted as 20 min  3) to ensure the growth of conformal and complete PDA coating. It has been reported that in fresh dopamine solution the PDA thickness increases linearly during the initial stage, after which the growth slows down and a lot of PDA nanoparticles formed in the solution deposits on the surface [15]. In present work this short-immersion method was used to ensure the PDA’s thickness and smoothness. XPS is able to provide chemical information of a surface. As shown in Fig. 2a, the XPS survey spectra of three PDA films deposit in phosphate buffer at different pH clearly show the peaks originated from C1s, O1s, and N1s, suggesting the successful deposition of PDA films. High resolution C1s spectra were further collected and shown in Fig. 2b–d, which are peak-fitted into three components corresponding to CAH/CAC (284.6 eV), CAO/CAN (286.0 eV), and C@O (288.5 eV) [15]. The C@O peak at 288.5 eV

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could be partially attributed to the oxidized quinone form in PDA film, while that at 284.6 eV corresponds to CAC/CAH groups. As the protein attachment on a surface is largely influenced by the surface roughness, we investigated the surface morphologies of three PDA films. As shown as AFM images in Fig. 3, the bare silicon shows a Root Mean Square roughness (Rq) of 0.412 nm. After the PDA deposition, the roughness increases slightly to a Rq value of 0.887, 0.965 and 0.842 nm, respectively for film deposited at pH 6.4, 7.4, and 8.5. All Rq values are calculated by using the AFM software. The surface morphology and roughness of three PDA films are similar with one another, possibly suggesting that the pH value does not greatly influence the PDA film rough in initial growth stage and short immersion method is suitable to prepare smooth PDA films [15]. 3.2. Kinetics of protein attachment on PDA film By using BSA as a model protein, we first investigated the kinetics of protein attachment on PDA thin film prepared in Tris buffer at pH 8.5 for 20 min growth. As shown in Fig. 4a, when BSA solution with different concentration flows onto the PDA surface, SPR angle dramatically increases at the initial stage, and after that the increase slows down until reaching a plateau after 2900 s incubation. According to the SPR principle, the SPR angle change is proportionally correlated to the protein attached on the surface with a ratio of 1 ng mm
2 per 120 m° and the final surface density of BSA on the PDA film was calculated to be 2.5, 2.17, and 1.53 ng mm
2 respectively for 100, 50, and 25 lg mL
1 BSA solution after 2900 s reaction [20]. It is worth noting that the small SPR decrease upon buffer changing (from BSA solution to blank PBS) is caused by the difference of refractive index between BSA solution and blank PBS. Due to the quantitative property of SPR measurement, it is possible to study the kinetics of protein attachment on PDA film. A simple tandem reaction could be proposed for protein attachment on a reactive surface, namely, the first step is the adsorption of protein on the surface, followed by reaction between the adsorbed protein and the reactive species on the surface, resulting in covalent binding of the protein via Schiff’s base formation or Michael addition [19,20]. The driving forces of protein adsorption on PDA may include electrostatic attraction, hydrophobic interaction, and/or others [20]. The processing of second reaction results in the shift of adsorption equilibrium in first step and more protein will adsorb/attach on the surface. Generally the adsorption of a substance at a liquid/solid interface can be described by Langmuir adsorption kinetics [19,20]:

RðtÞ ¼ Req 1
e
ðka Cþkd Þt




ð1Þ

where R(t), Req, ka and kd represent the time dependent surface density (or concentration) of adsorbed proteins, the equilibrium value for R at a certain bulk concentration C, the association rate constant, and dissociation rate constant for the adsorption, respectively. If taking into account the equilibrium shift caused by the second step, the kinetics of the whole tandem reaction can be described by Eq. (2)

RðtÞ ¼ Req
A1 e
a1 ðt
t0 Þ
A2 e
a2 ðt
t0 Þ

Fig. 1. SPR reflectivity vs. angle simulation curves of 50 nm gold film coated with PDA film with varying thickness.

ð2Þ

As shown in Fig. 4a, the in situ SPR binding curves were fitted by using Eq. (2) and the fitting curves (circle lines) are in reasonably good agreement with the experimental SPR curves (R2 = 0.9999, 0.9991, and 0.9997 for 20, 50, and 100 lg mL
1, respectively). Fig. 4b shows the plot of BSA concentration vs. final SPR angle shift obtained by systematic SPR experiments. It could be observed that higher concentration of BSA results in higher angle shift, and the SPR shift reach a plateau at concentration higher than

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Fig. 2. XPS survey spectra (a) and high-resolution C1s spectra (b–d) of PDA film deposited in freshly prepared 2 mg mL
1 dopamine solution in phosphate buffer at pH 6.4 (b), pH 7.4 (c), and pH 8.5 (d).

Fig. 3. AFM images of clean silicon (a), PDA film deposited in Tris buffer at pH 6.4 (20 min  3, b), 7.5 (20 min  3, c), and 8.5 (20 min, d). The scanned area is 1 lm  1 lm for each image.

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where Ca represents the final surface concentration of adsorbed protein BSA after reaching an equilibrium, and A0, B0, and K are the concentration of specific sites on the film surface, concentration of BSA in aqueous solution, and equilibrium constant of this adsorption process. In this model no lateral interaction between the adsorbed proteins is taken into account. Fitting the experimental data with Eq. (3) obtains the red curve in Fig. 4b, which is reasonably fitting with the data points. The value of equilibrium constant K is estimated to be 2.8  106 M
1.

8.5), where the dissolved oxygen acts as an oxidant [3]. Although some claimed that the slightly alkaline environment is necessary for PDA growth, it has been confirmed that PDA film could be successfully deposited even if the pH value was tuned to neutral or slightly acidic. Therefore it is quite interesting to investigate the influence of growing pH on the protein attachment of the resulting PDA surface. As shown in Fig. 5a, the in situ SPR binding curves were recorded when a 200 lg mL
1 BSA solution was used as a model protein to interact with PDA films deposited in Tris buffer with different pH value. From the binding curves it is observed that PDA film grown in higher pH value (8.5) exhibits higher protein immobilization capacity. Additional experiments further show the same trend by using 500 lg mL
1 BSA solution (Fig. 5b). Besides Tris solution, other solution such as PB buffer was also widely used for PDA polymerization. As shown in Fig. 5c and d, when PB buffer was used for PDA growth, the pH value shows the same trend as for Tris buffer, i.e., PDA deposited in higher pH possesses higher protein immobilization capacity. The SPR angle shifts caused by BSA solution with different concentration on different PDA films were plotted and shown in Fig. 6. It is clearly unveiled that for a given type of buffer, the SPR shift either for 200 or 500 lg mL
1 BSA increases with the increase of pH value. More interestingly, it is shown that for 200 lg mL
1 BSA, the protein attachment capacity of PDA films deposited in Tris buffer, regardless its pH value, is higher than that in PB buffer; however, for 500 lg mL
1 BSA solution, the opposite trend, i.e., higher protein attachment capacity was found on PDA films deposited in PB buffer. This may implies that both the equilibrium constant K (as in Eq. (3)) and the concentration of specific sites (i.e., A0 in Eq. (3)) for PDA films deposited in PB buffer are higher than these in Tris buffer. There is small difference in protein affinity for two PDA films. It is not surprising if we consider the fact that Tris buffer contains abundant amine groups due to the presence of Tris(hydroxymethyl)aminomethane component, which may compete with the protein to covalently bind to PDA film and consume active (quione) groups. It is reported that there is an equilibrium between catechol and quinone state in PDA film and the quinone groups is believed to possess the intrinsic ability to participate in nucleophilic addition reactions with the sulfhydryl or amino groups of proteins, enabling covalent binding of proteins [9,11,27]. Therefore these results possibly suggest that higher media pH shifts the catechol/quinone equilibrium toward quinone, which contributes to bioconjugation reactions and thus more protein could be attached. Considering the presence of catechol/quinone equilibrium in PDA film, the stability of PDA film in term of protein immobilization capacity was investigated. According to our SPR investigation (data not shown), the protein immobilization capacity of PDA film, regardless deposited either in Tris buffer or PB buffer at pH 8.5, shows negligible change upon immersing in PBS or storing in ambient atmosphere for 24 h, which suggests that the reactive quinone groups on PDA surface is quite stable and the catechol/quinone equilibrium in PDA film is not significantly deviated from its thermodynamic equilibrium point in response to environmental change. The catechol/quinone equilibrium in PDA film is mainly determined by the polymerization condition, and mild postpolymerization process cannot significantly shift this equilibrium. Further research is ongoing in our lab to chemically/electrochemically modulate the reactivity of PDA film.

3.3. Effect of growing pH and media on the protein attachment

3.4. Fluorescence quenching on PDA films

Various methods have been established to grow PDA films and the most used one is to initiate spontaneous self-polymerization of dopamine in Tris solution buffered at slightly alkaline pH (e.g., pH

To further investigate the protein-PDA film interaction, fluorescent protein microarray was printed by spotting fluorescent antibody on PDA-modified glass slides, and the fluorescent intensity

Fig. 4. (a) Representative in situ SPR binding curves (solid lines) and fitting curves (circles) of BSA solution with different concentration in 0.01 M PBS on PDA film deposited in Tris buffer at pH 8.5; (b) plot of SPR angle shift vs. BSA concentration (black points) and fitting curves (solid red line). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

200 lg mL
1. It is worth noting that the SPR measurement possess high reproducibility with less 5% relative standard derivation. Although only one SPR measurement was carried out for each concentration in Fig. 4b, the trend is reliable. According to the Langmuir adsorption, the surface coverage (or concentration) of the adsorbed protein at equilibrium could be obtained by Eq. (3) as follows.

C a ¼ A0 B0 K=ð1 þ B0 KÞ

ð3Þ

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Fig. 5. In situ SPR binding curves of 200 (a and c) or 500 lg mL
1 (b and d) BSA in 0.01 M PBS on PDA film deposited on SPR gold chips in freshly prepared 2 mg mL
1 dopamine in Tris buffer (a and b) or PB buffer (c and d). The arrows indicate the moments at which the solution was changed from 0.01 M PBS to BSA solution, or reversely.

Fig. 6. SPR angle increases upon BSA attachment on PDA films deposited in PB buffer or Tris buffer with different pH value. The concentration of BSA solution is 200 or 500 lg mL
1 in 0.01 M PBS, and the incubation time is ca. 2900 s.

of resultant microarray was measured for quantitative comparison. As shown as the representative fluorescence images in Fig. 7a–c, all the 5  5 fluorescent microarrays show regular roundish shape and excellent homogeneity in fluorescent intensity with low spot-to-spot deviation, indicating that the attachment density of

fluorescent protein on each spot is spatially homogeneous, and thus PDA film is very suitable for protein microarray as a universal functional layer. Fluorescent quantitation (Fig. 7d) suggests that the fluorescent intensity of microarray decreases with the pH value of PB buffer used for PDA growth, which is sharply contradictory to the SPR measurements discussed above (i.e., Figs. 5 and 6). Further experiments show that for fluorescent microarray printed on PDA film deposited in PB buffer at pH 7.4, the fluorescent intensity decreases with the deposition time (Fig. 8, entry a–d). By using GPTS-modified glass slide, a commercial available microarray substrate with reactive epoxy as a reference (entry g), it is found that the fluorescent intensity for microarray on PDA film with short deposition time (e.g., 20 min  3) is comparable or even higher than that on GPTS-modified slides when a same fluorescent ink was used, suggesting great potential of PDA thin film for fluorescent protein microarray [23,24]. With prolonged deposition time (6, 12, and 24 h), however, the fluorescent intensity decreases dramatically to less than 20% of original intensity. For PDA film grown in Tris buffer, the same trend was found (entry e, f in Fig. 8). We speculate that these observations are caused by the fluorescence quenching effect and thickness-dependent quenching efficiency of PDA film. It has been confirmed that PDA contains a large number of conjugated polymer chains with macrocycles, and colloidal PDA microsphere has been used as an excellent fluorescence quencher for homogeneous detection of biomolecules [28,29]. Although PDA film exhibits background fluorescence

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4. Conclusion In summary, PDA-protein interaction was systematically studied by using SPR and fluorescent microarray. The kinetics of protein immobilization on PDA film was in situ investigated. The protein loading capacity of PDA film was found to be slightly influenced by both the buffer pH and buffer type used for film growth, and higher pH results in slightly higher protein immobilization capacity. PDA film was stable in term of protein attachment even if immersed in PBS for 24 h. The fluorescence of a labelled protein was significantly quenched (up to 80% quenching efficiency) when attached on thick PDA film. This work may help clarify the longstanding ambiguity on protein immobilization on PDA and deepen our understanding on the PDA-protein interaction, and also offer a valuable guide for efficient protein attachment on PDA film in various bio-related applications. Acknowledgement We would like to gratefully acknowledge the financial support from National Natural Science Foundation of China (No. 21273173), Fundamental Research Funds for the Central Universities (XDJK2015B014), and Chongqing Key Laboratory for Advanced Materials and Technologies of Clean Energies. Fig. 7. Fluorescent image (a–c) and fluorescent intensity (d) of antibody microarray printed on PDA film, which was deposited on glass slides in freshly prepared 2 mg mL
1 dopamine in phosphate buffer at pH 6.4 (a), 7.4 (b) or 8.5 (c) for 20 min  3, all scale bars are 500 lm in a–c.

Fig. 8. Fluorescent intensity of antibody microarray printed on PDA-modified glass (entry a–f) and on GPTS-modified glass (entry g). PDA film was prepared by immersing glass slides in 2 mg mL
1 dopamine solution in phosphate buffer for 20 min  3 (a), 6 (b), 12 (c), 24 h (d) or in Tris buffer for 20 min  3 (e), and 12 h (f), respectively.

comparable to that of clean glass slides under present imaging condition, PDA polymer has also been explored as biocompatible fluorescent label for cell imaging and as a unique dye in dye-sensitized solar cell due to the presence of conjugated polymer chains [30– 33]. Therefore, it is not surprising that PDA film is able to quench the fluorescence of neighbouring fluorophores via possible energy transfer and/or electron transfer process. In present work, the quenching efficiency of PDA film seems to be positively correlated to the thickness of PDA film, possibly due to the density of conjugated chain in PDA film increases with film thickness. Higher pH value (as in Fig. 7) or longer deposition time (as in Fig. 8) results in thicker PDA film, and in turn higher quenching efficiency and lower fluorescent intensity.

References [1] K.H.A. Lau, C. Ren, S.H. Park, I. Szleifer, P.B. Messersmith, An experimental– theoretical analysis of protein adsorption on peptidomimetic polymer brushes, Langmuir 28 (2012) 2288–2298. [2] Y. Dang, C.-M. Xing, M. Quan, Y.-B. Wang, S.-P. Zhang, S.-Q. Shi, Y.-K. Gong, Substrate independent coating formation and anti-biofouling performance improvement of mussel inspired polydopamine, J. Mater. Chem. B 3 (2015) 4181–4190. [3] H. Lee, S.M. Dellatore, W.M. Miller, P.B. Messersmith, Mussel-inspired surface chemistry for multifunctional coatings, Science 318 (2007) 426–430. [4] H. Chen, L. Zhao, D. Chen, W. Hu, Stabilization of gold nanoparticles on glass surface with polydopamine thin film for reliable LSPR sensing, J. Colloid Interf. Sci. 460 (2015) 258–263. [5] W. Hu, G. He, H. Zhang, X. Wu, J. Li, Z. Zhao, Y. Qiao, Z. Lu, Y. Liu, C.M. Li, Polydopamine-functionalization of graphene oxide to enable dual signal amplification for sensitive surface plasmon resonance imaging detection of biomarker, Anal. Chem. 86 (2014) 4488–4493. [6] H. Zhang, X. Liu, G. He, X. Zhang, S. Bao, W. Hu, Bioinspired synthesis of nitrogen/sulfur co-doped graphene as an efficient electrocatalyst for oxygen reduction reaction, J. Power Sour. 279 (2015) 252–258. [7] M.S. Akin, M. Yilmaz, E. Babur, B. Ozdemir, H. Erdogan, U. Tamer, G. Demirel, Large area uniform deposition of silver nanoparticles through bio-inspired polydopamine coating on silicon nanowire arrays for practical SERS applications, J. Mater. Chem. B 2 (2014) 4894–4900. [8] N. Holten-Andersen, A. Jaishankar, M.J. Harrington, D.E. Fullenkamp, G. DiMarco, L. He, G.H. McKinley, P.B. Messersmith, K.Y.C. Leei, Metalcoordination: using one of nature’s tricks to control soft material mechanics, J. Mater. Chem. B 2 (2014) 2467–2472. [9] H. Lee, J. Rho, P.B. Messersmith, Facile conjugation of biomolecules onto surfaces via mussel adhesive protein inspired coatings, Adv. Mater. 21 (2009) 431–434. [10] S. Hong, Y.S. Na, S. Choi, I.T. Song, W.Y. Kim, H. Lee, Non-covalent self-assembly and covalent polymerization co-contribute to polydopamine formation, Adv. Funct. Mater. 22 (2012) 4711–4717. [11] D.R. Dreyer, D.J. Miller, B.D. Freeman, D.R. Paul, C.W. Bielawski, Elucidating the structure of poly(dopamine), Langmuir 28 (2012) 6428–6435. [12] J. Liebscher, R. Mrowczynski, H.A. Scheidt, C. Filip, N.D. Hadade, R. Turcu, A. Bende, S. Beck, Structure of polydopamine: a never-ending story?, Langmuir 29 (2013) 10539–10548 [13] Y. Ding, L.-T. Weng, M. Yang, Z. Yang, X. Lu, N. Huang, Y. Leng, Insights into the aggregation/deposition and structure of a polydopamine film, Langmuir 30 (2014) 12258–12269. [14] C. Zhang, Y. Ou, W.-X. Lei, L.-S. Wan, J. Ji, Z.-K. Xu, CuSO4/H2O2-induced rapid deposition of polydopamine coatings with high uniformity and enhanced stability, Angew. Chem. Int. Ed. 55 (2016) 3054–3057. [15] R.A. Zangmeister, T.A. Morris, M.J. Tarlov, Characterization of polydopamine thin films deposited at short times by autoxidation of dopamine, Langmuir 29 (2013) 8619–8628. [16] F. Bernsmann, V. Ball, F. Addiego, A. Ponche, M. Michel, J.J. de Almeida Gracio, V. Toniazzo, D. Ruch, Dopamine-melanin film deposition depends on the used oxidant and buffer solution, Langmuir 27 (2011) 2819–2825.

130

D. Chen et al. / Journal of Colloid and Interface Science 477 (2016) 123–130

[17] R. Ouyang, J. Lei, H. Ju, Surface molecularly imprinted nanowire for protein specific recognition, Chem. Commun. 44 (2008) 5761–5763. [18] W. Hu, Y. Liu, Z. Lu, C.M. Li, Poly oligo(ethylene glycol) methacrylate-coglycidyl methacrylate Brush Substrate for Sensitive Surface Plasmon Resonance Imaging Protein Arrays, Adv. Funct. Mater. 20 (2010) 3497–3503. [19] W. Hu, Z. Lu, Y. Liu, C.M. Li, In situ surface plasmon resonance investigation of the assembly process of multiwalled carbon nanotubes on an alkanethiol selfassembled monolayer for efficient protein immobilization and detection, Langmuir 26 (2010) 8386–8391. [20] W.H. hu, C.M. Li, X. Cui, H. Dong, Q. Zhou, In situ studies of protein adsorptions on poly(pyrrole-co-pyrrole propylic acid) film by electrochemical surface plasmon resonance, Langmuir 23 (2007) 2761–2767. [21] W. Hu, Y. Liu, T. Chen, Y. Liu, C.M. Li, Hybrid ZnO nanorod-polymer brush hierarchically nanostructured substrate for sensitive antibody microarrays, Adv. Mater. 27 (2015) 181–185. [22] W. Hu, X. Li, G. He, Z. Zhang, X. Zheng, P. Li, C.M. Li, Sensitive competitive immunoassay of multiple mycotoxins with non-fouling antigen microarray, Biosens. Bioelectron. 50 (2013) 338–344. [23] W. Hu, Y. Liu, Z. Zhu, H. Yang, C.M. Li, Randomly oriented ZnO nanorods as advanced substrate for high-performance protein microarrays, ACS Appl. Mater. Interf. 2 (2010) 1569–1572. [24] W. Hu, Y. Liu, H. Yang, X. Zhou, C.M. Li, ZnO nanorods-enhanced fluorescence for sensitive microarray detection of cancers in serum without additional reporter-amplification, Biosens. Bioelectron. 26 (2011) 3683–3687. [25] Y. Deng, X.Y. Zhu, T. Kienlen, A. Guo, Transport at the air/water interface is the reason for rings in protein microarrays, J. Am. Chem. Soc. 128 (2006) 2768– 2769.

[26] F. Bernsmann, A. Ponche, C. Ringwald, J. Hemmerle, J. Raya, B. Bechinger, J.-C. Voegel, P. Schaaf, V. Ball, Characterization of dopamine-melanin growth on silicon oxide, J. Phys Chem. C 113 (2009) 8234–8242. [27] R. Luo, L. Tang, S. Zhong, Z. Yang, J. Wang, Y. Weng, Q. Tu, C. Jiang, N. Huang, In vitro investigation of enhanced hemocompatibility and endothelial cell proliferation associated with quinone-rich polydopamine coating, ACS Appl. Mater. Interf. 5 (2013) 1704–1714. [28] W. Qiang, W. Li, X. Li, X. Chen, D. Xu, Bioinspired polydopamine nanospheres: a superquencher for fluorescence sensing of biomolecules, Chem. Sci. 5 (2014) 3018–3024. [29] Q. Liu, Z. Pu, A.M. Asiri, A.O. Al-Youbi, X. Sun, Polydopamine nanospheres: a biopolymer-based fluorescent sensing platform for DNA detection, Sens. Actuat. B – Chem. 191 (2014) 567–571. [30] H.J. Nam, B. Kim, M.J. Ko, M. Jin, J.M. Kim, D.-Y. Jung, A new mussel-inspired polydopamine sensitizer for dye-sensitized solar cells: controlled synthesis and charge transfer, Chem. Eur. J. 18 (2012) 14000–14007. [31] X. Zhang, S. Wang, L. Xu, L. Feng, Y. Ji, L. Tao, S. Li, Y. Wei, Biocompatible polydopamine fluorescent organic nanoparticles: facile preparation and cell imaging, Nanoscale 4 (2012) 5581–5584. [32] X. Chen, Y. Yan, M. Muellner, M.P. van Koeverden, K.F. Noi, W. Zhu, F. Caruso, Engineering fluorescent poly(dopamine) capsules, Langmuir 30 (2014) 2921– 2925. [33] A. Yildirim, M. Bayindir, Turn-on fluorescent dopamine sensing based on in situ formation of visible light emitting polydopamine nanoparticles, Anal. Chem. 86 (2014) 5508–5512.