Protein adsorption behavior on reduced graphene oxide and boron-doped diamond investigated by electrochemical impedance spectroscopy

Carbon 152 (2019) 354e362

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Protein adsorption behavior on reduced graphene oxide and boron-doped diamond investigated by electrochemical impedance spectroscopy Yixuan Huang a, Aiga Hara b, Chiaki Terashima b, Akira Fujishima b, Madoka Takai a, * a b

Department of Bioengineering, The University of Tokyo, Tokyo, 113-8656, Japan Photocatalysis International Research Center, Tokyo University of Science, Chiba, 278-8510, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 April 2019 Received in revised form 20 May 2019 Accepted 5 June 2019 Available online 8 June 2019

As advanced and promising carbon materials, we found reduced graphene oxide (rGO) and boron-doped diamond (BDD) demonstrated special impedance response regarding protein adsorption compared with other materials. According to the results of electrochemical impedance spectroscopy (EIS), rGO showed two time constants (two semicircles in Nyquist plot) when exposed to model protein bovine serum albumin (BSA) solutions; BDD showed decreased impedance response when exposed to high concentration of BSA. They demonstrated special but different impedance response upon BSA adsorption. Based on fitting results of EIS data and hypothesis of protein adsorption, protein adsorption model on the interface of protein solution and carbon electrode surfaces have been established, which is applicable to rGO and BDD surface, and is further expected to be applicable to other carbon materials. Besides the indepth understanding of protein adsorption behavior, this study also indicates a potential way to control the orientations of protein molecules on solid surface. © 2019 Elsevier Ltd. All rights reserved.

1. Introduction Carbon materials have been extensively explored due to their favorable mechanical, electrical, electrochemical, optical, chemical and thermal properties. Numerous carbon materials have been designed and investigated for diverse areas such as composite materials [1], electroanalysis [2], energy conversion and storage [3], sensors [4] and biosensors [5], adsorption of molecules and ions [6,7], drug delivery systems [8], field emission devices [9]. Among novel carbon materials, reduced graphene oxide (rGO) and borondoped diamond (BDD) have emerged as attractive ones. Owing to the unique structural and electronic properties, rGO has been utilized in varies of electrochemical applications, such as sensor [10,11], fuel cell supports [12], supercapacitance [13,14]. Moreover, it has not been until recently that rGO and BDD are explored as novel biocompatible materials. The rich functional groups on the rGO surface enable protein molecules to efficiently adsorb onto rGO surface from biological fluids. It is believed that rGO interacts with protein molecules through hydrophobic interaction, electrostatic

* Corresponding author. E-mail address: [email protected] (M. Takai). https://doi.org/10.1016/j.carbon.2019.06.023 0008-6223/© 2019 Elsevier Ltd. All rights reserved.

forces, hydrogen bonding and p
p stacking interaction due to the typical sp2 carbon structure [15]. More recently, BDD electrodes have been used in advanced biosensing applications due to its less biofouling and lower capacitance [16,17]. Contrary to rGO of sp2carbon, BDD of sp3-carbon structure is expected to resist protein adsorption on BDD surface from biological fluids [18]. Considering the opposite property of rGO and BDD regarding protein adsorption, the comparative analysis of protein adsorption behavior on rGO and BDD is expected to be of great significance. It is believed that any foreign material in contact with biological fluids will experience protein adsorption immediately. Protein adsorption behavior at solution/solid interface is known to be very complicated and have attracted considerable interest due to the application in chemical and medical industries [19], which has been studied using varies of techniques such as quartz crystal microbalance [20], infrared spectroscopy [21], radiolabeling, and electrochemical methods [22,23]. Among these technologies, although unable to directly measure the amount of adsorbed protein, electrochemical impedance spectroscopy (EIS), a label-free, real-time and in-situ technique, can provide more information about the processes occurring at surfaces, especially from the abnormal Nyquist plots. Generally, Nyquist plots are fitted to an Equivalent

Y. Huang et al. / Carbon 152 (2019) 354e362

Electrical Circuit (EEC) to extract quantitative information on the solution/electrode interface [24]. Some researchers have focused on the phenomenon of protein adsorption on rGO surface [15,25,26], but rare of them took advantage of EIS analysis. We reported an indepth illustration of protein adsorption behavior on carbon materials through comparative investigation of rGO and BDD by powerful EIS analysis for in-situ tests, and X-ray photoelectron spectroscopy (XPS) for ex-situ tests. In this research, bovine serum albumin (BSA) was selected as the model protein since its biophysical structures have been commonly studied. In the study, we found rGO and BDD demonstrated special impedance response regarding protein adsorption compared with other materials. Taking advantage of these unique phenomena is potential to be a breakthrough and provide a new perspective for the study of protein adsorption process. 2. Experimental 2.1. Materials Phosphate buffered saline (PBS, pH 7.4), sodium dodecyl sulfate (SDS), and sodium sulfate (Na2SO4) were purchased from Wako Pure Chemicals Industries (Osaka, Japan). Potassium ferricyanide (K3[Fe(CN)6]), potassium ferrocyanide (K4[Fe(CN)6]), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich, USA. Graphite was purchased from CoorsTek, Inc., Tokyo, Japan. Glassy carbon plates were purchased from ALS Co., Ltd., Tokyo, Japan. 2.2. Electrode preparation Polycrystalline boron-doped diamond thin films were grown on Si (111) substrates using a microwave plasma chemical vapor deposition (CVD) system equipped with a microwave plasma CVD reactor (Cornes Technologies Ltd., Model AX6500). The details preparation have been described previously [27,28]. Graphene oxide was synthesized from graphite by the modified Hummers' method [14]. All electrochemical experiments were performed on an electrochemical analyzer (ALS Co., Ltd., Tokyo, Japan). For fabrication of rGO modified glassy carbon electrodes (GCEs), firstly, GCEs (d ¼ 3 mm, from ALS Co., Ltd.) were polished with 0.3 mm and 0.05 mm Alumina slurry respectively then rinsed and sonicated in deionized (DI) water for 5 min. Graphene oxide (GO) modified GCE (GO/GCE) was prepared by casting 10 ml GO dispersion (0.2 mg/ml) onto the cleaned GCE surface and allowed to dry in mild condition. Then, GO was reduced to rGO through electrochemical reduction by recording 10 times of cyclic voltammetry in a potential range from 0 V to
1.5 V in 0.5 M Na2SO4 solution [29], followed by thoroughly rinsing with DI water, and the obtained electrode was denoted as rGO/GCE. The BDD plates (boron doping level:2.1  1021 cm
3, resistivity: 10
3 U cm) were electrically connected with copper wires by tin and packaged by epoxy glue to prepare electrochemical measurable BDD electrodes [30]. During the protein adsorption tests, fouled BDD electrodes were recovered by 10 min ultrasonication in 1 wt% of SDS solution.

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with 45 mg/ml BSA in PBS (pH 7.4) at room temperature for 24 h (BSA-rGO and BSA-BDD). After the treatments, the plates were rinsed with DI water and vacuum dried overnight. Then, XPS measurements were performed to record the core-level spectra of bare-rGO, bare-BDD, BSA-rGO and BSA-BDD. Atomic concentration ratios of respective plates were calculated through peak intensity of C 1s, N 1s, and O 1s. 2.4. Electrochemical characterization The electrochemical measurements were conducted in PBS (pH 7.4) with 5 mM K4[Fe(CN)6]/K3[Fe(CN)6] (1:1) as a redox probe. Na2SO4 was dissolved with DI water at the concentration of 0.5 M. Effects of protein adsorption were simulated by adding specific amounts of BSA to the PBS. All electrochemical measurements were obtained using ALS electrochemical analyzer with AgjAgCl as a reference electrode and Pt wire as a counter electrode. Measurements were repeated at least three times to confirm reproducibility. Cyclic voltammograms (CVs) were performed at the scan rate of 100 mV/s for all the experiments. Electrochemical impedance spectroscopies (EISs) were performed in buffer solution in the absence and presence of specific concentrations of BSA. The EISs were run at open circuit potential, the equilibrium potential of the redox couple, using a frequency range from 0.1 MHz to 0.01 Hz with 5 mV alternating voltage. EISs were set to run consecutively to record the continuous change of impedances. 2.5. EEC fitting To model protein adsorption at the electrode surface, the most commonly used EEC is the modified Randles circuit with only one time constant (resistor and capacitor in parallel), shown in Fig. 1. There is a solution resistance (Rs) to model the migration of charge through the solution, a double-layer capacitance (Cdl) that models the electric double layer on the electrode surface, a charge-transfer resistance (Rct) to model the charge-transfer reaction, and a Warburg impedance (W) which models the diffusion process. Although researchers adopted the circuits with a second time constant in their studies to model the protein adsorption process [24,31], their EIS data just demonstrated only one time constant, thus the fitting results were not convincing. However, different from other materials, rGO showed two time constants (two semicircles in Nyquist plot) when exposed to model protein BSA solutions. It is appropriate to utilize two-time-constant equivalent circuit to study protein adsorption behavior on rGO surface, and more quantitative information can be extracted from complicated EECs. In this study, based on fitting results of EIS data and hypothesis of the protein adsorption process, protein adsorption models for rGO and BDD surfaces have been established respectively with the extended EECs based on the Randles circuit. All impedance diagrams were fitted with the equivalent circuits

2.3. Surface characterization using XPS and SCA The rGO modified glassy carbon plates and BDD plates were used as substrates for X-ray photoelectron spectroscopy (XPS, JPS9010MC, JEOL, Tokyo, Japan) and static contact angle (SCA, CA-W automatic contact angle meter, Kyowa Interface Science, Saitama, Japan) measurements. For XPS measurements, the plates were treated in PBS (pH 7.4) at room temperature for 24 h (bare-rGO and bare-BDD), or treated

Fig. 1. Typical equivalent circuit used for the simulation of protein adsorption process on electrode surface.

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by the zView program. The square of the standard deviation between the original data and the calculated spectrum was less than 0.0001. Moreover, the weighted Sum of Squares, which is proportional to the average percentage error between the original data points and the fitting values, was less than 0.005. 3. Results and discussion

Table 1 Atomic concentration ratios of different plates. Samples

Bare-rGO BSA-rGO Bare-BDD BSA-BDD BSA (theor.)

Atomic concentration ratios (%) C 1s

N 1s

O 1s

92.33 68.2 98.57 84.44 64

0 13.22 0 4.79 17

7.67 18.58 1.43 10.77 19

3.1. Surface characterizations using XPS and SCA XPS measurements were performed to record the spectra of C 1s, N 1s, O 1s, S 2p3/2 and B 1s of different plates. However, none of the plates showed the signal of S 2p3/2 and B 1s, indicating the extremely low content of S in BSA and B in BDD. Therefore, S and B can be neglected when considering the surfaces’ composition. Fig. 2 shows the spectra of C 1s, N 1s and O 1s of bare-rGO, BSA-rGO, bareBDD and BSA-BDD plates, respectively. Major changes in the surface composition of rGO and BDD plates took place upon treatment with BSA. The appearance of a new N 1s signal indicates the existence of BSA on the plates. Moreover, the decreased C 1s signal and increased O 1s signal also indicates the occurrence of BSA adsorption on the plates. Table 1 shows the atomic concentration ratios of different plates calculated from peak intensities. Theoretical values of BSA's elemental composition were calculated from BSA's molecular formula (C2935H4617N781O898S39). The featured elemental composition of BSA was estimated to be C (64%), N(17%), O(19%), which was also in consistent with experimental results of other studies that characterize BSA's elemental composition by XPS [32,33]. According to the increased ratio of N element, the coverage rates of irreversible BSA adsorption on rGO and BDD plates are estimated to be 76.4% and 27.7%, respectively. Adsorbed BSA on rGO surface is about three times as much as that on BDD surface, indicating the huge difference between rGO and BDD surface regarding protein adsorption. The SCAs of water on rGO and BDD were 75.6 and 89.8 , respectively. It is well known that the effect of wettability of a surface is an important parameter in protein adsorption [34]. Generally, hydrophilic surfaces tend to better resist protein

adsorption than hydrophobic surfaces. However, this comment does not support the XPS results in this study. BDD surface is more hydrophobic than rGO surface, whereas protein adsorption on BDD surface is much less than that on rGO surface. During the process of protein adsorption on surfaces, hydrophobic interactions, electrostatic attraction, van der Waals’ forces, and hydrogen bonding have been demonstrated as the most important driving forces [35]. When the synergic interactions listed above are higher than the entropic and hydration repulsions for a surface, protein molecules tend to adsorb on the surface. This accounts for the higher amount of protein adsorption on rGO surface than BDD surface. BDD shows less protein adsorption due to its sp3 structure which has relatively weak interactions (only hydrophobic interaction) with protein molecules. However, owing to the sp2 carbon structure and rich surface chemistry on rGO, protein adsorption can be triggered through hydrophobic interaction, electrostatic force, hydrogen bonding and p
p stacking interaction between rGO and protein molecules. The XPS results indicate that rGO and BDD may have different protein adsorption behavior on the solution-solid interface, and it is further investigated through EIS measurements. 3.2. Impedance response of rGO The EIS data of rGO/GC electrode exposed to different concentration of BSA was recorded. Representative Nyquist plots of rGO without protein adsorption (Fig. 3a) and rGO exposed to various concentration of BSA (Fig. 3b) are shown with equivalent fit in the

Fig. 2. C 1s, N 1s and O 1s spectra of rGO/GC and BDD plates after 24 h incubation in PBS with and without BSA. (A colour version of this figure can be viewed online.)

Y. Huang et al. / Carbon 152 (2019) 354e362

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Fig. 3. a) Nyquist plot with the equivalent fit of rGO in the blank PBS solution containing 5 mM Fe(CN)64
/Fe(CN)3
6 . b) Nyquist plots with the equivalent fit of rGO exposed to different concentration of BSA solutions. (A colour version of this figure can be viewed online.)

frequency range from 0.1 MHz to 0.01 Hz. EIS data of rGO just showed one time constant (one semicircle) in the Nyquist plot in the absence of protein. However, different from other electrode materials, rGO demonstrated second time constant when exposed to protein solution, indicating and confirming the existence of second process on the interface between rGO and protein solution. 3.3. Modeling and fitting of rGO data The rGO surface can efficiently adsorb protein due to rich function groups on its surface and sp2 structure of graphene. The synergic effects of hydrophobic interactions, electrostatic forces, hydrogen bonding, and p
p stacking interaction lead to tight adsorption of a large amount of protein on rGO surface, which is also confirmed by XPS measurements. Therefore, the adsorbed protein on rGO surface may have a severe impact on the charge transfer process, serving as a porous insulating layer on rGO surface. Nyquist plot of rGO surface without any protein adsorption was well fitted with the modified Randles circuit, but when rGO was exposed to protein solution, the second semicircle appeared in Nyquist plot and the modified Randles circuit was no longer applicable in this condition. Therefore, an additional resistor and capacitor in parallel were introduced in the EEC to model the interface of rGO surface/protein solution. The model (Fig. 4a) and corresponding EEC (Fig. 4d) of porous protein layer on the electrode/protein solution interface have been established on the basis of the modified Randles circuit. As is shown in Fig. 4c, charge transfer between electrode and electrolyte is assumed to be blocked on protein adsorbed site, according to the significant increase of charge transfer impedance upon protein adsorption. Protein layer is assumed to be a porous insulating layer and can serve as a capacitive element. In addition, high density of protein adsorption on rGO surface leads to a small area of the interface, thus solution resistance within the interface of the protein layer is not negligible. A solution resistance Ris of interspace of protein layer is introduced to the modified Randles circuit in series with the RC circuit and Warburg impedance (Fig. 4c), because the second process appears in low-frequency range and is overlapped with the Warburg impedance. The electric double layer in the interfacial region of electrode and electrolyte can be modeled as several parallel layers (Fig. 4b). The inner layer called as compact layer and the outer layer that extends to the bulk of solution is the diffuse layer. Total thickness of the double layer should be the sum of the thickness of compact layer and diffuse layer. The compact layer is defined as the locus of centers of the nearest hydrated ions, thus the thickness of the compact layer can be assumed to be lower than 1 nm, since the radius of most of the hydrated ions are less than 1 nm [36,37]. Moreover, the characteristic thickness of diffuse layer

can be calculated from concentration of the bulk electrolyte, since the extension of diffuse layer depends on the ionic strength of the electrolyte. As the concentration of electrolyte (PBS) used in the experiments is relatively high (~0.1 mol/L), the calculated thickness is as low as 0.96 nm [38]. Therefore, the total thickness of the double layer was estimated to be less than 2 nm, which is much lower compared with the size of the protein molecular (BSA: 4 nme14 nm). As charges are either charging the capacitive protein layer or traveling through interspace among adsorbed protein molecules, Cp and Ris were added in series with RC circuit of electrode surface with adsorbed protein molecules. Constant Phase Element (CPE) was incorporated to account for a lack of homogeneity of the electrode surface and protein layer, and the capacitors of Cp and Cdl were implemented as CPEp and CPEdl, as is shown in Fig. 4d. When the EEC is applied to modeling protein adsorption layer on rGO surface, the elements in EEC are given corresponding physical significances. Rs (solution resistance), Rct (charge transfer resistance of interspace of the electrode), CPEdl (electric double layer capacitance of inhomogeneous interspace) and ZW (Warburg impedance of interspace of the electrode) share similar physical significances with that in Randles circuit, while Ris (solution resistance of interspace) and CPEp (capacitance of inhomogeneous protein layer) are introduced to model the second process on rGO surface. EIS data of protein adsorbed rGO has been fitted with EEC in Fig. 4d. Firstly, we utilized Instant Fit method to produce initial values of the elements in different frequency ranges. Before fitting with the whole circuit, all parameters were set to ‘þ, Free’, which means that the best fitting value for the elements should be calculated but constrained to be positive. EIS data has been well fitted using the EEC with a square of the standard deviation as low as 0.00003, indicating the suitability and accuracy of the protein adsorption EEC model. Representative fitting results are shown in Table 2. The values of Cdl and Cp were calculated from the fitting results of CPEdl and CPEp [39].

3.4. Protein adsorption behavior on rGO surface According to the model of adsorption layer in Fig. 4a, the elements in EEC (Fig. 4b) can be characterized by physical parameters of the electrolyte/electrode interface. Ris (Solution resistance of interspace area) and Rct (Charge transfer resistance of interspace on rGO surface) can be expressed as:

Ris ¼

rs d s

;

(1)

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Fig. 4. Schematic of a) model of porous insulating protein layer simplified as cuboids on electrode's surface; model of the compact layer, diffuse layer and diffusion layer on solution/ electrode (e.g. negatively charged electrode) interface without b) and with c) protein adsorption, with corresponding elements labeled in the schematic. d) Schematic of EEC applied to modeling rGO surface without and with protein adsorption. BSA is modeled as blue ellipsoid. (A colour version of this figure can be viewed online.)

Table 2 Parameters obtained by fitting EIS data of rGO exposed to specific amount of BSA. Symbol

Rs/U Cdl/F ndl Rct/U Cp/F np Ris/U

Concentration of BSA (mg/ml) 0

0.045

0.45

4.5

45

69.6 2.12  10
6 0.663 23 n/a n/a n/a

82.1 3.73  10
6 0.659 455 2.19  10
3 0.976 579

63.6 3.48  10
6 0.690 412 1.55  10
3 0.999 739

69.5 2.74  10
6 0.695 450 1.40  10
3 0.983 1210

95.8 2.26  10
6 0.669 514 1.28  10
3 0.985 1257

the resistivity of solution, rct is the charge transfer resistance per unit surface area. After simply rearranging Eq. (1) and Eq. (2), the ratio of Ris to Rct was deduced to be proportional to d. Moreover, rs and rct should be constant during the measurements, so the thickness of protein layer on the electrode surface can be qualitatively derived from Ris/ Rct, as is shown in Eq. (4).

d¼

df Rct ¼

rct ; s

(2)

where s is total interspace area (non-adsorbed area) on the electrode surface, d is the thickness of protein adsorption layer and rs is

Ris rct , ; Rct rs

Ris ; Rct

(3)

(4)

Moreover, the relative thickness of the protein layer can also be expressed from the view of the capacitance of protein layer, since Cp (capacitance of protein layer) has the following relationship with εp (permittivity of protein layer) and A (total surface area of protein layer). In addition, A can be calculated from Rct of electrode surface

Y. Huang et al. / Carbon 152 (2019) 354e362

with protein adsorption and R0ct of electrode surface without protein adsorption since the total area of the electrode surface is constant.

εp A ; d

Cp ¼ A¼

(5)

rct rct
; R0ct Rct

(6)

Therefore, the relative thickness of the protein layer can also be express as:

 d¼

1 R0ct Cp

 df

1 R0ct Cp




 1 ,εp rct ; Rct Cp

(7)




 1 ; Rct Cp

(8)

The quantitative information of EEC elements has been extracted from the fitting process so the thickness change of the protein layer can be qualitatively obtained by EIS data. Therefore, compared with other EIS analysis related to protein adsorption of different electrode materials, EIS analysis of rGO surfaces can provide more information on protein solution/solid interface due to the two time constant observed by EIS. Moreover, BSA (pI ¼ 4.7) molecules bear negative charges in a neutral environment (pH ¼ 7.4), thus a monolayer can be established on the solid surface due to the repulsive force among surface adsorbed protein molecules as well as the repulsive force between adsorbed protein and free protein in solution [40]. Considering the monolayer adsorption of BSA layer, the thickness change of BSA layer can be attributed to the ‘side-on’ or ‘end-on’ oriented protein molecules, referring to elliptically shaped protein respectively attached with its long or short axis to the surface [41]. Clearly, the BSA monolayer thickness is higher in the case of ‘end-on’ oriented protein molecules than that of ‘sideon’ oriented protein molecules. Accordingly, this analysis provides a new perspective on protein adsorption behavior, which can indirectly obtain orientations of adsorbed protein molecules in situ. The EIS was run consecutively and data at 1 h was used because the system has become relatively stable at the point. As is shown in Fig. 5, rGO electrodes incubated in different concentration of BSA solutions for 1 h demonstrates different thickness, indicating the adsorption orientations change with protein concentrations. According to the deduction of physical parameters of the electrolyte/ electrode interface, the relative thickness of the protein layer was expressed from the view of resistance and capacitance respectively. Both plots showed similar trends of thickness variation towards different BSA concentrations, indicating the convergences between them and providing evidence for the protein adsorption model on

359

rGO surface. Latour [40] has pointed out the Langmuir adsorption isotherm is inappropriately applied to characterize a protein adsorption process, while he also gave a hypothesis of actual protein adsorption behavior based on the review by Rabe et al. [19]. Our results strongly support the hypothesis, throwing light on the whole protein adsorption behavior from the point of view of protein layer's thickness change. A simplified illustration of protein adsorption behavior is shown in Fig. 5, utilizing ellipsoid to simulate the structure of model protein BSA. When incubated in low concentration of BSA solution (~0.045 mg/ml), the low thickness of protein adsorption layer indicates the ‘side-on’ orientation of the adsorbed protein. Once the protein adsorbs onto rGO surface, it begins to unfold and spread out and/or rolled from ‘end-on’ to ‘side-on’ orientation, in order to increase the ‘footprint’ on the surface thus minimize its free energy [42]. Moreover, the degree of spreading of a protein on the surface depends on the combination of the internal stability of the protein, the interaction strength of the protein-surface, and adsorbed protein-protein interactions. Low protein concentration corresponds to a low adsorption rate. When adsorption rate is much lower than orientation change rate, sufficient time and space are provided for adsorbed protein to maximize its ‘footprint’, thus the adsorbed protein molecules show up as spread ‘side-on’ orientation with apparently low thickness. When incubated in the medium concentration of BSA solution (0.045 mg/ml ~ 4.5 mg/ml), the adsorption rate could be as high as the change rate of orientation. The neighboring adsorption sites of adsorbed protein are occupied by other adsorbing protein molecules, and the protein-protein interaction tends to inhibit the ability of an adsorbed protein from spreading out on the surface. Therefore, protein molecules can largely retain their native-state structure with random orientations. The overall thickness of the protein layer falls in between that of ‘side-on’ and ‘end-on’ orientations. When incubated in the high concentration of BSA solution (4.5 mg/ml ~), adsorption sites on rGO surface are quickly occupied by protein molecules due to the very high adsorption rate. Adsorbed protein molecules are forced to be ‘end-on’ orientation due to the repulsion from neighboring protein molecules to enable more protein molecules to adsorb on rGO surface, thus finally showed up as a saturated ‘end-on’ protein layer with the highest thickness. What's more, the relative thickness of the protein layer rarely changed when incubated in highly concentrated BSA solution (45 mg/ml), also indicating the saturation of ‘end-on’ protein layer on rGO surface. Accordingly, protein molecules adsorbed on rGO surface were depicted as a densely packed monolayer due to the strong adsorption of protein on rGO surface.

Fig. 5. Relative thickness of BSA monolayer on rGO surface derived from resistance a) and capacitance b). Protein molecules adsorbed on rGO surface were depicted as a densely packed monolayer. (A colour version of this figure can be viewed online.)

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3.5. Impedance response of BDD The EIS data of BDD electrodes exposed to different concentration of BSA was also recorded by the same operational process as rGO electrodes. Compared with rGO electrodes, which showed two time constants, EIS data of BDD electrodes was as normal as other materials, with only one time constant in Nyquist plot in the presence of protein. It is important to note that the second process also happens on other materials' electrode-protein interface but unable to be observed. The interspace of protein layer on other electrodes’ surface is not as small as that of rGO surface, leading to a negligible solution resistance within the interspace area thus the second time constant cannot be observed. However, surprisingly and interestingly, BDD electrodes demonstrated decreased impedance response when incubated in the high concentration of BSA solution (4.5 mg/ml ~), while other electrodes showed increased impedance with increased concentration and reach saturation, taking the shape of a Langmuirlooking isotherm, overwhelmingly. Combined with the discussion of rGO related to protein adsorption behavior, it is assumed that the unique impedance response of BDD electrode is attributed to the condition of the protein adsorption layer on the BDD surface. 3.6. Modeling and fitting of BDD data To extract quantitative information on the solution/BDD interface, it is necessary to choose a proper EEC that can model the adsorption process precisely. It is inappropriate to apply the same EEC as rGO to modeling BDD because of their different surface properties and impedance response. Interactions between protein and rGO surface are so strong that a large number of protein molecules are firmly attached to rGO surface and desorption rate can be very low, thus charge transfer process on rGO surface can be severely influenced by adsorbed protein. A protein ‘layer’ is formed on rGO surface. Comparison of impedance change of rGO and BDD (Fig. S1) indicates that different from rGO, charge transfer property of BDD is slightly influenced by adsorbed protein. The reasons could be the weak interaction between protein and BDD surface, as well as only small amount of protein molecules are adsorbed on BDD surface. Results from XPS measurements also show that tightly adsorbed protein molecules on rGO surface are much more than that of BDD surface. Moreover, the stability of adsorbed protein can be easily tested by shaking the cell with protein solution and then measure the impedance immediately. After shaking, the impedance

of BDD decreased a lot and then gradually increased to the original value. However, rGO's impedance didn't show any change, indicating the reversible/loose binding (easy desorption of protein) between protein and BDD surface but irreversible/tight binding (hard desorption of protein) to rGO surface. Therefore, compared with protein ‘layer’, a monolayer of protein ‘island’ can be more appropriate to describe the condition of adsorbed protein molecules on the BDD surface. Considering that only one time constant can be observed in BDD's Nyquist plot, EEC with only one resistor and capacitor was utilized to model the protein adsorption process on the BDD surface. When the EEC is applied to modeling protein islands on BDD surface, the elements in EEC are given corresponding physical significances, with Rs (solution resistance), Rct (charge transfer resistance of interspace of the electrode), Cdl (electric double layer capacitance of interspace) and ZW (Warburg impedance of interspace of the electrode) defined in EEC. The capacitors of Cdl was implemented as CPEdl because of the inhomogeneity of the BDD surface. EIS data of protein adsorbed BDD was fitted using EEC in Fig. 6b with a square of the standard deviation as low as 0.00007. Representative fitting results are shown in Table 3. Cdl was calculated from the fitting result of CPEdl [39]. According to the Nyquist plots of BSA adsorbed BDD in Fig. 7a, Rct (charge transfer resistance of interspace on BDD surface) varied with protein concentrations. Interestingly, a decreased impedance response can be observed when BDD was exposed to a high concentration of BSA solution. 3.7. Protein adsorption behavior on BDD surface Based on the model of protein islands on the BDD surface in Fig. 6a, the elements in EEC can be characterized by physical parameters of the electrolyte/electrode interface. As A (total surface area of protein islands) can be calculated from Rct of electrode surface with protein adsorption and R0ct of electrode surface without protein adsorption by Eq. (6), A can be qualitatively derived from Rct and R0ct as expressed in Eq. (9).

 Af

 1 1 ;
R0ct Rct

(9)

Relative adsorbed area of protein islands on BDD surface was plotted as a function of the concentration of protein in Fig. 7b. Normally, the amount of protein molecules adsorbed on the solid

Fig. 6. a) Model of porous insulating protein islands on BDD surface. b) Schematic of EEC applied to modeling BDD surface without and with protein adsorption. BSA is modeled as blue ellipsoid. (A colour version of this figure can be viewed online.)

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Table 3 Parameters obtained by fitting EIS data of BDD exposed to specific amount of BSA. Symbol

Rs/U Cdl/F ndl Rct/U

Concentration of BSA (mg/ml) 0

0.00045

0.0045

0.045

0.45

4.5

45

48.2 7.23  10
7 0.881 22.3

44.3 1.19  10
6 0.913 48.5

32.8 1.40  10
6 0.931 91.3

49.2 1.49  10
6 0.939 185.4

45.4 1.53  10
6 0.943 231.3

39.9 1.51  10
6 0.916 95.5

38.1 1.60  10
6 0.919 92.5

Fig. 7. a) Nyquist plots of BDD exposed to different concentration of BSA solutions. Rct from equivalent fitting was plotted with corresponding concentration of protein. b) Relative adsorbed area of protein islands on BDD surface as a function of concentration of protein. Protein molecules adsorbed on BDD surface were depicted as a loosely packed monolayer. (A colour version of this figure can be viewed online.)

surface would increase with the concentration of protein in solution and reach saturation at some concentration. When all surface sites are filled with protein molecules, a monolayer coverage is formed, which means the adsorbed amount of protein molecules will not increase (and not decrease) with further increase in concentration of protein solution [19]. Therefore, it is proper to hypothesize that the decrease of the adsorbed area on BDD could be attributed to changes in orientation of adsorbed protein molecules. As stated previously, the monolayer adsorption of BSA was presented as ‘side-on’ or ‘end-on’ oriented protein molecules, referring to elliptically shaped protein respectively attached with its long or short axis to the surface. The phenomenon not only results in different thickness of protein monolayer but also leads to a different adsorbed area of a single protein molecule on the surface. When the surface is saturated with adsorbed protein molecules, ‘end-on’ oriented protein molecules occupy lower surface area than ‘side-on’ oriented protein molecules, thus resulting in higher interspace's area and lower charge transfer resistance. A simplified illustration of protein adsorption behavior is also shown in Fig. 7b. In relatively low concentration of BSA solution (~0.45 mg/ml), charge transfer resistance (Rct) increased with the concentration of BSA, indicating the increasing protein adsorbed area. The protein adsorption behavior is only controlled by the concentration of protein in solution, that is, the adsorption rate of protein in solution. However, a special phenomenon can be observed in high concentration of BSA solution (4.5 mg/ml ~) that impedance decreased a lot compared with that of lower protein's concentration, and the impedance rarely changed when incubated in more concentrated BSA solution (45 mg/ml), indicating saturation of adsorbed protein molecules on BDD surface. Owing to the very high adsorption rate, the neighboring adsorption sites of adsorbed protein are quickly occupied by other adsorbing protein molecules, and the protein-protein interactions tended to stop the adsorbed protein from spreading out on the surface. Therefore, protein molecules can largely retain their native-state structure and be forced to be ‘end-on’ orientation due to the repulsion from neighboring protein molecules, thus finally showed up as saturated

‘end-on’ protein islands with higher interspace's area and lower charge transfer resistance. Accordingly, protein molecules adsorbed on the BDD surface were depicted as a loosely packed monolayer due to the weak adsorption of protein on the BDD surface. As advanced carbon materials, rGO and BDD have totally different surface structure and property, leading to different impedance response and protein adsorption behavior on respective surfaces. However, they share a similar orientation's changing trend of adsorbed protein molecules when exposed to solution with different protein concentrations, indicating the universality of the established model for protein adsorption on carbon materials. 4. Conclusion Protein adsorption behavior on rGO and BDD surfaces were mainly investigated by EIS. Two kinds of equivalent circuit were utilized to model the physical processes occurring in solution/solid interface respectively according to the surface property and impedance response to protein adsorption. After fitting with EECs, qualitative thickness's changes and adsorbed surface area of adsorbed protein molecules were extracted from EIS data of rGO and BDD by simple deduction. Variations of thickness and adsorbed area with the protein concentration indicates the changes of orientation of adsorbed protein molecules, providing a new perspective on study of protein adsorption behavior. When exposed to BSA at neutral circumstance, rGO and BDD surfaces demonstrate densely and loosely packed monolayer of protein molecules respectively. Although rGO and BDD have different protein adsorption behavior due to their surface structure and property, they shared similar variation trend of protein's orientations towards protein's concentrations. The results can be extended to other carbon materials. If rGO (easy for protein adsorption) and BDD (resist protein adsorption) are considered as two extreme conditions that demonstrate unique impedance response, other carbon materials may fall in the middle and they may also share similar protein adsorption behavior regarding to changes of orientation: ‘side-on’ orientation in low concentration of protein

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solution; ‘end-on’ orientation in high concentration of protein solution. Besides the in-depth understanding of protein adsorption behavior, the study also indicates a potential way to control the protein orientation on solid surfaces.

[19] [20]

Acknowledgment [21]

Yixuan Huang was supported by the University of Tokyo Doctoral Student Special Incentives Program (SEUT-RA). This work was supported by Leading-Edge Industry Design Project of Saitama Prefecture 2018e2019. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.carbon.2019.06.023.

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