In Situ Monitoring of Protein Adsorption on a Nanoparticulated Gold

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In situ monitoring of protein adsorption on a nanoparticulated gold film by attenuated total reflection surface-enhanced infrared absorption spectroscopy Bo Jin, Wen-Jing Bao, Zeng-Qiang Wu, and Xing-Hua Xia Langmuir, Just Accepted Manuscript • DOI: 10.1021/la300819u • Publication Date (Web): 24 May 2012 Downloaded from http://pubs.acs.org on May 31, 2012

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In situ monitoring of protein adsorption on a nanoparticulated gold film by attenuated total reflection surface-enhanced infrared absorption spectroscopy Bo Jin, Wen-Jing Bao, Zeng-Qiang Wu, Xing-Hua Xia* State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, China. ABSTRACT: In situ surface enhanced infrared absorption spectroscopy (SEIRAS) with an attenuated total reflection (ATR) configuration has been used to monitor the adsorption kinetics of bovine hemoglobin (BHb) on an Au nanoparticles (NPs) film. The IR absorbance for BHb molecules on gold nanoparticles film deposited on Si hemispherical optical window is about 58 times higher than that on a bare Si optical window and the detection sensitivity has been improved by three orders of magnitude. From the IR signal as function of adsorption time, the adsorption kinetics and thermodynamics can be explored in situ. It is found that both the electrostatic interaction and the coordination bonds between BHb residues and Au NPs film surface affect the adsorption kinetics. The maximum adsorption can be obtained in solution pH 7.0 (closing to the isoelectric point of the protein) due to the electrostatic interaction among proteins. In addition, the isotherm of BHb adsorption follows well the Freundlich adsorption model.

INTRODUCTION Adsorption of proteins with dual hydrophilic and hydrophobic properties on materials surfaces is a common phenomenon which is often encountered in bionanotechnology and has been used to fabricate biosensors, bioelectronics devices and biofuel cells.1 This adsorption process is mainly driven by the weak supermolecular interactions including Van der Waals forces, hydrophilic and hydrophobic interaction, electrostatic interactions and hydrogen bonds, which could cause conformation change and even denaturation of the biomolecules in some cases.2-5 For example, it has been reported that the morphology, chemical structure, electronic property of the surface and interfacial electric field affect the kinetics and mechanism of protein adsorption.6-10 Obviously, understanding of protein adsorption at materials surfaces would help to improve practical performance of bio-chip,11-13 biosensors,14 biological fuel cell,15-22 drug delivery23 and new hybrid materials synthesis.24 Up to now, various methods have been developed to monitor the protein adsorption process at materials sur-

faces, including surface plasmon resonance (SPR),25-27 ellipsometry method,28 quartz crystal microbalance (QCM)9 and electrochemical methods.29 Although these methods are sensitive and can provide the adsorption kinetics of biological molecules, they cannot provide the conformation, orientation and structural information of the immobilized protein molecules at materials surfaces. In addition, these methods are usually affected by the environmental alterations. Infrared spectroscopy has been proved to be a powerful surface analysis technique for identifying the molecular structures. It has been applied to analyze protein secondary structure, i.e., a variety of infrared spectra of proteins adsorbed on germanium surface has been collected by using a FTIR in attenuated total reflection (ATR) mode. 30,31 Surface enhanced infrared absorption spectroscopy (SEIRAS), as an in-situ analysis technology with high sensitivity, has become a powerful tool to study the adsorption/desorption processes, conformation and function of biological molecules occurring at materials surfaces. SEIRAS is usually achieved on a rough noble metal film deposited on silicon prism in the attenuated total reflection mode. The signal enhancement is restricted to the immediate vicinity of the surface and drops exponentially because of the optical near-field effect.32-34 This technique offers an opportunity to monitor the materials surfaces without interference from the environment.35,36 Up to now, SEIRAS-ATR has been successfully applied in qualitative analysis of biological species ranging from small biomolecules to genetic and proteomic targets.37,38 Ataka et al.39 have studied the adsorption behavior of cytochrome c (cyt c) on carboxylic acid and hydroxyl terminated self-assembled monolayers on gold surfaces and found orientation related direct electrochemistry of the immobilized cyt c. Jiang et al.40 have found that the hindrance of electron transfer of adsorbed cyt c on the bare gold electrode was from the adsorption orientations in which most of the helices were parallel to the surface with a flatter orientation of the heme. In this study, a thin gold nanoparticles (NPs) film was deposited on a hemispherical silicon surface via the Galvanic exchange reaction.41-46 The adsorption of bovine hemoglobin (BHb) on this gold film was then monitored

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in situ using the SEIRAS-ATR. The adsorption isotherm of BHb was plotted and fitted by Langmuir adsorption equation and Freundlich adsorption equations. Subsequently, the influence of solution pH on the adsorption kinetics of BHb was studied and the interactions involved in the adsorption process were discussed.

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with an average particle size of about 100 nm. The gold nanoparticles are closely packed but yet separated to form numbers of nano-gaps, which is expected to produce large infrared signal enhancement. Another advantage of the chemically deposited Au NPs film is the strong adhesion of the gold NPs to the silicon substrate, because the preformed Si-H bond will facilitate the formation of silicide between Si and Au deposit.49 This property significantly improves the stability of the resultant Au NPs film, as is very important for biomimetic devices. Such nanoparticulated gold films have been reported to produce enhanced IR and Raman scattering signals.42,46 X-ray diffraction characterization shows that the deposited gold film is of typical face centred cubic structure including (111), (200), (220), and (311) orientations at diffraction angle of 38.32o, 44.48o, 65.04o, and 77.86o, respectively (Figure 1c).

EXPERIMENTAL SECTION Materials and reagents: The hemispherical Si (111) ATR prism (36 mm in diameter) was purchased from Alkor Technologies (Russia). Bovine hemoglobin was from Biological Products Corporation (Shanghai, China) and used without further purification. Other reagents were of analytical grade and used as received. All aqueous solutions were prepared using ultrapure water (Milli-Q, Millipore, USA).

IR signal enhancement of the Au nanoparticles film. SEIRAS-ATR spectra of BHb saturation immobilized on a chemically deposited Au NPs film and a bare Si prism were compared to show the signal enhancement caused by the Au NPs film. We can see from Figure 2 that two obvious peaks appear at 1600-1700 cm-1 and 1480-1575 cm-1. The sharp peak at 1654 cm-1 is attributed to the amide I vibration, which is due to the C=O stretching vibrations of the peptide groups.50 The amide II vibration, located at 1546 cm-1, corresponds to the N-H stretching vibration, together with a lesser contribution from the C-N and C=C stretching vibration.50 The secondary structure of protein can be generally obtained from the amide I region as it contains structural information involved in hydrogen bond of peptide chain and is highly sensitive to structural changes. The amide I region of BHb, located at 1654 cm-1, exhibits a typical character shared by α-helix-rich proteins.51 It can be predicted from the spectrum that the secondary structure of BHb adsorbed on the Au NPs film is well maintained. The bands at 1450 cm-1 and 1396 cm-1 are associated with the C-H bending vibration and mixed C-N and C-H bending vibration. The peaks at 1304 cm-1 and 1225 cm-1, named amide III, belong to the combined N-H in-plane bending and C-H stretching vibration.52 We calculated the intensity of amide II vibration, and its intensity on gold NPs film is 58 times larger than that on the Si prism, showing a remarkable enhancement while maintaining the natural structure of BHb.

Deposition of gold nanoparticles film on silicon surface: The hemispherical silicon prism was rinsed by sonication in anhydrous ethanol and ultrapure water respectively after polishing with alumina power (0.3 μm). Native silicon oxide layer was removed by immersing the surface of the silicon prism in 10% HF for 5 min, and then in 40% NH4F for 3 min, followed by rinsing in ultrapure water. This process is known to result in Si-H surfaces. Prior to the deposition, a plating solution containing 0.015M HAuCl4+0.15M Na2SO3+0.05M Na2S2O3+0.05M NH4Cl was prepared.46-48 Deposition of Au nanoparticles film was performed at 60 oC simply by dropping a mixture of 1.0 mL plating solution + 0.5 mL 2% HF onto the hydrogenterminated Si surface and maintained in the dark for 3 min. Ultrapure water was added to end the reaction finally. Instruments: Infrared spectra were measured with a Bruke Tensor27 Fourier transform spectrometer in the attenuated total reflection mode. P-polarized IR radiation was totally reflected at the gold nanoparticles film/solution interface with an incident angle θ=70o and was detected with a liquid-N2-cooled MCT detector. All the spectra were plotted in absorbance unit relative to a baseline which was recorded by immersing the surface in pure buffer for 30 min before further measurement. The O-H stretching of water is automatically subtracted by the IR software. The spectral range was 4000~1000 cm-1 at 4 cm-1 resolution. Atomic force microscopy (AFM) imaging was performed on an Agilent 5500 AFM/SPM system using tapping mode under ambient conditions. Scanning electron microscopy (FE-SEM, S-4800, Hitachi, Japan) at an accelerating voltage of 10 kV was used to characterize the morphology of the deposited gold films. X-ray diffraction pattern was obtained on an X’Pert X-ray diffraction spectrometer (Philips, USA). RESULTS AND DISCUSSION Characterization of Au nanoparticles film. The morphology of the SEIRAS-active Au NPs film was investigated with SEM and AFM. As shown in Figure 1, the chemically deposited gold film has an island structure

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Figure 3. .The ATR-SEIRA spectrum of 10 nM BHb adsorbed on the Au NPs film in 20 mM PBS at pH 7.0. The time for adsorption was 1 h.

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The deposited Au nanoparticles film not only improves the signal to noise ratio, but also lessens the solution interference. As shown in Figure 3, the amide bonds of BHb from a concentration of 10 nM still can been distinguished after adsorbed for 1 h, while on a bare Si surface, only BHb with a concentration over 3 μM could been detected (not shown). Obviously, the Au NPs film leads to 3 orders of magnitude better detection sensitivity than the bare Si prism.

Integral area (1546cm )

Adsorption kinetics of BHb. The adsorption kinetics of BHb on the Au NPs film surface under neutral condition was monitored by in situ SEIRAS-ATR. Figure 4 shows the evolution of SEIRA spectra as function of adsorption time. The intensity of amide bonds of BHb increases with adsorption time. The amide II vibration of protein is structure-insensitive, so its intensity is usually used to calculate the total quantity of protein.53 Pitt et al.54 reported that the area of amide II peak on a Ge prism had a linear relationship with the amount of dry protein. From this relationship, the amount of proteins adsorbed at the interface could be estimated. In our case, the band area of amide II region centered at 1546 cm-1 was plotted as function of adsorption time (Inset of Figure 4). It is clear that the adsorption kinetics starts very fast at its early stage, then slows down, and finally reaches a steady state. A monolayer of BHb is achieved within about 30 min.

Figure 1. .(a) SEM image (b) AFM image and (c) X-ray diffraction pattern of the gold NPs film deposited on a silicon o semispherical optical surface at 60 C for 3 min.

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Figure 2. .The ATR-SEIRA spectra of 0.5 mg•mL BHb in 20 mM PBS (pH 7.0) absorbed on the Au NPs film (a) and a bare Si prism (b) for 1h.

Figure 4. .Series of ATR-SEIRA spectra for the adsorption of -1 BHb on the Au NPs film from a solution of 0.5 mg•mL BHb + 0.02 M PBS (pH 7.0) at different adsorption time, showing the adsorption process of the protein. Inset shows the plot of -1 the integral for the amide II (1554 cm ) band versus time.

Adsorption isotherm of BHb. Adsorption isotherm is the relationship of distribution between two phases when the adsorption process of a solute reaches equilibrium on

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Langmuir the two-phase interface at a certain temperature, which reflects the nature of interactions between the adsorbates and the surface. All the IR spectra in Figure 5 were collected after adsorption for 30 min, so that all the infrared spectra can be regarded as saturated protein adsorption on the gold nanofilm/solution interface. From Figure 5, it can be clearly seen that the adsorption capacity of BHb on the gold NPs film gains with the increase of protein concentration in solution. However, increase of the adsorption amount of BHb on gold NPs film surface is slightly slower than the increase of protein concentration in solution, indicating that there is no simple quantitative relationship between these two parameters.

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where Ce is the equilibrium concentration of BHb in the solution, qe is the equilibrium adsorption amount of BHb on Au NPs film, qm is the saturated protein adsorption amount, K is the adsorption equilibrium constant, KF and n are constants related with the Au NPs film and BHb at a particular temperature.

Figure 5. (a) The SEIRA-ATR spectra of BHb adsorbed on the Au NPs film from different concentrations of protein. (b) The adsorption isotherm for BHb on Au NPs film.

Seen from Figure 6, the Freundlich fitting equation, with a linear correlation coefficient of 0.998, shows a better agreement with experiment data than the Langmuir fitting results (R2=0.976). Thus, the Freundlich adsorption model considering the interactions among adsorbed molecules and the non-uniformity of adsorption site can give a better description for the adsorption process of BHb on Au NPs film. According to Eq.(2), KF and n can be calculated as 1.20 and 1.50 respectively from the fitting curve, indicating a relatively easy process for BHb adsorption on gold NPs film.

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Figure 6. .Plots of surface concentration of BHb on the NPs film as function of solution concentration of the protein simulated by using Langmuir (a, triangles) and Freundlich isotherm (b, cycles).

Influence of solution pH on adsorption behavior of BHb. The protein molecules are mostly non-uniformly charged as the presence of ionized side chains. As a result, the surface charges and their distributions of proteins are related to solution pH values, which will certainly affect the adsorption kinetics if the electrostatic interactions play the determining role. Since the isoelectric point (pI) of BHb is 7.0,55 the protein is positively charged at solution pH lower than the pI, while it is negatively charged at solution pH higher than its pI. 7.0. At pH=pI, the total

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charge of protein will be zero. As shown in Figure 7, significant difference in adsorption kinetics is observed for solution pH=6.0, 7.0 and 8.0 from the normalized IR signal. This difference is mainly due to the alteration in electrostatic interactions between BHb molecules and the gold surface. As we measured before, the potential of zero charge (PZC) of porous Au electrode/PBS is near -0.10V (vs. SCE).56 In the present measurements, protein adsorption was performed at open circuit potential (OCP measured at 0.275V vs. SCE), indicating that surface of Au NPs film carries excess positive charges. Therefore, a fast adsorption kinetics of BHb is observed at solution pH 8.0 due to the electrostatic interactions between negatively charged BHb and the positively charged Au surface. In contrast, with positively charged BHb at pH 6.0, electrostatic repulsion makes the adsorption process slow down, thus longer adsorption time is required to reach adsorption equilibrium.

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Figure 8. .The ATR-SEIRA spectra of the BHb adsorbed on Au NPs film (a) at pH 6.0 (solid) followed by at pH 7.0 (dash); (b) at pH 8.0 (solid) followed by pH 7.0 (dash).

Electrostatic effects not only influence the adsorption rate of proteins, but affect the interaction among proteins as well. As with the same charge, the electrostatic repulsion between BHb may decrease its surface concentration at the nano-film/solution interface. As shown in Figure 8, the infrared spectra of saturation adsorbed BHb at pH 6.0 or pH 8.0 were first recorded. When the pH value of the solution was adjusted to 7.0, an increase in protein signal is observed (Figure 8), indicating that the surface coverage of BHb on gold surface gains as a result of elimination of static interaction among proteins.

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CONCLUSION In summary, surface enhanced infrared absorption spectroscopy is a sensitive technique for in-situ studying the adsorption process of proteins. The chemically deposited Au nanoparticles on Si surface exhibit good biocompatibility and high enhancement to infrared absorption to proteins. Results show that both the electrostatic interaction and the coordination bonds between BHb residues and Au NPs film surface affect the adsorption kinetics. The maximum adsorption can be obtained in solution pH 7.0. Due to these irreversible interactions and nonuniformed surface structure of gold nanoparticles, the adsorption process of BHb on gold nanoparticles film can be better described by the Freundlich adsorption equation.

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Figure 7. .Plot of surface coverage of BHb adsorption at various pH versus time; at pH 6.0 (●), pH 7.0 (▲) and pH 8.0 (■). The surface coverage is gained by assuming the saturation adsorption of BHb on the Au NPs film surface to be 100%. The data were obtained by integrating the band area for amide II of the IR spectra.

However, in solution pH 6.0, BHb can still adsorb on the positively charged Au NPs film surface, indicating that there are other primary forces driving the adsorption of BHb. This is confirmed by the fact that desorption of previously adsorbed BHb molecules on the gold nanoparticles film did not occur when we immersed the modified electrode into 1.0 M KCl solution for 3 h. Covalent interaction between Au atom and –NH2/-SH has been widely proven since the lone pair electrons of nitrogen and sulfur atom can be injected into the empty d orbital of Au atom to form weak coordination bonds. Therefore, surfacelocated hydrophilic amino acid residues of BHb, such as Lys, Glu, Cys, His and Arg, make it possible for proteins to interact with the gold surface despite of the existence of electrostatic repulsion.

AUTHOR INFORMATION Corresponding Author *E-mail:[email protected] (X. H. Xia)

ACKNOWLEDGMENT This work was supported by the Grants from the National 973 Basic Research Program (2012CB933804), the National Natural Science Foundation of China (NSFC, No. 21035002), the National Science Fund for Creative Research Groups (21121091) and the Natural Science Foundation of Jiangsu province (BK2010009).

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