Bovine Serum Albumin Adsorption on TiO2 Nanoparticle Surfaces

Subscriber access provided by UNIVERSITY OF THE SUNSHINE COAST

Article 2

Bovine Serum Albumin Adsorption on TiO Nanoparticle Surface: Effects of pH and Co-Adsorption of Phosphate on Protein-Surface Interactions and Protein Structure Zhenzhu Xu, and Vicki H. Grassian J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b07525 • Publication Date (Web): 07 Sep 2017 Downloaded from http://pubs.acs.org on September 15, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Physical Chemistry C is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Bovine Serum Albumin Adsorption on TiO2 Nanoparticle Surface: Effects of pH and CoAdsorption of Phosphate on Protein-Surface Interactions and Protein Structure Zhenzhu Xu1 and Vicki H. Grassian1,2* 1

Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093, USA 2 Departments of Nanoengineering and Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA 92093, USA

Abstract Protein adsorption on nanoparticle surfaces plays a critical role in biological systems and bovine serum albumin (BSA) is a useful model protein to study due to its high abundance and similar properties as its human variant. Herein, a quantitative understanding of the interaction between titanium dioxide (TiO2) nanoparticle (22 nm average diameter) and BSA was carried out to explore the effect of pH on surface coverage and adsorbed protein structure. Experiments were conducted under different pH conditions (pH 7.4, 4.5 and 2.0) that simulate the pH of blood, lung and stomach fluids, respectively. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy was used for in-situ adsorption characterization and protein secondary structure analysis. In addition, thermogravimetric analysis (TGA) was used to provide quantitative determination of the surface coverage. These results show that BSA adsorption on TiO2 highly depends on pH as well as the presence of salts. Furthermore, it is also shown that coadsorbed phosphate ion reduces the amount denaturation of BSA on TiO2 at acidic pH. Thus, the results of this study provide new insights into understanding protein behavior on nanoparticle surfaces at different pH in the presence and absence of co-adsorbed phosphate.

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Introduction The interaction of proteins with metal oxide nanoparticles plays an essential role in understanding the environmental health and safety (NanoEHS) of nanomaterials.1 TiO2 nanoparticles are some of the most commonly used metal oxide nanomaterials that are used in consumer products. TiO2 is the main component in pigment manufactory and coating material,2-3 and it is also commonly used in cosmetic products and food additives4 often in the form of nanoparticles. Although the broad application of TiO2 nanoparticles brings great benefits, it also raises potential concerns of any negative effects of TiO2 on human health and environment.5-7 As in previous studies, it has been shown that nanoparticles, once taken into biological fluids can adsorb proteins.8-10 Additionally, nanoparticles could migrate with adsorbed biological molecules between different organs.11 Thus, the biocompatibility of these TiO2 nanoparticles become critically important and the evaluation of the impact of nanoparticle on protein structure is of great interest. Since bovine serum albumin (BSA) is a widely used model protein, due to its high abundance, low cost and similar properties as its human variant it is useful to study the detailed interactions that occur upon adsorption so as to better understand protein-surface interaction.12-13 BSA has a molecular weight of 66 kDa,14 and isoelectric point (IEP) of 4.7.15-16 The forms of BSA are pH dependent. BSA stays in the normal form (N-form) from pH 9.0 to pH 4.5 and will abruptly change to the fast form (F-form) when pH decreases to 4.0. Between pH 4.5 and pH 4.0 BSA exists in an N-F transitional form. Below pH 3.5, BSA adopts a more extended form (E-form) and expanded to an oblate spheroid shape.17 The dimensions of N-from, F-form and E-from BSA are 8.0 × 8.0 × 3.0 Å, 4.0 × 4.0 × 12.9 Å, and 2.1 × 2.1 × 25.0 Å, respectively.17-18 Furthermore, proteins can undergo conformational changes upon surface adsorption.8,

19-21

pH can influence protein adsorption on nanoparticle surfaces.22 It has been


Page 2 of 30

Page 3 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


reported that protein adsorption is optimal at its isoelectric point23-24 as the protein-protein interaction is minimized and electrostatic repulsion is reduced, which enables compact protein molecules to pack onto the surface to the greatest extent.25-26 Phosphate in blood and in buffer solution provides a pH-stable environment.27 Phosphate is also known to readily adsorb to the surface of most metal oxide nanoparticles and therefore most likely co-adsorbs with BSA on nanoparticle surfaces.27-29 In fact, studies have shown that the presence of phosphate could block active sites on nanoparticle surface and reduces protein surface coverage.28,

30-31

However, there have been no studies on the impact of co-adsorbed

phosphate on protein adsorption and protein structure especially as a function of pH. Since both BSA form and phosphate speciation show pH-dependent adsorption, including pH-dependent speciation and pH-dependent conformation, a systematic study of BSA behavior at different pH in the presence of co-adsorbed phosphate is of great interest. In this study, attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy was used to monitor BSA adsorption onto TiO2 (22 nm) nanoparticle surface32 as a function of pH with and without the co-adsorption of phosphate. ATR-FTIR is a technique that provides real-time, in-situ characterization.33-35 It is based on the total internal reflection of IR radiation that happens between the two interfaces and uses the evanescent wave that penetrates into the media of interest to detect protein-nanoparticle interactions.33 Secondary structure analysis is applied to the infrared spectra with a focus on the protein amide I region (1600-1700 cm-1) to give information of protein conformational changes upon adsorption.36 Additionally, time course measurements were done to better understand the co-adsorption process. Complementing the ATR-FTIR spectroscopy data, thermogravimetric analysis (TGA) gives a quantitative characterization of the surface coverage of BSA on TiO2 nanoparticles.37-38 All these 3 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

experiments, taken together, give a comprehensive understanding of protein-nanoparticle interactions and the effect of pH, salts and co-adsorbed phosphate on protein structure. Experimental Methods Zeta potential measurements. Zetasizer Nano ZS90 was used to determine the zeta potential of TiO2 nanoparticles as a function of pH. The Smoluchowski equation was used to convert the measured nanoparticle electrophoretic mobility to zeta potential.39 First, three types of stock suspension were made by mixing 10 mg of TiO2 with 10 mL of Optima water (Fisher Chemical), BSA (1 mg/mL) solution, and BSA (1 mg/mL) phosphate buffer solution respectively. Phosphate buffer was made of 0.02 M disodium orthophosphate (Na2HPO4) and 0.02 M potassium dihydrogen chloride (KH2PO4)27-28. Then, nine types of samples were made by titrating each stock suspension to three different pH (pH 7.4, 4.5 and 2.0) with 1 M hydrochloric acid (HCl) and 0.8 M sodium hydroxide (NaOH). Triplicate measurements were done for each sample and the average value with standard deviation was reported in supporting information (Figure S4). Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy. Spectra are obtained with a model of Nicolet 6700 FTIR spectrometer (Thermo-Fisher). Protein solution are freshly made to make sure they are at the desired pH when being used. Parafilm was also used to reduce water evaporation from the solution. pH of the stock solution was measured after the experiment to make sure it fell within ± 0.2 pH units of its initial value. For solution phase spectra of native BSA, 10 mg/mL BSA solutions at pH 7.4, 4.5, and 2.0 are used. For surface adsorption studies, first, TiO2 nanoparticle thin film was made by depositing 1mL of 2.5 mg/mL TiO2 suspension on an AMTIR crystal through plate and allowing the crystal to dry overnight. Then, Optima water was flowed over the thin film for 15 min with a horizontal flow


Page 4 of 30

Page 5 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


to remove the loosely bound nanoparticles and to collect the background spectrum. Next, 1 mg/mL of BSA solution (pH 7.4, 4.5 and 2.0) was run over the surface for 90min for adsorption experiment. Following the adsorption experiment, the protein-coated surface was washed with water for another 90 min at the same pH as the corresponding adsorption experiment. Spectra are taken every 10 min throughout the whole experiment. The experiment at circumneutral pH was repeated with D2O as the solvent using deuterium chloride (DCl) and deuterated sodium hydroxide (NaOD) solution to adjust the pD of BSA solution. Other experimental conditions were controlled remained the same. The pD of the solution was measured with a normal pH meter and then corrected by using the equation pD=pH+0.4.40 The solution phase spectra of BSA are subtracted using a background spectra of pure water on the ATR crystal. The time-dependent spectra in the presence of BSA are subtracted with the background spectra of water on the nanoparticles to get the spectra of adsorbed BSA on TiO2 nanoparticle surface. For each time-dependent spectral series, all spectra were normalized to the intensity of the amide I band after 90-minutes. The amide I band of the BSA was fit to a Gaussian-Lorentzian shape with five protein secondary structural components. The band position of each secondary components was located by referring to both the literature reported wavenumber range8,

20, 41

and the secondary derivative of the spectra. Spectra of BSA (10

mg/mL) in solution were fit first and then used as the initial guess to fit the adsorbed BSA spectra at the respective pH. All spectra are modified to a linear baseline and maximum peak height of amide I band was normalized to 1.0 for direct comparison. Thermogravimetric Analysis (TGA) of BSA Adsorption. Samples are prepared by mixing 2.5 mg BSA with 1mL of a 1 mg/mL BSA and adjusted to the pH of interest (pH 7.4, 4.5 and 2.0) with HCl and NaOH. Then, the samples were sonicated for 20 min to create an even suspension. 5 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Next, samples are stored at 4°C for 24 hours for incubation. After incubation, each sample was centrifuged at 10,000 rpm for 20 min and washed with pure water after removing the supernatant. After washing for three times, samples are placed in a fume hood for two to three days to make sure they are completely dried. Pyris 1 TGA, Perkin-Elmer was used to run the TGA experiment by heating the dry sample to 700°C at a rate of 5°C/min. The weight of the sample was recorded as a function temperature. The temperature range from 100°C to 600°C was used to monitor weight loss to quantify BSA adsorption on TiO2 nanoparticles. Results and Discussion ATR-FTIR spectroscopy of BSA in solution and adsorbed on TiO2 at pH 7.4, 4.5 and 2.0. ATR-FTIR spectra of BSA in solution at a concentration of 10mg/mL at three pH (pH 7.4, 4.5 and 2.0) are shown in Figure 1.42 The two characteristic bands of BSA, amide I band and amide II band, are centered at 1651 cm-1 and 1548 cm-1 respectively and are observed for all three pH. The amide I band comprises of the symmetric stretching of C=O as the major component and CN bending mode as a minor component. The amide II band consists of C-N stretching mode of N-H bending mode in peptide backbone of protein. The two peaks showing up at 1455 cm-1 and 1397 cm-1 are assigned to the CH2 scissoring and C-O carboxylate stretching respectively. At pH 2.0, the peak at 1711 cm-1 is attributed to the C=O stretching of protonated carboxylic group at acidic pH.43 In general, spectra at pH 7.4 and pH 4.5 are similar to each other in regard to the peak shape and positions. This is consistent with the fact that BSA remains in its N-form in the range of these two pHs.


Page 6 of 30

Page 7 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Normalized ATR-FTIR spectra of 10 mg/mL BSA solution at pH 7.4, 4.5 and 2.0.

ATR-FTIR offers a helpful method to monitor protein adsorption on nanoparticle surfaces through in-situ characterization. Additionally, in order to explore the impact of phosphate on BSA adsorption, infrared spectra were recorded with and without the presence of phosphate (Figure 2a and b respectively). Figure 2 displays the ATR spectra from 1000 cm-1 to 1800 cm-1of BSA adsorption on TiO2 (22 nm) nanoparticles at pH 7.4, 4.5 and 2.0. This region includes both amide I and amide II bands for BSA and characteristic bands for phosphate.

Figure 2. Normalized ATR-FTIR spectra of 1 mg/mL BSA adsorption as a function of time on TiO2 (22 nm) (a) without phosphate at pH 7.4 (top), pH 4.5 (middle) and pH 2.0 (bottom), (b) with phosphate at pH 7.4 (top), pH 4.5 (middle) and pH 2.0 (bottom). Spectra are shown for four different time points: 10, 30, 60, and 90 minutes (red, green, blue and purple colored spectra, respectively).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The three different pH values are chosen to imitate the pH of human blood, lung fluid, and stomach fluid, respectively.44 Spectra are normalized by normalizing the highest peak to 1 in each figure to exclude the factor of the different refractive index at different experimental conditions. BSA adsorption on TiO2 highly depends on pH, which is true in both sets of experiments with and without the presence of phosphate. For experiment without the presence of phosphate (Figure 2a), BSA amide I peak remains its position upon adsorption at pH 7.4 and pH 4.5. However, at pH 2.0, amide I band of adsorbed BSA diminishes with time and a broad band at around 1630 cm-1 gradually grows up. This phenomenon indicates that BSA undergoes complete denaturation upon adsorption on TiO2 surface at very acidic pH and the peak at 1630 cm-1 is assigned to the NH3+ groups in the highly protonated BSA on the surface.32 Another band near 1410 cm-1 is also observed for BSA adsorption on TiO2 at pH 2.0. This band is due to a combination of multiple vibrational modes but mainly arises from the symmetric stretching mode of COO- in BSA bonded to the hydroxylated TiO2 surface.33, 45-47 Thermogravimetric analysis (TGA) was also carried out to provide quantitative information of BSA adsorption on TiO2 at the three pHs (Table S2). TGA results show that BSA surface coverage is the highest at pH 4.5. This is the pH that is closest to the isoelectric point of BSA (pH 4.7) at which BSA adsorption is greatest. In a previously published paper,32 we have demonstrated that the solution pH impacts protein adsorption differently on SiO2 versus TiO2. TiO2 nanoparticle has hydroxyl groups (-OH) on its surfaces once hydrated that can interact with the adsorbed protein. The pre-adsorbed hydroxyl groups are diminished at low pH and replaced with Ti-OH2+ or Ti-OH+-Ti groups,48 which leads to less adsorbed protein at pH 2.0 than pH 7.4. 8 ACS Paragon Plus Environment

Page 8 of 30

Page 9 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


When phosphate is present, both BSA amide bands are observed as is phosphate bands at lower wavenumber (Figure 2b). The phosphate bands are due to phosphate adsorbed on the surface of the TiO2 nanoparticle as shown by the frequencies of the absorption bands when compared to experiments of phosphate adsorption and literature values for phosphate adsorbed on metal oxide surfaces.28, 49-50 The bands around 1115 cm-1 and 1077 cm-1 comprise a set of doublets which are attributed to the asymmetric and symmetric P-O stretching vibration in phosphate.49 The growth of both protein and phosphate absorption bands with time indicates that BSA and phosphate are co-adsorbed on TiO2 surface. BSA adsorption on TiO2 in the presence of phosphate also shows significant pH dependence. The ratio of the integrated intensity of the amide I band of BSA and phosphate band differs depending on pH with the ratio being the largest at pH 4.5. Additionally, BSA adsorption reaches saturation coverage much faster at pH 4.5. Several differences are observed between BSA adsorption with and without phosphate. The most significant difference occurs at pH 2.0 when BSA completely denatures on TiO2 surface in the absence of phosphate whereas this is not observed in the presence of phosphate. This phenomenon suggests that co-adsorbed phosphate significantly alters protein adsorption at low pH and changes the surface interaction such that denaturation does not occur. Besides the pronounced impact of phosphate on BSA adsorption at acidic pH, phosphate also shows influence on the conformation of adsorbed BSA at pH 7.4 and pH 4.5. The ratio between the amide I and amide II bands is commonly used to give a qualitative assessment of the conformational change of BSA upon adsorption.19 It could be observed in Figure 2 that amide I/II ratio is less than one for both pH 7.4 and 4.5 when phosphate is absent and is larger than one when phosphate is present. As the change of amide I/II ratio is attributed to protein secondary



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 30

structure change, it could be derived that the conformation of adsorbed BSA differs between experiments with and without of presence phosphate at the same pH. Secondary structure analysis of BSA in solution and adsorbed on TiO2. BSA conformational change upon adsorption was analyzed by assessing the secondary structure information of solution phase BSA and adsorbed BSA on TiO2 surface. For this purpose, the amide I band in each spectrum was curve fit with five protein secondary structural motifs; β-sheets/turns (16851663 cm-1), α-helices (1655-1650 cm-1), random chains (1648-1644 cm-1), extended chains/βsheets (1639-1621 cm-1) and side chain moieties (1616-1600 cm-1).8,

20, 41

The secondary

derivative spectra were also referred to refine the peak of maximum of each secondary component. The normalized curve fit amide I bands are presented in Figure 3 and the percent ratio of each secondary contents is summarized in Table 1. The main secondary structure, α-helix, is reported to be around 60-65% of the total secondary content, but some reports give values that vary by around 10%.18, 41, 51-54 In this study, a secondary structural content change that is greater than 10% is considered to be significant as based on viability in the reported literature. Deuterated water has no interferences in the amide I region since the D2O bending mode occurs around 1200 cm-1.55 Therefore, experiments of BSA adsorption on phosphate coated surface were done in Optima water (H2O) and deuterated water (D2O) respectively for the circumneutral pH (pD). The D2O curve fitting (Figure S1) and secondary structure analysis results are compared with those of experiment with H2O (Table S1). Since both H2O and D2O give similar results (all values agree within + 1%), this indicates that the bending mode at 1645 cm-1 is being subtracted out when H2O is used giving the same structural fitting components.


Page 11 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Background subtracted and normalized BSA amide I band for secondary structural analysis curve fitting for structural analysis: (a) Adsorbed BSA on TiO2 (22nm) nanoparticles without the presence of phosphate at pH 7.4 (top), pH 4.5 (middle) and pH 2.0 (bottom); (b) Adsorbed BSA on TiO2 (22nm) nanoparticles in the presence of phosphate at pH 7.4 (top), pH 4.5 (middle) and pH 2.0 (bottom). The black lines represent the original experimental spectrum and the light blue/green/red dotted lines represent the overall fit at pH 7.4 and pH 2.0, respectively. Component bands are given for β-sheets/turns (dark blue), α-helices (dark pink), random chains (dark red), extended chains/β-sheets (light pink), and side chain moieties (lime).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 30

Table 1. The Secondary Structure Content (%) in BSA Determined via Curve Fitting for BSA in Solution and after Adsorption onto the Nanoparticle Surfaces; TiO2 (22 nm) and Phosphate Coated TiO2 (22 nm).

pH

7.4

4.5

2.0

secondary structure

adsorbed BSA on 22 nm TiO2 (∆)a

solution phase BSA

adsorbed BSA on phosphate coated 22 nm TiO2 (∆)a

β-sheets/turns 5 9 (+4) 6 (+1) α-helices 68 51 (-17) 52 (-16) Random chains 4 13 (+9) 17 (+13) Extended chains/β-sheets 20 23 (+3) 23 (+3) Side chain moieties 3 4 (+1) 3 (+0) β-sheets/turns 9 7 (-2) 4 (-5) α-helices 65 41 (-24) 42 (-23) Random chains 7 20 (+14) 19 (+12) Extended chains/β-sheets 17 25 (+8) 29 (+12) Side chain moieties 2 6 (+4) 6 (+4) β-sheets/turns 6 2 (-4) 5 (-1) α-helices 60 30 (-30) 48 (-12) Random chains 9 8 (-1) 15 (-6) Extended chains/β-sheets 19 24 (+15) 26 (+7) Side chain moieties 6 36 (+30) 6 (+0) a ∆ = difference between adsorbed and solution phase structure content

In solution, BSA helical content decreases with decreasing pH, which is consistent with that reported in the literature.18, 54 Compared to solution phase BSA, BSA loses α-helical content and gains random chain and sheet structures upon adsorption at the different pH. The increase of sheet contents suggests that adsorbed BSA unfolds and thus expands its structure on nanoparticle surface. For BSA adsorption in the absence of phosphate, the α-helix content of adsorbed BSA decreases with decreasing pH. At pH 2.0, BSA loses half of its α-helix content on TiO2 surface compared to its molecules in solution, accompanying a 30% gain of the side chain structure. This large conformational change of BSA indicates that BSA completely denatures upon adsorption at acidic pH, which is consistent with the result of the observed spectra. A recent study of BSA adsorption on vapor-phase-grown TiO2 indicates that low pH facilitates adsorption-induced BSA conformational change by decreasing BSA helix content to a greater extent without causing 12 ACS Paragon Plus Environment

Page 13 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


protein denaturation.56 This study used a slightly higher acidic pH which is pH 3.0 and not the lower value of 2.0 that was used in the current study. Furthermore, the type of studied TiO2 differed which has been shown to have an impact on its toxicity to biological molecules.57 In the presence of phosphate, phosphate shows limited influence on the secondary structure of adsorbed BSA at pH 7.4 and 4.5. However, at pH 2.0, there is a significant difference in BSA secondary structure in the presence of phosphate compared to in the absence. BSA retains much more helical content upon adsorption. In contrast, the side chain components for adsorbed BSA in the presence of phosphate is dramatically lower than its comparison. This result demonstrates that the effect of phosphate on BSA conformational change upon adsorption is pH dependent. Bar plots of BSA shown in Figure 4 give a graphical presentation of BSA secondary structure as a function of pH. It is easily visualized in Figure 4 that both solution phase BSA and adsorbed BSA without the presence of phosphate have less α-helical content as pH decreases whereas for adsorbed BSA in the presence of phosphate the lowest α-helix ratio is achieved at pH 4.5. At pH 2.0, the comparison between two adsorption experiments clearly shows that phosphate protects BSA from denaturation by reducing its conformational change.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 30

Figure 4. Bar plot of the secondary structure content (%) in BSA determined via curve fitting for BSA in solution and after adsorption onto the TiO2 (22 nm) nanoparticle surfaces, Ads (no P), and phosphate coated TiO2 (22 nm) nanoparticle surfaces, Ads (with P), at (a) pH 7.4; (b) pH 4.5 and (c) pH 2.0.

One possible reason for the lack of protein denaturation in the presence of phosphate may be due to the fact that phosphate adsorbs on active sites on the TiO2 surface. Both phosphate and BSA have been reported to bond to TiO2 surfaces through terminal hydroxyl groups, and the adsorbed species depend on pH.29, 45, 50 Phosphate, once bonded to TiO2 surface at lower pH, could form a bridging bidentate complex, (TiO)2PO2 and herein reduces the available Ti-OH sites for BSA to attach to.45, 49-50, 58 It could be seen from Figure 2b that at pH 2.0, phosphate occupies a large proportion of binding size on TiO2 surface, according to the small amide I to phosphate absorbance ratio in the ATR spectrum. The occupation of active sites by adsorbed phosphate prevents BSA from interacting with TiO2 surface to the enough extent and fully


Page 15 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


expanded its structure on the surface. As a result, BSA conformational change is reduced and denaturation does not occur. To confirm these unique interactions, similar experiments were carried out substituting phosphate with NaCl. Figure S2 shows that the presence of NaCl in BSA solution could also protect BSA from denaturing on TiO2 surface at acidic pH. To verify the mechanisms, studies with a pre-treatment of the TiO2 surface were carried out with phosphate (Figure S3a) and NaCl (Figure S3b). Figure S3 shows that BSA in fact keeps the amide I peak on phosphate pre-treated TiO2 surface, whereas completely loses it on NaCl pre-treated TiO2 surface. This result indicates that NaCl prevents BSA denaturation by adjusting solution ionic strength,59 and thus, must be present in the solution with BSA at the same time. On the other hand, phosphate adsorbs on TiO2 surface and prevents BSA denaturation on nanoparticle surface via its interaction with coadsorbed BSA molecules. Kinetics of BSA and phosphate adsorption on TiO2 to probe reversible nature of the adsorption process. The normalized integrated absorbance of BSA amide II band (1547 cm-1)60 and phosphate band (1080 cm-1)28 are used to determine the adsorption kinetics and relative affinity of both species on TiO2 nanoparticle surfaces (Figure 5). In this figure, the amide II band and phosphate band were chosen as their band shape and position remain unchanged throughout the experiment for all pH. Especially for amide II band at pH 2.0, unlike amide I band, its band position is not affected by the denaturation of BSA. Figure 5 indicates that both adsorption kinetics and adsorbate affinity towards the surface highly depend on pH. Moreover, the existence of phosphate significantly influences BSA adsorption kinetics and BSA affinity towards TiO2 surface. BSA adsorption shows no sign of equilibrium within 90 min when phosphate is absent, which is true for all pH (Figure 5a). On the other hand, BSA adsorption equilibrium was 15 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 30

achieved at pH 4.5 and pH 2.0 within 90 min when phosphate is present (Figure 5b). This result indicates that phosphate impacts BSA kinetics onto TiO2 surface at low pH.

Some other

differences can also be observed between Figure 5a and Figure 5b. For example, BSA adsorption kinetics curve at pH 7.4 is very similar to that at pH 4.5 in the absence of phosphate. However, when phosphate is present, BSA adsorption kinetics curve at pH 7.4 is more similar to that at pH 2.0. Besides BSA, phosphate adsorption kinetics also largely depends on pH. Figure 5b shows that achievement of phosphate adsorption equilibrium is the fastest at pH 7.4 and lowest at pH 4.5. It can also be seen from Figure 5b that the kinetics curve of BSA adsorption is very similar to that of phosphate adsorption at pH 4.5 and 2.0 whereas is distinctively different from the other at pH 7.4.

Figure 5. Plots of the normalized integrated absorbance for the adsorbed BSA amide II band (at 1547 cm-1) and adsorbed phosphate band (at 1080 cm-1) during adsorption onto TiO2 (22 nm) (time 0 to 90 min) and desorption experiments (time 90 to 180 min) plotted as a function of time. (a) BSA adsorption experiments are done without phosphate at pH 7.4 (top), pH 4.5 (middle) and pH 2.0 (bottom); (b) BSA adsorption experiments are done with phosphate at pH 7.4 (top), pH 4.5 (middle) and pH 2.0 (bottom). Desorption studies are done with solutions that contain no BSA or phosphate at the same pH as the corresponding adsorption experiments. The solid and open circle markers represent the integrated area of the BSA amide II band and the phosphate bands, respectively.


Page 17 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Desorption experiments were carried out by changing the solution to pure water of different pH. The desorption region in Figure 5 shows that phosphate could significantly change the affinity of BSA towards TiO2 surface depending on pH. When phosphate is absent (Figure 5a), the normalized integrated absorbance for BSA remains at 1 for pH 7.4 and pH 2.0 and only shows a slight decrease at pH 4.5 throughout the whole desorption experiment. These results suggest that BSA residuals are strongly bonded to TiO2 surface under these conditions. However, when phosphate is present (Figure 5b), BSA shows almost 30% loss with water at pH 2.0, which indicates the interaction between BSA and surface is not as strong in the presence of phosphate. Interestingly, the existence of phosphate does not always decrease the affinity of BSA towards TiO2 surface. At pH 4.5, BSA normalized integrated absorbance decreases more when phosphate is absent, whereas the opposite is true for pH 2.0. And at pH 7.4, no significant difference was observed for BSA behavior between experiment with and without of presence of phosphate. Phosphate also shows different bonding strength towards TiO2 surface at different pH. The normalized integrated absorbance of phosphate decreases to the most extent at pH 7.4 while the least at pH 4.5. In general, neither BSA nor phosphate completely desorbs from the surface, but phosphate absorptions always decrease more than for BSA absorptions at the same pH. The different influence of phosphate on BSA behavior at different pH is due to several factors including the speciation of phosphate and the charge on BSA as a function of pH. The influence of phosphate on the behavior of BSA becomes more obvious with decreasing pH. At pH 7.4, BSA behavior is relative dependent from that of phosphate for both adsorption and desorption region. This is due to the electrostatic repulsion between the two species at this pH. At pH 7.4, BSA is negatively charged and phosphate ions are present mostly as HPO42- and H2PO4- in the aqueous phase. The interaction between BSA and phosphate is less unfavorable



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 30

and the impact of phosphate on BSA is weak. Therefore, BSA behavior is relatively dependent. As pH decreases to 4.5, BSA becomes less negative and phosphate ions become to H2PO4dominant. The interaction between phosphate and BSA increases. In this case, the adsorption kinetics of BSA becomes similar to that of phosphate. When pH continuously decreases to 2.0, BSA gives very similar behavior to that of phosphate for both adsorption and desorption experiment, which indicates BSA and phosphate are strongly interacting with each other. At pH 2.0, BSA is highly positively charged while phosphate exists in H3PO4 and H2PO4-. The electrostatic attraction between BSA and phosphate is very strong. And as BSA does not undergo complete denaturation on TiO2 surface when phosphate is present, its interaction with TiO2 surface is not as strong and when phosphate desorbs from the surface, BSA also desorbs due to its strong interaction with phosphate ions. ATR-FTIR spectroscopy of adsorbed BSA on TiO2 with pH adjustment. To further our understanding of the effects of pH on adsorbed protein conformation, we investigated how pH changes after surface adsorption influence the adsorbed protein conformation. Figure 6 shows the conformational changes of adsorbed protein on the surface as a function of pH. Both ATRFITR spectra and the secondary structure analysis are included. Two sets of experiments are shown. In the first experiment (Figure 6a and c), BSA adsorption on the TiO2 nanoparticle surface was done at pH 7.4. Following adsorption at pH 7.4, the BSA coated TiO2 nanoparticle surface was then exposed to aqueous solution of pH 2.0 for 90 min then the solution pH was changed to 7.4 for 90 min. In the second experiment (Figure 6b and d), BSA was initially at pH 2.0 and then the BSA coated TiO2 surface was exposed to pH 7.4 water and then again to pH 2.0.


Page 19 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Conformation changes of adsorbed BSA on TiO2 (22 nm) nanoparticle surface as a function of pH. (a): ATR-FTIR spectra of TiO2 (22 nm) nanoparticle exposed to 1 mg/mL of BSA at pH 7.4 for 90 min, then solution at pH 2.0 for 90 min and then with solution at pH 7.4 for 90 min. Secondary structural content (%) of each spectrum is shown in (c); (b): ATR-FTIR spectra of TiO2 (22 nm) nanoparticle exposed to 1 mg/mL of BSA at pH 2.0 for 90 min, then with solution at pH 7.4 for 90 min, and then with solution at pH 2.0 for 90 min. Secondary structural content (%) of each spectrum is shown in (d).

The results of these two sets of experiments can be seen in the ATR spectra collected by adjusting the solution pH. In Figure 6a, the amide I band of adsorbed BSA gets much broaden at acidic pH. In addition, the amide I/II ratio increases and then decreases when the pH changes. This shows that solution pH affects the conformation of adsorbed BSA. In Figure 6b, the effect of solution pH is even more significant. Following low pH adsorption, exposing adsorbed BSA to a solution pH 7.4 shows that BSA can go from a denatured state back to a more folded conformation. Furthermore, when the pH of the solution is decreased again to 2.0, the amide I peak still remains, although its relative intensity increases. Figure 6a and b, taken together, 19 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 30

shows that protein structure could be adjusted on TiO2 surface by changing the pH but denaturation does not at physiological pH. Secondary structure analysis for amide I band also gives important information about the conformational change of the adsorbed protein on nanoparticle surface (Figure 6c and d). It can be easily seen that α-helix and side chain content fluctuate with pH. In general, adsorbed BSA has less helix content and more side chain content at pH 2.0 than pH 7.4 which suggests the adsorbed BSA has a more disordered structure at low pH. This conclusion is further confirmed by the fact that the completely denatured BSA has the lowest helix content and the highest side chain content among all conditions. The pH dependence of the conformation of adsorbed BSA can be explained by the flexible structure of BSA. BSA is a soft protein,18 which means it has relatively high structural flexibility and can have different orientations when adsorbed onto nanoparticle surface. The flexible structure of BSA enables the further changes of its secondary structure after adsorption. However, it also noteworthy that the BSA secondary structure content does not exactly go back to the original values with pH, which indicates that BSA structural change induced by pH is not completely reversible. Conclusions The interaction of protein with metal oxide nanoparticles plays a critical role in the environmental health and safety of nanomaterials. This study shows that protein conformation changes as a function of pH for both protein molecules in solution and adsorbed on nanoparticle surfaces. BSA adsorption experiments with and without phosphate co-adsorption were compared. The impact of phosphate on protein adsorption and protein structure as a function of pH is summarized in Figure 7. Experimental results show that co-adsorbed phosphate on TiO2 surface could not only affect protein conformational change upon adsorption but also influence 20 ACS Paragon Plus Environment

Page 21 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


protein adsorption kinetics. Especially, phosphate can prevent BSA from denaturation on TiO2 surface at very acidic pH. The following desorption experiment also shows that phosphate could even change the affinity of BSA towards nanoparticle surfaces. These results reveal the important impact of phosphate on protein adsorption and give the potential of using co-adsorbed components to prevent protein denaturation in environmental and health and safety aspects. In addition, this work also explored the structural change of BSA after adsorption and shows that structure of adsorbed protein can be further changed, for multiple times, by adjusting the environmental pH, which gives important insights of the role of pH in protein structure modification. Taken together, this study provides a systematic approach to understanding the effect of pH and co-adsorbed phosphate on protein-surface interaction and protein structural changes upon adsorption.

Figure 7. A summary of the impact of phosphate on protein adsorption and protein structure as a function of pH. At pH 2.0, the branches of the adsorbed protein with the absence of phosphate represent the amino acid side chains of the denatured BSA on the surface. For the denatured BSA the major increase upon adsorption is the side chain moieties. The adsorbed proteins without branches are the proteins that unfolded but not completely denatured on TiO2 surface. For these proteins, the major increase upon adsorption is the β-structures.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 30

Supporting Information Description. Supporting information includes the following figures and tables: (i) ATR-FTIR data and the corresponding secondary structure analysis for BSA adsorption on TiO2 in deuterated water (1mg/mL in D2O) in the presence of phosphate at pD 7.4 (Figure S1 and Table S1); (ii) ATR-FTIR spectra of BSA adsorption on TiO2 in the presence of phosphate or sodium chloride (Figure S2 and S3); (iii) Zeta potential of TiO2 solution as a function of pH (Figure S4); (iv) TGA determined surface coverage of BSA adsorbed on TiO2 as a function of pH (Table S2). Author Information Corresponding Author: *E-mail: [email protected]. Tel.: 858-534-2499. ORCID: Zhenzhu Xu: 0000-0003-2889-2146 Vicki H. Grassian: 0000-0001-5052-0045 Notes: The authors declare no competing financial interest. Acknowledgment This material is based upon work supported by the National Science Foundation under Grant CHE1606607. Zeta potential measurements were done using Zetasizer Nano ZS90 in Professor Michael J. Sailor laboratory at the University of California, San Diego in the Department of Chemistry and Biochemistry with assistance and training provided by Nicole Chan. Thermal gravimetric analysis was done using the instrumentation in Professor Allan Guymon laboratory 22 ACS Paragon Plus Environment

Page 23 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


at the University of Iowa in the Department of Chemical and Biochemical Engineering. The authors also acknowledge helpful discussions with Brittany E. Givens and Dr. Natalia I. Gonzalez-Pech. References 1.

Thomas, C. R.; George, S.; Horst, A. M.; Ji, Z.; Miller, R. J.; Peralta-Videa, J. R.; Xia,

T.; Pokhrel, S.; Mädler, L.; Gardea-Torresdey, J. L. Nanomaterials in the Environment: From Materials to High-Throughput Screening to Organisms. ACS Nano 2011, 5, 13-22 2.

Shi, H.; Magaye, R.; Castranova, V.; Zhao, J. Titanium Dioxide Nanoparticles: A Review

of Current Toxicological Data. Part. Fibre Toxicol. 2013, 10, 15. 3.

Suttiponparnit, K.; Jiang, J.; Sahu, M.; Suvachittanont, S.; Charinpanitkul, T.; Biswas, P.

Role of Surface Area, Primary Particle Size, and Crystal Phase on Titanium Dioxide Nanoparticle Dispersion Properties. Nanoscale Res. Lett. 2011, 6, 27. 4.

Weir, A.; Westerhoff, P.; Fabricius, L.; Hristovski, K.; Von Goetz, N. Titanium Dioxide

Nanoparticles in Food and Personal Care Products. Environ. Sci. Technol. 2012, 46, 2242-2250. 5.

Oberdörster, G.; Oberdörster, E.; Oberdörster, J. Nanotoxicology: An Emerging

Discipline Evolving from Studies of Ultrafine Particles. Environ. Health Perspect. 2005, 113, 823-839. 6.

Warheit, D. B.; Sayes, C. M.; Reed, K. L.; Swain, K. A. Health Effects Related to

Nanoparticle Exposures: Environmental, Health and Safety Considerations for Assessing Hazards and Risks. Pharmacol. Ther. 2008, 120, 35-42. 7.

Lee, J.; Mahendra, S.; Alvarez, P. J. Nanomaterials in the Construction Industry: A

Review of Their Applications and Environmental Health and Safety Considerations. ACS Nano 2010, 4, 3580-3590. 23 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

8.

Page 24 of 30

Roach, P.; Farrar, D.; Perry, C. C. Interpretation of Protein Adsorption: Surface-Induced

Conformational Changes. J. Am. Chem. Soc. 2005, 127, 8168-8173. 9.

Hu, W.; Peng, C.; Lv, M.; Li, X.; Zhang, Y.; Chen, N.; Fan, C.; Huang, Q. Protein

Corona-Mediated Mitigation of Cytotoxicity of Graphene Oxide. ACS Nano 2011, 5, 3693-3700. 10.

Gebauer, J. S.; Malissek, M.; Simon, S.; Knauer, S. K.; Maskos, M.; Stauber, R. H.;

Peukert, W.; Treuel, L. Impact of the Nanoparticle–Protein Corona on Colloidal Stability and Protein Structure. Langmuir 2012, 28, 9673-9679. 11.

Jani, P. U.; McCarthy, D. E.; Florence, A. T. Titanium Dioxide (Rutile) Particle Uptake

from the Rat GI Tract and Translocation to Systemic Organs after Oral Administration. Int. J. Pharm. 1994, 105, 157-168. 12.

Dominguez-Medina, S.; Blankenburg, J.; Olson, J.; Landes, C. F.; Link, S. Adsorption of

a Protein Monolayer Via Hydrophobic Interactions Prevents Nanoparticle Aggregation under Harsh Environmental Conditions. ACS Sustainable Chem. Eng. 2013, 1, 833-842. 13.

Brewer, S. H.; Glomm, W. R.; Johnson, M. C.; Knag, M. K.; Franzen, S. Probing BSA

Binding to Citrate-Coated Gold Nanoparticles and Surfaces. Langmuir 2005, 21, 9303-9307. 14.

Reed, R.; Putnam, F.; Peters, T. Sequence of Residues 400–403 of Bovine Serum

Albumin. Biochem. J. 1980, 191, 867-868. 15.

Salis, A.; Boström, M.; Medda, L.; Cugia, F.; Barse, B.; Parsons, D. F.; Ninham, B. W.;

Monduzzi, M. Measurements and Theoretical Interpretation of Points of Zero Charge/Potential of BSA Protein. Langmuir 2011, 27, 11597-11604. 16.

Rezwan, K.; Studart, A.; Vörös, J.; Gauckler, L. Change of ζ Potential of Biocompatible

Colloidal Oxide Particles upon Adsorption of Bovine Serum Albumin and Lysozyme. J. Phys. Chem. B 2005, 109, 14469-14474.


Page 25 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


17.

Carter, D. C.; Ho, J. X. Structure of Serum Albumin. Adv. Protein Chem. 1994, 45, 153-

203. 18.

Jachimska, B.; Pajor, A. Physico-Chemical Characterization of Bovine Serum Albumin

in Solution and as Deposited on Surfaces. Bioelectrochemistry 2012, 87, 138-146. 19.

Roach, P.; Farrar, D.; Perry, C. C. Surface Tailoring for Controlled Protein Adsorption:

Effect of Topography at the Nanometer Scale and Chemistry. J. Am. Chem. Soc. 2006, 128, 3939-3945. 20.

Shang, L.; Wang, Y.; Jiang, J.; Dong, S. pH-Dependent Protein Conformational Changes

in Albumin: Gold Nanoparticle Bioconjugates: A Spectroscopic Study. Langmuir 2007, 23, 2714-2721. 21.

Moulin, A.; O'shea, S.; Badley, R.; Doyle, P.; Welland, M. Measuring Surface-Induced

Conformational Changes in Proteins. Langmuir 1999, 15, 8776-8779. 22.

Szewczuk-Karpisz, K.; Wiśniewska, M.; Myśliwiec, D. Albumin Adsorption Influence

on the Stability of the Mesoporous Zirconia Suspension. J. Ind. Eng. Chem. 2015, 32, 113-119. 23.

Demanèche, S.; Chapel, J.-P.; Monrozier, L. J.; Quiquampoix, H. Dissimilar pH-

Dependent Adsorption Features of Bovine Serum Albumin and α-Chymotrypsin on Mica Probed by AFM. Colloids Surf., B 2009, 70, 226-231. 24.

Quiquampoix, H.; Ratcliffe, R. A

31

P NMR Study of the Adsorption of Bovine Serum

Albumin on Montmorillonite Using Phosphate and the Paramagnetic Cation Mn2+: Modification of Conformation with pH. J. Colloid Interface Sci. 1992, 148, 343-352. 25.

Phillips, L. G. Structure-Function Properties of Food Proteins; Academic Press: San

Diego, U.S.A., 2013.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

26.

Page 26 of 30

Rezwan, K.; Meier, L. P.; Rezwan, M.; Vörös, J.; Textor, M.; Gauckler, L. J. Bovine

Serum Albumin Adsorption onto Colloidal Al2O3 Particles: A New Model Based on Zeta Potential and UV−Vis Measurements. Langmuir 2004, 20, 10055-10061. 27.

Wei, T.; Kaewtathip, S.; Shing, K. Buffer Effect on Protein Adsorption at Liquid/Solid

Interface. J. Phys. Chem. C 2009, 113, 2053-2062. 28.

Moulton, S.; Barisci, J.; McQuillan, A.; Wallace, G. ATR-IR Spectroscopic Studies of

the Influence of Phosphate Buffer on Adsorption of Immunoglobulin G to TiO2. Colloids Surf., A 2003, 220, 159-167. 29.

Jeyachandran, Y.; Mielczarski, E.; Rai, B.; Mielczarski, J. Quantitative and Qualitative

Evaluation of Adsorption/Desorption of Bovine Serum Albumin on Hydrophilic and Hydrophobic Surfaces. Langmuir 2009, 25, 11614-11620. 30.

Pradier, C.; Karman, F.; Telegdi, J.; Kalman, E.; Marcus, P. Adsorption of Bovine Serum

Albumin on Chromium and Molybdenum Surfaces Investigated by Fourier-Transform Infrared Reflection-Absorption Spectroscopy (FT-IRRAS) and X-Ray Photoelectron Spectroscopy. J. Phys. Chem. B 2003, 107, 6766-6773. 31.

Jeyachandran, Y.; Mielczarski, J.; Mielczarski, E.; Rai, B. Efficiency of Blocking of

Non-Specific Interaction of Different Proteins by BSA Adsorbed on Hydrophobic and Hydrophilic Surfaces. J. Colloid Interface Sci. 2010, 341, 136-142. 32.

Givens, B. E.; Xu, Z.; Fiegel, J.; Grassian, V. H. Bovine Serum Albumin Adsorption on

SiO2 and TiO2 Nanoparticle Surfaces at Circumneutral and Acidic pH: A Tale of Two Nano-Bio Surface Interactions. J. Colloid Interface Sci. 2017, 493, 334-341.


Page 27 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


33.

Mudunkotuwa, I. A.; Al Minshid, A.; Grassian, V. H. ATR-FTIR Spectroscopy as a Tool

to Probe Surface Adsorption on Nanoparticles at the Liquid–Solid Interface in Environmentally and Biologically Relevant Media. Analyst 2014, 139, 870-881. 34.

Baker, M. J.; Trevisan, J.; Bassan, P.; Bhargava, R.; Butler, H. J.; Dorling, K. M.;

Fielden, P. R.; Fogarty, S. W.; Fullwood, N. J.; Heys, K. A. Using Fourier Transform IR Spectroscopy to Analyze Biological Materials. Nat. Protoc. 2014, 9, 1771-1791. 35.

Yang, H.; Yang, S.; Kong, J.; Dong, A.; Yu, S. Obtaining Information About Protein

Secondary Structures in Aqueous Solution Using Fourier Transform IR Spectroscopy. Nat. Protoc. 2015, 10, 382-396. 36.

Steiner, G.; Tunc, S.; Maitz, M.; Salzer, R. Conformational Changes During Protein

Adsorption. FT-IR Spectroscopic Imaging of Adsorbed Fibrinogen Layers. Anal. Chem. 2007, 79, 1311-1316. 37.

Lehman, S. E.; Mudunkotuwa, I. A.; Grassian, V. H.; Larsen, S. C. Nano–Bio

Interactions of Porous and Nonporous Silica Nanoparticles of Varied Surface Chemistry: A Structural, Kinetic, and Thermodynamic Study of Protein Adsorption from RPMI Culture Medium. Langmuir 2016, 32, 731-742. 38.

Saha, B.; Das, G. Malachite Nanoparticle: A New Basic Hydrophilic Surface for pH-

Controlled Adsorption of Bovine Serum Albumin with a High Loading Capacity. J. Phys. Chem. C 2009, 113, 15667-15675. 39.

Zhang, Y.; Yang, M.; Park, J. H.; Singelyn, J.; Ma, H.; Sailor, M. J.; Ruoslahti, E.;

Ozkan, M.; Ozkan, C. A Surface-Charge Study on Cellular-Uptake Behavior of F3-PeptideConjugated Iron Oxide Nanoparticles. Small 2009, 5, 1990-1996.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

40.

Page 28 of 30

Murayama, K.; Tomida, M. Heat-Induced Secondary Structure and Conformation

Change of Bovine Serum Albumin Investigated by Fourier Transform Infrared Spectroscopy. Biochemistry 2004, 43, 11526-11532. 41.

Murayama,

K.;

Wu,

Y.;

Czarnik-Matusewicz,

B.;

Ozaki,

Y.

Two-

Dimensional/Attenuated Total Reflection Infrared Correlation Spectroscopy Studies on Secondary Structural Changes in Human Serum Albumin in Aqueous Solutions: pH-Dependent Structural Changes in the Secondary Structures and in the Hydrogen Bondings of Side Chains. J. Phys. Chem. B 2001, 105, 4763-4769. 42.

Schwinte, P.; Voegel, J.-C.; Picart, C.; Haikel, Y.; Schaaf, P.; Szalontai, B. Stabilizing

Effects of Various Polyelectrolyte Multilayer Films on the Structure of Adsorbed/Embedded Fibrinogen Molecules: An ATR-FTIR Study. J. Phys. Chem. B 2001, 105, 11906-11916. 43.

Mudunkotuwa, I. A.; Grassian, V. H. Histidine Adsorption on TiO2 Nanoparticles: An

Integrated

Spectroscopic,

Thermodynamic,

and

Molecular-Based

Approach

toward

Understanding Nano-Bio Interactions. Langmuir 2014, 30, 8751-8760. 44.

Schmaljohann, D. Thermo-and pH-Responsive Polymers in Drug Delivery. Adv. Drug

Delivery Rev. 2006, 58, 1655-1670. 45.

Kang, Y.; Li, X.; Tu, Y.; Wang, Q.; Ågren, H. On the Mechanism of Protein Adsorption

onto Hydroxylated and Nonhydroxylated TiO2 Surfaces. J. Phys. Chem. C 2010, 114, 1449614502. 46.

Lee, K. E.; Gomez, M. A.; Elouatik, S.; Demopoulos, G. P. Further Understanding of the

Adsorption Mechanism of N719 Sensitizer on Anatase TiO2 Films for DSSC Applications Using Vibrational Spectroscopy and Confocal Raman Imaging. Langmuir 2010, 26, 9575-9583.


Page 29 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


47.

Mudunkotuwa, I. A.; Grassian, V. H. Citric Acid Adsorption on TiO2 Nanoparticles in

Aqueous Suspensions at Acidic and Circumneutral pH: Surface Coverage, Surface Speciation, and Its Impact on Nanoparticle− Nanoparticle Interactions. J. Am. Chem. Soc. 2010, 132, 1498614994. 48.

Connor, P. A.; Dobson, K. D.; McQuillan, A. J. Infrared Spectroscopy of the

TiO2/Aqueous Solution Interface. Langmuir 1999, 15, 2402-2408. 49.

Gong, W. A Real Time in Situ ATR-FTIR Spectroscopic Study of Linear Phosphate

Adsorption on Titania Surfaces. Int. J. Miner. Process. 2001, 63, 147-165. 50.

Connor, P.; McQuillan, A. J. Phosphate Adsorption onto TiO2 from Aqueous Solutions:

An in Situ Internal Reflection Infrared Spectroscopic Study. Langmuir 1999, 15, 2916-2921. 51.

Gülseren, Đ.; Güzey, D.; Bruce, B. D.; Weiss, J. Structural and Functional Changes in

Ultrasonicated Bovine Serum Albumin Solutions. Ultrason. Sonochem. 2007, 14, 173-183. 52.

Bouhekka, A.; Bürgi, T. In Situ ATR-FTIR Spectroscopy Study of Adsorbed Protein:

Visible Light Denaturation of Bovine Serum Albumin on TiO2. Appl. Surf. Sci. 2012, 261, 369374. 53.

Peters Jr, T. All About Albumin: Biochemistry, Genetics, and Medical Applications;

Academic Press: San Diego, U.S.A., 1995. 54.

Takeda, K.; Wada, A.; Yamamoto, K.; Moriyama, Y.; Aoki, K. Conformational Change

of Bovine Serum Albumin by Heat Treatment. J. Protein Chem. 1989, 8, 653-659. 55.

Innocenzi, P.; Malfatti, L.; Carboni, D.; Takahashi, M. Sol-to-Gel Transition in Fast

Evaporating

Systems

Observed

by

in

Situ

Time-Resolved

ChemPhysChem 2015, 16, 1933-1939.


Infrared

Spectroscopy.


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

56.

Page 30 of 30

Márquez, A.; Berger, T.; Feinle, A.; Hüsing, N.; Himly, M.; Duschl, A.; Diwald, O.

Bovine Serum Albumin Adsorption on TiO2 Colloids: The Effect of Particle Agglomeration and Surface Composition. Langmuir 2017, 33, 2551-2558. 57.

Clément, L.; Hurel, C.; Marmier, N. Toxicity of TiO2 Nanoparticles to Cladocerans,

Algae, Rotifers and Plants-Effects of Size and Crystalline Structure. Chemosphere 2013, 90, 1083-1090. 58.

Hong, Y.; Yu, M.; Lin, J.; Cheng, K.; Weng, W.; Wang, H. Surface Hydroxyl Groups

Direct Cellular Response on Amorphous and Anatase TiO2 Nanodots. Colloids Surf., B 2014, 123, 68-74. 59.

Ludwig, D. B.; Carpenter, J. F.; Hamel, J. B.; Randolph, T. W. Protein Adsorption and

Excipient Effects on Kinetic Stability of Silicone Oil Emulsions. J. Pharm. Sci. 2010, 99, 17211733. 60.

Tantipolphan, R.; Rades, T.; McQuillan, A.; Medlicott, N. Adsorption of Bovine Serum

Albumin (BSA) onto Lecithin Studied by Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy. Int. J. Pharm. 2007, 337, 40-47.

Table of Contents Image


Bovine Serum Albumin Adsorption on TiO2 Nanoparticle Surfaces

Recommend Documents