Adsorption of Bovine Serum Albumin and Lysozyme on Functionalized

Aug 27, 2014 - Functionalization of multiwalled carbon nanotubes by microwave irradiation for lysozyme attachment: comparison of covalent and adsorpti...
0 downloads 6 Views 3MB Size
Article pubs.acs.org/JPCC

Adsorption of Bovine Serum Albumin and Lysozyme on Functionalized Carbon Nanotubes Peng Du,† Jian Zhao,†,‡ Hamid Mashayekhi,† and Baoshan Xing*,† †

Stockbridge School of Agriculture, University of Massachusetts, Amherst, Massachusetts 01003, United States College of Environmental Science and Engineering, Key Laboratory of Ministry of Education, Ocean University of China, Qingdao 266100, P. R. China



S Supporting Information *

ABSTRACT: In this work, we examined adsorption of bovine serum albumin (BSA) and lysozyme (LYZ) on carboxylated (CM), hydroxylated (HM), and graphitized (GM) multiwall carbon nanotubes (CNTs). All adsorption isotherms were fitted well with the Langmuir model. The maximum adsorption capacities (mg/g) followed the order HM > CM > GM for both BSA and LYZ, which positively related to the surface areas of the three CNTs. However, after surface area normalization, adsorption capacities (mg/m2) followed the order HM > GM > CM for BSA and GM > CM > HM for LYZ, indicating that functional groups and hydrophobicity of CNTs also contributed to protein adsorption. In addition, adsorption of LYZ (81 800−90 700 mg/ g) was at least 300 times higher than that of BSA (132−266 mg/g) for all the three CNTs. BSA molecules on CNTs surface mainly showed a monolayer adsorption while LYZ adsorption was through multilayers. Moreover, BSA at the tested concentrations was able to disperse the three CNTs. However, no significant dispersion was observed for all the three CNTs in the presence of LYZ at the same concentrations. The results revealed that α-helix structure of both the proteins diminished after interacting with the three CNTs. This research will be helpful to clarify the mechanism of protein adsorption on functionalized CNTs and would be of importance for using CNTs in biomedical and pharmaceutical fields.

1. INTRODUCTION Carbon nanotubes (CNTs) are becoming one of the most popular research focuses due to their outstanding chemical and physical properties, such as well-ordered structure, large surface area, excellent chemical and thermal stability, and rich electronic properties.1 The potential applications of CNTs are mainly in biomedical, chemical, engineering, and pharmaceutical fields, such as drug delivery, biosensor, gene therapy, antimicrobial agents, nanoinjection, and wastewater treatment.2 Due to a wide array of potential applications, the toxicity of CNTs is of considerable interest.3 CNTs can cross the membrane barrier by energy-independent penetration and energy-dependent endocytosis and then accumulate mainly in human cell endosomes and lysosomes.4 Several factors were shown to affect the toxicity of CNTs, such as metal impurities,5 agglomeration,6 layer numbers,7 shape,8,9 length,9 and surface modification.10 It is reported that CNTs functionalized by acid treatment are more hydrophilic and show less aggregation in © XXXX American Chemical Society

human cell lysosomes and cytoplasm and insignificant cell viability changes.11 Functionalized CNTs are described as the graphitized CNTs coated with chemical functional groups, polymers, or biomacromolecules (e.g., DNA, proteins)12 in order to improve their chemical and biological properties. Compared to graphitized CNTs, functionalized CNTs have higher bioavailability and stability for effective use in biomedical and chemical applications.13 The unparalleled optical and electrical properties of functionalized CNTs are excellent for bioimaging.14 In addition, functionalized CNTs can also improve their drug delivery efficiency by the specific recognition sites between the functional groups and the target cells.15 Vardharajula et al. reported that functionalized CNTs could reduce cytotoxic Received: May 7, 2014 Revised: August 25, 2014

A

dx.doi.org/10.1021/jp5044943 | J. Phys. Chem. C XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry C

Article

Table 1. Fitting Results to Adsorption Isotherms of BSA and LYZ on the Three CNTs

BSA

type

model

HM

Langmuir Langmuir Q1 Langmuir Langmuir Q1 Langmuir Langmuir Q1 Langmuir Langmuir Q1 Langmuir Langmuir Q1 Langmuir Langmuir Q1

CM

GM

LYZ

HM

CM

GM

R2

Q0 (mg/g)

Q1 (mg/g)

(Q0 + Q1) (mg/g)

Q1/(Q0 + Q1) × 100 (%)

+

0.925 0.995

238.2 ± 11.99 207.8 ± 5.317

− 58.37 ± 4.373

238.2 ± 11.99 266.2 ± 9.690

− 21.93

1.045 1.168

− 541.8

+

0.878 0.985

134.2 ± 4.901 91.51 ± 4.062

− 61.71 ± 3.977

134.2 ± 4.901 153.2 ± 8.039

− 40.28

0.818 0.934

− 389.7

− 740.5

+

0.984 0.993

130.2 ± 1.047 115.2 ± 4.082

− 16.97 ± 4.638

130.2 ± 1.047 132.2 ± 8.720

− 12.84

1.113 1.130

− 278.0

− 528.3

+

0.992 0.993

90700 ± 6100 88130 ± 5895

− 0a

90700 ± 6100 88130 ± 5895

− −

397.8 386.5

429.5 −

601.4 −

+

0.995 0.996

84670 ± 4602 82090 ± 4346

− 0a

84670 ± 4602 82090 ± 4346

− −

516.3 500.5

309.0 −

432.6 −

+

0.995 0.996

81780 ± 4303 79290 ± 3776

− 0a

81780 ± 4303 79290 ± 3776

− −

699.0 677.7

220.4 −

308.6 −

(Q0 + Q1)/Asurf (mg/m2)

model Ib (mg/g)

model IIc (mg/g) − 1029

The fitting value is approximately 0. bThe minimum saturated adsorption on the three CNTs. cThe maximum saturated adsorption on the three CNTs.

a

effect by improving their dispersion and biocompatibility.12 However, there is still a lack of information about the impact of CNTs on human health, especially the interactions of CNTs with common proteins. Thus, determining the interaction mechanisms between proteins and functionalized CNTs will greatly help to understand the health risk of CNTs at a fundamental level. Bovine serum albumin (BSA), which is a model protein due to its relatively high structural stability, has a wide range of applications in molecular biology.16,17 Previous research showed that the hydrophobic pocket of BSA would get exposed after being adsorbed on CNTs. Furthermore, the hydrophobic and aromatic residues in the binding pocket of BSA would interact with the hydrophobic surface of CNTs.18 Jeyachandran et al. calculated the adsorption energy for BSA molecules on hydrophilic surface of GeO2 (GeOH) and hydrophobic surface of polystyrene (PS),19 and showed that the hydrophilic surface of BSA molecules would be attracted to the hydrophilic surface of GeOH while the hydrophobic binding pocket of BSA could be attracted to the hydrophobic PS. Compared to BSA, lysozyme is a more stable protein.20,21 Calvaresi et al. used computer simulation to calculate the adsorption site of LYZ on CNTs. Amino acid residues Y20− Y23 and W111−R114 were determined to act like tweezers to clamp the CNTs.22 Most researches mainly focused on the interaction between proteins and different types of graphitized CNTs, such as single-walled CNTs, multiwalled CNTs,23 and CNTs with different diameters.24 However, reports are rare on the adsorption mechanisms of BSA and LYZ on functionalized CNTs. The possible secondary structure change of proteins caused by the functional groups of CNTs is unclear. In this study, we aimed to study the adsorption of BSA and LYZ on functionalized CNTs in phosphate buffer. Then, the dispersion of functionalized CNTs by these two proteins was investigated. The secondary structural changes of BSA and LYZ on the CNT surface and the effects of functional groups on CNTs were further analyzed. Our research will be useful for better understanding the mechanism of protein adsorption on functionalized CNTs and would provide useful information for the biomedical application of functionalized CNTs.

2. EXPERIMENTAL METHODS 2.1. Materials. Three types of multiwall CNTs (purity >95%), including hydroxylized (HM), carboxylized (CM), and graphitized (GM), were purchased from Chengdu Organic Chemistry Co., Chinese Academy of Sciences. All the three CNTs were characterized and their properties including surface area, total and surface elemental composition, and surface functional groups are shown in Table S1 in the Supporting Information. BSA and LYZ lyophilized powders were obtained from Sigma-Aldrich Co. The characteristics of BSA and LYZ such as isoelectric point (pI), molecular weight, and total hydrophobic residues on surface are listed in Table S2 in the Supporting Information. Phosphate buffer (5 mM, pH 7.0), with the addition of NaN3 (200 mg/L, finial concentration) as a biocide, was used as the background solution throughout the whole experiments. The protein concentration assay set for the measurement of protein was purchased from Bio-Rad Co. 2.2. Adsorption Experiments. All adsorption isotherms were obtained by a batch equilibration technique at 25 ± 1 °C in 40 mL glass vials.25 The adsorption experiments were conducted with 12 concentration points including a blank sample, and each point was run in duplicate. For BSA adsorption, 20 ± 0.5 mg of CNTs (HM, CM, or GM) was added into a 40 mL vial. Then, 40 ± 0.3 mL of BSA solutions ranging from 0 to 600 mg/L was added. For LYZ adsorption, the CNT mass and LYZ (0−13 000 mg/L) volume added were 3.0 ± 0.2 mg and 40 ± 0.3 mL, respectively. All vials were shaken at 150 rpm for 72 h to reach adsorption equilibrium. Then, all the vials were centrifuged at 3000 rpm for 25 min, and the protein concentration in the supernatant was diluted into the range of the calibration curve and then determined by the Bradford method at 595 nm using a UV spectrometer.26 2.3. Isotherm Model Fitting. Two types of Langmuir models were used to fit the adsorption isotherms of proteins on CNTs. Type I was a reversible Langmuir-type isotherm model,27,28 which has been applied in BSA adsorption on titanium surface at pH 8.0 and 9.0.29 Type II was an irreversible adsorption plus reversible Langmuir-type adsorption, which has been observed for the adsorption of carboxylic acids containing multiple carboxyl groups on stainless steel and adsorption of αB

dx.doi.org/10.1021/jp5044943 | J. Phys. Chem. C XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry C

Article

Figure 1. Adsorption isotherms of BSA and LYZ on the three CNTs. (A) For BSA adsorption isotherms, the protein concentration was in the range of 0−600 mg/L and the three types of CNTs concentrations were 500 mg/L. (B) For LYZ adsorption isotherms, the protein concentration was in the range of 0−13 000 mg/L and the three types of CNTs concentrations were 75 mg/L.

lactoalbumin at pH = 4.5 and β-casein at pH = 6.0 on titanium surface with a higher sorption affinity.30−33 Type II model showed an intercept Q1 on y-axis which was considered as the adsorption maximum contributed by chemical bonding.31 The model equations of Type I and Type II are as follows: Qe =

determine the interfacial tension between BSA solution and corn oil. The concentration of BSA ranged from 0 to 13 000 mg/L. The data were automatically recorded every 3 s and the final data were the average number of records in 15 min. The CMC of LYZ was not measured in this study because no obvious dispersion of CNTs was observed in section 2.4. The critical micelle concentration (CMC) of BSA was obtained based on the aggregation of BSA molecules at high concentrations and calculated according to the critical change of interfacial tension.34 2.6. Secondary Structure Changes of Protein after Adsorption on CNTs As Determined by FTIR and CD Spectrometers. A PerkinElmer Spectrum One Fourier transform infrared (FTIR) spectrometer, equipped with a lithium tantalite detector and one-reflection horizontal attenuated total reflectance (ATR) accessory with a diamond/ZnSe internal reflection element crystal, was used to collect all ATR-FTIR spectra. The concentrations of CNTs (HM, CM, or GM) and BSA were 500 and 600 mg/L, respectively. To start, 40 mL of 600 mg/L BSA was added into a vial that already had 20 mg of CNTs (HM, CM, or GM) while 13 000 mg/L of LYZ and 3.0 mg of CNTs were used. The samples were shaken at 150 rpm for 72 h and then left undisturbed for 24 h. After centrifugation at 3000 rpm for 30 min, the supernatant of the mixture samples was dropped on the crystal plate. The samples were detected after being completely dried. All spectra were obtained by collecting at least 200 scans with a spectral resolution of 4 cm−1 and a scan speed of 0.5 cm/s and subtracting with the blank (each type of CNTs in phosphate buffer in the absence of protein). The baselines of all the spectra were corrected automatically using PerkinElmer spectrum software, and the root-mean-square noise of all the spectra was calculated. Seven or nine major peaks were determined by second derivative in the spectrum range 1600−1700 cm−1 for the analysis of protein secondary structure.35 Finally, the amide I overlap peaks (ranging from 1600 to 1700 cm−1) were separated using OriginPro 9.0 software and the peak area was calculated based on Gaussian function. Secondary structures of proteins before and after CNT treatment were also examined by a circular dichroism (CD) spectrometer (Jasco 815). The secondary structure of proteins was measured over the range of 190−240 nm by 0.2 nm intervals in 0.1 cm path length cell. The scan rate was set up at 100 nm/min, and each spectrum was from the average of 20 scans.18

Q 0bCe 1 + bCe

(I)

where Q0 (mg/g) is the maximum adsorption capacity by physical bonding, Qe (mg/g) is equilibrium concentration adsorbed on CNTs, Ce (mg/L) is equilibrium protein concentration in solution, and b (L/mg) is adsorption coefficient. Qe =

Q 0bCe 1 + bCe

+ Q1

(II)

where Q1 (mg/g) is the adsorption maximum by chemical bonding. In order to study the arrangement of protein molecules on the surface of the three CNTs, the theoretical maximum and minimum adsorption capacities (Table 1) were calculated based on both surface areas of CNTs (Table S1) and the threedimension size of proteins (Table S2). 2.4. Turbidity and Surface Charge of the Three CNTs in the Presence of BSA and LYZ. In the CNTs−protein solutions, the protein (BSA or LYZ) concentrations were varied from 0 to 13 000 mg/L, with a fixed CNT concentration at 500 mg/L in 40 mL vials. The vials were shaken at 150 rpm for 72 h, and then all the samples were allowed to stand for 24 h. The turbidity of supernatants was measured at 800 nm by a UV−vis spectrometer. Surface charges of CNT−protein hybrids in the vials were also determined by a ZetaPlus zeta potential analyzer (Brookhaven Instruments Co.), and the data were obtained by averaging the results from six measurements. All samples were run in duplicate for both turbidity and surface charge measurements. For the dispersed CNTs in the presence of protein, transmission electron microscopy (TEM, JEOL 100CX) was used to observe their morphologies. The CNTs−protein samples were prepared by dispersing CNTs in protein solutions at room temperature, and the supernatants were diluted if needed. All the samples were dropped on 400 square mesh copper grids and air-dried for 3 days, then the samples were observed using TEM. 2.5. Critical Micelle Concentration (CMC) Determination. Drop shape analyzer (DSA100, Kruss) was used to C

dx.doi.org/10.1021/jp5044943 | J. Phys. Chem. C XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry C

Article

Figure 2. TEM images of BSA and LYZ on GM: (A) BSA adsorption on GM; (B) LYZ adsorption on GM. TEM image in panel B clearly shows the adsorption corona (blue arrow) for LYZ on GM.

3. RESULTS AND DISCUSSION 3.1. Adsorption of BSA and LYZ on Functionalized CNTs and Factor Regulated. Adsorption isotherms of BSA by functionalized CNTs and their fitting results by two types of isotherm models are shown in Figure 1 and Table 1, respectively. For BSA adsorption on all the three CNTs, type II model (RHM2 = 0.995, RCM2 = 0.985, and RGM2 = 0.993) fitted better than type I (RHM2 = 0.925, RCM2 = 0.878, and RGM2 = 0.984). Therefore, the fitting results from type II were used to obtain the adsorption maxima of BSA on the three CNTs. The adsorption capacity of BSA followed an order HM > CM > GM, which was the same as the surface area order of the three CNTs (Table S1). However, after surface area normalization, the adsorption capacity order was HM > GM > CM. The different order of adsorption maxima and surface-areanormalized capacity indicated that the different functional groups on CNTs might also affect BSA adsorption besides surface area. This result is supported by our previous study, in which adsorption capacities of bulk and nano oxide particles for BSA were controlled by the surface area and functional group of the particles.27 Chemical bonds may be formed between functional groups of CNTs and BSA, and this was supported by the good fitting of isotherms by type II Langmuir model (Table 1). For BSA adsorption on CM, the percentage of chemical adsorption (Q1/(Q0 + Q1)%, 40.28%) was much higher than those of the other two CNTs (21.93% for HM and 12.84% for GM), indicating the stronger covalent bonding between carboxyl groups on CNTs and amine groups of BSA.29,36,37 Piao et al. also showed that the carboxyl groups on single-wall CNTs could interact with amine groups of amino acids to form an amide bond (−C(O)NH− group) by using FTIR.37 However, for BSA sorption on HM, the hydroxyl groups can only generate ester bonds with carboxyl groups on BSA in acidic condition, and thus the chemical adsorption percentage was lower than CM in neutral phosphate buffer in the present work. The oxygen content of GM was much lower than HM and GM according to the data of both elemental composition analysis and X-ray photoelectron spectroscopy (XPS) (Table S1). Thus, BSA on GM had the weakest covalent bonding and lowest percentage of chemical adsorption. For LYZ adsorption, the fitting results from both type I (RHM2 = 0.992, RCM2 = 0.995, and RGM2 = 0.995) and type II (RHM2 = 0.993, RCM2 = 0.996, and RGM2 = 0.996) were similar, indicating that the covalent bonds did not contribute the dominant LYZ adsorption. The covalent bonds could also be formed between CM and amine groups of LYZ. The unobvious chemical adsorption contribution was probably because of the multilayer adsorption and the extremely high adsorption capacities. As a result, most of the adsorbed LYZ molecules

did not directly interact with CM. The multilayer adsorption of LYZ will be discussed later. Hydrophobic interaction, π−π interaction, hydrogen binding, and electrostatic attraction were probably responsible for the adsorption of LYZ on CNTs.22,38−40 As shown in Table 1, the order of maximum adsorption capacities (Q0 + Q1) of LYZ was HM > CM > GM, which is also positively related to the surface area of the three CNTs. However, the surface-area-normalized adsorption capacity followed an order of GM > CM > HM, which is negatively related to the oxygen contents of the three CNTs (Table S1). This suggested that the hydrophobic and π−π interactions could be the dominant driving forces for LYZ sorption on the three CNTs. It should be noted that the maximum adsorption capacity of LYZ was around 280 times higher than those of BSA on each type of CNTs, indicating that LYZ on CNTs surface may not be a monolayer. The theoretical maximum and minimum saturated adsorptions of each protein on the three CNTs based on monolayer adsorption were calculated and are listed in Table 1. We assumed that the surface arrangement was based on the three dimensions of the proteins, and the schematic diagram is shown in Figure S1 in the Supporting Information. The experimental adsorption capacity of BSA (Q0 + Q1) for each type of CNTs was less than its theoretical minimum adsorption capacity, suggesting a monolayer adsorption of BSA on CNTs. The lower experimental adsorption capacities may be due to the electrostatic repulsion between BSA molecules (−16 mV) and CNTs (−8, −22, and −20 mV for GM, CM, and HM, respectively) and among BSA molecules in phosphate buffer (pH = 7). However, the experimental adsorption capacities for LYZ on the three CNTs were much higher than the theoretical maximum adsorption capacities. Although the adsorption isotherms of LYZ were fitted well by Langmuir model which is based on the assumption of monolayer adsorption, multilayer adsorption may actually occur according to the extremely higher experimental adsorption capacities. We further observed the morphologies of CNTs in the presence of proteins (Figure 2, and Figure S2 in the Supporting Information). A LYZ “corona” is clearly shown on the surface of GM (Figure 2B), CM (Figure S2A), and HM (Figure S2B); however, no “corona” was observed on CNTs in the presence of BSA (Figure 2A, Figure S6A,B). Therefore, multilayer adsorption of LYZ may occur on the three CNTs, as “corona”.2,41−43 Our result is highly consistent with that of Kim et al., in which BSA monolayer adsorption and LYZ multilayer adsorption were found on polystyrene and silica by infrared−visible sum frequency generation (SFG) vibrational spectroscopy.38 In fact, for LYZ, the two possible hydrophobic sites located on the two sides of LYZ proteins were reported as the potential D

dx.doi.org/10.1021/jp5044943 | J. Phys. Chem. C XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry C

Article

Figure 3. TEM images of GM dispersed by BSA and dispersion of three CNTs in the presence of BSA or LYZ. (A) GM in the phosphate buffer; (B) BSA in phosphate buffer; (C) GM in the presence of BSA; (D) dispersion of three CNTs in the presence of BSA or LYZ. In panel D, the concentrations of CNTs and proteins in the solutions were 500 and 0−13 000 mg/L (initial concentration), respectively.

−10 and −20 mV for all CNTs (Figure S4 in the Supporting Information) and electrostatic repulsion could be another driving force for the dispersion of CNTs. The dispersion of CNTs in the presence of BSA was further observed using TEM (Figure 3A−C, and Figure S6 in the Supporting Information). The aggregation of CNTs was clearly observed in the absence of BSA (Figure 3A). However, individual CNTs were observed for all the three CNTs (Figure 3C, Figure S6A,B), and the edge of these individual CNTs was unclear due to BSA coating. In addition, when the BSA concentration was higher than 1000 mg/L, a slight decrease of CNT dispersion was observed (Figure 3D). Water−oil interfacial tensions were measured as a function of BSA concentrations (Figure S5 in the Supporting Information). A critical interfacial tension change was observed when BSA concentration was around 950−1050 mg/L, indicating the occurrence of BSA aggregation. Our previous study also observed that BSA molecules could form ringlike aggregate due to hydrophobic interaction using an atomic force microscope.47 The formation of aggregates may decrease the dispersion performance of BSA, thereby leading to the above slight dispersion decrease. Interestingly, the overall dispersion of the three CNTs in the presence of BSA followed GM > CM > HM, which has no correlation with the maximum adsorption capacity or surfacearea-normalized adsorption capacity (Table 1) of the three CNTs, indicating that the dispersion was not a dominant factor that affects the adsorption capacity. CM and HM had higher amounts of functional groups than GM as indicated by the oxygen percentages (Table S1). The functional groups of CM and HM may interact with the hydrophilic surface of BSA via hydrogen bonding, thus exposing the hydrophobic core to the surface. This interaction could lower the hydrophilic property of functionalized CNTs and BSA hybrids19 and may be responsible for the observed lower dispersibility of functionalized CNTs in the presence of BSA compared to GM. Moreover, the best dispersion of all the three CNTs reached at the BSA concentration of around 1000 mg/L. Then the

adsorption sites by molecular dynamics simulations.22 One site is located at the hydrophobic core in the α-domain and the other site is located at a groove between the α- and β-domains. There are also some hydrophobic clusters on the surface of the β-domain. No matter which site interacts with CNTs, the remaining one would potentially stack with other LYZ molecules at the high concentration of LYZ. However, the multilayer adsorption mechanism of LYZ is not completely clear at this point and further study needs to be conducted. In addition, chemical binding could only occur on the first adsorption layer, and therefore, multilayer adsorption and high adsorption capacity of LYZ could also be the reasons for the insignificant chemical adsorption of LYZ on CNTs. 3.2. Dispersion of the Three CNTs in the Presence of BSA and LYZ. In the presence of LYZ, no obvious dispersion was observed for all the three CNTs (Figure 3D), which may be due to the highly hydrophobic surface of LYZ,44 suggesting that LYZ was not hydrophilic enough to disperse the CNTs. This result is opposite to a previous study showing that LYZ could improve the dispersion of single-wall CNTs.45 This obvious dispersion performance is due to the assistance of sonication, which could deconstruct the structure of LYZ molecules. However, the dispersion of CNTs in the presence of BSA was obviously higher than those in the presence of LYZ. The dispersion of GM dramatically increased with increasing BSA concentration and reached the highest dispersion level at a BSA concentration of around 700 mg/L. The same trend was shown for CM and HM, but the highest dispersion was observed when the concentration of BSA was around 1000 mg/ L (Figure 3D). BSA molecule contains both hydrophobic and hydrophilic residues on the surface (Table S2). After being adsorbed on CNTs, the hydrophobic residues of BSA molecules would interact with the CNTs surface and expose the hydrophilic residues outside to water (Figure S3A in the Supporting Information). Therefore, the steric repulsive force could prevent the aggregation of CNTs.46 In addition, the zeta potential values of BSA-adsorbed CNTs were in the range of E

dx.doi.org/10.1021/jp5044943 | J. Phys. Chem. C XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry C

Article

Figure 4. IR spectra of BSA (A) and LYZ (B) adsorbed on the three CNTs. The spectrum of each bound protein was obtained by subtracting the spectrum of the treated CNTs in phosphate solution.

Table 2. Ratio of Amide I and Amide II (AI/AII) and the Percentage of Secondary Structure of BSA and LYZ in the Presence or Absence of the Three CNTs Determined by ATR-IR Multipeak Fitting Program and CD Spectrometer ATR-IR multipeak fitting (%)

ATR-IR

CD spectra (%)

samples

AI/AII

β antiparallel

β-turn

α-helix

unordered

β-sheet

α-helix

β-strand

unordered

BSA BSA-HM BSA-CM BSA-GM LYZ LYZ-HM LYZ-CM LYZ-GM

1.465 1.589 1.579 1.349 1.481 1.389 1.455 1.461

0.36 0.57 0 0.62 2.00 1.85 2.14 2.12

11.78 18.03 19.56 21.88 41.60 41.35 44.49 43.94

59.93 54.94 43.96 41.55 8.81 7.77 8.12 7.85

6.77 5.11 11.81 14.97 17.70 18.81 16.57 16.67

21.15 21.34 24.67 20.97 29.89 30.22 28.68 29.42

36.38 36.17 28.23 30.43 6.36 4.23 4.23 4.21

9.68 10.48 22.38 17.75 44.99 46.99 46.29 48.81

53.94 53.35 40.39 51.82 48.65 48.78 49.48 46.98

The peaks at 1656 and 1542 cm−1 were identified as amide I (CO stretching vibration) and amide II (mainly N−H bending vibration, coupled to CO and CC stretching), respectively.27,39 These two peaks are widely used to study the secondary structure of proteins. Other major peaks can be assigned in the folowing regions: 2871 cm−1 (CH2 stretching), 2369 cm−1 (P−H phosphine), 1287 cm−1 (C−O stretching from COOH), 1130 cm−1 (aliphatic C−N), and 1603 cm−1 (C−N stretching).27 The ratio of amide I and II indicates the structural stability of proteins, and the ratios of proteins before and after adsorption are shown in Table 2. For BSA sorption on the three CNTs, the ratio order followed HM > CM > BSA > GM, suggesting that the functional groups of CNTs could directly interfere with the N−H stretching by hydrogen bonding and covalent bonding or interact with the side chain of the backbone in order to increase the ratio of amide I and II by decreasing the amide II bind strength.39 For LYZ samples, the ratios were closer to each other and the order followed LYZ> MG > MC > MH (Figure 4B). However, we cannot explain the observed ratio order currently. Multilayer adsorption may be related to this order and further investigation is required. 3.4. Secondary Structural Changes of BSA and LYZ after Adsorption on the Three CNTs. The secondary structural changes of BSA and LYZ after adsorption on the three CNTs were investigated using both FT-IR and CD spectrometers. Secondary structures of BSA and LYZ were quantitatively analyzed using an IR spectra multipeak fitting program (Figure 5). The peaks ranging from 1600 to 1700 cm−1 are β-antiparallel (1693 ± 2 and 1689 cm−1), β-turn (1682 ± 1, 1670 ± 1, and 1662 ± 1 cm−1), α-helix (1654

dispersions were decreased with increasing BSA concentrations from 1000 to 13 000 mg/L, and the decreasing percentages of GM, CM, and HM were calculated as 19%, 15%, and 13%, respectively. The deceasing percentage of functionalized CNTs (CM and HM) in the presence of BSA was less than that of GM, probably because of the functional group-induced arrangement of BSA molecules on CNT surface (Figure S3). For GM, the hydrophobic residues/core of BSA would be adsorbed on the GM surface while leaving the hydrophilic residues exposed in aqueous solution. For HM and CM, the surface functionality of the two CNTs reduced the hydrophilic residue number of BSA at the exposed aqueous interface. Therefore, the surface of functionalized CNTs and BSA hybrids could be more hydrophobic and tended to aggregate together with increasing BSA concentrations. 3.3. ATR-IR Spectra of Proteins after Sorption on the Three CNTs. ATR-IR spectra of free proteins and proteins bound on the three CNTs are presented in Figure 4. The spectra of bound proteins were obtained by subtracting the IR spectra of pure CNTs from those of protein−CNTs hybrids. The sharp peak at 3312 cm−1, assigned as N−H stretching (mainly from −NH2), was shown in the spectra of all BSA samples except for that adsorbed on CM (Figure 4A), suggesting the formation of peptide bonds between −NH2 on BSA molecules and −COOH on CM. For LYZ, the sharp peak at 3312 cm−1 was observed in all samples including the one bound on CM. This is probably because of multilayer adsorption of LYZ on CM, and that the percentage of −NH2 groups that participated in the reaction was much lower than BSA. F

dx.doi.org/10.1021/jp5044943 | J. Phys. Chem. C XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry C

Article

Figure 5. Secondary derivative spectra of BSA and LYZ after adsorption on HM (left), CM (middle), and GM (right). Original IR peak (red), αhelix (cyan), β-turn (brownish red), antiparallel (green), β-sheet (pink), and random coil (black).

cm−1), unordered (1645 cm−1), and β-sheet (1637 ± 1, 1624 ± 62, and 1615 ± 1 cm−1), respectively (Figure 5), which are highly consistent with previous studies.36,39,48,49 However, previous study demonstrated that IR spectra multipeak fitting program cannot completely separate the β-sheet and β-turn in the range of 1685−1633 cm−1 and extended chains and β-sheet in the range of 1632−1621 cm−1.39,48 So in this section, we are mainly discussing for the percentage of α-helix structure change. The fitting result showed that α-helical content of BSA decreased from 59.93% to 54.94%, 43.96%, and 41.55% after being adsorbed on HM, CM, and GM, respectively. The deconstruction of BSA molecules indicated that CNTs could directly or indirectly interfere with the hydrogen bond of the N−H group with CO group on the α-helix from the backbones and side chains in order to make BSA protein more readily adsorb on the surface of the CNTs. Our result agreed with a previous study showing that the loss of helical structure on hydrophobic surface was greater than those on hydrophilic surface.48 In addition, because of the dynamic adsorption and desorption of BSA on CNTs, the BSA protein could refold after desorption from CNTs.24,40 However, for the CM, the formation of amine bond could prevent the BSA protein back to refold, indicating the loss of α-helical structure on CM (27%) is close to the loss on GM (30%). For LYZ, the α-helix decreased slightly from 8.81% to 7.77%, 8.12%, and 7.85% after adsorbed on HM, CM, and GM, respectively, indicating the much more stable structure of LYZ than BSA. Previous study proved that the adsorption site of LYZ was the hydrophobic pocket that is located downside of LYZ with random structure

and loops.22,50,51 This adsorption process may also indicate the slight changes of the secondary structure of LYZ. CD spectra of the free and bound proteins are shown in Figure S7 in the Supporting Information. The α-helical content of BSA decreased from 36.38% to 36.17%, 28.23%, and 30.43% after being adsorbed on HM, CM, and GM, respectively, which are in agreement with the FTIR multipeak fitting results. The similar results showed that the α-helical content of BSA reduced 32% to 16% on totally hydrophobic silica surface.48 CD results also showed a slightly loss of α-helix on HM and greater loss on GM and CM. However, for LYZ adsorption, the results suggested the slight loss of α-helical structure on three CNTs and slight gain on β-structure. The lower secondary changes of LYZ than BSA may be due to the relative stable structure of LYZ as indicated by lower adiabatic compressibility (Table S2).

4. CONCLUSIONS This study demonstrated the adsorption properties and mechanisms of BSA and LYZ on functionalized CNTs. The maximum adsorption capacity followed the order of LYZ-HM > LYZ-CM > LYZ-GM ≫ BSA-HM > BSA-CM > BSA-GM. However, surface-area-normalized adsorption capacities followed LYZ-GM > LYZ-CM > LYZ-HM ≫ BSA-HM > BSAGM > BSA-CM. The different orders of adsorption capacity and surface-area-normalized adsorption capacities attested that both surface area and functional groups of CNTs affected the protein adsorption. BSA molecules on CNTs followed a monolayer adsorption manner while LYZ molecules could form multilayers on CNT surface, which led to extremely high G

dx.doi.org/10.1021/jp5044943 | J. Phys. Chem. C XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry C

Article

Carbon Nanotubes Introduced into the Abdominal Cavity of Mice Show Asbestos-like Pathogenicity in a Pilot Study. Nat. Nanotechnol. 2008, 3, 423−428. (10) Fubini, B.; Ghiazza, M.; Fenoglio, I. Physico-chemical Features of Engineered Nanoparticles Relevant to Their Toxicity. Nanotoxicology 2010, 4, 347−363. (11) Porter, A. E.; Gass, M.; Bendall, J. S.; Muller, K.; Goode, A.; Skepper, J. N.; Midgley, P. A.; Welland, M. Uptake of Noncytotoxic Acid-Treated Single-Walled Carbon Nanotubes into the Cytoplasm of Human Macrophage Cells. ACS Nano 2009, 3, 1485−1492. (12) Vardharajula, S.; Ali, S. Z.; Tiwari, P. M.; Eroglu, E.; Vig, K.; Dennis, V. A.; Singh, S. R. Functionalized Carbon Nanotubes: Biomedical Applications. Int. J. Nanomed. 2012, 7, 5361−5374. (13) Ge, C. C.; Meng, L.; Xu, L. G.; Bai, R.; Du, J. F.; Zhang, L. L.; Li, Y.; Chang, Y. Z.; Zhao, Y. L.; Chen, C. Y. Acute Pulmonary and Moderate Cardiovascular Responses of Spontaneously Hypertensive Rats after Exposure to Single-wall Carbon Nanotubes. Nanotoxicology 2012, 6, 526−542. (14) Bhunia, S. K.; Saha, A.; Maity, A. R.; Ray, S. C.; Jana, N. R. Carbon Nanoparticle-based Fluorescent Bioimaging Probes. Sci. Rep. 2013, 3, 1473. (15) Jain, K. K. Advances in Use of Functionalized Carbon Nanotubes for Drug Design and Discovery. Expert Opin. Drug Discovery 2012, 7, 1029−1037. (16) Xiao, Y.; Isaacs, S. N. Enzyme-linked Immunosorbent Assay (ELISA) and Blocking with Bovine Serum Albumin (BSA)Not All BSAs are Alike. J. Immunol. Methods 2012, 384, 148−151. (17) Mesapogu, S.; Jillepalli, C.; Arora, D. Restriction Enzymes and Their Role in Microbiology, in Analyzing Microbes; Arora, D. K., Das, S., Sukumar, M., Eds.; Springer: Berlin, 2013; pp 31−36. (18) Zhao, X. C.; Liu, R. T.; Chi, Z. X.; Teng, Y.; Qin, P. F. New Insights into the Behavior of Bovine Serum Albumin Adsorbed onto Carbon Nanotubes: Comprehensive Spectroscopic Studies. J. Phys. Chem. B 2010, 114, 5625−5631. (19) Jeyachandran, Y. L.; Mielezarski, E.; Rai, B.; Mielczarski, J. A. Quantitative and Qualitative Evaluation of Adsorption/Desorption of Bovine Serum Albumin on Hydrophilic and Hydrophobic Surfaces. Langmuir 2009, 25, 11614−11620. (20) Zhao, J.; Wang, Z. Y.; Ghosh, S.; Xing, B. S. Phenanthrene Binding by Humic Acid−Protein Complexes as Studied by Passive Dosing Technique. Environ. Pollut. 2014, 184, 145−153. (21) Chalikian, T. V.; Totrov, M.; Abagyan, R.; Breslauer, K. J. The Hydration of Globular Proteins as Derived from Volume and Compressibility Measurements: Cross Correlating Thermodynamic and Structural Data. J. Mol. Biol. 1996, 260, 588−603. (22) Calvaresi, M.; Hoefinger, S.; Zerbetto, F. Probing the Structure of Lysozyme-Carbon-Nanotube Hybrids with Molecular Dynamics. Chem.Eur. J. 2012, 18, 4308−4313. (23) Wang, Z. Y.; Zhao, J.; Li, F. M.; Gao, D. M.; Xing, B. S. Adsorption and Inhibition of Acetylcholinesterase by Different Nanoparticles. Chemosphere 2009, 77, 67−73. (24) Mu, Q. X.; Liu, W.; Xing, Y. H.; Zhou, H. Y.; Li, Z. W.; Zhang, Y.; Ji, L. H.; Wang, F.; Si, Z. K.; Zhang, B.; Yan, B. Protein Binding by Functionalized Multiwalled Carbon Nanotubes is Governed by the Surface Chemistry of Both Parties and the Nanotube Diameter. J. Phys. Chem. C 2008, 112, 3300−3307. (25) Zhao, J.; Wang, Z. Y.; Mashayekhi, H.; Mayer, P.; Chefetz, B.; Xing, B. S. Pulmonary Surfactant Suppressed Phenanthrene Adsorption on Carbon Nanotubes through Solubilization and Competition As Examined by Passive Dosing Technique. Environ. Sci. Technol. 2012, 46, 5369−5377. (26) Bradford, M. M. Rapid and Sensitive Method for Quantitation of Microgram Quantities of Protein Utilizing Principle of Protein-Dye Binding. Anal. Biochem. 1976, 72, 248−254. (27) Song, L.; Yang, K.; Jiang, W.; Du, P.; Xing, B. S. Adsorption of Bovine Serum Albumin on Nano and Bulk Oxide Particles in Deionized Water. Colloids Surf., B 2012, 94, 341−346.

adsorption capacities (81 800−90 700 mg/g) of the three CNTs for LYZ. Moreover, BSA molecules could disperse all the three CNTs in phosphate buffer. However, LYZ molecules were unable to disperse CNTs. Finally, the secondary structure of BSA and LYZ were changed upon adsorption on the three CNTs according to both FTIR and CD results. GM had higher structural change of α-helix than the other two functionalized CNTs, and the α-helix of BSA was easier to be diminished by CNTs than LYZ. The information on interactions between proteins and CNTs will be of importance in biomedical and chemical applications, and will help to gain the knowledge of toxicity and risk assessment.



ASSOCIATED CONTENT

S Supporting Information *

The supporting information includes the characteristics of CNTs, both BSA and LYZ proteins, modes I and II for adsorption simulation, the TEM images of BSA-HM, BSA-CM, LYZ-HM, and LYZ-CM hybrids, the surface charge of three CNTs in two proteins, and the CD spectra of two proteins in the presence of three CNTs. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*Tel.: +1 413 545 5212. E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This project was supported by the USDA AFRI Hatch program (MAS00978) and NSF of China (41120134004). The authors gratefully thank Emily Cole at UMASS Amherst for editing the manuscript.



REFERENCES

(1) Iijima, S. Helical Microtubules of Graphitic Carbon. Nature 1991, 354, 56−58. (2) Lynch, I.; Dawson, K. A. Protein-nanoparticle Interactions. Nano Today 2008, 3, 40−47. (3) Ali-Boucetta, H.; Al-Jamal, K.; Kostarelos, K. Cytotoxic Assessment of Carbon Nanotube Interaction with Cell Cultures, in Biomedical Nanotechnology; Hurst, S. J., Ed.; Humana Press: New York, 2011; pp 299−312. (4) Firme, C. P.; Bandaru, P. R. Toxicity Issues in the Application of Carbon Nanotubes to Biological Systems. Nanomed-Nanotechnol. 2010, 6, 245−256. (5) Pulskamp, K.; Diabate, S.; Krug, H. F. Carbon Nanotubes Show No Sign of Acute Toxicity But Induce Intracellular Reactive Oxygen Species in Dependence on Contaminants. Toxicol. Lett. 2007, 168, 58−74. (6) Belyanskaya, L.; Weigel, S.; Hirsch, C.; Tobler, U.; Krug, H. F.; Wick, P. Effects of Carbon Nanotubes on Primary Neurons and Glial Cells. Neurotoxicology 2009, 30, 702−711. (7) Jia, G.; Wang, H. F.; Yan, L.; Wang, X.; Pei, R. J.; Yan, T.; Zhao, Y. L.; Guo, X. B. Cytotoxicity of Carbon Nanomaterials: Single-wall Nanotube, Multi-wall Nanotube, and Fullerene. Environ. Sci. Technol. 2005, 39, 1378−1383. (8) Palomaki, J.; Valimaki, E.; Sund, J.; Vippola, M.; Clausen, P. A.; Jensen, K. A.; Savolainen, K.; Matikainen, S.; Alenius, H. Long, Needle-like Carbon Nanotubes and Asbestos Activate the NLRP3 Inflammasome through a Similar Mechanism. ACS Nano 2011, 5, 6861−6870. (9) Poland, C. A.; Duffin, R.; Kinloch, I.; Maynard, A.; Wallace, W. A. H.; Seaton, A.; Stone, V.; Brown, S.; MacNee, W.; Donaldson, K. H

dx.doi.org/10.1021/jp5044943 | J. Phys. Chem. C XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry C

Article

Single-Walled Carbon Nanotubes. Environ. Sci. Technol. 2010, 44, 2412−2418. (47) Zhao, J.; Wang, Z. Y.; Zhao, Q.; Xing, B. S. Adsorption of Phenanthrene on Multilayer Graphene as Affected by Surfactant and Exfoliation. Environ. Sci. Technol. 2013, 48, 331−339. (48) 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. (49) Hamdani, S.; Joly, D.; Carpentier, R.; Tajmir-Riahi, H. A. The Effect of Methylamine on the Solution Structures of Human and Bovine Serum Albumins. J. Mol. Struct. 2009, 936, 80−86. (50) Horn, D. W.; Tracy, K.; Easley, C. J.; Davis, V. A. Lysozyme Dispersed Single-Walled Carbon Nanotubes: Interaction and Activity. J. Phys. Chem. C 2012, 116, 10341−10348. (51) Karajanagi, S. S.; Vertegel, A. A.; Kane, R. S.; Dordick, J. S. Structure and Function of Enzymes Adsorbed onto Single-Walled Carbon Nanotubes. Langmuir 2004, 20, 11594−11599.

(28) Wang, F.; Yao, J.; Sun, K.; Xing, B. S. Adsorption of Dialkyl Phthalate Esters on Carbon Nanotubes. Environ. Sci. Technol. 2010, 44, 6985−6991. (29) Imamura, K.; Shimomura, M.; Nagai, S.; Akamatsu, M.; Nakanishi, K. Adsorption Characteristics of Various Proteins to a Titanium Surface. J. Biosci. Bioeng. 2008, 106, 273−278. (30) Imamura, K.; Kawasaki, Y.; Awadzu, T.; Sakiyama, T.; Nakanishi, K. Contribution of Acidic Amino Residues to the Adsorption of Peptides onto a Stainless Steel Surface. J. Colloid Interface Sci. 2003, 267, 294−301. (31) Nagayasu, T.; Imamura, K.; Nakanishi, K. Adsorption Characteristics of Various Organic Substances on the Surfaces of Tantalum, Titanium, and Zirconium. J. Colloid Interface Sci. 2005, 286, 462−470. (32) Imamura, K.; Kawasaki, Y.; Nagayasu, T.; Sakiyama, T.; Nakanishi, K. Adsorption Characteristics of Oligopeptides Composed of Acidic and Basic Amino Acids on Titanium Surface. J. Biosci. Bioeng. 2007, 103, 7−12. (33) Nagayasu, T.; Yoshioka, C.; Imamura, K.; Nakanishi, K. Effects of Carboxyl Groups on the Adsorption Behavior of Low-MolecularWeight Substances on a Stainless Steel Surface. J. Colloid Interface Sci. 2004, 279, 296−306. (34) Blesic, M.; Marques, M. H.; Plechkova, N. V.; Seddon, K. R.; Rebelo, L. P. N.; Lopes, A. Self-Aggregation of Ionic Liquids: Micelle Formation in Aqueous Solution. Green Chem. 2007, 9, 481−490. (35) Jiang, W.; Yang, K.; Vachet, R. W.; Xing, B. S. Interaction between Oxide Nanoparticles and Biomolecules of the Bacterial Cell Envelope As Examined by Infrared Spectroscopy. Langmuir 2010, 26, 18071−18077. (36) Dembereldorj, U.; Ganbold, E.; Seo, J.; Lee, S. Y.; Yang, S. I.; Joo, S. Conformational Changes of Proteins Adsorbed onto ZnO Nanoparticle Surfaces Investigated by Concentration-dependent Infrared Spectroscopy. Vib. Spectrosc. 2012, 59, 23−28. (37) Piao, L.; Liu, Q.; Li, Y. Interaction of Amino Acids and SingleWall Carbon Nanotubes. J. Phys. Chem. C 2011, 116, 1724−1731. (38) Kim, J.; Somorjai, G. A. Molecular Packing of Lysozyme, Fibrinogen, and Bovine Serum Albumin on Hydrophilic and Hydrophobic Surfaces Studied by Infrared-Visible Sum Frequency Generation and Fluorescence Microscopy. J. Am. Chem. Soc. 2003, 125, 3150−3158. (39) Roach, P.; Farrar, D.; Perry, C. C. Interpretation of Protein Adsorption: Surface-induced Conformational Changes. J. Am. Chem. Soc. 2005, 127, 8168−8173. (40) Wang, Z. Y.; Zhao, J.; Song, L.; Mashayekhi, H.; Chefetz, B.; Xing, B. S. Adsorption and Desorption of Phenanthrene on Carbon Nanotubes in Simulated Gastrointestinal Fluids. Environ. Sci. Technol. 2011, 45, 6018−6024. (41) Cedervall, T.; Lynch, I.; Lindman, S.; Berggård, T.; Thulin, E.; Nilsson, H.; Dawson, K. A.; Linse, S. Understanding the Nanoparticle−Protein Corona Using Methods to Quantify Exchange Rates and Affinities of Proteins for Nanoparticles. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2050−2055. (42) Lindman, S.; Lynch, I.; Thulin, E.; Nilsson, H.; Dawson, K. A.; Linse, S. Systematic Investigation of the Thermodynamics of HSA Adsorption to N-iso-propylacrylamide/N-tert-butylacrylamide Copolymer Nanoparticles. Effects of Particle Size and Hydrophobicity. Nano Lett. 2007, 7, 914−920. (43) Mahmoudi, M.; Simchi, A.; Imani, M.; Hafeli, U. O. Superparamagnetic Iron Oxide Nanoparticles with Rigid Cross-linked Polyethylene Glycol Fumarate Coating for Application in Imaging and Drug Delivery. J. Phys. Chem. C 2009, 113, 8124−8131. (44) Cao, J.; Pham, D. K.; Tonge, L.; Nicolau, D. V. Predicting Surface Properties of Proteins on the Connolly molecular Surface. Smart Mater. Struct. 2002, 11, 772−777. (45) Matsuura, K.; Saito, T.; Okazaki, T.; Ohshima, S.; Yumura, M.; Iijima, S. Selectivity of Water-soluble Proteins in Single-walled Carbon Nanotube Dispersions. Chem. Phys. Lett. 2006, 429, 497−502. (46) Saleh, N. B.; Pfefferle, L. D.; Elimelech, M. Influence of Biomacromolecules and Humic Acid on the Aggregation Kinetics of I

dx.doi.org/10.1021/jp5044943 | J. Phys. Chem. C XXXX, XXX, XXX−XXX