Accepted Manuscript Title: Protein Adsorption Behaviors of Carboxymethylated Bacterial Cellulose Membranes Author: Qinghua Lin Yudong Zheng Guojie Wang Xiangning Shi Tao Zhang Jie Yu Jian Sun PII: DOI: Reference:
S0141-8130(14)00759-4 http://dx.doi.org/doi:10.1016/j.ijbiomac.2014.11.011 BIOMAC 4727
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
24-8-2014 13-11-2014 17-11-2014
Please cite this article as: Q. Lin, Y. Zheng, G. Wang, X. Shi, T. Zhang, J. Yu, J. Sun, Protein Adsorption Behaviors of Carboxymethylated Bacterial Cellulose Membranes, International Journal of Biological Macromolecules (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.11.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highlights
1.
Carboxymethylation largely changed the surface of BC membrane.
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2.
The hydrophobicity of CBC increased with the increase of the DS and pH;
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3.
Protein adsorption showed opposite performance around its isoelectric point.
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Protein Adsorption Behaviors of Carboxymethylated
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Bacterial Cellulose Membranes
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Qinghua Lin, Yudong Zheng*, Guojie Wang*, Xiangning Shi, Tao Zhang, Jie Yu, Jian Sun
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School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, PR China
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Corresponding author: Prof. Yudong Zheng, Prof. Guojie Wang E-mail: [email protected]; [email protected] Phone: +86-010-62330802, Fax: +86-010-62332336
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Abstract
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Carboxymethylated Bacterial Cellulose (CBC) membranes with different degrees of substitution
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(DS) were prepared by surface carboxymethylation of bacterial cellulose, which were characterized by
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FTIR, contact angles and zeta potential. Both the contact angels and zeta potentials increased with the
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increase of degrees of carboxymethylation. Protein adsorption behaviors of CBC membranes were
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investigated with bovine serum albumin (BSA) as model protein and the adsorption quantity increased
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gradually with the increase of the DS. The protein adsorption was not only governed by the chemical
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structure of CBC but also by the electrostatic interaction between CBC and BSA. The protein was
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found to be adsorbed on CBC membrane at pH lower its isoelectric point (pI) while no protein was
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adsorbed at pH above its pI. The protein adsorption on the CBC membranes was also characterized by
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scanning electron microscopy (SEM).
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Key word: Carboxymethylated; Bacterial cellulose; Surface; Protein adsorption.
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1. Introduction Protein adsorption behavior has been paid more and more attention in current research fields including bio-nanotechnology, biocompatibility evaluation and biological response between materials
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and biological organisms. As a matter of fact, such behavior is governed by the chemical structures of
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material surfaces, accordingly, providential surface modification may provide appropriate behaviors
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of protein-surface interactions needed [1, 2].
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Bacterial cellulose (BC), synthesized in abundance by Acetobacter xylinum, possesses unique
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physical and mechanical properties as well as high purity and uniformity with wide applications in
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industry and biomedical field, such as paper products [3], fuel cells [4], acoustics [5], wound dressings
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[6], blood vessel prosthesis [7], tissue regeneration materials [8] and other biomedical devices [9-11].
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Besides, BC has been attracting attention as a raw material for preparation of advanced materials due
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to its advantageous properties that are imparted by the unique structure [12-17]. BC derivative
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modified by various chemical substitutions with staggered degrees of functional groups could open
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further possibilities for new applications in different fields, due to its microfibrous structure and
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chemical activity; for instance, surface acetylation of bacterial cellulose was prepared to modify its
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physical properties， while preserving the microfibrillar morphology [18]. Diethylenetriamine BC and
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amidoximated BC was prepared for adsorptive removal of Cu (II) and Pb (II) [19]. Phosphorylated
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bacterial cellulose had been prepared for selective adsorption of different proteins, and it was found to
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be effective as an adsorbent for proteins than that of phosphorylated plant cellulose [20]. Quaternary
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ammonium bacterial cellulose (QABC) and Quaternary ammonium plant cellulose (QAPC) had also
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been prepared as adsorbents for proteins. QABC showed a higher adsorption capacity for hemoglobin
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compared with QAPC, but a lower adsorption capacity for thymol blue [21]. Sodium
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carboxymethylcellulose (SCMC) scaffolds were prepared by solvent-evaporation technique, which
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were beneficial to the partial thickness wound healing [22].
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Based on excellent performance of BC, CBC is looking forward to be another promising biomedical
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material in tissue repair or hemostasis dressing. So it is important and necessary to understand the
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relationship between CBC-protein interactions. However, there were few reports on the protein
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absorption behaviors of CBC membrane and its potential role in regulating the protein adsorption
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quantity. In this study, different carboxymethyl substitutions (DS) of CBC membranes were prepared 3
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by modifying BC with the reaction of carboxymethylation. The contact angle and zeta potential of
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CBC membranes were investigated. The effects of the DS of CBC membranes and pH values on
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protein adsorption behaviors were also examined in order to figure out the controllable protein
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adsorption. Besides, the reaction kinetics and adsorption isotherm were also evaluated. SEM images
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clearly showed the difference in protein adsorption of the CBC membranes with different degrees of
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carboxymethylation.
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2. Experimental
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2.1 Materials
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The BC used in this study was the gel-like cellulose membrane formed by A. xylinum AGR 60,
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kindly supplied by the Hainan Yida Food Co, Ltd, washing with distilled water. As a pre-treatment,
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BC membranes were immersed in 0.1mol/L sodium hydroxide solution for 60 min at 90℃ water bath
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in order to remove the bacterial cell debris, then thoroughly washed to neutral by de-ionized water. The
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following BSA (Wako Pure Chemical Industries, Osaka, Japan) reagent for the adsorption
69
experiments was used as received. All other reagents were reagent grade and were used as received.
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2.2 Preparation of carboxymethylated bacterial cellulose membrane
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CBC membranes were prepared from BC membranes according to the preparation scheme as Fig. 1 shows [23]. After pre-treatment (Water bath 55 for 1h), BC was cut into disks (d=15mm) with a
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trephine, then soaking in 10% sodium hydroxide solution for 2h before adding various amounts of
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ethanol and chloroacetic acid. Finally, all the mixture was treated in a water bath at the conditions
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pre-determined. Preparation process of CBC was examined under various conditions in order to obtain
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membranes with different degrees of substitution.
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Fig 1.
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Fig. 1 The preparation scheme of CBC 4
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The degree of substitution of BC was determined as follows: after carboxymethylation, three CMC
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disks were crushed to form slurry by using an Ultrasonic Cell Crusher. After vacuum drying at 110℃
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(weighed and recorded as m), CBC powder was dissolved in 50mL water, adding 30mL, 0.105mol/L
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hydrochloric acid solution, let set for an hour. Then added 1~2 drops of phenolphthalein, using
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0.095mol/L sodium hydroxide solution as titrant until the mixed solution turned pink. Volume of
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sodium hydroxide solution consumed was recorded as V, the DS of CBC was then calculated
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according to the following formula [24]:
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Where B= (0.095×V-0.105×30) ×10-3 (mL), represents the number of NaOH mol reacting with the
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substituted functional groups. Number 58 represents molecular weight of group –CH2COO- (g/mmol).
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Number 162 represents the millimole of substituted glucose unit (g/mmol). m (g) represents the dried
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quantity of CBC.
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2.3 FT-IR and Contact angles of CBC membrane
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Chemical structure information was tested by Fourier Transform Infrared Spectroscopy (FTIR,
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NICOLET 750) with a range of frequency from 4500 cm -1 to 450 cm-1 and a resolution of 4 cm-1.
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Contact angles (CA) on the carboxymethylated bacterial cellulose membrane were measured with
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an optical contact angle meter (OCA20, Dataphysics Inc) at ambient temperature. Water drop volumes
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were 3µl. pH of acid environment is 4.0 and pH of alkaline environment is 7.8.
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2.4 Zeta potential after carboxymethylation Zeta-potential (ζ-potential) of the BC and CBC has a great influence on the adsorption
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performance via electrostatic interaction and they were tested using a Zeta Potential and Submicron
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Particle Size Analyzer (DelsaTM Nano C).
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2.5 Adsorption of BSA on the carboxymethylated bacterial cellulose. Bovine serum albumin (BSA) was used to study the adsorbability of BC and CBC. BSA solutions
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with a certain concentration were prepared using PBS with a pH of 3.0±0.2. The pH values were
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adjusted by adding acetic acid solution. 10 cm3 of pre-determined concentration BSA aqueous solution
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and 1 disk of the adsorbent (BC or CBC) were mixed in a stoppered glass tube and shaken at 120 rpm
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in a thermostat-regulated shaker at 30℃. After different adsorption time, CBC was taken out, and then 5
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the concentration of BSA in the stoppered glass tube was determined by UV-VIS spectrophotometry
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(HP 8453, USA) via the absorbance at 276 nm. So, the adsorption amounts and capacities could be
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calculated by comparing the concentration before and after adsorption. Experiments: effect of
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adsorbate concentration on adsorption, effect of pH on adsorption and effect of different DS on
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adsorption were all carried out in the similar way. The calibration curves and equations of absorbance
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versus BSA concentrations in working condition were obtained by linear fit.
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2.6 Morphology
The microstructure of BC, CBC4, CBC24 and CBC36 were observed by scanning electron
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microscopy (SEM, Apollo 300, and 10 KV). All of the samples were freeze-dried and then coated with
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a thin layer of gold using a sputter coater before SEM examination.
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3. Results and discussion
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3.1 Preparation and the DS of CBC membrane
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Many factors could affect the carboxymethylation process, so different reaction conditions need to be discussed. Table 1 show various reaction conditions for preparing different DS of CBCs and DS
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test results.
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Table 1 Reaction conditions for preparing CBC along with the DS test results
CBC1
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Conditions for carboxymethylation
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DS test parameters and results
mass(
Vethanol(mL
mClCH2CO
Water bath
VNaOH(wt
Reaction
VHCl(
V(mL
g)
)
ONa(g)
Temperature(℃)
10%, mL)
time(h)
mL)
)
162B
DS
0.896
10
5.38
50
24.21
1
30
30.4
0.0648
0.074
10
5.28
50
23.77
4
30
30.4
0.0648
0.076
20
4.79
55
21.55
12
30
30.6
0.0972
0.127
20
5.33
55
23.99
20
30
30.7
0.1134
0.134
25
5.19
60
23.34
24
30
30.7
0.1134
0.138
30
5.07
60
22.83
36
30
30.9
0.1458
0.184
30
5.11
60
22.99
42
30
30.9
0.1458
0.182
6
CBC4
0.880 4
CBC12
0.798 1
CBC20
0.888 4
CBC24
0.864 4
CBC36
0.845
CBC42
0.851
7 5 6
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Obviously, there are many factors that can affect the DS of carboxymethylation. For BC membrane,
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the reaction reagents participated in the reaction were difficult to permeate into the nanoscale network
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structure of BC, therefore the exchange of inner free water in the network with the reaction reagents
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solution became the major influence factor. That is to say, the DS will increase as the reaction time
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increases which is consistent with the test results. However, when the reaction carries on the more than
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42 hours, rather than the DS continues to increase, it decreases slightly. The existent of the nanoscale
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network lead to the carboxymethyl substitution would only happen on the surface of BC membranes or
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a little more inside it, so it is no rush that the maximum DS was only 0.184.
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3.2 Characterization of CBC membrane by FTIR, Contact Angles and Zeta potential
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Fig. 2 shows the FTIR spectra of BC and CBC with different DS. For BC, there are three absorption peaks at 3370, 2900 and 1060 cm-1 corresponding to the stretching vibrations of the group (-OH),
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(-CH2) and (C-O) respectively. Compared the FTIR spectra of original BC with those of CBC, new
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absorption peaks appeared at 1595cm-1 and 1410 cm-1 assigned as C=O stretching vibration of
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carboxymethyl group after carboxymethylation, indicated the successful branch joint of the
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carboxymethyl group at bacterial cellulose membrane molecular chain and the result is consistent with
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the previous report [25].
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Fig 2.
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Fig. 2 FTIR spectra of BC and CBC with different DS
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Fig 3.
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Fig. 3 Influence of pH conditions on the contact angle of BC and CBC
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It is well-known that the hydrophobicity and electrostatic interaction are the main factors that affect
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protein adsorption [26]. Contact angles results shows the CAs of the BC membranes was always about
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8° no matter how the solution pH fluctuated. In alkaline environment with pH=10.5, the CAs of 8
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CBC12, CBC24 and CBC36 are 10°, 12° and 15°, respectively (Fig. 3), obviously, indicates
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carboxymethylation gradually reduces the hydrophilic performance of BC membrane. But when the
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solution pH turned to acid， the CA of CBC membranes ranged from 11° to 25° , which may be
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attributed to the ion exchange process (-CH2COO(-)Na(+) transformed into -CH2COO(-)H(+)) on
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carboxymethyl group when solution pH turned from alkaline to acid. Groups -CH2COO(-)Na(+) could
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entirely dissociate in aqueous solution which leaded to these molecular chains stretching in the water
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while groups -CH2COO(-)H(+) generated from -CH2COO(-)Na(+) could not, they curled up together
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through intermolecular force and then gradually reducing the hydrophilicity of CBC membranes.
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Fig. 4 Surface potential of BSA at different pH and CBC membranes with different DS
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Fig 5.
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Fig. 5 The schematic diagram of the changing zeta potential after carboxymethylation
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Fig. 4 shows the surface potential of BSA at different pH and CBC membrane with different DS.
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With the increase of the DS, the surface potential of CBC increases. It is well known that the stability
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of particles dispersed outside solid surface could be influenced by the solid surface charge. 9
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Carboxymethylation completely changed the surface electric charge of BC. Then CBC functions as an
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ion exchanger. As zeta potential is considered to be the potential where sliding surface exists. The
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sliding surface gradually closes to the solid surface with the increase of carboxymethyl groups which
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leads to the increase of Zeta potential value (Fig .5). As a matter of fact, the value of zeta potential is
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not big even if the degree of substitution at a relatively high level..
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3.3 Protein adsorption behaviors on the CBC membrane with different
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DS
As CBC membrane should function as an ion exchanger, the pH of the aqueous phase is one of the
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most important factors in protein adsorption. Adsorption profiles of BSA (isoelectric point, 4.5~6.0)
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on different DS CBC as a function of pH are shown in Fig. 6 (a). BSA was adsorbed on CBC at pHs
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below the corresponding pIs. At such pH values BSA are positively charged and adsorbed on the CBC
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anionic adsorbent. The maximum adsorption quantity increased with the increase of the DS. When
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pHs upon the corresponding pIs, the adsorption quantity turn to close to zero. This phenomenon
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indicated that proteins which were adsorbed on CBC via electrostatic interaction could be desorbed by
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contacting with aqueous alkali solution. The reason may be that the proteins are negatively charged
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and electrically repelled against carboxymethyl group of CBC. As a result of no electrostatic
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interaction and excellent hydrophilicity, BC had the lowest adsorption capacity; besides, swelling
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increased the superficial area of CBC. Almost all the BSA adsorbed on CBC was desorbed at pH
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10.35.
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Fig. 6 (a) Adsorption profiles of BSA on CBC with different DS as a function of pH; BSA, C= 2.0mg/mL; volume = 15cm3; adsorption time=24h. (b) Protein adsorption quantity changes over time, BSA, C= 1.0mg/mL; volume = 15cm3; pH= 4.35. (c) The fitting result of first order reaction kinetics model. (d) Adsorption profiles of BSA on BC and CBC membranes isotherms of BSA on CBCs, BSA, volume = 10mL; pH= 3.0; T= 30℃; adsorption time=24h
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Fig. 6 (b) shows the relationship between time and protein adsorption at pH=4.35. Adsorption
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quantity increases as time goes on but the equilibrium time longer also as they were 5, 37, 44 and 50h
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respectively, besides, the initial and equilibrium absorption capacity increases with the increase of DS.
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BC membrane had low adsorption quantity which may due to the abundant pore structures and high
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surface area that made BSA quickly penetrate into these pores through physical absorption. The
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adsorption data before equilibrium absorption were correlated with the first order reaction kinetics
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model using the following mathematical expression:
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Where
denotes the initial concentration of BSA which is 1mg/mL in this experiment, t is the
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reaction time, kt represents the gradient. As shown in Fig .6 (c), ln(a-c) was plotted against time, got
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lines with gradients -1.03×10-4, -11.5×10-4, -15.8×10-4 and -19.8×10-4 respectively， which indicated
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the absorption process accorded with the first order reaction. Fig. 6 (d) showed the adsorption of BSA using CBC. The adsorption amount of BSA increased by
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increasing the equilibrium concentration and approached constant values. In addition, the adsorption
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amount of BSA increased with increase of the carboxymethylation percentage of the CBC membrane.
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The equilibrium experimental data were then correlated with the Langmuir isotherm model to
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determine the maximum adsorption capacities of BSA on CBC membrane (qmax). The Langmuir
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model assumes monolayer adsorption as expressed by the following mathematical expression:
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Where q denotes the amount of BSA adsorbed on CBC, Ce is the equilibrium concentration of BSA
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in the aqueous solution, and K is the adsorption equilibrium constant [20]. The theoretical adsorption isotherms calculated from the Langmuir isotherm model are depicted in
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Fig. 6. It is obvious that the experimental values of adsorption of BSA agreed with the calculated data
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of monolayer adsorption via electrostatic interaction. The qmax values of BSA on CBCs with
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carboxymethylation time 0, 4, 24 and 36h were evaluated as 0.031, 0.152, 0.174 and 0.217mg/mL,
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respectively.
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Fig 7.
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Fig. 7 Relationship between DS and surface excess, BSA, C= 1.6mg/mL; volume = 15cm3; pH= 3.0.
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The relationship between DS and surface excess had been calculated as Fig. 7 shown. It is well
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known that the surface adsorption capacity directly affects the application of the materials in tissue
232
engineering. The surface excess of CBC adds as the DS increases, the maximum value is
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approximately 0.45mg/mm2 when the DS up to 0.184, higher than that of PBC(phosphorylated
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bacterial cellulose) and QABC(quaternary ammonium bacterial cellulose) [20, 21]. It provides the
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possibility to control the protein adsorption amount to adapt to the amount needs in the application of
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tissue engineering by adjusting the DS of CBC.
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3.4 Protein adsorption revealed by SEM
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Fig. 8 shows the SEM images of BC and CBC before and after protein adsorption. BC showed an
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intensive 3-dimension network structure of the nano-fibrils and nanoporosity (Fig. 8 (a)). This unique
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nano-structure result in a large surface area which provide the possibility to hold a large amount of
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biomacromolecules. After carboxymethylation, a little portion nano-fibrils on the surface of CBC
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membrane tended to aggregate or combine into bundles (Fig. 8 (b)), and the nano-structure of CBC
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seemed more loosely than that of BC.Fig .8 (c) (d) (e) (f) shows the surface microtopography of BC,
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CBC4, CBC24 and CBC36 after protein adsorption. It is quite clear that the protein adsorption
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increases with the increase of the DS, which consistent with experimental data previous presented.
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Obviously, BSA aggregated on the surface of BC and CBC, and it seemed the rising DS contributed to
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this phenomenon, beyond that, it was not hard to find that a little protein adsorbed by the BC 13
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membrane penetrated into the network as Fig .8(c) shown. Proteins adsorbed on the surface of CBC4
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(Fig .8(d)) showed a comparatively loose aggregation compared with those of CBC24 and CBC36 (Fig
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.8(e) (f)) on which protein aggregated together into clots and adsorbed as a whole on the surface of
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CBC.
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Fig. 8 SEM images of (a) BC, (b) CBC, (c) BC adsorbed with BSA, (d) CBC4 adsorbed with BSA, (e)
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CBC24 adsorbed with BSA and (f) CBC36 adsorbed with BSA.BSA: C= 1.0mg/mL; volume = 15cm3;
256
pH= 4.35.
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4. Conclusions
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Carboxymethylated bacterial cellulose membranes with different degrees of carboxymethyl
259
substitution (DS) were prepared and the protein adsorption behaviors on the surface of CBC
260
membranes were investigated using the BSA as the model protein. Overall, the adsorption quantity
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increased gradually with the increase of the DS. It is found that the protein adsorption was not only
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governed by the chemical structure of CBC but also by the electrostatic interaction between CBC and
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BSA, since the protein could be adsorbed on CBC membrane at a lower pH than its isoelectric point
264
(pI) while it could be hardly adsorbed at pH above its pI, where the pH values controlled the charges of
265
CBC and BSA. Meanwhile, the adsorption isotherms showed that the protein adsorption behaviors
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corresponded with Langmuir monolayer adsorption model. The protein adsorption behaviors
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controlled by the degrees of carboxymethyl substitution and environmental pH endowed the CBC
268
membranes with smart properties to adjust the protein adsorption. The prepared CBC membranes,
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which are intelligent materials for protein adsorption, can find their wide applications in biomedical
270
fields.
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5. Acknowledgements
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This study is financially supported by National Natural Science Foundation of China Project (Grant
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No.51073024 and Grant No. 51273021), and the National Science and Technology Support Project of
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China (Grant No. 2011BAK15B04).
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Table Captions
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Table 1 Reaction conditions for preparing CBC along with the DS test results
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309 Figure Captions
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Fig. 1 The preparation scheme of CBC
312
Fig. 2 FTIR spectra of BC and CBC with different DS
313
Fig. 3 Influence of pH conditions on the contact angle of BC and CBC
314
Fig. 4 Surface potential of BSA at different pH and CBC membrane with different DS
315
Fig. 5 The schematic diagram of the changing zeta potential after carboxymethylation
316
Fig. 6 (a) Adsorption profiles of BSA on CBC with different DS as a function of pH; BSA, C=
317
2.0mg/mL; volume = 15cm3. (b) Protein adsorption quantity changes over time, BSA, C= 1.0mg/mL;
318
volume = 15cm3; pH= 4.35. (c) The fitting result of first order reaction kinetics model. (d) Adsorption
319
isotherms of BSA on CBCs, BSA, volume = 10mL; pH= 3.0; T= 30℃.
320
Fig. 7 Relationship between DS and surface excess, BSA, C= 1.6mg/mL; volume = 15cm3; pH= 3.0.
321 322 323
Fig. 8 SEM images of (a) BC, (b) CBC, (c) BC adsorbed with BSA, (d) CBC4 adsorbed with BSA, (e) CBC24 adsorbed with BSA and (f) CBC36 adsorbed with BSA.BSA: C= 1.0mg/mL; volume = 15cm3; pH= 4.35.
Ac ce p
324
te
d
M
an
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cr
ip t
310
19
Page 19 of 28
Table 1 Reaction conditions for preparing CBC along with the DS test results Sample
Conditions for carboxymethylation m(g)
Vethanol(mL)
mClCH2COONa
DS test parameters and results
Water bath
VNaOH(wt
Reaction
Temperature(℃)
10%, mL)
time(h)
VHCl(mL)
V(mL)
162B
DS
0.8966
10
5.38
50
24.21
1
30
30.4
0.0648
0.074
CBC4
0.8804
10
5.28
50
23.77
4
30
30.4
0.0648
0.076
CBC12
0.7981
20
4.79
55
21.55
12
30
30.6
0.0972
0.127
CBC20
0.8884
20
5.33
55
23.99
20
30
30.7
0.1134
0.134
CBC24
0.8644
25
5.19
60
23.34
24
30
30.7
0.1134
0.138
CBC36
0.8457
30
5.07
60
22.83
36
30
30.9
0.1458
0.184
CBC42
0.8515
30
5.11
60
22.99
42
30
30.9
0.1458
0.182
ip t
CBC1
us
cr
324
Ac ce p
te
d
M
an
325
20
Page 20 of 28
325 326
Fig. 1 The preparation scheme of CBC
Ac ce p
te
d
M
an
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cr
ip t
327
21
Page 21 of 28
ip t cr us
327 328
Fig. 2 FTIR spectra of BC and CBC with different DS
Ac ce p
te
d
M
an
329
22
Page 22 of 28
ip t cr us
Fig. 3 Influence of pH conditions on the contact angle of BC and CBC
an
329 330
Ac ce p
te
d
M
331
23
Page 23 of 28
ip t cr us
331 332
Fig. 4 Surface potential of BSA at different pH and CBC membrane with different DS
Ac ce p
te
d
M
an
333
24
Page 24 of 28
ip t
Fig. 5 The schematic diagram of the changing zeta potential after carboxymethylation
cr
333 334
Ac ce p
te
d
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an
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335
25
Page 25 of 28
ip t cr us an d
te
340
Fig. 6 (a) Adsorption profiles of BSA on CBC with different DS as a function of pH; BSA, C= 2.0mg/mL; volume = 15cm3. (b) Protein adsorption quantity changes over time, BSA, C= 1.0mg/mL; volume = 15cm3; pH= 4.35. (c) The fitting result of first order reaction kinetics model. (d) Adsorption isotherms of BSA on CBCs, BSA, volume = 10mL; pH= 3.0; T= 30℃.
Ac ce p
336 337 338 339
M
335
26
Page 26 of 28
ip t cr us
340 341
Fig. 7 Relationship between DS and surface excess, BSA, C= 1.6mg/mL; volume = 15cm3; pH= 3.0.
Ac ce p
te
d
M
an
342
27
Page 27 of 28
cr
ip t
342
Fig. 8 SEM images of (a) BC, (b) CBC, (c) BC adsorbed with BSA, (d) CBC4 adsorbed with BSA, (e) CBC24 adsorbed with BSA and (f) CBC36 adsorbed with BSA.BSA: C= 1.0mg/mL; volume = 15cm3; pH= 4.35.
an
344 345 346
us
343
Ac ce p
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Page 28 of 28
S0141-8130(14)00759-4 http://dx.doi.org/doi:10.1016/j.ijbiomac.2014.11.011 BIOMAC 4727
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
24-8-2014 13-11-2014 17-11-2014
Please cite this article as: Q. Lin, Y. Zheng, G. Wang, X. Shi, T. Zhang, J. Yu, J. Sun, Protein Adsorption Behaviors of Carboxymethylated Bacterial Cellulose Membranes, International Journal of Biological Macromolecules (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.11.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Highlights
1.
Carboxymethylation largely changed the surface of BC membrane.
3
2.
The hydrophobicity of CBC increased with the increase of the DS and pH;
4
3.
Protein adsorption showed opposite performance around its isoelectric point.
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2
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1
Page 1 of 28
5
Protein Adsorption Behaviors of Carboxymethylated
7
Bacterial Cellulose Membranes
8
Qinghua Lin, Yudong Zheng*, Guojie Wang*, Xiangning Shi, Tao Zhang, Jie Yu, Jian Sun
ip t
6
School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, PR China
11 12 13 14
Corresponding author: Prof. Yudong Zheng, Prof. Guojie Wang E-mail: [email protected]; [email protected] Phone: +86-010-62330802, Fax: +86-010-62332336
15
Abstract
an
us
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9 10
Carboxymethylated Bacterial Cellulose (CBC) membranes with different degrees of substitution
17
(DS) were prepared by surface carboxymethylation of bacterial cellulose, which were characterized by
18
FTIR, contact angles and zeta potential. Both the contact angels and zeta potentials increased with the
19
increase of degrees of carboxymethylation. Protein adsorption behaviors of CBC membranes were
20
investigated with bovine serum albumin (BSA) as model protein and the adsorption quantity increased
21
gradually with the increase of the DS. The protein adsorption was not only governed by the chemical
22
structure of CBC but also by the electrostatic interaction between CBC and BSA. The protein was
23
found to be adsorbed on CBC membrane at pH lower its isoelectric point (pI) while no protein was
24
adsorbed at pH above its pI. The protein adsorption on the CBC membranes was also characterized by
25
scanning electron microscopy (SEM).
26
Key word: Carboxymethylated; Bacterial cellulose; Surface; Protein adsorption.
Ac ce p
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16
2
Page 2 of 28
27 28
1. Introduction Protein adsorption behavior has been paid more and more attention in current research fields including bio-nanotechnology, biocompatibility evaluation and biological response between materials
30
and biological organisms. As a matter of fact, such behavior is governed by the chemical structures of
31
material surfaces, accordingly, providential surface modification may provide appropriate behaviors
32
of protein-surface interactions needed [1, 2].
ip t
29
Bacterial cellulose (BC), synthesized in abundance by Acetobacter xylinum, possesses unique
34
physical and mechanical properties as well as high purity and uniformity with wide applications in
35
industry and biomedical field, such as paper products [3], fuel cells [4], acoustics [5], wound dressings
36
[6], blood vessel prosthesis [7], tissue regeneration materials [8] and other biomedical devices [9-11].
37
Besides, BC has been attracting attention as a raw material for preparation of advanced materials due
38
to its advantageous properties that are imparted by the unique structure [12-17]. BC derivative
39
modified by various chemical substitutions with staggered degrees of functional groups could open
40
further possibilities for new applications in different fields, due to its microfibrous structure and
41
chemical activity; for instance, surface acetylation of bacterial cellulose was prepared to modify its
42
physical properties， while preserving the microfibrillar morphology [18]. Diethylenetriamine BC and
43
amidoximated BC was prepared for adsorptive removal of Cu (II) and Pb (II) [19]. Phosphorylated
44
bacterial cellulose had been prepared for selective adsorption of different proteins, and it was found to
45
be effective as an adsorbent for proteins than that of phosphorylated plant cellulose [20]. Quaternary
46
ammonium bacterial cellulose (QABC) and Quaternary ammonium plant cellulose (QAPC) had also
47
been prepared as adsorbents for proteins. QABC showed a higher adsorption capacity for hemoglobin
48
compared with QAPC, but a lower adsorption capacity for thymol blue [21]. Sodium
49
carboxymethylcellulose (SCMC) scaffolds were prepared by solvent-evaporation technique, which
50
were beneficial to the partial thickness wound healing [22].
51
Ac ce p
te
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33
Based on excellent performance of BC, CBC is looking forward to be another promising biomedical
52
material in tissue repair or hemostasis dressing. So it is important and necessary to understand the
53
relationship between CBC-protein interactions. However, there were few reports on the protein
54
absorption behaviors of CBC membrane and its potential role in regulating the protein adsorption
55
quantity. In this study, different carboxymethyl substitutions (DS) of CBC membranes were prepared 3
Page 3 of 28
by modifying BC with the reaction of carboxymethylation. The contact angle and zeta potential of
57
CBC membranes were investigated. The effects of the DS of CBC membranes and pH values on
58
protein adsorption behaviors were also examined in order to figure out the controllable protein
59
adsorption. Besides, the reaction kinetics and adsorption isotherm were also evaluated. SEM images
60
clearly showed the difference in protein adsorption of the CBC membranes with different degrees of
61
carboxymethylation.
62
2. Experimental
63
2.1 Materials
us
cr
ip t
56
The BC used in this study was the gel-like cellulose membrane formed by A. xylinum AGR 60,
65
kindly supplied by the Hainan Yida Food Co, Ltd, washing with distilled water. As a pre-treatment,
66
BC membranes were immersed in 0.1mol/L sodium hydroxide solution for 60 min at 90℃ water bath
67
in order to remove the bacterial cell debris, then thoroughly washed to neutral by de-ionized water. The
68
following BSA (Wako Pure Chemical Industries, Osaka, Japan) reagent for the adsorption
69
experiments was used as received. All other reagents were reagent grade and were used as received.
70
2.2 Preparation of carboxymethylated bacterial cellulose membrane
M
d
te
71
an
64
CBC membranes were prepared from BC membranes according to the preparation scheme as Fig. 1 shows [23]. After pre-treatment (Water bath 55 for 1h), BC was cut into disks (d=15mm) with a
73
trephine, then soaking in 10% sodium hydroxide solution for 2h before adding various amounts of
74
ethanol and chloroacetic acid. Finally, all the mixture was treated in a water bath at the conditions
75
pre-determined. Preparation process of CBC was examined under various conditions in order to obtain
76
membranes with different degrees of substitution.
77 78
Ac ce p
72
Fig 1.
79
80 81
Fig. 1 The preparation scheme of CBC 4
Page 4 of 28
The degree of substitution of BC was determined as follows: after carboxymethylation, three CMC
83
disks were crushed to form slurry by using an Ultrasonic Cell Crusher. After vacuum drying at 110℃
84
(weighed and recorded as m), CBC powder was dissolved in 50mL water, adding 30mL, 0.105mol/L
85
hydrochloric acid solution, let set for an hour. Then added 1~2 drops of phenolphthalein, using
86
0.095mol/L sodium hydroxide solution as titrant until the mixed solution turned pink. Volume of
87
sodium hydroxide solution consumed was recorded as V, the DS of CBC was then calculated
88
according to the following formula [24]:
cr
ip t
82
Where B= (0.095×V-0.105×30) ×10-3 (mL), represents the number of NaOH mol reacting with the
91
substituted functional groups. Number 58 represents molecular weight of group –CH2COO- (g/mmol).
92
Number 162 represents the millimole of substituted glucose unit (g/mmol). m (g) represents the dried
93
quantity of CBC.
an
2.3 FT-IR and Contact angles of CBC membrane
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94
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89 90
Chemical structure information was tested by Fourier Transform Infrared Spectroscopy (FTIR,
96
NICOLET 750) with a range of frequency from 4500 cm -1 to 450 cm-1 and a resolution of 4 cm-1.
d
95
Contact angles (CA) on the carboxymethylated bacterial cellulose membrane were measured with
98
an optical contact angle meter (OCA20, Dataphysics Inc) at ambient temperature. Water drop volumes
99
were 3µl. pH of acid environment is 4.0 and pH of alkaline environment is 7.8.
101
Ac ce p
100
te
97
2.4 Zeta potential after carboxymethylation Zeta-potential (ζ-potential) of the BC and CBC has a great influence on the adsorption
102
performance via electrostatic interaction and they were tested using a Zeta Potential and Submicron
103
Particle Size Analyzer (DelsaTM Nano C).
104 105
2.5 Adsorption of BSA on the carboxymethylated bacterial cellulose. Bovine serum albumin (BSA) was used to study the adsorbability of BC and CBC. BSA solutions
106
with a certain concentration were prepared using PBS with a pH of 3.0±0.2. The pH values were
107
adjusted by adding acetic acid solution. 10 cm3 of pre-determined concentration BSA aqueous solution
108
and 1 disk of the adsorbent (BC or CBC) were mixed in a stoppered glass tube and shaken at 120 rpm
109
in a thermostat-regulated shaker at 30℃. After different adsorption time, CBC was taken out, and then 5
Page 5 of 28
the concentration of BSA in the stoppered glass tube was determined by UV-VIS spectrophotometry
111
(HP 8453, USA) via the absorbance at 276 nm. So, the adsorption amounts and capacities could be
112
calculated by comparing the concentration before and after adsorption. Experiments: effect of
113
adsorbate concentration on adsorption, effect of pH on adsorption and effect of different DS on
114
adsorption were all carried out in the similar way. The calibration curves and equations of absorbance
115
versus BSA concentrations in working condition were obtained by linear fit.
cr
117
2.6 Morphology
The microstructure of BC, CBC4, CBC24 and CBC36 were observed by scanning electron
us
116
ip t
110
microscopy (SEM, Apollo 300, and 10 KV). All of the samples were freeze-dried and then coated with
119
a thin layer of gold using a sputter coater before SEM examination.
120
3. Results and discussion
122
3.1 Preparation and the DS of CBC membrane
M
121
an
118
Many factors could affect the carboxymethylation process, so different reaction conditions need to be discussed. Table 1 show various reaction conditions for preparing different DS of CBCs and DS
124
test results.
125
Table 1 Reaction conditions for preparing CBC along with the DS test results
CBC1
te
Conditions for carboxymethylation
Ac ce p
Sample
d
123
DS test parameters and results
mass(
Vethanol(mL
mClCH2CO
Water bath
VNaOH(wt
Reaction
VHCl(
V(mL
g)
)
ONa(g)
Temperature(℃)
10%, mL)
time(h)
mL)
)
162B
DS
0.896
10
5.38
50
24.21
1
30
30.4
0.0648
0.074
10
5.28
50
23.77
4
30
30.4
0.0648
0.076
20
4.79
55
21.55
12
30
30.6
0.0972
0.127
20
5.33
55
23.99
20
30
30.7
0.1134
0.134
25
5.19
60
23.34
24
30
30.7
0.1134
0.138
30
5.07
60
22.83
36
30
30.9
0.1458
0.184
30
5.11
60
22.99
42
30
30.9
0.1458
0.182
6
CBC4
0.880 4
CBC12
0.798 1
CBC20
0.888 4
CBC24
0.864 4
CBC36
0.845
CBC42
0.851
7 5 6
Page 6 of 28
Obviously, there are many factors that can affect the DS of carboxymethylation. For BC membrane,
127
the reaction reagents participated in the reaction were difficult to permeate into the nanoscale network
128
structure of BC, therefore the exchange of inner free water in the network with the reaction reagents
129
solution became the major influence factor. That is to say, the DS will increase as the reaction time
130
increases which is consistent with the test results. However, when the reaction carries on the more than
131
42 hours, rather than the DS continues to increase, it decreases slightly. The existent of the nanoscale
132
network lead to the carboxymethyl substitution would only happen on the surface of BC membranes or
133
a little more inside it, so it is no rush that the maximum DS was only 0.184.
136
cr
us
135
3.2 Characterization of CBC membrane by FTIR, Contact Angles and Zeta potential
an
134
ip t
126
Fig. 2 shows the FTIR spectra of BC and CBC with different DS. For BC, there are three absorption peaks at 3370, 2900 and 1060 cm-1 corresponding to the stretching vibrations of the group (-OH),
138
(-CH2) and (C-O) respectively. Compared the FTIR spectra of original BC with those of CBC, new
139
absorption peaks appeared at 1595cm-1 and 1410 cm-1 assigned as C=O stretching vibration of
140
carboxymethyl group after carboxymethylation, indicated the successful branch joint of the
141
carboxymethyl group at bacterial cellulose membrane molecular chain and the result is consistent with
142
the previous report [25].
144 145 146
d
te
Ac ce p
143
M
137
Fig 2.
7
Page 7 of 28
ip t cr us
147 148
Fig. 2 FTIR spectra of BC and CBC with different DS
an
149 150
M
Fig 3.
Ac ce p
te
d
151
152 153
Fig. 3 Influence of pH conditions on the contact angle of BC and CBC
154
It is well-known that the hydrophobicity and electrostatic interaction are the main factors that affect
155
protein adsorption [26]. Contact angles results shows the CAs of the BC membranes was always about
156
8° no matter how the solution pH fluctuated. In alkaline environment with pH=10.5, the CAs of 8
Page 8 of 28
CBC12, CBC24 and CBC36 are 10°, 12° and 15°, respectively (Fig. 3), obviously, indicates
158
carboxymethylation gradually reduces the hydrophilic performance of BC membrane. But when the
159
solution pH turned to acid， the CA of CBC membranes ranged from 11° to 25° , which may be
160
attributed to the ion exchange process (-CH2COO(-)Na(+) transformed into -CH2COO(-)H(+)) on
161
carboxymethyl group when solution pH turned from alkaline to acid. Groups -CH2COO(-)Na(+) could
162
entirely dissociate in aqueous solution which leaded to these molecular chains stretching in the water
163
while groups -CH2COO(-)H(+) generated from -CH2COO(-)Na(+) could not, they curled up together
164
through intermolecular force and then gradually reducing the hydrophilicity of CBC membranes.
167
Fig. 4 Surface potential of BSA at different pH and CBC membranes with different DS
Ac ce p
165 166
te
d
M
an
us
cr
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157
Fig 5.
168 169
Fig. 5 The schematic diagram of the changing zeta potential after carboxymethylation
170
Fig. 4 shows the surface potential of BSA at different pH and CBC membrane with different DS.
171
With the increase of the DS, the surface potential of CBC increases. It is well known that the stability
172
of particles dispersed outside solid surface could be influenced by the solid surface charge. 9
Page 9 of 28
Carboxymethylation completely changed the surface electric charge of BC. Then CBC functions as an
174
ion exchanger. As zeta potential is considered to be the potential where sliding surface exists. The
175
sliding surface gradually closes to the solid surface with the increase of carboxymethyl groups which
176
leads to the increase of Zeta potential value (Fig .5). As a matter of fact, the value of zeta potential is
177
not big even if the degree of substitution at a relatively high level..
179
3.3 Protein adsorption behaviors on the CBC membrane with different
cr
178
ip t
173
DS
As CBC membrane should function as an ion exchanger, the pH of the aqueous phase is one of the
181
most important factors in protein adsorption. Adsorption profiles of BSA (isoelectric point, 4.5~6.0)
182
on different DS CBC as a function of pH are shown in Fig. 6 (a). BSA was adsorbed on CBC at pHs
183
below the corresponding pIs. At such pH values BSA are positively charged and adsorbed on the CBC
184
anionic adsorbent. The maximum adsorption quantity increased with the increase of the DS. When
185
pHs upon the corresponding pIs, the adsorption quantity turn to close to zero. This phenomenon
186
indicated that proteins which were adsorbed on CBC via electrostatic interaction could be desorbed by
187
contacting with aqueous alkali solution. The reason may be that the proteins are negatively charged
188
and electrically repelled against carboxymethyl group of CBC. As a result of no electrostatic
189
interaction and excellent hydrophilicity, BC had the lowest adsorption capacity; besides, swelling
190
increased the superficial area of CBC. Almost all the BSA adsorbed on CBC was desorbed at pH
191
10.35.
193
an
M
d
te
Ac ce p
192
us
180
Fig 6.
10
Page 10 of 28
ip t cr us an M
194
Fig. 6 (a) Adsorption profiles of BSA on CBC with different DS as a function of pH; BSA, C= 2.0mg/mL; volume = 15cm3; adsorption time=24h. (b) Protein adsorption quantity changes over time, BSA, C= 1.0mg/mL; volume = 15cm3; pH= 4.35. (c) The fitting result of first order reaction kinetics model. (d) Adsorption profiles of BSA on BC and CBC membranes isotherms of BSA on CBCs, BSA, volume = 10mL; pH= 3.0; T= 30℃; adsorption time=24h
200
Fig. 6 (b) shows the relationship between time and protein adsorption at pH=4.35. Adsorption
Ac ce p
te
d
195 196 197 198 199
201
quantity increases as time goes on but the equilibrium time longer also as they were 5, 37, 44 and 50h
202
respectively, besides, the initial and equilibrium absorption capacity increases with the increase of DS.
203
BC membrane had low adsorption quantity which may due to the abundant pore structures and high
204
surface area that made BSA quickly penetrate into these pores through physical absorption. The
205
adsorption data before equilibrium absorption were correlated with the first order reaction kinetics
206
model using the following mathematical expression:
207 208
Where
denotes the initial concentration of BSA which is 1mg/mL in this experiment, t is the
209
reaction time, kt represents the gradient. As shown in Fig .6 (c), ln(a-c) was plotted against time, got
11
Page 11 of 28
210
lines with gradients -1.03×10-4, -11.5×10-4, -15.8×10-4 and -19.8×10-4 respectively， which indicated
211
the absorption process accorded with the first order reaction. Fig. 6 (d) showed the adsorption of BSA using CBC. The adsorption amount of BSA increased by
213
increasing the equilibrium concentration and approached constant values. In addition, the adsorption
214
amount of BSA increased with increase of the carboxymethylation percentage of the CBC membrane.
215
The equilibrium experimental data were then correlated with the Langmuir isotherm model to
216
determine the maximum adsorption capacities of BSA on CBC membrane (qmax). The Langmuir
217
model assumes monolayer adsorption as expressed by the following mathematical expression:
us
cr
ip t
212
an
218
221
Where q denotes the amount of BSA adsorbed on CBC, Ce is the equilibrium concentration of BSA
d
220
M
219
in the aqueous solution, and K is the adsorption equilibrium constant [20]. The theoretical adsorption isotherms calculated from the Langmuir isotherm model are depicted in
223
Fig. 6. It is obvious that the experimental values of adsorption of BSA agreed with the calculated data
224
of monolayer adsorption via electrostatic interaction. The qmax values of BSA on CBCs with
225
carboxymethylation time 0, 4, 24 and 36h were evaluated as 0.031, 0.152, 0.174 and 0.217mg/mL,
226
respectively.
Ac ce p
227
te
222
Fig 7.
12
Page 12 of 28
ip t cr us
Fig. 7 Relationship between DS and surface excess, BSA, C= 1.6mg/mL; volume = 15cm3; pH= 3.0.
230
The relationship between DS and surface excess had been calculated as Fig. 7 shown. It is well
231
known that the surface adsorption capacity directly affects the application of the materials in tissue
232
engineering. The surface excess of CBC adds as the DS increases, the maximum value is
233
approximately 0.45mg/mm2 when the DS up to 0.184, higher than that of PBC(phosphorylated
234
bacterial cellulose) and QABC(quaternary ammonium bacterial cellulose) [20, 21]. It provides the
235
possibility to control the protein adsorption amount to adapt to the amount needs in the application of
236
tissue engineering by adjusting the DS of CBC.
M
d
te
Ac ce p
237
an
228 229
3.4 Protein adsorption revealed by SEM
238
Fig. 8 shows the SEM images of BC and CBC before and after protein adsorption. BC showed an
239
intensive 3-dimension network structure of the nano-fibrils and nanoporosity (Fig. 8 (a)). This unique
240
nano-structure result in a large surface area which provide the possibility to hold a large amount of
241
biomacromolecules. After carboxymethylation, a little portion nano-fibrils on the surface of CBC
242
membrane tended to aggregate or combine into bundles (Fig. 8 (b)), and the nano-structure of CBC
243
seemed more loosely than that of BC.Fig .8 (c) (d) (e) (f) shows the surface microtopography of BC,
244
CBC4, CBC24 and CBC36 after protein adsorption. It is quite clear that the protein adsorption
245
increases with the increase of the DS, which consistent with experimental data previous presented.
246
Obviously, BSA aggregated on the surface of BC and CBC, and it seemed the rising DS contributed to
247
this phenomenon, beyond that, it was not hard to find that a little protein adsorbed by the BC 13
Page 13 of 28
membrane penetrated into the network as Fig .8(c) shown. Proteins adsorbed on the surface of CBC4
249
(Fig .8(d)) showed a comparatively loose aggregation compared with those of CBC24 and CBC36 (Fig
250
.8(e) (f)) on which protein aggregated together into clots and adsorbed as a whole on the surface of
251
CBC.
ip t
248
an
us
cr
252
M
253
Fig. 8 SEM images of (a) BC, (b) CBC, (c) BC adsorbed with BSA, (d) CBC4 adsorbed with BSA, (e)
255
CBC24 adsorbed with BSA and (f) CBC36 adsorbed with BSA.BSA: C= 1.0mg/mL; volume = 15cm3;
256
pH= 4.35.
257
4. Conclusions
258
Carboxymethylated bacterial cellulose membranes with different degrees of carboxymethyl
259
substitution (DS) were prepared and the protein adsorption behaviors on the surface of CBC
260
membranes were investigated using the BSA as the model protein. Overall, the adsorption quantity
261
increased gradually with the increase of the DS. It is found that the protein adsorption was not only
262
governed by the chemical structure of CBC but also by the electrostatic interaction between CBC and
263
BSA, since the protein could be adsorbed on CBC membrane at a lower pH than its isoelectric point
264
(pI) while it could be hardly adsorbed at pH above its pI, where the pH values controlled the charges of
265
CBC and BSA. Meanwhile, the adsorption isotherms showed that the protein adsorption behaviors
266
corresponded with Langmuir monolayer adsorption model. The protein adsorption behaviors
267
controlled by the degrees of carboxymethyl substitution and environmental pH endowed the CBC
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membranes with smart properties to adjust the protein adsorption. The prepared CBC membranes,
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which are intelligent materials for protein adsorption, can find their wide applications in biomedical
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fields.
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5. Acknowledgements
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This study is financially supported by National Natural Science Foundation of China Project (Grant
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No.51073024 and Grant No. 51273021), and the National Science and Technology Support Project of
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China (Grant No. 2011BAK15B04).
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6. References
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Table Captions
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Table 1 Reaction conditions for preparing CBC along with the DS test results
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309 Figure Captions
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Fig. 1 The preparation scheme of CBC
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Fig. 2 FTIR spectra of BC and CBC with different DS
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Fig. 3 Influence of pH conditions on the contact angle of BC and CBC
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Fig. 4 Surface potential of BSA at different pH and CBC membrane with different DS
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Fig. 5 The schematic diagram of the changing zeta potential after carboxymethylation
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Fig. 6 (a) Adsorption profiles of BSA on CBC with different DS as a function of pH; BSA, C=
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2.0mg/mL; volume = 15cm3. (b) Protein adsorption quantity changes over time, BSA, C= 1.0mg/mL;
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volume = 15cm3; pH= 4.35. (c) The fitting result of first order reaction kinetics model. (d) Adsorption
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isotherms of BSA on CBCs, BSA, volume = 10mL; pH= 3.0; T= 30℃.
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Fig. 7 Relationship between DS and surface excess, BSA, C= 1.6mg/mL; volume = 15cm3; pH= 3.0.
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Fig. 8 SEM images of (a) BC, (b) CBC, (c) BC adsorbed with BSA, (d) CBC4 adsorbed with BSA, (e) CBC24 adsorbed with BSA and (f) CBC36 adsorbed with BSA.BSA: C= 1.0mg/mL; volume = 15cm3; pH= 4.35.
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Table 1 Reaction conditions for preparing CBC along with the DS test results Sample
Conditions for carboxymethylation m(g)
Vethanol(mL)
mClCH2COONa
DS test parameters and results
Water bath
VNaOH(wt
Reaction
Temperature(℃)
10%, mL)
time(h)
VHCl(mL)
V(mL)
162B
DS
0.8966
10
5.38
50
24.21
1
30
30.4
0.0648
0.074
CBC4
0.8804
10
5.28
50
23.77
4
30
30.4
0.0648
0.076
CBC12
0.7981
20
4.79
55
21.55
12
30
30.6
0.0972
0.127
CBC20
0.8884
20
5.33
55
23.99
20
30
30.7
0.1134
0.134
CBC24
0.8644
25
5.19
60
23.34
24
30
30.7
0.1134
0.138
CBC36
0.8457
30
5.07
60
22.83
36
30
30.9
0.1458
0.184
CBC42
0.8515
30
5.11
60
22.99
42
30
30.9
0.1458
0.182
ip t
CBC1
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325 326
Fig. 1 The preparation scheme of CBC
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Fig. 2 FTIR spectra of BC and CBC with different DS
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Fig. 3 Influence of pH conditions on the contact angle of BC and CBC
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Fig. 4 Surface potential of BSA at different pH and CBC membrane with different DS
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Fig. 5 The schematic diagram of the changing zeta potential after carboxymethylation
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Fig. 6 (a) Adsorption profiles of BSA on CBC with different DS as a function of pH; BSA, C= 2.0mg/mL; volume = 15cm3. (b) Protein adsorption quantity changes over time, BSA, C= 1.0mg/mL; volume = 15cm3; pH= 4.35. (c) The fitting result of first order reaction kinetics model. (d) Adsorption isotherms of BSA on CBCs, BSA, volume = 10mL; pH= 3.0; T= 30℃.
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Fig. 7 Relationship between DS and surface excess, BSA, C= 1.6mg/mL; volume = 15cm3; pH= 3.0.
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Fig. 8 SEM images of (a) BC, (b) CBC, (c) BC adsorbed with BSA, (d) CBC4 adsorbed with BSA, (e) CBC24 adsorbed with BSA and (f) CBC36 adsorbed with BSA.BSA: C= 1.0mg/mL; volume = 15cm3; pH= 4.35.
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