Investigation of the effects of starch on the physical and biological properties of polyacrylamide (PAAm)/starch nanofibers
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Here, we report the development of a new polyacrylamide (PAAm)/starch nanofibers’ blend system and highlight its potential as substrate for efficient enzyme immobilization. PAAm was synthesized and blended with starch. The final blend was then electrospun into nanofibers. The response surface methodology was used to analyze the parameters that control nanofiber’s diameter. Electrospun mat was then modified either by cross-linking or phytase immobilization using silane coupling agent and glutaraldehyde chemistry. Physico-chemical properties of blends were investigated using spectroscopic and thermal studies. The evaluation of immobilized enzyme kinetics on both pure and the starch blended PAAm nanofibers was performed using Michaelis–Menten kinetic curves. Fourier transform infrared spectroscopy results along with differential scanning and X-ray diffraction confirmed that blending was successfully accomplished. TGA analysis also demonstrated that the presence of starch enhances the thermal degradability of PAAm nanofibers. Finally, it was shown that addition of starch to PAAm increases the efficacies of enzyme loading and, therefore, significantly enhances the activity as well as kinetics of the immobilized enzyme on electrospun blend mats.
KeywordsStarch Nanofibers Polyacrylamide Enzyme immobilization Biological property
There has been a great progress in the preparation, function, and evaluation of biomaterials concerning multifaceted practicability of them. Thanks to the outstanding properties and the key role in diversified areas of performance, a wide range of biomaterials has been increasingly used in biological applications. The acknowledgement that investigation of the impact of natural biopolymer-biomaterial along with its relevant mechanical and biological experiments comprising enzymatic survey can be amply efficient stimulates the authors to pursue this novel subject.
Chemical and physical properties of starch have been widely studied due to its flexibility for a variety of applications because of its biodegradability and economical availability (Whistler et al. 1984; Mano et al. 2003; Kaushik et al. 2010). The advantage that glycosidic bonds between monosaccharides provide polysaccharides with good biocompatibility and availability properties makes it suitable to be used as cell carriers in tissue engineering (Bačáková et al. 2014). Polysaccharides are even able to improve mechanical properties of natural fibers (Curvelo et al. 2001; Kaushik et al. 2010). It was observed that various blends of polysaccharide component show good potential to be used in numerous biomedical applications (Shelke et al. 2014). Polysaccharides such as starch modified nanofibrous scaffolds are capable of promoting cellular activities through increasing bioactivity and cell retention (Bačáková et al. 2014). Biocompatibility and cell cultural properties of scaffolds obtained from blended starch nanofibers can significantly intensify the cells viability (Gomes et al. 2006; Salgado et al. 2004). Starch-based scaffolds can diminish diffusion constraints and mechanically stimulate the marrow stroma cells to develop the bone-like mineralized tissue (Gomes et al. 2006). Gomes et al. (2006) showed that starch-based scaffolds affect not only the sequential development of osteoblastic cells but also the functionality of tissues formed in vitro. To our knowledge, the role of natural polysaccharide nanofibers as a bed for enzyme immobilization and its effects on physical and biological properties has not been thoroughly investigated. So here, we aim to study the role of starch in blended nanofibers and further study its potential ability on improving enzymatic performance.
Electrospinning is such a commonly used, convenient, versatile, and economically effective process that creates electrified jets from various polymer solutions including even polymer blends, composites, etc. (Ramakrishna et al. 2005). In this process, the solution is contained in a capillary with a syringe tip and a collecting device. Nanofibers will be stretched out from the solution by applying high voltage to the polymer jet (Ramakrishna et al. 2005). Nanofibers have many remarkable advantages and superior mechanical properties in comparison with other fibers. The properties of nanofibers including their very large surface area-to-volume ratio and porous morphology in nanoscale range make them feasible to be employed in numerous applications such as in drug delivery and tissue engineering or as a nanofibrous mat and a proper bed for enzyme immobilization (Ramakrishna et al. 2005; Amini et al. 2013; Lee et al. 2005; Mohebali et al. 2016; Shelke et al. 2016; Oktay et al. 2015; Leung and Ko 2011).
Enzyme immobilization on nanofibrous mat not only does reform instability and non-reusability of the enzyme, but also enhances enzyme’s reaction time which is an efficient way to eliminate its defects (Amini et al. 2013; Wang et al. 2009; Wagner et al. 2015). Efficacy of enzyme such as: biocompatibilities, etc., entirely depends on the bed employed for immobilization (Amini et al. 2013; Wang et al. 2009; Amini et al. 2012; Ebadi et al. 2015). Of many prominent biological properties of nanofibrous nanowebs is its ability to be biodegradable by microorganisms. It is supposed that starch compounded nanofibers make a prospective candidate as a bed in biological applications and enzyme immobilization (Shelke et al. 2014).
Fabrication of nanofibers out of pure starch and without blending with other synthetic high-molecular-weight polymers is very challenging. Here, we used the blending technology to prepare multi-functional nanofibers to meet our specific objectives. We chose to work with polyacrylamide (PAAm) which is one of the most important superabsorbent polymers. PAAm has a very routine synthesis protocol and it is known as one of the economical biocompatible polymers. PAAm was selected to be compounded with starch. It is supposed that its ability to absorb a large amount of water to form a stable hydrogel makes PAAm a promising candidate for developing biological applications and immobilization of aqueous enzyme’s solutions (Amini et al. 2013).
We employed design of experiments (DOE) method to investigate the effect of addition of starch (interconnected factor) on the diameter of PAAm nanofibers (selected response). DOE fits experimental results to mathematical equations representing some models based on a combination of values to predict the process (Chow and Yap 2008). Response surface methodology (RSM) is an empirical-based technique of gathering statistical and mathematical models to design, model, and analyze experiments (Chow and Yap 2008; Montgomery 1997; Yalcinkaya et al. 2010; Bas and Boyac 2007; Yördem et al. 2008; Rabbi et al. 2012). RSM can be used to determine optimum operating conditions by detecting the relation of an influenced objective response and several variables (Chow and Yap 2008; Montgomery 1997; Yalcinkaya et al. 2010; Bas and Boyac 2007; Yördem et al. 2008; Rabbi et al. 2012). It determines the effect of independent variables on a selected process (Yördem et al. 2008; Heydari et al. 2013). Therefore, RSM is utilized to investigate the effects of PAAm concentration and starch content on nanofibers’ diameter. The aim of this study is to investigate the impact of starch on the process ability and characterization of physical and biological properties of native starch-based nanofibrous systems.
Acrylamide monomer (AM), (71.08 g/mol, Merck) was used for polyacrylamide (PAAm) synthesis. Potassium peroxodisulfate (KPS), (270.33 g/mol, Merck) was used as a radical initiator for the synthesis reaction of PAAm. 3-Aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) were bought from Sigma-Aldrich Co and PanreacQuimica San Co. for cross-linking reaction of nanofibers, respectively. Wheat starch was a general food grade prepared from the market. Formic acid was purchased from Riedel–de Haen as a solvent for electrospinning. Sodium phytate (P3168) was bought from Sigma-Aldrich Co. as a substrate of the phytase. Escherichia coli DH5α and primers for PCR were purchased from Sinaclone Co. The sequence encoding Aspergillus niger Phytase (P34752) was synthesized by ShineGene Molecular Biotech (Shanghai, China). Other analytical grade chemicals were obtained from Merck Co.
Experimental design and resulted responses
wt% of PAAm (g/ml)
Starch compounding (%)
Average fibers diameter (nm)
206.1 ± 15
218.3 ± 18
241.2 ± 17
144.9 ± 15
208.7 ± 20
218.3 ± 12
200.7 ± 16
242.0 ± 25
201.7 ± 14
122.1 ± 10
120.2 ± 14
230.7 ± 20
206.0 ± 19
50 g of acrylamide was dissolved in 200 ml distilled water. The solution was then air-degassed and heated up to 80 °C beneath nitrogen atmosphere. Radical initiator and oxidant, KPS (0.5% wt concentration to acrylamide), was added to the solution and free radical polymerization was started after initiator was added to the monomers. After 2 h, the polymerization was stopped and the resultant product was precipitated with acetone and preserved in vacuum desiccators.
We made starch/PAAm solutions according to the experimental design (Table 1) by dissolving starch and PAAm separately in formic acid. Solutions were heated up to 50 °C and stirred for 3 h. Then, they were mixed up together and stirred for 48 h at room temperature. The polymer concentration was varied from 1.5 to 4% (w/v, with respect to the solvent) and the percentage of added starch changed from 20 to 50% (w/w, with respect to the polymer concentration). The polymer blend solutions were injected into 1 ml syringe which was fixed horizontally on a syringe pump (model: JMS SP-500). The flow rate of the solutions was adjusted at 0.1 ml/h. The electrode of the high voltage–power supply (model: Gold Star PZ-121) was clamped to the metal needle tip and a 15 kV voltage was applied to generate the electrical field. The tip-to-collector distance was adjusted to 20 cm. To spin the solutions, the functional parameters: feed rate, applied voltage, and tip-to-collector distance were verified, and the uniform nanofibers were obtained. A 100 cm2 grounded static vertical collector covered by a piece of aluminum foil was used for the fiber deposition. The complete electrospinning apparatus was enclosed under a vacuum hood and an electrospinning process was carried out at room temperature.
Cloning and expression of phytase
The recombinant vector PUC57 containing Aspergillus niger Phytase was transformed to the competent cell of E. coli. After colony PCR screening, plasmids were purified from the positive clones by the alkaline lysis method and then digested with HindIII/BamhI. The phytase gene was inserted into pFPMTMFα as an expression and also as a shuttle vector. pFPMTMFα comprising phytase was transformed into E. coli DH5α. All the cloning steps were done according to Sambrook et al. (1989). The phytase was transformed into Pichiapastoresis.
The pre-culture was grown in a 250 ml flask containing 50 ml of YPD (1% yeast extract, 2% peptone, and 2% glucose) at 37 °C for an overnight (16–18 h) on a rotary shaker at 250 rpm. The main culture medium (1% yeast extract, 2% peptone, 1% glycerol) was inoculated with 5–10% (v/v) from pre-culture after washing by sterile water. The main cultivation for enzyme production was performed for 72 h under the same conditions as with the pre-culture. The cells were harvested by centrifugation at 2500×g for 10 min. The extracellular activity was determined in the cell-free supernatant.
Phytase activity assessment
We test the phytase assessment by measuring the amount of inorganic phosphate liberated from sodium phytate (Heinonen and Lahti 1981). Different amounts of phytase in a solution were evaluated and 3 U/ml was identified as the optimum phytase concentration in an enzymatic aqueous solution. The assay mixture contains 0.5 ml of 2.5 mM Na-phytate (in 0.1 M pH 5.0 Na-acetate buffer) and 0.475 ml of 0.1 M Na-acetate buffer (pH 5.0). All nanofibers were immersed in the assay mixture and were incubated for 30 min at 50 °C. The reaction was stopped by keeping nanofibers out from the mixture and adding 2 ml of color reagent solution (25% ammonium molybdate solution 5% + sulfuric acid 5 N + acetone). After 45 s, color was developed and 0.1 ml citrate 1 M was added to each test tube to fix the color. Finally, absorbance was read at 380 nm. The activity of phytase before and after immobilization process was assessed to determine the amount of bound enzymes. One unit (1 U) of phytase was defined as the amount of phytase which releases 1 μmol inorganic phosphate per minute at the assay condition.
Cross-linking and enzyme immobilization
Blended PAAm/starch nanofibers were modified through cross-linking modification to attain biological applications. Of many approaches for cross-linking the surface of nanofibers, we selected an approach to raise stability and to create appropriate links for bonding using APTES and GA, respectively (Amini et al. 2013; Frone et al. 2013). Samples were immersed in the 20 v/v% solutions of APTES in methanol at 50 °C for 4 h and 1 v/v% solutions of the GA in methanol at 70 °C for another 4 h, while shaking, respectively. To remove residual links from non-reacted APTES and GA factors, they were well washed with NaAc buffer which keeps pH constant at 5.0. Through this approach, flexible and appropriate nanofibrous mats were obtained. The nanofibers were immersed in 2 ml synthesized phytase solution at 4 °C overnight while shaking. After removing nanofibers out of enzyme solution, un-attached enzymes were washed three times with NaAc buffer and their activities were assessed.
New blended starch nanofibers retained their physical shape after keeping in the enzyme solution for a long time which endorses high stability after cross-linking. In contrast, nanofibers obtained from pure PAAm nanofibers did not retain their structure without the same cross-linking method, and they were gradually destructed after maintaining in enzyme or buffer solution. This phenomenon can be explained by considering the high hydrophilic nature of PAAm.
The morphology of the electrospun blended PAAm/starch nanofibers was characterized with a high-resolution scanning electron microscopy (SEM) (Hitachi S4160); and ImageJ software was used for measurement of the fibers’ diameter. Design Expert software (version 7, Stat-Ease, Inc., Minneapolis, MN 55413) was used for evaluation of the fibers’ diameter based on the affected nanofibers variables. Fourier transform infrared spectroscopy (FT-IR, model: Nicolet IR100) was used to study differentiations of spectra for starch, PAAm, and blended PAAm/starch which could indicate the changes in the chemical structure upon blending. Thermal analysis of the fibers was done using differential scanning calorimetry (DSC) (NETZSCH 200 F3) and thermal gravimetric analysis (TGA) (Linseis PT-10). DSC measurement allows one to evaluate the thermal behavior differences of the nanofibers and determining the influence of starch on PAAm nanofibers, before and after cross-linking modification. About 11 mg of nanofibers were heated up at 10 °C/min from room temperature to 400 and 800 °C at 10 °C/min under nitrogen atmosphere for DSC and TGA analysis, respectively. Wide angle X-ray diffraction (XRD, model: Siemens D500) measurements were performed to obtain crystalline structure information. The X-ray generator was equipped with an iron tube operating at 35 kV and 25 mA. The wavelength of irradiation was ~0.194 nm. XRD diffractograms were acquired at room temperature over a 2θ range of 5°–40° at 1.2°/min. The enzyme activity was evaluated according to Henion and Lathi protocol including some modification. The nanofibrous mat was all prepared at the same size of 1 cm2. Optical density (OD) of immobilized phytase mixtures was measured via a UV–Vis Spectrometer (Micro plate reader, model: NanoQuant, infinite M200 Pro) at the absorbance of 380 nm. Michaelis–Menten kinetics for immobilized and free phytase were measured for different phytic acid concentrations as substrate.
Results and discussion
As it is shown in Fig. 1e, fabrication of pure starch nanofibers is quite challenging and leads to the bead shape structure. Blending with other synthetic high-molecular-weight polymers such as PAAm is the answer. At a definite weight fraction of the blended PAAm/starch solution, increasing weight fraction of the starch proportionally leads to a reduction in the weight fraction of the PAAm. Subsequently, a reduction of the PAAm concentration in the solution apparently can reduce the viscosity of the solution and can conduce to decline of the nanofibers’ diameter. This can be obviously inferred from pursuing the resultant values of the diameter of a 2.75 wt% solution with a PAAm:starch blending proportion of (80:20) to (50:50), respectively.
Diameter analysis via RSM method
ANOVA results of suggested model
P > F
Lack of fit
In starch spectra, a strong and broad peak at 3423.18 (cm−1) is attributed to the characteristic absorption peak of the stretching vibration of O–H. The peaks at ~1084.07 (cm−1), 1158.40 (cm−1) and 1019.60 (cm−1) are related to the vibration of C–O in C–O–H groups. The peak at 2928.39 (cm−1) is ascribed to the asymmetric stretching of C–H whose intensity vibration is significantly lessened in the blend format. The peak at 1646.55 cm−1 is attributed to the adsorbed water. The peak at about 1007.87 (cm−1) is ascribed to C–O–H stretching. The peaks at 857 (cm−1) and 763.81 (cm−1) are related to the C–H absorbance of d-glucopyranosyl ring stretching.
Peaks at 1665.92 (cm−1) and 2961.55 (cm−1) imply the presence of PAAm, and peaks at 796.40 (cm−1), 1019.60 (cm−1), and 1170.67 (cm−1) are attributed to the starch in the blended nanofibers. FT-IR investigation demonstrates that blending was carried out in a satisfactory way due to the presence of all the starch and PAAm bonds in the blended nanofibers.
TGA analysis results
T50% mass loss (°C)
Inflect temp. (°C)
Mass change (%) between 300 and 600 °C
Blend with immobilized enzyme
Comparing mass loss temperatures of the PAAm/starch nanofibers and cross-linked ones can barely specify that the cross-linking modification significantly increases the nanofibers thermal stability. However, immobilization procedures and putting nanofibers in the aqueous enzyme solution for 18 h at 4 °C decrease the thermal stability (in comparison with cross-linked nanofibers). This is indorsed by comparing 50% mass loss temperatures (T50% mass loss) of the PAAm/starch nanofibers, cross-linked PAAm/starch nanofibers, and immobilized phytase on the PAAm/starch nanofibers, which are 348, 500, and 411 °C, respectively. Cross-linking improved thermal stability by about 43% measured at T50% mass loss and also it modified 18% mass residue (in comparison with blended nanofibers) which could be regarded as an evidence for proper cross-linking procedure. Nevertheless, holding nanofibers at 4 °C for 18 h while shaking in enzyme solution reduces thermal degradation of them (17.7% lower thermal stability compared to cross-linked nanofibers before immobilization).
According to TGA results, it is proved that the addition of starch reduces the thermal stability, since it increases the degradation of PAAm nanofibers as reported previously (Kaushik et al. 2010). A starch blended PAAm nanofibers of 2.75 wt% (containing 35% starch relative to PAAm) increases thermal degradation of pure PAAm nanofibers by 19.44%, in accordance with the first derivative peak temperature. Considering the intrinsic biodegradability of wheat starch, as thermal degradation of a polymer begins with chain cleavage, starch incorporation could lead to improving the thermal degradability of polymeric nanofibers according to the purpose which is using polymeric nanofibers in biological digestive application.
Decomposition kinetics of nanofibers can be inspected by observing the slopes of TGA curves of the PAAm and the blended starch ones. The sharper slope of the curve of the PAAm nanofibers indicates that its thermal decomposition is progressed at a higher speed than the blended nanofibers’ one which suddenly starts at 310 °C. Blended nanofibers originated from two components; hence, its thermal degradation behavior is a function of both starch and PAAm. Subsequently, it would result in a broad degradation peak and a lower speed.
Differential scanning calorimetry analysis
Due to the slight change of the heat capacity, a weaker heat flow signal of the blended starch material is generated in comparison with other polymers (Liu et al. 2009). Therefore, measurement of glass transition temperature (Tg) of the starch by DSC is difficult. Moreover, the multiple phase transitions during the heating process and the instability due to evaporation of water included in starch make the study of thermal behavior and crystallography of starch materials more difficult (Liu et al. 2009). Liu et al. evaluated Tg of starch containing different amounts of moisture by a high-speed DSC (above 50 °C/min) (Liu et al. 2009). Wheat starch DSC-related peak with different moisture occurs in the range of 60–105 °C which contains about 0.8% lipids and 28% amylase (Lionetto et al. 2005).
In pure PAAm, despite the fact that the peak centered at 273 °C, no significant melting point is observed due to the poor crystalline structure of PAAm which is proved by XRD analysis thereafter. In blended nanofibers, the presence of two peaks is observed and the rather huge exothermic peak centered at 100 °C is related to the starch and the peak which is shifted at 280 °C belongs to the PAAm (Amini et al. 2013; Lionetto et al. 2005; Zeng et al. 2011), although its conjunction shows the finest inter mixture of two phases and satisfactory compounding. The Tg of the pure PAAm nanofibers was measured at 189 °C. Disappearing of Tg in the X-linked blend nanofibers is the evidence of satisfactory cross-linking treatment of nanofibers. Both starch and PAAm peaks are sustained after X-linking with the difference of enthalpy (ΔH) of the starch caused by losing its moisture.
In accordance with the literature, pure PAAm nanofibers did not show any significant crystalline pattern over a 2θ range of 5°–40° (Tang et al. 2008). As reported in the literature, X-ray diffractograms of the wheat starch samples show typical of amorphous systems with sharp peaks centered at 2θ around 15°–18°–23° (Wang et al. 2011b; Zeng et al. 2011; Lionetto et al. 2005). Although, when it is blended with other polymers and electrospun to nanofibers, the crystalline structure of resultant fibers has been destroyed and the intensity of the peaks is decreased substantially (in comparison with its pure powder type). Moreover, followed by being solved in formic acid during solution preparation, the orientation of the starch’s chains may be disordered. Even more, it is observed that the amount of the starch in accordance with PAAm in the solution is a lot less. It is supposed that solvent rapid vaporization, electrical driving force, and stretching of fibers imposed by electrospinning procedure do not lead to orientated arrangement of the chains and crystalline structure (Khan et al. 2009).
It has been shown that neither type of fibers shows crystalline structure. Observing XRD patterns of both PAAm and starch blended PAAm nanofibers indicates that they have almost the same non-crystalline pattern. We hypothesize that there is no more crystalline phase existed; however, it may also be possible to claim that there might be less than 5 wt% from which it could be inferred that the dominant phase of samples is amorphous. Accordingly, it can be deduced that blending was successfully accomplished as the resultant nanofibers are in single phase.
Immobilized enzyme activity measurements
Kinetic data for free and immobilized phytase
Immobilized on PAAm nanofibers
Immobilized on PAAm/starch blend nanofibers
Here, we report that the immobilization improves the kinetics of enzyme activity. In addition, it is proved that the presence of starch could slightly fix the reaction rate and it also provides improved activity in comparison with enzyme immobilization on pure PAAm nanofibers.
All biological assessments show satisfactory covalent attachment of the phytase. Besides, it is proved that the presence of appropriate amount of the starch could even repair biological properties.
Here, we have synthesized polyacrylamide (PAAm), blended with different amounts of wheat starch and fabricated randomly oriented nanofibers. The SEM images and RSM diameter analysis have shown that more percentage of starch addition—proportionally against the percentage of PAAm—could lead to the reduction of the resultant nanofibers’ diameter. The blend was successfully made as the FT-IR results had proved the perfect structure. The thermal properties of PAAm nanofibers were shown to be improved by the presence of starch which enhanced the thermal degradability of PAAm nanofibers. Moreover, we have shown that cross-linking had synergetic effects in addition to surface modification which eventually enhanced thermal stability as well as preventing undesirable transformation; therefore, it increased the stability of nanofibers in unfriendly environment. This paper shows the promising potential of enzyme immobilization of starch/PAAm nanofibers. Overall, it was shown that the addition of starch could significantly increase the immobilized enzyme activity. We conclude that starch-aiding nanofibers improve kinetics activity of immobilized enzyme and also they surpassingly operate as a promising substrate for enzyme immobilization.
The authors would like to thank Shahid Beheshti University Resources for their great help and support.
- Bačáková L, Novotná K, Pařízek M (2014) Polysaccharides as cell carriers for tissue engineering: the use of cellulose in vascular wall reconstruction. Physiol Res 63:29–47Google Scholar
- Daughton CG (1988) Quantitation of acrylamide (and polyacrylamide): critical review of methods for trace determination/formulation analysis & future-research recommendations. California Public Health Foundation, BerkeleyGoogle Scholar
- Dwivedi P, Narvi SS, Tewari RP (2012) Chitosan modification through natural route: development of Ag/CS-g-PAAm using Curcuma longa. J Chin Med Res Dev (JCMRD) 1:23–27Google Scholar
- Montgomery GC (1997) Design and analysis of experiments. Wiley, New YorkGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USAGoogle Scholar
- Wagner I, Nagy ZK, Vass P, Feher C, Barta Z, Vigh T, Sóti PL, Harasztos AH, Pataki H, Balogh A, Verreck G, Assche IV, Marosi G (2015) Stable formulation of protein-type drug in electrospun polymeric fiber followed by tableting and scaling-up experiments. Polym Adv Technol 26:1461–1467CrossRefGoogle Scholar
- Wang H, Wan W, Jiang S, Jiang S, Zhai L, Jiang Q (2011a) Poly(vinyl alcohol)/oxidized starch fibres via electrospinning technique: fabrication and characterization. Iran Polym J 20(7):551–558Google Scholar
- Wang H, Wang W, Jiang S, Jiang S, Zhai L, Jiang Q (2011b) Poly(vinyl alcohol)/oxidized starch fibres via electrospinning technique: fabrication and characterization. Iran Polym J 20:551–558Google Scholar
- Whistler RL, Bemiller JN, Paschall EF (1984) Starch chemistry and technology. Academic, New YorkGoogle Scholar
- Yalcinkaya SE, Yildiz N, Sacak M, Calimli A (2010) Preparation of polystyrene/montmorillonite nanocomposite: optimization by response surface methodology (RSM). Turk J Chem 34:581–592Google Scholar
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