Fibers and Polymers 2012, Vol.13, No.8, 994-998
DOI 10.1007/s12221-012-0994-y
Manufacturing Polymethyl Methacrylate Nanofibers as a Support for Enzyme Immobilization Navid Amini, Saeedeh Mazinani1*, Seyed-Omid Ranaei-Siadat2**, Mohammadreza Kalaee3, Kaveh Niknam2, and Vida Adlfar2 Department of Polymer Engineering, South Tehran Branch, Islamic Azad University, 1777613651, Tehran, Iran Amirkabir Nanotechnology Research Institute (ANTRI), Amirkabir University of Technology, 15875-4413, Tehran, Iran 2 NanoBiotechnology Engineering Lab., Department of Biotechnology, Faculty of Energy Engineering and New Technologies, Shahid Beheshti University, GC, Tehran, Iran 3 Polymer Engineering Group, Department of Engineering, Qom University of Technology, Qom, Iran (Received November 22, 2011; Revised March 12, 2012; Accepted March 20, 2012) 1
Abstract: Nanofibers have a great potential for enzyme immobilization application due to their large surface area to volume ratio besides their porous structure. In this work, we produce polymethyl methacrylate (PMMA) nanofibers via electrospinning method in dimethylformamide (DMF) as solvent. Thereafter, we employ a chemical method on final PMMA nanofiberous web to covalently immobilize acetylcholinesterase (AChE) enzyme on membrane surface. Morphology and tensile properties of nanofibers are studied as first steps of characterization to make sure of obtaining a properly stable membrane for enzyme carrying application. Thereafter, the stability and activity of immobilized enzymes as two main characteristic parameters are tested and reported for different applications such as biosensor manufacturing. Keywords: PMMA, Electrospinning, Acetylcholinesterase enzyme, Immobilization
high surface area to volume ratio and porous structure which has made them a proper candidate for enzyme immobilization application. Besides, it does not have the disadvantages of other nanomaterials such as requiring to pass the dispersion and recycling procedure. AChE (acetylcholinesterase) (EC 3.1.1.7) is a serine hydrolyser which catalyses the hydrolysis of the neurotransmitter acetylcholine [22]. Carbamates and organophosphates are potent inhibitors of AChE which carbamylate or phosphorylate the serine residue of the active-site triad which causes blocking the nerve signal transference into the postsynaptic membrane. These agents have been widely used for agricultural pesticides. This shows the importance of developing biosensors based on AChE to detect the inhibitors mentioned previously. One of the most important factors in biosensor fabrication is the effective enzyme immobilization on the membrane surface in the way that certifies suitable enzyme activity and reusability [23]. Different methods for AChE immobilization have been so far reported: adsorption, sol-gel encapsulation and enzyme cross linking [23,24]. Stoilova et al. immobilized AChE on polyacrylonitrile nanofibrous mats treated with chitosan and showed that immobilized enzyme have greater activity and thermal stability than free enzyme [25]. Moradzadegan et al. co-spun AChE with PVA (polyvinyl alcohol) and BSA (bovine serum albumin) as a method to stabilize the enzymes. They showed that the immobilized AChE included improved stability while retaining a considerable activity at lower pH values in comparison with free enzymes [26]. In this work, electrospinning is employed for PMMA nanofiberous membrane manufacturing applicable for nanobiosensors development. Here, we employ PMMA (polymethyl
Introduction Nanofibers are appropriate candidates for many applications owing to their large surface area, their superior mechanical performance besides high porosity [1-3]. Among different methods for nanofiber manufacturing, electrospinning has gained much attention in the last decade because of its versatility to spinning a wide range of polymeric fibers, and its ability to consistently produce fibers in the submicron range. It is difficult to achieve nanofibers by using standard mechanical fiber-spinning techniques [4], and offering a relatively inexpensive and simple method for nanofibers formation [5]. Electrospun nanofibers have been successfully applied in various fields such as: tissue engineering scaffolds [6-12], drug delivery [13-15], wound dressing [16-18], filtration [16], nano-sensors [17-20], etc. [19-21]. Immobilization of enzymes on inert, insoluble materials is an active area of research for improving the functionality and performance of enzymes for bioprocessing applications. Moreover, immobilized enzymes offer several advantages such as reusability, better controlling of reaction and more stability compared to enzymes soluble in the medium [4]. The results of immobilization, including the performance of immobilized enzymes, strongly depend on the properties of supports which are usually referred to as material types, compositions, structures, and etc. Different nano-structured materials have been so far used as supports, such as mesoporous silica, nanotubes, nanoparticles, and nanofibers [1]. Among these nanomaterials, nanofibers show extremely **Corresponding author:
[email protected] **Corresponding author:
[email protected] 994
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methacrylate) nanofibers for immobilization of AChE enzyme for the first time. We use optical microscopy imaging technique for morphological analysis of the resulting PMMA membrane besides image analysis as the first step. In addition, tensile properties of PMMA nanofibers are measured to characterize the enzyme support and to show the successful cross linking confirmation after enzyme immobilization. Activity of the immobilized enzyme is measured after several times washing to prove the successful enzyme immobilization. We also compare the thermal stability and the kinetics of immobilized enzyme with those of free enzymes before immobilization to show the effective role of immobilization.
Experimental Materials PMMA was purchased from Aldrich Co. including the molecular weight of Mw=60,000. APTS (3-aminopropyltriethoxysilane) and ATCh (acetylthiocholine) were bought from Sigma-Aldrich Co. and GA (glutaraldehyde) from Panreac Quimica Sau Co. Pichia pastoris was purchased from Invitrogen Co. All other chemicals were analytical grade and they were provided from Merck Co. Electrospinning of PMMA PMMA was dissolved in DMF (dimethylformamide) at 8 wt% concentration for 24 h at room temperature. The electrospinning set-up consisted of syringe pump (SP-500 model), high voltage DC power supply (Gold Star PZ-121 model), syringe with flat-end metal needle and a metal collector which was grounded and fully covered with aluminum foil. Positive electrode was connected to the needle and the negative one to the collector. A 20 kV voltage was applied to generate the electrical field between the needle and collector of 15 cm distance. Nanofibrous mats were then collected on aluminum foil. Enzyme Production Acetylcholinesterase gene sequence from Drosophila melanogaster was codon optimized based on pichia pastoris codon usage. The gene was synthesized and cloned in to pPinkα-HC and then was transformed in Pichia pastorisusing electroporation method. The enzyme was expressed and secreted in to the medium actively. The enzyme solution was purified on procainamide-sepharose 4B affinity gel. Acetylcholinesterase activity was assayed using acetylthiocholine as the substrate and using Ellman’s method Cross Linking and Enzyme Immobilization At the beginning, 20 wt% of APTS solution in aqueous media was prepared and the PMMA membrane was immersed in the solution at 80 oC for 4 h while shaking. Then the membrane was left in a 5 wt% GA solution in aqueous media at 120 oC for an additional 4 h. Then the membrane was
Figure 1. Schematic of enzyme immobilization on PMMA nanofibers showing both covalent attachment and physical absorption of AChE.
brought out of the solution and well washed using distilled water to remove any residual unreacted GA and APTS. Thereafter, the membrane was left in the enzyme solution overnight in cold room at 4 oC while shaking. Figure 1 shows schematic procedure of enzyme immobilization on PMMA nanofibers. This schematic can clearly explain how AChE enzyme is immobilized on PMMA nanofibers’ surface via both covalent attachment and physical absorption after nanofibers cross linking by APTS & GA. The successful cross linking of PMMA fibers will be proved by mechanical test results as follows. Measurements and Characterizations The morphologies of PMMA nanofibers were examined via optical microscopy method. Samples were spun on a lamella and were assessed using optical microscopy. Tensile properties of nanofibers were measured with tensile testmachine model Santam SMT-20 at 15 mm gauge length and 2 mm/min cross head speed at 27 oC with 53 % relative humidity. The specimens were cut in a typical size of 20 mm length and 5 mm width and they were prepared at a thickness of about 100 µm. Measuring the enzymes activity was performed according to Ellman’s reagent. We first prepared a 100 ml phosphate buffer of 25 mM; and PH=7.4 to produce Ellman’s solution. We then dissolved 37.5 mg sodium carbonate and we added 91 mg DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)) followed by raising the volume to 1000 ml. The membranes were all prepared at the same size of 1.5×3 cm and the approximate weight of 0.003 g. Then, we put each membrane in the 980 µl of Ellman’s and 20 µl of 1 mM ATCh as the substrate. After 1 min, the membranes were examined and the optical density (OD) of the solution was measured via UV-vis photometer at 412 nm. We measured the enzyme activity at different concentrations of substrate (ATCh) to employ
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michaelis-menten kinetic modeling method. Thermal stability of the immobilized enzyme was carried out at 55 oC for both free and also the immobilized forms of enzymes on nanofiberous membrane.
Results and Discussion Enzyme Support Characterization Figure 2 shows the optical images of final electrospun PMMA nanofibers via optical microscopy on as-spun fibers (Figure 2(a)) besides nanofiberous membrane followed by enzyme immobilization (Figure 2(b)). We used this technique for fast assessment of nanofibers' morphologies before and after enzyme immobilization and to avoid employing more complicated methods such as scanning electron microscopy (SEM). We could make the observations by collecting the nanofibers on transparent collection substrate. Average fiber diameter is obtained about 293 nm followed by employing image analysis technique on the fibers' images. Figure 3 shows PMMA nanofiber diameter distribution after analysis. As it is clearly shown in the image, fine beadless fibers are obtained at employed polymer concentration. Figure 1(b) shows the optical microscopy image of PMMA nanofibers
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after AChE immobilization. As it is obviously shown, the enzyme immobilization procedure does not affect PMMA nanofibers morphology and the nanofibers completely retain their 3D structure. Therefore, the resulting membrane after enzyme immobilization could pass further characterization steps. In addition to morphological characterization, mechanical properties were also assessed to evaluate the membrane properties. Figure 4 shows the mechanical properties and stress-strain curves for PMMA nanofibers with (a) and without (b) AChE. Due to the amorphous structure of PMMA and electrospinning induced crystallization, the nanofibers behavior is quite brittle before immobilization, and hence the nanofibers show a very small value of elongation at break. However, as it is clearly shown in the curves and the reported values given in the Table 1, the tensile modules of PMMA nanofibers significantly increase. This is obtained followed by the cross linking step for enzyme immobilization. This also comes as an evidence of successful cross linking by forming the chemical bonds amongst the polymer chains which results in increasing the modulus and mechanical strength of nanofiberous membrane after cross linking (The schematic of cross linking and enzyme immobilization was previously shown in Figure 1). Enzyme Activity We used PMMA nanofibers for enzyme immobilization application as the main objective of this work. Figure 5 illustrates the assay solution ODs for immobilized AChE on PMMA nanofibers. We washed the membrane 3 times in a vial with phosphate buffer by a vortex. The assay of immobilized enzyme on the PMMA nanofibers after 10 uses
Figure 2. PMMA nanofibers morphology (a) as-spun PMMA nanofiber and (b) PMMA nanofiber after cosslinking & AChE immobilization.
Figure 4. Stress strain curve for (a) as-spun PMMA nanofiber and (b) PMMA nanofiber after AChE immobilization. Table 1. Tensile properties of PMMA nanofibers
Figure 3. Nanofiber diameter distribution.
Nanofiber type PMMA PMMA/AChE
E (MPa) 31.65±0.34 43.4±0.21
εb (%) 2.8±0.11 2.6±0.17
σ0 (MPa) 0.8±0.02 1.01±0.01
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Figure 5. Relative activity of immobilized enzyme.
Table 2. Km for free and immobilized AChE Enzyme Free Immobilized
Km 0.0259 mM 0.0272 mM
shows only about 12 % decrease which illustrates excellent immobilization and successful covalent attachment of AChE to acrylate groups of polymeric nanofibers. This was also previously confirmed via mechanical experiments test results. In addition, the immobilized AChE retains 98.6 % of its activity after 30 days in 4 oC as experimented thereafter. Table 2 shows the kinetic values obtained from MichaelisMenten curve (the equations are not given here). As it is obvious, the Km (obtained followed by plotting the Michaelis-Menten curves, the results are not shown here) of immobilized enzyme does not show clear difference from its free form and it only shows a slight increase which is an evidence that the immobilization has not significantly changed the enzyme kinetic and it only causes a very small decrease in enzyme activity. Enzyme Thermal Stability Figure 6 shows the thermal stability for free and immobilized AChE enzymes, respectively. It is obviously shown that immobilization increases the thermal stability of AChE enzyme. Covalent attachment of enzymes to polymeric nanofibers surface (as done successfully and previously proved) causes undesirable enzymes conformations which do not propose considerable stability of the enzyme under unfriendly probable environmental temperature increases [27]. Note that the scale of the y-axis in two graphs is not the same so it is not possible to compare the numbers given on these graphs. However, we could compare the half times of these two forms of AChE (free and immobilized). Half time calculation for free enzyme at temperature of 55 oC is less than one minute but that of the immobilized enzyme is much higher and it is about 95 seconds. This result shows the
Figure 6. Thermal stability for (a) free AChE and (b) immobilized AChE.
successful immobilization of AChE; since the immobilization process properly increases the enzyme stability as the temperature increases.
Conclusion Polymethyl methacrylate nanofibers were successfully fabricated via electrospinning method. Very fine and beadless fibers were obtained with 8 wt% of PMMA content in DMF solvent. We employed these nanofibers for immobilization of acetylcholinesterase enzyme. Optical microscopic images showed that the cross linking with enzyme did not change the final morphology of nanofibers. A stress-strain curve and enhancing the mechanical strength and modulus depicted successful cross linking of polymer after enzyme immobilization. Finally the activity of the immobilized enzyme after 10 uses showed good reusability of the immobilized AChE which is correspondent to the proper enzyme immobilization. The thermal stability of the enzymes also showed improvement after immobilization comparing to the free form.
Acknowledgement The authors would like to thank Shahid Beheshti University for the great help and support to do this work.
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