Materials Science and Engineering C 50 (2015) 251–256
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Boronate affinity nanoparticles for RNA isolation Aykut Toprak a, Cansu Görgün a, Cansu İlke Kuru a, Ceren Türkcan a, Murat Uygun b, Sinan Akgöl a,⁎ a b
Ege University, Faculty of Science, Department of Biochemistry, 35100 Bornova, Izmir, Turkey Adnan Menderes University, Koçarlı Vocational and Training School, 09010 Aydın, Turkey
a r t i c l e
i n f o
Article history: Received 10 April 2014 Received in revised form 3 October 2014 Accepted 10 November 2014 Available online 12 November 2014 Keywords: RNA Boronate affinity Nanoparticle
a b s t r a c t In this presented paper, boronic acid incorporated poly(HEMA) based nanoparticles were synthesized for RNA adsorption. For this purpose, poly(HEMA) nanoparticles were synthesized by using the surfactant free emulsion polymerization technique. Then, nanoparticles were modified with 3-(2-imidazoline-1-yl)propyl(triethoxysilane) (IMEO) and functionalized with phenylboronic acid (PBA). Prepared nanoparticles were characterized with SEM, FTIR and zeta-size. Optimum RNA adsorption conditions were investigated with different pHs, temperatures and initial RNA concentrations in order to determine the maximum RNA adsorption onto poly(HEMA)-IMEO-PBA nanoparticles. It was also studied that, synthesized nanoparticles could be used for 5 successive reuses and adsorption capacity of the nanoparticles decreased only about 5% at the end of the 5 cycles. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Recently, nanoparticle applications have received great attention in various biotechnological applications due to their unusual chemical and physical properties. Nanoparticles with various shapes and functionalities have been synthesized and used in different kinds of biotechnological applications such as purification of biomolecules, separation or sorting of cells, immobilization of proteins and enzymes, medical diagnosis, sensor applications, immunoassays, magnetic resonance imaging and controlled drug delivery studies [1–5]. Nanoparticles serve high surface areas and consequently they can bind high amount of biomolecules. Because of their unique features, nanoparticles can easily be used for the separation or purification of the biomolecules from complex mediums. Also, nanoparticles can eliminate the time consumptive purification steps in biomolecule separation studies [6]. RNA plays a critical role in the transformation of the genetic information and new roles of RNA have also been discovered over the past ten years. In order to understand the functions and the properties of the RNA, its biochemical and structural characterizations have great importance. When viewed from this aspect, pure and non-denatured RNA is very essential. On the other hand, recently used RNA purification techniques induce loss of time and the final RNA preparation is in denature form. Different RNA purification strategies have been developed, in order to overcome these drawbacks. Specially, affinity chromatographic techniques have been applied for the purification of the RNA from different mediums [7–14]. One of the efficiently used affinity techniques is the boronate affinity chromatography and it has been used for the isolation of biomolecules such as nucleotides, RNA, glycated proteins and ⁎ Corresponding author at: Ege University Biochemistry Department, Izmir, Turkey. E-mail address: [email protected] (S. Akgöl).
http://dx.doi.org/10.1016/j.msec.2014.11.033 0928-4931/© 2014 Elsevier B.V. All rights reserved.
glycol-enzymes [15]. In this affinity technique, diol moieties which are in a cis configuration form a complex with boronic acid group. With this interaction, a negatively charged cyclic boronate ester takes place [16]. In this work, p(HEMA) nanoparticles carrying boronic acid group were synthesized for purification or recognition of RNA. For this purpose, poly(HEMA) nanoparticles were synthesized by the surfactant free emulsion polymerization technique and then characterized. Nanoparticles were then modified with IMEO. Boronic acid group was introduced into the nanoparticle structure by the interaction of the IMEO modified nanoparticles with phenyl boronic acid (PBA). Conditions of RNA adsorption onto poly(HEMA)-IMEO-PBA nanoparticles were optimized by using different pHs, temperatures and initial RNA concentrations. Finally reusability of the poly(HEMA)-IMEO-PBA nanoparticles was investigated for 5 sequential cycles. 2. Experimental 2.1. Materials RNA (from Saccharomyces cerevisiae), hydroxyethyl methacrylate (HEMA), ethylene glycol dimethacrylate (EGDMA), potassium persulfate and phenylboronic acid (PBA) were supplied from Sigma (St. Louis, USA). All other chemicals were of analytical grade and used without further purification. 2.2. Synthesis of poly(HEMA) nanoparticles Poly(HEMA) nanoparticles were synthesized by using the surfactant free emulsion polymerization technique [17,18]. Briefly, 0.5 g of poly(vinyl alcohol) (PVA) was dissolved in 100 mL of deionized water and used
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as a stabilizer. Then, 0.6 mL of HEMA and 0.3 mL of EGDMA were added to the initial dispersion mixture as monomers and were mixed by using an ultrasonic water bath for 30 min. Potassium persulfate (KPS) was used as an initiator with the concentration of 20 mg/mL. In order to eliminate the dissolved oxygen from the polymerization mixture, nitrogen gas was blown through the medium. Polymerization reaction was taken place in a shaking water bath at 70 °C with the shaking rate of 150 rpm for 7 h by using a polymerization reactor. At the end of the polymerization process, the synthesized polymers were washed with ethanol and distilled water for several times in order to remove the unreacted monomers and initiators. 2.3. Modification of poly(HEMA) nanoparticles Prepared poly(HEMA) nanoparticles were firstly functionalized with the silane group bearing molecule 3-(2-imidazoline-1yl)propyl(triethoxysilane) (IMEO). The silane groups easily react with the hydroxyl group bearing surface [i.e. poly(HEMA)] [19]. For this purpose, poly(HEMA) particles were mixed with IMEO with the molar ratio of 2:1 at 25 °C for 24 h. Silanized nanoparticles were collected by the help of centrifuge and washed with distilled water [20]. Incorporation of the IMEO onto poly(HEMA) structure is schematically summarized in Fig. 1. In this presented work, phenyl boronic acid (PBA) was used as a boronate affinity ligand. With the attachment of IMEO, the surface of the nanoparticle was functionalized by imidazoline groups. Nitrogen atoms of the imidazoline group can easily interact with the boron of the PBA via a simple coordination bond. By the incorporation of the
PBA to the nanoparticle structure, boronate affinity nanoparticles were prepared. For this, 200 μL of IMEO activated nanoparticle solution was mixed with 0.25 mg/mL of phenylboronic acid solution (in MES buffer, 25 mM, pH 6.0). Then this solution was stirred for 2 h and centrifuged at 10,000 rpm for 20 min. Supernatant solution was stored for the calculation of the binding amount of PBA. Incorporation of PBA onto poly(HEMA)-IMEO structure was also studied with different conditions such as medium pH, temperature and initial PBA concentration. For this, pH of the binding solution was changed between 5.0 and 10.0. The effect of temperature on the PBA binding was studied with the temperature range of 4–55 °C. Initial PBA concentration was changed between 0.6 and 8.0 mg/mL in order to show the effect of PBA concentration on the PBA binding. Bonded PBA was calculated by using the following formula: Q¼
C i −C f V m 10−3
:
ð1Þ
Here, Q is the bonded amount of PBA (mg/g); Ci and Cf are the initial and final PBA concentrations, respectively (mg/mL); V is the volume of the binding solution (mL); and m is the mass of the poly(HEMA)-IMEO nanoparticle (g). 2.4. Characterization of nanoparticles Scanning electron microscope (SEM) was used for investigation of the surface morphology and shape of the synthesized nanoparticle and it was also used for the determination of the nanoparticle size. For
Fig. 1. Schematic presentation for the incorporation of IMEO onto poly(HEMA) nanoparticles.
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this, prepared nanoparticles were covered with a thin layer of gold and SEM photographs of the poly(HEMA) nanoparticles were taken by using SEM devices (Philips, XL-30S FEG, the Netherlands). The average size and size distribution of the synthesized nanoparticles were determined by using a Zeta Sizer (Malvern Instruments, 3000 HAS, England). Incorporation of IMEO onto the poly(HEMA) nanoparticles was investigated by using a FTIR spectrophotometer (Shimadzu, FTIR 8000, Japan). For this, 0.1 g of dried nanoparticle was mixed with 0.1 g of IR grade KBr and then pressed into a pellet form and FTIR spectrum was recorded.
2.5. RNA adsorption studies RNA adsorption onto poly(HEMA)-IMEO-PBA nanoparticle was studied in batch wise experimental set-up. In this part, 100 μL of poly(HEMA)-IMEO-PBA nanoparticles and 350 μL aqueous solution of RNA were mixed with 0.5 mL of buffer solution and stirred with the rate of 100 rpm at room temperature for 1 h. At the end of this adsorption period, nanoparticles were centrifuged (14,000 rpm for 20 min) and aqueous phase was stored for the determination of the final RNA concentration. Initial and final RNA concentrations were determined
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by using a UV–vis spectrophotometer (Shimadzu, UV-1601, Japan) at 260 nm. The adsorbed amount of RNA was calculated by using Eq. (1). In order to enhance the specificity of the boronate affinity nanoparticle towards to RNA, some medium conditions were changed. The most important parameter which affects the specificity is the medium pH. For this, the effect of pH on the RNA adsorption onto poly(HEMA)-IMEOPBA nanoparticle was investigated by using different buffer systems and for this purpose pH of the adsorption medium was changed between 6.0 and 9.5. The effect of initial RNA concentration on the adsorption efficiency was also studied with the concentration range of 0.02– 0.5 mg/mL. The medium temperature of the adsorption mixture was changed between 4 °C and 50 °C, in order to investigate the temperature effect on the RNA adsorption. 2.6. Desorption and reusability studies Adsorbed RNA was desorbed from the nanoparticles by using 25 mM of sodium borate buffer (pH 9.0, in 0.5 M NaCl) at room temperature. For this, nanoparticles were transferred in a desorption medium and stirred for 1 h. At the end of the desorption period, nanoparticles were precipitated and desorbed amount of RNA was determined spectrophotometrically. Reusability profile of the poly(HEMA)-IMEO-PBA
Fig. 2. Schematic illustration of the adsorption process of RNA molecules onto poly(HEMA)-IMEO-PBA nanoparticles.
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nanoparticle was also studied for 5 successive adsorption/desorption cycles. 3. Results and discussion 3.1. Characterization of poly(HEMA)-IMEO-PBA nanoparticles Poly(HEMA) nanoparticles were synthesized by the surfactant free emulsion polymerization technique and then functionalized with
IMEO and PBA. These borate group bearing nanoparticles were used for the affinity adsorption of RNA from its aqueous solutions. RNA adsorption process is schematically illustrated in Fig. 2. The structure and the surface morphology of the poly(HEMA)IMEO-PBA nanoparticles are demonstrated in Fig. 3A. As seen here, the synthesized poly(HEMA)-IMEO-PBA nanoparticles were spherical in shape and their size was found to be around 100 nm. The average particle size and size distribution of the poly(HEMA)IMEO-PBA nanoparticles are demonstrated in Fig. 3B. As shown in
Fig. 3. A) SEM images of poly(HEMA) nanoparticles. B) Zeta-size and size distribution of poly(HEMA) nanoparticles. C) FTIR spectrum of p(HEMA) nanoparticles and poly(HEMA)-IMEOPBA nanoparticles.
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Fig. 3B., the average particle size of the nanoparticle was calculated about 111 nm with 1.15 polydispersity index. The FTIR spectra of poly(HEMA), IMEO and poly(HEMA)-IMEO are shown in Fig. 3C. As seen here, incorporation of IMEO onto the poly(HEM) structure was realized by some characteristic peaks. Briefly, the vibration band around 1243 cm−1 belonged to the Si–O–C group, and this was the most realistic evidence for the incorporation of the IMEO onto poly(HEMA) structure. Also, IMEO's C–N vibration band around 1655 cm−1 also occurred in the FTIR spectrum of poly(HEMA)-IMEO structure. Within all these results it can be concluded that, IMEO was successfully incorporated to the polymeric structure of the poly(HEMA). 3.2. PBA binding onto poly(HEMA)-IMEO nanoparticles In order to reach the maximum PBA binding onto poly(HEMA)IMEO nanoparticles, the effects of medium pH, temperature and initial PBA concentration were investigated. The effect of pH on the PBA binding is demonstrated in Fig. 4A. As shown here, maximum PBA binding was observed at pH 9.0 by using 25.0 mM of borate buffer and maximum PBA binding was found to be 606.34 mg PBA/g polymer. The isoelectric point of PBA is 8.9 and PBA was in maximum ionized form and tetragonal structure and these properties could increase the adsorption of PBA [17]. Fig. 4B. shows the effect of temperature on the PBA binding. As seen here, the bonded amount of PBA was slightly decreased from 620.41 mg/g polymer to 578.29 mg/g polymer with a temperature increase from 4 to 50 °C. The effect of initial PBA concentration onto PBA binding is demonstrated in Fig. 4C and it is easily concluded from this figure that, PBA binding capacity of the poly (HEMA)-IMEO nanoparticle increased with increasing initial PBA concentration. With these results from the PBA binding studies, maximum PBA binding onto the nanoparticles was found to be pH 9.0 borate buffer, 25 °C medium temperature and 0.25 mg/mL PBA concentration. 3.3. RNA adsorption studies RNA adsorption studies were carried out with different pHs, temperatures and various initial RNA concentrations, in order to determine the maximum RNA adsorption conditions onto poly(HEMA)-IMEO-PBA nanoparticles. The effect of medium pH on RNA adsorption is shown in Fig. 5A and as seen here, maximum RNA adsorption was observed at pH 8.5 carbonate buffer (25 mM). Above and below of this pH value, the adsorbed amount of RNA was significantly decreased. Because of the isoelectric point of the PBA, all the affinity matrix was negatively charged at higher pH values that 8.9 and therefore, electrostatic repulsive forces took place between poly(HEMA)-IMEO-PBA nanoparticles and RNA which had a negative character. RNA adsorption studies were carried out with different RNA concentrations, and findings are demonstrated in Fig. 5B. As seen in the figure, the RNA adsorption capacity of the poly(HEMA)-IMEO-PBA nanoparticles increased with increasing RNA concentration. By using these adsorption data, RNA adsorption isotherms were calculated. The most used model isotherms are Langmuir and Freundlich isotherms and are represented as following equations, Eqs. (2) and (3), respectively [21].
1 ¼ qe
1 Q max
lnqe ¼
þ
1
Q max b
1 ð ln C e Þ þ lnK f : n
1 Ce
ð2Þ
ð3Þ
Here, Langmuir isotherm constant is represented as b, and KF and n are the Freundlich isotherm constant and Freundlich exponent, respectively. The value of 1/n is an indicator for surface heterogeneity of the
Fig. 4. Effects of pH (A), medium temperature (B) and initial PBA concentration (C) on the PBA bonding onto poly(HEMA)-IMEO nanoparticles (incubation time: 60 min, pH: 9.0; initial PBA concentration: 0.25 mg/mL, temperature: 25 °C).
adsorption matrix and ranging between 0 and 1. At heterogeneous systems, this value is close to the zero. qe is the theoretical monolayer saturation capacity of adsorption matrix. Table 1 shows the constants and parameters of the Langmuir and Freundlich isotherms. As seen here, Freundlich isotherm fits the experimental data best by comparing the R2 values of isotherms. It can be concluded with this result that, the RNA adsorption system onto the nanoparticles was heterogeneous and the adsorption process was reversible [22,23]. The effect of medium temperature on the adsorption of RNA onto the poly(HEMA)-IMEO-PBA nanoparticles is demonstrated in Fig. 5C. As clearly seen in this figure, the adsorbed amount of RNA significantly decreased with the increasing temperature. This decrease can be attributed to the disruptive thermodynamic influences which can weaken the interactions between the poly(HEMA)-IMEO-PBA nanoparticles and RNA molecules. Another reason of this decrease is the three dimensional
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affinity nanoparticle was found to be 147.04 mg/g by using 0.5 mg/mL initial RNA concentration in pH 8.5 carbonate buffer (25 mM) at 25 °C. 3.4. Desorption and reusability studies In order to regenerate the poly(HEMA)-IMEO-PBA nanoparticles, adsorbed RNA was desorbed by using 25 mM of sodium borate buffer (pH 9.0, in 0.5 M NaCl). Because the nanoparticles were negatively charged at pH 9.0, electrostatic repulsive interactions took place between the nanoparticles and RNA molecules. Desorption agent was prepared in a NaCl solution and it was also used for breaking the electrostatic interactions. Desorption rate was found to be 95% by using this desorption agent. The reusability profile of the poly(HEMA)-IMEO-PBA nanoparticles was investigated for 5 successive reuses and the RNA adsorption capacity of the poly(HEMA)-IMEO-PBA nanoparticles decreased only about 5.0% at the end of the 5 reuses. 4. Conclusion Polymers in nanometer size have been intensively used as a support material for adsorption of biomolecules because of their unique physical and chemical properties and high surface areas. These polymers can be easily functionalized with various ligands and agents with minor modifications. In this presented paper, poly(HEMA) nanoparticles were synthesized and derivatized with IMEO and functionalized with PBA. Prepared nanoparticles were characterized by FTIR, SEM and zeta-size analyses. These functional nanoparticles were used for the adsorption of RNA as a boronic acid affinity system. Incorporation of IMEO ad PBA onto the polymer structure was optimized and optimum RNA adsorption conditions were studied with various pHs, temperatures and initial RNA concentrations. Maximum RNA adsorption onto the poly(HEMA)IMEO-PBA nanoparticles was found to be 56.07 mg/g nanoparticle. These boronic acid functionalized nanoparticles were also used for 5 successive reuse and it was found that, there was no significant decrease in their RNA adsorption capacity. Finally it can be concluded that, these new boronic acid affinity nanoparticles can be used for adsorption or purification of RNA for preparative studies. References
Fig. 5. Effects of medium pH (A), initial RNA concentration (B) and medium temperature (C) on the RNA adsorption onto poly(HEMA)-IMEO-PBA nanoparticles (incubation time: 90 min, temperature: 25 °C, pH: 8.5, initial RNA concentration: 0.5 mg/mL).
changes which are taken place at high temperature. Additionally it is well known that, when dealing with the ionic interactions the adsorption capacity of the adsorption matrix generally decreased because of the exothermic nature of the adsorption process [1,24,25]. At the end of the optimization studies for the RNA adsorption, the maximum amount of adsorbed RNA onto the synthesized boronate
Table 1 Kinetic constants of Langmuir and Freundlich isotherms. Langmuir adsorption isotherm
Freundlich adsorption isotherm
qmax = 1501 mg/g b = 0.343 mL/mg R2 = 0.53
KF = 1995 mg/g 1/n = 0.573 R2 = 0.968
[1] B. Akduman, M. Uygun, D.A. Uygun, M. Antalik, J. Nanoparticle Res. 15 (2013) 1564. [2] N. Kaya, D.A. Uygun, S. Akgöl, A. Denizli, Appl. Biochem. Biotechnol. 169 (2013) 2153. [3] D.A. Uygun, M.E. Çorman, N. Öztürk, S. Akgöl, A. Denizli, Mater. Sci. Eng. C 30 (2010) 1285. [4] M. Mandal, S. Kundu, S.K. Ghosh, S. Panigrahi, T.K. Sau, S.M. Yusuf, T. Pal, J. Colloid Interface Sci. 286 (2005) 187. [5] J. Bao, W. Chen, T. Liu, Y. Zhu, P. Jin, L. Wang, J. Liu, Y. Wei, Y. Li, ACS Nano 1 (2007) 293. [6] C. Okoli, M. Boutonnet, L. Mariey, S. Jaras, G. Rajarao, J. Chem. Technol. Biotechnol. 86 (2011) 1386. [7] L.E. Easton, Y. Shibata, P.J. Lukavsky, RNA 16 (2010) 647. [8] R.T. Batey, J.S. Kieft, RNA 13 (2007) 1384. [9] P.A. Sharp, Cell 136 (2009) 577. [10] J.F. Milligan, D.R. Groebe, G.W. Witherell, O.C. Uhlenbeck, Nucleic Acids Res. 15 (1987) 8783. [11] S.R. Price, N. Ito, C. Oubridge, J.M. Avis, K. Nagai, J. Mol. Biol. 249 (1995) 398. [12] J.A. Doudna, Methods Mol. Biol. 74 (1997) 365. [13] O.C. Uhlenbeck, RNA 1 (1995) 4. [14] P.J. Lukavsky, J.D. Puglisi, RNA 10 (2004) 889. [15] S. Senel, S.T. Çamli, M. Tuncel, A. Tuncel, J. Chromatogr. B 769 (2002) 283. [16] R. Tuytten, F. Lemiere, E.L. Esmans, W.A. Herrebout, B.J. van der Veken, B.U.W. Maes, E. Witters, R.P. Newton, E. Dudley, Anal. Chem. 79 (2007) 6662. [17] C. Türkcan, S. Akgöl, A. Denizli, Mater. Sci. Eng. C 33 (2013) 4498. [18] N. Öztürk, N. Bereli, S. Akgöl, A. Denizli, Colloids Surf. B: Biointerfaces 67 (2008) 14. [19] G.J. Kluth, M.M. Sung, R. Maboudian, Langmuir 13 (1997) 3775. [20] N. Ozturk, M.E. Gunay, S. Akgol, A. Denizli, Biotechnol. Prog. 23 (2007) 1149. [21] M. Uygun, D.A. Uygun, E. Özçalışkan, S. Akgöl, A. Denizli, J. Chromatogr. B 887–888 (2012) 73. [22] F. Plieva, P. Ekström, I.Y. Galaev, B. Mattiasson, Soft Matter 4 (2008) 2418. [23] N.E. Labrou, A. Karagouni, Y.D. Clonis, Biotechnol. Bioeng. 48 (1995) 278. [24] E.B. Altıntaş, A. Denizli, J. Chromatogr. B 832 (2006) 216. [25] S. Akgöl, N. Bereli, A. Denizli, Macromol. Biosci. 5 (2005) 786.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Boronate affinity nanoparticles for RNA isolation Aykut Toprak a, Cansu Görgün a, Cansu İlke Kuru a, Ceren Türkcan a, Murat Uygun b, Sinan Akgöl a,⁎ a b
Ege University, Faculty of Science, Department of Biochemistry, 35100 Bornova, Izmir, Turkey Adnan Menderes University, Koçarlı Vocational and Training School, 09010 Aydın, Turkey
a r t i c l e
i n f o
Article history: Received 10 April 2014 Received in revised form 3 October 2014 Accepted 10 November 2014 Available online 12 November 2014 Keywords: RNA Boronate affinity Nanoparticle
a b s t r a c t In this presented paper, boronic acid incorporated poly(HEMA) based nanoparticles were synthesized for RNA adsorption. For this purpose, poly(HEMA) nanoparticles were synthesized by using the surfactant free emulsion polymerization technique. Then, nanoparticles were modified with 3-(2-imidazoline-1-yl)propyl(triethoxysilane) (IMEO) and functionalized with phenylboronic acid (PBA). Prepared nanoparticles were characterized with SEM, FTIR and zeta-size. Optimum RNA adsorption conditions were investigated with different pHs, temperatures and initial RNA concentrations in order to determine the maximum RNA adsorption onto poly(HEMA)-IMEO-PBA nanoparticles. It was also studied that, synthesized nanoparticles could be used for 5 successive reuses and adsorption capacity of the nanoparticles decreased only about 5% at the end of the 5 cycles. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Recently, nanoparticle applications have received great attention in various biotechnological applications due to their unusual chemical and physical properties. Nanoparticles with various shapes and functionalities have been synthesized and used in different kinds of biotechnological applications such as purification of biomolecules, separation or sorting of cells, immobilization of proteins and enzymes, medical diagnosis, sensor applications, immunoassays, magnetic resonance imaging and controlled drug delivery studies [1–5]. Nanoparticles serve high surface areas and consequently they can bind high amount of biomolecules. Because of their unique features, nanoparticles can easily be used for the separation or purification of the biomolecules from complex mediums. Also, nanoparticles can eliminate the time consumptive purification steps in biomolecule separation studies [6]. RNA plays a critical role in the transformation of the genetic information and new roles of RNA have also been discovered over the past ten years. In order to understand the functions and the properties of the RNA, its biochemical and structural characterizations have great importance. When viewed from this aspect, pure and non-denatured RNA is very essential. On the other hand, recently used RNA purification techniques induce loss of time and the final RNA preparation is in denature form. Different RNA purification strategies have been developed, in order to overcome these drawbacks. Specially, affinity chromatographic techniques have been applied for the purification of the RNA from different mediums [7–14]. One of the efficiently used affinity techniques is the boronate affinity chromatography and it has been used for the isolation of biomolecules such as nucleotides, RNA, glycated proteins and ⁎ Corresponding author at: Ege University Biochemistry Department, Izmir, Turkey. E-mail address: [email protected] (S. Akgöl).
http://dx.doi.org/10.1016/j.msec.2014.11.033 0928-4931/© 2014 Elsevier B.V. All rights reserved.
glycol-enzymes [15]. In this affinity technique, diol moieties which are in a cis configuration form a complex with boronic acid group. With this interaction, a negatively charged cyclic boronate ester takes place [16]. In this work, p(HEMA) nanoparticles carrying boronic acid group were synthesized for purification or recognition of RNA. For this purpose, poly(HEMA) nanoparticles were synthesized by the surfactant free emulsion polymerization technique and then characterized. Nanoparticles were then modified with IMEO. Boronic acid group was introduced into the nanoparticle structure by the interaction of the IMEO modified nanoparticles with phenyl boronic acid (PBA). Conditions of RNA adsorption onto poly(HEMA)-IMEO-PBA nanoparticles were optimized by using different pHs, temperatures and initial RNA concentrations. Finally reusability of the poly(HEMA)-IMEO-PBA nanoparticles was investigated for 5 sequential cycles. 2. Experimental 2.1. Materials RNA (from Saccharomyces cerevisiae), hydroxyethyl methacrylate (HEMA), ethylene glycol dimethacrylate (EGDMA), potassium persulfate and phenylboronic acid (PBA) were supplied from Sigma (St. Louis, USA). All other chemicals were of analytical grade and used without further purification. 2.2. Synthesis of poly(HEMA) nanoparticles Poly(HEMA) nanoparticles were synthesized by using the surfactant free emulsion polymerization technique [17,18]. Briefly, 0.5 g of poly(vinyl alcohol) (PVA) was dissolved in 100 mL of deionized water and used
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as a stabilizer. Then, 0.6 mL of HEMA and 0.3 mL of EGDMA were added to the initial dispersion mixture as monomers and were mixed by using an ultrasonic water bath for 30 min. Potassium persulfate (KPS) was used as an initiator with the concentration of 20 mg/mL. In order to eliminate the dissolved oxygen from the polymerization mixture, nitrogen gas was blown through the medium. Polymerization reaction was taken place in a shaking water bath at 70 °C with the shaking rate of 150 rpm for 7 h by using a polymerization reactor. At the end of the polymerization process, the synthesized polymers were washed with ethanol and distilled water for several times in order to remove the unreacted monomers and initiators. 2.3. Modification of poly(HEMA) nanoparticles Prepared poly(HEMA) nanoparticles were firstly functionalized with the silane group bearing molecule 3-(2-imidazoline-1yl)propyl(triethoxysilane) (IMEO). The silane groups easily react with the hydroxyl group bearing surface [i.e. poly(HEMA)] [19]. For this purpose, poly(HEMA) particles were mixed with IMEO with the molar ratio of 2:1 at 25 °C for 24 h. Silanized nanoparticles were collected by the help of centrifuge and washed with distilled water [20]. Incorporation of the IMEO onto poly(HEMA) structure is schematically summarized in Fig. 1. In this presented work, phenyl boronic acid (PBA) was used as a boronate affinity ligand. With the attachment of IMEO, the surface of the nanoparticle was functionalized by imidazoline groups. Nitrogen atoms of the imidazoline group can easily interact with the boron of the PBA via a simple coordination bond. By the incorporation of the
PBA to the nanoparticle structure, boronate affinity nanoparticles were prepared. For this, 200 μL of IMEO activated nanoparticle solution was mixed with 0.25 mg/mL of phenylboronic acid solution (in MES buffer, 25 mM, pH 6.0). Then this solution was stirred for 2 h and centrifuged at 10,000 rpm for 20 min. Supernatant solution was stored for the calculation of the binding amount of PBA. Incorporation of PBA onto poly(HEMA)-IMEO structure was also studied with different conditions such as medium pH, temperature and initial PBA concentration. For this, pH of the binding solution was changed between 5.0 and 10.0. The effect of temperature on the PBA binding was studied with the temperature range of 4–55 °C. Initial PBA concentration was changed between 0.6 and 8.0 mg/mL in order to show the effect of PBA concentration on the PBA binding. Bonded PBA was calculated by using the following formula: Q¼
C i −C f V m 10−3
:
ð1Þ
Here, Q is the bonded amount of PBA (mg/g); Ci and Cf are the initial and final PBA concentrations, respectively (mg/mL); V is the volume of the binding solution (mL); and m is the mass of the poly(HEMA)-IMEO nanoparticle (g). 2.4. Characterization of nanoparticles Scanning electron microscope (SEM) was used for investigation of the surface morphology and shape of the synthesized nanoparticle and it was also used for the determination of the nanoparticle size. For
Fig. 1. Schematic presentation for the incorporation of IMEO onto poly(HEMA) nanoparticles.
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this, prepared nanoparticles were covered with a thin layer of gold and SEM photographs of the poly(HEMA) nanoparticles were taken by using SEM devices (Philips, XL-30S FEG, the Netherlands). The average size and size distribution of the synthesized nanoparticles were determined by using a Zeta Sizer (Malvern Instruments, 3000 HAS, England). Incorporation of IMEO onto the poly(HEMA) nanoparticles was investigated by using a FTIR spectrophotometer (Shimadzu, FTIR 8000, Japan). For this, 0.1 g of dried nanoparticle was mixed with 0.1 g of IR grade KBr and then pressed into a pellet form and FTIR spectrum was recorded.
2.5. RNA adsorption studies RNA adsorption onto poly(HEMA)-IMEO-PBA nanoparticle was studied in batch wise experimental set-up. In this part, 100 μL of poly(HEMA)-IMEO-PBA nanoparticles and 350 μL aqueous solution of RNA were mixed with 0.5 mL of buffer solution and stirred with the rate of 100 rpm at room temperature for 1 h. At the end of this adsorption period, nanoparticles were centrifuged (14,000 rpm for 20 min) and aqueous phase was stored for the determination of the final RNA concentration. Initial and final RNA concentrations were determined
253
by using a UV–vis spectrophotometer (Shimadzu, UV-1601, Japan) at 260 nm. The adsorbed amount of RNA was calculated by using Eq. (1). In order to enhance the specificity of the boronate affinity nanoparticle towards to RNA, some medium conditions were changed. The most important parameter which affects the specificity is the medium pH. For this, the effect of pH on the RNA adsorption onto poly(HEMA)-IMEOPBA nanoparticle was investigated by using different buffer systems and for this purpose pH of the adsorption medium was changed between 6.0 and 9.5. The effect of initial RNA concentration on the adsorption efficiency was also studied with the concentration range of 0.02– 0.5 mg/mL. The medium temperature of the adsorption mixture was changed between 4 °C and 50 °C, in order to investigate the temperature effect on the RNA adsorption. 2.6. Desorption and reusability studies Adsorbed RNA was desorbed from the nanoparticles by using 25 mM of sodium borate buffer (pH 9.0, in 0.5 M NaCl) at room temperature. For this, nanoparticles were transferred in a desorption medium and stirred for 1 h. At the end of the desorption period, nanoparticles were precipitated and desorbed amount of RNA was determined spectrophotometrically. Reusability profile of the poly(HEMA)-IMEO-PBA
Fig. 2. Schematic illustration of the adsorption process of RNA molecules onto poly(HEMA)-IMEO-PBA nanoparticles.
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nanoparticle was also studied for 5 successive adsorption/desorption cycles. 3. Results and discussion 3.1. Characterization of poly(HEMA)-IMEO-PBA nanoparticles Poly(HEMA) nanoparticles were synthesized by the surfactant free emulsion polymerization technique and then functionalized with
IMEO and PBA. These borate group bearing nanoparticles were used for the affinity adsorption of RNA from its aqueous solutions. RNA adsorption process is schematically illustrated in Fig. 2. The structure and the surface morphology of the poly(HEMA)IMEO-PBA nanoparticles are demonstrated in Fig. 3A. As seen here, the synthesized poly(HEMA)-IMEO-PBA nanoparticles were spherical in shape and their size was found to be around 100 nm. The average particle size and size distribution of the poly(HEMA)IMEO-PBA nanoparticles are demonstrated in Fig. 3B. As shown in
Fig. 3. A) SEM images of poly(HEMA) nanoparticles. B) Zeta-size and size distribution of poly(HEMA) nanoparticles. C) FTIR spectrum of p(HEMA) nanoparticles and poly(HEMA)-IMEOPBA nanoparticles.
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Fig. 3B., the average particle size of the nanoparticle was calculated about 111 nm with 1.15 polydispersity index. The FTIR spectra of poly(HEMA), IMEO and poly(HEMA)-IMEO are shown in Fig. 3C. As seen here, incorporation of IMEO onto the poly(HEM) structure was realized by some characteristic peaks. Briefly, the vibration band around 1243 cm−1 belonged to the Si–O–C group, and this was the most realistic evidence for the incorporation of the IMEO onto poly(HEMA) structure. Also, IMEO's C–N vibration band around 1655 cm−1 also occurred in the FTIR spectrum of poly(HEMA)-IMEO structure. Within all these results it can be concluded that, IMEO was successfully incorporated to the polymeric structure of the poly(HEMA). 3.2. PBA binding onto poly(HEMA)-IMEO nanoparticles In order to reach the maximum PBA binding onto poly(HEMA)IMEO nanoparticles, the effects of medium pH, temperature and initial PBA concentration were investigated. The effect of pH on the PBA binding is demonstrated in Fig. 4A. As shown here, maximum PBA binding was observed at pH 9.0 by using 25.0 mM of borate buffer and maximum PBA binding was found to be 606.34 mg PBA/g polymer. The isoelectric point of PBA is 8.9 and PBA was in maximum ionized form and tetragonal structure and these properties could increase the adsorption of PBA [17]. Fig. 4B. shows the effect of temperature on the PBA binding. As seen here, the bonded amount of PBA was slightly decreased from 620.41 mg/g polymer to 578.29 mg/g polymer with a temperature increase from 4 to 50 °C. The effect of initial PBA concentration onto PBA binding is demonstrated in Fig. 4C and it is easily concluded from this figure that, PBA binding capacity of the poly (HEMA)-IMEO nanoparticle increased with increasing initial PBA concentration. With these results from the PBA binding studies, maximum PBA binding onto the nanoparticles was found to be pH 9.0 borate buffer, 25 °C medium temperature and 0.25 mg/mL PBA concentration. 3.3. RNA adsorption studies RNA adsorption studies were carried out with different pHs, temperatures and various initial RNA concentrations, in order to determine the maximum RNA adsorption conditions onto poly(HEMA)-IMEO-PBA nanoparticles. The effect of medium pH on RNA adsorption is shown in Fig. 5A and as seen here, maximum RNA adsorption was observed at pH 8.5 carbonate buffer (25 mM). Above and below of this pH value, the adsorbed amount of RNA was significantly decreased. Because of the isoelectric point of the PBA, all the affinity matrix was negatively charged at higher pH values that 8.9 and therefore, electrostatic repulsive forces took place between poly(HEMA)-IMEO-PBA nanoparticles and RNA which had a negative character. RNA adsorption studies were carried out with different RNA concentrations, and findings are demonstrated in Fig. 5B. As seen in the figure, the RNA adsorption capacity of the poly(HEMA)-IMEO-PBA nanoparticles increased with increasing RNA concentration. By using these adsorption data, RNA adsorption isotherms were calculated. The most used model isotherms are Langmuir and Freundlich isotherms and are represented as following equations, Eqs. (2) and (3), respectively [21].
1 ¼ qe
1 Q max
lnqe ¼
þ
1
Q max b
1 ð ln C e Þ þ lnK f : n
1 Ce
ð2Þ
ð3Þ
Here, Langmuir isotherm constant is represented as b, and KF and n are the Freundlich isotherm constant and Freundlich exponent, respectively. The value of 1/n is an indicator for surface heterogeneity of the
Fig. 4. Effects of pH (A), medium temperature (B) and initial PBA concentration (C) on the PBA bonding onto poly(HEMA)-IMEO nanoparticles (incubation time: 60 min, pH: 9.0; initial PBA concentration: 0.25 mg/mL, temperature: 25 °C).
adsorption matrix and ranging between 0 and 1. At heterogeneous systems, this value is close to the zero. qe is the theoretical monolayer saturation capacity of adsorption matrix. Table 1 shows the constants and parameters of the Langmuir and Freundlich isotherms. As seen here, Freundlich isotherm fits the experimental data best by comparing the R2 values of isotherms. It can be concluded with this result that, the RNA adsorption system onto the nanoparticles was heterogeneous and the adsorption process was reversible [22,23]. The effect of medium temperature on the adsorption of RNA onto the poly(HEMA)-IMEO-PBA nanoparticles is demonstrated in Fig. 5C. As clearly seen in this figure, the adsorbed amount of RNA significantly decreased with the increasing temperature. This decrease can be attributed to the disruptive thermodynamic influences which can weaken the interactions between the poly(HEMA)-IMEO-PBA nanoparticles and RNA molecules. Another reason of this decrease is the three dimensional
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affinity nanoparticle was found to be 147.04 mg/g by using 0.5 mg/mL initial RNA concentration in pH 8.5 carbonate buffer (25 mM) at 25 °C. 3.4. Desorption and reusability studies In order to regenerate the poly(HEMA)-IMEO-PBA nanoparticles, adsorbed RNA was desorbed by using 25 mM of sodium borate buffer (pH 9.0, in 0.5 M NaCl). Because the nanoparticles were negatively charged at pH 9.0, electrostatic repulsive interactions took place between the nanoparticles and RNA molecules. Desorption agent was prepared in a NaCl solution and it was also used for breaking the electrostatic interactions. Desorption rate was found to be 95% by using this desorption agent. The reusability profile of the poly(HEMA)-IMEO-PBA nanoparticles was investigated for 5 successive reuses and the RNA adsorption capacity of the poly(HEMA)-IMEO-PBA nanoparticles decreased only about 5.0% at the end of the 5 reuses. 4. Conclusion Polymers in nanometer size have been intensively used as a support material for adsorption of biomolecules because of their unique physical and chemical properties and high surface areas. These polymers can be easily functionalized with various ligands and agents with minor modifications. In this presented paper, poly(HEMA) nanoparticles were synthesized and derivatized with IMEO and functionalized with PBA. Prepared nanoparticles were characterized by FTIR, SEM and zeta-size analyses. These functional nanoparticles were used for the adsorption of RNA as a boronic acid affinity system. Incorporation of IMEO ad PBA onto the polymer structure was optimized and optimum RNA adsorption conditions were studied with various pHs, temperatures and initial RNA concentrations. Maximum RNA adsorption onto the poly(HEMA)IMEO-PBA nanoparticles was found to be 56.07 mg/g nanoparticle. These boronic acid functionalized nanoparticles were also used for 5 successive reuse and it was found that, there was no significant decrease in their RNA adsorption capacity. Finally it can be concluded that, these new boronic acid affinity nanoparticles can be used for adsorption or purification of RNA for preparative studies. References
Fig. 5. Effects of medium pH (A), initial RNA concentration (B) and medium temperature (C) on the RNA adsorption onto poly(HEMA)-IMEO-PBA nanoparticles (incubation time: 90 min, temperature: 25 °C, pH: 8.5, initial RNA concentration: 0.5 mg/mL).
changes which are taken place at high temperature. Additionally it is well known that, when dealing with the ionic interactions the adsorption capacity of the adsorption matrix generally decreased because of the exothermic nature of the adsorption process [1,24,25]. At the end of the optimization studies for the RNA adsorption, the maximum amount of adsorbed RNA onto the synthesized boronate
Table 1 Kinetic constants of Langmuir and Freundlich isotherms. Langmuir adsorption isotherm
Freundlich adsorption isotherm
qmax = 1501 mg/g b = 0.343 mL/mg R2 = 0.53
KF = 1995 mg/g 1/n = 0.573 R2 = 0.968
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