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Au nanoparticles
Materials Science and Engineering C 30 (2010) 311–315
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Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Capture and release of genomic DNA by PEI modified Fe3O4/Au nanoparticles Hanwen Sun a,⁎, Xinjun Zhu b, Lianying Zhang a,b, Yong Zhang c, Dunqing Wang a a b c
Dezhou Institute of Advanced Materials, Dezhou University, Dezhou 253023, PR China Department of Life Science, Dezhou University, Dezhou 253023, PR China Department of physics, Dezhou University, Dezhou 253023, PR China
a r t i c l e
i n f o
Article history: Received 20 August 2009 Received in revised form 19 October 2009 Accepted 12 November 2009 Available online 26 November 2009 Keywords: Fe3O4/Au nanoparticles Polyethylenimine DNA capture and release Extraction
a b s t r a c t Polyethylenimine (PEI) modified Fe3O4/Au nanoparticles were synthesized in aqueous solution and characterized by photo correlation spectroscopy (PCS) and vibrating sample magnetometer (VSM). The so-obtained Fe3O4/Au-PEI nanoparticles were capable of efficient electrostatic capture of DNA. The maximum amount of genomic DNA captured on 1.0 mg Fe3O4/Au-PEI nanoparticles was 90 μg. The DNA release behavior was studied and the DNA recovery from Fe3O4/Au-PEI nanoparticles approached 100% under optimal conditions. DNA extraction from mammalian cells using Fe3O4/Au-PEI nanoparticles was successfully performed. Up to approximately 43.1 μg of high-purity (OD260/OD280 ratio = 1.81) genomic DNA was extracted from 10 mg of liver tissue. The results indicated that the prepared Fe3O4/Au-PEI nanoparticles could be successfully used for DNA capture and release. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.
1. Introduction In recent years, the magnetic particles have emerged as a promising new platform in biomedical applications, such as protein and enzyme immobilization [1,2], magnetic resonance imaging (MRI) [3], hybridization and detection of DNA [4,5] etc. The magnetic particles were composite particles composed of iron oxide nanoparticles and various natural or synthetic polymers such as polystyrene, poly (ethylene glycol), poly (vinyl alcohol), chitosan and so on [6]. In order to add new properties (inertness, protection of the magnetic core against oxidation), an external inert shell such as Au has been widely attempted [7–9]. The unique Au surface is safe in both animals and humans [10], and exhibit an absorption band in the visible region due to its surface plasmon phenomenon, allowing the colorimetry of biomolecules with high-selectivity. Moreover, the Au coating renders sites for strong binding with various proteins (e.g. enzyme, biotin and so on) through their mercapto groups [11,12]. Thus, magnetic nanoparticles covered with an Au shell would provide all the characteristics of the Au element suitable for biological application and deliver magnetic properties to be utilized for further manipulation with an external magnetic field. However, the DNA/oligonucleotides cannot be attached on magnetic/Au nanoparticles directly. The conventional methods for this purpose can be divided into two classes: first, the DNA is conjugated with biotin and then the biotinylated DNA is captured by avidin-activated magnetic/Au nanoparticles [13]; and secondly, the
⁎ Corresponding author. Tel./fax: +86 534 8989506. E-mail address: [email protected] (H. Sun).
DNA is thiol modified, and then captured by magnetic/Au nanoparticles [14]. These protocols are time consuming and labor intensive. Polyethylenimine (PEI) is a water soluble cationic polymer which contains amino and imino groups in each polymer chain. When the polymer PEI is dispersed in aqueous solution, each polymer chain is positively charged by amino and imino groups[15]. These amino and imino groups are expected to adsorb onto the surface of magnetic/Au nanoparticles. Recently, S. Seino et al. [16] had synthesized PEI modified Fe2O3/Au nanoparticles and found that the PEI modification could greatly improve the dispersibility of Fe2O3/Au nanoparticles. However, the characteristics of DNA binding onto PEI modified magnetic/Au nanoparticles have still not been sufficiently investigated. In this paper, PEI modified Fe3O4/Au nanoparticles were synthesized and characterized by PCS and VSM, respectively. The DNA capture and release behavior using these magnetic nanoparticles was studied and a simple and convenient method to extract genomic DNA from mammalian cells was also established. 2. Experimental 2.1. Materials and methods Polyethylenimine (PEI, 30% in water), analytical grade, was purchased from Tokyo Chemical Industry Co. Ltd, Japan. Ferrous chloride tetrahydrate (FeCl2∙ 4H2O), ferric chloride hexahydrate (FeCl3∙6H2O), AuCl3·HCl·4H2O (Au ≥ 47.8%), analytical grade, were purchased from Shanghai Chemical Reagents Company (China) and used without further treatment. Nitrogen (99.99%) was available from Dezhou LongLi Company, China. Genomic DNA (Fish Sperm) was
0928-4931/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2009.11.005
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supplied by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd, China. All the other chemicals were of analytical grade. Stirrer (IKA Company, Germany) was used to synthesize the Fe3O4/Au-PEI nanoparticles. The size and ζ-potential of Fe3O4/Au-PEI nanoparticles were detected by photo correlation spectroscopy (PCS, Zetasizer Nano ZS, Malvern Instruments Ltd.). Magnetic measurement was recorded on a vibrating sample magnetometer (VSM, Princeton Applied Research Model 155) at 25 °C. UV spectrophotometer (BioMate) was used to determine DNA concentration. 2.2. Synthesis of Fe3O4/Au-PEI nanoparticles The Fe3O4/Au nanoparticles were prepared according to the method described by Cui et al. [17]. The Fe3O4/Au-PEI nanoparticles were synthesized by mixing the aqueous dispersion of 40 mg Fe3O4/ Au nanoparticles with 2 mL PEI for 6 h at room temperature. The Fe3O4/Au-PEI nanoparticles were collected magnetically and washed three times with distilled water to remove the excess PEI, and at last the Fe3O4/Au-PEI nanoparticles were redispersed in distilled water. 2.3. DNA capture on Fe3O4/Au-PEI nanoparticles 1.0 mg Fe3O4/Au-PEI nanoparticles were mixed with 2 mL DNA (25, 50, 100, 200, 300 and 400 μg) in binding buffer (PBS, pH 5.0, 5.8, 6.6 and 7.4), and incubated for 10 min at room temperature. The Fe3O4/Au-PEI–DNA complexes were collected magnetically and washed three times with binding buffer, and the wash solution was flow-through collected separately. Captured DNA was quantified as the total DNA amount minus the amount of DNA in the initial supernatant fraction and the washed fractions. 2.4. DNA release from Fe3O4/Au-PEI nanoparticles 1.0 mg Fe3O4/Au-PEI nanoparticles with 80 μg DNA in the form of sediment were transferred to a clean 5 mL centrifugal tube with 3 mL elution buffer. The sealed tube was placed in a water bath maintaining temperature at 37 °C. At specified collection times, 100 µL of sample was taken from the tube and DNA concentration was measured. The samples in the 5 mL tube were replenished with 100 µL fresh elution buffer at 37 °C. Triplicate samples were analyzed. The total released DNA Mi at time i was calculated from Eq. (1) Mi = CiV + ∑Ci−1 Vs:
ð1Þ
Where Ci is the concentration of DNA in the elution buffer at time i, V is the total volume of elution buffer (3 mL) and Vs is the sample volume (0.1 mL).
agarose gel in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8.0). The Electrophoresis was run at 110 V for 40 min. 3. Results and discussion 3.1. Character of Fe3O4/Au-PEI nanoparticles The PEI modification was carried out in distilled water (pH ≈ 7.0), in which the Fe3O4/Au nanoparticles were natively charged with the ζ-potential was −24.7 mV. Thus, the PEI could adsorb onto the surface of Fe3O4/Au nanoparticles via electrostatic attraction. Fig. 1 shows the ζ-potential changes of Fe3O4/Au nanoparticles before and after PEI modification. The isoelectric point (IEP) of the Fe3O4/Au nanoparticles was located at pH 5.7. After PEI modification, the IEP shifted to a higher pH value of 7.6 and a highly positive surface charge in a wider range of pH (from 2.0 to 7.0) was observed, indicating that the Fe3O4/Au-PEI nanoparticles were well-dispersed in this pH range. The ζ-potential of the Fe3O4/Au-PEI nanoparticles was much larger than that without PEI modification, moreover, the pH range with highly positive ζ-potential turned wider, which makes the Fe3O4/AuPEI nanoparticles have the ability to bind DNA molecules. The particle size was measured by PCS. As can be seen from Fig. 2, the average diameter of Fe3O4/Au-PEI nanoparticles was 74 nm, which was slightly larger than that of the Fe3O4/Au nanoparticles (68 nm). This result revealed that the modification process did not significantly result in the agglomeration and the size change of the particles. The magnetic property of the Fe3O4/Au nanoparticles before and after PEI modification was studied by vibration sample magnetometer (VSM). As can be seen from Fig. 3, the Fe3O4/Au-PEI nanoparticles behaved superparamagnetically with saturated magnetization (Ms) of 39 emu/g, which was slightly lower than that of the naked Fe3O4/Au nanoparticles (48 emu/g). 3.2. DNA capture The DNA-binding capacity of Fe3O4/Au-PEI nanoparticles is examined using DNA at different concentrations. The amount of DNA captured on the nanoparticles increased linearly with increasing DNA concentrations (Fig. 4). The effect of pH of binding buffer on the DNA capture was shown in Fig. 4. The amount of DNA captured on the nanoparticles increased with decreasing pH. The maximum capture capacity was observed at pH 5.0. As can be seen from Fig. 1, with the pH increasing in the range of 5.0–7.4, the positive charge density of the Fe3 O 4 /Au-PEI
2.5. Extraction of genomic DNA from mammalian cells 100 μL of liver homogenate (10% homogenate of rat liver prepared in 0.32 M buffered sucrose, pH 7.5) and 100 μL of 1% (w/v) sodium dodecyl sulfate (SDS) solution were added in 1.5 mL Eppendorf tube by gentle inversion two or three times and incubated at room temperature for 1 min. After incubation, Fe3O4/Au-PEI nanoparticles dispersed in 1.0 mL binding buffer (PBS, pH 5.0) were added into the cell lysate. The suspension was mixed by inversion and allowed to stand at room temperature for 2 min. The Fe3O4/Au-PEI–DNA complexes were collected magnetically, and the supernatant was removed. Then the Fe3O4/Au-PEI–DNA complexes were washed three times with binding buffer. Finally, the magnetic nanoparticles were resuspended in 100 μL of TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.2), and the Fe3O4/Au-PEI–DNA complexes were eluted by incubation at 45 °C for 20 min. The extracted DNA was determined by UV spectrophotometry at 260 nm and gel electrophoresis using 0.8%
Fig. 1. The ζ-potential of Fe3O4/Au nanoparticle (a) before and (b) after PEI modification.
H. Sun et al. / Materials Science and Engineering C 30 (2010) 311–315
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Fig. 2. The particle size of (a) Fe3O4/Au and (b) Fe3O4/Au-PEI nanoparticles.
Tris–HCl buffer (pH 7.4 and 8.2) and PBS buffer (pH 5.0) were used as elution buffer in this study. As can be seen from Fig. 6, the captured DNA released largely in elution buffer pH 7.4 and 8.2, while only a small quantity of DNA (b8%) could desorb from the Fe3O4/Au-PEI nanoparticles at pH 5.0. The ζ-potential data for the Fe3O4/Au-PEI
nanoparticles showed a steady decrease in positive surface charge (or a steady increase in negative surface charge) with increasing pH value of elution buffer in the range of 5.0–8.2, which could result in a decrease of electrostatic attraction between Fe3O4/Au-PEI nanoparticles and DNA molecules. Therefore, the DNA could be released from nanoparticles easily. From Fig. 6, it can also be seen that the DNA releasing reached equilibrium within 20 min. The influence of salt concentration and temperature of elution buffer on DNA release were also studied. The release yield of DNA was increased with increasing salt concentration. In the elution buffer pH 8.2, the equilibrium of DNA release was achieved at salt concentration 1.5 M, and in the elution buffer pH 7.4, the equilibrium of DNA release was achieved at salt concentration 2.0 M (Fig. 7a). It may be attributed to the fact that the active site for DNA binding decreased with increasing salt concentration. The effect of temperature on DNA release was determined in Tris–HCl buffer with the salt concentration 2.0 M in the range of 20–60 °C. As it can be seen from Fig. 7b, the amount of DNA released increased with increasing temperature. The release yield was almost 100% for the pH 8.2 elution solution with a salt concentration 2.0 M at 50 °C. Data obtained suggests that nearly all DNA could released from the Fe3O4/Au-PEI nanoparticles.
Fig. 3. Magnetization curve of (a) Fe3O4/Au and (b) Fe3O4/Au-PEI nanoparticles.
Fig. 4. The amount of DNA captured on 1.0 mg of Fe3O4/Au-PEI nanoparticles.
nanoparticles decreased. This may lead to a reduction of active DNAbinding sites, thereby resulting in the decrease of DNA capture capacity. The Fe3O4/Au-PEI nanoparticles (1.0 mg) were capable of capturing 90 μg of DNA at pH 5.0. This result was much higher than previous reports [18,19]. This may because of higher amount of PEI adsorbed on Fe3O4/Au nanoparticles through static interaction in this report. The adsorption kinetics of DNA on Fe3O4/Au-PEI nanoparticles is studied using 1.0 mg Fe3O4/Au-PEI nanoparticles incubated with 100 μg DNA at pH 5.0. (Fig. 5). To confirm a complete equilibrium between DNA and Fe3O4/Au-PEI nanoparticles, these experiments were examined until the adsorption time was 20 min. All the adsorptions tended toward equilibrium within 1 min. 3.3. DNA release
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Fig. 5. The adsorption kinetics of DNA on Fe3O4/Au-PEI nanoparticles.
3.4. Extraction of genomic DNA In order to determine the optimal amount of Fe3O4/Au-PEI nanoparticles added into 100 μL of liver homogenate, several amounts of Fe3O4/Au-PEI nanoparticles were studied at ranges of 100–800 μg. As shown in Fig. 8, the amount of extracted DNA increased significantly while the amount of Fe3O4/Au-PEI nanoparticles increased from 100 to 600 μg. The yield of DNA could reach to 43.1 μg per 10 mg liver tissue judged by UV spectrophotometry at 260 nm. The average OD260/OD280 ratio was 1.81, indicating that the DNA was of good quality with negligible protein contamination. Also, as can be seen in the gel picture, no low-molecular weight bands or smear were detected, indicating the absence of RNA contamination. 4. Conclusion In summary, the PEI modified Fe3O4/Au nanoparticles were successfully synthesized in aqueous solution. The PEI modification made the Fe3O4/Au nanoparticles have a much larger ζ-potential, while no resulting in obviously changes in particles size. The Fe3O4/ Au-PEI nanoparticles could capture DNA via a simple protocol with the highest capture value of 90 μg/mg. The DNA release test showed that the release behavior was affected greatly by the pH, salt concentration and temperature of the elution buffer, and the DNA recovery could approach 100% under optimal conditions. Besides, the
Fig. 6. DNA release from Fe3O4/Au-PEI nanoparticles.
Fig. 7. Effect of (a) salt concentration and (b) temperature of elution buffer on DNA release.(The salt concentration in elution buffer was adjusted by adding NaCl to achieve the required concentration).
Fe3O4/Au-PEI nanoparticles were successfully used for genomic DNA extraction from liver homogenate. The results in this work indicated that the prepared Fe3O4/Au-PEI nanoparticles could be successfully used for DNA capture, release, and DNA extraction with high yield and purity.
Fig. 8. Figure agarose gel electrophoresis: effect of amount of magnetic particles on DNA extraction (M: Lambda DNA/Hind III Marker, 1: 100, 2: 200, 3: 400, 4: 600, 5: 800 μg of Fe3O4/Au-PEI nanoparticles).
H. Sun et al. / Materials Science and Engineering C 30 (2010) 311–315
Acknowledgments The Nature Science Foundation of Shandong Province (no. Q2006F01) is acknowledged for supporting this research. This work was also financially supported by Key Lab of Biophysics in Universities of Shandong. References [1] S. Wang, Y. Zhou, W. Sun, Mat. Sci. Eng. C 29 (2006) 1196. [2] Y.C. Li, Y.S. Lin, P.J. Tsai, P.J. Chen, Y.C. Chen, Anal. Chem. 79 (2007) 7519. [3] F.H. Wang, I.H. Lee, N. Holmström, T. Yoshitake, D.K. Kim, M. Muhammed, J. Frisén, L. Olson, C. Spenger, J. Kehr, Nanotechnology 17 (2006) 1911. [4] J. Prodelalová, B. Rittich, A. Spanová, K. Petrová, M.J. Benes, J. Chromatogr. A 1056 (2004) 43. [5] M.E. Park, J.H. Chang, Mat. Sci. Eng. C 27 (2007) 1232. [6] A.K. Gupta, M. Gupta, Biomaterials 26 (2005) 3995.
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[7] M. Pita, J.M. Abad, C. Vaz-Dominguez, C. Briones, E. Mateo-Martí, J.A. Martín-Gago, P. Morales Mdel, V.M. Fernández, J. Colloid. Interface Sci. 321 (2008) 484. [8] H.L. Liu, J.H. Wu, J.H. Min, J.H. Lee, Y.K. Kim, J. Nanosci. Nanotechnol. 9 (2009) 754. [9] V. Salgueirino-Maceira, M.A. Correa-Duarte, M.A. Lopez-Quintela, J. Rivas, J. Nanosci. Nanotechnol. 9 (2009) 3684. [10] H.L. Liu, C.H. Sonn, J.H. Wu, K.-M. Lee, Y.K. Kim, Biomaterials 29 (2008) 4003. [11] D.L. Huber, Small 1 (2005) 482. [12] C.C. Berry, J. Mater. Chem. 15 (2005) 543. [13] W.M. Hassen, C. Chaix, A. Abdelghani, F. Bessueille, D. Leonard, N. JaffrezicRenault, Sens. Actuators B, Chem. 134 (2008) 755. [14] T.L. Chang, C.Y. Tsai, C.C. Sun, R. Uppala, C.C. Chen, C.H. Lin, P.H. Chen, Microelectron. Eng. 83 (2006) 1630. [15] T. Uchikoshi, T. Hisashige, Y. Sakka, J. Ceram. Soc. Jpn. 110 (2002) 840. [16] S. Seino, Y. Matsuoka, T. Kinoshita, T. Nakagawa, T.A. Yamamoto, J. Magn. Magn. Mater. 321 (2009) 1404. [17] Y.L. Cui, Y.N. Wang, W.L. Hui, Z.F. Zhang, X.F. Xin, C. Chen, Biomed. Microdevices 7 (2005) 153. [18] T. Nakagawa, R. Hashimoto, K. Maruyama, T. Tanaka, H. Takeyama, T. Matsunaga, Biotechnol. Bioeng. 94 (2006) 862. [19] C.L. Chiang, C.S. Sung, T.F. Wu, C.Y. Chen, C.Y. Hsu, J. Chromatogr. B 822 (2005) 54.
Contents lists available at ScienceDirect
Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Capture and release of genomic DNA by PEI modified Fe3O4/Au nanoparticles Hanwen Sun a,⁎, Xinjun Zhu b, Lianying Zhang a,b, Yong Zhang c, Dunqing Wang a a b c
Dezhou Institute of Advanced Materials, Dezhou University, Dezhou 253023, PR China Department of Life Science, Dezhou University, Dezhou 253023, PR China Department of physics, Dezhou University, Dezhou 253023, PR China
a r t i c l e
i n f o
Article history: Received 20 August 2009 Received in revised form 19 October 2009 Accepted 12 November 2009 Available online 26 November 2009 Keywords: Fe3O4/Au nanoparticles Polyethylenimine DNA capture and release Extraction
a b s t r a c t Polyethylenimine (PEI) modified Fe3O4/Au nanoparticles were synthesized in aqueous solution and characterized by photo correlation spectroscopy (PCS) and vibrating sample magnetometer (VSM). The so-obtained Fe3O4/Au-PEI nanoparticles were capable of efficient electrostatic capture of DNA. The maximum amount of genomic DNA captured on 1.0 mg Fe3O4/Au-PEI nanoparticles was 90 μg. The DNA release behavior was studied and the DNA recovery from Fe3O4/Au-PEI nanoparticles approached 100% under optimal conditions. DNA extraction from mammalian cells using Fe3O4/Au-PEI nanoparticles was successfully performed. Up to approximately 43.1 μg of high-purity (OD260/OD280 ratio = 1.81) genomic DNA was extracted from 10 mg of liver tissue. The results indicated that the prepared Fe3O4/Au-PEI nanoparticles could be successfully used for DNA capture and release. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.
1. Introduction In recent years, the magnetic particles have emerged as a promising new platform in biomedical applications, such as protein and enzyme immobilization [1,2], magnetic resonance imaging (MRI) [3], hybridization and detection of DNA [4,5] etc. The magnetic particles were composite particles composed of iron oxide nanoparticles and various natural or synthetic polymers such as polystyrene, poly (ethylene glycol), poly (vinyl alcohol), chitosan and so on [6]. In order to add new properties (inertness, protection of the magnetic core against oxidation), an external inert shell such as Au has been widely attempted [7–9]. The unique Au surface is safe in both animals and humans [10], and exhibit an absorption band in the visible region due to its surface plasmon phenomenon, allowing the colorimetry of biomolecules with high-selectivity. Moreover, the Au coating renders sites for strong binding with various proteins (e.g. enzyme, biotin and so on) through their mercapto groups [11,12]. Thus, magnetic nanoparticles covered with an Au shell would provide all the characteristics of the Au element suitable for biological application and deliver magnetic properties to be utilized for further manipulation with an external magnetic field. However, the DNA/oligonucleotides cannot be attached on magnetic/Au nanoparticles directly. The conventional methods for this purpose can be divided into two classes: first, the DNA is conjugated with biotin and then the biotinylated DNA is captured by avidin-activated magnetic/Au nanoparticles [13]; and secondly, the
⁎ Corresponding author. Tel./fax: +86 534 8989506. E-mail address: [email protected] (H. Sun).
DNA is thiol modified, and then captured by magnetic/Au nanoparticles [14]. These protocols are time consuming and labor intensive. Polyethylenimine (PEI) is a water soluble cationic polymer which contains amino and imino groups in each polymer chain. When the polymer PEI is dispersed in aqueous solution, each polymer chain is positively charged by amino and imino groups[15]. These amino and imino groups are expected to adsorb onto the surface of magnetic/Au nanoparticles. Recently, S. Seino et al. [16] had synthesized PEI modified Fe2O3/Au nanoparticles and found that the PEI modification could greatly improve the dispersibility of Fe2O3/Au nanoparticles. However, the characteristics of DNA binding onto PEI modified magnetic/Au nanoparticles have still not been sufficiently investigated. In this paper, PEI modified Fe3O4/Au nanoparticles were synthesized and characterized by PCS and VSM, respectively. The DNA capture and release behavior using these magnetic nanoparticles was studied and a simple and convenient method to extract genomic DNA from mammalian cells was also established. 2. Experimental 2.1. Materials and methods Polyethylenimine (PEI, 30% in water), analytical grade, was purchased from Tokyo Chemical Industry Co. Ltd, Japan. Ferrous chloride tetrahydrate (FeCl2∙ 4H2O), ferric chloride hexahydrate (FeCl3∙6H2O), AuCl3·HCl·4H2O (Au ≥ 47.8%), analytical grade, were purchased from Shanghai Chemical Reagents Company (China) and used without further treatment. Nitrogen (99.99%) was available from Dezhou LongLi Company, China. Genomic DNA (Fish Sperm) was
0928-4931/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2009.11.005
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supplied by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd, China. All the other chemicals were of analytical grade. Stirrer (IKA Company, Germany) was used to synthesize the Fe3O4/Au-PEI nanoparticles. The size and ζ-potential of Fe3O4/Au-PEI nanoparticles were detected by photo correlation spectroscopy (PCS, Zetasizer Nano ZS, Malvern Instruments Ltd.). Magnetic measurement was recorded on a vibrating sample magnetometer (VSM, Princeton Applied Research Model 155) at 25 °C. UV spectrophotometer (BioMate) was used to determine DNA concentration. 2.2. Synthesis of Fe3O4/Au-PEI nanoparticles The Fe3O4/Au nanoparticles were prepared according to the method described by Cui et al. [17]. The Fe3O4/Au-PEI nanoparticles were synthesized by mixing the aqueous dispersion of 40 mg Fe3O4/ Au nanoparticles with 2 mL PEI for 6 h at room temperature. The Fe3O4/Au-PEI nanoparticles were collected magnetically and washed three times with distilled water to remove the excess PEI, and at last the Fe3O4/Au-PEI nanoparticles were redispersed in distilled water. 2.3. DNA capture on Fe3O4/Au-PEI nanoparticles 1.0 mg Fe3O4/Au-PEI nanoparticles were mixed with 2 mL DNA (25, 50, 100, 200, 300 and 400 μg) in binding buffer (PBS, pH 5.0, 5.8, 6.6 and 7.4), and incubated for 10 min at room temperature. The Fe3O4/Au-PEI–DNA complexes were collected magnetically and washed three times with binding buffer, and the wash solution was flow-through collected separately. Captured DNA was quantified as the total DNA amount minus the amount of DNA in the initial supernatant fraction and the washed fractions. 2.4. DNA release from Fe3O4/Au-PEI nanoparticles 1.0 mg Fe3O4/Au-PEI nanoparticles with 80 μg DNA in the form of sediment were transferred to a clean 5 mL centrifugal tube with 3 mL elution buffer. The sealed tube was placed in a water bath maintaining temperature at 37 °C. At specified collection times, 100 µL of sample was taken from the tube and DNA concentration was measured. The samples in the 5 mL tube were replenished with 100 µL fresh elution buffer at 37 °C. Triplicate samples were analyzed. The total released DNA Mi at time i was calculated from Eq. (1) Mi = CiV + ∑Ci−1 Vs:
ð1Þ
Where Ci is the concentration of DNA in the elution buffer at time i, V is the total volume of elution buffer (3 mL) and Vs is the sample volume (0.1 mL).
agarose gel in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8.0). The Electrophoresis was run at 110 V for 40 min. 3. Results and discussion 3.1. Character of Fe3O4/Au-PEI nanoparticles The PEI modification was carried out in distilled water (pH ≈ 7.0), in which the Fe3O4/Au nanoparticles were natively charged with the ζ-potential was −24.7 mV. Thus, the PEI could adsorb onto the surface of Fe3O4/Au nanoparticles via electrostatic attraction. Fig. 1 shows the ζ-potential changes of Fe3O4/Au nanoparticles before and after PEI modification. The isoelectric point (IEP) of the Fe3O4/Au nanoparticles was located at pH 5.7. After PEI modification, the IEP shifted to a higher pH value of 7.6 and a highly positive surface charge in a wider range of pH (from 2.0 to 7.0) was observed, indicating that the Fe3O4/Au-PEI nanoparticles were well-dispersed in this pH range. The ζ-potential of the Fe3O4/Au-PEI nanoparticles was much larger than that without PEI modification, moreover, the pH range with highly positive ζ-potential turned wider, which makes the Fe3O4/AuPEI nanoparticles have the ability to bind DNA molecules. The particle size was measured by PCS. As can be seen from Fig. 2, the average diameter of Fe3O4/Au-PEI nanoparticles was 74 nm, which was slightly larger than that of the Fe3O4/Au nanoparticles (68 nm). This result revealed that the modification process did not significantly result in the agglomeration and the size change of the particles. The magnetic property of the Fe3O4/Au nanoparticles before and after PEI modification was studied by vibration sample magnetometer (VSM). As can be seen from Fig. 3, the Fe3O4/Au-PEI nanoparticles behaved superparamagnetically with saturated magnetization (Ms) of 39 emu/g, which was slightly lower than that of the naked Fe3O4/Au nanoparticles (48 emu/g). 3.2. DNA capture The DNA-binding capacity of Fe3O4/Au-PEI nanoparticles is examined using DNA at different concentrations. The amount of DNA captured on the nanoparticles increased linearly with increasing DNA concentrations (Fig. 4). The effect of pH of binding buffer on the DNA capture was shown in Fig. 4. The amount of DNA captured on the nanoparticles increased with decreasing pH. The maximum capture capacity was observed at pH 5.0. As can be seen from Fig. 1, with the pH increasing in the range of 5.0–7.4, the positive charge density of the Fe3 O 4 /Au-PEI
2.5. Extraction of genomic DNA from mammalian cells 100 μL of liver homogenate (10% homogenate of rat liver prepared in 0.32 M buffered sucrose, pH 7.5) and 100 μL of 1% (w/v) sodium dodecyl sulfate (SDS) solution were added in 1.5 mL Eppendorf tube by gentle inversion two or three times and incubated at room temperature for 1 min. After incubation, Fe3O4/Au-PEI nanoparticles dispersed in 1.0 mL binding buffer (PBS, pH 5.0) were added into the cell lysate. The suspension was mixed by inversion and allowed to stand at room temperature for 2 min. The Fe3O4/Au-PEI–DNA complexes were collected magnetically, and the supernatant was removed. Then the Fe3O4/Au-PEI–DNA complexes were washed three times with binding buffer. Finally, the magnetic nanoparticles were resuspended in 100 μL of TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.2), and the Fe3O4/Au-PEI–DNA complexes were eluted by incubation at 45 °C for 20 min. The extracted DNA was determined by UV spectrophotometry at 260 nm and gel electrophoresis using 0.8%
Fig. 1. The ζ-potential of Fe3O4/Au nanoparticle (a) before and (b) after PEI modification.
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Fig. 2. The particle size of (a) Fe3O4/Au and (b) Fe3O4/Au-PEI nanoparticles.
Tris–HCl buffer (pH 7.4 and 8.2) and PBS buffer (pH 5.0) were used as elution buffer in this study. As can be seen from Fig. 6, the captured DNA released largely in elution buffer pH 7.4 and 8.2, while only a small quantity of DNA (b8%) could desorb from the Fe3O4/Au-PEI nanoparticles at pH 5.0. The ζ-potential data for the Fe3O4/Au-PEI
nanoparticles showed a steady decrease in positive surface charge (or a steady increase in negative surface charge) with increasing pH value of elution buffer in the range of 5.0–8.2, which could result in a decrease of electrostatic attraction between Fe3O4/Au-PEI nanoparticles and DNA molecules. Therefore, the DNA could be released from nanoparticles easily. From Fig. 6, it can also be seen that the DNA releasing reached equilibrium within 20 min. The influence of salt concentration and temperature of elution buffer on DNA release were also studied. The release yield of DNA was increased with increasing salt concentration. In the elution buffer pH 8.2, the equilibrium of DNA release was achieved at salt concentration 1.5 M, and in the elution buffer pH 7.4, the equilibrium of DNA release was achieved at salt concentration 2.0 M (Fig. 7a). It may be attributed to the fact that the active site for DNA binding decreased with increasing salt concentration. The effect of temperature on DNA release was determined in Tris–HCl buffer with the salt concentration 2.0 M in the range of 20–60 °C. As it can be seen from Fig. 7b, the amount of DNA released increased with increasing temperature. The release yield was almost 100% for the pH 8.2 elution solution with a salt concentration 2.0 M at 50 °C. Data obtained suggests that nearly all DNA could released from the Fe3O4/Au-PEI nanoparticles.
Fig. 3. Magnetization curve of (a) Fe3O4/Au and (b) Fe3O4/Au-PEI nanoparticles.
Fig. 4. The amount of DNA captured on 1.0 mg of Fe3O4/Au-PEI nanoparticles.
nanoparticles decreased. This may lead to a reduction of active DNAbinding sites, thereby resulting in the decrease of DNA capture capacity. The Fe3O4/Au-PEI nanoparticles (1.0 mg) were capable of capturing 90 μg of DNA at pH 5.0. This result was much higher than previous reports [18,19]. This may because of higher amount of PEI adsorbed on Fe3O4/Au nanoparticles through static interaction in this report. The adsorption kinetics of DNA on Fe3O4/Au-PEI nanoparticles is studied using 1.0 mg Fe3O4/Au-PEI nanoparticles incubated with 100 μg DNA at pH 5.0. (Fig. 5). To confirm a complete equilibrium between DNA and Fe3O4/Au-PEI nanoparticles, these experiments were examined until the adsorption time was 20 min. All the adsorptions tended toward equilibrium within 1 min. 3.3. DNA release
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Fig. 5. The adsorption kinetics of DNA on Fe3O4/Au-PEI nanoparticles.
3.4. Extraction of genomic DNA In order to determine the optimal amount of Fe3O4/Au-PEI nanoparticles added into 100 μL of liver homogenate, several amounts of Fe3O4/Au-PEI nanoparticles were studied at ranges of 100–800 μg. As shown in Fig. 8, the amount of extracted DNA increased significantly while the amount of Fe3O4/Au-PEI nanoparticles increased from 100 to 600 μg. The yield of DNA could reach to 43.1 μg per 10 mg liver tissue judged by UV spectrophotometry at 260 nm. The average OD260/OD280 ratio was 1.81, indicating that the DNA was of good quality with negligible protein contamination. Also, as can be seen in the gel picture, no low-molecular weight bands or smear were detected, indicating the absence of RNA contamination. 4. Conclusion In summary, the PEI modified Fe3O4/Au nanoparticles were successfully synthesized in aqueous solution. The PEI modification made the Fe3O4/Au nanoparticles have a much larger ζ-potential, while no resulting in obviously changes in particles size. The Fe3O4/ Au-PEI nanoparticles could capture DNA via a simple protocol with the highest capture value of 90 μg/mg. The DNA release test showed that the release behavior was affected greatly by the pH, salt concentration and temperature of the elution buffer, and the DNA recovery could approach 100% under optimal conditions. Besides, the
Fig. 6. DNA release from Fe3O4/Au-PEI nanoparticles.
Fig. 7. Effect of (a) salt concentration and (b) temperature of elution buffer on DNA release.(The salt concentration in elution buffer was adjusted by adding NaCl to achieve the required concentration).
Fe3O4/Au-PEI nanoparticles were successfully used for genomic DNA extraction from liver homogenate. The results in this work indicated that the prepared Fe3O4/Au-PEI nanoparticles could be successfully used for DNA capture, release, and DNA extraction with high yield and purity.
Fig. 8. Figure agarose gel electrophoresis: effect of amount of magnetic particles on DNA extraction (M: Lambda DNA/Hind III Marker, 1: 100, 2: 200, 3: 400, 4: 600, 5: 800 μg of Fe3O4/Au-PEI nanoparticles).
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Acknowledgments The Nature Science Foundation of Shandong Province (no. Q2006F01) is acknowledged for supporting this research. This work was also financially supported by Key Lab of Biophysics in Universities of Shandong. References [1] S. Wang, Y. Zhou, W. Sun, Mat. Sci. Eng. C 29 (2006) 1196. [2] Y.C. Li, Y.S. Lin, P.J. Tsai, P.J. Chen, Y.C. Chen, Anal. Chem. 79 (2007) 7519. [3] F.H. Wang, I.H. Lee, N. Holmström, T. Yoshitake, D.K. Kim, M. Muhammed, J. Frisén, L. Olson, C. Spenger, J. Kehr, Nanotechnology 17 (2006) 1911. [4] J. Prodelalová, B. Rittich, A. Spanová, K. Petrová, M.J. Benes, J. Chromatogr. A 1056 (2004) 43. [5] M.E. Park, J.H. Chang, Mat. Sci. Eng. C 27 (2007) 1232. [6] A.K. Gupta, M. Gupta, Biomaterials 26 (2005) 3995.
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