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Isolation of DNA using magnetic nanoparticles coated with dimercaptosuccinic acid
Analytical Biochemistry 447 (2014) 114–118
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Isolation of DNA using magnetic nanoparticles coated with dimercaptosuccinic acid Ji Hyun Min a,b, Mi-Kyung Woo b,c, Ha Young Yoon a,b, Jin Woo Jang b,c, Jun Hua Wu b, Chae-Seung Lim b,c,⇑, Young Keun Kim a,b,⇑ a b c
Department of Materials Science and Engineering, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-713, Republic of Korea Pioneer Research Center for Biomedical Nanocrystals, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-713, Republic of Korea Department of Laboratory Medicine, College of Medicine, Korea University Guro Hospital, Seoul 152-703, Republic of Korea
a r t i c l e
i n f o
Article history: Received 27 August 2013 Received in revised form 10 November 2013 Accepted 17 November 2013 Available online 27 November 2013 Keywords: DNA isolation Magnetic nanoparticle Dimercaptosuccinic acid Superparamagnetism
a b s t r a c t Lately, the isolation of DNA using magnetic nanoparticles has received increased attention owing to their facile manipulation and low costs. Although methods involving their magnetic separation have been extensively studied, there is currently a need for an efficient technique to isolate DNA for highly sensitive diagnostic applications. We describe herein a method to isolate and purify DNA using biofunctionalized superparamagnetic nanoparticles synthesized by a modified polyol method to obtain the desired monodispersity, followed by surface modification with meso-2,3-dimercaptosuccinic acid (DMSA) containing carboxyl groups for DNA absorption. The DMSA-coated magnetic nanoparticles (DMSA-MNPs) were used for the isolation of DNA, with a maximum yield of 86.16%. In particular, we found that the isolation of DNA using small quantities of DMSA-MNPs was much more efficient than that using commercial microbeads (NucliSENS-easyMAG, BioMérieux). Moreover, the DMSA-MNPs were successfully employed in the isolation of genomic DNA from human blood. In addition, the resulting DNA–nanoparticle complex was directly subjected to PCR amplification without prior elution, which could eventually lead to simple, rapid, sensitive and integrated diagnostic systems. Ó 2013 Elsevier Inc. All rights reserved.
The isolation of DNA is an essential process in molecular biology and a fundamental step for initiating other downstream activities such as sequencing, amplification, hybridization, ligation, cloning, and biodetection. Several researchers have studied DNA isolation for potential applications such as diagnosis of diseases, pathogen detection, and gene therapy [1–5]. Various methods have been employed to separate DNAs from crude biological samples; such methods have been generally classified into either fluid-phase or solid-phase categories [1,2]. The fluid-phase extraction methods involving a phenol/chloroform mixture and cetyltrimethylammonium bromide (CTAB)1 usually comprise complicated steps, including centrifugation, precipitation, and filtration. Moreover, this processing is time-consuming, labor-intensive, and toxic [1–3]. Hence, alternative isolation methods have been proposed using a
⇑ Corresponding authors. Fax: +82 2 2626 1465 (C.-S. Lim), Fax: +82 2 928 3584 (Y.K. Kim). E-mail addresses: [email protected] (C.-S. Lim), [email protected] (Y.K. Kim). 1 Abbreviations used: DMSA, meso-2,3-dimercaptosuccinic acid; DMSA-MNPs, DMSA-coated magnetic nanoparticles; DW, deionized water; PEG-PPG-PEG, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol); VSM, vibrating sample magnetometer. 0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.11.018
variety of solid-phase supports (e.g., silica, glass fibers, carbon nanotubes, and magnetic particles) [6–8]. Recent advances in the development of integrated systems to isolate DNA have employed the solid-phase supports [9], among which magnetic nanoparticles (MNPs) have received most attention because of their easy manipulation and cost-effectiveness [10,11]. In addition, separation techniques involving MNPs offer significant benefits over conventional methods owing to shorter processing times, sustainable costs, limited use of chemicals, ease of automation, and minor physical or chemical damage. The absorption of DNA molecules on solid supports is driven through hydrogen-bonding and hydrophobic and electrostatic interactions, which are intimately associated with the surface condition of the solid supports [12,13]. In this regard, many studies have focused on the development of new solid supports and/or surface modification protocols to enhance the isolation and/or recovery efficiency of DNA molecules on the carrier surface. For example, MNPs have been surface-modified using functional groups (–CO2H, –OH, and –NH2) [13–17], whereas hybrid magnetic particles have been coated with inorganic materials (e.g., silica and gold) for achieving high loading (isolation) efficiency [12,16,18– 20]. Nevertheless, the current requirements of highly sensitive diagnosis and miniaturization have propelled the development of
Isolation of DNA using magnetic nanoparticles coated with DMSA / J.H. Min et al. / Anal. Biochem. 447 (2014) 114–118
more effective DNA isolation systems. Consequently, there is currently tremendous interest in exploring novel solid supports and surface-modification methods. In this work, we propose surface-modified superparamagnetic nanoparticles for an effective isolation of DNA. Unlike most of the previous studies, the MNPs used herein were prepared in the form of solid supports by an organic-phase synthesis known to facilitate the production of superparamagnetic nanoparticles with desired monodispersity [21,22]. The surface of the resultant monosized MNPs was functionalized using meso-2,3-dimercaptosuccinic acid (DMSA). The DMSA molecule possesses both carboxylate and thiol groups, which are able to bond effectively with DNA molecules [23]. The resulting complex could be used for DNA absorption because of its high affinity for MNPs, stability in aqueous solution, and easy bioconjugation. This complex can be used in various bio-based applications such as MRI agents, nano-carriers for cancer therapy, and investigation of cell responses [24–26]. In the present work, the DMSA-coated MNPs were evaluated for their DNA isolation efficiency, and the results were compared with the efficiency of other surface-modified nanoparticles, including commercially available beads. Materials and methods Preparation of DMSA-coated magnetic nanoparticles The synthesis of MNPs was carried out by a modified polyol method [27]. Iron acetylacetonate (Fe(acac)3), 1,2-hexadecanediol, and poly(ethylene glycol)-block-poly(propylene glycol)-blockpoly(ethylene glycol) (PEG-PPG-PEG) were used as an iron precursor, a reducing agent, and a surfactant, respectively. The materials were mixed in octyl ether and heated to 300 °C with continuous magnetic stirring. The mixture was then cooled down to room temperature followed by the precipitation of the nanoparticles using ethanol. Then, the precipitates were washed using centrifugation; finally, they were extracted by an external magnetic field. To accomplish the surface modification of the MNPs, 1 mmol DMSA in 5 mL of dimethyl sulfoxide (DMSO) was added to 3 mg MNPs in 1 mL of ethanol. Then, the mixture was sonicated at 4 °C for 6 h, and the resultant product was washed with ethanol by centrifugation. Finally, the precipitated nanocomplex was dissolved in deionized water (DW). All the chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). The MNPs were analyzed using transmission electron microscopy (TEM, JEOL 2010F), zeta potential measurement, vibrating sample magnetometer (VSM, Lakeshore 7300), and Fourier transform infrared spectroscopy (FTIR, PerkinElmer Spectrum GX). DNA complexation and isolation by the nanoparticles We evaluated the efficiency of DNA isolation by the DMSAcoated MNPs (DMSA-MNPs), and compared the results with those obtained using the fresh Fe3O4 MNPs and commercial beads. The amplified PCR products (700 bp), which were used as the DNA source, were obtained from the control primers in the TOPO TA Cloning kit (Invitrogen, Carlsbad, CA, USA). The absorption of DNA molecules on the magnetic carriers was carried out as follows: 1 lg of the PCR products in 15 lL DW was first dissolved in 15 lL of a binding buffer containing 18.2 wt% polyethylene glycol 8000 (Sigma–Aldrich, St. Louis, MO, USA) and 4.0 M NaCl. Then, 250 lg of the MNPs or DMSA-MNPs was added. After incubating the resulting mixture at room temperature for 10 min, the complexes formed by the magnetic supports and DNA molecules were separated from the solution using an external magnetic field. Subsequently, the complexes were rinsed several
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times with 30 lL of 70–30% ethanol-water and dried briefly. Next, the DNA molecules on the magnetic supports were eluted using a TE buffer (10 mM Tris–HCl, 1 mM EDTA in pH 7.8). In other words, 30 lL TE buffer was added to the dried complexes, followed by incubation at 60 °C for 10 min. Then, the MNPs were removed using a magnetic field, and the eluted DNA molecules were collected by centrifugation for quantitation purposes. For comparison studies, commercially available magnetic beads (NucliSENS-easyMAG, BioMérieux, Durham, NC, USA) were used according to the manufacturer’s instructions. An easyMAG Lysis Buffer and two types of wash solutions (easyMAG Extraction Buffer 1 and 2) were used for DNA extraction. First, 500 lg of the PCR products in DW was mixed with the lysis buffer, and the complexes were formed using commercial beads. DNA molecules were successively washed with 100 lL of wash solution 1 and 100 lL of wash solution 2, followed by the elution of DNA molecules with 30 lL of the buffer (NucliSENS easyMAG Extraction buffer 3). The other procedures used were the same as those for the MNPs. The evaluation of the isolated DNA molecules from the MNPs, DMSA-MNPs, and commercial beads was performed using agarose gel electrophoresis and UV spectrophotometry. The inputted and eluted DNA molecules from each solid support were loaded in 1.5% agarose gel containing 0.5 mg ethidium bromide per milliliter and electrophoresed at a voltage of 150 V for 15 min. The DNA bands were visualized using a gel image system (ChemiDOC XRS+, Molecular Imager ChemicDocTM XRS Imaging System, BioRad Laboratories Inc., Hercules, CA, USA), while the quantity was analyzed using the AlphaEaseFC (Genetic Technologies Inc., Miami, FL, USA). In addition, the quality of the isolated DNA molecu–Vis measurements. Isolation of genomic DNA from human blood and direct PCR amplification in beta actin gene The DMSA-MNPs were used for isolating human genomic DNA molecules from human blood. First, various volumes of human blood (2, 4, and 8 lL) were added separately to 200 lL of a lysis buffer solution (3.0 M NaI solution). Then, a binding buffer solution (18.2 wt% polyethylene glycol 8000 and 4.0 M NaCl) was added, followed by incubation with 3 lg of DMSA-MNPs at room temperature for 10 min. The samples were washed several times with 30 lL of 70–30% ethanol–water and dried briefly. The resultant mixture was used directly as a PCR template without any purification or elution. The human b-actin from blood was amplified using oligonucleotide primers: b-actin-F1, 50 -GAGAAAATCTGGCACCACAC-30 ; b-actin-R1, 50 -GATGTCCACGTCACACTTCA-30 . The concentration of the primers was 0.1 lM in 100 lL of the reaction mixture (10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP), containing 10 lL of DNA and 2.5 units of TaKaRa Ex Taq polymerase (Takara Bio Inc., Shiga, Japan). The reaction mixtures were cycled 30 times. Each cycle included denaturation at 94 °C for 30 s, annealing at 64 °C for 30 s, and extension at 72 °C for 30 s in a PCR Thermal Cycler TP600 (Takara Bio Inc., Shiga, Japan). The 16S rRNA-amplified products were size-fractionated using agarose gels (1.5%) by electrophoresis with ethidium bromide (0.5 mg/mL). Results and discussion The MNPs were synthesized by a modified polyol method as described in our previous work [27]. As shown in Fig. 1a, the nanoparticles are monodispersed and spherical with an average diameter of 8.4 nm and a narrow distribution (±1.1 nm). The high resolution, as shown in the inset of Fig. 1a, reveals that the nanoparticles possess distinct lattices and, therefore, high crystallinity.
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Isolation of DNA using magnetic nanoparticles coated with DMSA / J.H. Min et al. / Anal. Biochem. 447 (2014) 114–118
Fig.1. Morphology and hysteresis curve of magnetic nanoparticles. (a) TEM image (inset: high resolution image); (b) magnetic hysteresis curve (inset: magnification of hysteresis curve around zero field).
The corresponding magnetic properties were measured using VSM. As depicted in Fig. 1b, the magnetic hysteresis loop exhibits typical superparamagnetic behavior with near zero coercivity, thus preventing the self-aggregation of magnetic particles. On the contrary, commercially available beads had soft ferromagnetic traits with a coercivity of 180 Oe, indicating that after the applied magnetic field was removed, a residual magnetization (remanence) was present in the MNPs causing undesired spontaneous aggregation. As PEG-PPG-PEG was used as a surfactant in the synthetic process, the fresh nanoparticles were coated with the block copolymer containing hydroxyl groups [28]. In order to modify the nanoparticle surface with carboxyl groups for DNA complexation, DMSA solution was added to the nanoparticles dispersed in ethanol. Even after the surface modification, the nanoparticles possessed good superparamagnetic behavior, indicating that the coating process had a negligible impact on the magnetic properties, and the resulting DMSA-MNPs were resistant against aggregation. The surface property of the fresh MNPs and DMSA-MNPs was examined using zeta potential measurements. The zeta potential of the fresh nanoparticles was 20.1±3.7 mV while that of the DMSA-MNPs was 51.4±7.2 mV, implying that the surface was, unambiguously, more negatively charged, and thus, the coating process was effective. The surface condition was further inspected using FTIR (Fig. 2). The FTIR spectra of the fresh nanoparticles, as anticipated, showed a strong peak at 1115 cm, which was assigned to the C– O–C stretching vibration, and a characteristic peak for the C–H bending vibration at 1570 cm, which corresponded to the coating
Fig.2. FTIR spectra of (a) DMSA-coated magnetic nanoparticles and (b) fresh magnetic nanoparticles.
of PEG-PPG-PEG on the nanoparticle surface [28]. Nevertheless, no peaks for the C–O–C bonding appeared around 1120 cm in the DMSA-MNPs, suggesting that the polymer was replaced with DMSA after coating. Instead, the DMSA-MNPs displayed two characteristic bands at 1610 and 1390 cm, consistent with the previous reports [29–30]. The bands were derived from the symmetric and asymmetric stretches of the carboxyl groups (–CO2H), revealing that the DSMA molecules were successfully loaded onto the nanoparticle surface [30]. Subsequently, we tested the DMSA-MNPs against the fresh MNPs for the isolation of DNA. For this, 1 lg of the PCR products was used as the DNA source, and 250 lg of the DMSA-MNPs or fresh MNPs were added to isolate the DNA molecules. The outcome of the electrophoresis of the DMSA-MNPs was much brighter than that of the fresh nanoparticles, indicating that the yield of DNA isolation of the DMSA-MNPs was significantly higher than that of the fresh MNPs. This was because the carboxyl groups of DMSA on the nanoparticle surface were connected to the DNA molecules through hydrogen bonding, thereby efficiently isolating DNA molecules [13,14]. The enhanced yield of the DMSA-MNPs demonstrated that DMSA was successfully coated onto the MNPs; thus, the carboxyl groups on the surface of the MNPs increased the efficiency of absorption. In addition, the optical density ratio, OD 260/ OD 280, was 1.8, indicating that the DNA was of good quality. Next, the DNA-isolation efficiency of the DMSA-MNPs was compared to that of commercially available beads by investigating the isolation yields as a function of concentration. As the commercial beads, the NucliSENS-easyMAG (BioMerieux) was employed for this comparison study, which has been widely used for the automated extraction of DNA from clinical samples [31]. To evaluate the isolation efficiency, 500 ng of the amplified PCR products was used as the target DNA in each experiment. Fig. 3 shows two sets of the DNA-isolation yields: DMSA-MNPs and commercially available beads. The amount of solid magnetic beads used was in the range 2–128 mg. As shown in Fig. 3b, the DMSA-MNPs had a maximum isolation yield of 86.16%, which was higher than that of commercially available beads (83.41%). In particular, even with small quantities of DMSA-MNPs, the isolation of DNA was much more efficient than that of commercially available beads. To obtain 500 ng of DNA, 2–4 lg DMSA-MNPs provided an isolation yield of 60–70%, while commercially available beads provided a lower isolation yield with the same quantities. Though 16 lg DMSAMNPs provided a DNA isolation yield of 80%, at least 128 lg of commercially available beads had to be used to obtain the same yield. However, the isolation yields of the DMSA-MNPs decreased with increasing amount of the particle after the saturation point. Such trends for the DNA isolation yields could be observed because excess amounts of magnetic supports made it difficult for the
Isolation of DNA using magnetic nanoparticles coated with DMSA / J.H. Min et al. / Anal. Biochem. 447 (2014) 114–118
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of the PCR products of the genomic DNA when 3 lg DMSA-MNPs was used in the human blood series of 2, 4, and 6 lL. In all the samples, the b-actin genes from the genomic DNA were successfully amplified by the PCR process. This demonstrated that the DMSAMNPs were not only useful for isolating the genomic DNA from human whole blood but also for amplifying the human genes by a simplified PCR procedure. Conclusion
Fig.3. Comparison of DNA isolation efficiency of small quantities of DMSA-MNPs with that of commercial beads. Here, 1 lg DNA was bound separately with DMSAMNPs as well as commercial beads in various quantities. DNA isolated from these particles was verified using agarose gel analysis (a) and isolation yields were plotted as a function of the quantity of particles (b).
separation of DNA molecules from the DNA-magnetic support complexes, for instance, the disruption of DNA elution from magnetic supports and interference of excess magnetic supports in collecting DNA by applying a magnetic field [17]. Finally, the DMSA-MNPs were used for isolating genomic DNAs from human whole blood, followed by PCR amplification. First, samples of lysis solution were prepared from a human blood series (2, 4, and 6 lL), and 3 lg of the DMSA-MNPs was added to each lysis solution for binding with the genomic DNA molecules. Then, the DNA-bound DMSA-MNPs were isolated using an external magnetic field. The isolated DNA complexes could be amplified by PCR modulation for detecting specific genes. In particular, the resultant DNA-bound DMSA-MNPs complexes were used directly for PCR modulation without prior elution, which simplified the entire process of gene detection. Fig. 4 presents the results of electrophoresis
Fig.4. Agarose gel electrophoresis showing PCR amplification of b-actin gene (621 bp) in genomic DNA, isolated from human whole blood using 3 lg DMSAMMPs. Lines 1, 2, and 3 correspond to results obtained from 2, 4, and 8 lL human blood, respectively; Line M is DNA molecular mass maker.
We have successfully prepared DMSA-coated MNPs for the isolation of DNA by a modified polyol method. The TEM observations revealed that the DMSA-MNPs were monodispersed and highly crystalline with a narrow size distribution while the VSM results verified their superparamagnetic behavior, a desirable trait for bio-based applications. The MNPs were coated with DMSA for the isolation of DNA. We demonstrated that the DMSA-MNP complexes were highly efficient in binding to and thereby isolating the DNA molecules from crude biological samples. In particular, the DMSA-MNPs were much more useful for the isolation of DNA, even in relatively small quantities, compared to commercially available beads. Furthermore, the DMSA-MNPs were directly applied to the isolation of human genomic DNA without prior elution or purification, thereby leading to a simple, rapid, and time-saving analysis for the detection of diseases. Consequently, even small quantities of the biofunctionalized nanoparticles could be potentially utilized for a highly efficient isolation of DNA from biological samples. Their other biomedical applications include highly sensitive biosensing and automatic diagnostic systems. Acknowledgments This research was supported by the Pioneer Research Center Program (NRF-2011-0002128), the Leap Research Program (NRF2010-0017950), and the General Research Program (2013R1A1A2013842) by the National Research Foundation of Korea. J.H. Min acknowledges financial support by Korea University. References [1] S.C. Tan, B.C. Yiap, DNA, RNA, and protein extraction: the past and the present, J. Biomed. Biotechnol. 2009 (2009) 574398. [2] S. Berensmeier, Magnetic particles for the separation and purification of nucleic acids, Appl. Microbiol. Biotechnol. 73 (3) (2006) 495–504. [3] J. Corchero, A. Villaverde, Biomedical applications of distally controlled magnetic nanoparticles, Trends Biotechnol. 27 (8) (2009) 468–476. [4] S.C. Dixon, J. Horti, Y. Guo, E. Reed, W.D. Figg, Methods for extracting and amplifying genomic DNA isolated from frozen serum, Nat. Biotechnol. 16 (1) (1998) 91–94. [5] N.A. Saunders, Application of nanomaterials to arrays for infectious disease diagnosis, Nanomedicine 6 (2) (2011) 271–280. [6] D.A. Dederich, G. Okwuonu, T. Garner, A. Denn, A. Sutton, M. Escotto, A. Martindale, O. Delgado, D.M. Muzny, R.A. Gibbs, M.L. Metzker, Glass bead purification of plasmid template DNA for high throughput sequencing of mammalian genomes, Nucleic Acids Res. 30 (7) (2002). [7] I.I. Shakhmaeva, E.R. Bulatov, O.V. Bondar, D.V. Saifullina, M. Culha, A.A. Rizvanov, T.I. Abdullin, Binding and purification of plasmid DNA using multilayered carbon nanotubes, J. Biotechnol. 152 (3) (2011) 102–107. [8] Z.M. Saiyed, M. Parasramka, S.D. Telang, C.N. Ramchand, Extraction of DNA from agarose gel using magnetic nanoparticles (magnetite or Fe3O4), Anal. Biochem. 363 (2) (2007) 288–290. [9] S.M. Azimi, G. Nixon, J. Ahern, W. Balachandran, A magnetic bead-based DNA extraction and purification microfluidic device, Microfluid Nanofluidics 11 (2) (2011) 157–165. [10] K. Aguilar-Arteaga, J.A. Rodriguez, E. Barrado, Magnetic solids in analytical chemistry: a review, Anal. Chim. Acta 674 (2) (2010) 157–165. [11] R. Hao, R.J. Xing, Z.C. Xu, Y.L. Hou, S. Gao, S.H. Sun, Synthesis, functionalization, and biomedical applications of multifunctional magnetic nanoparticles, Adv. Mater. 22 (25) (2010) 2729–2742. [12] K. Kang, J. Choi, J.H. Nam, S.C. Lee, K.J. Kim, S.W. Lee, J.H. Chang, Preparation and characterization of chemically functionalized silica-coated magnetic nanoparticles as a DNA separator, J. Phys. Chem. B 113 (2) (2009) 536–543.
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Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Isolation of DNA using magnetic nanoparticles coated with dimercaptosuccinic acid Ji Hyun Min a,b, Mi-Kyung Woo b,c, Ha Young Yoon a,b, Jin Woo Jang b,c, Jun Hua Wu b, Chae-Seung Lim b,c,⇑, Young Keun Kim a,b,⇑ a b c
Department of Materials Science and Engineering, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-713, Republic of Korea Pioneer Research Center for Biomedical Nanocrystals, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-713, Republic of Korea Department of Laboratory Medicine, College of Medicine, Korea University Guro Hospital, Seoul 152-703, Republic of Korea
a r t i c l e
i n f o
Article history: Received 27 August 2013 Received in revised form 10 November 2013 Accepted 17 November 2013 Available online 27 November 2013 Keywords: DNA isolation Magnetic nanoparticle Dimercaptosuccinic acid Superparamagnetism
a b s t r a c t Lately, the isolation of DNA using magnetic nanoparticles has received increased attention owing to their facile manipulation and low costs. Although methods involving their magnetic separation have been extensively studied, there is currently a need for an efficient technique to isolate DNA for highly sensitive diagnostic applications. We describe herein a method to isolate and purify DNA using biofunctionalized superparamagnetic nanoparticles synthesized by a modified polyol method to obtain the desired monodispersity, followed by surface modification with meso-2,3-dimercaptosuccinic acid (DMSA) containing carboxyl groups for DNA absorption. The DMSA-coated magnetic nanoparticles (DMSA-MNPs) were used for the isolation of DNA, with a maximum yield of 86.16%. In particular, we found that the isolation of DNA using small quantities of DMSA-MNPs was much more efficient than that using commercial microbeads (NucliSENS-easyMAG, BioMérieux). Moreover, the DMSA-MNPs were successfully employed in the isolation of genomic DNA from human blood. In addition, the resulting DNA–nanoparticle complex was directly subjected to PCR amplification without prior elution, which could eventually lead to simple, rapid, sensitive and integrated diagnostic systems. Ó 2013 Elsevier Inc. All rights reserved.
The isolation of DNA is an essential process in molecular biology and a fundamental step for initiating other downstream activities such as sequencing, amplification, hybridization, ligation, cloning, and biodetection. Several researchers have studied DNA isolation for potential applications such as diagnosis of diseases, pathogen detection, and gene therapy [1–5]. Various methods have been employed to separate DNAs from crude biological samples; such methods have been generally classified into either fluid-phase or solid-phase categories [1,2]. The fluid-phase extraction methods involving a phenol/chloroform mixture and cetyltrimethylammonium bromide (CTAB)1 usually comprise complicated steps, including centrifugation, precipitation, and filtration. Moreover, this processing is time-consuming, labor-intensive, and toxic [1–3]. Hence, alternative isolation methods have been proposed using a
⇑ Corresponding authors. Fax: +82 2 2626 1465 (C.-S. Lim), Fax: +82 2 928 3584 (Y.K. Kim). E-mail addresses: [email protected] (C.-S. Lim), [email protected] (Y.K. Kim). 1 Abbreviations used: DMSA, meso-2,3-dimercaptosuccinic acid; DMSA-MNPs, DMSA-coated magnetic nanoparticles; DW, deionized water; PEG-PPG-PEG, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol); VSM, vibrating sample magnetometer. 0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.11.018
variety of solid-phase supports (e.g., silica, glass fibers, carbon nanotubes, and magnetic particles) [6–8]. Recent advances in the development of integrated systems to isolate DNA have employed the solid-phase supports [9], among which magnetic nanoparticles (MNPs) have received most attention because of their easy manipulation and cost-effectiveness [10,11]. In addition, separation techniques involving MNPs offer significant benefits over conventional methods owing to shorter processing times, sustainable costs, limited use of chemicals, ease of automation, and minor physical or chemical damage. The absorption of DNA molecules on solid supports is driven through hydrogen-bonding and hydrophobic and electrostatic interactions, which are intimately associated with the surface condition of the solid supports [12,13]. In this regard, many studies have focused on the development of new solid supports and/or surface modification protocols to enhance the isolation and/or recovery efficiency of DNA molecules on the carrier surface. For example, MNPs have been surface-modified using functional groups (–CO2H, –OH, and –NH2) [13–17], whereas hybrid magnetic particles have been coated with inorganic materials (e.g., silica and gold) for achieving high loading (isolation) efficiency [12,16,18– 20]. Nevertheless, the current requirements of highly sensitive diagnosis and miniaturization have propelled the development of
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more effective DNA isolation systems. Consequently, there is currently tremendous interest in exploring novel solid supports and surface-modification methods. In this work, we propose surface-modified superparamagnetic nanoparticles for an effective isolation of DNA. Unlike most of the previous studies, the MNPs used herein were prepared in the form of solid supports by an organic-phase synthesis known to facilitate the production of superparamagnetic nanoparticles with desired monodispersity [21,22]. The surface of the resultant monosized MNPs was functionalized using meso-2,3-dimercaptosuccinic acid (DMSA). The DMSA molecule possesses both carboxylate and thiol groups, which are able to bond effectively with DNA molecules [23]. The resulting complex could be used for DNA absorption because of its high affinity for MNPs, stability in aqueous solution, and easy bioconjugation. This complex can be used in various bio-based applications such as MRI agents, nano-carriers for cancer therapy, and investigation of cell responses [24–26]. In the present work, the DMSA-coated MNPs were evaluated for their DNA isolation efficiency, and the results were compared with the efficiency of other surface-modified nanoparticles, including commercially available beads. Materials and methods Preparation of DMSA-coated magnetic nanoparticles The synthesis of MNPs was carried out by a modified polyol method [27]. Iron acetylacetonate (Fe(acac)3), 1,2-hexadecanediol, and poly(ethylene glycol)-block-poly(propylene glycol)-blockpoly(ethylene glycol) (PEG-PPG-PEG) were used as an iron precursor, a reducing agent, and a surfactant, respectively. The materials were mixed in octyl ether and heated to 300 °C with continuous magnetic stirring. The mixture was then cooled down to room temperature followed by the precipitation of the nanoparticles using ethanol. Then, the precipitates were washed using centrifugation; finally, they were extracted by an external magnetic field. To accomplish the surface modification of the MNPs, 1 mmol DMSA in 5 mL of dimethyl sulfoxide (DMSO) was added to 3 mg MNPs in 1 mL of ethanol. Then, the mixture was sonicated at 4 °C for 6 h, and the resultant product was washed with ethanol by centrifugation. Finally, the precipitated nanocomplex was dissolved in deionized water (DW). All the chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). The MNPs were analyzed using transmission electron microscopy (TEM, JEOL 2010F), zeta potential measurement, vibrating sample magnetometer (VSM, Lakeshore 7300), and Fourier transform infrared spectroscopy (FTIR, PerkinElmer Spectrum GX). DNA complexation and isolation by the nanoparticles We evaluated the efficiency of DNA isolation by the DMSAcoated MNPs (DMSA-MNPs), and compared the results with those obtained using the fresh Fe3O4 MNPs and commercial beads. The amplified PCR products (700 bp), which were used as the DNA source, were obtained from the control primers in the TOPO TA Cloning kit (Invitrogen, Carlsbad, CA, USA). The absorption of DNA molecules on the magnetic carriers was carried out as follows: 1 lg of the PCR products in 15 lL DW was first dissolved in 15 lL of a binding buffer containing 18.2 wt% polyethylene glycol 8000 (Sigma–Aldrich, St. Louis, MO, USA) and 4.0 M NaCl. Then, 250 lg of the MNPs or DMSA-MNPs was added. After incubating the resulting mixture at room temperature for 10 min, the complexes formed by the magnetic supports and DNA molecules were separated from the solution using an external magnetic field. Subsequently, the complexes were rinsed several
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times with 30 lL of 70–30% ethanol-water and dried briefly. Next, the DNA molecules on the magnetic supports were eluted using a TE buffer (10 mM Tris–HCl, 1 mM EDTA in pH 7.8). In other words, 30 lL TE buffer was added to the dried complexes, followed by incubation at 60 °C for 10 min. Then, the MNPs were removed using a magnetic field, and the eluted DNA molecules were collected by centrifugation for quantitation purposes. For comparison studies, commercially available magnetic beads (NucliSENS-easyMAG, BioMérieux, Durham, NC, USA) were used according to the manufacturer’s instructions. An easyMAG Lysis Buffer and two types of wash solutions (easyMAG Extraction Buffer 1 and 2) were used for DNA extraction. First, 500 lg of the PCR products in DW was mixed with the lysis buffer, and the complexes were formed using commercial beads. DNA molecules were successively washed with 100 lL of wash solution 1 and 100 lL of wash solution 2, followed by the elution of DNA molecules with 30 lL of the buffer (NucliSENS easyMAG Extraction buffer 3). The other procedures used were the same as those for the MNPs. The evaluation of the isolated DNA molecules from the MNPs, DMSA-MNPs, and commercial beads was performed using agarose gel electrophoresis and UV spectrophotometry. The inputted and eluted DNA molecules from each solid support were loaded in 1.5% agarose gel containing 0.5 mg ethidium bromide per milliliter and electrophoresed at a voltage of 150 V for 15 min. The DNA bands were visualized using a gel image system (ChemiDOC XRS+, Molecular Imager ChemicDocTM XRS Imaging System, BioRad Laboratories Inc., Hercules, CA, USA), while the quantity was analyzed using the AlphaEaseFC (Genetic Technologies Inc., Miami, FL, USA). In addition, the quality of the isolated DNA molecu–Vis measurements. Isolation of genomic DNA from human blood and direct PCR amplification in beta actin gene The DMSA-MNPs were used for isolating human genomic DNA molecules from human blood. First, various volumes of human blood (2, 4, and 8 lL) were added separately to 200 lL of a lysis buffer solution (3.0 M NaI solution). Then, a binding buffer solution (18.2 wt% polyethylene glycol 8000 and 4.0 M NaCl) was added, followed by incubation with 3 lg of DMSA-MNPs at room temperature for 10 min. The samples were washed several times with 30 lL of 70–30% ethanol–water and dried briefly. The resultant mixture was used directly as a PCR template without any purification or elution. The human b-actin from blood was amplified using oligonucleotide primers: b-actin-F1, 50 -GAGAAAATCTGGCACCACAC-30 ; b-actin-R1, 50 -GATGTCCACGTCACACTTCA-30 . The concentration of the primers was 0.1 lM in 100 lL of the reaction mixture (10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP), containing 10 lL of DNA and 2.5 units of TaKaRa Ex Taq polymerase (Takara Bio Inc., Shiga, Japan). The reaction mixtures were cycled 30 times. Each cycle included denaturation at 94 °C for 30 s, annealing at 64 °C for 30 s, and extension at 72 °C for 30 s in a PCR Thermal Cycler TP600 (Takara Bio Inc., Shiga, Japan). The 16S rRNA-amplified products were size-fractionated using agarose gels (1.5%) by electrophoresis with ethidium bromide (0.5 mg/mL). Results and discussion The MNPs were synthesized by a modified polyol method as described in our previous work [27]. As shown in Fig. 1a, the nanoparticles are monodispersed and spherical with an average diameter of 8.4 nm and a narrow distribution (±1.1 nm). The high resolution, as shown in the inset of Fig. 1a, reveals that the nanoparticles possess distinct lattices and, therefore, high crystallinity.
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Fig.1. Morphology and hysteresis curve of magnetic nanoparticles. (a) TEM image (inset: high resolution image); (b) magnetic hysteresis curve (inset: magnification of hysteresis curve around zero field).
The corresponding magnetic properties were measured using VSM. As depicted in Fig. 1b, the magnetic hysteresis loop exhibits typical superparamagnetic behavior with near zero coercivity, thus preventing the self-aggregation of magnetic particles. On the contrary, commercially available beads had soft ferromagnetic traits with a coercivity of 180 Oe, indicating that after the applied magnetic field was removed, a residual magnetization (remanence) was present in the MNPs causing undesired spontaneous aggregation. As PEG-PPG-PEG was used as a surfactant in the synthetic process, the fresh nanoparticles were coated with the block copolymer containing hydroxyl groups [28]. In order to modify the nanoparticle surface with carboxyl groups for DNA complexation, DMSA solution was added to the nanoparticles dispersed in ethanol. Even after the surface modification, the nanoparticles possessed good superparamagnetic behavior, indicating that the coating process had a negligible impact on the magnetic properties, and the resulting DMSA-MNPs were resistant against aggregation. The surface property of the fresh MNPs and DMSA-MNPs was examined using zeta potential measurements. The zeta potential of the fresh nanoparticles was 20.1±3.7 mV while that of the DMSA-MNPs was 51.4±7.2 mV, implying that the surface was, unambiguously, more negatively charged, and thus, the coating process was effective. The surface condition was further inspected using FTIR (Fig. 2). The FTIR spectra of the fresh nanoparticles, as anticipated, showed a strong peak at 1115 cm, which was assigned to the C– O–C stretching vibration, and a characteristic peak for the C–H bending vibration at 1570 cm, which corresponded to the coating
Fig.2. FTIR spectra of (a) DMSA-coated magnetic nanoparticles and (b) fresh magnetic nanoparticles.
of PEG-PPG-PEG on the nanoparticle surface [28]. Nevertheless, no peaks for the C–O–C bonding appeared around 1120 cm in the DMSA-MNPs, suggesting that the polymer was replaced with DMSA after coating. Instead, the DMSA-MNPs displayed two characteristic bands at 1610 and 1390 cm, consistent with the previous reports [29–30]. The bands were derived from the symmetric and asymmetric stretches of the carboxyl groups (–CO2H), revealing that the DSMA molecules were successfully loaded onto the nanoparticle surface [30]. Subsequently, we tested the DMSA-MNPs against the fresh MNPs for the isolation of DNA. For this, 1 lg of the PCR products was used as the DNA source, and 250 lg of the DMSA-MNPs or fresh MNPs were added to isolate the DNA molecules. The outcome of the electrophoresis of the DMSA-MNPs was much brighter than that of the fresh nanoparticles, indicating that the yield of DNA isolation of the DMSA-MNPs was significantly higher than that of the fresh MNPs. This was because the carboxyl groups of DMSA on the nanoparticle surface were connected to the DNA molecules through hydrogen bonding, thereby efficiently isolating DNA molecules [13,14]. The enhanced yield of the DMSA-MNPs demonstrated that DMSA was successfully coated onto the MNPs; thus, the carboxyl groups on the surface of the MNPs increased the efficiency of absorption. In addition, the optical density ratio, OD 260/ OD 280, was 1.8, indicating that the DNA was of good quality. Next, the DNA-isolation efficiency of the DMSA-MNPs was compared to that of commercially available beads by investigating the isolation yields as a function of concentration. As the commercial beads, the NucliSENS-easyMAG (BioMerieux) was employed for this comparison study, which has been widely used for the automated extraction of DNA from clinical samples [31]. To evaluate the isolation efficiency, 500 ng of the amplified PCR products was used as the target DNA in each experiment. Fig. 3 shows two sets of the DNA-isolation yields: DMSA-MNPs and commercially available beads. The amount of solid magnetic beads used was in the range 2–128 mg. As shown in Fig. 3b, the DMSA-MNPs had a maximum isolation yield of 86.16%, which was higher than that of commercially available beads (83.41%). In particular, even with small quantities of DMSA-MNPs, the isolation of DNA was much more efficient than that of commercially available beads. To obtain 500 ng of DNA, 2–4 lg DMSA-MNPs provided an isolation yield of 60–70%, while commercially available beads provided a lower isolation yield with the same quantities. Though 16 lg DMSAMNPs provided a DNA isolation yield of 80%, at least 128 lg of commercially available beads had to be used to obtain the same yield. However, the isolation yields of the DMSA-MNPs decreased with increasing amount of the particle after the saturation point. Such trends for the DNA isolation yields could be observed because excess amounts of magnetic supports made it difficult for the
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of the PCR products of the genomic DNA when 3 lg DMSA-MNPs was used in the human blood series of 2, 4, and 6 lL. In all the samples, the b-actin genes from the genomic DNA were successfully amplified by the PCR process. This demonstrated that the DMSAMNPs were not only useful for isolating the genomic DNA from human whole blood but also for amplifying the human genes by a simplified PCR procedure. Conclusion
Fig.3. Comparison of DNA isolation efficiency of small quantities of DMSA-MNPs with that of commercial beads. Here, 1 lg DNA was bound separately with DMSAMNPs as well as commercial beads in various quantities. DNA isolated from these particles was verified using agarose gel analysis (a) and isolation yields were plotted as a function of the quantity of particles (b).
separation of DNA molecules from the DNA-magnetic support complexes, for instance, the disruption of DNA elution from magnetic supports and interference of excess magnetic supports in collecting DNA by applying a magnetic field [17]. Finally, the DMSA-MNPs were used for isolating genomic DNAs from human whole blood, followed by PCR amplification. First, samples of lysis solution were prepared from a human blood series (2, 4, and 6 lL), and 3 lg of the DMSA-MNPs was added to each lysis solution for binding with the genomic DNA molecules. Then, the DNA-bound DMSA-MNPs were isolated using an external magnetic field. The isolated DNA complexes could be amplified by PCR modulation for detecting specific genes. In particular, the resultant DNA-bound DMSA-MNPs complexes were used directly for PCR modulation without prior elution, which simplified the entire process of gene detection. Fig. 4 presents the results of electrophoresis
Fig.4. Agarose gel electrophoresis showing PCR amplification of b-actin gene (621 bp) in genomic DNA, isolated from human whole blood using 3 lg DMSAMMPs. Lines 1, 2, and 3 correspond to results obtained from 2, 4, and 8 lL human blood, respectively; Line M is DNA molecular mass maker.
We have successfully prepared DMSA-coated MNPs for the isolation of DNA by a modified polyol method. The TEM observations revealed that the DMSA-MNPs were monodispersed and highly crystalline with a narrow size distribution while the VSM results verified their superparamagnetic behavior, a desirable trait for bio-based applications. The MNPs were coated with DMSA for the isolation of DNA. We demonstrated that the DMSA-MNP complexes were highly efficient in binding to and thereby isolating the DNA molecules from crude biological samples. In particular, the DMSA-MNPs were much more useful for the isolation of DNA, even in relatively small quantities, compared to commercially available beads. Furthermore, the DMSA-MNPs were directly applied to the isolation of human genomic DNA without prior elution or purification, thereby leading to a simple, rapid, and time-saving analysis for the detection of diseases. Consequently, even small quantities of the biofunctionalized nanoparticles could be potentially utilized for a highly efficient isolation of DNA from biological samples. Their other biomedical applications include highly sensitive biosensing and automatic diagnostic systems. Acknowledgments This research was supported by the Pioneer Research Center Program (NRF-2011-0002128), the Leap Research Program (NRF2010-0017950), and the General Research Program (2013R1A1A2013842) by the National Research Foundation of Korea. J.H. Min acknowledges financial support by Korea University. References [1] S.C. Tan, B.C. Yiap, DNA, RNA, and protein extraction: the past and the present, J. Biomed. Biotechnol. 2009 (2009) 574398. [2] S. Berensmeier, Magnetic particles for the separation and purification of nucleic acids, Appl. Microbiol. Biotechnol. 73 (3) (2006) 495–504. [3] J. Corchero, A. Villaverde, Biomedical applications of distally controlled magnetic nanoparticles, Trends Biotechnol. 27 (8) (2009) 468–476. [4] S.C. Dixon, J. Horti, Y. Guo, E. Reed, W.D. Figg, Methods for extracting and amplifying genomic DNA isolated from frozen serum, Nat. Biotechnol. 16 (1) (1998) 91–94. [5] N.A. Saunders, Application of nanomaterials to arrays for infectious disease diagnosis, Nanomedicine 6 (2) (2011) 271–280. [6] D.A. Dederich, G. Okwuonu, T. Garner, A. Denn, A. Sutton, M. Escotto, A. Martindale, O. Delgado, D.M. Muzny, R.A. Gibbs, M.L. Metzker, Glass bead purification of plasmid template DNA for high throughput sequencing of mammalian genomes, Nucleic Acids Res. 30 (7) (2002). [7] I.I. Shakhmaeva, E.R. Bulatov, O.V. Bondar, D.V. Saifullina, M. Culha, A.A. Rizvanov, T.I. Abdullin, Binding and purification of plasmid DNA using multilayered carbon nanotubes, J. Biotechnol. 152 (3) (2011) 102–107. [8] Z.M. Saiyed, M. Parasramka, S.D. Telang, C.N. Ramchand, Extraction of DNA from agarose gel using magnetic nanoparticles (magnetite or Fe3O4), Anal. Biochem. 363 (2) (2007) 288–290. [9] S.M. Azimi, G. Nixon, J. Ahern, W. Balachandran, A magnetic bead-based DNA extraction and purification microfluidic device, Microfluid Nanofluidics 11 (2) (2011) 157–165. [10] K. Aguilar-Arteaga, J.A. Rodriguez, E. Barrado, Magnetic solids in analytical chemistry: a review, Anal. Chim. Acta 674 (2) (2010) 157–165. [11] R. Hao, R.J. Xing, Z.C. Xu, Y.L. Hou, S. Gao, S.H. Sun, Synthesis, functionalization, and biomedical applications of multifunctional magnetic nanoparticles, Adv. Mater. 22 (25) (2010) 2729–2742. [12] K. Kang, J. Choi, J.H. Nam, S.C. Lee, K.J. Kim, S.W. Lee, J.H. Chang, Preparation and characterization of chemically functionalized silica-coated magnetic nanoparticles as a DNA separator, J. Phys. Chem. B 113 (2) (2009) 536–543.
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