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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
Department of Materials Science and Engineering, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-713, Republic of Korea
b
Pioneer Research Center for Biomedical Nanocrystals, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-713, Republic of Korea
c
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 a b s t r a c t

Article history: Lately, the isolation of DNA using magnetic nanoparticles has received increased attention owing to their
Received 27 August 2013 facile manipulation and low costs. Although methods involving their magnetic separation have been
Received in revised form 10 November 2013 extensively studied, there is currently a need for an efficient technique to isolate DNA for highly sensitive
Accepted 17 November 2013
diagnostic applications. We describe herein a method to isolate and purify DNA using biofunctionalized
Available online 27 November 2013
superparamagnetic nanoparticles synthesized by a modified polyol method to obtain the desired mono-
dispersity, followed by surface modification with meso-2,3-dimercaptosuccinic acid (DMSA) containing
Keywords:
carboxyl groups for DNA absorption. The DMSA-coated magnetic nanoparticles (DMSA-MNPs) were used
DNA isolation
Magnetic nanoparticle
for the isolation of DNA, with a maximum yield of 86.16%. In particular, we found that the isolation of
Dimercaptosuccinic acid DNA using small quantities of DMSA-MNPs was much more efficient than that using commercial micro-
Superparamagnetism beads (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 variety of solid-phase supports (e.g., silica, glass fibers, carbon nano-
and a fundamental step for initiating other downstream activities tubes, and magnetic particles) [6–8]. Recent advances in the devel-
such as sequencing, amplification, hybridization, ligation, cloning, opment of integrated systems to isolate DNA have employed the
and biodetection. Several researchers have studied DNA isolation solid-phase supports [9], among which magnetic nanoparticles
for potential applications such as diagnosis of diseases, pathogen (MNPs) have received most attention because of their easy manipu-
detection, and gene therapy [1–5]. Various methods have been em- lation and cost-effectiveness [10,11]. In addition, separation tech-
ployed to separate DNAs from crude biological samples; such niques involving MNPs offer significant benefits over conventional
methods have been generally classified into either fluid-phase or methods owing to shorter processing times, sustainable costs, lim-
solid-phase categories [1,2]. The fluid-phase extraction methods ited use of chemicals, ease of automation, and minor physical or
involving a phenol/chloroform mixture and cetyltrimethylammo- chemical damage.
nium bromide (CTAB)1 usually comprise complicated steps, includ- The absorption of DNA molecules on solid supports is driven
ing centrifugation, precipitation, and filtration. Moreover, this through hydrogen-bonding and hydrophobic and electrostatic
processing is time-consuming, labor-intensive, and toxic [1–3]. interactions, which are intimately associated with the surface con-
Hence, alternative isolation methods have been proposed using a dition 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
⇑ Corresponding authors. Fax: +82 2 2626 1465 (C.-S. Lim), Fax: +82 2 928 3584 recovery efficiency of DNA molecules on the carrier surface. For
(Y.K. Kim). example, MNPs have been surface-modified using functional
E-mail addresses: malarim@korea.ac.kr (C.-S. Lim), ykim97@korea.ac.kr groups (–CO2H, –OH, and –NH2) [13–17], whereas hybrid magnetic
(Y.K. Kim). particles have been coated with inorganic materials (e.g., silica and
1
Abbreviations used: DMSA, meso-2,3-dimercaptosuccinic acid; DMSA-MNPs,
gold) for achieving high loading (isolation) efficiency [12,16,18–
DMSA-coated magnetic nanoparticles; DW, deionized water; PEG-PPG-PEG, poly(eth-
ylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol); VSM, vibrat- 20]. Nevertheless, the current requirements of highly sensitive
ing sample magnetometer. diagnosis and miniaturization have propelled the development of

0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ab.2013.11.018
Isolation of DNA using magnetic nanoparticles coated with DMSA / J.H. Min et al. / Anal. Biochem. 447 (2014) 114–118 115

more effective DNA isolation systems. Consequently, there is cur- times with 30 lL of 70–30% ethanol-water and dried briefly. Next,
rently tremendous interest in exploring novel solid supports and the DNA molecules on the magnetic supports were eluted using a
surface-modification methods. TE buffer (10 mM Tris–HCl, 1 mM EDTA in pH 7.8). In other words,
In this work, we propose surface-modified superparamagnetic 30 lL TE buffer was added to the dried complexes, followed by
nanoparticles for an effective isolation of DNA. Unlike most of incubation at 60 °C for 10 min. Then, the MNPs were removed
the previous studies, the MNPs used herein were prepared in the using a magnetic field, and the eluted DNA molecules were col-
form of solid supports by an organic-phase synthesis known to lected by centrifugation for quantitation purposes.
facilitate the production of superparamagnetic nanoparticles with For comparison studies, commercially available magnetic beads
desired monodispersity [21,22]. The surface of the resultant mono- (NucliSENS-easyMAG, BioMérieux, Durham, NC, USA) were used
sized MNPs was functionalized using meso-2,3-dimercaptosucci- according to the manufacturer’s instructions. An easyMAG Lysis
nic acid (DMSA). The DMSA molecule possesses both carboxylate Buffer and two types of wash solutions (easyMAG Extraction Buffer
and thiol groups, which are able to bond effectively with DNA mol- 1 and 2) were used for DNA extraction. First, 500 lg of the PCR
ecules [23]. The resulting complex could be used for DNA absorp- products in DW was mixed with the lysis buffer, and the com-
tion because of its high affinity for MNPs, stability in aqueous plexes were formed using commercial beads. DNA molecules were
solution, and easy bioconjugation. This complex can be used in var- successively washed with 100 lL of wash solution 1 and 100 lL of
ious bio-based applications such as MRI agents, nano-carriers for wash solution 2, followed by the elution of DNA molecules with
cancer therapy, and investigation of cell responses [24–26]. In 30 lL of the buffer (NucliSENS easyMAG Extraction buffer 3). The
the present work, the DMSA-coated MNPs were evaluated for their other procedures used were the same as those for the MNPs.
DNA isolation efficiency, and the results were compared with the The evaluation of the isolated DNA molecules from the MNPs,
efficiency of other surface-modified nanoparticles, including com- DMSA-MNPs, and commercial beads was performed using agarose
mercially available beads. 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
Materials and methods
and electrophoresed at a voltage of 150 V for 15 min. The DNA
bands were visualized using a gel image system (ChemiDOC
Preparation of DMSA-coated magnetic nanoparticles
XRS+, Molecular Imager ChemicDocTM XRS Imaging System, Bio-
Rad Laboratories Inc., Hercules, CA, USA), while the quantity was
The synthesis of MNPs was carried out by a modified polyol
analyzed using the AlphaEaseFC (Genetic Technologies Inc., Miami,
method [27]. Iron acetylacetonate (Fe(acac)3), 1,2-hexadecanediol,
FL, USA). In addition, the quality of the isolated DNA molecu–Vis
and poly(ethylene glycol)-block-poly(propylene glycol)-block-
measurements.
poly(ethylene glycol) (PEG-PPG-PEG) were used as an iron precur-
sor, a reducing agent, and a surfactant, respectively. The materials
were mixed in octyl ether and heated to 300 °C with continuous Isolation of genomic DNA from human blood and direct PCR
magnetic stirring. The mixture was then cooled down to room tem- amplification in beta actin gene
perature followed by the precipitation of the nanoparticles using
ethanol. Then, the precipitates were washed using centrifugation; The DMSA-MNPs were used for isolating human genomic DNA
finally, they were extracted by an external magnetic field. To molecules from human blood. First, various volumes of human
accomplish the surface modification of the MNPs, 1 mmol DMSA blood (2, 4, and 8 lL) were added separately to 200 lL of a lysis
in 5 mL of dimethyl sulfoxide (DMSO) was added to 3 mg MNPs buffer solution (3.0 M NaI solution). Then, a binding buffer solution
in 1 mL of ethanol. Then, the mixture was sonicated at 4 °C for (18.2 wt% polyethylene glycol 8000 and 4.0 M NaCl) was added,
6 h, and the resultant product was washed with ethanol by centri- followed by incubation with 3 lg of DMSA-MNPs at room temper-
fugation. Finally, the precipitated nanocomplex was dissolved in ature for 10 min. The samples were washed several times with
deionized water (DW). All the chemicals were purchased from Sig- 30 lL of 70–30% ethanol–water and dried briefly. The resultant
ma-Aldrich (St. Louis, MO, USA). The MNPs were analyzed using mixture was used directly as a PCR template without any purifica-
transmission electron microscopy (TEM, JEOL 2010F), zeta poten- tion or elution. The human b-actin from blood was amplified using
tial measurement, vibrating sample magnetometer (VSM, Lake- oligonucleotide primers: b-actin-F1, 50 -GAGAAAATCTGGCACCA-
shore 7300), and Fourier transform infrared spectroscopy (FTIR, CAC-30 ; b-actin-R1, 50 -GATGTCCACGTCACACTTCA-30 . The concen-
PerkinElmer Spectrum GX). tration 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
DNA complexation and isolation by the nanoparticles
TaKaRa Ex Taq polymerase (Takara Bio Inc., Shiga, Japan). The reac-
tion mixtures were cycled 30 times. Each cycle included denatur-
We evaluated the efficiency of DNA isolation by the DMSA-
ation at 94 °C for 30 s, annealing at 64 °C for 30 s, and extension
coated MNPs (DMSA-MNPs), and compared the results with those
at 72 °C for 30 s in a PCR Thermal Cycler TP600 (Takara Bio Inc.,
obtained using the fresh Fe3O4 MNPs and commercial beads. The
Shiga, Japan). The 16S rRNA-amplified products were size-fraction-
amplified PCR products (700 bp), which were used as the DNA
ated using agarose gels (1.5%) by electrophoresis with ethidium
source, were obtained from the control primers in the TOPO TA
bromide (0.5 mg/mL).
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 Results and discussion
first dissolved in 15 lL of a binding buffer containing 18.2 wt%
polyethylene glycol 8000 (Sigma–Aldrich, St. Louis, MO, USA) and The MNPs were synthesized by a modified polyol method as de-
4.0 M NaCl. Then, 250 lg of the MNPs or DMSA-MNPs was added. scribed in our previous work [27]. As shown in Fig. 1a, the nano-
After incubating the resulting mixture at room temperature for particles are monodispersed and spherical with an average
10 min, the complexes formed by the magnetic supports and diameter of 8.4 nm and a narrow distribution (±1.1 nm). The high
DNA molecules were separated from the solution using an external resolution, as shown in the inset of Fig. 1a, reveals that the nano-
magnetic field. Subsequently, the complexes were rinsed several particles possess distinct lattices and, therefore, high crystallinity.
116 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. of PEG-PPG-PEG on the nanoparticle surface [28]. Nevertheless, no
As depicted in Fig. 1b, the magnetic hysteresis loop exhibits typical peaks for the C–O–C bonding appeared around 1120 cm in the
superparamagnetic behavior with near zero coercivity, thus pre- DMSA-MNPs, suggesting that the polymer was replaced with
venting the self-aggregation of magnetic particles. On the contrary, DMSA after coating. Instead, the DMSA-MNPs displayed two char-
commercially available beads had soft ferromagnetic traits with a acteristic bands at 1610 and 1390 cm, consistent with the previ-
coercivity of 180 Oe, indicating that after the applied magnetic ous reports [29–30]. The bands were derived from the symmetric
field was removed, a residual magnetization (remanence) was and asymmetric stretches of the carboxyl groups (–CO2H), reveal-
present in the MNPs causing undesired spontaneous aggregation. ing that the DSMA molecules were successfully loaded onto the
As PEG-PPG-PEG was used as a surfactant in the synthetic pro- nanoparticle surface [30].
cess, the fresh nanoparticles were coated with the block copolymer Subsequently, we tested the DMSA-MNPs against the fresh
containing hydroxyl groups [28]. In order to modify the nanoparti- MNPs for the isolation of DNA. For this, 1 lg of the PCR products
cle surface with carboxyl groups for DNA complexation, DMSA was used as the DNA source, and 250 lg of the DMSA-MNPs or
solution was added to the nanoparticles dispersed in ethanol. Even fresh MNPs were added to isolate the DNA molecules. The outcome
after the surface modification, the nanoparticles possessed good of the electrophoresis of the DMSA-MNPs was much brighter than
superparamagnetic behavior, indicating that the coating process that of the fresh nanoparticles, indicating that the yield of DNA iso-
had a negligible impact on the magnetic properties, and the result- lation of the DMSA-MNPs was significantly higher than that of the
ing DMSA-MNPs were resistant against aggregation. The surface fresh MNPs. This was because the carboxyl groups of DMSA on the
property of the fresh MNPs and DMSA-MNPs was examined using nanoparticle surface were connected to the DNA molecules
zeta potential measurements. The zeta potential of the fresh nano- through hydrogen bonding, thereby efficiently isolating DNA mol-
particles was 20.1±3.7 mV while that of the DMSA-MNPs was ecules [13,14]. The enhanced yield of the DMSA-MNPs demon-
51.4±7.2 mV, implying that the surface was, unambiguously, strated that DMSA was successfully coated onto the MNPs; thus,
more negatively charged, and thus, the coating process was effec- the carboxyl groups on the surface of the MNPs increased the effi-
tive. The surface condition was further inspected using FTIR ciency of absorption. In addition, the optical density ratio, OD 260/
(Fig. 2). The FTIR spectra of the fresh nanoparticles, as anticipated, OD 280, was 1.8, indicating that the DNA was of good quality.
showed a strong peak at 1115 cm, which was assigned to the C– Next, the DNA-isolation efficiency of the DMSA-MNPs was com-
O–C stretching vibration, and a characteristic peak for the C–H pared to that of commercially available beads by investigating the
bending vibration at 1570 cm, which corresponded to the coating 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 auto-
mated 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 avail-
able 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 max-
imum isolation yield of 86.16%, which was higher than that of com-
mercially 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 DMSA-
MNPs 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.
Fig.2. FTIR spectra of (a) DMSA-coated magnetic nanoparticles and (b) fresh Such trends for the DNA isolation yields could be observed because
magnetic nanoparticles. 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) 114–118 117

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 sam-
ples, the b-actin genes from the genomic DNA were successfully
amplified by the PCR process. This demonstrated that the DMSA-
MNPs were not only useful for isolating the genomic DNA from hu-
man whole blood but also for amplifying the human genes by a
simplified PCR procedure.

Conclusion

We have successfully prepared DMSA-coated MNPs for the iso-


lation 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 com-
plexes 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 purifica-
tion, thereby leading to a simple, rapid, and time-saving analysis
Fig.3. Comparison of DNA isolation efficiency of small quantities of DMSA-MNPs for the detection of diseases. Consequently, even small quantities
with that of commercial beads. Here, 1 lg DNA was bound separately with DMSA- of the biofunctionalized nanoparticles could be potentially utilized
MNPs as well as commercial beads in various quantities. DNA isolated from these for a highly efficient isolation of DNA from biological samples.
particles was verified using agarose gel analysis (a) and isolation yields were
Their other biomedical applications include highly sensitive bio-
plotted as a function of the quantity of particles (b).
sensing and automatic diagnostic systems.

separation of DNA molecules from the DNA-magnetic support Acknowledgments


complexes, for instance, the disruption of DNA elution from mag-
netic supports and interference of excess magnetic supports in col- This research was supported by the Pioneer Research Center
lecting DNA by applying a magnetic field [17]. Program (NRF-2011-0002128), the Leap Research Program (NRF-
Finally, the DMSA-MNPs were used for isolating genomic DNAs 2010-0017950), and the General Research Program
from human whole blood, followed by PCR amplification. First, (2013R1A1A2013842) by the National Research Foundation of Kor-
samples of lysis solution were prepared from a human blood series ea. J.H. Min acknowledges financial support by Korea University.
(2, 4, and 6 lL), and 3 lg of the DMSA-MNPs was added to each ly-
sis solution for binding with the genomic DNA molecules. Then, the
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