Download Magnetic Nanoparticles as separators of nucleic acids

Katarzyna NIEMIROWICZ – Department of Experimental Pharmacology, Medical University
of Białystok; Agnieszka Z. WILCZEWSKA – Institute of Chemistry, University of Białystok; Halina
CAR – Department of Experimental Pharmacology, Medical University of Bialystok, Poland
Please cite as: CHEMIK 2013, 67, 10, 836–841
Introduction
Molecular biology deals with the explanation of how the
functioning of living organisms is subject to the characteristics of
particles, and especially biopolymers such as nucleic acids and proteins.
Along with the development and advancement of molecular biology
techniques, it was possible to develop several methods of separation
of macromolecules using different physicochemical phenomena [1, 2].
These methods are based mainly on the properties of macromolecules,
such as surface charge, the presence of functional groups and the
generation of hydrophobic interactions [3]. Nanotechnology is an
interdisciplinary science and its robust growth is directly related to the
advances in medical science. There are high hopes associated with
the use of magnetic nanoparticles (MNP – Magnetic Nanoparticles) in
molecular biology. These display a number of features that enable them
to provide a basis for their use as separation tools. In addition to the
possessed surface charge and related electro-kinetic properties, they
are characterized by magnetic properties which facilitate the separation
process, and thereby the puriication of the resulting conjugate
to a constant magnetic nano-phase. Another important feature of the
nanostructures is a large surface area which allows the immobilization
of many biologically active molecules at the same time [4÷6]. MNP
are used in many biomedical applications, including the controlled
delivery of drugs (DDS – Drug Delivery Systems) [7], targeted therapy
(TT – Target Therapy) [8] and at the level of diagnostic separation in
the sorting of cells [9], in isolation and detection of bacteria [10, 11] and
also as nano-biosensors to detect exogenous substances [12].
This paper presents the synthesis and application of magnetic nanoparticles functionalized by aminopropyltrimethoxysilane (APTMS)
for the immobilization of DNA. The analysis of the conjugates of DNAnanoparticle was performed by a spectroscopy of diffuse relectance
in infrared (DRIFTS – Diffuse Relectance Infrared Fourier Transform
Spectroscopy).
Material and methods
The hydrated salts of iron chloride (II) and (III), 25% solution of
an ammonium base, 3-aminopropyltrimethoxysilane (APTMS) were
purchased from Sigma-Aldrich company, water was deionized with
a Milli-Q system (Millipore), a yeast species of Saccharomyces cerevisiae
was used for testing. A magnetic separation was carried out with
neodymium magnet.
After obtaining a ferroluid suspension, APTMS was added (0.5 ml).
The reaction was conducted under argon in a slightly alkaline medium
in the presence of ultrasound. The synthesized nanoparticles were
analyzed by infrared spectroscopy (FTIR) Figure 1.
Fig. 1. FT-IR spectra of aminosilane nanoparticles Abbreviations: [%T]
– transmittance FT-IR (KBr, ν, cm-1): 3428 – stretching N–H bonds;
1115 – stretching C-O bonds; 1035 – stretching Si–O bonds;
693 – stretching Fe–O bonds
Isolating DNA from Saccharomyces cerevisiae
Sodium chloride (4 g) and sodium dodecyl sulfate (30 mg) were
dissolved in deionized water (90 ml). Then, 20 g of baker’s yeast
(Saccharomyces cerevisiae) were added. The mixture was incubated
for 15 min at 60°C in a water bath, then cooled in an ice bath for
5 min, sonicated for 1 min and iltered. Then, 5 drops of pineapple juice
were added to the iltrate and dispensed into test tubes (10 ml). In the
inal stage, chilled ethanol (95%) was added to each tube. DNA was
knocked off in the form of ine white strands [14].
Immobilization of DNA on the surface of MNP
Magnetic nanoparticles functionalized with o-aminopropyl (30 mg)
groups were suspended by sonication in deionized water (10 ml) and
then added to the extract containing isolated nucleic acid (10 ml). The
nanoparticles were separated from the solution using an external magnetic
ield. The moistened substance was decanted and the nanoparticles were
transferred via Pasteur pipette to aluminum foil, left to dry and analyzed
by diffuse relectance spectroscopy DRIFTS (Fig. 2).
The synthesis of magnetic nanoparticles MNP
For the synthesis of magnetic nanoparticles based on iron oxide,
a modiication of a known Massart method was used [13]. The basis
of the method is a co-precipitation of hydrated iron chloride (II) and
iron (III) in a 1:2 molar ratio of the reactants in a basic medium, which
contained a 25% aqueous solution of an ammonium base. The synthesis
was carried out in aqueous conditions in an argon atmosphere in
deionized water. During the synthesis, a sonication was used.
The MNP surface modiication of APTMS nanostructures
The nanoparticles obtained in the irst step were subjected
to a magnetic separation using a magnet, and puriied by washing
several times with ethanol and dried in an oven at 50°C. The cleaned
MNP (500 mg) was re-suspended in ethanol as a result of sonication.
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Fig. 2. DRIFTS spectra of MNP-DNA conjugate. Abbreviations:
KM –units of Kubelk-Munk = log inverse relectance (log1/R) versus
wavenumber. Opis widma: FT-IR (DRIFTS, ν, cm-1): 3415 – stretching
N-H bonds; 1660 – stretching C=O bonds; 1632 – bending N–H bonds;
1600 – stretching C=C of the imidazole ring ; 1225 – assymetric stretching PO2 bonds; 1178 – stretching C–O bonds; 1031 – stretching Si–O
bonds; 643 – stretching Fe–O stretching
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Magnetic Nanoparticles as separators of nucleic acids
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Results and discussion
The separation and puriication of DNA is a key stage for the most
of the procedures used in molecular biology. The main purpose of this
step is to obtain a stable biological material of high quality and purity,
regardless of its origin. When choosing a suitable method of isolation one
must take into account many aspects such as: the nature and origin of
the material, the type of nucleic acid (DNA, RNA) and the inal purpose
of the isolated acid [15]. Most isolated DNA is genomic or plasmid.
This paper presents the use of synthesized magnetic nanoparticles
for the immobilization of deoxyribonucleic acid (DNA) for MNP with
amino groups on their surface.
The co-precipitation reaction, and then polycondensation of
3-aminopropyltrimethoxysilane, resulted in obtaining magnetic
nanoparticles with terminal amino groups. The synthesized
nanoparticles were analyzed in the infrared spectral in KBr using
a Nicolet Magna IR 550 Series II spectrophotometer. It was
possible to identify intense signals at 1115 and 1035 cm-1, derived
respectively from the vibrations of stretched C–O and Si–O bonds.
In the spectrum there was a signal of vibration of the stretched N–H
bonds at 3428 cm-1 and the signal at 693 cm-1 corresponding to the
vibrations of the stretched Fe–O bonds.
To isolate DNA, a precipitation method with the yeast suspension
was used. Using sodium chloride and the SDS surfactant caused
a breakdown of cell membranes, nuclear envelope, organelle
membranes and other intracellular membranes. The incubation at
60°C accelerated the process of disintegration of biological
membranes and the denaturation of DNA-ase, which are the enzymes
naturally occurring in cells, able to digest DNA. In the next step,
the use of sonication led to a mechanical destruction of cell walls
and to the release of cell contents into the mixture. An addition of
fresh pineapple juice containing proteolytic enzymes, consumed the
proteins present in the iltered mixture. The concentrated ethanol
resulted in the precipitation of DNA from the solution.
The next stage was to perform the immobilization of DNA on
the aminosilane magnetic nanoparticles (Fig. 3). An originally cloudy
solution, under the inluence of an external magnetic ield, gained
clariication which conirms that there was an attachment of DNA
to MNP . The immobilization mechanism was based on a non-covalent
interaction of the surface amino groups of nanoparticles with the
functional groups of DNA. Most likely, a hydrogen bond was developed
between the electronegative nitrogen atoms of NH2 groups and the
hydrogen atoms of the functional groups of deoxyribonucleic acid. In
addition, it can be assumed that the surface charges on the surface of the
nanoparticles and of the nucleic acid played a role in the immobilization.
Aminosilane nanoparticles contain a positive surface charge, while
the nucleic acid has a negative charge. It is due to the presence of
ortophosphoric acid residues in its structure.
The obtained conjugate of MNP – DNA was analyzed by the
infrared relection method (Fig. 2). It revealed the presence of
signals characteristic to nucleic acids. These included signals at:
1660–1632 cm– 1 of C=O stretching vibrations and bending vibrations
of NH, 1600 cm– 1 stretching vibration of C=C of the imidazole ring.
The PO2 phosphate groups were identiied on the basis of the signals
at 1225 cm– 1 characteristic of the asymmetric stretching vibrations and
at 1096 cm– 1, corresponding to the symmetric stretching vibrations. In
addition, some characteristic signals were observed for the nanoparticles
at 1031 cm– 1 corresponding to the stretching vibrations of the Si –O and
the stretching vibrations of Fe–O bonds at 643 cm-1. The described
method allows to carry out a rapid and simple immobilization of
DNA without the need for speciic surface complexes of the ligands
such as streptavidin – biotin system [16, 17]. The technique based
on the use of silica microspheres was developed by Chen et al [18].
The resulting microspheres were used as the adsorbent used in the
extraction of DNA from plant material to the solid phase. There are
many procedures describing the method of DNA isolation, which
result in obtaining a RNA-free DNA, proteins and other compounds
that may interfere in subsequent analyzes. Isolation tends to break the
nucleic – protein complex (DNP) and to extract DNA in a possibly
unaltered state, biologically active and chemically stable. Due to the
size and sensitivity of the chromosomal DNA it is virtually impossible
to conduct its isolation in an unaltered state as a part of the DNA
undergoes a mechanical damage. The traditional methods of isolation
are all multi-step and labor-intensive processes. They require the use
of several reagents, as well as a high precision from the person carrying
out the process. When performing an isolation, a lot of mistakes can be
made, which may lead to the destruction of or damage to the structure
of DNA, or result in an incomplete isolation and obtaining a material that
is not suitable for further research.
Summary and Conclusions
As a result of the reaction of the co-precipitation of iron oxide,
and then the poly-condensation of 3-aminopropyltrimethoxysilane,
magnetic nanostructures were obtained of a core-shell type with
amino groups on their surface. A large surface area of nanoparticles
is also a large area of possible impacts of magnetic nanoparticles of
the amino groups with functional groups contained in the chains of
deoxyribonucleic acid, which in turn allows the adsorption of DNA from
a solution. The application of the obtained nanoparticles provides an
opportunity to use them as ixed magnetic sorbents used for separating
nucleic acids (by adsorption) during their isolation from the solution.
The proposed working method of isolating nucleic acids may provide
a basis for setting new standards in diagnostic molecular biology.
Acknowledgement
This study was performed under the project “Studying, researching,
commercializing – of the doctoral support program at UMB”, Measure 8.2.1 of the
Human Capital Operational Programme, co-inanced by the European Union under
the European Social Fund Programme
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Fig. 3. Immobilization of DNA on aminosilane nanoparticles. Abbreviations: MNP – magnetic nanoparticles; DNA – deoxyribonucleic acid;
MNP-DNA – conjugate magnetic nanoparticles – deoxyribonucleic acid
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Katarzyna NEMIROWICZ – M.Sc., is a graduate (chemistry) of the Faculty
of Biology and Chemistry, University of Bialystok (2011) and Laboratory
Medicine at the Medical University of Bialystok (2012). At present she is the
second year PhD student in the Department of Experimental Pharmacology,
Medical University of Bialystok, Faculty of Medicine. Since February 2013 she
has been a principal investigator the grant Prelude. She is also the beneiciary
of a program “I study, investigate, commercialize” – UMB doctoral support
program funded by the European Union under the European Social Fund as
well as the scholar of Polpharma Science Foundation Scholarship. Research
interests: organic synthesis, nanotechnology and targeted therapy. She is the
author and co-author of ive research papers, 13 papers and communications
runs at national and international conferences, as well as a patent application.
e-mail: [email protected], phone: +85 748 55 54
Agnieszka Z. WILCZEWSKA – Ph.D, Doctoral thesis defended at the
Faculty of Chemistry of the University of Warsaw in 1998. Currently, she works
at the Faculty of Biology and Chemistry, University of Bialystok, Department
of Chemistry of Natural Products. Scientiic interest: vinyl polymers, MADIX
polymerization and nanotechnology.
e-mail: [email protected]
Halina CAR – M.D., Ph.D., is a graduate of the Faculty of Medicine, Medical
University of Bialystok (1987). She obtained the degree of Doctor of Medicine
in 1990, and defended her habilitation thesis in 2007. In the years 1987÷2010
she worked in the Department of Pharmacology, Medical University of
Bialystok. For the last three years has been a head of the Department of
Experimental Pharmacology, Medical University of Bialystok. She works as
Voivodshipl Consultant in Clinical Pharmacology. Research interests: learning
and memory, neurodegeneration and possibilities of their therapy, tumor
processes in the brain, targeted therapy. She is the author and co-author of 58
scientiic articles published in the international medical journals and 80 papers
and communications presented at national and international conferences, as
well as a patent application
e-mail: [email protected], [email protected], phone:+85 748 55 54
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