Isolation of Nucleic Acids from Different Biological Objects with Silica—Magnetite Nanoparticles
Now, modified magnetic particles are widely used in different biological and
 medical applications (enzyme and protein immobilization, cells separation
 and purification, MRI, targeted drug delivery, etc.). The aim of the present
 study is to reveal the ability of synthesized...
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| Published in: | Наносистеми, наноматеріали, нанотехнології |
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| Date: | 2008 |
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| Language: | English |
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Інститут металофізики ім. Г.В. Курдюмова НАН України
2008
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| Cite this: | Isolation of Nucleic Acids from Different Biological Objects
 with Silica—Magnetite Nanoparticles / N.N. Volkova, O.N. Derjabin, N.O. Dudchenko // Наносистеми, наноматеріали, нанотехнології: Зб. наук. пр. — К.: РВВ ІМФ, 2008. — Т. 6, № 3. — С. 1009-1018. — Бібліогр.: 12 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1860089361670864896 |
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| author | Volkova, N.N. Derjabin, O.N. Dudchenko, N.O. |
| author_facet | Volkova, N.N. Derjabin, O.N. Dudchenko, N.O. |
| citation_txt | Isolation of Nucleic Acids from Different Biological Objects
 with Silica—Magnetite Nanoparticles / N.N. Volkova, O.N. Derjabin, N.O. Dudchenko // Наносистеми, наноматеріали, нанотехнології: Зб. наук. пр. — К.: РВВ ІМФ, 2008. — Т. 6, № 3. — С. 1009-1018. — Бібліогр.: 12 назв. — англ. |
| collection | DSpace DC |
| container_title | Наносистеми, наноматеріали, нанотехнології |
| description | Now, modified magnetic particles are widely used in different biological and
medical applications (enzyme and protein immobilization, cells separation
and purification, MRI, targeted drug delivery, etc.). The aim of the present
study is to reveal the ability of synthesized silica-modified magnetic particles
to isolate DNA from the biological tissues in comparison with common
method used the non-magnetic particles. Magnetite (Fe3O4) particles are prepared
via co-precipitation of Fe+2 and Fe+3 with NH4OH in aqueous solution.
Silica—magnetite nanocomposites are prepared via tetraethoxysilane hydrolyzation
in alcohol—water—ammonia mixture. The average core size of synthesized
magnetic nanoparticles is about 15 nm (according to the TEM data).
Application of these compounds for DNA isolation from different biological
objects showed significant time-savings, overall higher yields, lower RNA
contamination and better polymerase chain reaction (PCR) amplification
compared to commercial available silica non-magnetic particles (Promega).
High efficiency of nucleic-acid purification by silica—magnetite particles is
confirmed in molecular assays with reverse transcriptase—polymerase chain
reaction (RT—PCR) assays of RNA- and DNA-virus diseases of plants, avian,
cattle and estimation of bacterial spectrum in dairy products (probiotics).
Модифіковані магнетні частинки зараз широко використовуються для різних біологічних та медичних застосувань (іммобілізація ензимів та білків,
виділення та очищення клітин, ЯМР, направлена доставка ліків та ін.).
Метою цього дослідження було показати здатність синтезованих магнетних
частинок, модифікованих кремнеземом, виділяти ДНК з різних біологічних тканин у порівнянні із стандартною методою з використанням немагнетних частинок. Магнетитові (Fe3O4) частинки було одержано шляхом співосадження Fe+2 та Fe+3 за допомогою NH4OH у воднім розчині. Силікамагнетитові нанокомпозити були одержані шляхом гідролізації тетраетоксисилана у спирто-водяно-амонійній суміші. Середній розмір ядра синтезованих магнетних наночастинок був біля 15 нм (за даними просвітлювальної
електронної мікроскопії). Застосування цих сполук для виділення ДНК зрізних біологічних тканин виявило значне заощадження часу, загальний
більш високий вихід ДНК, більш низьку кількість домішок РНК та кращу
ампліфікацію полімеразної ланцюгової реакції (ПЛР) у порівнянні з традиційними силіційовими немагнетними частинками (Promega). Високу
ефективність очищення нуклеїнових кислот за допомогою силіка-магнетитових частинок було підтверджено в молекулярно-біологічних тестах, що
були виконані на основі зворотньої транскрипції з подальшою полімеразною ланцюговою реакцією (ЗТ—ПЛР) щодо виявлення РНК та ДНК вірусних захворювань рослин, птахів, сільськогосподарських тварин та оцінки
якости молочних продуктів (пробіотиків).
Модифицированные магнитные частицы сейчас широко используются в
различных биологических и медицинских приложениях (иммобилизация
энзимов и белков, выделение и очистка клеток, ЯМР, направленная доставка лекарств и т.д.). Целью настоящего исследования было показать
способность синтезированных магнитных частиц, модифицированных
кремнеземом, выделять ДНК из различных биологических тканей по
сравнению со стандартным методом с использованием немагнитных частиц. Магнетитовые (Fe3O4) частицы были получены путем соосаждения
Fe+2 и Fe+3 с помощью NH4OH в водном растворе. Кремний-магнетитовые
нанокомпозиты были приготовлены путем гидролизации тетраетоксисилана в спирто-водно-аммониевой смеси. Средний размер ядра синтезированных магнитных наночастиц был около 15 нм (по данным просвечивающей электронной микроскопии). Применение этих соединений для
выделения ДНК из различных биологических тканей выявило значительную экономию времени, общий более высокий выход ДНК, более
низкое количество примесей РНК и лучшую амплификацию полимеразной цепной реакции (ПЦР) по сравнению с коммерчески доступными
кремниевыми немагнитными частицами (Promega). Высокая эффективность очистки нуклеиновых кислот с помощью кремний-магнетитовых
частиц была подтверждена в молекулярно-биологических тестах, выполняемых на основе обратной транскрипции с последующей полимеразной
цепной реакцией (ОТ—ПЦР) по выявлению РНК и ДНК вирусных заболеваний растений, птиц, сельскохозяйственных животных и оценке качества молочных продуктов (пробиотиков).
|
| first_indexed | 2025-12-07T17:22:02Z |
| format | Article |
| fulltext |
1009
PACS numbers: 81.07.Nb, 81.16.Fg, 82.37.Rs, 82.39.-k, 82.45.-h, 87.14.-g, 87.15.Tt
Isolation of Nucleic Acids from Different Biological Objects
with Silica—Magnetite Nanoparticles
N. N. Volkova, O. N. Derjabin, and N. O. Dudchenko
Institute for Applied Problems of Physics and Biophysics, N.A.S. of Ukraine,
3 Sluzhbova Str.,
03142 Kyyiv, Ukraine
Now, modified magnetic particles are widely used in different biological and
medical applications (enzyme and protein immobilization, cells separation
and purification, MRI, targeted drug delivery, etc.). The aim of the present
study is to reveal the ability of synthesized silica-modified magnetic particles
to isolate DNA from the biological tissues in comparison with common
method used the non-magnetic particles. Magnetite (Fe3O4) particles are pre-
pared via co-precipitation of Fe+2
and Fe+3
with NH4OH in aqueous solution.
Silica—magnetite nanocomposites are prepared via tetraethoxysilane hydro-
lyzation in alcohol—water—ammonia mixture. The average core size of syn-
thesized magnetic nanoparticles is about 15 nm (according to the TEM data).
Application of these compounds for DNA isolation from different biological
objects showed significant time-savings, overall higher yields, lower RNA
contamination and better polymerase chain reaction (PCR) amplification
compared to commercial available silica non-magnetic particles (Promega).
High efficiency of nucleic-acid purification by silica—magnetite particles is
confirmed in molecular assays with reverse transcriptase—polymerase chain
reaction (RT—PCR) assays of RNA- and DNA-virus diseases of plants, avian,
cattle and estimation of bacterial spectrum in dairy products (probiotics).
Модифіковані магнетні частинки зараз широко використовуються для різ-
них біологічних та медичних застосувань (іммобілізація ензимів та білків,
виділення та очищення клітин, ЯМР, направлена доставка ліків та ін.).
Метою цього дослідження було показати здатність синтезованих магнетних
частинок, модифікованих кремнеземом, виділяти ДНК з різних біологіч-
них тканин у порівнянні із стандартною методою з використанням немаг-
нетних частинок. Магнетитові (Fe3O4) частинки було одержано шляхом спі-
восадження Fe+2 та Fe+3 за допомогою NH4OH у воднім розчині. Силіка-
магнетитові нанокомпозити були одержані шляхом гідролізації тетраеток-
сисилана у спирто-водяно-амонійній суміші. Середній розмір ядра синтезо-
ваних магнетних наночастинок був біля 15 нм (за даними просвітлювальної
електронної мікроскопії). Застосування цих сполук для виділення ДНК з
Наносистеми, наноматеріали, нанотехнології
Nanosystems, Nanomaterials, Nanotechnologies
2008, т. 6, № 3, сс. 1009—1018
© 2008 ІМФ (Інститут металофізики
ім. Г. В. Курдюмова НАН України)
Надруковано в Україні.
Фотокопіювання дозволено
тільки відповідно до ліцензії
1010 N. N. VOLKOVA, O. N. DERJABIN, and N. O. DUDCHENKO
різних біологічних тканин виявило значне заощадження часу, загальний
більш високий вихід ДНК, більш низьку кількість домішок РНК та кращу
ампліфікацію полімеразної ланцюгової реакції (ПЛР) у порівнянні з тра-
диційними силіційовими немагнетними частинками (Promega). Високу
ефективність очищення нуклеїнових кислот за допомогою силіка-магнети-
тових частинок було підтверджено в молекулярно-біологічних тестах, що
були виконані на основі зворотньої транскрипції з подальшою полімераз-
ною ланцюговою реакцією (ЗТ—ПЛР) щодо виявлення РНК та ДНК вірус-
них захворювань рослин, птахів, сільськогосподарських тварин та оцінки
якости молочних продуктів (пробіотиків).
Модифицированные магнитные частицы сейчас широко используются в
различных биологических и медицинских приложениях (иммобилизация
энзимов и белков, выделение и очистка клеток, ЯМР, направленная дос-
тавка лекарств и т.д.). Целью настоящего исследования было показать
способность синтезированных магнитных частиц, модифицированных
кремнеземом, выделять ДНК из различных биологических тканей по
сравнению со стандартным методом с использованием немагнитных час-
тиц. Магнетитовые (Fe3O4) частицы были получены путем соосаждения
Fe+2
и Fe+3
с помощью NH4OH в водном растворе. Кремний-магнетитовые
нанокомпозиты были приготовлены путем гидролизации тетраетоксиси-
лана в спирто-водно-аммониевой смеси. Средний размер ядра синтезиро-
ванных магнитных наночастиц был около 15 нм (по данным просвечи-
вающей электронной микроскопии). Применение этих соединений для
выделения ДНК из различных биологических тканей выявило значи-
тельную экономию времени, общий более высокий выход ДНК, более
низкое количество примесей РНК и лучшую амплификацию полимераз-
ной цепной реакции (ПЦР) по сравнению с коммерчески доступными
кремниевыми немагнитными частицами (Promega). Высокая эффектив-
ность очистки нуклеиновых кислот с помощью кремний-магнетитовых
частиц была подтверждена в молекулярно-биологических тестах, выпол-
няемых на основе обратной транскрипции с последующей полимеразной
цепной реакцией (ОТ—ПЦР) по выявлению РНК и ДНК вирусных заболе-
ваний растений, птиц, сельскохозяйственных животных и оценке качест-
ва молочных продуктов (пробиотиков).
Key words: silica—magnetite nanoparticles, DNA isolation, magnetic particles.
(Received November 21, 2007)
1. INTRODUCTION
The superior sensitivity of nucleic acid amplification technique en-
ables diagnosis of infectious diseases an early stage before positive se-
rologic results confirm an infection. These molecular methods have
become a standard application in clinical laboratory in recent years. In
addition to diagnosis of infectious diseases, the determination of virus
load has gained increasing importance in medical and veterinarian vi-
rology laboratory. Although the introduction of real-time polymerase
ISOLATION OF NUCLEIC ACIDS FROM DIFFERENT BIOLOGICAL OBJECTS 1011
chain reaction (PCR) has led to considerable progress in automating
the amplification and detection steps of molecular biological tech-
nique, a nuclear acid isolation remains very labour-intensive when per-
formed with traditional phenol—chloroform extraction and ethanol
precipitation methods. Additionally, these methods are too compli-
cated, time-consuming, and hazardous and produce on the last stage
denaturated nucleic acids.
Modified magnetic particles now are widely used in different bio-
logical and medical applications (enzyme and protein immobilization,
cells separation and purification, MRI, targeted drug delivery, etc.) [1,
2]. Due to the strong magnetic properties and low toxicity of magnetic
particles, their applications in biotechnology and medicine have gained
significant attention. Basically, all types of magnetic particles consist
of magnetic core with inorganic or organic shell. The target molecules
or cells are captured on magnetic particles coated with a target-specific
surface, and separated from unbound components by the application of
magnetic field. The need for quick bacterial plasmid DNA and virus
DNA/RNA preparation methods has increased the flood molecular
protocols requiring highly purified genetic templates [1—3].
Magnetic separation of DNA offer benefits over usual method due to
rapid processing time, reduced chemical needs, the ease of separation
[1]. Thus, the aim of the present study was to reveal the ability of syn-
thesized silica-modified magnetic particles to isolate DNA from dif-
ferent biological tissues in comparison with common method based on a
non-magnetic sorbents.
2. MATERIALS AND METHODS
Materials. Ferric chloride hexahydrate, ferrous sulphate tetrahy-
drate, tetraethoxysilane were purchased from Sigma Chemical Co.
Agarose L (low electroendosmoid) was from Amersham Biosciences
(Uppsala, Sweden). Reagents for use in DNA isolation and analysis
were of molecular biology grade. Ribonuclease A was obtained from
‘Sigma’. All other chemicals and solvents used were of analytical
grade. The water used throughout this work was the reagent-grade wa-
ter produced by Milli-Q Ultra-Pure-Water Purification System.
Preparation of Silica—Magnetite Nanocomposites. The magnetite
particles were prepared via co-precipitation of Fe+2
and Fe+3
with
NH4OH in aqueous solution under normal conditions. Stock solutions
of 1 M FeCl3⋅6H2O and 2 M FeSO4⋅4H2O were prepared as a source of
iron by dissolving the respective chemicals in deionised water under
stirring. Stock solution of 1 M NH4OH was prepared by dilution of con-
centrated NH4OH solution. The reagents solutions were mixed quickly
in reaction vessel, and 50 ml of ammonium solution was added drop-by-
drop to reaction mixture under slow mechanical stirring. After the re-
1012 N. N. VOLKOVA, O. N. DERJABIN, and N. O. DUDCHENKO
action completing, magnetic particles were lightly dispersed using ul-
trasound disperser, three times rinsed with deionised water to remove
the residual surfactant and unreacted reagents.
Obtained magnetite was coated with silica via tetraethoxysilane hy-
drolyzation in alcohol—water—ammonia mixture. Thereto, obtained
magnetic particles were dispersed in 25 ml of water using ultrasound
disperser. 100 ml of ethanol, 2 ml of concentrated NH4OH were added
to the reaction mixture at slow mechanical stirring. After that, 3 ml of
tetraethoxysilane (TEOS) were added drop-by-drop to the reaction
mixture. The hydrolysis of TEOS was carried out for 20 hours under
normal conditions.
The resultant product was thoroughly rinsed with deionised water
three times to remove the residual surfactant and unreacted reagents,
and collected by magnetic separation using a permanent magnet. The
silica—magnetite nanocomposite (MAGNAT) was stored in deionised
water at a concentration of 10 mg/ml.
Characterization of Magnetic Nanoparticles. The size and morphol-
ogy of magnetic nanoparticles were observed by transmission electron
microscopy (TEM) using PEM-U (Sumy, Ukraine). Magnetic measure-
ments were performed using magnetometer with Coulomb sensor (Tver
University, Russia). X-ray diffraction measurements performed using
diffractometer DRON-UM1 in filtered emission CoKα with recording
Bragg—Brentano geometry.
Magnetic Response Characteristics. Magnetic response of synthesized
magnetite nanocomposites was measured by monitoring an optical
density of the magnetite adsorbent suspended in water at 600 nm. A
spectrophotometer cuvette holder with attached neodymium (S36
grade) magnet was used.
Binding Capacity of Magnetite Nanocomposites. Binding capacity of
engineered nanoparticles was tested against Marker DNA standards
with different molecular mass. Binding and recovery of Marker DNA
fragments (Lambda DNA/Hind III with 125-23.130 bp and
phiX174/Hae III with 72-1.353 bp) were titrated into 2000 μg concen-
trations of nanoparticles and nonmagnetic commercial absorbent.
Binding was performed in binding buffer for DNA purification, elu-
tion of absorbed nucleic was carried out in deionised water. Eluted
DNA was quantified by absorbance at 260 nm.
Purification of Plasmid DNA by Silica—Magnetite Nanocomposites.
E.coli cells expressing the plasmid pGL3-Conrol-Vector were grown to
log phase in culture media containing 100 μg/ml ampicillin. Bacterial
cells were harvested from 3 ml of cell culture and treate4d with 0.05 M
Tris-HCL μg/ml ribonuclease A. Cell lysis was performed with 0.2 M
NaOH containing 1% of dodecil sulphate. Genomic DNA and other con-
taminants were precipitated by addition of 6M guanidine-hydrochlo-
ride, pH 5.5. After centrifugation, the cell lysates were used for plasmid
DNA purification with synthesized nanocomposites. Binding and elu-
ISOLATION OF NUCLEIC ACIDS FROM DIFFERENT BIOLOGICAL OBJECTS 1013
tion of plasmid DNA were performed with common procedure and
chemicals. The concentration of purified nucleic acids was calculated
using absorbance at 260 nm.
Purification of Total DNA from Plants, Bacterial and Mammal Tis-
sues by Silica—Magnetite Nanocomposites. Procedures of DNA ex-
traction from different biological samples were the same as it was writ-
ten above but included the stage of tissue homogenization before DNA
extraction and usage of tissue specific buffer systems.
Isolation of Total DNA from Milk Food Products by Silica—Magnetite
Nanocomposites. Procedure of DNA extraction from milk product
samples were the same as it was written as it is indicated above.
Total RNA Extraction by Silica—Magnetite Nanocomposites from
Avian and Mammal Tissue Samples. Purification of total RNA from
avian tissue, embryonated eggs and porcine blood cells were performed
with usage a commercial buffer system kits for RNA extraction (‘Am-
pliSens’, Russia). Mononuclear blood cells were harvested by centrifu-
gation and washed two times with Hank’s solution. Cell pellets were
mixed with extracting buffer and suspension of silica—magnetite
nanoparticles in dose of 10 μl (stoke concentration 10 mg/ml) were
added to each samples. Sedimentation was carried out by neodymium
(S36 grade) magnet (‘Promega’, USA). Repeated procedure of suspen-
sion/sedimentation was made. Elution of absorbed nucleic acids was
carried out in deionised water. Eluted RNA was quantified by absorb-
ance at 260 nm. All above manipulations were done in 4°C. Purified
RNA immediately was used for reaction of reverse transcription.
Reverse Transcriptase Reaction (a Single cDNA Synthesis). Reaction
mixture included 1 μg of total RNA, 0.5 μg oligo dT18 primer and incu-
bated in microtubes for 5 min in 70°C, cooled on ice. Then 1 mM dNTP,
10 mM Tris buffer, 40 unite of RNAse inhibitor were added to each re-
action mixture and 5 min incubation in 37°C was followed. Finally, 200
units of M-MulLV enzyme were added to reaction cocktail and it was
incubated for 60 min in 37°C. Then, inactivation of enzyme was made
during 10 min in 70°C. Synthesized cDNA was stored in −20°C.
PCR Amplification. The PCR procedure had been carried out with
primers targeting the insertion elements IS900 of Map. The mass of
amplicons is 800 bp. The purified DNA (0 μl) was mixed with cocktail
including PCR buffer (10 mM Tris-HCL, pH 8.3) with 50 mM KCL, 1.5
mM MgCL, 5.0 pm/ml of each primers, 200 μM of each dNTP, 2.5 U
Tag DNA polymerase. Cycling conditions were 20 cycles of 95°C for 1
min, 60°C for 1 min, 72°C for 1 min. Molecular mass of amplified DNA
fragments were detected by electrophoresis in 1% agarose with 0.5
μg/ml ethidium bromide and 1 kb molecular mass standards (Sigma).
The running buffer was TAE (49 mM Tris, 20 mM acetic acid, 1 mM
EDTA, pH 8.0). Electrophoresis was carried out at 90 V for 1 hour.
Visualisation PCR products was performed by UV illumination.
1014 N. N. VOLKOVA, O. N. DERJABIN, and N. O. DUDCHENKO
3. RESULTS AND DISCUSSION
The size and morphology of magnetic particles were characterized by
TEM (data not shown). It shows that the size of magnetic nanoparticles
is about 15 nm. X-ray diffraction (XRD) measurements show that the
magnetic core of the synthesized particles consists of magnetite
(Fe3O4). Six characteristic peaks for Fe3O4 in XRD pattern (data are not
shown) were observed for magnetic nanoparticles. These peaks reveal
that the resultant particles were pure Fe3O4.
The superparamagnetic properties of the magnetic particles were
verified by magnetization curve measurements. Saturation magneti-
zation of silica-modified magnetite particles was 37 emu/g (A⋅m2/kg).
This saturation magnetization of magnetic particles makes them sus-
ceptible to magnetic field and therefore makes the solid and liquid
phases separate easily.
Magnetic response of silica—magnetite nanoparticles has been ana-
lysed. Magnetic response was measured by placing nanoparticles in
buffer solution in spectrophotometer cuvette attaching to magnet on it
outside wall. Optical density of particles suspensions was measured at
600 nm over time. More than 90% of magnetic nanoparticles at con-
centration above 0.1 mg/ml were removed from buffer solution in less
than 10 seconds after magnetic field applying. Sedimentation of 90%
of nanoparticles at concentration 0.01 mg/ml were observed 25 sec-
onds after applying of magnetic field.
The mechanism of this process could be envisaged in the following
way. At the first stage of sedimentation, a few particles magnetize and
self-attract to form a critical particle mass that moves toward magnet.
At the required particle concentration for most molecular-biological
applications, efficient removal of particles is accomplished in under 30
seconds (Promega DNA kits). In case of our nanoparticles, about
15 seconds is enough for optical clearing of solution. The standard
variant of magnet was used for these investigations (Promega’s Mag-
neSilTM magnetic stand, which incorporate S36 grade neodymium rare
earth magnet).
Binding Capacity of Magnetite Nanocomposites. Ionic strength and
pH are the crucial factors estimating processes binding and elution of
nucleic acids by silica magnetite nanobeads. Absorption capacity of
nanoparticles could be modulated in wide ranges by the ionic strength
of binding buffer system, which is used for DNA purification. DNA is a
polyanionic molecular due to presence of phosphate groups and inter-
acts with positively charged functional groups on silica—magnetite par-
ticles surface [3]. In order to determine the ion strength effect on of
synthesized nanocomposites, we tested a several NaCl concentration
ranges of 0—4 M in binding buffer. It had been found that presence of
2M NaCl and above concentrations resulted in maximal binding of
ISOLATION OF NUCLEIC ACIDS FROM DIFFERENT BIOLOGICAL OBJECTS 1015
plasmid DNA and marker small DNA fragments. In this study, the in-
fluence of binding buffer pH on DNA absorption of nanocomposites had
been estimated also. As expected, pH of binding solution had no effect
on plasmid DNA absorption by silica—magnetite nanoparticles. These
results are in agreement with data obtained by Chen-Li Chiang [1, 6] for
silica—magnetite nanoparticles with more diameter size (about 31 nm).
As revealed, the synthesized nanoparticles possessed an increased re-
covery small DNA marker fragments over commercial nonmagnetic ma-
terials (Fig. 1). This recovery was inversely related to DNA size and
much higher recovery of smaller marker DNA was registered. Final re-
covery of small fragments was a primary function of binding since these
DNA were efficiently eluted from nanocomposites in water. The recov-
ery of lambda marker DNA fragments was a function of both binding
capacity and elution efficiency of synthesized nanocomposites. The lar-
ger fragments of DNA have a reduced binding capacity at these condi-
tions and reduced efficiency of elution in water at room temperature.
Plasmid DNA Purification by Silica—Magnetite Nanocomposites.
Obtained magnetic particles were tested for DNA isolation from E.coli
cultures, which had been transfected with some gene-engineering con-
structions. A set of experiments with bacterial cell lysates for measur-
ing of absorption capacity of synthesized nanoparticles were per-
formed. The increasing amounts of nanoabsorbent were added to bac-
terial cell lysates prepared from 10 ml cultures of E.coli containing the
high copy number of pGL3-Control Vector (plasmid DNA). The tradi-
tional silica absorbent of nucleic acids was used as a control. The result
of silica magnetite nanoparticles usage was an isolation of 80 μg of
plasmid DNA at 2.2 mg particles added to lysate. In contrast, 5.0 mg
commercial tradition silica absorbent was required for isolation of
equivalent amount of plasmid DNA from a 10 ml culture. The results
demonstrated that absorbing capacity of magnetic particles was sig-
Fig. 1. Recovery of small (72-1.353 bp) marker DNA fragments from mag-
netic nanocomposites and commercial silica adsorbent.
1016 N. N. VOLKOVA, O. N. DERJABIN, and N. O. DUDCHENKO
nificantly higher than that of traditional non-magnetic silica carriers.
Application of Silica—Magnetite Nanoparticles in Molecular Diagno-
sis of Cattle and Avian Viral Diseases. The worldwide occurrence and
re-occurrence of trans-boundary viral diseases like classical swine fever
indicates that there is an acute need for the development of high-
capacity, powerful and reliable methods for detecting a causative viral
and bacterial agent before they could spread to large populations and
cause a tremendous loss. During the last one and a half decade, more
then 40 nested polymerase chain reaction assays have been developed for
variety of DNA and RNA viruses. False negative and positive results are
avoided now by the using of special tools, practices and internal controls
for purification nucleic acids and technique of amplifications.
In this study, we tested the possibility of synthesized nanocompo-
sites for high-effective purification of native total RNA from porcine
and avian tissues for diagnosis of avian Bronchitis virus and virus of
classic swine fever in domestic and wild populations [1, 2]. Results of
reverse transcriptase—polymerase chain reaction (RT—PCR) for mo-
lecular diagnosis of viral diseases are presented in Fig. 2. The same re-
sults were found in RT—PCR assay of health and infected avian an em-
bryonated eggs by bronchitis virus [1, 2].
Comparative analysis of data obtained with usage of silica—
magnetite nanocomposites and commercial silica absorbent demon-
strated a reduction of false negative samples in diagnosis these viral
diseases and made procedure of RNA purification much more easy,
fast and simple.
Application of Silica—Magnetite Nanoparticles in Molecular Diag-
nosis of Anthrax. Bacillus anthracis is the etiologic agent of anthrax,
an acute fatale disease among mammals. It was thought to differ from
Fig. 2. The agarose gel electrophoresis of the RT—PCR products, which were
obtained from tissues of health and infected swine. Lines 1, 2, 3–health ani-
mals; M–DNA markers; lines 4, 5, 6–amplified fragments of viral DNA.
ISOLATION OF NUCLEIC ACIDS FROM DIFFERENT BIOLOGICAL OBJECTS 1017
Bacillus cereus, an opportunistic pathogen and cause of food poison-
ing, by presence of plasmids pXO1 and XO2, which encode the lethal
toxin complex and poly-D-glutamic acid capsule, respectively.
In this set of experiments, the silica—magnetite nanoparticles were
used in differential molecular diagnostic of Bacillus anthracis and
non-B-anthracis bacteria. With PCR-based technique it was conformed
the presence of both plasmids which encode capsule and toxin of Bacil-
lus anthracis in one bacterial strain which earlier was conceded as Ba-
cillus cereus and was known as weak pathogen (Fig. 3).
This work confirmed the fact that non-B-anthracis bacteria could
possess the anthrax toxin genes and explained their high pathogenic
Fig. 3. The agarose gel electrophoresis of the PCR amplified DNA fragments
from bacterial vaccine strains, Bacillus cereus (anthracoides) and bacterial
vaccine strains with protective antigen. Lines from 1 to 8–vaccine strains
(weak immunogens); line 9–Bacillus anthracoides within capsule; line M–
DNA markers; lines from 10 to 13–vaccine strains with protective antigen
(high immunogenic properties).
a b
Fig. 4. The agarose gel electrophoresis of the PCR amplified virus DNA frag-
ments from sugar beet (a) and Bifidobacterium DNA from probiotic tablets
(b). Line M–DNA markers; line 1–commercial absorbent; line 2–magnetite
nanoparticles. Line M–DNA markers; line 1–negative control; lines from 2
to 5–Bifidobacterium strains.
1018 N. N. VOLKOVA, O. N. DERJABIN, and N. O. DUDCHENKO
properties in causing a severe inhalation anthrax-like illness. There-
fore, the presence of amplified DNA fragments with molecular mass of
capsule antigen has proved the virulence of this bacteria strain and a
potential dangerous of same vaccine drugs made on base of Bacillus
anthracoides for personals and cattle.
Isolation and Identification of DNA in Plant and Dairy Products. In
this experimental set, the nanoparticles were hydrolyzed and carboxyl
groups on there surface were inducted by oxidation. The carboxyl-
functionalized silica-magnetite nanocomposites were tested for bind-
ing of Bifidobacterium and Lactobacterium DNA from crude lysates of
different probiotic tablets or from culture cell lyophilisates [1]. The
binding capacity of nanoparticles was higher then traditional commer-
cial silica absorbent. The efficiency of DNA purification was con-
firmed by results of PCR amplification with specific primers for these
bacterial strains (Fig. 4, b).
Comparative analysis of viral DNA detection in sugar beet was per-
formed with usage of commercial nonmagnetic and synthesized silica-
magnetite nanoparticles. As it followed from data performed in Fig. 4,
a, the binding capacity and efficiency of tested nanocomposites were
much more high then traditional DNA absorbent.
Thus, the data obtained have proved the high efficiency of synthesized
silica-modified magnetite particles for high purification of vital and bac-
terial native DNA/RNA for nucleic acid amplification technique.
Acknowledgements. This work was partially funded by Scientific-
Technology Centre in Ukraine (STCU project # 3074 ‘SQUID-magne-
tometry system to control magnetic contrast agents and targeted
transport of medications with magnetic carriers’).
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| id | nasplib_isofts_kiev_ua-123456789-76191 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 1816-5230 |
| language | English |
| last_indexed | 2025-12-07T17:22:02Z |
| publishDate | 2008 |
| publisher | Інститут металофізики ім. Г.В. Курдюмова НАН України |
| record_format | dspace |
| spelling | Volkova, N.N. Derjabin, O.N. Dudchenko, N.O. 2015-02-08T17:42:14Z 2015-02-08T17:42:14Z 2008 Isolation of Nucleic Acids from Different Biological Objects
 with Silica—Magnetite Nanoparticles / N.N. Volkova, O.N. Derjabin, N.O. Dudchenko // Наносистеми, наноматеріали, нанотехнології: Зб. наук. пр. — К.: РВВ ІМФ, 2008. — Т. 6, № 3. — С. 1009-1018. — Бібліогр.: 12 назв. — англ. 1816-5230 PACS numbers: 81.07.Nb,81.16.Fg,82.37.Rs,82.39.-k,82.45.-h,87.14.-g,87.15.Tt https://nasplib.isofts.kiev.ua/handle/123456789/76191 Now, modified magnetic particles are widely used in different biological and
 medical applications (enzyme and protein immobilization, cells separation
 and purification, MRI, targeted drug delivery, etc.). The aim of the present
 study is to reveal the ability of synthesized silica-modified magnetic particles
 to isolate DNA from the biological tissues in comparison with common
 method used the non-magnetic particles. Magnetite (Fe3O4) particles are prepared
 via co-precipitation of Fe+2 and Fe+3 with NH4OH in aqueous solution.
 Silica—magnetite nanocomposites are prepared via tetraethoxysilane hydrolyzation
 in alcohol—water—ammonia mixture. The average core size of synthesized
 magnetic nanoparticles is about 15 nm (according to the TEM data).
 Application of these compounds for DNA isolation from different biological
 objects showed significant time-savings, overall higher yields, lower RNA
 contamination and better polymerase chain reaction (PCR) amplification
 compared to commercial available silica non-magnetic particles (Promega).
 High efficiency of nucleic-acid purification by silica—magnetite particles is
 confirmed in molecular assays with reverse transcriptase—polymerase chain
 reaction (RT—PCR) assays of RNA- and DNA-virus diseases of plants, avian,
 cattle and estimation of bacterial spectrum in dairy products (probiotics). Модифіковані магнетні частинки зараз широко використовуються для різних біологічних та медичних застосувань (іммобілізація ензимів та білків,
 виділення та очищення клітин, ЯМР, направлена доставка ліків та ін.).
 Метою цього дослідження було показати здатність синтезованих магнетних
 частинок, модифікованих кремнеземом, виділяти ДНК з різних біологічних тканин у порівнянні із стандартною методою з використанням немагнетних частинок. Магнетитові (Fe3O4) частинки було одержано шляхом співосадження Fe+2 та Fe+3 за допомогою NH4OH у воднім розчині. Силікамагнетитові нанокомпозити були одержані шляхом гідролізації тетраетоксисилана у спирто-водяно-амонійній суміші. Середній розмір ядра синтезованих магнетних наночастинок був біля 15 нм (за даними просвітлювальної
 електронної мікроскопії). Застосування цих сполук для виділення ДНК зрізних біологічних тканин виявило значне заощадження часу, загальний
 більш високий вихід ДНК, більш низьку кількість домішок РНК та кращу
 ампліфікацію полімеразної ланцюгової реакції (ПЛР) у порівнянні з традиційними силіційовими немагнетними частинками (Promega). Високу
 ефективність очищення нуклеїнових кислот за допомогою силіка-магнетитових частинок було підтверджено в молекулярно-біологічних тестах, що
 були виконані на основі зворотньої транскрипції з подальшою полімеразною ланцюговою реакцією (ЗТ—ПЛР) щодо виявлення РНК та ДНК вірусних захворювань рослин, птахів, сільськогосподарських тварин та оцінки
 якости молочних продуктів (пробіотиків). Модифицированные магнитные частицы сейчас широко используются в
 различных биологических и медицинских приложениях (иммобилизация
 энзимов и белков, выделение и очистка клеток, ЯМР, направленная доставка лекарств и т.д.). Целью настоящего исследования было показать
 способность синтезированных магнитных частиц, модифицированных
 кремнеземом, выделять ДНК из различных биологических тканей по
 сравнению со стандартным методом с использованием немагнитных частиц. Магнетитовые (Fe3O4) частицы были получены путем соосаждения
 Fe+2 и Fe+3 с помощью NH4OH в водном растворе. Кремний-магнетитовые
 нанокомпозиты были приготовлены путем гидролизации тетраетоксисилана в спирто-водно-аммониевой смеси. Средний размер ядра синтезированных магнитных наночастиц был около 15 нм (по данным просвечивающей электронной микроскопии). Применение этих соединений для
 выделения ДНК из различных биологических тканей выявило значительную экономию времени, общий более высокий выход ДНК, более
 низкое количество примесей РНК и лучшую амплификацию полимеразной цепной реакции (ПЦР) по сравнению с коммерчески доступными
 кремниевыми немагнитными частицами (Promega). Высокая эффективность очистки нуклеиновых кислот с помощью кремний-магнетитовых
 частиц была подтверждена в молекулярно-биологических тестах, выполняемых на основе обратной транскрипции с последующей полимеразной
 цепной реакцией (ОТ—ПЦР) по выявлению РНК и ДНК вирусных заболеваний растений, птиц, сельскохозяйственных животных и оценке качества молочных продуктов (пробиотиков). en Інститут металофізики ім. Г.В. Курдюмова НАН України Наносистеми, наноматеріали, нанотехнології Isolation of Nucleic Acids from Different Biological Objects with Silica—Magnetite Nanoparticles Article published earlier |
| spellingShingle | Isolation of Nucleic Acids from Different Biological Objects with Silica—Magnetite Nanoparticles Volkova, N.N. Derjabin, O.N. Dudchenko, N.O. |
| title | Isolation of Nucleic Acids from Different Biological Objects with Silica—Magnetite Nanoparticles |
| title_full | Isolation of Nucleic Acids from Different Biological Objects with Silica—Magnetite Nanoparticles |
| title_fullStr | Isolation of Nucleic Acids from Different Biological Objects with Silica—Magnetite Nanoparticles |
| title_full_unstemmed | Isolation of Nucleic Acids from Different Biological Objects with Silica—Magnetite Nanoparticles |
| title_short | Isolation of Nucleic Acids from Different Biological Objects with Silica—Magnetite Nanoparticles |
| title_sort | isolation of nucleic acids from different biological objects with silica—magnetite nanoparticles |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/76191 |
| work_keys_str_mv | AT volkovann isolationofnucleicacidsfromdifferentbiologicalobjectswithsilicamagnetitenanoparticles AT derjabinon isolationofnucleicacidsfromdifferentbiologicalobjectswithsilicamagnetitenanoparticles AT dudchenkono isolationofnucleicacidsfromdifferentbiologicalobjectswithsilicamagnetitenanoparticles |