Magnetic beads
Single-Step Purification of Recombinant Thermus aquaticus DNA Polymerase
Using DNA-Aptamer Immobilized Novel Affinity Magnetic Beads
DNA aptamer [Thiol-T-Pol-Apta] specific for Taq-
polymerase was obtained from Thermo Electron, (Dreieich,
Germany). The sequence of DNA-aptamer for Taq-polymerase
enzyme was 5′-HS-(CH 2 ) 6 -TTTTTTGGCGGA-GCGATCATCT-
CA-GAGCATTCTTAG-CGTTTTGTTCTT-GTCTATGA. Am-
ersham E. coli GST-Gene Fusion Expression system was
obtained from Amersham Pharmacia Biotech (Piscataway, NJ).
Isopropyl β- D -thiogalactopyranoside (IPTG), polyvinyl alcohol
(PVA), toluene, acetonitrile (ACN), and trifluoroacetic acid
(TFA) were supplied from Sigma (St Louis, MO) and used as
received. Methyl methacrylate (MMA), glycidyl methacrylate
(methacrylic acid 2,3-epoxypropyl isopropyl ether; GMA),
EGDMA, and R-R′-azoisobisbutyronitrile (AIBN) were obtained
from Fluka (Switzerland), and the monomers were distilled
under reduced pressure in the presence of hydroquinone and
stored at 4 °C until use. All other chemicals were of analytical
grade and were purchased from Merck (Darmstadt, Germany).
The water used in the present work was purified using a
Barnstead system (Dubuque, IA).
Preparation of Affinity Magnetic Beads. The magnetic
beads were prepared in two sequential steps. In the first step,
ferric-poly(GMA-MMA-EGDMA) beads were prepared via
suspension polymerization, which was carried out in an aqueous
dispersion medium containing FeCl 3 (0.3 M, 400 mL, used as
a precursor for the thermal iron oxide precipitation in the beads).
The organic phase contained GMA (7.5 mL), MMA (7.5 mL),
EGDMA (7.5 mL, as cross-linker), and 5.0% PVA (20 mL, as
stabilizer) were mixed together with 0.2 g of AIBN as initiator
in 20 mL of toluene. The polymerization reactor was placed in
a water bath and heated to 65 °C. The reactor was then equipped
with a mechanical stirrer, nitrogen inlet, and reflux condenser.
The polymerization reaction was maintained at 70 °C for 2.0 h
and then at 80 °C for 1.0 h. After the reaction, the resultant
beads were filtered under suction and washed with distilled water
and ethanol.
In the second step, magnetic poly(GMA-MMA-EGDMA)
beads were prepared by conventional coprecipitation reaction
of iron oxide in the beads. For the coprecipitation reaction, 5.0
g og FeCl 2 was dissolved in purified water (100 mL) and then
transferred into a reactor containing ferric-poly(GMA-MMA-
EGDMA) beads (15 g) in NH 3 /H 2 O (50 mL, 25% w/v). The
reactor was equipped with reflux condenser and refluxed under
nitrogen atmosphere at 40 and 50 °C for 2 h and then at 90 °C
for 2 h with continuous stirring. Finally, the synthesized
magnetic beads were separated from the reaction medium,
washed in ethanol solution (70%; 250 mL) for 3 h, and then
washed with purified water. During the thermal precipitation
reaction, the epoxy groups of the magnetic beads were also
converted into amino groups via ammonolysis reaction due to
the presence of ammonia in the medium (27). Glutaric dialde-
hyde activation of the amino groups of the supports was carried
out as described previously (28). A schematic representation
of the chemistry of the preparation of magnetic affinity beads
is presented in Figure 1.
DNA-thiol-aptamer specific for Taq-polymerase was im-
mobilized covalently via its thiol group to the activated magnetic
poly(GMA-MMA-EGDMA) beads. The beads were washed six
times with phosphate buffer (1.0 mL, 50 mM; pH 8.0) by a
magnetic separation technique (i.e., the beads were trapped in
a magnetic field) and after the aspiration of the solution, they
were then resuspended in fresh buffer solution. The coupling
reaction was initiated by addition of DNA-aptamer to the
activated magnetic beads suspension (50 mg of magnetic beads
in a total of 500 μL reaction medium), which was then vortexed
thoroughly and incubated for different time points and conditions
(pH between 4.0 and 11.0; DNA-aptamer concentration between
50 and 500 μg/mL) under 50 rpm shaking. After 6 h of reaction,
the immobilization medium was then removed and DNA-
aptamer immobilized magnetic beads were washed three times
with phosphate buffer (1.0 mL, 50 mM, pH 8.0). The amounts
of DNA-aptamer in the initial and final mixture were measured
at 260 nm by using a double beam UV-vis spectrophotometer
(model 1601, Shimadzu, Tokyo, Japan).
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Preparation of Ferrites /Fe 3 O 4 The FeCl 3 .6H 2 O (5.4 g) and urea (3.6 g) were dissolved in water (200 mL) at 85 to 90 o C for 2 h. The solution turned brown in color. The resultant reaction mixture was cooled to room temperature to which was added FeSO 4 .7H 2 O (2.8 g) and then 0.1 M NaOH until pH 10. The molar ratio of Fe (III) to Fe (II) in the above system was nearly 2.00. The obtained hydroxides were treated by ultrasound in the sealed flask at 30 to 35 o C for 30 min. After ageing for 5 h the obtained black powder of Fe 3 O 4 was washed, and dried under vacuum.
Iron oxide particles were synthesized as described by Dallas et al.,
(2007) with slight modification. Briefly, 1.25 g of FeSO 4 .7H 2 O and
0.6 g KOH (Merck, India) was dissolved in 30 ml H 2 O of prefixed
temperature (50°C). The mixture was refluxed for 1 h. The as-
prepared magnetic solids were insoluble in aqueous or organic
media. They were isolated by centrifugation, washing several times
with water and drying. The particles thus obtained exhibited a strong
magnetic response.
Ferric chloride,
1-ethyl 3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(EDAC), sodium hydroxide, acetic anhydride, iron(II) chloride
tetrahydrate 97%, iron(III) chloride hexahydrate 99%, nitric acid,
(3-aminopropyl)trimethoxysilane, 1-butanol, octane, toluene, metha-
nol,
L
-aspartic acid, tetramethylammonium bromide, 11-mercap-
toundecanoic acid, and sodium tetrahydridoborate were obtained
from Sigma-Aldrich (St. Louis, MO),
Preparation of Fe 3 O 4 Magnetic Nanoparticles. Magnetic
nanoparticles Fe 3 O 4 were prepared by the hydrothermal copre-
cipitation of ferric and ferrous ions using NaOH as the base,
described by Kouassi et al. 27 Typically iron(II) chloride and iron-
(III) chloride (1-2) were dissolved in Nanopure water at the
concentration of 0.25 M iron ions and chemically precipitated at
room temperature (25 °C) by adding 1 M NaOH at a constant of
pH 10. The precipitates were heated at 80 °C for 35 min under
continuous mixing and washed four times in water and several
times in ethanol. During washing, the magnetic nanoparticles were
separated from the supernatant using a magnetic separator of
trength greater than 20 megaoersted (MOe), and the particles
were finally dried in a vacuum oven at 70 °C.
Surface Modification of Fe 3 O 4 Magnetic Nanoparticles
with APTMS. Magnetic nanoparticles (1 g) were washed with
99.5% methanol and twice with Nanopure water and soaked in 10
mL of 3 mM APTMS solution in a toluene/methanol (1:1 v/v)
mix. The suspension was then transferred into a three-necked flask
with a water-cooled condenser and temperature controller with a
nitrogen gas flow at 80 °C for 20 h under vigorous stirring.
Silanization was bound to occur at the surfaces of the particles
bearing hydroxyl groups, which in the presence of an organic
solvent results in the formation of an APTMS coating with a large
density of amines. 20 The particles were recovered by applying an
external magnetic field after the silanization process and washed
three times with methanol and dried at 50 °C in a vacuum oven.
LAA Functionalization of Magnetic Nanoparticles. One
gram of magnetic nanoparticles was immersed in 50 mL of 0.1 M
LAA solution prepared in nitric acid at pH ∼2. The mixture was
sonicated for 15 min and vigorously stirred for 10 h at room
temperature. An external magnetic field was applied to recover
the particles and washed two times with Nanopure water. The
process is expected to ensure the chemisorption of LAA, bearing
carboxyl and amino groups on the surface of the particles.