456 Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 6
Kauffmann et al.
16sRNA quantification [27]. Girault H. et al. used magnetic
beads on based immunoaffinity CE for total serum IgE assay
[28]. Wang Y. et al. reported using n-octadecyltrichlorosi-
lane bonded silica MPs packed inside a capillary for the
[35]. Hydrogen peroxide (30%) was from Vel (Leuven,
Belgium). The background electrolyte (BGE) was made of
phosphate buffer (PB) (pH 7.4) (final concentration of
phosphate 12.5 mM) containing hydrogen peroxide (100
ꢀM) and sulfated-ꢀ-cyclodextrin (sulfated-ꢀ-CD) at 20
mg/ml (Sigma, Belgium). Aminopropyltriethoxysilane
(APTS) was from Chisso, Ltd. (Minimata, Japan).
Glutaraldehyde 25% wt (GA) was from Aldrich (Belgium).
All the solutions were prepared with reverse osmosis
purified (ROP) water and filtrated through a 0.45 ꢀm
membrane filter before injection (Sartorius, Germany).
separation of
a mixture containing thiourea, toluene,
naphthalene, fluorine, and anthracene by CE [29].
In this work, we prepared HRP immobilized magnetic
nanoparticles (HRP-MNPs) and HRP immobilized micro-
particles (HRP-MMPs) for using in-line as a microreactor in
CE for enzymatic transformation of paracetamol (APAP).
HRP is a good model enzyme mimicking “in vivo”
peroxidase systems (myeloperoxidase and cyclo-oxygenases)
and to some extent the CYP450 system [30]. The HRP-
MNPs were injected and magnetically retained in a short
reaction zone in the capillary of the CE setup. Subsequently,
the substrate APAP was injected in the microreactor based
CE. The expected advantages of such a configuration are (i)
a low biocomponent and analyte consumption, (ii) a high
biocatalytic conversion efficiency, (iii) excellent time
resolution between reaction and detection, (iv) on-line
identification of bioreaction products (v) readily and
reproducibly microreactor renewing. This set up could be a
useful tool for in vitro drug biotransformation studies. As
model analyte, paracetamol was selected because it is one of
the world’s most widely used pain relievers and because the
oxidation of APAP by HRP in the presence of H2O2 was
quite extensively studied in the literature [30-33]. A
thorough report on paracetamol metabolism and tolerability
was recently reported [30]. The HRP/H2O2 system generated
the APAP free radical, N-acetyl-p-benzosemiquinone imine
(NAPSQI), which rapidly polymerized giving rise to APAP
dimers, trimers and tetramers [31-33]. It was reported that a
minor reaction pathway occurred due to NAPSQI
disproportionation to form N-acetyl-p-benzoquinone imine
(NAPQI) and APAP [32]. In humans, APAP metabolization
occurred mainly via the CYP450 system (CYP2E1) giving
rise, by a two electrons, two protons transfer, to NAPQI
which is highly reactive towards endogenous thiols. At high
concentrations of APAP, glutathione reserves became
depleted allowing NAPQI to react with liver cell proteins
eventually causing tissue necrosis [30]. Human peroxidases
such as myeloperoxidase and the peroxidase function of
cyclo-oxygenase (COX isoenzymes) have also been reported
to be involved in APAP metabolization [34] but their in vivo
contribution to the production of NAPQI has not been
determined. Dimer and further polymers are produced by
peroxidases but their urinary output has not been determined
yet [30].
Enzyme Immobilization
The magnetic particles had amino binding sites allowing
proteins or nucleic acids to be conjugated to the particles by
a bifunctional agent. The magnetic microparticles (MMP)s
were functionalized with APTS, as described previously
[18]. APTS-functionalized MMPs (10 mg) were then reacted
with 1mL 2.5% GA solution (in 12.5 mM PB, pH 7.4) for
1 h and rinsed thoroughly with ROP water. Then, 1 mL HRP
solution (3 mg/mL) was reacted with the GA-activated
particles in 12.5 mM PB pH 7.4 for 1 h at room temperature.
The suspension was rinsed with ROP water (5 ꢁ 1 mL) and
the resulting HRP-MMPs were stored in 1 mL PB (2mM, pH
7.4).
The magnetic nanoparticles (MNPs, 1 mL) were washed
firstly with phosphate buffer (5 ꢁ 1 mL), then reacted with
1 mL of a 2.5 wt.% GA solution (12.5 mM PB, pH 7.4) at
room temperature for 30 min. After washing with PB (5 x 1
mL) the GA treated MNPs were reacted with 1 mL HRP
(3mg/mL) solution during 1 h at room temperature to obtain
the HRP-MNPs. The suspension was washed with PB
(5 ꢁ 1 mL) and stored in 1 mL PB (12.5 mM, pH 7.4) at 4 °C
(final concentration 10 mg/mL). All the washing steps were
performed by trapping the magnetic particles to the vial wall
with a magnet and releasing them after completion.
Visible spectrophotometry was applied for the
determination of the amount of HRP immobilized onto the
MNPs. After the immobilization of HRP, the suspension was
diluted 20 times and the absorbance was measured. The
amount of immobilized HRP was determined by measuring
the initial and final concentration of HRP by referring to a
calibration curve of HRP realized in PB of pH 7.4 (ꢀ = 402
nm) and was found to be equal to 0.20 ꢀmol/g. The same
method was used to determine the quantity of HRP
immobilized onto the MMPs (0.24 ꢀmol/g i.e. ~ 10 ꢀg/mg).
An immobilization yield of 86 ꢀg/mg was reported for
trypsin by using GA as bifunctional reagent onto amino
magnetized beads [24]. This high amount could be attributed
to the distinct nature of the enzyme used (trypsin comparing
to HRP) and to the higher specific surface area of small size
beads (50 nm comparing to 300 nm).
EXPERIMENTAL SECTION
Materials and Reagents
Horseradish peroxidase (HRP) (EC 1.11.1.7 type 11,
240U/mg), was from Sigma (Bornem, Belgium).
Superparamagnetic silica microparticles (MMPs) (5 ꢀm)
were duly provided by Fuji Silysia Chemical Ltd. (Kasugai,
Japan). The superparamagnetic Amino-Adembeads (MNPs)
(300 nm) were purchased from Ademtech (Pessac, France).
It consisted of a MNP suspension at a concentration of 10
mg/mL. Acetaminophen (APAP) was purchased from
SERVA (New York, USA). NAPQI was from Sigma
(Bornem, Belgium), it was dissolved in pH 7.4 just prior use
Apparatus
The capillary electrophoresis instrument (Thermo
Separation Products, Spectraphoresis 100) was equipped
with a UV detector. The detector wavelength was set at
245nm. Fused silica capillaries (Composite Metal Services
Ltd, UK) of 75 μm i.d. and 75 cm total length (50 cm from
the inlet with 42 cm from the magnets to the detector) were