Fabrication of novel hierarchically ordered porous magnetic nanocomposites for bio-catalysis

Article abstract of DOI:10.1039/c0cc01747g

Novel hierarchically ordered porous magnetic nanocomposites with interconnecting macroporous windows and meso-microporous walls containing well dispersed magnetic nanoparticles have been fabricated and used as a support to immobilise lipase for the effici

Full text of DOI:10.1039/c0cc01747g

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Fabrication of novel hierarchically ordered porous magnetic
nanocomposites for bio-catalysisw
a
b
c
Tapas Sen,* Ian James Bruce and Tim Mercer
Received 4th June 2010, Accepted 26th July 2010
DOI: 10.1039/c0cc01747g
Novel hierarchically ordered porous magnetic nanocomposites
with interconnecting macroporous windows and meso-microporous
walls containing well dispersed magnetic nanoparticles have been
fabricated and used as a support to immobilise lipase for the
efficient hydrolysis of ester.
of uniform diameter B400 nm using silica gel in an acidic
medium (aqueous hydrochloric acid) containing a tri-block
copolymer (F127: EO107PO70EO107) as a surfactant, n-butanol
as a co-solvent and a mixture of ferrous and ferric chlorides as
a source of iron. The silica gel filled polystyrene monolith
composite was dried at 60 1C overnight. Templates were
removed from the composite by washing with toluene followed
by calcination at 500 1C for 8 h in the presence of air.
When analysed, the nanocomposites exhibited an XRD
pattern (Fig. 1a) corresponding to a single strong peak
(2y = 2.06) in the low angle region (2y values of 1.5 to 10)
and multiple weak peaks in the high angle region (2y values
of 10 to 70). The low angle peak corresponds to inter-planar
spacing of 4.3 nm due to the presence of mesoporosity without
any indication of mesopore ordering. The multiple high angle
Magnetic nanocomposites consisting of a mesoporous silica shell
surrounding a magnetic core are an interesting class of nano-
1–6
composites with high surface area and magnetic properties that
have recently been exploited in various applications e.g. magnetic
5
bio-separation, controlled drug delivery
4,6
and magnetic
6
7
contrasting. Recently, Zhao and coworkers have reported the
fabrication of a magnetic core–shell zeolite for the immobilization
of protein (trypsin). Porous materials from microporous
(
pore diameter o 2 nm) to mesoporous (2 nm o pore diameter
o 50 nm) to macroporous (pore diameter > 50 nm) have evolved
over the last two decades in
8, 9
as a result of extensive research
order to overcome the difficulty of the diffusional restriction of
biomolecules, i.e. protein and enzymes, through the micro/
10,11
mesopores. We have reported
pure silica nanocomposites
containing micro, meso and macroporosity with hierarchical
ordering using a template assisted synthesis. However, it should
be noted that such materials have never been used for applications.
Immobilization of enzymes in mesoporous, macroporous and
12
mesocellular silica foams has been studied for bio-catalysis.
However, it was found that such materials lacked ordering of the
13
pore structure. Dyal et al. first reported magnetic (maghemite)
nanoparticles for the immobilization of lipase (Candida rugosa)
and used them for the hydrolysis of ester (p-nitrophenyl
5,14
palmitate). We have previously reported
magnetic meso-
porous nanocomposites, but no reports are available on the
fabrication of hierarchically ordered porous magnetic nano-
composites and their application in bio-catalysis. Herein we
report the template assisted fabrication of novel hierarchically
ordered porous magnetic nanocomposites and their potential
use as a support for bio-catalysis, i.e. immobilization of lipase
for the hydrolysis of ester (p-nitrophenyl palmitate).
The novel nanocomposites were synthesized (see ESIw)
by filling the interstitial sites of a polystyrene latex monolith
a
Centre for Materials Science, School of Forensic and Investigative
Sciences, University of Central Lancashire, Preston, UK PR1 2HE.
E-mail: tsen@uclan.ac.uk; Fax: +44(0)1772894981;
Tel: +44(0)1772894371
Department of Biosciences, University of Kent, Canterbury,
UK CT2 7NJ
School of Computing Engineering and Physical Sciences,
b
c
University of Central Lancashire, Preston, UK PR1 2HE
w Electronic supplementary information (ESI) available: Detailed
experimental protocol of fabrication of the nanocomposites, surface
modification, magnetic measurement, bio-catalysis, Fig. S1 to S4 and
Table S1. See DOI: 10.1039/c0cc01747g
Fig. 1 (a) XRD pattern of the hierarchically ordered porous
magnetic nanocomposites with expanded regions of low angle
(2y values of 1.5 to 10) and high angle (2y values of 10 to 70); (b)
Room temperature (RT) magnetic data of the nanocomposites.
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1
5
peaks relate to iron oxides (magnetite or maghemite) nano-
particles. The weak intensity of the XRD peaks in the high
angle region could be due to the nanocrystalline nature of the
iron oxides. When magnetic measurements (see ESIw) were
carried out on a powder form of the crushed nanocomposites
at RT, negligible hysteresis (Fig. 1b) was observed which is
indicative of the superparamagnetic state found in iron oxide
1
6
particles of size below B15 nm. The measured saturation
magnetisation value of B13 emu/g is much lower than the
Scheme 1 Strategy for the immobilization of lipase on the novel
1
7
value reported for bulk magnetite (B92 emu/g). A lower
value is consistent with both the dilution of magnetic particles
hierarchically ordered porous magnetic nanocomposites (HOPMN).
1
within the bulk and with surface effects becoming more
8
diameter of 3.4 nm. T-plot analysis data suggested the
2
À1
dominant as the particle size decreases in the superpara-
14
presence of a small micropore area (Smicropores = 48 m g ).
The mesopore diameter is consistent with the XRD
data(d = 4.3 nm) and similar to the pure silica analog reported
1
magnetic range. Furthermore, we have previously reported
6
a similar observation when a large amount of diamagnetic
silica was present in the magnetic materials.
1
1
earlier. Lipase (Candida rugosa) immobilization was carried
out by chemical conjugation and the method (see ESIw) has
been presented schematically in reaction Scheme 1.
Scanning electron micrographs (SEM) of the nano-
composites exhibited an ordered macroporous structure
consisting of macropore voids of diameter B350 nm with
interconnecting windows of diameter B150 nm (see Fig. 2a
and b). The macroporous window size obtained from trans-
mission electron micrographs (TEM) of the nanocomposites
Surface amine density of the aminopropyl triethoxy silane
(APTS) functionalised hierarchically ordered porous magnetic
À1
nanocomposite was measured to be 64 nmol mg by an
1
9,20
established colourimetric assay
(see ESIw). The lipase
(
see Fig. 2d) was consistent with that obtained from the SEM
(Candida rugosa) concentration in non-functionalised and
functionalised nanocomposites has been calculated using the
pre-established calibration curve (see Fig. S3 in ESIw) and the
data. TEM micrographs also exhibited disordered (worm-hole)
mesopores covered with iron oxide nanoparticles (black dots).
The iron oxide nanoparticles were uniformly distributed
throughout the nanocomposites i.e. including the region around
the macroporous windows. These iron oxide particles could
potentially be used for lipase immobilisation or as magnetic
centers for magnetic separation. When such materials were
À1
values were calculated to be 32.5 mg mg (non-functionalised
À1
form) and 38.2 mg mg (functionalised form), respectively.
The lipase (Candida rugosa) immobilized functionalised
form of the hierarchically ordered porous magnetic nano-
composite exhibited a catalytic conversion value of 5.9 Â
1
0,11
À3
À1
À1
synthesized
without the presence of iron chlorides, a distinct
10 mmol min mg of enzyme for the hydrolysis of ester
(p-nitrophenyl palmitate) to alcohol (p-nitrophenol) and
acid (palmitic acid) and the value was found to be nearly half
amorphous region was observed to form around the inter-
connecting windows. The mechanism of formation of such
10,11
À3
À1
À1
porous nanocomposites has been well established.
The BET surface area of the novel nanocomposites was
2
measured to be 196 m g with the hysteresis loop (see Fig. S2
(3.9 Â 10 mmol min mg of enzyme) when the lipase
immobilized non-functionalised form (see ESI, Table S1w) was
used as a support. The values were found to be 10 order in
À1
À2
in the ESIw) indicating the presence of mesopores of BJH pore
magnitude compared to the value obtained using pure lipase
(
Candida rugosa) in a homogeneous medium. A similar result
3
1
was reported by Dyal et al. when maghemite nanoparticles
were used as a support.
The kinetics of hydrolysis of 4-nitrophenyl palmitate using
lipase immobilized nanocomposites (functionalised and non-
functionalised forms) has been presented in Fig. 3. The rate of
hydrolysis was observe to increase with time for both materials,
however, the catalytic efficiency of lipase immobilized functiona-
lised nanocomposites was observed to be around two fold higher
in value compared to lipase immobilized non-functionalised
nanocomposite. The catalytic efficiency was unaffected upon
the 2nd cycle on the lipase immobilized functionalised nano-
composite whereas a dramatic reduction was observed when the
non-functionalised form was used as a support.
The above observation suggests that the lipase was loosely
bonded (physically adsorbed) to the non-functionalised nano-
composite and leached into the solution during the first
catalytic cycle whereas it was chemically bonded to the
functionalised surface as shown in reaction Scheme 1. The
enzyme loaded nanocomposites were stable at 4 1C stored
inside the freezer for the period of at least a month as we have
performed duplicate experiments using singly prepared
Fig. 2 Electron micrographs of the hierarchically ordered magnetic
nanocomposites: SEM (a & b); TEM (c & d).
6
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porous magnetic silica nanocomposites have been fabricated
and used as a bio-catalytic support for the hydrolysis of
p-nitrophenyl palmitate.
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Chem. Commun., 2010, 46, 6807–6809 | 6809