One pot glucose detection by [FeIII(biuret-amide)] immobilized on mesoporous silica nanoparticles: An efficient HRP mimic

Article abstract of DOI:10.1039/c2cc30970j

An [Fe^III(biuret-amide)] complex has been immobilized onto mesoporous silica nanoparticles via Cu(i) catalyzed azide-alkyne click chemistry. This hybrid material functions as an efficient peroxidase mimic and was successfully used for the quantitative determination of hydrogen peroxide and glucose via a one-pot colorimetric assay.

Full text of DOI:10.1039/c2cc30970j

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One pot glucose detection by [FeIII(biuret-amide)] immobilized on
mesoporous silica nanoparticles: an efficient HRP mimicw
Bharmana Malvi, Chakadola Panda, Basab B. Dhar and Sayam Sen Gupta*
Received 10th February 2012, Accepted 5th March 2012
DOI: 10.1039/c2cc30970j
An [FeIII(biuret-amide)] complex has been immobilized onto
mesoporous silica nanoparticles via Cu(I) catalyzed azide–alkyne
click chemistry. This hybrid material functions as an efficient
peroxidase mimic and was successfully used for the quantitative
determination of hydrogen peroxide and glucose via a one-pot
colorimetric assay.
two step process: first incubation of glucose with glucose oxidase
(GOX) at pH 7.4 to generate H2O2 and then subsequent incuba-
tion of this glucose reaction mixture with NPs at low pH (B4) to
oxidize a peroxidase substrate like 3,30,5,50-tetramethylbenzidine
(TMB) to generate a visual signal.6b,7,8a Thus there is a need
for the development of robust materials that can be used as
biosensors9 and in immunoassays at physiological pH of 7.4.
We hereby report the synthesis of a mesoporous silica
nanoparticles (MSNs) based hybrid material (Fe-MSN) that
mimics the enzyme HRP and functions as a biosensor at
physiological pH. This hybrid material contains a robust small
molecule peroxidase mimic [FeIII(biuret-amide)] developed in
our group10 that is covalently attached to azide containing
MSN particles by Cu(I) catalyzed azide alkyne cycloaddition
reaction (CuAAC).11 Fe-MSN has several attributes that
make it very attractive: (i) the small molecule peroxidase
mimic [FeIII(biuret-amide)] in Fe-MSN exhibits excellent
reactivity and very high stability, especially at low pH and
high ionic strength;10 (ii) the co-condensation method used to
synthesize the azide grafted material allows the attachment of
[FeIII(biuret-amide)] catalyst mostly inside the MSN pore
which limits its interaction with enzymes like GOX during
one-pot glucose detection;12 (iii) usage of MSN particles having
1% azide groups also allows reasonable site-isolation of the
catalyst and this is expected to reduce intermolecular degradation
of the catalyst as has been shown before;11c,13 and (iv) the silanol
groups on the outer surface of MSN can be further functionalized
to incorporate targeting ligands such as antigens/antibodies
for usage in immunoassays like ELISA. The synthesized Fe-MSN
nanoparticles were characterized by several techniques such as
FT-IR, EPR, ICP and nitrogen adsorption–desorption experi-
ments. We also show that these hybrid MSN particles can be
used as a biosensor for the quantitative detection of H2O2 and
glucose in a one-pot method.
Peroxidases are ubiquitous in nature and are known to
activate hydrogen peroxide to perform a variety of chemical
reactions such as oxidations.1 They have been extensively
studied for many industrial applications such as bleaching
inducers, for example, in the detergent and pulp industries.2
Although, peroxidases have potential commercial applications
in many different areas, the most developed field for their
commercial application is analytical diagnostics.1,3 For example,
horseradish peroxidase (HRP), a heme-containing peroxidase, is
widely used for detection of glucose and in immunoassays where
the key step is the peroxidase catalyzed conversion of a non-
luminescent substrate into a luminescent one, thus amplifying
the signal many fold.4 However, limited stability of these
enzymes (gets denatured upon heating or chemical changes)
and the relatively low productivities (preparation, purification
and storage are time consuming) are important features which
hinder the commercial application of HRP. Hence, for several
decades a significant amount of research has been carried out
to make systems that mimic HRP. Small molecule analogs that
have been reported include synthetic porphyrins, sulfonated
phthalocyanines and Fe-TAML’s.5 Recently, various nano-
materials including Fe3O4 magnetic NPs,6 positively charged
gold NPs,7 cupric oxide NPs,8a ceria NPs,8b graphene oxide,8c
CNTs,8d carbon nanodots8e and folate-polyoxometalate hybrid
spheres8f have been reported to possess peroxidase like activity
and have been used for the visual detection of H2O2 and glucose.
But these NPs display peroxidase activity only in acidic pH, which
makes their application as biosensor unwieldy. For example,
quantitative detection of glucose by these NPs involves a
The azide functionalized mesoporous silica nanoparticles
(N3-MSN) were prepared by one pot co-condensation of
TEOS and 3-azidopropyl triethoxysilane (AzPTES) in the
molar ratio of 99 : 1 (Scheme 1).14 After template removal,
the resultant material was analyzed by a variety of analytical
techniques (ESIw). The amount of azide groups grafted on the
surface of N3-MSN was determined to be 0.12 mmol gꢀ1 by
elemental analysis and TGA (Fig. S6, ESIw). XRD, TEM and
SEM showed formation of well-ordered two-dimensional
hexagonal mesoporous particles with a spherical morphology
CReST Unit, Chemical Engineering Div.,
National Chemical laboratory, Dr. Homi Bhabha Road, Pune,
411008, India. E-mail: ss.sengupta@ncl.res.in;
Fax: +91 20 25902621; Tel: +91 20 25902747
w Electronic supplementary information (ESI) available: Experimental
details and characterizations of MSN’s and catalysts. See DOI:
10.1039/c2cc30970j
c
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Chem. Commun., 2012, 48, 5289–5291 5289

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Fig. 1 Typical images of: (a) TMB and H2O2 in the absence of Fe-MSN,
(b) TMB and Fe-MSN in the absence of H2O2, (c) TMB and H2O2 in the
presence of Fe-MSN, (d) TMB and H2O2 in the presence of N3-MSN and
(e) TMB and H2O2 in the presence of propargyl alcohol clicked MSN
particles (PrOH-MSN). Experimental conditions: 10 mL of TMB solution,
747 mL of de-ionized water, 133 mL of phosphate buffer (150 mM, pH 7),
200 mL H2O2 solution (10 mM) and 10 mL of catalyst solution.
Scheme 1 Synthesis of [FeIII(biuret-amide)] functionalized MSN.
Both the materials did not produce blue color in the solution
even after 3 h of incubation time. Thus the blue color of
the solution originates from the catalytic activity of the
[FeIII(biuret-amide)] complex anchored on the MSN particles.
Since the catalytic activity of the Fe-MSN is dependent on the
concentration of the hydrogen peroxide in the solution, this
method can be used for the quantitative estimation of hydrogen
peroxide. Fig. 2 shows the change in absorbance at 650 nm with
increase in the concentration of the hydrogen peroxide in
solution. The calibration graph of the absorbance at 650 nm
to concentration of hydrogen peroxide is linear in the range
1.0 ꢂ 10ꢀ4 to 5.0 ꢂ 10ꢀ3 M with a detection limit at 1.0 ꢂ 10ꢀ5 M.
To investigate the peroxidase like activity of Fe-MSN, we
studied the apparent steady-state kinetic parameters for the
reaction. The kinetic data were fitted perfectly to a typical
Michaelis–Menten curve (Fig. S8 and S9, ESIw). The Km
(Michaelis constant) and Vmax (maximal reaction velocity)
values determined from the Michaelis–Menten curve are given
in Table 1. The Km value of the Fe-MSN with H2O2 as the
substrate was significantly higher than that of HRP6a (Table 1)
consistent with the observation that a higher concentration of
H2O2 was required to observe maximal activity for the Fe-MSN
whereas the Km value of the Fe-MSN with TMB as the substrate
was about 10 times lower than that of HRP, suggesting that the
Fe-MSN have a higher affinity for TMB than HRP.6a
having particle size of 100 ꢁ 10 nm (Fig. S2 and S3, ESIw).
Nitrogen adsorption–desorption showed the multi-point BET
surface area, pore diameter and pore volume to be 724 m2 gꢀ1
,
2.4 nm and 0.93 cc gꢀ1 respectively (Fig. S4(a), Table S1, ESIw).
These values are consistent with those of the other organo-
functionalized mesoporous silica nanoparticles reported earlier.14
Since the MSN particles had the organoazide group attached to
their surface, the metal complex [FeIII(biuret-amide)] having a
pendent alkyne group was synthesized so that it can be attached
to the N3-MSN using CuAAC. The detailed synthetic scheme
and characterization of the [FeIII(biuret-amide)]–alkyne catalyst
are described in ESI.w
The azidopropyl labelled MSN nanoparticle, N3-MSN, was
then subjected to CuAAC with the [FeIII(biuret-amide)]–alkyne
complex using CuSO4 and sodium ascorbate as described in the
literature.11a,c,15 Efficient removal of copper during the washing
has been observed by the absence of copper in ICP analysis. The
extent of click reaction was estimated by IR spectroscopy11a
(Fig. S5, ESIw), TGA and ICP analysis and the amount of
catalyst incorporated was estimated by ICP to be 0.048 mmol
of [FeIII(biuret-amide)] per gram of Fe-MSN. TGA showed
incorporation of 0.056 mmol gꢀ1 [FeIII(biuret-amide)] complex
per gram of Fe-MSN (Fig. S6, ESIw). The EPR spectrum of
Fe-MSN displayed a resonance at g = 4.2 and 1.96 which is
characteristic of the [FeIII(biuret-amide)] complex (Fig. S7, ESIw).
Hence TGA, ICP, EPR and IR spectroscopy data indicate
successful incorporation of the [FeIII(biuret-amide)] complex onto
the surface of MSN particles via CuAAC reaction. The N2
adsorption–desorption isotherm of Fe-MSN displayed a similar
profile to its parent N3-MSN, which indicates that the material
did not undergo any physical change during the course of the
click reaction and its subsequent workup (Fig. S4(b), ESIw).
To investigate the peroxidase like activity of the Fe-MSN
particles, the catalytic oxidation of peroxidase substrate
3,30,5,50-tetramethylbenzidine (TMB) in the presence of
hydrogen peroxide was tested (Fig. 1). Catalytic oxidation of
TMB by Fe-MSN in the presence of H2O2 yields a blue
colored solution. The blue color of the solution results from
the product formed by the oxidation of TMB, which gives a
maximum absorbance at 650 nm in the UV-Vis spectrum. To
confirm that the blue color is formed because of the catalytic
activity of the [FeIII(biuret-amide)] complex anchored on the
surface of MSN’s, a series of control experiments were carried
out using azide functionalized MSN particles (N3-MSN)
and propargyl alcohol clicked MSN particles (PrOH-MSN).
Further, we extended the peroxidase like activity of the
Fe-MSN particles for the detection of glucose by coupling the
above catalytic reaction with glucose oxidation reaction by
glucose oxidase (GOX). The GOX oxidizes glucose to form
Fig. 2 Linear calibration plot between the absorbance at 650 nm and
concentration of H2O2 at pH 7. The inset shows the dependence of
the absorbance at 650 nm on the concentration of H2O2 in the range
0.1 mM to 10 mM.
c
5290 Chem. Commun., 2012, 48, 5289–5291
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Table 1 Comparison of kinetic parameters of TMB oxidation by
Fe-MSN and HRP
to be 5.77 mM (Fig. 3 and ESIw). Thus, this study demonstrates
that Fe-MSN can be used for the analysis of real samples.
In conclusion, we have reported immobilization of the
[FeIII(biuret-amide)] complex on mesoporous silica nano-
particles using Cu(I) catalyzed azide alkyne click chemistry.
This organic–inorganic hybrid material functions as an efficient
peroxidase mimic and has been used successfully for the
colorimetric assay of H2O2 and glucose in one-pot. Further,
the presence of silanol groups on the outer surface of Fe-MSN
allows easy functionalization to install targeting ligands so
that these materials can be used for immunoassays. Such work
is ongoing in our laboratory.
Catalyst
Fe-MSN
Substrate
Vmax/M sꢀ1
Km/M
TMB
H2O2
TMB
H2O2
1.01 ꢂ 10ꢀ8
1.41 ꢂ 10ꢀ6
10.0 ꢂ 10ꢀ8
8.71 ꢂ 10ꢀ8
1.58 ꢂ 10ꢀ5
5.2 ꢂ 10ꢀ1
4.34 ꢂ 10ꢀ4
3.70 ꢂ 10ꢀ3
HRP6a
S. S. G. acknowledges DST, New Delhi (Grant No: SR/S1/PC-
56/2008) and DAE, New Delhi (BRNS; Grant no. 2009/37/33/
BRNS) for funding. B. M., C. P., and B. B. D. acknowledge
CSIR (New Delhi) for fellowships/associateship. We also
acknowledge Prof. Ranjan Das, TIFR, Mumbai for help with
EPR measurements.
Notes and references
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and concentration of glucose at pH 7.4. The inset shows the dependence
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0.01 mM to 3 mM. (J) Glucose in mice blood plasma analysis.
gluconic acid and H2O2 quantitatively; the H2O2 thus produced
is then utilized by Fe-MSN to oxidize TMB to produce a blue
colored solution. Using this methodology, the calibration graph
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described before. In Fe-MSN nanoparticles, most of the
[FeIII(biuret-amide)] catalysts are anchored inside the 2.3 nm
pore channels which allows access to small molecules such as
H2O2 and TMB while large molecules like glucose oxidase are
excluded since they cannot enter the pore channels because of
their large size. As a result, multistep reactions occur at two
different parts of the material, i.e. generation of H2O2 from
glucose using GOX occurs outside the pore channels while the
oxidation of TMB by the [FeIII(biuret-amide)] complex takes
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lactose showed the specificity of the developed method towards
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with mice blood plasma containing 5.88 mM glucose
(ESIw). The average concentration of glucose in mice blood
plasma was determined by the above described methodology
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Chem. Commun., 2012, 48, 5289–5291 5291