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pH-Sensitive Water-Soluble Nanospheric Imprinted
Hydrogels Prepared as Horseradish Peroxidase
Mimetic Enzymes
By Zhiyong Chen, Li Xu, Yuan Liang, and Meiping Zhao*
Natural enzymes are biomolecules that catalyze chemical
reactions with remarkable specificity under very mild condi-
tions.[1] The construction of artificial enzymes with similar high
selectivity and catalytic activity, especially novel compounds or
materials with improved stabilities over natural enzymes, has
been an intriguing field for many years.[2–4] Molecularly
imprinted polymers (MIPs) have been successfully employed
to generate catalytically active binding sites for the mimicking of a
myriad of enzymes.[5–7] With transition-state analogues (TSAs) as
the templates, highly crosslinked, soluble, imprinted microgel
catalysts and soluble imprinted nanogels with a single cavity per
particle for carbonate hydrolysis have been reported by
Resmini[8,9] and Wulff, respectively.[10,11] In these homogeneous
phase models, mass transfer was significantly facilitated in
contrast to the traditional insoluble imprinted polymers, though
the catalytic activity is still modest in aqueous media.
In previous work, we have tried to use the substrate as the
template and chloro[3,7,12,17-tetramethyl-8,13-divinylporphyrin-
2,18-dipropanoato(2-)]iron(III) (hemin) as a cofunctional mono-
mer to act as the catalytic center. The resultant MIP successfully
mimicked natural peroxidase in the oxidation reaction of
homovanillic acid (HVA) to its fluorescent biphenyl dimer,
indicating a promising alternative way to generate enzyme
mimics.[12,13] However, the catalytic activity of the imprinted
polymer was restricted because of its limited solubility in water.
To improve this solubility, we further prepared a terpolymer of
acrylamide, 4-vinylpyridine, and hemin, which yielded a
soluble enzyme system in dimethylsulfoxide (DMSO)-Tris–HCl
(5:95, v/v) buffer. This homogeneous catalyst showed greater
catalytic activity in the DMSO–Tris–HCl buffer than in pure
Tris–HCl buffer.[14] Though there is no problem for the
above-imprinted polymer to function in the DMSO-containing
buffer, it is more intriguing to prepare a homogeneously
imprinted catalyst with high catalytic activity in pure aqueous
solution. Another difficult issue in designing imprinted catalysts
is to mimic the induced fit often encountered in natural
enzymes.[15] Actually, when a catalyst and a stimuli-responsive
polymer are integrated into the same material, novel functions
[*] Dr. M. P. Zhao, Dr. Z. Y. Chen, Dr. L. Xu, Y. Liang
Beijing National Laboratory for Molecular Sciences (BNLMS)
Key Laboratory of Bio-organic Chemistry and Molecular Engineering
of Ministry of Education
College of Chemistry and Molecular Engineering, Peking University
Beijing 100871 (P.R. China)
E-mail: mpzhao@pku.edu.cn
DOI: 10.1002/adma.200903122
1488 ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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that are different from those of the component materials may be
generated.[16] These intelligent gels can not only catalyze a
chemical reaction but their catalysis can also be switched on and
off by external stimuli. Recently, several attempts have been
reported to obtain stimuli-sensitive imprinted hydrogels that
change their rebinding abilities toward the template molecule in
response to temperature,[17] pH,[18] and photo irradiation.[19] We
have previously developed poly-N- isopropylacrylamide
(pNIPAM)-based protein-imprinted polymer gels that can
sensitively respond not only to external stimuli, such as
temperature and salt concentration, but also to the corresponding
template protein with significant volume shrinking.[20] However,
the employment of a stimuli-responsive polymer in the
preparation of a catalytic MIP with typical enzyme-like
characteristics has thus far not yet been reported. So in this
study, we attempt to improve the water solubility of the imprinted
catalyst and to mimic the dual functions of natural enzymes by
combining a stimuli-responsive hydrogel and a molecularly
imprinted catalyst in the same material.
pNIPAM normally possesses a lower critical solution
temperature (LCST) of 32 8C. At higher temperatures, the
polymer appears as a hydrophobic globule which can be separated
from the polymerization solution. Below the LCST, the
polymer switches to a hydrophilic random coil which is
soluble in water.[21,22] In our previous work, copolymerization
of NIPAM, acrylamide (AAm) and methacrylic acid with
N,N0 -methylenebisacrylamide (MBAAm) as a crosslinker resulted
in a thermal- and salt-sensitive polymer gel that exhibited a
remarkable volume change at temperatures above 40 8C or at
sodium chloride concentrations above 1.0 M.[20] Based on this, an
imprinted tetrapolymer of NIPAM, 4-vinylpyridine (4-Vpy),
hemin, and AAm cross-linked with ethylene glycol dimethacry-
late (EDMA) with HVA as a template was synthesized at 70 8C by
precipitation from the solvents. After cooling, the polymer was
precipitated by adding a 3 M sodium chloride solution in water.
The total monomer concentration in the polymerization solution
was kept below 2% to prevent coagulation. Since too much
crosslinker gave unstable dispersions, the amount of EDMA used
in our experiments was below 5 wt% with respect to the total
monomer. After removal of the template, the particles were
washed and freeze-dried. The re-suspended aqueous mixtures
were filtered through a 0.45 mm membrane to remove the large
aggregates.[21] Thus the solubility of the residual hydrogel fraction
was greatly improved.
The morphology of the obtained imprinted hydrogel was
observed by environmental scanning electron microscopy
(ESEM). A spherical shape with diameters in the range of
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particle size distributions were very narrow. The non-imprinted

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gel particles showed a slightly larger size and similar size
distribution, indicating that the influence of the template in
the polymer synthesis on the polymer morphology was negligible.
HVA can be oxidized to a biphenyl dimer by hydrogen peroxide
with peroxidase as the catalyst. The dimer shows a strong and
stable fluorescent emission with excitation and emission
wavelengths at 315 and 425 nm, respectively.[23] This provides
a very sensitive and convenient platform for evaluating the
catalytic activity of horseradish peroxidase (HRP) mimics.
The pH is one of the major factors that influences the catalytic
activity of enzymes. Many artificial enzymes show distinctly
different pH conditions compared to their natural counterparts.
For example, when hemin itself is used as a catalyst,[2] the
optimum pH value was about 11.0. While for natural HRP,
the optimum pH is 8.5 in 0.1 M Tris-HCl buffer.[2,23] Previously,
we have successfully shifted the optimum pH of the insoluble
catalytic polymer to pH 8.3.[12] So in this study, we further
investigated the influence of pH on the catalytic rate of the
pNIPAM-based imprinted hydrogels. As shown in Figure 2, the
catalytic rates of the polymer gel rapidly increase when
the solution pH approaches pH 8.0–9.0 from either the acidic
side or the alkaline side. Such trend is similar to previously
reported results.[12] However, a detailed comparison shows that
for the previously prepared insoluble-polymer catalytic system,
the maximal catalytic rate at pH 8.3 was only 2.5-fold of that at
pH 7.5 or 9.0. But for the imprinted hydrogel presented in this
study, an abrupt increase of about 10-fold of the catalytic rate was
observed within the same pH range with a maximum at 8.5. To
find out the possible reasons for such dramatic increase, we
further examined the morphology of the hydrogel nanospheres
under different pH conditions with ESEM. It was found that the
hydrogel is in a largely swollen state under both acidic and
alkaline conditions as compared to its relatively compact state
at pH 8.0 (see Fig. 1B and 1C). Since the ESEM measurements
were all conducted at 4 8C, we further measured the dependence
of the diameter of the imprinted hydrogel nanospheres on the pH
of the solution by dynamic light scattering (DLS) at 25 8C.
The diameter values as a function of pH are shown in Figure 3.
As can be seen, the hydrodynamic radius (D) of the imprinted

Figure 1. Comparison of the ESEM images of pH-sensitive water-soluble

imprinted hydrogels under different pH conditions. a) pH ¼ 8.0;
b) pH ¼ 2.0; c) pH ¼ 11.0.

several hundreds of nanometers can be seen from the ESEM

images, as shown in Figure 1a. This is quite different from either
the insoluble catalytic MIP[12] or the DMSO-soluble imprinted
hydrogel[14] prepared in our previous work. As shown in our
previous paper,[14] the terpolymer of 4-vinylpyridine, hemin, and
acrylamide has irregular shapes and forms a turbid suspension in
pure water. Here, by incorporation of NIPAM into the polymer,
regular nanospheres were obtained and clear solutions in water
were obtained.
The weight average molar mass Mw and Mw/Mn ratio of the
Figure 2. pH effect on the catalytic rate of the imprinted hydrogels in
obtained imprinted and non-imprinted polymer gels were Tris-HCl buffer (50 mM). The concentrations of catalyst and H2O2 were
measured by static light scattering at 25 8C. The experimental 2.64
107 M (final hemin concentration) and 5.36
103 M, respectively.
results are shown in Table S1 in the Supporting Information. The The concentration of HVA was 2.74
104 M.

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vicinity of the active binding sites. The decrease in catalytic rate at

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higher or lower pH may result from a decrease in the

Figure 3. The hydrodynamic particle size of imprinted microgels
measured by dynamic light scattering.
hydrogels is approximately 250 nm between pH 7 and 8 and
increases to around 450 and 570 nm at pH 2 and 11, respectively.
This pH-responsive variation of the gel particle size should be
attributed to the ionic monomers copolymerized with NIPAM. At
increasing pH values the carboxyl groups of hemin are
deprotonated so that a negative electrostatic repulsion is
enhanced within the polymer. When this repulsion overcomes
the attractive forces such as hydrogen bonding or hydrophobic
interaction, the gel network swells. While under acidic conditions
the pyridine residue of 4-Vpy in the polymer are protonated and
the positive repulsion forces increase so that the polymer gel also
swells. In the range between pH 7 to 8, the negative and positive
charges are balanced, so the gel particles turn to a compact
spherical state.
Aqueous electrophoresis was employed to determine the zeta
potentials of the polymer gel. Based on the zeta potential data, the
pI of the imprinted hydrogel was found to be 7.8. Thus, these
nanospherical gels showed enhanced hydrophilic behavior in
aqueous media at lower and higher pH, but near the isoelectric
point of the polymer, they became more hydrophobic and
compact. Such property is quite important in modulating the
catalytic characteristics of the enzyme-like polymer. By control-
ling the pH, the network charges can be eliminated, so that the
catalytic activity is greatly promoted by excluding water from the
hydrophobic affinity in the swollen gel for the substrate. So by
copolymerization of NIPAM with the ionic comonomers 4-Vpy
and hemin, we successfully obtained a polymer gel whose
catalytic activity may be conveniently controlled by changing
the pH of the solution.
In our previous studies,[12–14] we have proven that hemin
combined with AAm and 4-Vpy can provide multiple-site
interactions (hydrogen bonding and coordination interaction)
with template HVA when preparing peroxidase-like imprinted
polymers. The kinetics of the oxidation reactions of HVA to its
dimer catalyzed by the pNIPAM-based tetrapolymer hydrogel
were investigated in Tris-HCl buffer (pH 8.5, 50 mM) according to
established methods.[24] The reactions can be well described by
conventional Michaelis–Menten kinetics and a Lineweaver–Burk
plot is shown in Figure S1 in the Supporting Information. The
Michaelis–Menten constant (Km), the maximal velocity (Vmax),
and turnover number (kcat) of the water-soluble, imprinted
nanospherical hydrogel (MIPw) and the corresponding blank
polymer (NIPw) prepared in this study were all determined and
are shown in Table 1. We used ( p-hydroxyphenyl) propionic acid
( p-HPPA) as a substrate analogue to evaluate the selectivity of the
imprinted catalyst. For the purpose of comparison, the kinetic
data of the previously prepared insoluble (MIPis) and DMSO
(5%)-soluble (MIPdmso) imprinted polymers are also listed in
Table 1.
Km represents the affinity of a given enzyme towards the
substrate. Smaller Km values thus indicate a stronger affinity
between the enzyme and the substrate. From Table 1, the Km
value for MIPw to the template (HVA) was about one third of that
for NIPw, while the Kcat value for the former was about 2.4-fold of
that for the latter, indicating a distinct positive contribution of the
molecular imprinting process to the catalytic activity of the
enzyme-like MIP. A further support for the imprinting effect can
be observed from the difference in Km values observed between
the alternative substrate ( p-HPPA) and the template substrate
(HVA) by the same MIPw and NIPw as described above. The lower
Kcat and larger Km of MIPw for p-HPPA demonstrate the
specificity of the catalytic enzyme towards its template. Notably,
there is no distinct difference between MIPw and NIPw in their
catalytic activity toward the nonspecific p-HPPA substrate.
The catalytic efficiency of the imprinted catalyst can be
estimated by the ratio of the values of Vmax and Km,[25,26] which

Table 1. Kinetic parameters of the oxidation reaction of HVA catalyzed by the imprinted and non-imprinted polymer gels.

Polymer Substrate Km [M] Vmax [s1] kcat [M1 s1] Vmax /Km (
104)

MIPw [a] HVA (1.83  0.18)
104 17.30  0.49 (6.55  0.35)
107 9.45
NIPw [a] HVA (5.50  0.25)
104 8.16  0.19 (2.75  0.47)
107 1.48
MIPw [a] p-HPPA (7.93  0.40)
104 5.49  0.16 (2.08  0.06)
107 0.69
NIPw [a] p-HPPA (7.40  0.39)
104 6.97  0.19 (2.35  0.47)
107 0.94
MIPis [b] HVA 3.18
103 31.85 6.41
106 1.00
MIPdmso [c] HVA (3.94  0.18)
104 12.32  0.59 (3.05  0.13)
106 3.12

[a] Tris-HCl buffer (pH 8.5, 50 mM); The concentration of H2O2 is 5.36
103 M. The concentrations of HVA vary from 1.34
103 to 1.10
104 M. The concentrations of MIPw
and NIPw are 2.64
107 M and 2.97
107 M (final hemin concentration), respectively. Data are the mean  SD of experiments performed in triplicate. [b] Data from reference
[12]. The catalyst was the highly crosslinked insoluble imprinted polymer. [c] Data from reference [14]. The catalyst was the highly crosslinked imprinted polymer soluble in
DMSO-Tris–HCl (5:95, v/v).

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were calculated and are listed in the last column of Table 1.
Clearly, MIPw in pure Tris-HCl buffer showed a much greater
catalytic efficiency than both the highly crosslinked insoluble
polymer (MIPis) and the highly crosslinked DMSO-soluble
imprinted nanogels (MIPdmso) under similar conditions. This
remarkable improvement should be attributed to several
important factors. First, MIPw behaves as a homogeneous
catalyst in the pure Tris–HCl system, thus the accessibility of the
active sites is greatly enhanced. More importantly, NIPAM may
create a strong hydrophobic environment in the imprinted
nanostructure in the resultant tetrapolymer. When the imprinted
nanospherical gel shrinks to the smallest size at pH values close
to the pI of the polymer, the hydrophobic affinity between the
imprinted active sites and the substrate may be strengthened,
thus accelerating the catalytic process. The reversible affinity of
the hydrophobic molecules by NIPAM polymers has been
previously reported and used for reversible affinity chromato-
graphy.[27] In Wang’s work,[16] it was observed that longer alkyl
chains may produce a strong hydrophobic attraction between the
gel and the substrate. In our experiment, we chose EDMA instead
of MBAAm as the crosslinker because of its higher hydro-
phobicity than the latter.
At pH values close to its pI, the polymer gel shrinks to a
relatively compact state, facilitating the recovery of the imprinting
cavity. The enhanced hydrophobic affinity may induce and
strengthen the specific interaction offered by other functional
monomers, so that the effective HVA concentration within the gel
was increased, which improved the overall catalytic rate. Besides,
the swelling or shrinking equilibrium can be reached within a few
minutes for nanospheric hydrogels instead of a few hours for
macroscopic gels with the same chemical structure.[28] So,
imprinted hydrogel nanospheres can rapidly regulate their
conformation in catalysis processes.
In conclusion, a novel catalytic system based on an imprinted
tetrapolymer of 4-vinylpyridine, hemin, acrylamide, and
N-isopropylacrylamide cross-linked by ethylene glycol dimetha-
crylate with homovanillic acid as the template molecule was
developed as an enzyme mimic of horseradish peroxidase. By
taking advantage of its thermal- and salt-responsive properties,
the polymer was prepared and separated as a solid polymer, while
applied as a water-soluble nanospheric hydrogel. The imprinted
gel particles showed a remarkably sensitive response to the pH
conditions. Under optimal pH conditions, the imprinted gel
achieved an excellent hydrophobic microenvironment within the
imprinted nanostructures, which significantly increased the
catalytic efficiency. The results of this study demonstrate that
incorporation of stimuli-sensitive monomers into a catalytic,
molecularly imprinted system can be an effective way to modulate
the microenvironment around the catalytic centers in artificial
enzymes.
Experimental
Preparation of Molecularly Imprinted Hydrogels: Polymer hydrogels were
prepared by precipitation polymerization using a modification of previous
methods [21,22]. The monomer concentration was about 1% (w/w) to
prevent coagulation. In a typical experiment, after the dissolution of NIPAM
(200 mg, 1.77 mmol), hemin (13 mg, 0.02 mmol), AAm (5.6 mg,
Adv. Mater. 2010, 22, 1488–1492 ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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0.08 mmol), 4-Vpy (8 mL, 0.08 mmol), HVA (3.5 mg, 0.02 mmol), EDMA
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(10 mL, 0.05 mmol), and 10 mg of azobisisobutyronitrile (AIBN) in 20 mL of
a mixture of DMSO and water (7:3, v/v), the mixture was bubbled with
nitrogen for 10 min and heated to 70 8C to start the polymerization
reaction. The reaction proceeded under stirring (400 rpm) for 48 h at 70 8C.
After cooling, the polymer was precipitated by the addition of sodium
chloride dissolved in water (3 M), centrifuged at 12 000 rpm for 30 min and
resuspended in a solution of 10% acetic acid in methanol (v/v) to extract
the template [5]. The solution was gently stirred overnight. The particles
were centrifuged again and thoroughly dialyzed (molecular weight cut off
3 500; Visking membrane, Carl Roth, Karlsruhe, Germany) for 3 days
against distilled water. Finally, the particles were freeze-dried in vacuum at
50 8C in an Alpha1-4 freezer (Leybold, Cologne, Germany) for at least
48 h. The non-imprinted control polymer (NIP) was generated in the same
way without the addition of the HVA template.
Characterization of the Obtained Molecularly Imprinted Hydrogels: The
particle morphology was observed using environmental scanning electron
microscopy (ESEM) (FEI Quanta 200FEG). The particles containing water
were mounted without drying on metal stubs and measured under a low
vacuum (ca. 103 atm.) and relatively low temperature (near 4 8C). Details
of the static light scattering (SLS) study, dynamic light scattering (DLS)
study, aqueous electrophoresis, and the determination of the hemin
content present in the polymers are described in the Supporting
Information.
Catalytic Reaction with the Imprinted Hydrogel Nanospheres as
Catalyst: The catalytic reactions (total volume, 2.0 mL) were carried out
in Tris-HCl buffer (50 mM, pH 8.5, 1.7 mL), stirred with the solution of the
polymers (0.1 mg mL1, 0.1 mL) at 25 8C. The final concentrations were
fixed at 5.0
103 g L1 (hemin concentration, 2.64
107 M) for the
polymer and 5.26
103 M for H2O2, with HVA concentrations varying
from 1.30
103 M to 1.10
104 M. The dynamic curve of the dimeriza-
tion reaction of HVA catalyzed by the MIP was recorded with a
luminescence spectrometer (LS-50B, Perkin Elmer, USA) and the
pseudo-first-order rate constants were obtained from linear plotting of
the fluorescence intensity versus time.
Acknowledgements
This work was supported by the National Natural Science Foundation of
China (90717002 and 30872109) and the National 863 Program
(2006AA10Z435). Supporting Information is available online from Wiley
InterScience or from the author.
Received: September 10, 2009
Published online: December 28, 2009
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