Azobenzene-containing molecularly imprinted polymer microspheres with photoresponsive template binding properties

Article abstract of DOI:10.1039/c0jm02898c

The first successful preparation of azobenzene (azo)-containing molecularly imprinted polymer (MIP) microspheres with photoresponsive template binding properties is described. A methacrylate azo functional monomer with a pyridine group was used for this purpose, and its good solubility in acetonitrile allowed the implementation of molecular imprinting via precipitation polymerization, leading to azo-containing MIP microspheres (number-average diameter = 1.33 μm, polydispersity index = 1.15) with obvious molecular imprinting effects towards the template 2,4-dichlorophenoxyacetic acid (2,4-D), rather fast template rebinding kinetics, and appreciable selectivity over structurally related compounds. The binding association constant K_a and apparent maximum number N_max for high-affinity sites of the imprinted polymer in the dark environment were determined by Scatchard analysis to be 2.3 × 10⁴ M^-1 and 10.0 μmol g^-1, respectively. Most importantly, the binding affinity of the imprinted sites in azo-containing MIP microspheres was found to be photoresponsive towards the template, which decreased upon UV light irradiation (as revealed by the resulting lower K _a value for high-affinity sites and reduced specific bindings), whereas it could be recovered during the subsequent thermal (or visible light-induced) back-isomerization. Furthermore, this photoregulation process proved to be highly repeatable under photoswitching conditions. The Royal Society of Chemistry.

Full text of DOI:10.1039/c0jm02898c

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PAPER
Azobenzene-containing molecularly imprinted polymer microspheres with
photoresponsive template binding properties†
*
Liangjing Fang, Sujing Chen, Ying Zhang and Huiqi Zhang
Received 1st September 2010, Accepted 1st November 2010
DOI: 10.1039/c0jm02898c
The first successful preparation of azobenzene (azo)-containing molecularly imprinted polymer (MIP)
microspheres with photoresponsive template binding properties is described. A methacrylate azo
functional monomer with a pyridine group was used for this purpose, and its good solubility in
acetonitrile allowed the implementation of molecular imprinting via precipitation polymerization,
leading to azo-containing MIP microspheres (number-average diameter ¼ 1.33 mm, polydispersity
index ¼ 1.15) with obvious molecular imprinting effects towards the template 2,4-
dichlorophenoxyacetic acid (2,4-D), rather fast template rebinding kinetics, and appreciable selectivity
over structurally related compounds. The binding association constant Ka and apparent maximum
number Nmax for high-affinity sites of the imprinted polymer in the dark environment were determined
by Scatchard analysis to be 2.3 ꢀ 104 Mꢁ1 and 10.0 mmol gꢁ1, respectively. Most importantly, the
binding affinity of the imprinted sites in azo-containing MIP microspheres was found to be
photoresponsive towards the template, which decreased upon UV light irradiation (as revealed by the
resulting lower Ka value for high-affinity sites and reduced specific bindings), whereas it could be
recovered during the subsequent thermal (or visible light-induced) back-isomerization. Furthermore,
this photoregulation process proved to be highly repeatable under photoswitching conditions.
appealing physical and chemical characteristics make MIPs very
Introduction
promising candidates for many applications, including separa-
tion, chemical sensors, antibody mimics, biomimetic catalysis,
drug delivery, drug development, and so on.1–7
Molecular imprinting is a facile and versatile approach for
preparing artificial receptors with tailor-made molecular recog-
nition binding sites.1–7 A typical molecular imprinting process
usually involves the formation of a complex between a template
molecule and a functional monomer via either non-covalent
interactions (such as hydrogen bonds, ionic interactions,
hydrophobic interactions, and metal-ion chelating interactions)
or reversible covalent bonds (e.g., boronate ester, ketal and
acetal, or Schiff base), which is subsequently copolymerized with
a crosslinking monomer in the presence of a suitable porogenic
solvent. After polymerization, the template is removed from the
obtained crosslinked polymer network, leading to a molecularly
imprinted polymer (MIP) with binding cavities complementary
to the shape, size and functionality of the template. The virtues of
the MIPs include the outstanding affinity and selectivity towards
the template molecules as well as their high mechanical stability
and remarkable resistance to extreme pH conditions, organic
solvents, metal ions, and autoclave treatment. Such highly
Despite the tremendous progress made in the molecular
imprinting field, many challenges still remain to be addressed,
especially in the design of advanced MIP materials mimicking the
biological receptors.8–10 It is well known that the biological
receptors have many fascinating characteristics such as their high
responsivity towards external stimuli (e.g., the temperature). The
presently developed MIPs, however, are normally difficult to
deform for regulating their binding properties because a high
fraction of a crosslinker is usually used in molecular imprinting
to stabilize the binding sites. Recently, the rational use of certain
special co-functional monomers (e.g., N-isopropylacrylamide
(NIPAAm), acrylic acid, and azobenzene (azo) monomers) in
molecular imprinting, together with the appropriate choice of the
amounts of the crosslinkers used, makes it possible to obtain
responsive MIPs towards different stimuli such as temperature,
pH, and light.11
Photoirradiation has become one of the most frequently
adopted external stimuli for responsive materials because light is
a clean energy and can be manipulated precisely, rapidly and
remotely.12,13 Numerous well-established photoresponsive
molecular systems are now available.14–22 Among them, azo-
containing systems have attracted great attention because they
Key Laboratory of Functional Polymer Materials (Nankai University),
Ministry of Education, Department of Chemistry, Nankai University,
Tianjin, 300071, P. R. China. E-mail: zhanghuiqi@nankai.edu.cn; Fax:
+86 2223507193; Tel: +86 2223507193
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c0jm02898c
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can exhibit excellent photoinduced fast and reversible isomeri-
zation between the trans and cis-isomers of the azo moieties upon
exposure to UV or visible light, which can trigger significant
changes in the geometry and dipole moment of the azo chro-
mophore.14–22
properties under photoirradiation conditions were characterized
in detail.
Experimental
Recent years have witnessed considerable interest in photo-
responsive MIP materials.11,23–28 Such advanced functional MIP
materials have been mainly prepared by introducing a functional
azo moiety into the selective binding sites of the MIPs. The
configuration of the azo groups can be regulated by light illu-
mination, which brings about a large alteration of the spatial
arrangement of the binding functionalities in the binding sites,
thus leading to a significant change in the strength of the host–
guest interactions. Minoura and coworkers described the first
preparation of azo-containing MIP membranes with photo-
regulated template binding properties by using p-phenyl-
azoacrylanilide as the functional monomer.23,24 The groups of
Lam and Yu reported the fabrication of a photoresponsive MIP
for the photoregulated release and uptake of caffeine by using 4-
((4-methacryloyloxy)phenylazo)benzoic acid as the functional
monomer.25 Later, Lam and coworkers also prepared photo-
responsive molecularly imprinted hydrogels for the photo-
regulated release and uptake of pharmaceuticals in the aqueous
media by using a water-soluble azo functional monomer 4-((4-
methacryloyloxy)phenylazo)benzenesulfonic acid.26 Schmitzer
Materials
Ethylene glycol dimethacrylate (EGDMA, Alfa Aesar, 98%) was
purified by distillation under vacuum. Acetonitrile (Tianjin
Jiangtian Chemicals, China, analytical grade (AR)) was refluxed
over calcium hydride (CaH2) and then distilled. Tetrahydrofuran
(THF, Tianjin Jiangtian Chemicals, 99%) was refluxed over
sodium and then distilled. N,N-Dimethylformamide (DMF,
Tianjin Jiangtian Chemicals, 99.5%) was dried with anhydrous
magnesium sulfate (MgSO4) and then distilled under vacuum.
Methanol (Tianjin Jiangtian Chemicals, AR) was distilled prior
to use. Triethylamine (TEA, Tianjin Jiangtian Chemicals, 99%)
was dried with anhydrous sodium sulfate (Na2SO4) and then
distilled. Azobisisobutyronitrile (AIBN, Tianjin Jiangtian
Chemicals, Chemical purity (CP)) was recrystallized from
ethanol and stored at ꢁ18 ꢂC before use. Thionyl chloride
(Tianjin Jiangtian Chemicals, 99.5%) was purified by distillation
just prior to use. Methacrylic chloride was obtained by the
reaction between methacrylic acid and thionyl chloride. Meth-
acrylic anhydride was prepared by reacting methacrylic acid and
methacrylic chloride in the presence of an aqueous solution of
and coworkers developed
a
new azo monomer di-
and successfully
sodium hydroxide, following
a
literature procedure.31
(ureidoethylenemethacrylate)azobenzene
4-(4-Hydroxyphenylazo)pyridine was prepared following
a previously reported procedure (Scheme S1 in the supporting
information†).32 4-Aminopyridine (Shanghai Jingchun Chemical
Company, China, AR), 4-dimethylaminopyridine (DMAP,
Merck, AR), 2,4-dichlorophenoxyacetic acid (2,4-D, Alfa Aesar,
prepared a photoresponsive MIP by using it as both the func-
tional monomer and crosslinker.27 Takeuchi and coworkers
synthesized a photoresponsive porphyrin-imprinted polymer by
using a novel azo functional monomer bearing a diaminopyri-
dine group.28 While all the above reports have demonstrated
that the photoresponsive azo-containing MIPs are of great
potential in many applications such as smart separation,
extraction, and assays as well as efficient drug delivery systems,
their rather limited physical formats (i.e., bulk polymer
membranes,23,24 bulk monoliths,25,27,28 and bulk hydrogels26)
have significantly influenced their applications. In particular,
MIP particles with irregular shapes and relatively large sizes
(normally tens of micrometers in diameter) are normally
obtained after the time-consuming and laborious grinding and
sieving of the bulk MIPs, which are inappropriate for such
applications as binding assays and drug delivery systems because
the best physical format for such applications is spherical
beads.29,30 Moreover, the binding sites inside the MIP particles
with relatively large sizes are inaccessible, thereby significantly
lowering the template loading capacities of the MIP particles.
Therefore, the development of azo-containing MIP microspheres
with photoresponsive template binding properties remains an
important goal.
Scheme 1 Schematic illustration for the preparation of azo-containing
MIP microspheres with photoresponsive binding sites.
In this paper, we describe for the first time the successful
preparation of azo-containing MIP microspheres with photo-
responsive template binding properties via precipitation poly-
merization by using an acetonitrile-soluble azo functional
monomer with a pyridine group (i.e., 4-((4-methacryloyloxy)-
phenylazo)pyridine, Schemes 1 and 2). The chemical structures,
morphologies, particle sizes and size distributions, template
rebinding properties and substrate selectivity of the obtained
azo-containing MIP microspheres as well as their binding
Scheme 2 The chemical structures of the azo functional monomer
MAzoPy, the template molecule 2,4-D, and the related test compounds of
the template (i.e., DPAc and POAc).
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98%), 2,4-dichlorophenylacetic acid (DPAc, Acros, 99%), phe-
noxyacetic acid (POAc, Acros, 98 + %), and all the other reagents
were commercially available and used as received. The chemical
structures of 2,4-D, DPAc and POAc are shown in Scheme 2.
Fourier transform infrared (FT-IR) spectra of the azo-con-
taining MIP/NIP microspheres were obtained using a Nicolet
Magna-560 FT-IR spectrometer.
The elemental analyses of the MIP/NIP microspheres were
carried out to determine their chemical compositions and
crosslinking densities by using Elementar Vario EL (Germany).
By assuming that the obtained MIP/NIP microspheres contain x
moles of the bound MAzoPy unit (molecular formula:
C15H13N3O2) and y moles of the bound EGDMA unit (molec-
ular formula: C10H14O4), the following equations can be derived
for the weight fractions of carbon (CC) and nitrogen (CN):
Preparation of the methacrylate azo functional monomer with
a pyridine group 4-((4-methacryloyloxy)phenylazo)pyridine
(MAzoPy) (Scheme 2 and Scheme S1 in the supporting
information†)
To a solution of 4-(4-hydroxyphenylazo)pyridine (2.3901 g, 12
mmol) in dried THF (150 mL), methacrylic anhydride (3.57 mL,
24 mmol), 4-dimethylaminopyridine (DMAP, 0.1320 g, 1.2
mmol), and triethylamine (TEA, 1.67 mL, 12 mmol) were added
ꢀ
ꢁ
CC ¼ ð15xþ10yÞMC= xMMAzoPy þ yMEGDMA
;
ꢀ
ꢁ
CN ¼ 3xMN= xMMAzoPy þ yMEGDMA
ꢂ
successively. After being stirred at 40 C for 24 h, the reaction
where MC is the atomic weight of carbon, MN the atomic weight
of nitrogen, MMAzoPy the molecular weight of MAzoPy, and
MEGDMA the molecular weight of EGDMA. The molar fractions
of the bound EGDMA unit in the MIP/NIP microspheres (i.e., y/
(x + y), which can also be utilized to express the crosslinking
densities of the MIP/NIP microspheres) can thus be obtained by
introducing CC and CN values (determined by elemental analysis)
into the above two equations.
mixture was poured into a large amount of water (300 mL) and
extracted with chloroform. The obtained organic phase was
dried over Na2SO4 and the chloroform removed using a rotary
evaporator, leading to a brown solid product (3.12 g, yield: 95%).
ꢂ
m.p. 144–145 C (determined by polarizing optical microscope
(POM) with a heating rate of 10 ꢂC minꢁ1); UV-vis (acetonitrile):
lmax/nm (3/dm3 molꢁ1 cmꢁ1) ¼ 318 (18800), around 432 (700); 1H
NMR (CDCl3): d (ppm) ¼ 8.80 (d, J ¼ 4.8 Hz, 2H, Ar-H), 8.01
(d, J ¼ 8.8 Hz, 2H, Ar-H), 7.69 (d, J ¼ 4.8 Hz, 2H, Ar-H), 7.31
(d, J ¼ 8.8 Hz, 2H, Ar-H), 6.39 (s, 1H, CH2 ¼ C-), 5.81 (s, 1H,
CH2 ¼ C-), 2.08 (s, 3H, -CH3).
The morphologies, particle sizes, and size distributions of the
azo-containing MIP/NIP microspheres were determined with
a scanning electron microscope (SEM, Shimadzu SS-550). All of
the SEM size data reflected an average of about 100 particles
each, which were calculated by using the following formulas:
Preparation of azo-containing MIP and non-imprinted polymer
(NIP) microspheres
k
k
k
k
X
X
X
X
Dn ¼
niDi=
ni; Dw
¼
niD4i =
niD3i ; U ¼ Dw=Dn
The azo-containing MIP microspheres were prepared via
precipitation polymerization using MAzoPy, 2,4-D, EGDMA,
AIBN, and acetonitrile as the functional monomer, template,
crosslinker, initiator, and porogenic solvent, respectively,
according to the following procedure: MAzoPy (0.4005 g, 1.5
mmol), 2,4-D (0.3316 g, 1.5 mmol), and dried acetonitrile (75
mL) were added into a one-neck round-bottom flask (100 mL)
successively; a clear homogeneous solution was obtained after
the mixture was stirred at ambient temperature for 3 h in the dark
(the azo monomer was kept in the trans-rich state, Scheme 1).
EGDMA (0.85 mL, 4.5 mmol) and AIBN (0.0086 g, 0.0525
mmol) were then added into the above system. The reaction
mixture was bubbled with argon for 20 min in an ice bath and the
flask then sealed and immersed in a thermostated oil bath at 60
ꢂC. After 48 h of polymerization at 60 ꢂC in the dark, the
resulting polymer particles were collected by centrifugation of
the reaction mixture, which were subsequently purified through
Soxhlet extraction with methanol–acetic acid (9 : 1 v/v, 48 h) and
acetonitrile (48 h) successively until no template could be
detected in the extraction solution. After being dried at 40 ꢂC
under vacuum for 24 h, khaki colored azo-containing MIP
microspheres were obtained in a yield of 66%.
i¼1
i¼1
i¼1
i¼1
where Dn is the number-average diameter, Dw the weight-average
diameter, U the size distribution index, k the total number of the
measured particles, Di the particle diameter of the determined
microspheres, and ni the particle number of the microspheres
with a diameter Di.
A UV-vis scanning spectrophotometer (TU1900, Beijing Pur-
kinje General Instrument Co., Ltd) was utilized to obtain the
UV-vis spectra of the samples at 25 ꢂC. The photochemical
isomerization of the MAzoPy solution was investigated by irra-
diating them firstly with a 365 nm UV lamp (12 W) and then with
a 450 nm visible light lamp (18 W, wavelength range: 400–550
nm, lmax ¼ 450 nm). The thermal back-isomerization of the
MAzoPy solution was studied by its exposure to 365 nm UV light
(12 W) for 210 s, and its subsequent time spent in the dark at 25
ꢂC, with the UV-vis spectra of the MAzoPy solution measured at
different times.
The concentrations of the template molecule 2,4-D and its
related compounds were quantified with a high-performance
liquid chromatograph (HPLC, Scientific System Inc., USA)
equipped with a UV-vis detector. The wavelength used for the
determination of 2,4-D, DPAc, and POAc was 284, 272, and 270
nm, respectively. A mixture of methanol and 0.5% aqueous
solution of acetic acid (4/1 v/v) was used as the mobile phase at
The corresponding azo-containing NIP microspheres (khaki
color) were also prepared and purified under identical conditions
except that the template was omitted (yield: 65%).
a flow rate of 1 mL minꢁ1
.
The binding kinetics of the template molecule 2,4-D with the
azo-containing MIP/NIP microspheres was evaluated by batch
adsorption experiments, where 5 mg of MIP/NIP microspheres
were incubated with a solution of 2,4-D in acetonitrile (0.5 mL,
Characterization
1H NMR spectra were recorded on a Varian Unity plus-400
spectrometer (400 MHz).
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ꢂ
0.05 mM) at 25 C in the dark for different times. After centri-
Results and discussion
fugation, the amounts of the template remaining in the super-
natants (expressed as F) were determined by HPLC and those
bound to the MIP/NIP microspheres (B) could thus be obtained
by subtracting F from the initial template concentration.
Equilibrium binding experiments were performed by incu-
bating a 2,4-D solution in acetonitrile (0.5 mL, 0.05 mM) with
different amounts of azo-containing MIP/NIP microspheres at
25 ꢂC for 6 h either in the dark or under the irradiation of 365 nm
UV light (16 W). The amounts of the template bound to the
MIP/NIP microspheres were then quantified with HPLC.
The binding isotherm of the azo-containing MIP microspheres
was studied with Scatchard analysis:33 MIP microspheres (5 mg)
were incubated with a series of 2,4-D solutions in acetonitrile
(C ¼ 0.04–1.0 mM, 0.5 mL) at 25 ꢂC for 6 h either in the dark or
under the irradiation of 365 nm UV light (16 W). After centri-
fugation, the amounts of the template bound to the MIP
microspheres (B) were determined by HPLC. The Scatchard
equation used is B/F ¼ (Nmax ꢁ B)Ka, where Ka and Nmax
represent the binding association constant and apparent
maximum number of the binding sites, respectively.
Over the past few years, we have been working in the fields of
molecular imprinting and photoresponsive azo-containing
functional materials.5,8,17,34–38 In this paper, we combine the
above two fields together and aim to develop azo-containing
MIP microspheres with photoresponsive template binding
properties. The spherical physical format and rather small sizes
of such photoresponsive azo-containing MIP microspheres make
them highly promising in many potential applications such as
smart separation, extraction, and assays, efficient drug delivery
systems, and intelligent chemical carriers (which can release
chemical reagents according to the requirements under light
irradiation). To the best of our knowledge, there has been no
report to date on the azo-containing spherical MIP particles with
photoresponsive template binding properties.
Precipitation polymerization has proven very versatile for
preparing MIP micro-/sub-microspheres because of its easy
operation and lack of need for any surfactant or stabi-
lizer.30,35,36,39–41 Spherical MIP particles with a diameter ranging
from 100 nm to several micrometers can be readily obtained by
tuning the polymerization conditions, including the kind of
solvents used, the amounts of the initiator and solvent used, and
the polymerization temperature and time, which are highly useful
for different application purposes. In particular, it has been well
established that the choice of an appropriate solvent is pivotal for
the successful preparation of MIP microspheres via precipitation
polymerization, and acetonitrile has proven to be the most
commonly utilized solvent for this purpose.30,35,39,41 Therefore, it
can be anticipated that the development of an acetonitrile-
soluble azo functional monomer should make it possible to
prepare spherical azo-containing MIP particles via precipitation
polymerization. In this context, it is worth mentioning that the
groups of Lam and Yu also attempted to prepare photo-
responsive azo-containing MIP particles via precipitation poly-
merization, but only a bulk MIP monolith was obtained due to
the use of an inappropriate solvent (in their case, a mixture of
DMF and acetonitrile was used as the polymerization solvent
instead of pure acetonitrile because their azo functional mono-
mer was not soluble in acetonitrile itself).25
The binding selectivity of the azo-containing MIP/NIP
microspheres was evaluated by measuring their rebinding
capacities towards 2,4-D and its structurally related compounds
(DPAc and POAc, Scheme 2): 5 mg of MIP/NIP microspheres
were incubated with 0.5 mL of a mixed solution of 2,4-D, DPAc,
and POAc in acetonitrile (C2,4-D, DPAc or POAc ¼ 0.05 mM) at 25
ꢂC for 6 h in the dark and the amounts of 2,4-D and the related
compounds bound to the MIP/NIP microspheres were quantified
by HPLC.
The studies on the photoregulated release and uptake of the
template 2,4-D and its analogues by the azo-containing MIP
microspheres were performed by alternately switching on and
turning off the UV light irradiation on the mixtures of MIP
microspheres and a solution of analytes: A series of samples were
prepared by adding 5.0 mg of azo-containing MIP microspheres
and a mixed solution of 2,4-D, DPAc, and POAc in acetonitrile
(0.5 mL, C2,4-D, DPAc or POAc ¼ 0.05 mM) into plastic Eppendorf
tubes (2 mL), respectively, which were subsequently sealed and
put into an incubator equippꢂed with a 365 nm UV lamp (16 W).
After their incubation at 25 C in the dark for 6 h, one sample
was taken out from the incubator and centrifuged to determine
the amounts of the analytes bound by the MIP microspheres (by
HPLC). The UV light was then switched on in the incubator to
irradiate the remaining samples immediately after the first
sample was taken out. After 3 h of UV light irradiation with
It is also known that 4-vinylpyridine is a widely used func-
tional monomer in the molecular imprinting field, which can
form noncovalent interactions with many template molecules
bearing various functional groups such as phenol,35,42 hydroxyl,43
or carboxylic acid.35,36,44 Therefore, a methacrylate azo func-
tional monomer with a pyridine unit (i.e., MAzoPy, Scheme 2)
was designed for our purpose, which was readily prepared by
ꢂ
incubation for the samples at 25 C, the UV light was switched
reacting
methacrylic
anhydride
with
4-(4-hydroxy-
off and the second sample was taken out for the determination of
the binding of the analytes. The remaining samples were then
phenylazo)pyridine in the presence of a very effective acylation
catalyst DMAP in quite good yields (Scheme S1 in the sup-
porting information†), and its purity was satisfactory, as deter-
mined via both thin layer chromatography and 1H NMR
spectroscopy. To our delight, this azo functional monomer
proved to be fully soluble in acetonitrile and could thus be
directly utilized to prepare azo-containing MIP microspheres via
precipitation polymerization for a wide range of templates.
The UV-vis spectrum of the solution of MAzoPy in acetoni-
trile exhibited one strong absorption band around 318 nm and
another very weak one around 432 nm, which are typical for the
ꢂ
incubated at 25 C in the dark for another 18 h, and the third
sample was taken out for the determination of the binding of the
analytes. The UV light was then switched on again to irradiate
the remaining samples immediately at 25 ꢂC after the third
sample was taken out. The above photoswitching procedures
(i.e., UV light on for 3 h and UV light off for 18 h alternately)
were repeated until all of the other samples were measured.
The studies on the photoregulated release and uptake of the
template 2,4-D and its analogues by the azo-containing NIP
microspheres were carried out similarly.
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azo compounds and can be ascribed to the p / p* and n / p*
electron transitions of the N]N bond, respectively (Fig. 1).45 By
irradiation with 365 nm UV light the studied azo monomer
solution underwent significant trans to cis photoisomerization
(Fig. 1a). The intensity of the p / p* transition band around
318 nm decreased rapidly, whereas that of the n / p* transition
band around 432 nm slightly increased. The existence of isobestic
points at 271 and 366 nm was characteristic for the existence of
two distinct absorbing species in equilibrium with each other and
at the same time proved that no side reaction took place during
the photoisomerization process in the range studied.45 Irradi-
ating the thermodynamically less stable cis-MAzoPy with visible
light led to the cis to trans back-isomerization of MAzoPy in
solution (Fig. 1b). However, the finally recovered absorbance of
trans-MAzoPy was lower than that before UV irradiation with
the recovery of the trans-isomer being 83%. Similar phenomena
were also observed previously by others.46,47 Its real cause is not
totally clear yet, and further investigation is ongoing to provide
a reasonable explanation. Nevertheless, the photochemical
isomerization became completely reversible upon subsequent
cycles of UV and visible light irradiation (Fig. S1 in the sup-
porting information†). In the absence of visible light (i.e., in the
dark), the thermal back-isomerization of the MAzoPy solution
was slow, but near to complete recovery could be reached after
a rather long time (Fig. 1c).
With this acetonitrile-soluble and highly photoresponsive azo
functional monomer MAzoPy in hand, we started to test whether
it could be utilized in molecular imprinting for the direct prep-
aration of azo-containing MIP microspheres via precipitation
polymerization. A herbicide 2,4-D (Scheme 2) was chosen here as
the model template molecule because it could form hydrogen
bonding with the pyridine group of MAzoPy in acetonitrile.35 In
addition, EGDMA, AIBN, and acetonitrile were used as the
crosslinker, initiator, and porogenic solvent, respectively. The
molar
ratio
of
the
reactant
combination
2,4-
D : MAzoPy : EGDMA : AIBN was 1 : 1 : 3 : 0.035 and the
volume of the utilized acetonitrile was 97% relative to that of the
whole reaction system. Note that the molar ratio of the template
to MAzoPy is much higher than that used in a normal molecular
imprinting system, where an excess of functional monomer is
usually required in order to shift the equilibrium towards the
complex formation. This could be attributed to the characteris-
tics of precipitation polymerization, where a very large amount
of porogenic solvent (typically $95% of the total reaction
volume) is normally used, which leads to most of the template
dissolving into the porogenic solvent instead of interacting with
the functional monomer to form
a
prepolymerization
complex.30,35,39–41 Therefore, increasing the amount of template
would be necessary to improve the formation of a prepolymeri-
zation complex as well as the imprinting efficiency.48,49 As
a reference, the corresponding azo-containing NIP particles were
also prepared similarly by omitting the templateꢂin the reaction
system. All the reactions were performed at 60 C in an argon
atmosphere in the dark (in order to keep the azo moieties in the
trans-rich state, Scheme 1), and the resulting polymer particles
were thoroughly purified through Soxhlet extraction, leading to
khaki colored MIP and NIP particles with their yields being 66
and 65%, respectively. The khaki color of the MIP and NIP
particles indicated that the azo functional moieties were
successfully incorporated into the resulting polymers.
The morphologies, particle sizes, and size distributions of the
obtained azo-containing MIP and NIP particles were firstly
characterized via SEM (Fig. 2), and the results showed that
spherical azo-containing MIP and NIP particles were success-
fully prepared via precipitation polymerization. The number-
average diameters (Dn) of the MIP and NIP beads were 1.33 and
1.28 mm, respectively, and they had a polydispersity index (U) of
1.15 and 1.11, respectively. The rather similar diameters of the
obtained azo-containing MIP and NIP microspheres revealed
that the template molecule had little influence on the particle
growth during the precipitation polymerization in this case,
which is somewhat different from previous reports.40,41
Fig. 1 UV-vis spectral changes in dependence of time for the solution of
MAzoPy in acetonitrile (C ¼ 0.05 mM) at 25 ꢂC: (a) upon irradiation
with 365 nm UV light; (b) upon irradiating the solution (which has been
irradiated with 365 nm UV light for 210 s) with visible light; (c) the
thermal back-isomerization of the azo monomer solution after its being
irradiated for 210 s.
Fig. 3 presents the FT-IR spectra of the obtained azo-con-
taining MIP and NIP microspheres, from which it can be clearly
seen that the MIP and NIP microspheres have rather similar
chemical structures. The presence of three significant peaks
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and NIP microspheres were readily available as they could be
simply expressed by the molar fractions of the bound EGDMA
unit in the obtained polymers. The molar fractions of the bound
EGDMA unit in both the MIP and NIP microspheres were
determined to be 74%, which were in excellent consistency with
that of EGDMA in the initial comonomer mixture (75%), sug-
gesting that EGDMA had rather similar reactivity with MAzoPy
in the studied reaction system. Moreover, the above results also
demonstrated that the crosslinking densities of the azo-contain-
ing MIP and NIP microspheres were high enough for stabilizing
the shapes of the imprinted cavities.
The binding kinetics of the template molecule 2,4-D with the
azo-containing MIP and NIP microspheres was evaluated by
batch adsorption experiments, which were carried out by incu-
bating a certain amount of polymers in a dilute solution of 2,4-D
in acetonitrile in the dark. Fig. 4 shows the adsorption uptake of
the polymers versus the incubation time. It can be seen clearly
that both the MIP and NIP microspheres reached their binding
equilibriums at a time of about 1 h, indicating the occurrence of
very fast template rebinding processes.
Equilibrium binding experiments were then performed to
study the template rebinding properties of the azo-containing
MIP and NIP microspheres by incubating a 2,4-D solution in
acetonitrile with different amounts of polymers at ambient
temperature for 6 h in the dark. Fig. 5 shows that the azo-con-
taining MIP microspheres bound more 2,4-D than the NIP
microspheres in a wide range of polymer concentrations. For
example, in a dilute solution of 2,4-D in acetonitrile, while 16 mg
of MIP microspheres bound 81% of 2,4-D, an equivalent amount
of the corresponding control bound only 64%. This, together
with the rather similar diameters of the MIP and NIP micro-
spheres (which should lead to their similar specific surface areas
and nonspecific template bindings), clearly reveals the successful
generation of selective binding sites in the obtained MIP particles
and thus the successful molecular imprinting process.
Fig. 2 Scanning electron micrographs of the azo-containing MIP (a)
and NIP (b) microspheres.
around 1733 (C]O stretching), 1252 and 1144 cmꢁ1 (C–O–C
stretching) supported the existence of poly(EGDMA) in the
obtained MIP and NIP microspheres. The characteristic peaks
corresponding to the C]N stretching (1587 cmꢁ1) and C]C
stretching (1460 cmꢁ1) in the pyridine rings could also be
observed, revealing that poly(MAzoPy) was also present in the
MIP and NIP microspheres. Furthermore, the presence of small
peaks around 1637 cmꢁ1 (corresponding to the C]C stretching
mode) demonstrated that less than 100% of the bound EGDMA
molecules were crosslinked in the MIP and NIP microspheres.50
The azo-containing MIP and NIP microspheres were further
studied via elemental analysis to determine their chemical
compositions, from which the crosslinking densities of the MIP
To get more insight into the binding characteristics of the azo-
containing MIP microspheres, further studies were carried out
using Scatchard analysis. The results showed that the Scatchard
plot of the azo-containing MIP microspheres determined in the
dark could be fitted into two straight lines (Fig. 6a), suggesting
that the binding sites in the azo-containing MIP microspheres
Fig. 4 Binding kinetics of 2,4-D on the azo-containing MIP (filled
symbols) and NIP (open symbols) microspheres. 5 mg of MIP/NIP
microspheres were incubated with a solution of 2,4-D in acetonitrile (0.5
mL, 0.05 mM) at 25 ꢂC in the dark for different times.
Fig. 3 FT-IR spectra of the azo-containing MIP (a) and NIP (b)
microspheres.
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Table 1 The isothermal binding parameters of the azo-containing MIP
microspheres determined in the dark and under the irradiation of UV
light, respectively
High-affinity binding Low-affinity binding
sites a
a
sites
ka ꢀ10ꢁ4 Nmax /mmol ka ꢀ10ꢁ4 Nmax /mmol
Sample
Mꢁ1
gꢁ1
Mꢁ1
gꢁ1
MIP in the dark
MIP under UV light
irradiation
2.3
1.6
10.0
10.9
0.19
0.17
29.4
35.9
a
Ka and Nmax represent the binding association constant and apparent
maximum number of the high- and low-affinity binding sites,
respectively, which were obtained by Scatchard analysis.
Fig. 5 Equilibrium bindings of 2,4-D (C ¼ 0.05 mM) on different
amounts of azo-containing MIP (filled symbols) and NIP (open symbols)
microspheres in the dark (circle) and under UV light irradiation
(triangle), respectively.
The binding selectivity of the MIPs is often determined by
comparing the binding of the template with those of its
analogues, which affords an indication of the cross-reactivity of
the MIPs towards selected molecules. The bindings of the azo-
containing MIP and NIP microspheres towards 2,4-D were thus
compared to two structurally related compounds, DPAc and
POAc (Scheme 2), which have the same functionality (i.e.,
carboxyl group) but differ either in the distance between the
functional group and the benzene ring or in the numbers of
substituents on the benzene ring. As can be seen clearly from
Fig. 7, besides binding 2,4-D, the MIP microspheres also
adsorbed DPAc and POAc, suggesting the existence of certain
cross-binding reactivity. Nevertheless, the MIP microspheres
showed significantly lower binding capacities towards DPAc and
POAc than towards 2,4-D, thus demonstrating the high selec-
tivity of the MIP microspheres towards 2,4-D. It is worth
mentioning here that while the presence of certain cross-binding
reactivity in the MIPs might be undesirable for such applications
as sensors, this could actually be an advantage in sample treat-
ment because a class of template analogues could also be
removed or enriched efficiently.
were heterogeneous and their affinities could be approximated by
two binding association constants (Ka) corresponding to the
high- and low-affinity sites, as usually observed for the MIPs
prepared via the noncovalent molecular imprinting approach.51
We are particularly interested in the high-affinity sites because
they are mainly responsible for the specific binding of the MIP
(i.e., the binding difference between the MIP and its corre-
sponding NIP52). In the present work, the Ka and Nmax values of
the high-affinity sites for the azo-containing MIP microspheres
were evaluated from the Scatchard plot and found to be 2.3 ꢀ 104
Mꢁ1 and 10.0 mmol gꢁ1, respectively (Table 1). In addition, the Ka
and Nmax values of the low-affinity sites for the azo-containing
MIP microspheres were also determined, which were 0.19 ꢀ 104
Mꢁ1 and 29.4 mmol gꢁ1, respectively.
On the basis of the above results, it can be concluded that azo-
containing MIP microspheres could indeed be readily prepared
via precipitation polymerization by using an acetonitrile-soluble
azo functional monomer with a pyridine group (i.e., MAzoPy),
which showed obvious molecular imprinting effects towards the
template, rather fast template rebinding kinetics, and appreciable
Fig. 7 Selective bindings of the azo-containing MIP (filled columns) and
NIP (open columns) microspheres towards 2,4-D, DPAc and POAc in
their mixed solution in acetonitrile (C2,4-D, DPAc or POAc ¼ 0.05 mM) at 25
ꢂC in the dark.
Fig. 6 Scatchard plots of the azo-containing MIP microspheres deter-
mined in the dark (a) and under the irradiation of UV light (b), respec-
tively.
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selectivity over structurally related compounds. In the following
sections, a series of experiments were further carried out to check
whether the obtained azo-containing MIP microspheres could
show photoresponsive template binding properties.
The Scatchard analysis was also carried out by incubating
a certain amount of azo-containing MIP microspheres with
a series of template solutions with different concentrations under
the irradiation of UV light. In an analogue with that determined
in the dark (Fig. 6a), the Scatchard plot of the azo-containing
MIP microspheres obtained under the irradiation of UV light
could also be fitted into two straight lines (Fig. 6b), from which
the Ka and Nmax values of the high-affinity sites for the MIP
microspheres were evaluated to be 1.6 ꢀ 104 Mꢁ1 and 10.9 mmol
gꢁ1, respectively, while those of the low-affinity sites for the MIP
microspheres were evaluated to be 0.17 ꢀ 104 Mꢁ1 and 35.9 mmol
gꢁ1, respectively (Table 1). It can be seen clearly that the Ka value
of the high-affinity sites for the azo-containing MIP micro-
spheres obtained under the irradiation of UV light was obviously
lower than that determined in the dark (Ka ¼ 2.3 ꢀ 104 Mꢁ1),
while their Nmax values were rather similar (Table 1). These
results strongly demonstrated that the affinity of the binding sites
in the azo-containing MIP microspheres towards the template
indeed decreased under the irradiation of UV light, which agreed
well with their decreased specific template bindings under UV
light irradiation. In addition, an increase in the Nmax value of the
low-affinity sites was also observed for the azo-containing MIP
microspheres under the irradiation of UV light in comparison
with that determined in the dark (Table 1). This, together with
the almost similar Ka values of the low-affinity sites for the azo-
containing MIP microspheres determined both in the dark and
under UV light irradiation, revealed that the nonspecific
template bindings of the MIP microspheres increased under UV
light irradiation, which corresponded well with the increase in the
nonspecific template binding capacities of the NIP microspheres
under UV light irradiation (Fig. 5).
The equilibrium binding experiments were performed by
incubating a 2,4-D solution in acetonitrile with different amounts
of polymers at ambient temperature for 6 h under the irradiation
of UV light for the above purpose. As can be seen clearly from
Fig. 5, the equilibrium binding capacities of the azo-containing
MIP microspheres decreased upon exposure to 365 nm UV light
in a wide range of polymer concentrations, suggesting that the
template binding properties of the azo-containing MIP micro-
spheres were indeed photoresponsive. It is worth mentioning
here that in contrast with the azo-containing MIP microspheres,
a certain increase in the equilibrium binding capacities was
observed for the azo-containing NIP microspheres under the
irradiation of UV light, in particular in the case of low polymer
concentrations. This phenomenon might be stemmed from the
dipole difference between the trans- and cis-azo isomers (4.4
Debye) and the slight polarity increase for the resulting cis-azo
isomer under UV light irradiation.15 This UV light-induced
polarity increase for the azo groups on the surfaces of the NIP
microspheres might promote an increase in the interactions
between NIP particles and template molecules, thus leading to
their increased nonspecific template binding capacities. More-
over, this effect should be amplified in the dilute solutions of NIP
microspheres because UV light could penetrate more deeply into
the dilute solutions and thus showed more influence on the
binding properties of the NIP particles. Note that a similar
increase in the nonspecific template bindings should also be
present for the azo-containing MIP microspheres under the
irradiation of UV light, which was, however, covered up by the
more significant decrease of the template bindings due to
the photoresponsive binding properties of the MIP microspheres.
Therefore, to illustrate the influence of UV light irradiation on
the binding properties of the azo-containing MIP microspheres
more accurately, the specific bindings of the MIP microspheres
under UV light irradiation were derived by subtracting the
template bindings of the NIP microspheres from those of the
MIP microspheres, and they were compared with those obtained
in the dark (Fig. S2 in the supporting information†). The results
clearly showed that the specific template bindings of the azo-
containing MIP microspheres decreased greatly upon UV light
irradiation. For instance, in a dilute solution of 2,4-D in aceto-
nitrile, 16 mg of MIP microspheres showed a specific template
binding of 17% (relative to the initial 2,4-D concentration) in the
dark, while it decreased to 4% upon UV light irradiation. This
might be ascribed to the trans to cis photoisomerization of the
azo chromophores within the binding sites of the MIP micro-
spheres, which could lead to the alteration of the spatial
arrangement of their binding functionalities and thus the affinity
change of the binding sites, as schematically illustrated in Scheme
1. In this context, it is worth mentioning that the use of cis-
MAzoPy as the functional monomer in the above molecular
imprinting system (i.e., to perform the self-assembly of MAzoPy
and the template as well as the precipitation polymerization
under the irradiation of UV light) should also lead to azo-con-
taining MIP microspheres with photoresponsive template
binding properties.
The photoresponsive template binding properties of the azo-
containing MIP microspheres were further confirmed by their
photoregulated release and uptake of the template molecule.
Fig. 8a shows the change of the binding capacities of azo-con-
taining MIP/NIP microspheres towards 2,4-D and its analogues
(DPAc and POAc) under repetitive photoswitching conditions.
The experiments were performed by firstly incubating a series of
samples (which were composed of 5 mg of azo-containing MIP/
NIP microspheres and 0.5 mL of a solution containing 2,4-D,
DPAc, and POAc in acetonitrile (C2,4-D, DPAc or POAc ¼ 0.05
ꢂ
mM)) at 25 C in the dark for 6 h until the equilibrium analyte
bindings were reached. As expected, the azo-containing MIP
microspheres showed significantly higher binding capacities
towards 2,4-D than towards DPAc and POAc, revealing again the
high selectivity of the MIP microspheres towards the template.
The UV light irradiation on the above mixed solutions with
equilibrium analyte bindings led to the obvious release of the
template from the azo-containing MIP microspheres into the
solutions, and the template bindings of the MIP microspheres
decreased from 54 to 48% after 3 h of UV light irradiation. The
subsequent thermal back-isomerization of the azo-containing
MIP microspheres in the dark caused an increase in the template
binding from 48 to 52% (our experimental results showed that the
visible light-induced back-isomerization of the azo-containing
MIP microspheres could also be used to cause the template uptake
from the solutions). Repeating the photoswitching cycles resulted
in the release and uptake of 2,4-D in quantities very similar to
those of the previous cycles, which clearly demonstrated the
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reversibility of the binding siteconfiguration andsubstrate affinity
in the course of photoswitching of azo chromophores. Note that
although the azo-containing MIP microspheres were also able to
bring about some degree of photoregulated release and uptake for
the structural analogues of the template (i.e., DPAc and POAc),
their extents were significantly lower than that of 2,4-D under the
similar experimental conditions. This also indicated that the
binding sites in the azo-containing MIP microspheres possessed
a specific affinity for the template 2,4-D. It is worth pointing out
here that a slight photoregulated release and uptake of the ana-
lytes were also observed for the azo-containing NIP microspheres
under repetitive photoswitching conditions, but they were in the
opposite direction in comparison with the MIP microspheres. For
example, the bindings of the azo-containing NIP microspheres
towards 2,4-D increased from 41 to 43% upon UV light irradia-
tion (just like that observed in Fig. 5), while it dropped back to
41% upon switching off the UV light. This could also be attributed
to the polarity changes of the azo moieties on the surfaces of NIP
microspheres under the photoswitching conditions. To exclude
the influence of this photoinduced nonspecific binding changes,
the dependence of the specific analyte bindings of the azo-con-
taining MIP microspheres on photoswitching conditions were
also presented (Fig. 8b), which demonstrated the photoregulated
release and uptake of the template by the azo-containing MIP
microspheres more clearly. Based on the above results, it can be
concluded that the azo-containing MIP microspheres indeed
show obvious photoresponsive template binding properties and
the affinity of the binding sites in the MIP microspheres towards
the template can be easily tuned by the simple photoswitching. To
our knowledge, this is the first demonstration of azo-containing
MIP microspheres with photoregulated release and uptake abili-
ties towards the template molecule.
Conclusions
This paper demonstrates for the first time that azo-containing
MIP microspheres with photoresponsive template binding
properties can be easily prepared via precipitation polymeriza-
tion by using an acetonitrile-soluble azo functional monomer
with a pyridine group. The resulting azo-containing MIP
microspheres had a number-average diameter of 1.33 mm and
a polydispersity index of 1.15, and they showed obvious molec-
ular imprinting effects towards the template, fast template
rebinding kinetics, and an appreciable selectivity over structur-
ally related compounds. Furthermore, they have also proven to
exhibit obvious photoresponsive binding properties towards the
template molecule. Considering the great versatility of precipi-
tation polymerization in the preparation of uniform spherical
MIP particles with different diameters (ranging from 100 nm to
several micrometers) and the general applicability of MAzoPy in
the preparation of MIPs for a wide range of templates (those
with various functional groups such as phenol, hydroxyl, or
carboxylic acid), we believe that the findings we report here
represent a facile, general, and efficient way for fabricating
advanced photoresponsive spherical azo-containing MIP beads
with desired particle sizes. Furthermore, we also foresee that such
spherical azo-containing MIP particles should be of tremendous
potential in many applications such as smart separation,
extraction, and assays, intelligent chemical carriers, and efficient
drug delivery systems.
Acknowledgements
The authors gratefully acknowledge the financial supports from
National Natural Science Foundation of China (20744003,
20774044, 20974048), Natural Science Foundation of Tianjin
(06YFJMJC15100), a supporting program for New Century
Excellent Talents, Ministry of Education (NCET-07-0462), the
project sponsored by SRF for ROCS, SEM, and a Start-funding
from Nankai University.
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