CHINESE JOURNAL OF ANALYTICAL CHEMISTRY

Volume 48, Issue 7, July 2020

Online English edition of the Chinese language journal

Cite this article as: Chinese J. Anal. Chem., 2020, 48(7): e20069–e20074 REVIEW

Stimuli-responsive Polymers-based Two-dimensional

Photonic Crystals Biosensors
CUI Chun-Guo1, YOU Ai-Mei2, SHI Kai-Yao3,*, LIU RUI4, LU Yu-Yuan5, ZHANG Qiang2,*
1
Department of Breast Surgery, the Third Hospital of Jilin University, Changchun 130033, China
2
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun
130022, China
3
Provincial Key Laboratory for Gene Diagnosis of Cardiovascular Disease, Jilin Provincial Engineering Laboratory for Endothelial Function
and Genetic Diagnosis, Department of Cardiology, the Third Hospital of Jilin University, Changchun 130033, China
4
Drug Engineering Research Center, Harbin University of Commerce, Harbin 150076, China
5
State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences,
Changchun 130022, China

Abstract: Stimuli-responsive polymers-based two-dimensional photonic crystals (2DPCs) are comprised of two main components,
stimuli-responsive hydrogels and structured microparticles. Microparticles can self-assemble into ordered structures in two
dimensional spacing on the surface of hydrogels. The ordered structure allows structural colors that can be used as indicator
phenomena to detect various analytes. The changes in colors are associated with the concentration change of analytes. Therefore, the
concentration of the analytes can be determined using a simple colorimetric comparison. 2DPCs have attracted much attention due to
their sensing applications. This article covers a brief general introduction of 2DPC and highlights many milestone examples as
biosensors to inspire more innovative research work in the near future.

Key Words: Photonic crystals; Biosensor; Hydrogels; Two-dimensional ordered structure

1 Two-dimensional photonic crystals
Photonic crystals are comprised of spatially arranged periodic
dielectric materials, which can modulate electromagnetic waves
and provide unique reflection at specific wavelengths[1,2]. In
nature, there are many photonic crystals type periodically
structured surfaces observed, such as the Morpho rhetenor
butterfly[3], peacock[4], sea mouse[5], Eupholus magnificus
insect[6] and opals[7]. Two-dimensional photonic crystals
(2DPCs) are comprised of periodic elements in two spatial
directions with intermittent variation in refractive indices, which
creates a photonic stop-band[8]. Unlike dyes which exhibit
colors because the chromophores selectively absorb light at
certain wavelengths, 2DPCs yield structural colors due to the
reflection of certain light wavelengths in the visible range. The
wavelength of the reflected light is dependent on the ordered
________________________
Received 23 December 2019; accepted 30 April 2020
*Corresponding author. Email: shiky@jlu.edu.cn; qiang.zhang@ciac.ac.cn
This work was supported by the National Natural Science Foundation of China (No. 21604091).
Copyright © 2020, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved.
DOI: 10.1016/S1872-2040(20)60033-0
lattice structure and photonic stop-band of 2DPCs. The
interference light can be described using the well-known Bragg
equation, modified for the photonic crystals, as shown in
equation as follows:
mλ = 31/2dsinθ[9] (1)
where, m is the diffraction order, λ is the wavelength in air, d is
the nearest neighboring particle spacing, and θ is the angle
between the incident light and the diffraction crystal planes.
2DPCs are primarily produced by complex top-down methods
such as photolithography and etching techniques[10]. The
properties can be manipulated by tuning the order, size, and
defects of the nanostructures of 2DPCs. However, the
conventional top-down processes suffer from high-cost and
non-scalable synthesis.
Self-assembly methods have been developed and considered
as the potential alternatives to conventional top-down process[11].
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Firstly, colloidal particles self-assemble into ordered 2D
hexagonal crystal arrays on the water surface[12]. Then, these
arrays will be transferred to the glass substrate, and the
polymerization of these monomers will take place on the top of
these 2D particle arrays. The sensor will be obtained by peeling
it off the glass slide[13]. The sensor is comprised of a hydrogel
matrix and 2D ordered nanoparticles arrays, as shown in
Fig.1[13]. When an analyte is present in the system, it will
selectively bind with the functional hydrogels, which will make
the hydrogel swell or shrink. This process will change the 2D
nanoparticle spacing. If the spacing (d) changes, it can be
predicted from the Bragg equation that a peak-shift of diffracted
wavelength should take place, thus the colorimetric readout of
the sensor will change in the presence of the analyte. Therefore,
the amount of analyte in the system will be directly related to
the color change and thus the analyte concentration can be
determined by using a simple colorimetric comparison method.
This review focuses on stimuli-responsive polymer-based
2DPCs biosensors, and we will mainly highlight their
applications in analysis and detection of target analytes.
2DPCs exhibit a promising prospect as sensors due to their
unique properties such as low cost, ease of instrumental
readout, simple operation, and portability. However, for
three-dimensional photonic crystals (3DPCs), the ordered
arrays are always embedded in the hydrogel matrix. Thus, the
cross-linked network can only allow ions and small molecules
to pass through the hydrogel. So, the 3DPCs are confined to
detect small molecules and ions. The 3D ordered array system
also requires exclusion of ions. In addition, the hydrogel
polymerization and the introduction of recognition units
processes can partially disordered the 3D array, limiting their
applications in practical. Therefore, 2DPCs are appropriate
platforms for detecting not only ions and small moleculs but
also biomacromolecules and even microorganism. The
recognition cites are on the surfaces of the 2DPCs, so the
analytes can directly bind with the functional cites on the
surfaces of 2DPCs, resulting in changes of the optical property
of 2DPCs. The aim of this review is to familiarize the reader
with recent developments of 2DPCs in the area of sensing and
to encourage further research in this field to overcome some
of its challenges and also to inspire new innovations for these
Fig.2 (A) Preparation of 2D array hydrogel avidin sensor; (B) Dependence of diffraction wavelength on avidin concentration[14]
materials.
2 Stimuli-responsive polymer based 2DPCs
biosensors
Firstly, we take an example of 2DPCs sensor for protein
sensing. A 2DPC was prepared by polymerizing a hydrogel on
top of a close-packed hexagonal 2D particle array[14]. The
polystyrene (PS) particles were fixed in the crosslinked
hydrogel networks during the process of polymerization. Then
the hydrogel matrix was functionalized by attaching biotin
groups, as shown in Fig.2A. The structure of 2DPCs was
verified by scanning electron microscopy (SEM), and only
single layer ordered PS particles were observed on the surface
of hydrogels. The 2DPCs were used to sense avidin due to the
strong affinity of biotin to avidin with a dissociation constant
(Kd) in the order of about 10−14 mol/L. Indeed, this is one of
the strongest non-covalent interactions known in nature[15].
Avidin has four extraordinarily high-affinity binding sites for
biotin, which forms crosslinks and make the hydrogel shrink.
The shrinkage of hydrogels led to the decrease of PS distance
between PS particles and caused blue shift of 2D array
diffraction. As shown in Fig.2, the blue shifts of diffraction
wavelength were found when the avidin concentration
increased from 0 to 1 mg/mL. The diffraction colors changed
from red to yellow-green with the increasing concentrations in
the range of 0–0.1 mg/mL (Fig.2B).
A 2DPCs-based concanavalin A (Con A) sensor has been
reported by Asher and coworkers[16], which was prepared using
mannose carbohydrate functionalized hydrogels and PS
Fig.1 Schematic structure and detection diagram of 2DPCs[13]
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colloidal arrays. The Con A has homotetramer of four
identical subunits that could bind to mannose groups by
hydrogen bonding and van der Waals interactions. Following
a similar mechanism, the binding of Con A to the mannose
groups of hydrogel increases the hydrogel cross-linking, such
that the 2D array particle spacing decreases, causing a
diffraction blue shift (Fig.3A). As shown in Fig.3B, the
2DPCs exhibit wavelength blue shifts of 50 nm with Con A
concentrations increasing from 0 to 2 mg/mL. And the color
of the 2DPCs changed from red to green in the concentration
range. Thus, the sensor acted as an indicator of Con A that
could be used to roughly measure Con A concentrations by
visually observing diffraction color changes. The PS particle
spacing was measured by monitoring the Debye diffraction
ring diameter. It decreased from 1160 nm to 1060 nm with the
Con A concentration increasing from 0 to 1 mg/mL. The PS
particle spacing kept constant in the presence of other proteins
like Ricinus communis and BSA, which proved the changes of
PS particle distance resulting from lectin-carbohydrate
interactions. The same group also further explored the
carbohydrate functionalized 2DPCs to selectively detect lectin
proteins[17]. Authors synthesized a series of hydrogels with
different carbohydrate groups through free radical copoly-
merization. The peak of diffraction wavelength of the 2D PC
PAAm-AAc-Lactose-80 sensor exhibited a shift of 40 nm as
the ricin concentrations increased from 0 to 1.0 mg/mL.
Meanwhile, the diffraction color changed from red to green as
the ricin concentrations increased from 0 to 1.0 mg/mL. The
Fig.3 (A) Mechanism of Con A sensor; (B) Dependence of normalized diffraction spectra of 2DPCs hydrogel sensors upon Con A
concentration[16]
Fig.4 (A) Schematic representation of UCCH biosensor structure and detection of urea and urease inhibitor; (B) Dependence of particle spacing
of UCCH upon urea concentration[19]
ricin also made the 2D particle spacing decrease because
multivalent ricin binding to the hydrogel lactose increased the
cross-link density of the hydrogel. This shrinks of the
hydrogel led to decrease of 2D array particle spacing. The
range of spacing decrease was dependent on the content of
lactose in hydrogels.
Besides the examples of detection of biomacromolecules,
another distinguishing example has been reported by Asher
group who utilized the 2DPCs to selectively detect Candida
albicans (C. albicans)[13]. The 2DPCs hydrogel was prepared
by crosslinking of Con A. There were massive mannan groups
on the surface of C. albicans cells that multivalently and
selectively bond to Con A-based 2DPCs through biocognition
between the lectin of Con A and the mannan of C. albicans
cells. The crosslinking caused by C. albicans led to shrinkage
of Con A-based hydrogel, which reduced distance of the
2DPC particle, resulting in blue-shifts of the diffracted light.
The visual color changes of 2DPC were observed in the C.
albicans detecting process. The detection limit reached as low
as 32 CFU/mL for C. albicans. The 2DPCs exhibited an
excellent selectivity toward C. albicans over E. coli because
of the lack of mannan on E. coli surface[18].
A label-free urease coupled colloidal crystal hydrogel
(UCCH) biosensor has been developed to detect urea and
urease inhibitor[19]. The UCCH biosensor was fabricated by
embedding a 2D PS array in the surface of the urease
functionalized hydrogel, as shown in Fig.4A. The recognition
element urease was simply immobilized in the hydrogel
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via covalent coupling with the carboxylates of the acrylic acid

monomer. When the UCCH biosensor was immersed into urea
solution, the enzymatic catalysis of urea by urease could yield
ammonium (NH4+) and bicarbonate (HCO3-). These ions could
reduce the electrostatic repulsion between the carboxylate
groups of the hydrogel, shrink the UCCH volume and lead to
an evident blue-shift in the diffraction wavelength (Fig.4B).
At the same time, once urea and urease inhibitor PPD
coexisting in aqueous solution, the urease inhibitor would
intervene in the hydrolysis reaction of urea and reduced the
degree of the blue-shift. Owing to the periodic ordered
structure of the 2D colloidal crystal template, Debye ring
could also be observed. The volumetric change of the UCCH
could also alter the diameter of Debye diffraction ring, which
could insure the accuracy of the measurement results. The
naked-eye detection of urea and urease inhibitor could be
realized via observing the change in structure color and the
Fig.5 Fabrication of 2DPC-DNA hydrogel film: (A) Schematic
Debye ring diameter of the UCCH biosensor, which was more
illustration of synthesis of DNA hydrogel copolymers; (B)
convenient than conventional spectrometer analysis. The Preparation of 2DPC-DNA hydrogel film; (C) pH-responsive
smart 2D UCCH biosensor could get rid of the dependence on changes of 2DPC-DNA hydrogel film between shrinkage and
the equipment and provided simple and portable detection swelling state, resulting in particle spacing changes of
technology, meanwhile, the label-free and visual advantages 2DPC-DNA hydrogel[20]
might make it become a potential platform for onsite analysis.
In addition to the commonly used hydrogels, DNA chelation between Cys and Ag+ could open the C-Ag+-C
cross-linked hydrogels with structural and functional structure[21,22]. Therefore, Ag+/Cys was another stimulating
information have attracted significant attention. Shape- factor to regulate the particle spacing of 2DPC-DNA hydrogel
memory DNA hydrogel films serving as both external stimuli films. The particle spacing increased as increasing Cys
concentration, as a result, the diameter of Debye diffraction
recognition units and actuating units have been incorporated
ring decreased. By introduction of Ag+ in the system again,
with monolayer regularly ordered PS colloidal nanoparticles
the reversible deformation of DNA hydrogel film exhibited an
to construct responsive 2D photonic crystals (2DPC-DNA), as
increased diameter of Debye diffraction ring.
shown in Fig.5[20]. The fabrication was a simple heating-
A series of acetylcholinesterase (AChE)-functionalized
cooling process, DNA hydrogel film was fabricated through
2DPCs were prepared by Qi et al.[23,24] to construct sensing
cooling down the 95 °C DNA homogeneous solution that in a
platform. With AchE as the recognition agent, an easy and
film mold to room temperature, and the 2DPCs arrays was
general method was developed for the colorimetric detection
prepared by transferring the PS colloidal nanoparticles to the
of organophosphates such as nerve agents (sarin) and their
surface of the hydrogel film and embedding onto the surface mimic (dipterex). As shown in Fig.6, firstly, the 2DPCs array
via a fast heating-cooling process. The cytosine-rich i-motif was prepared according to the air/water interface self-assembly
sequence in the DNA hydrogel served as the responsive unit to method, and then transferred to a glass slide and naturally dried
H + , and the self-complementary sequence served as the at room temperature. Secondly, the polyacrylamide-acrylic acid
shape-memory unit. The responsiveness of the 2DPC-DNA (PAM-AA) 2DPC hydrogel was prepared via photo-
hydrogel film was due to the formation of i-motif structure in polymerization and then peeled off from the glass slide.
acidic conditions and the dissociation of i-motif structure to Finally, AChE was immobilized on the 2DPC hydrogel with
yield a random coil under neutral and alkaline conditions. N-ethyl-N’-(3-diethylaminopropyl) carbodiimide (EDC) as a
Therefore, environmental pH changes could actuate the condensation agent. The carboxyl groups in PAM-AA
reversible shrinking and swelling of the shape-memory DNA hydrogel could interact with amino groups in AChE in the
hydrogel films and in turn modulate the lattice spacing and presence of EDC. The AChE-functionalized 2DPC could not
Debye diffraction ring of the 2DPC array. The structural color only display the structural color changes but also the Debye
changed from blue-green-dominated coloration to yellow- ring diameter changes in the presence of different
dominated coloration of the 2DPC-DNA hydrogel films concentrations of analytes. Linear relationships between the
between pH 5.0 and 8.0. Furthermore, it was discovered that logarithm of the analyte concentration and the particle spacing
Ag+/Cys could induce the formation/ dissociation of i-motif of the AChE-functionalized 2DPC were observed, and the
structures at neutral conditions, as Ag+ could combine with limits of detection were 7.7 × 10‒15 mol/L for Dipterex and
cytosine in DNA strand to form C-Ag+-C structure, and the 6.7 × 10‒17 mol/L for sarin, respectively.
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self-assembly method, and the 2D crystalline colloidal array

Fig.6 Fabrication of AChE-functionalized 2DPC and its response to
analytes[23]
Molecularly imprinting technique is drawing increasing
attentions due to its competence in producing synthetic
polymers with highly specific recognition sites. With the
integration of molecularly imprinting technique and 2DPC
method, Meng’s group reported the 2D molecularly imprinted
photonic crystal hydrogel (MIPCH) to screen antibiotics
oxytetracycline (OTC) in milk[25]. As shown in Fig.7, the
2DPC array was prepared using the air/water interface
was subsequently lifted up with a clean glass slide and
naturally dried at room temperature. Then, the pre-
polymerization solution of template OTC, monomers (AM
and AA) and 2,2-diethoxyacetophenone (DEAP) were
infiltrated into the interstitial space of PS template, and the
OTC imprinted 2D MIPCH was obtained after UV
polymerization. Finally, the imprinted cavity for the target
molecules formed after the remove of OTC templates in a
mixture of acetic acid and SDS solution. The nanocavities in
the surface of the PC hydrogel could be regarded as the
recognition element and provided specific binding sites for
OTC, just like the biological receptor counterparts. The
density of cross-linkers had a significant influence on the
structure, strength and swelling ratio of hydrogel. And more
importantly, it could affect the sensitivity of the imprinted
2DPC hydrogel[26]. The particle spacing changes of the 2DPC
hydrogels could be calculated by measuring the change of the
Debye ring diameter. When the 2D MIPCH was immersed
into the OTC solutions, the particle spacing increased from
794 nm to 887 nm (94 nm) with OTC concentration increasing
from 0 to 60 μmol/L, followed with the structural color of the
2D MIPCH sensors shifting from blue through green to orange
and then to red, as shown in Fig.7B. But the contrast group of the
non-imprinted 2DPC hydrogel did not display obvious response

Fig.7 (A) Illustration of preparing OTC imprinted hydrogel films; (B) Analysis of OTC by OTC imprinted and non-imprinted hydrogels,
respectively; (C) Response of OTC imprinted hydrogel to OTC, TC, DOC and CTC[25]
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to OTC, owing to lack of specific binding sites as the
imprinted ones. As a result, the particle spacing of
non-imprinted 2DPC hydrogels increased only 26 nm, and the
structural color remained blue. The selectivity of the OTC
imprinted hydrogel was also tested using OTC and its
structural analogues (Tetracycline (TC), doxycycline
hydrochloride (DOC) and chlortetracycline hydrochloride
(CTC)). As shown in Fig.7C, the particle spacing of OTC
imprinted hydrogel only changed a little to the three analogues
(TC, CTC, and DOC), in contrast to the obvious response to
OTC. More importantly, the detection of OTC in milk sample
was achieved at the same concentration range of OTC
standard. With increasing OTC concentration in milk sample,
the particle spacing of OTC imprinted 2D MIPCH sensor
increased about 92 nm. What’s more, the same group
employed this method to fabricate a molecularly imprinted
colloidal array to detect clindamycin hydrochiloride (CLI)[27].
Thus, it was reasonable to come to a conclusion that the
molecularly imprinted 2DPC hydrogel would be promising to
be utilized to identify trace amount of analytes in real food
samples.
3 Conclusions
The purpose of this review is to gather the achievements of
2DPCs sensor to help the newcomers to have a general
overview of the current research state. Besides aforesaid
examples, 2DPCs also has been used to determine pH, metal
cations, carbohydrates, proteins, surfactants, anionic drugs,
humidity, and ammonia[16,28‒31]. 2DPCs sensor based on
stimuli-responsive polymers exhibit significant advantages,
such as easy preparation, low cost, and wide detection range.
2DPCs are particularly suitable for sensing biomacro-
molecules and even microorganism, which surmounts the
diffusion limits in comparison of 3DPCs. Compared with
other commercial techniques, 2DPCs-based sensor exhibits
many advantages, such as lower cost, easy use, and visual
colorimetric detection. In particular, most sensors have been
prepared based on simple process, which can be carried out in
general research labs without the need for expensive
instruments and strict processing conditions. The research of
2DPCs is an interdiscipline that gets involved with biology,
nanotechnology, polymer chemistry, organic synthesis, etc.
There are tremendous opportunities in both fundamental and
applied science.
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