Colloids and Surfaces B: Biointerfaces 197 (2021) 111433

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Photoconjugation of temperature- and pH-responsive polymer with silica

nanoparticles for separation and enrichment of bacteria
Hongwei Zheng a, b, Haiyue Gong a, Limin Cao b, Hong Lin b, *, Lei Ye a, *
a
Division of Pure and Applied Biochemistry, Department of Chemistry, Lund University, Box 124, 221 00, Lund, Sweden
b
Food Safety Laboratory, College of Food Science & Engineering, Ocean University of China, Qingdao, 266003, China

A R T I C L E I N F O A B S T R A C T

Keywords: A new photoconjugation approach was developed to prepare nanoparticle-supported boronic acid polymer for
Photoconjugation effective separation and enrichment of bacteria. The photo-activated polymer immobilization was demonstrated
Reversible addition fragmentation chain by coupling an azide-modified copolymer of N-isopropylacrylamide and glycidyl methacrylate to a per­
transfer polymerization
fluorophenyl azide-modified silica surface. The thermoresponsive polymer was synthesized using reversible
Boronate affinity
Perfluorophenyl azide
addition fragmentation chain transfer polymerization followed by conversion of the pendant epoxides into azide
Polymer groups. The perfluorophenyl azide-modified silica nanoparticles were synthesized by an amidation reaction
Bacteria between amino-functionalized silica and pentafluorobenzoyl chloride, and a subsequent treatment with sodium
Bioseparation azide. Bacteria-capturing boronic acid was conjugated to the silica-supported polymer chains via Cu(I)-catalyzed
azide-alkyne cycloaddition (CuAAC) click reaction. The particle size, morphology and organic content of the
composite nanoparticles were characterized systematically. The capability of the nanocomposite to bind Gram-
positive and Gram-negative bacteria was investigated. The nanocomposite exhibited high binding capacities for
E. coli (13.4 × 107 CFU/mg) and S. epidermidis (7.66 × 107 CFU/mg) in phosphate buffered saline. The new
photoconjugation strategy enables fast and straightforward grafting of functional polymers on surface, which
opens many new opportunities for designing functional materials for bioseparation and biosensing.

1. Introduction [7], stimuli-responsive hydrogels [8], sensors [9,10], bioconjugates [11]

Rapid identification, monitoring and detection of pathogenic bacte­
ria is essential for tackling bacterial threats including foodborne disease,
biohazards and hospital-acquired infections [1,2]. Modern detection
techniques based on measurement of spectroscopic, immunological or
genetic signature of bacteria are fast and sensitive, allowing detection of
even a single cell in some cases [3,4]. However, these analytical methods
still have not been fully exploited in practical applications due to that
the trace level of target bacteria cannot be separated from other inter­
ference substances in biological samples [4,5]. Therefore, selective
isolation and enrichment of target bacteria remains a critical step in
detection of bacteria. The traditional, commonly used methods such as
centrifugation, filtration, and affinity separation based on antibody,
lectin and vancomycin, etc. involve complicated procedure, and are
time-consuming. The affinity materials are normally associated with
high cost and poor stability [4,6].
Recently, boronate affinity techniques have been utilized in a num­
ber of biochemical applications including chromatographic separation
and multifunctional nanomaterials [12], where the reversible covalent
bond between boronic acid and the cis-diol structure in glycoproteins
and nucleosides enables specific molecular recognition [13–15]. The
presence of poly- and oligo-saccharides on the surface of microbial cells
constitutes the base of using boronate affinity to capture bacteria
[16–18]. A series of boronate affinity materials has been reported for
bacteria analysis [17–19]. To increase the binding strength, multiple
boronic acids are attached to poly(amidoamine) (PAMAM) dendrimers,
highly branched polyethyleneimine (PEI) and multifunctional polymers
to increase the number and density of boronate affinity ligands [20,21].
Polymers are ideal building blocks for nanofabrication and engi­
neering of functional materials, and living/controlled radical polymer­
ization (CRP) has been proven as a powerful tool for preparation of well-
defined polymers that are compatible with biological settings [22–25].
To immobilize polymer chains on supporting materials, both “grafting
from” and “grafting to” methods are widely used [26]. Surface-initiated
radical polymerization is the most commonly used “grafting-from”
method. By combining click chemistry with surface-initiated atom

* Corresponding authors.
E-mail addresses: linhong@ouc.edu.cn (H. Lin), lei.ye@tbiokem.lth.se (L. Ye).

https://doi.org/10.1016/j.colsurfb.2020.111433
Received 23 June 2020; Received in revised form 27 August 2020; Accepted 17 October 2020
Available online 4 November 2020
0927-7765/© 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
H. Zheng et al.
transfer radical polymerization (SI-ATRP), Ye and co-workers reported a
two-step, consecutive SI-ATRP to graft block copolymer brushes on silica
nanoparticles followed by click-conjugation of repetitive boronic acids
to the polymer brushes [27]. Although the “grafting from” method gives
a high chain density on surface, it requires a pre-attachment of initiator
on the surface of supporting materials, and is more difficult to produce
polymer products with well-controlled chain length and molecular
weight distribution. The controllability of surface-initiated CRP may
also be affected by potential side reactions [28]. Conversely, the
“grafting to” method conjugates a premade functionalized polymer to a
substrate. Although this method normally results in a lower grafting
density, it requires fewer reaction steps, and allows to immobilize
well-defined polymer products because the polymer synthesis can be
carried out in homogeneous solution under optimal reaction conditions
[29]. The “grafting-to” method therefore broadens significantly the
scope of the functional polymers and substrates. Among a variety of
reported surface grafting reactions, click chemistry is the mostly used
due to its high coupling efficiency [30,31]. Using thiol-ene click chem­
istry, Liu and coworkers synthesized a thiol-terminated polymer by
reversible addition-fragmentation chain transfer (RAFT) polymeriza­
tion, and grafted the polymer chains to acrylate-modified magnetic
nanoparticles [31]. In spite of its high selectivity and mild reaction
condition, the click chemistry-based conjugation requires a “clickable”
group to be added to a polymer prior to its immobilization. The addi­
tional reaction step increases the complexity of material synthesis and is
often difficult to accomplish. Apart from click chemistry, most of the
other grafting methods require a harsh reaction condition such as high
temperature and a long reaction time [31]. Therefore, there is an
emergent need to explore new grafting methods to realize fast and
straightforward polymer conjugation on solid nanoparticles.
In the context of photolabelling/functionalization processes, the
need for a scalable method to link polymers to supporting materials has
Scheme 1. Synthesis of Si@poly(NIPAm-co-GMA)@PCAPBA nanocomposite via photoconjugation and CuAAC click reaction, and schematic of bacterial binding to
the nanocomposite.
2
Colloids and Surfaces B: Biointerfaces 197 (2021) 111433
led us to investigate the use of perfluorophenyl azide (PFPA) as a
chemical modifier. PFPAs are known for their highly photoreactive
azide group that generate surface-bound nitrene to react with organic
materials possessing CH, NH and C– – C groups [32]. Moreover, the
simple introduction of azide at the para position of pentafluorophenyl
group is an attractive feature, which makes PFPAs widely exploited re­
agent for labelling of biomolecules, crosslinking of polymers, and
functionalization of materials [33,34]. In our group, PFPA-based pho­
toconjugation was previously used to prepare molecular recognition
materials from molecularly imprinted polymers and magnetic nano­
particles [35]. The nondestructive feature of the photoconjugation made
it possible to construct highly specific affinity material from optimized
nanoparticle building blocks. Hence, the drawbacks of conventional
conjugation methods, especially the complicated modification processes
to introduce reactive groups and tedious reaction steps, can be avoided.
In this work, a new photoconjugation procedure was developed to
allow direct coupling of functional polymers to inorganic nanoparticles
using a commercially available perfluorophenyl reagent. A boronic acid-
tagged and core-shell type nanoparticles, as depicted in Scheme 1, was
prepared and used to demonstrate the general applicability of the new
conjugation method. The nanocomposite was designed to bind bacteria
by virtue of multiple boronic acid ligands attached on a flexible polymer
backbone. Taking advantage of RAFT polymerization, a thermores­
ponsive copolymer was synthesized from N-isopropylacrylamide and
glycidyl methacrylate, and used as a versatile intermediate to be linked
to PFPA-modified silica nanoparticles. To synthesize PFPA-modified
nanoparticles, amino-functionalized silica was reacted with penta­
fluorobenzyl chloride, followed by reaction with sodium azide. After the
photoconjugation, high density of boronic acid was added to the poly­
mer chains via CuAAC click reaction. The composite materials collected
after different reaction steps were characterized using dynamic light
scattering (DLS), scanning electron microscopy (SEM), transmission
H. Zheng et al.
electron microscopy (TEM), Fourier transform infrared spectroscopy
(FT-IR), elemental analysis and thermogravimetric analysis (TGA).
Finally, the bacteria binding characteristics of the composite nano­
particles were studied to reveal the importance of the flexible polymer
chains and the abundance of the immobilized boronic acids. The feasi­
bility of using the nanoparticle-supported polymer to capture bacteria in
complex sample (25 % milk) was also demonstrated.
2. Experimental section
2.1. Materials
Tetraethylorthosilicate (TEOS), (3-aminopropyl)-triethoxysiliane
(APTES), pentafluorobenzoyl chloride (PFBCl), N-isopropylacrylamide
(NIPAm), glycidyl methacrylate (GMA), cumyl dithiobenzoate (CDB), 3-
aminophenylboronic acid (APBA) hemisulfate salt (≥ 95 %), propargyl
chloroformate, dimethyl sulfoxide-d6 (DMSO-d6), propargylamine, so­
dium azide (NaN3), copper (II) sulfate (CuSO4), sodium ascorbate,
Alizarin Red S (ARS), triethylamine, dichloromethane and methanol
were purchased from Sigma-Aldrich and used without further purifica­
tion. Glutaraldehyde 50 % in aqueous solution was obtained from VWR
Chemicals. 2,2′ -Azobis(2-methylpropionitrile) (AIBN, 98 %), yeast
extract granulated and agar were obtained from Merck. AIBN was re-
crystallized from methanol before use. Tryptone was purchased from
Duchefa Biochemie. Acetone, N,N-dimethylformamide (DMF), ethanol,
ammonium hydroxide (25 %), sodium hydrogen carbonate (NaHCO3),
sodium hydroxide, potassium chloride, sodium chloride, di-sodium
hydrogen phosphate, potassium dihydrogen phosphate and ammonium
chloride were purchased from Fisher Scientific. Ultrapure water (18.0
MΩ cm) was obtained from an ELGA LabWater System (Vivendi Water
Systems Ltd). 3-(prop-2-ynyloxycarbonylamino)phenyl-boronic acid
(PCAPBA) was synthesized according to our previously published pro­
cedure [36]. Milk was purchased from a local market in Lund, and was
stored at 4 ℃ before use.
2.2. Preparation of perfluorophenyl azide-functionalized silica
nanoparticles (Si@PFPA)
Silica nanoparticles were synthesized by “one-step” Stöber procedure
with slight modifications [37]. Typically, a mixture of water (16.5 mL),
methanol (50 mL) and ammonium hydroxide (25 %, 11.2 mL) were
introduced into a 500 mL round bottom flask. After addition of 65 mL
methanol containing 6.9 mL TEOS, the mixture was magnetically stirred
at 20 ℃ for 12 h. The silica particles were separated by centrifugation at
12,000 rpm (19,802 g) for 5 min to remove the unreacted TEOS and
ammonia, followed by thorough washing with water and methanol, and
dried in a vacuum desiccator.
The silica nanoparticles (1.5 g) were dispersed in 50 mL of 1% APTES
solution prepared in anhydrous toluene. The suspension was magneti­
cally stirred at reflux temperature for 24 h. The particles were then
isolated by centrifugation, washed thoroughly with acetone and meth­
anol, and dried in a vacuum desiccator. The product was denoted as
Si@NH2.
Si@NH2 nanoparticles (300 mg) and 1.5 mL of triethylamine were
dispersed in 15 mL of dichloromethane. To the suspension, PFBCl (0.5
mL, 3.47 mmol) dissolved in 1.5 mL of dichloromethane was added
dropwise. The reaction mixture was protected from light and stirred at
room temperature for 3 h. The nanoparticles were collected by centri­
fugation, washed with dichloromethane, and dried in a vacuum desic­
cator overnight. The PFBCl-functionalized silica nanoparticles are
denoted as Si@PFBCl.
A mixture of 100 mg of Si@PFBCl particles and 0.98 g of NaN3 in
acetone (20 mL) and water (7 mL) was refluxed at 90 ℃ for 2 h. The
particles were then isolated by centrifugation, washed with water and
acetone, and dried in a vacuum desiccator. The obtained per­
fluorophenyl azide-functionalized silica nanoparticles are denoted as
3
Colloids and Surfaces B: Biointerfaces 197 (2021) 111433
Si@PFPA.
2.3. Detection of amine groups on Si@NH2
The amine groups on Si@NH2 particles were detected using a pre­
viously reported method [38]. Briefly, a 0.35 % (w/v) ninhydrin solu­
tion in absolute ethanol was freshly prepared. Si@NH2 particles were
dried at 80 ℃ for 8 h, from which 25 mg was taken and dispersed in 4 mL
of ethanol, followed by ultrasound sonication for 30 min. Next, the
suspension was mixed with 1 mL of ninhydrin solution and sonicated for
another 10 min. The sample was heated in a water bath at 65 ℃ for 30
min. After cooling to ambient temperature, the sample was centrifuged
at 8000 rpm (8801 g) for 30 min to remove the particles. Finally, the
supernatant (appropriately 1 mL) was analyzed by UV–vis spectrometry.
2.4. Synthesis of poly(NIPAm-co-GMA)
Poly(NIPAm-co-GMA) was synthesized using RAFT polymerization
by varying the molar ratio of NIPAm: GMA from 5: 1 to 3: 1. As one
example, NIPAm (165.2 mg, 1.46 mmol) and GMA (41.51 mg, 0.29
mmol) (with a molar ratio of NIPAm: GMA = 5: 1) were dissolved in
methanol (2 mL) in a flask. AIBN (0.62 mg, 0.0037 mmol) as an initiator
and CDB (3.26 mg; 0.012 mmol) as a RAFT agent were added to the flask
(the ratio of monomer: RAFT agent: initiator ≈ 450: 4: 1). The mixture
was cooled by an ice-water bath, deoxygenated under reduced pressure,
and nitrogen-purged for 15 min. Then, the flask was sealed and kept at
70 ℃ under a nitrogen atmosphere for polymerization for 24 h with
vigorous stirring. After the reaction, the mixture was exposed to air and
cooled to ambient temperature. The product was placed in a dialysis
tube (MWCO 1000 Da) and kept in water for 3 days (with the water
replaced 5–6 times per day). Finally, the polymer product was dried by
rotary evaporation at 40 ℃ to completely remove water.
2.5. Synthesis of poly(NIPAm-co-GMA)@N3
Poly(NIPAm-co-GMA) (80 mg), NaN3 (78 mg) and ammonium
chloride (64.5 mg) was mixed in 7.5 mL of DMF. After sonication for 10
min, the mixture was stirred at 60 ℃ for 24 h. After the reaction, the
product was dialyzed for 3 days in water (with the water replaced 5–6
times per day), and dried by rotary evaporation at 40 ℃.
2.6. Preparation of Si@poly(NIPAm-co-GMA)@N3 by photoconjugation
Si@PFPA particles (20 mg) and poly(NIPAm-co-GMA)@N3 (80 mg)
were homogenized in 3 mL of acetone by sonication. The mixture was
transferred into a glass Petri dish (diameter =3 cm), and the solvent was
evaporated under reduced pressure. The dry particles were photo­
activated through a 318 nm optical filter under a 450 W mercury vapor
lamp for 7 min. The distance between the lamp and the filter was 5 cm
while the distance between the optical filter and the particles was 2.5
cm. The particles were then collected, washed with methanol and water,
and dried in a vacuum desiccator.
As a control, a mixture of Si@PFPA particles and poly(NIPAm-co-
GMA)@N3 was prepared. Without the step of photoactivation, the
mixture was processed in the same way as mentioned earlier. The
product was used as a reference to study the role of the
photoconjugation.
2.7. Preparation of Si@poly(NIPAm-co-GMA)@PCAPBA by click
chemistry
Si@poly(NIPAm-co-GMA)@N3 (50 mg) was dispersed in 10 mL of
methanol-water solution (v/v, 1:1) by sonication. Subsequently, 2 mL of
PCAPBA solution in methanol-water (v/v, 1:1) (54 mg/mL) was added.
The mixture was sonicated for 10 min followed by addition of 20 μL of
CuSO4 (100 mM) and 100 μL of sodium ascorbate (100 mM). After
H. Zheng et al.
purging with nitrogen for 10 min, the reaction mixture was sealed and
shaken on a rocking table at ambient temperature for 24 h. The particles
were then isolated by centrifugation and washed thoroughly with 50 %
methanol-water solution. The obtained particle was denoted as Si@poly
(NIPAm-co-GMA)@PCAPBA.
2.8. Bacteria cultivation
S. epidermidis and E. coli were cultured in Luria-Bertani (LB) medium.
The bacteria strains were grown in 5 mL of medium at 37 ℃ for 12 h
with shaking at 180 rpm. After cultivation, the bacteria cells were
collected by centrifugation at 4500 rpm for 8 min, washed three times
with PBS (0.01 M, pH 7.4) and finally suspended in 1 mL of PBS. The
quantification of bacteria relied on a calibration plot between the con­
centration of bacteria and the optical density of the culture measured at
600 nm (OD600). Prior to binding experiments, bacteria samples were
diluted with PBS to give an OD600 value of 0.3.
2.9. Bacteria binding with Si@poly(NIPAm-co-GMA)@PCAPBA
Si@poly(NIPAm-co-GMA)@PCAPBA particles (1 mg) were dispersed
in a solution (1 mL) of E. coli or S. epidermidis (OD600 = 0.3) in PBS (0.01
M) with pH from 7.0 to 8.5. The samples were gently shaken at 20 ℃ for
30 min, and then centrifuged at 2000 rpm (270 g) for 6 s, followed by
standing for 30 min. After this sedimentation step, the supernatant was
collected to measure the OD600, from which the concentration of the
unbound bacteria was calculated.
The adsorption capacities (Q) of the composites were calculated
using the equation:
(C0 − Ct )V
Q=
m Si@PFPA with poly(NIPAm-co-GMA)@N3 was achieved by photo­
where C0 (CFU/mg) is the initial concentration of the bacteria suspen­
sion, Ct (CFU/mg) is the concentration of bacteria in supernatant, V
(mL) is the volume of the bacteria sample and m (mg) is the mass of the
composites.
To investigate the influence of temperature on bacteria binding, the
same operation procedure was used except that the temperature was set
at 40 ℃ in the binding step.
2.10. Bacterial binding in complex samples
To study bacteria binding in complex samples, 10 mL of 25 % cow
milk (wt/wt) was spiked with 103 CFU/mL of E. coli and S. epidermidis
separately. After adjusting the sample pH to 8.0, 5 mg of the composite
particles in 400 μL of PBS was added. The mixture was stirred at 20 ℃
for 30 min, then left standing for 40 min to settle the particles. The
supernatant was then collected to quantify the amount of unsettled
bacteria. As a control, 400 μL of PBS was used to replace the composite
particles.
2.11. Characterization
Attenuated total reflection (ATR) infrared analysis was performed on
a Thermo Fisher-Scientific FT-IR instrument (Thermo Fisher-Scientific
Inc., Waltham, MA, USA). Scanning-electron microscopy (SEM) char­
acterization was performed on a JEOL scanning electron microscope
(SEM, JSM-6700 F, JEOL, Japan). Transmission-electron microscopy
(TEM) was performed with Tecnai Spirit BioTWIN electron microscope
(FEI Company, Hillsboro, OR, USA). Dynamic light scattering (DLS)
analysis was performed on a temperature-controlled Zetasizer Nano ZS
particle size analyzer (Malvern Instruments, UK). The absorbance and
transmittance analyses were performed on Cary 60 UV–vis spectro­
photometer (Agilent Technologies, USA). Thermal gravimetric analysis
(TGA) was performed in air, and the samples were heated at a rate of 10
4
Colloids and Surfaces B: Biointerfaces 197 (2021) 111433
℃/min. Gel permeation chromatography (GPC) was performed on a
Viscothek GPCmax instrument equipped with a PFG column
(300 mm × 8 mm; 5 μm particle size) and a RI detector from PSS (Mainz,
Germany). DMF containing 10 mM LiBr was used as mobile phase at a
flow rate of 0.75 mL/min at 60 ℃. Polystyrene was used as standard for
calibration. Elemental analysis (C, H, N, Si, F, B) was performed by
Mikroanalytisches Laboratorium Kolbe (Germany). 1H NMR spectrum
was obtained on a Bruker DR X400 spectrometer at 400.13 MHz using
DMSO-d6 as solvent (δ =2.5 ppm).
Different nanoparticles (1 mg) were dispersed in a solution (1 mL) of
E. coli or S. epidermidis (OD600 = 0.3) in PBS (0.01 M, pH 7.4). The
mixture was gently shaken at 20 ℃ for 30 min, and then mixed with 1
mL of glutaraldehyde solution (5% v/v; PBS, 0.01 M, pH 7.4). After
shaking at 20 ℃ for 1 h, the sample was isolated by centrifugation and
washed thoroughly with PBS (0.01 M, pH 7.4). Finally, the sample was
resuspended in 1 mL of PBS and analyzed by TEM.
3. Results and discussion
The procedure for preparation of the boronate affinity nano­
composite, Si@poly(NIPAm-co-GMA)@PCAPBA, is shown in Scheme 1.
Briefly, the silica nanoparticles were synthesized by one-step Stöber
method followed by modification with APTES to give the amino-
functionalized silica nanoparticles, which were used to prepare the
precursor nanoparticle (Si@PFBCl) to be converted into the photoactive,
perfluorophenyl azide-functionalized silica nanoparticles (Si@PFPA).
Si@PFPA nanoparticles were obtained by reacting Si@PFBCl with so­
dium azide. The copolymer, poly(NIPAm-co-GMA) was synthesized by
RAFT polymerization after optimizing the monomer ratio and reaction
time. The copolymer was reacted with sodium azide to introduce mul­
tiple azide groups and to enhance its hydrophilicity. The conjugation of
chemical reaction under UV light, leading to the nanoparticle-supported
polymer, Si@poly(NIPAm-co-GMA)@N3. In the next step, a CuAAC click
reaction between the azide groups in Si@poly(NIPAm-co-GMA)@N3 and
PCAPBA resulted in the final bacteria-capturing material, Si@poly
(NIPAm-co-GMA)@PCAPBA, a new pH- and temperature- responsive
composite. The advantage of this synthetic procedure resides on the
simple photo-activated conjugation, which can be used to immobilize all
types of organic polymers on PFPA-modified surface.
3.1. Optimization and characterization of polymers
Thermoresponsive copolymers with varying amount of NIPAm and
GMA as monomers, CDB as RAFT agent and AIBN as initiator were first
synthesized in methanol. The choice of RAFT polymerization was made
based on its functional group tolerance and general applicability to
synthesize well-defined and water-soluble polymers [39,40]. NIPAm
and GMA were used as the monomers because their copolymerization
was expected to produce a hydrophilic “smart”, temperature-responsive
polymer in which the epoxy groups can be utilized to introduce different
affinity ligands.
Considering the compatibilities of NIPAm and GMA with water,
different ratios between NIPAm and GMA were studied for polymer
synthesis to endow the copolymer an appropriate hydrophilicity. When
the molar ratio of NIPAm: GMA was raised to 4: 1, the copolymer ob­
tained after 24 h reaction still exhibited poor miscibility with water (Fig.
S1). On the other hand, excessive reduction of GMA in the monomer
mixture was deemed to decrease the chemically convertible moieties in
the copolymer, leading to a low density of immobilized boronic acid
ligands. Apart from the monomer ratio, the length of the copolymer
chain also plays an important role in its solubility and temperature
response property. Therefore, the polymerization time was varied to
observe the effect on the hydrophilicity of polymers. As shown in Fig. S1,
the polymer products exhibited an increased hydrophilicity with
decreasing polymerization time. When the polymerization time was
H. Zheng et al.
shorter than 5 h, the product formed a homogeneous dispersion in
water. Notably, even the homopolymer prepared from a single mono­
mer, NIPAm, exhibited similar compatibility with water. Short reaction
time unfortunately resulted in smaller amount of polymer products (Fig.
S1). Considering both the hydrophilicity and the yield of polymer,
further synthesis was carried out using a NIPAm: GMA ratio of 5: 1 and
polymerization time of 2.5− 24 h. The yield of polymer obtained after
2.5, 5, 8, and 24 h reaction were approximately 6%, 14 %, 22 % and 48
%, respectively. The obtained polymers were reacted with NaN3 to
introduce pendent azide groups and at the same time to improve the
hydrophilicity. As shown in Fig. S2, after the modification, all the four
polymers became water-soluble and showed the characteristic IR band
for organic azide at 2100 cm− 1 (Fig. S3).
It is well known that pNIPAm exhibits a sharp phase transition and
undergoes dehydration and inter chain aggregation when the tempera­
ture increases above its lower critical solution temperature (LCST) at
around 32 ℃ [27]. The aggregation of pNIPAm can be monitored by
measuring the optical transmittance of the polymer solution. As shown
in Fig. 1, poly(NIPAm-co-GMA)@N3 prepared by 2.5− 24 h polymeri­
zation exhibited different temperature-responsive phase transition in
aqueous solution. The polymer synthesized by 24 h polymerization had
the lowest phase transition temperature. In the remaining experiments,
only the polymer prepared by 24 h polymerization was investigated. The
poly(NIPAm-co-GMA)@N3 prepared by 24 h polymerization has a mo­
lecular weight Mn of 27,000 g mol− 1 and a molecular weight distribution
of 1.5, as determined by GPC analysis (Fig. S4). In the 1H NMR spectrum
of poly(NIPAm-co-GMA), the terminal CH-CH2-O from GMA has two
resonance bands at 2.67 and 2.79 ppm, and the two methyl groups from
NIPAm give a single band at 1.02 ppm (Fig. S5) [41,42]. The relative
intensities of these NMR signals indicate that the final ratio of NIPAm:
GMA in poly(NIPAm-co-GMA) to be approximately 2.6: 1. The content
of NIPAm is lower than expected, which is likely caused by a lower
reactivity of NIPAm during the RAFT copolymerization.
3.2. Preparation and characterization of composite nanoparticles
In order to add pentafluorophenyl groups on nanoparticle surface,
silica nanoparticles were first functionalized with APTES to introduce
amino groups, which were used to react with pentafluorobenzyl chloride
to give the Si@PFBCl nanoparticles. The presence of amino groups on
Si@NH2 nanoparticles was confirmed by ninhydrin assay, where the
amino-functionalized nanoparticles generated a deep-blue color after
reacting with ninhydrin solution (Fig. S6). After modifying the
Fig. 1. Thermoresponsive phase transition monitored by optical transmittance
of poly(NIPAm-co-GMA)@N3 synthesized using (a) 24 h, (b) 8 h, (c) 5 h and (d)
2.5 h of polymerization.
5
Colloids and Surfaces B: Biointerfaces 197 (2021) 111433
nanoparticles with PFBCl, a subsequent reaction with sodium azide
converted Si@PFBCl into the photoactive Si@PFPA nanoparticles [32].
The chemical composition of the composite particles was analyzed by
FT-IR spectroscopy (Fig. 2). Several distinct IR absorption bands were
observed for silica at 1051 cm− 1 (asymmetric vibration of Si-O), 944
cm− 1 (asymmetric vibration of Si− OH), and 796 cm− 1 (symmetric vi­
bration of Si-O). After acylation reaction with PFBCl, two weak
adsorption bands at 1490 cm− 1 and 1495 cm− 1 appeared, which can be
ascribed to the skeleton vibration of benzene ring. Although the quantity
of the surface-bound PFBCl is not high, the characteristic IR bands are
clearly visible in contrast to the IR spectrum of Si@NH2. After further
reaction with NaN3, a new band at 2115 cm− 1 occurred, which can be
assigned to the surface-bound phenyl azide, confirming successful
immobilization of the photoactive group on the nanoparticle surface.
The polymer-functionalized silica nanoparticles were synthesized
through photoconjugation of poly(NIPAm-co-GMA)@N3 with Si@PFPA
(Scheme 1). To avoid excessive inter-particle crosslinking, the mass ratio
between Si@PFPA and poly(NIPAm-co-GMA)@N3 was chosen as 1: 4 to
make sure a complete coverage of polymer on the nanoparticle surface.
Prior to photoconjugation, the solvent was also removed to avoid side
reactions. The photoconjugation reaction was very efficient and finished
in less than 10 min. After the photoconjugation, the intensity of the IR
band at 2100 cm− 1 increased significantly (Fig. S7a), which was
attributed to the azide groups in the polymer. Besides, the IR bands in
the range of 1400 to 1750 cm− 1 of the composite nanoparticles also
changed dramatically after the photoconjugation with poly(NIPAm-co-
GMA)@N3 (Fig. S3). The above results confirm successful immobiliza­
tion of the copolymer on the PFPA-modified nanoparticles. In a control
experiment without the photoconjugation step, after Si@PFPA was
exposed to poly(NIPAm-co-GMA)@N3 and washed, the dry particles did
not give clear azide band (Fig. S7b).
The alkyne-tagged boronic acid was added to Si@poly(NIPAm-co-
GMA)@N3 by CuAAC reaction. As shown in Fig. S8, after the CuAAC
reaction the azide band on the composite nanoparticles almost dis­
appeared completely, suggesting successful addition of the boronic acid
to the immobilized polymer chains. Moreover, the B–O signal at 1332
cm− 1 also indicates successful immobilization of the boronic acid.
ARS was employed to detect the boronic acid on Si@poly(NIPAm-co-
GMA)@PCAPBA due to its dramatic color change and fluorescence
emission after forming ester with boronic acid [27]. As shown in Fig. S9,
after mixing with ARS, Si@poly(NIPAm-co-GMA)@PCAPBA displayed
an orange color different from all the other nanoparticles. Furthermore,
the mixture of Si@poly(NIPAm-co-GMA)@PCAPBA and ARS generated
a strong fluorescence emission centered at 575 nm (Fig. S10), indicating
formation of ester bond between ARS and the boronic acid in the com­
posite nanoparticles. As a comparison, the other particles (Si@NH2 and
Si@poly(NIPAm-co-GMA)@N3) mixed with ARS were not fluorescent.
Fig. 2. FT IR spectra of SiO2, Si@NH2, Si@PFBCl and Si@PFPA.
H. Zheng et al.
All these results confirm successful introduction of boronic acid into the
composite particles.
Elemental analysis of Si@poly(NIPAm-co-GMA)@PCAPBA revealed
that the content of C, H, N, Si, F and B in the composite particles were
25.4 %, 3.89 %, 4.03 %, 14.6 %, 0.15 % and 0.17 %, respectively. Based
on the boron content, the density of boronic acid ligands in Si@poly
(NIPAm-co-GMA)@PCAPBA is estimated to be ~ 0.157 mmol/g
particles.
The organic contents of the different nanoparticles (silica core,
Si@NH2, Si@PFBCl, Si@PFPA, Si@poly(NIPAm-co-GMA)@N3 and
Si@poly(NIPAm-co-GMA)@PCAPBA) were investigated by TGA anal­
ysis. As shown in Fig. S11, the weight loss (approximately 4–10 %)
below 250 ℃ can be attributed to the evaporation of residual water and
solvent. Above 700 ℃, ~ 0.3 % more weight loss occurred from silica
core to Si@NH2, which is attributed to the aminopropyl group in APTES.
Fig. 3. SEM images of (a) Si@NH2, (c)Si@PFPA, (e) Si@poly(NIPAm-co-GMA)@N3; TEM images of (b) Si@NH2, (d)Si@PFPA, (f) Si@poly(NIPAm-co-GMA)@N3.
6
Colloids and Surfaces B: Biointerfaces 197 (2021) 111433
Besides, the difference in weight loss (appropriately 1.1 wt%) between
Si@NH2 and Si@PFBCl comes from the immobilized pentafluorobenzyl
group. From Si@PFBCl to Si@PFPA, only a small weight loss (~ 0.70 %)
occurred due to conversion of the terminal fluoride into azide group. A
further weight loss (~ 50 %) was observed from Si@poly(NIPAm-co-
GMA)@N3, caused by thermo-decomposition of the immobilized
organic polymers. Based on the TGA curve, the amount of the polymer
conjugated on silica was calculated to be ~ 1.40 g/g silica. For the final
Si@poly(NIPAm-co-GMA)@PCAPBA particles, based on the content of
the inorganic material (29.5 %, from TGA analysis) and the density of
boronic acid in the composite particles (0.157 mmol/g particles), the
amount of boronic acid ligand immobilized on the silica nanoparticles is
estimated to be ~ 0.532 mmol/g silica.
H. Zheng et al.
3.3. Characterization of composite nanoparticles by electron microscopy
and dynamic light scattering
The size and morphology of silica, Si@NH2, Si@PFPA and Si@poly
(NIPAm-co-GMA)@N3 particles were analyzed using SEM, TEM and
DLS. The SEM image in Fig. S12 shows that the silica core has a uniform
and spherical shape. The hydrodynamic diameter of the silica core was
determined to be 195 nm (PDI: 0.002). After being modified with the
amino group, the nanoparticles were still uniform and spherical
(Fig. 3a), and their hydrodynamic diameter increased to 203 nm (PDI:
0.004). After introduction of PFBCl and its conversion into the photo­
active PFPA, the nanoparticles became less smooth (Fig. 3c, d), and the
hydrodynamic diameter of Si@PFPA increased to 217 nm (PDI: 0.312).
For the silica-polymer composite Si@poly(NIPAm-co-GMA)@N3, the
SEM (Fig. 3e) indicates that the nanocomposite particles became less
spherical. The TEM image (Fig. 3f) reveals the presence of the polymer
layer grafted on the inorganic nanoparticles. Due to the attachment of
the polymer chains, the hydrodynamic diameter of the nanoparticles
increased by ~ 90 nm (Fig. S12e).
3.4. Evaluation of Si@poly(NIPAm-co-GMA)@PCAPBA for bacteria
separation
To evaluate the practicability of Si@poly(NIPAm-co-GMA)
@PCAPBA as an affinity material for separation of bacteria, E. coil and
S. epidermidis were used as two models. To quantify the concentration of
the bacteria, two standard plots were established by comparing the
colony-forming unit (CFU) with the optical density (OD600) of the bac­
teria (Fig. S13). The boronic acids immobilized on Si@poly(NIPAm-co-
GMA)@PCAPBA were supposed to capture the bacteria through forming
boronic ester bonds with cis-diol compounds on the bacteria surface.
Besides the intended boronic acid-diol binding, other interactions
including ionic interaction, van de Waals force and hydrophobic effect,
etc. may also affect bacteria binding [21,43]. To find out the importance
of the immobilized boronic acid for bacteria binding, a control experi­
mental was performed using Si@poly(NIPAm-co-GMA)@N3 as a refer­
ence material. As shown in Fig. S14, the boronic acid-functionalized
Si@poly(NIPAm-co-GMA)@PCAPBA nanoparticles exhibited signifi­
cantly higher adsorption of the two bacteria than Si@poly(NIPAm-­
co-GMA)@N3, suggesting that the boronic acid played a critical role in
the bacteria binding. As binding with the boronic acid-modified nano­
particles were expected to cause bacteria aggregation, the colloidal
stabilities of E. coli and S. epidermidis were also studied before and after
Fig. 4. Sedimentation of bacterial cells and nanoparticles monitored by mea­
surement of optical absorbance at 600 nm. E. coli or S. epidermidis suspension (1
mL, OD600≈0.6) in PBS (0.01 M, pH 7.4) was mixed with or without Si@poly
(NIPAm-co-GMA)@PCAPBA nanoparticles (1 mg). A suspension of Si@poly
(NIPAm-co-GMA)@PCAPBA (1 mg/mL) in PBS was used as a control.
7
Colloids and Surfaces B: Biointerfaces 197 (2021) 111433
addition of the nanoparticles, by measuring the change of light absor­
bance of the bacterial suspensions. As shown in Fig. 4, while E. coli and
S. epidermidis themselves remained as a stable suspension, addition of
the boronic acid-modified nanoparticles caused the bacterial cells to
settle quickly due to their binding to the composite particles. After
mixing with Si@poly(NIPAm-co-GMA)@PCAPBA for 30 min, the
amount of E. coli cells settled was significantly more than S. epidermidis.
The faster sedimentation of E. coli caused by Si@poly(NIPAm-co-GMA)
@PCAPBA demonstrates that the boronic acid-modified nanoparticles
can bind Gram-negative bacteria more effectively, as observed in the
bacterial binding experiment (Fig. S14).
More detailed structure of the particle-mediated E. coli and
S. epidermidis aggregates was inspected using TEM (Fig. 5). Without the
boronic acid ligand, Si@poly(NIPAm-co-GMA)@N3 particles added to
the bacteria suspension were found to form closely packed nanoparticle
aggregates (Fig. 5a, c). When Si@poly(NIPAm-co-GMA)@PCAPBA was
added to the bacteria suspension, the boronic acid-modified nano­
particles bound readily to the bacterial cells to form particle-cell ag­
gregates (Fig. 5b, d). No significant disintegration of bacterial cells was
observed from the TEM images.
For the purpose of separating living bacteria, it is necessary to find
out if the boronic acid-modified nanoparticles impact bacteria viability.
As shown in Fig. S15, the growth curves of E. coli and S. epidermidis in the
presence of the different nanoparticles are similar to that observed in
PBS, suggesting that the functional groups in these materials (including
azide, primary amine and boronic acid) do not cause cell death. More­
over, after the particle-bound bacteria were transferred into a growth
medium, the turbidity of the culture medium increased significantly,
indicating that the particle-bound bacteria were able to proliferate (Fig.
S16).
The effect of temperature on bacterial binding provided by Si@poly
(NIPAm-co-GMA)@PCAPBA was further investigated. As shown in
Fig. 6a, at 40 ℃, the amount of E. coli and S. epidermidis bound to the
nanoparticles decreased slightly to 10.9 × 107 and 6.51 × 107 CFU/mg,
respectively. Compared to glycoprotein binding to nanoparticle-
supported polymer brushes, the bacteria binding does not display
obvious temperature effect. The small impact of temperature may be
attributed to the insignificant change of the polymer conformation, as
the polymer chains are very likely anchored on the particle surface
through multiple chemical bonds due to the photoconjugation. To
improve thermo-responsive property of bacterial binding, one possible
way is to utilize surface-grafted polymer brushes containing pNIPAm
and terminal boronic acids [44].
Fig. 6b shows the effect of pH on bacterial binding in the range of pH
7.0− 8.5. For E. coli, the highest bacterial binding to Si@poly(NIPAm-co-
GMA)@PCAPBA was observed at pH 8.0, while the binding of
S. epidermidis increased gradually when the pH was changed from
7.0–8.5. At pH 7.0, the boronic acid-modified composite nanoparticles
exhibit binding capacities for E. coli and S. epidermidis as 8.97 × 107 and
5.92 × 107 CFU/mg, respectively.
3.5. Separation of bacteria from milk samples
To further evaluate the feasibility of using Si@poly(NIPAm-co-GMA)
@PCAPBA to capture bacteria in real samples, the bacterial separation
was tested in milk samples spiked with E. coli and S. epidermidis. Cow
milk (4 × diluted) was spiked with E. coli and S. epidermidis at ~ 103 cell/
mL and used as test samples. After adjusting the pH of the test samples to
8.0, Si@poly(NIPAm-co-GMA)@PCAPBA particles were added. After
equilibrium adsorption, the number of unbound bacteria was quantified
by the colony counting method. As shown in Fig. 7, most of the bacterial
cells were captured by the boronic acid-modified particles and settled.
The nanoparticles exhibited more efficient binding towards E. coli than
S. epidermidis, which can be attributed to the different saccharide
structures on the cellular surfaces. Considering the abundance of inter­
fering substances in milk such as proteins, lipids, carbohydrates, etc., the
H. Zheng et al. Colloids and Surfaces B: Biointerfaces 197 (2021) 111433

Fig. 5. TEM images of E. coli mixed with (a) Si@poly(NIPAm-co-GMA)@N3 and (b) Si@poly(NIPAm-co-GMA)@PCAPBA, and S. epidermidis mixed with (c) Si@poly
(NIPAm-co-GMA)@N3 and (d) Si@poly(NIPAm-co-GMA)@PCAPBA.

Fig. 6. Effect of temperature and pH on bacterial binding with nanoparticles.

boronic acid-modified nanoparticles are considered highly effective for
bacterial adsorption and separation.
4. Conclusions
In this work, a novel pH-responsive nanocomposite was developed
for selective enrichment of living bacteria. The synthetic approach
combines controlled radical polymerization, high efficiency photo­
conjugation and click chemistry to produce the new bacteria-capturing
nanomaterial. The RAFT polymerization allowed a facile and versatile
control over the structure and function of the polymer intermediate, and
the CuAAC click chemistry enabled a straightforward coupling of the
boronic acid to the flexible polymer chains. Most importantly, the
photoconjugation chemistry made it possible to achieve simple and very
fast polymer immobilization on silica nanoparticles. Due to the high
density of affinity ligands appended on the flexible polymer chains, the
new affinity material exhibited excellent affinity towards bacteria. Using
silica as a model representing inorganic nanoparticles, a practical and
facile method to turn an inert surface into photoactive substrate has
been demonstrated. The synthetic procedure developed in this work

8
H. Zheng et al.
Fig. 7. Separation of E. coli and S. epidermidis from spiked 25 % milk sample using Si@poly(NIPAm-co-GMA)@PCAPBA. (a) remaining E. coli in the spiked milk after
treatment with the composite nanoparticles; (b) E. coli in the spiked milk; (c) remaining S. epidermidis in the spiked milk after treatment with the composite
nanoparticles; (d) S. epidermidis in the spiked milk.
provide a versatile approach for preparing polymer-based affinity ad­
sorbents for not only bacteria, but also other important biological tar­
gets, e.g. proteins, nucleic acids and viruses.
CRediT authorship contribution statement
Hongwei Zheng: Conceptualization, Methodology, Investigation,
Writing - original draft. Haiyue Gong: Methodology, Writing - review &
editing. Limin Cao: Supervision, Writing - review & editing. Hong Lin:
Supervision, Writing - review & editing. Lei Ye: Resources, Conceptu­
alization, Methodology, Supervision, Writing - review & editing.
Declaration of Competing Interest
The authors declare no competing finical interest.
Acknowledgments
This work was supported by the Swedish Research Council VR (grant
number 2019-04228). Hongwei Zheng thanks the China Scholarship
Council (CSC) for a visiting scholarship. Hongwei Zheng is a visiting
student in Lund University and a PhD candidate in Ocean University of
China.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.colsurfb.2020.111433.
References
[1] S. Hameed, L. Xie, Y. Ying, Conventional and emerging detection techniques for
pathogenic bacteria in food science: a review, Trends Food Sci. Tech. 81 (2018)
61–73, https://doi.org/10.1016/j.tifs.2018.05.020.
[2] S. Long, L. Miao, R. Li, F. Deng, Q. Qiao, X. Liu, A. Yan, Z. Xu, Rapid identification
of bacteria by membrane-responsive aggregation of a pyrene derivative, ACS Sens.
4 (2019) 281–285, https://doi.org/10.1021/acssensors.8b01466.
[3] C. Ho, N. Jean, C.A. Hogan, L. Blackmon, S.S. Jeffrey, M. Holodniy, N. Banaei, A.A.
E. Saleh, S. Ermon, J. Dionne, Rapid identification of pathogenic bacteria using
Raman spectroscopy and deep learning, Nat. Commun. 10 (2019) 1–8, https://doi.
org/10.1038/s41467-019-12898-9.
[4] C.A. Batt, Food pathogen detection, Science 316 (2007) 1579–1580, https://doi.
org/10.1126/science.1140729.
[5] K. Kant, M.A. Shahbazi, V.P. Dave, T.A. Ngo, V.A. Chidambara, L.Q. Than, D.
D. Bang, A. Wolff, Microfluidic devices for sample preparation and rapid detection
of foodborne pathogens, Biotechnol. Adv. 36 (2018) 1003–1024, https://doi.org/
10.1016/j.biotechadv.2018.03.002.
[6] K.A. Stevens, L.A. Jaykus, Bacterial separation and concentration from complex
sample matrices: a review, Crit. Rev. Microbiol. 30 (2004) 7–24, https://doi.org/
10.1080/10408410490266410.
[7] M. Golabi, F. Kuralay, E.W.H. Jager, V. Beni, A.P.F. Turner, Electrochemical
bacterial detection using poly (3-aminophenylboronic acid)-based imprinted
polymer, Biosens. Bioelectron. 93 (2017) 87–93, https://doi.org/10.1016/j.
bios.2016.09.088.
9
Colloids and Surfaces B: Biointerfaces 197 (2021) 111433
[8] W.L.A. Brooks, B.S. Sumerlin, Synthesis and applications of boronic acid-
containing polymers: from materials to medicine, Chem. Rev. 116 (2016)
1375–1397, https://doi.org/10.1021/acs.chemrev.5b00300.
[9] G. Mo, X. He, C. Zhou, D. Ya, J. Feng, C. Yu, B. Deng, A novel ECL sensor based on a
boronate affinity molecular imprinting technique and functionalized SiO2@ CQDs/
AuNPs/MPBA nanocomposites for sensitive determination of alpha-fetoprotein,
Biosens. Bioelectron. 126 (2019) 558–564, https://doi.org/10.1016/j.
bios.2018.11.013.
[10] J. Ye, Y. Chen, Z. Liu, A boronate affinity sandwich assay: an appealing alternative
to immunoassays for the determination of glycoproteins, Angew. Chem., Int. Ed. 53
(2014) 10386–10389, https://doi.org/10.1002/anie.201405525.
[11] J.P.M. António, R. Russo, C.P. Carvalho, P.M.S.D. Cal, P.M.P. Gois, Boronic acids as
building blocks for the construction of therapeutically useful bioconjugates, Chem.
Soc. Rev. 48 (2019) 3513–3536, https://doi.org/10.1039/C9CS00184K.
[12] A. Stubelius, S. Lee, A. Almutairi, The chemistry of boronic acids in nanomaterials
for drug delivery, Acc. Chem. Res. 52 (2019) 3108–3119, https://doi.org/
10.1021/acs.accounts.9b00292.
[13] Y. Chen, A. Huang, Y. Zhang, Z. Bie, Recent advances of boronate affinity materials
in sample preparation, Anal. Chim. Acta 1076 (2019) 1–17, https://doi.org/
10.1016/j.aca.2019.04.050.
[14] D. Li, Y. Chen, Z. Liu, Boronate affinity materials for separation and molecular
recognition: structure, properties and applications, Chem. Soc. Rev. 44 (2015)
8097–8123, https://doi.org/10.1039/C5CS00013K.
[15] Z. Liu, H. He, Synthesis and applications of boronate affinity materials: from class
selectivity to biomimetic specificity, Acc. Chem. Res. 50 (2017) 2185–2193,
https://doi.org/10.1021/acs.accounts.7b00179.
[16] J. Wang, J. Gao, D. Liu, D. Han, Z. Wang, Phenylboronic acid functionalized gold
nanoparticles for highly sensitive detection of Staphylococcus aureus, Nanoscale 4
(2012) 451–454, https://doi.org/10.1039/C2NR11657J.
[17] D. Dechtrirat, N. Gajovic-Eichelmann, F. Wojcik, L. Hartmann, F.F. Bier, F.
W. Scheller, Electrochemical displacement sensor based on ferrocene boronic acid
tracer and immobilized glycan for saccharide binding proteins and E. coli, Biosens.
Bioelectron. 58 (2014) 1–8, https://doi.org/10.1016/j.bios.2014.02.028.
[18] M. Golabi, F. Kuralay, E.W.H. Jager, V. Beni, A.P.F. Turner, Electrochemical
bacterial detection using poly (3-aminophenylboronic acid)-based imprinted
polymer, Biosens. Bioelectron. 93 (2017) 87–93, https://doi.org/10.1016/j.
bios.2016.09.088.
[19] Y. Tsuchido, R. Horiuchi, T. Hashimoto, K. Ishihara, N. Kanzawa, T. Hayashita,
Rapid and selective discrimination of gram-positive and gram-negative bacteria by
boronic acid-modified poly(amidoamine) dendrimer, Anal. Chem. 91 (2019)
3929–3935, https://doi.org/10.1021/acs.analchem.8b04870.
[20] H. Wang, Z. Bie, C. Lü, Z. Liu, Magnetic nanoparticles with dendrimer-assisted
boronate avidity for the selective enrichment of trace glycoproteins, Chem. Sci. 4
(2013) 4298–4303, https://doi.org/10.1039/C3SC51623G.
[21] H. Zheng, F. Han, H. Lin, L. Cao, T.R. Pavase, J. Sui, Preparation of a novel
polyethyleneimine functionalized sepharose-boronate affinity material and its
application in selective enrichment of foodborne pathogenic bacteria, Food Chem.
294 (2019) 468–476, https://doi.org/10.1016/j.foodchem.2019.05.023.
[22] J. Niu, D.J. Lunn, A. Pusuluri, J.I. Yoo, M.A. O’Malley, S. Mitragotri, H.T. Soh, C.
J. Hawker, Engineering live cell surfaces with functional polymers via
cytocompatible controlled radical polymerization, Nat. Chem. 9 (2017) 537–545,
https://doi.org/10.1038/nchem.2713.
[23] T. Pelras, C.S. Mahon, M. Müllner, Synthesis and applications of
compartmentalised molecular polymer brushes, Angew. Chem., Int. Ed. 57 (2018)
6982–6994, https://doi.org/10.1002/anie.201711878.
[24] A.J. Convertine, N. Ayres, C.W. Scales, A.B. Lowe, C.L. McCormick, Facile,
controlled, room-temperature RAFT polymerization of N-isopropylacrylamide,
Biomacromolecules 5 (2004) 1177–1180, https://doi.org/10.1021/bm049825h.
[25] X. Chen, X. Liu, C. Miao, P. Song, Y. Xiong, Ionic liquid-like inimer mediated RAFT
polymerization of N-isopropylacrylamide, Eur. Polym. J. 107 (2018) 229–235,
https://doi.org/10.1016/j.eurpolymj.2018.08.016.
[26] L. Moroni, M.K. Gunnewiek, E.M. Benetti, Polymer brush coatings regulating cell
behavior: passive interfaces turn into active, Acta Biomater. 10 (2014) 2367–2378,
https://doi.org/10.1016/j.actbio.2014.02.048.
H. Zheng et al.
[27] L. Jiang, M.E. Messing, L. Ye, Temperature and pH dual-responsive core-brush
nanocomposite for enrichment of glycoproteins, ACS Appl. Mater. Interfaces 9
(2017) 8985–8995, https://doi.org/10.1021/acsami.6b15326.
[28] J. Chen, M. Liu, C. Chen, H. Gong, C. Gao, Synthesis and characterization of silica
nanoparticles with well-defined thermoresponsive PNIPAM via a combination of
RAFT and click chemistry, ACS Appl. Mater. Interfaces 3 (2011) 3215–3223,
https://doi.org/10.1021/am2007189.
[29] J. Sutrisno, I. Pramudya, X. Aerken, A. Fuchs, Surface grafting of poly
(pentafluorostyrene) on the iron and iron oxide particles via reversible addition
fragmentation chain transfer (RAFT) polymerization, J. Appl. Polym. Sci. 134
(2017), https://doi.org/10.1002/app.44898.
[30] H. Liu, H. Chung, Self-healing properties of lignin-containing nanocomposite:
synthesis of lignin-graft-poly (5-acetylaminopentyl acrylate) via RAFT and click
chemistry, Macromolecules 49 (2016) 7246–7256, https://doi.org/10.1021/acs.
macromol.6b01028.
[31] R. Liu, P. Zhang, H. Dai, Synthesis of magnetic particles with well-defined living
polymeric chains via combination of RAFT polymerization and thiol-ene click
chemistry, J. Polym. Res. 23 (2016) 218, https://doi.org/10.1007/s10965-016-
1113-3.
[32] J.F.W. Keana, S.X. Cai, New reagents for photoaffinity labeling: synthesis and
photolysis of functionalized perfluorophenyl azides, J. Org. Chem. 55 (1990)
3640–3647, https://doi.org/10.1021/jo00298a048.
[33] H. Baruah, S. Puthenveetil, Y.A. Choi, S. Shah, A.Y. Ting, An engineered aryl azide
ligase for site-specific mapping of protein–protein interactions through photo-
cross-linking, Angew. Chem., Int. Ed. 47 (2008) 7018–7021, https://doi.org/
10.1002/anie.200802088.
[34] O. Norberg, L. Deng, M. Yan, O. Ramström, Photo-click immobilization of
carbohydrates on polymeric surfaces - a quick method to functionalize surfaces for
biomolecular recognition studies, Bioconjugate Chem. 20 (2009) 2364–2370,
https://doi.org/10.1021/bc9003519.
[35] C. Xu, K.M.A. Uddin, X. Shen, H.S.N. Jayawardena, M. Yan, L. Ye,
Photoconjugation of molecularly imprinted polymer with magnetic nanoparticles,
10
Colloids and Surfaces B: Biointerfaces 197 (2021) 111433
ACS Appl. Mater. Interfaces 5 (2013) 5208–5213, https://doi.org/10.1021/
am401042u.
[36] T. Suksrichavalit, K. Yoshimatsu, V. Prachayasittikul, L. Bülow, L. Ye, “Clickable”
affinity ligands for effective separation of glycoproteins, J. Chromatogr. A 1217
(2010) 3635–3641, https://doi.org/10.1016/j.chroma.2010.03.050.
[37] W. Stöber, A. Fink, E. Bohn, Controlled growth of monodisperse silica spheres in
the micron size range, J. Colloid Interface Sci. 26 (1968) 62–69, https://doi.org/
10.1016/0021-9797(68)90272-5.
[38] E. Soto-Cantu, R. Cueto, J. Koch, P.S. Russo, Synthesis and rapid characterization
of amine-functionalized silica, Langmuir 28 (2012) 5562–5569, https://doi.org/
10.1021/la204981b.
[39] D. Roy, J.N. Cambre, B.S. Sumerlin, Triply-responsive boronic acid block
copolymers: solution self-assembly induced by changes in temperature, pH, or
sugar concentration, Chem. Commun. (Camb.) (2009) 2106–2108, https://doi.
org/10.1039/B900374F.
[40] Y. Ma, Y. Zhang, M. Zhao, X. Guo, H. Zhang, Efficient synthesis of narrowly
dispersed molecularly imprinted polymer microspheres with multiple stimuli-
responsive template binding properties in aqueous media, Chem. Commun.
(Camb.) 48 (2012) 6217–6219, https://doi.org/10.1039/C2CC31932B.
[41] J. Li, Y. Zhou, Z. Luo, S. Zhu, A polyelectrolyte-containing copolymer with a gas-
switchable lower critical solution temperature-type phase transition, Polym. Chem.
10 (2019) 260–266, https://doi.org/10.1039/C8PY01265B.
[42] P. Li, R. Xu, W. Wang, X. Li, Z. Xu, K.W.K. Yeung, P.K. Chu, Thermosensitive poly
(N-isopropylacrylamide-co-glycidyl methacrylate) microgels for controlled drug
release, Colloids Surf. B Biointerfaces 101 (2013) 251–255, https://doi.org/
10.1016/j.colsurfb.2012.07.009.
[43] H. Gong, K. Zhang, C. Dicko, L. Bülow, L. Ye, Ag–polymer nanocomposites for
capture, detection, and destruction of bacteria, ACS Appl. Nano Mater. 2 (2019)
1655–1663, https://doi.org/10.1021/acsanm.9b00112.
[44] H. Ma, L. Jiang, S. Hajizadeh, H. Gong, B. Lu, L. Ye, Nanoparticle-supported
polymer brushes for temperature-regulated glycoprotein separation: investigation
of structure–function relationship, J. Mater. Chem. B Mater. Biol. Med. 6 (2018)
3770–3781, https://doi.org/10.1039/C8TB00627J.