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Journal of Chromatography A, xxx (2018) xxx–xxx

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Selective recognition and fast enrichment of anthocyanins by dummy

molecularly imprinted magnetic nanoparticles
Qian-Yu Zhao a,1 , Hai-Tian Zhao a,1 , Xin Yang a,∗ , Hua Zhang a , Ai-Jun Dong a , Jing Wang b,∗ ,
Bin Li c,∗
Department of Food Sciences and Engineering, School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin, 150001, PR China
Key Laboratory of Agro-Product Quality and Safety, Institute of Quality Standard & Testing Technology for Agro-Product, Chinese Academy of Agricultural
Sciences, Beijing, 100081, PR China
School of Pharmacy, Jiangxi University of Traditional Chinese Medicine, 818 Meiling Avenue, Nanchang, 330004, Jiangxi, PR China

a r t i c l e i n f o a b s t r a c t

Article history: In this work, a novel synthetic strategy for separation media which quickly specific recognize antho-
Received 22 May 2018 cyanins was developed by dummy molecular imprinting technology and magnetic separation technology.
Received in revised form 25 July 2018 Rutin, similar to cyanidin-3-O-rutinoside structure, was selected as a virtual template. The appropriate
Accepted 12 August 2018
molecular imprinting system was selected by NIP library screening method, 4-vinyl pyridine and ace-
Available online xxx
tonitrile as functional monomer and solvent respectively. The molecularly imprinted layer was formed
on the surface of the magnetic carrier to prepare dummy magnetic molecularly imprinted polymers.
The microstructures of prepared composites were characterized by scanning electron microscopy (SEM),
Magnetic molecularly imprinted polymers
Surface imprinting method
Fourier transform infrared spectroscopy (FT-IR), thermal gravimetric analysis (TGA) and X-ray diffrac-
Dummy template tion (XRD). Adsorption kinetics, isothermal adsorption curves and selective specificity were investigated
Cyanidin-3-O-rutinoside to further reveal the specific recognition mechanism of the adsorbents on anthocyanins. The dummy
molecularly imprinted polymers showed a short kinetic equilibrium time, high selectivity (comparing
with quercetin and naringin), and satisfying adsorption capacity for anthocyanins. The binding capac-
ity and the imprinting factor of dummy molecularly imprinted polymers can reach 15.69 mg g−1 and
2.05, respectively. In addition, the purity of cyanidin-3-O-rutinoside in the crude extract from Lonicera
caerulea increased to 89% after the adsorption of dummy magnetic molecular imprinted polymers. Many
favorable capabilities of the prepared molecularly imprinted polymers also provided the basis for further
application for separation and purification of anthocyanins in the future.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction According to the reported researches recently, more than 500

anthocyanins have been found from plants [8,9]. However, unsta-
Anthocyanins belong to flavonoid polyphenols, and anthocyani- ble structures as well as various analogues leads to very difficult
din forms glycosides with various sugars under natural conditions. isolation and purification of anthocyanins. Therefore, the rapid iso-
Anthocyanins, as an important class of water-soluble natural food lation and purification of anthocyanin monomer has always been
colorings, are safe, non-toxic, rich in resources, and have some a difficult problem to be solved. The traditional separation and
nutrition and pharmacological effects, such as the removal of free purification methods including column chromatography [10], high
radicals [1], anti-cancer [2], anti-inflammatory [3], inhibition of performance liquid [11], membrane separation [12], high-speed
lipid peroxidation [4], protection liver [5], prevention diabetes [6], countercurrent chromatography [13] and so on, cannot be accom-
protection vision [7] and more. plished in one go and requires multiple methods to work with each
other in order to obtain a relatively single anthocyanin component,
and have the following problems exist: the using of a large amount
of organic solvents, very low yield and the structure changed.
∗ Corresponding authors. Since anthocyanins are difficult to purify, many pharmacological
E-mail addresses: (X. Yang), w experiments only use anthocyanin crude extracts as experimental
(J. Wang), (B. Li). materials, which greatly hinder the anthocyanin structure-activity
Qianyu Zhao and Haitian Zhao contributed equally to this work and should be relationship research process [14]. Therefore, there is an urgent
considered co-first authors.
0021-9673/© 2018 Elsevier B.V. All rights reserved.

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Fig. 1. The chemical structure of cyanidin-3-O-rutinoside (A) and rutin (B).

requirment to develop a new and effective separation and purifi- the functional monomer, however the actual synthesis process of
cation method for anthocyanins. molecularly imprinted polymers is also affected by many factors.
Molecular imprinting technology (MIT) is a technology for the A new method, NIP screening library, is put forward which use
synthesis of molecular imprinting polymers (MIPs), a synthetic non-imprinted polymers (NIPs) to replace corresponding MIPs for
receptor with special molecular recognition function [15]. The pre- screening functional monomers and porogens [26]. NIPs and MIPs
pared polymers form a special “memory” to the template molecule have the same adsorption behavior, if the NIPs have interaction
both in the spatial structure and the chemical bond which can with the template molecule, and the corresponding MIPs will show
identify the target compound from the complex system in a short a very good imprinting effect. This method simulates the forma-
period of time. Because molecularly imprinted polymers have the tion of MIPs more accurately. The synthesis of NIP screening library
properties of special selectivity, easy preparation, simple operation skips the process of template elution and reduces costs. NIP Screen-
and low solvent consumption, MIPs can replace of the traditional ing Library one-time high-throughput screened multiple factors
chromatography stationary phases for the purification of active that affect the adsorption quantity of MIPs. In this paper, functional
ingredients. monomers and solvents were screened by NIP screening library
Currently, very little work has been reported on antho- simultaneously.
cyanins molecularly imprinted polymers because anthocyanins are In this paper, a novel adsorbent that can quickly isolate antho-
highly unstable and susceptible to pH, oxygen, temperature, light, cyanins is prepared by the combination of surface molecular
enzymes and other factors. Similarly, under the existing synthetic imprinting technique and dummy template molecular imprint-
molecular imprinting conditions (UV light, radiation or heat), it is ing strategy. Surface molecular imprinting technology makes the
difficult to ensure that the structure of anthocyanins is not dam- imprinting sites located in or near the surface, which increases the
aged. In addition, the expensive price of anthocyanin monomers effective imprinting sites, improves the imprinting capacity, accel-
increases the cost of MIPs. erates the mass transfer rate and facilitates the diffusion and elution
Dummy template molecular imprinting technology is the use of the template molecules. We select 4-vinyl pyridine as func-
of close structural analogs to replace the real template molecules tional monomer and acetonitrile as solvent according to NIP library
[16], which is developed to avoid template leak in trace detection. screening. 4-VP can form electrostatic attraction and ␲–␲* inter-
It was reported that dummy templates based on MIPs for nons- action with the dummy template rutin. The structure of C3R was
teroidal anti-inflammatory [17], bisphenol A [18], triazophos [19] similar to rutin. DMMIPs also recognized C3R by electrostatic inter-
have been reported. The resultant MIPs demonstrated highly selec- action and ␲–␲* interaction. These two forces were not disturbed
tive and sensitive recognition and determination abilities, which in water, which was a guarantee to recognize anthocyanins in
have great application potential in real sample. In recent years, it water. The optimized molar ratio of template-functional monomer-
is also applied to the imprinting of a class of template molecules crosslinker were 1:8:40 (shown in Fig. S1). Magnetic nanoparticles
that are expensive, unsafe, unstable and low solubility in synthe- are selected as the supporter because of its large specific surface
sis system [20,21]. Therefore, we plan to use the dummy template area, good compatibility and superparamagnetic. In the separation
molecular imprinting synthesis strategy to solve this problem. In process magnetic molecularly imprinted polymers (MMIPs) can be
dummy template molecular imprinting technology, how to choose quickly separated from liquid, which simplify the separation pro-
a virtual template is crucial. cess. The synthesis route of dummy magnetic molecular imprinted
In this study, we selected cyanidin-3-O-rutinoside (C3R) as a polymers (DMMIPs) is represented in Fig. 2.
research target, and rutin as a dummy template for synthesis of
MIPs (Fig.1) because they are all flavonoids and have similar C6 C3 C6
carbon skeletons and the number of hydroxyl groups on the car- 2. Experimental
bon skeleton and substitution position of these two substances are
the same. In addition, they have uniform type of linked sugar and 2.1. Instrumentation
glycosidic bond.
The type of functional monomer plays a crucial role in the A TU-1900 Double-beam UV–vis spectrophotometer (Persee,
imprinting process. The interaction between the template and the China) was used to measure the absorbance of rutin and C3R.
functional monomer directly determine the recognition perfor- Scanning electron micros-copy(SEM) was observed using a Quanta
mance and adsorption capacity of the polymers. The selection of the 200FEG (FEI, U. S.) which was employed to examine the size and
best functional monomer is a key step in the preparation of MIPs. morphology of the nanoparticles. Fourier transform infrared spec-
The usual methods for screening functional monomers including tra (FT-IR) were perform on an Avatar 360 (Nicolet, U. S.) instrument
UV spectroscopy [22], nuclear magnetic resonance [23], fluores- to verify whether the molecularly imprinted film was coated onto
cence spectroscopy [24] and computer simulation [25] and so on, the surface of vinyl-modified magnetic nanoparticles. The crystal
only can judge the strength of the interaction of the template and structure of the nanoparticles was characterized by a JCXA 733
X-ray diffractometer (Shimadzu, Japan). Thermal Gravity Analysis

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Fig. 2. The synthesis route of dummy magnetic molecular imprinted polymers (DMMIPs).

(TGA) was obtained from room temperature to 800 ◦ with a heating Chemical Reagent Co. (Shanghai, China). Cyanidin-3-O-rutinoside
rate of 10 ◦ C min−1 under a nitrogen atmosphere by a Netzsch STA (97%) was purchased from Seebio Chemical Reagent Co. (Shanghai,
449C (Germany) thermogravimetric analyzer. The magnetic prop- China).
erties were made using a LakeShore 7404 (USA) Vibrating sample
magnetometer (VSM). Nitrogen sorption isotherms were obtained
at −196 ◦ C on a 3H-2000PS1 Gas Sorption and Porosimetry system.
Samples were normally prepared for measurement after degassing
at 150 ◦ C under vacuum until a final pressure of 1 × 10-3 Torr was 2.3. Synthesis of vinyl-functionalized magnetic nanoparticles
reached. High-performance liquid chromatography (HPLC) analysis
was carried out using an Agilent 1200 series liquid chromatogra- An aqueous suspension of superparamagnetic magnetite
phy (U. S.), equipped with an Agilent G1315B UV–vis Detector and a nanoparticles was synthesized by a hydrothermal method. Briefly,
TC-C18 column (4.6 mm i.d. × 250 mm, Agilent Technologies, USA). 13.5 g of FeCl3 ·6H2 O and 36 g of NaAc were dissolved in 500 mL of
ethylene glycol, then the solution was transferred into an autoclave.
2.2. Chemicals and materials The reaction maintained 6 h at 200 ◦ C. After reaction finished, we
obtained black product by a magnet, used ethyl alcohol and water
All commercial reagents were analytical grade and used to wash product several times to remove unreacted reagent, dried
directly. Double distilled water was used in this study. Iron(III) under vacuum at 60 ◦ C for 12 h. The Fe3 O4 @SiO2 magnetic nanopar-
chloride hexahydrate (FeCl3 ·6H2 O, 99%) and methacrylic acid ticles were prepared according to a sol-gel method. 0.4 g of Fe3 O4
(MAA, 99.5%) were purchased from Tianli Chemical Reagent Co. were first dissolved in 200 mL of the mixture of isopropanol and
(Tianjing, China). Tetraethyl orthosilicate (TEOS, 99.5%), tetrahy- water (4:1, v/v). Followed by ultrasonic treatment for 15 min. Sub-
drofuran (THF, 99.5%), acetonitrile (MeCN, %) and acetone (AC, sequently, 6 mL ammonium hydroxide and 3 mL TEOS were added
99%) were purchased from Kemiou Chemical Reagent Co. (Tian- drop by drop, and the mixture was mechanically stirred at room
jing, China). Rutin (Ru, 98%), naringin (Nar, 95%), qurecetin (Qu, temperature for 12 h. After the resultant particles were isolated by
97%), 2-propenoic acid, 2-methyl-, 3-(trimethoxysilyl)propyl ester a magnet, washed three times with ethyl alcohol and water respec-
(MPS, 97%), methyl-2-propen-1-ol (MAOH, 99%), 4-vinylpyridine tively, dried under vacuum at 60 ◦ C for 12 h. Then used MPS to
(4-VP, 96%), 4- vinylbenzoic acid (4-VBA, 97%) and ethy- introduce vinyl groups on the surface of Fe3 O4 @SiO2 . Took 0.2 g
lene glycol dimethacrylate (EGDMA, 98%) were purchased from of Fe3 O4 @SiO2 into a three-neck flask which contained 200 mL
Aladdin (Shanghai, China). Acrylamide (AM, 99.9%) was pur- toluene. The mixture with ultrasonic treatment maintained 15 min
chased from Amresco Chemical Reagent Co. (Shanghai, China). to make nanoparticles disperse in toluene evenly. 4 mL MPS were
2,2-Azoisobutyronitrile (AIBN, 98%) was purchased from Xiya added dropwise into flask under successive mechanical stirring.
Chemical Reagent Co. (Shandong, China). Acrylic acid (AA, 99%) The reaction continued 12 h at room temperature. After reaction,
was purchased from Basf Chemical Reagent Co. (Tianjing, China). the mixture was collected by an external magnetic field. The pro-
Methacrylic acid (MAA, 98%) was purchased from Sinopharm cess of wash and dry were consistent with the above.

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2.4. Non-imprinted polymers combinatorial libraries at a concentration of 40 mg L−1 , respectively, at room tempera-
ture for 2 h. The samples were separated by external magnetic
Non-imprinted polymers combinatorial libraries included 24 field. The equilibrium concentrations of anthocyanins, quercetin
different polymers prepared by bulk polymerization which com- and naringin in the solution were measured at 520 nm, 375 nm
binations were reported in Table S1. In a glass vial, 0.6 mmol and 282 nm using a two-beam UV–vis spectrophotometer. The con-
functional monomer was dissolved in the solvent, and then added centration of the carbohydrates was measured by phenol-sulphate
3 mmol of cross-linker EGDMA and 0.1 mmol of initiator AIBN into colorimetry at 490 nm. The imprinting factor (IF) [28] was calcu-
solution. Subsequently deoxygenated via nitrogen for 10 min. The lated according to Eq. (2).
polymerization was at 60 ◦ C for 24 h. Used ethyl alcohol to wash
product three times to remove unreacted reagent and dried under IF = QMIPs /QNIPs (2)
vacuum at 60 ◦ C for 12 h. The capacity of non-imprinted polymers Where IF was the imprinting factor of molecularly imprinted, QMIPs
was evaluated by the adsorption of rutin. was the adsorption amount of molecularly imprinted polymers
(mg g−1 ), QNIPs was the adsorption amount of non-molecularly
2.5. Preparation of DMMIPs and DMNIPs imprinted polymers (mg g−1 ).
The selectivity coefficient (SC) [29]were calculated by the fol-
In this work, we used the surface molecular imprinted method lowing Eq. (3):
to synthesize DMMIPs with rutin as dummy template and 4-VP as
functional monomer. The pre-polymerization solutions were pre- SC = IF t /IF c (3)
pared by adding 0.075 mmol of dummy template in 15 mL of MeCN
in a glass vial, with a molar ratio template: functional monomer Where IFt and IFc were the imprinting factor for template and its
1:8. Pre-polymerization continued 6 h for sufficient self-assembly structure analogues.
between rutin and 4-VP. And then 40 mg of vinyl-functionalized Finally, the reusability and stability of DMMIPs were investi-
magnetic nanoparticles were added into pre-polymerization solu- gated by measuring the adsorption capacity for target molecule
tions. After 2 h, put 3 mmol of EGDMA and 0.1 mmol of AIBN in the adsorption-desorption cycles. Took 5 mg DMMIPs to 5 mL
into mixture. The mixture was purged with nitrogen for 10 min. 50 mg L−1 of C3R aqueous solution, placed on a thermostatic shaker
Then the polymerization process was performed at 60 ◦ C for 24 h, for 2 h. Under the action of an applied magnetic field to solid-liquid
imprinted polymers were collected through magnet separation. To separation, the cyanidin-3-O-rutinoside concentration in super-
remove the template, the obtained product was washed repeatedly natant was measured by UV spectrophotometry. The collected
with the mixture of methanol and acetic acid (9:1, v/v) until no rutin DMMIPs were eluted with the mixture of methanol / acetic acid
detected by UV–vis spectrophotometer. For comparison, the mag- (9:1, v/v) until C3R were not detected, and then the sample was
netic non-imprinted polymers (DMNIPs) were prepared with the vacuum dried at 40 ◦ C. Repeated the above steps 10 times.
same condition without adding dummy template.
2.7. Analytical applications in purification of crude anthocyanin
2.6. Adsorption experiments
200 mg MIPs were added to a 100 mL centrifuge tube followed
The experiment which studied the property of DMMIPs included by the addition of 20 mL of 100 mg L−1 40% crude anthocyanin prod-
four parts, adsorption isotherm, adsorption kinetic, specificity and uct from the lonicera caerulea. After adsorption for 2 h, the MIPs
reusability. The first part, the measurement of adsorption isotherm, with magnetic separation were washed three times with 5 mL of
was that added 5 mg of absorbent (i.e. DMMIPs and DMNIPs, respec- double distilled water and then were eluted with 1 mL of 1% acetic
tively) into solutions of C3R with different concentrations ranging acid/ methanol for 30 min. After magnetic separation, the eluted
from 5 mg L−1 to 50 mg L−1 (The solvent was 0.1 mol L−1 of citrate solution was filtered through a 0.22 microfiltration membrane for
buffer solution, pH = 4, temperature was 35 ◦ ), respectively. When HPLC-UV detection. HPLC conditions and the preparation methods
reached the adsorption equilibrium, the concentration of C3R in for crude anthocyanins were described in detail in the Supporting
the supernatant solution was measured by a UV–vis Spectropho- information.
tometer at 520 nm detection wavelength. The adsorption quantum
capacity [27] was calculated according to Eq. (1), thus we can fab- 3. Results and discussion
ricate adsorption isotherm curves.
3.1. Characterization of DMMIPs
Qe = (C0 − Ce )V/m (1)
Where Qe (mg g−1 ) is the equilibrium adsorbed amount of The morphology of Fe3 O4 , Fe3 O4 @SiO2 , DMMIPs, DMNIPs were
cyanidin-3-O-rutinoside, C0 (mg L−1 ) and Ce (mg L−1 ) are the initial characterized by SEM. It can be seen from the Fig. 3A that the mean
and equilibrium concentration of cyanidin-3-O-rutinoside, respec- diameter of the superparamagnetic Fe3 O4 nanoparticles prepared
tively. V (L) is the solution volume, m (g) is the weight of absorbent. by the hydrothermal method was about 170 nm and the particle
Secondly, adsorption kinetic, 5 mg of absorbent and 5 mL size distribution was uniform. The particle size of the microspheres
50 mg L−1 C3R solution (The solvent was 0.1 mol L−1 of citrate buffer was increased to about 400 nm (Fe3 O4 @SiO2 ), which indicated that
solution, pH = 4, temperature was 35 ◦ ) were mixed together. The hydrolytic TEOS coated on the surface of the Fe3 O4 particles suc-
content of C3R in supernatant solution was detected at differ- cessful to form microspheres with smooth surface, uniform size
ent time intervals (5–180 nm). And then corresponding adsorption dispersion. Compared with Fe3 O4 @SiO2 , the average size of the
kinetic curves were fabricated. magnetic molecularly imprinted particles (DMMIPs and DMNIPs)
Thirdly, adsorption specificity studies were used to evaluated did not change significantly, but the surface was rough. Because
the specificity of synthetic adsorbents. Quercetin (Que), naringin the reaction was carried out on the surface of the support, and the
(Nar), cyanidin-3-O-rutinoside (C3G), cyanidin (Cy), delphinidin molecularly imprinted layer was porous structure.
(Dp), pelargonidin (Pg), malvidin (Mv), peonidin (Pn), petunidin FT-IR spectra of Fe3 O4 , Fe3 O4 @SiO2 , Fe3 O4 @SiO2 @C = C,
(Pt), rutinose (Ruo), lactose (Lac), sucrose (Suc), glucose (Glu), DMMIPs, DMNIPs were shown in Fig. 3B. The peak at 575 cm−1 was
rhamnose (Rha), raffinose (Raf) were selected as structural analogs. attributed to the Fe O that was the characteristic absorption peak
5 mg of DMMIPs and DMNIPs were placed in a solution of analogs of Fe3 O4 , which proved the successful synthesis of Fe3 O4 magnetic

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Fig. 3. (A)SEM images, (B) FT-IR spectra, (C) TGA curves, (D) XRD patterns, (E) magnetization curves at 300 K.

particles (curve of Fe3 O4 ). In comparison with the curve of Fe3 O4 , of Fe3 O4 was 2.34% (does not contain water) when the tempera-
the new characteristic peaks at 951 cm−1 , 799 cm−1 and 1091 cm−1 ture reached 900 ◦ because the part of Fe3 O4 microspheres were
(curve of Fe3 O4 @SiO2 ) resulted from the stretching of Si OH, Si O, pyrolyzed into Fe2 O3 . From the curve of Fe3 O4 @SiO2 , the mass loss
and Si O Si, respectively, which proved that SiO2 layer was of Fe3 O4 @SiO2 was 5.59% in the temperature range of 200–900 ◦ ,
synthesized on the surface of Fe3 O4 . The typical peak at 1637 cm-1 due to the loss of silanol groups on the surface of Fe3 O4 @ SiO2 . The
for C C groups showed that MPS was grafted on the surface of high melting point of SiO2 made it keep steady in high temperature.
Fe3 O4 @ SiO2 (curve of Fe3 O4 @SiO2 @C C). The spectra of DMMIPs Therefore, the mass loss of SiO2 during the whole heating process
and DMNIPs (curve DMMIPs and DMNIPs) all showed typical was very low. In contrast to curve of Fe3 O4 @SiO2 , the thermal loss
stretching vibration of C O (1736 cm−1 ), CH3 (2987 cm−1 ), CH2 of Fe3 O4 @SiO2 @C C was 6.16%, which was 0.56% more than that
(2956 cm−1 ) and aromatic ring (1599 cm−1 ) which demonstrated of Fe3 O4 @ SiO2 . The reason was that Fe3 O4 @SiO2 was coated with
the cross-linker EGDMA and the functional monomer 4-VP were MPS. The weight of DMMIPs decreased fast from 250 ◦ to 400 ◦ , and
grafted successfully. Each of the above characteristic absorption the temperature was higher than 400 ◦ , the weight loss of DMMIPs
peaks indicated the successful synthesis of each reaction in the was not obvious, which originated from the imprinted polymers
preparation of DMMIPs and DMNIPs. on the surface of magnetic support. It improved that the layer of
The thermogravimetric analysis was used to determine the molecular polymers was synthesized successfully on the surface of
composition of magnetic microspheres coated with the polymers. the alkylated magnetic nanocomposite that occupied 28.89% of the
Fig. 3C gave the TGA curve of four kinds of particles, Fe3 O4 , magnetic molecularly imprinted particles approximately.
Fe3 O4 @SiO2 @C C and DMMIPs. The weight loss at the first 100 ◦ Fig. 3D showed the characterization of the Fe3 O4 , Fe3 O4 @SiO2 ,
was mainly due to the desorption of organic solvent and the evap- and DMMIPs crystal forms used X-ray diffraction patterns. The six
oration of water in the polymers structure. We mainly analyzed obvious diffraction peaks of 30.14◦ , 35.51◦ , 43.08◦ , 53.57◦ , 56.96◦
the data between 200 ◦ and 900 ◦ . In curve of Fe3 O4 , the weight loss and 62.62◦ shown in curve (a) correspond to the indices of crystal

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Table 1 slowly in the polymerization to provide a material with smaller

Data of nitrogen adsorption of nanoparticles.
pore size and larger surface area [32]. Large surface area of the poly-
Polymers SBET (m2 g−1 ) Vp (cm3 g−1 ) Dmean (nm) mers meant the more recognition sites and the higher adsorption
Fe3 O4 40.13 0.19 10.51 of the target. Therefore, we choosed acetonitrile as porogen.
Fe3 O4 @SiO2 15.88 0.047 6.28 Comparing the same solvents in the Fig. S7C, it can be obviously
DMMIPs 65.62 0.12 5.19 observed that the adsorption capacity of polymers with 4-VP and
DMNIPs 30.42 0.084 4.27 MA as functional monomers were significantly higher. 4-VP inter-
acted with the template molecule strongly that was mainly due
to its structural characteristics of molecules. 4-VP was a nitrogen-
face of (220), (311), (400), (422), (511), and (440), respectively, was containing heterocyclic compound with strong basicity which can
in agreement with standard Fe3 O4 XRD data [30]. After coated with be used as a hydrogen bond acceptor and also formed ␲-␲* conju-
the polymer layer, the characteristic peaks position did not change gation with the benzene ring. These features allowed it to interact
which proved Fe3 O4 crystal structure unchanged. with template molecules to form a stable host-guest complex. In
The magnetic properties of nanoparticles were studied using addition, the strength of the hydrogen bond was decided to the
VSM. The hysteresis loop of Fe3 O4 and Fe3 O4 @SiO2 , DMMIPs at electronegativity of the acceptor and donor. The higher the elec-
300 K were shown in Fig. 3E. The saturation magnetization values tron density of the acceptor, the stronger the hydrogen bond. It also
of the prepared Fe3 O4 , Fe3 O4 @SiO2 , DMMIPs were 67.34, 28.84 and was affected by the steric hindrance of the acceptor, the smaller the
20.01 emu g−1 , respectively. It can be seen that an increase in the steric hindrance, the stronger the hydrogen bonding. Therefore, MA
number of non-magnetic surface coatings caused a decrease in the formed stronger interaction with template molecules than other
magnetization value of nanoparticles. Three magnetic hysteresis functional monomers.
loops are all symmetrical to the origin, indicating the existence of The first five groups with good effects were selected from the
superparamagnetic behavior [31]. 24 combinations and the corresponding MIPs were synthesized to
Data of nitrogen adsorption of nanoparticles were shown in verify the accuracy of the NIP library screening method. The experi-
Table 1. After coating silica on the surface of Fe3 O4 , the spe- mental results were shown in Fig. S4. It verified that NIPs have some
cific surface area, pore volume, and average pore diameter of the interaction sites on the template molecule, and the correspond-
nanoparticles were significantly reduced because of increased the ing MIPs showed good imprinting effect on the template molecule,
particle size and the silica layer was relatively dense. However, the which can be used to screen functional monomers and porogen
specific surface area, pore volume and average pore diameter of the with NIP library. The imprinting factor was the index to evaluate
DMMIPs were increased significantly, indicating that the imprinted the molecular imprinting effect. The polymers with combination
membrane formed on the surface of the carrier with porous struc- of 4-VP-ACE had the best imprinting effect. The 4-VP molecular
ture, which mainly because the imprinting holes formed after structure is a rigid plane, and the recognition site formed is more
template molecules eluted, which also ensured a large adsorption stable. However, the MA conformation is easily changed so that
of DMMIPs to the target molecule. the molecularly imprinted holes cannot well match with the tar-
get compound and the specificity was reduced. Therefore, 4-VP and
3.2. Non-imprinted polymers combinatorial libraries acetonitrile were chosen as the functional monomer and porogen
Fig. S2A showed the adsorption capacity of 24 different poly-
mers in the NIP screening library. Comparison of the congener
functional monomer, the adsorption capacity of polymers with 3.3. The optimization of extraction conditions
acetonitrile as the porogenic agent were significantly higher than
others (Fig. S2B). In the experiment, we found an interesting phe- 3.3.1. The effect of pH
nomenon that the morphology of polymers formed in different The extent of C3R extraction depended on its interaction with
solvent were differently. The polymers’ morphological properties the pyridine groups of DMMIPs. Anthocyanins existed in different
(porosity and surface area) were determined by the type of solvent forms under different pH. To evaluate the effect of pH, working
during the polymerization. That was also the factor affected the solutions with different pH in the range of 2.0–7.0 were studied.
adsorption amount. The non-molecularly imprinted polymers syn- As shown in Fig. S5A, the highest adsorption capacity of C3R by
thesized in acetonitrile were dispersed in the solution uniformly DMMIPs was at pH 4.0 and lesser adsorption was observed at lower
as a white solution without precipitation. The NIPs synthesized and higher pH values, which may be due to the fact that when
with acetone were dispersed partially in the solution. Synthesized the pH of the extract solvent was close to pKa (The pKa of C3R is
with methanol, polymers’ morphology liked residue which were of 4.7), the anthocyanin was the most acidic and had the strongest
the worst dispersion in the solution and all precipitated. Synthe- electrostatic interaction with DMMIPs. Therefore, the pH 4.0 was
sized in tetrahydrofuran, NIPs were translucent viscous colloidal, selected as optimized pH.
good affinity for solvents. The difference of microstructure between
these polymers were observed by the scanning electron microscopy
for further study. In Fig. S3, the polymers formed in acetonitrile 3.3.2. The effect of temperature
was monodisperse sphericity which size was smallest. But the DMMIPs recognized anthocyanins through electrostatic inter-
polymers were formed in other solvents with varying degrees of actions, and temperature affected electrostatic forces. Therefore,
cross-linking. Because the force between the solvent molecules and the effects of different temperatures (5 ◦ –35 ◦ ) on the adsorption
the polymer chains in a good solvent was stronger than the force amount were investigated. It can be seen from the Fig. S5B that at
between the polymer chains. In poor solvents, the force between 35 ◦ , the adsorption of C3R by DMMIPs was highest, because the
the polymer chains increased, formed a larger aggregation. The molecular thermal motion was slower at low temperatures, slow-
phase separation of porogen and growing polymer occurred dur- ing down the mass transfer rate. When the temperature increased,
ing the polymerization thereby forming pores. The separation of the molecular thermal motion was intensified making the distance
porogen and low solubility phase happened in the early period between the molecules become large, and the electrostatic force
of polymerization which tended to form larger pores and lower was weakened, so the adsorption amount decreased. Therefore, the
surface area. In contrast, porogen and higher solubility separated 35 ◦ was selected as optimized temperature.

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Fig. 4. (A) Curve of adsorption kinetics, (B) the pseudo-first-order model, (C) the pseudo-second-order model, (D) segmented model.

3.3.3. The effect of solvent VP on the support surface in the absence of template. The physical
Solvent was also an important factor affecting DMMIPs bind- structure, electrostatic interaction and ␲–␲* interaction of DMMIPs
ing capacity. In order to study the effect of this factor, water and imprinting cavities matched the target molecules, which identi-
ethanol were mixed in different proportions (1:4, 2:3, 3:2, 4:1) fied C3R through physical and chemical forces and adsorbed C3R
as binding solvents, the results were shown in Fig. S5C. It could in the imprinted pores. In order to further study the adsorption
be observed that the binding performance of DMMIPs was best in mechanism of DMMIPs, the first-order adsorption kinetics model
pure water solution. Because water promoted the ionization of C3R (Fig. 4B) and the second-order adsorption kinetics model (Fig. 4C)
which enhanced the electrostatic force between C3R and DMMIPs. were used. The pseudo-first-order rate expression and the pseudo-
In addition, the hydrophobic group will spontaneously aggregate second-order rate expression were calculated according to Eqs. (4)
in water, shortening the distance between the pyridine functional and (5), respectively [34].
group of DMMIPs and the benzene ring of C3R, increasing the ␲–␲*  
log Q eq − Qt = logQ1cal − k1 t/2.303 (4)
interaction. Therefore, water was selected as optimized solvent.
t/Qt = 1/(k2 Q2cal ) + t/Q2cal (5)

3.4. Binding properties of DMMIPs Where t was the adsorption time (min), Qeq was the equilibrium
adsorption capacity (mg g−1 ), Qt was the adsorption capacity at
3.4.1. Adsorption kinetics different time intervals (mg g−1 ), Qcal was the theoretical adsorp-
Fig. 4A showed the adsorption kinetic curves of 50 mg L−1 C3R tion capacity, k1 was the first-order rate constant (min−1 ), k2 was
solution onto DMMIPs and DMNIPs. At 40 min, DMNIPs almost the rate constant of second-order adsorption (g mg−1 min−1 ).
reached saturation, and the adsorption capacity was 7.28 mg g−1 The fitting effect of two models is not very ideal according to
at this time. The maximum saturated adsorption capacity was coefficient of determination, R2 . We divided the data into two
7.58 mg g−1 at 180 min. Polypyridine compounds were formed on sections for analysis (Fig. 4D) and found that the adsorption was
the surface of DMNIPs, so DMNIPs can adsorb C3R. At 60 min, consistent with the pseudo- first-order model up to 40 min with
the adsorption capacity of molecularly imprinted polymers came a fitting curve of Y = −0.018 X + 1.30 and R2 = 0.98. After 40 min,
near to saturation and the adsorption rate was fast compared with the adsorption process was in accordance with the pseudo-second-
the molecularly imprinted polymers that were synthesized with order model, and the fitting curve was Y = 0.059 X + 0.28, R2 = 0.99.
bulk polymerization [33]. At this time, the adsorption amount of According to the slope and intercept of the linear plot, the adsorp-
DMMIPs was 15.59 mg g−1 , and the maximum saturated adsorption tion rate constants k1 (0.041 min−1 ) and k2 (0.012 g mg−1 min−1 )
capacity was 16.57 mg g−1 at 180 min. The experimental results of two kinds of adsorption were calculated respectively. These
showed that the adsorption amount of C3R by DMMIPs was 2.18 results suggested that the pseudo first order kinetic model pre-
times higher than that of DMNIPs. In the preparation of molecular dominated in the first 40 min and the pseudo second order kinetic
imprinting, DMMIPs added rutin as the dummy template, whereas model dominated after 40 min in the adsorption of target molecules
DMNIPs were polypyridines formed by the polymerization of 4- by prepared DMMIPs. The whole process was that the adsorbate

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Fig. 5. (A) Curve of adsorption isotherms, (B) fitted Langmuir equation of DMMIPs, (C) fitted Freundlich equation of DMMIPs, (D) the Scatchard plot for the DMMIPs and

diffused and accumulated to the surface of the adsorbent firstly Fig. 5B and C were the fit linear of the Langmuir model and Fre-
through the physical interaction, and then the adsorbate and the undlich model. Compared with the R2 of two fitting models, the
functional monomer in the cavity further occurred the electron and Langmuir model was suitable for the adsorption process for C3R.
the common electron transfer and was adsorbed to the recognition The Langmuir model reflected the monolayer adsorption of C3R on
hole. the surface of imprinting material, and all the adsorption sites had
the same adsorption energy and enthalpy change.
3.4.2. Adsorption isotherm The Scatchard equation was adopted to evaluate the binding
Fig. 5A showed the adsorption isotherms of C3R solutions on ability of the DMMIPs and DMNIPs. The Scatchard equation [35]
DMMIPs and DMNIPs. In the range of 0–40 mg L−1 C3R solutions, the was expressed as follows Eqs. (8) and (9), respectively.
adsorption amount of DMMIPs increased with the increase of C3R
Qeq /Ceq = Qmax /Kd − Qeq /Kd (8)
concentration. When the C3R concentration was over 40 mg L−1 ,
the increasing rate of DMMIPs adsorption tended to be balanced Ka = 1/Kd (9)
gradually. Adsorption was saturated because when the C3R con-
centration reached a certain value, all the specific imprinting sites where Qeq was the equilibrium adsorption capacity (mg g−1 ), Qmax
on the surface of the DMMIPs were occupied by the C3R molecules. was the apparent maximum binding amount (mg g−1 ), Ceq was the
Adsorption isotherms made clear how adsorbed molecules acted cyanidin-3-O-rutinoside concentration at equilibrium (mg mL−1 ),
on the sorbent surface. Therefore, it was very important to predict Kd was the equilibrium dissociation constant (g L−1 ), and Ka was
the adsorption mode of the imprinted material surface by using the equilibrium binding constant (L g −1 ).
the theoretical formula to simulate adsorption isotherms data. The As shown in Fig. 5D, the Scatchard plot for the DMMIPs were
Langmuir model and the Freundlich model were used. The empir- one straight line, which indicated that there was only one type of
ical linear equations for the Langmuir model and the Freundlich binding site in DMMIPs. The fitting result can be consistent with the
model were shown in Eqs. (6) and (7), respectively [34]. Langmuir plot results. According to the linear regression equation,
the Ka of DMMIPs and DMNIPs were calculated to be 0.057 L g −1
1/Qeq = 1/Qm + 1/(bQm Ceq ) (6) and 0.037 L g −1 , respectively. The Ka of DMMIPs was higher than
that of DMNIPs, which confirm the high affinity of the DMMIPs to
logQeq = logkf + (1/n)logCeq (7)
Where Qeq was the equilibrium adsorption capacity (mg Qm g−1 ),
was the saturated adsorption capacity of polymers (mg g−1 ), Ceq 3.4.3. Selectivity of DMMIPs
was cyanidin-3-O-rutinoside concentration (mg L−1 ), b was the In order to prove the high affinity and selectivity of DMMIPs for
Langmuir adsorption equilibriumconstant (L mg−1 ), kf was the Fre- cyanidin-3-O-rutinoside, flavonoids (Que and Nar), anthocyanins
undlich adsorption equilibrium constant (L mg−1 ), n was a constant. (C3G, Cy, Dp, Pg, Mv, Pn, Pt), carbohydrates (Ruo, Lac, Suc, Glu,

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Fig. 6. (A) Selective recognition of DMMIPs and MNIPs for cyanidin-3-O-rutinoside. (B) Reusability of the DMMIPs for ten adsorption and desorption times.

Fig. 7. (A) The HPLC chromatograms of the extract of Lonicera caerulea. The standard of cyanidin-3-O-rutinoside (a), the chromatogram of initial sample of the extract
of Lonicera caerulea (b), the chromatogram of the extract after being adsorbed by DMMIPs (c), the chromatogram of obtained cyanidin-3-O-rutinoside after elution from
DMMIPs (d). (B) Comparison of C3R selectivity and saturation uptake amount for DMMIPs with that of other benchmark materials.

Rha, Raf) were chosen as competitive molecules in this study. The tive elution process was particularly important in the application
selectivity of DMMIPs was evaluated by selective adsorption exper- of molecularly imprinted polymers. The results also proved that
iments and the results were displayed in Fig. 6A and Table S2. The DMMIPs still have good adsorption capacity for anthocyanins after
chemical structure of analogue was shown in Fig. S6. The adsorption repeatedly used.
amount of C3R (15.69 mg g−1 ) was significantly higher than other
structure analogues, which demonstrated that DMMIPs can recog- 3.5. Analytical applications in purification of crude anthocyanin
nize C3R specifically. The reason why the adsorption quantity of
carbohydrates by DMMIPs was very low was that carbohydrates Anthocyanins were used in many areas due to their special phys-
were neutral and can only be combined with DMMIPs through iological activity, especially antioxidation activities. However, the
hydrogen bonds, but the interaction was diminished in water. The complexity of natural substrates and the poor stability of antho-
remaining analogues were weakly acidic and had benzene rings, cyanins limited the isolation and purification of anthocyanins.
which can bind with DMMIPs in water, but still showed low adsorp- Therefore, in this work we developed a novel adsorption mate-
tion quantity. The size, shape, distribution of the characteristic rial that can rapidly and efficiently extract anthocyanins from the
functional groups of molecules did not match with the molecu- extract of the Lonicera caerulea. The chromatograms of the extract
larly imprinted three-dimensional pores that resulted in the weak before and after being adsorbed by DMMIPs, and the eluent solu-
adsorption ability of DMMIPs for them. tion from DMMIPs were exhibited in Fig. 7A. Compared with the
chromatograms of the extract before (curve b) and after (curve c)
3.4.4. Reusability being adsorbed by DMMIPs, we can see that the adsorption of C3R in
The reusability of molecularly imprinted polymers was an DMMIPs is significantly higher than other components in the crude
important property for application. Reusability of the DMMIPs and extract. We achieved the expected goal of separating and purify-
DMNIPs were shown in Fig. 6B. The adsorption capacity of DMMIPs ing anthocyanins. The purity of C3R in the eluate was increased to
only decreased by 15.3% after ten adsorption-elution cycles. The 89% (The content of anthocyanin in crude extract was only 40%).
experiment results showed that the three-dimensional pores of the It is indicated that the prepared DMMIPs had excellent selectiv-
imprinted polymers were very stable which were not destroyed ity for C3R and can be applied to separation and purification of
during the process of elution. The slight decrease of adsorption anthocyanins in real samples.
capacity was because that the three-dimensional cavities on the Three benchmark adsorbents (such as octa decylsilyl silicion
surface of imprinted polymers were blocked partly after repeated (ODS), polyamide resin (PAM), column chromatography silica gel
the template molecule binding and elution. Therefore, the effec- (SG)) were selected to compare with the prepared DMMIPs. The

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