Molecularly Imprinted Photonic Hydrogels for Visual Detection of Methylanthranilate in Wine

CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 47, Issue 9, September 2019 Online English edition of the Chinese language journal

Cite this article as: Chinese J. Anal. Chem., 2019, 47(9): 1330–1336

RESEARCH PAPER

Molecularly Imprinted Photonic Hydrogels for Visual Detection of Methylanthranilate in Wine WU Wei-Zhen1, HUANG Meng-Xia1, HUANG Qing-Da1, LYU Cai-Hua1, LAI Jia-Ping1,*, SUN Hui2,* 1 2

School of Chemistry & Environment, South China Normal University, Guangzhou 510006, China College of Environmental Science & Engineering, Guangzhou University, Guangzhou 510006, China

Abstract:

Methylanthranilate (MA) is a kind of spice that is illegally added in the wine in China. The determination of MA in wine

is an important part of quality control for wine production. Therefore, it is of great significance to develop a rapid method for detection of prohibited spice in wine. In this work, an inverse opal structural molecularly imprinted photonic hydrogels (MIPHs) membrane was fabricated by merging the advantages of photonic crystal and molecular imprinting techniques. The MIPHs membrane was prepared by using MA as template molecule, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as crosslinker, followed by a thermal polymerization at 60 ºC. Then the inverse opal structural MIPHs membrane was obtained after removing photonic crystal template and imprinted molecules. The MIPHs membrane exhibited a good selectivity property to MA, and it responded to MA within 6 min. The Bragg diffraction peak shift increased with the increase of MA concentration. A linear relationship was found between the ∆λ and the concentration of MA in the range of 0.1‒10.0 mM. And the limit of detection in the present work is about 31 μM. Furthermore, a color change of the MIPHs membrane could be observed by naked eyes. Therefore, the smart MIPHs membrane showed great potential in rapid and visual detection of MA in wine. Key Words:

Molecular imprinting; Photonic crystal; Visual detection; Methylanthranilate

1 Introduction Wine is more and more popular drink with the improvement of living standards. However, in recent years, the wine quality problems have attracted more and more attention due to the illegal additives. To reduce the cost and enhance the taste of wine, many illegal additives such as sweeteners, grape flavors and other contraband products are illegally added in the production of wine. However, the mandatory national standard GB15037-2006 “wine” has specified clearly that wine products cannot be added into any flavor. And grape flavor is mainly prepared by spices and additives, of which spices are mainly anthranilic acid carboxylates, such as methylanthranilate (MA)[1]. Therefore, the rapid and accurate detection of spice molecules in wine is an important item in wine quality control for wine production. At present, there are few reports about the detection

methods of spices in wine. Most of the procedures are judged by sensory evaluation method, which are not accuracy more or less[2]. And the chromatographic procedure is the standard detection method for detection of spices in wine at present[3]. Although the chromatographic detection is accurate, reliable and widely applicable, there are still many shortcomings, such as the need for more complex sample pretreatment process, more expensive instruments and cumbersome equipment, which are not suitable for rapid detection on-site, resulting in some limitations. Therefore, it is of great significance to develop a convenient, rapid, sensitive and specific method for on-site detection of spices in wine. Photonic crystals (PCs) are those materials with different dielectric constants (refractive indices) arranged in space to form ordered structures. Therefore, they are also called photonic bandgap materials, which have unique optical properties and are widely used in optical sensing[4–10].

________________________ Received 18 February 2019; accepted 29 May 2019 *Corresponding author. E-mail: [email protected]; [email protected] This work was supported by the National Natural Science Foundation of China (Nos. 21677053, 21876033). Copyright © 2019, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(19)61188-6

WU Wei-Zhen et al. / Chinese Journal of Analytical Chemistry, 2019, 47(9): 1330–1336

Molecularly imprinted polymers (MIPs) are a kind of materials with specific recognition for target molecules[11–13]. Here molecularly imprinted technology is introduced into photonic crystal sensing materials to construct a molecularly imprinted photonic hydrogels (MIPHs) membrane that possesses both photonic crystal characteristics and specific recognition ability of MIPs. In the process of molecular recognition, the hydrogel sensing membrane will cause the position shift of photonic band gap. In other words, the molecular recognition process can be directly performed by optical signal of photonic crystal without any signal conversion elements. Furthermore, the color change can be directly observed by naked eyes if the Bragg diffraction shift is large enough to achieve the purpose of visual detection. Therefore, there are some advantages using these sensing materials in on-site analysis and detection[14,15]. In recent years, Li et al[16,17] successfully applied this material to visually detect dopamine, oleic acid and so on. Subsequently, these materials were also successfully applied to fast identify and detect the environmental endocrine disruptors[18,19], amino acids[20,21], pesticides[22,23], antibiotics[24–28], explosives[29], metal ions[30,31], etc. It has proved the feasibility of this sensing material in the analysis and detection. However, there is no report about using molecular imprinting photonic crystal hydrogel sensing membrane for detection of spice molecules in wine. In this work, an intelligent molecularly imprinted photonic crystal hydrogel sensing membrane was developed using methylanthranilate as imprinted molecule for the rapid and visual detection of methylanthranilate in wine.

2 Experimental 2.1

and hydrogen peroxide (30%) were purchased from Guangzhou Chemical Reagent Co. Polymethyl methacrylates (PMMA) was purchased from local manufacturer. All organic solvents were of analytical purity. 2.2 2.2.1

Experimental methods Preparation of SiO2 microspheres and photonic crystal templates

The SiO2 microspheres were synthesized using the modified Stöber method. Typically, 16 mL of ethanol, 25 mL of deionized water and 9 mL of ammonium hydroxide were added into a 250-mL conical flask, successively. Then the mixture was stirred at 1100 rpm under ambient. After that, the homogeneous mixture of 3 mL of TEOS and 45 mL of ethanol was added into the flask and the resulting mixture was stirred at 400 rpm for 2 h. After centrifuging at 3500 rpm for 10 min, the precipitated microspheres were washed 3 to 4 times with ethanol and ultrasoniced dispersion in a certain amount of ethanol for the further use. The photonic crystal templates were prepared by means of the vertical deposition self-assembly method using the suspension SiO2 microspheres. Generally, 4 mL of the suspension SiO2 microspheres with an appropriate concentration was placed into 7-mL vial. The cleaned glass slides, which were activated in anthropophagous alkaloids solution, were put into each vial vertically for photonic crystal growth at 30 ºC and humidity of 45%. After the ethanol was evaporated completely, the photonic crystal templates were obtained on the glass slides, which arranged orderly and appeared an opal structure.

Instruments and reagents 2.2.2 Preparation of methylanthranilate MIPHs membrane

The equipments used here mainly included desktop high speed centrifuge (H1850, Xiangyi Instrument, Hunan), digital water bath thermostat (SHA-BA, Aohua Instrument, Changzhou), vortex shaker (XW-80A, Jingke Instrument, Shanghai), numerical control ultrasonic cleaner (KH-300DE, Hechuang Ultrasonic Instrument, Kunshan), pH meter (PHS-3C, Precision Science Instrument, Shanghai), scanning electron microscope (JSM-7001F, Electronics Corporation, Japan), fiber optic spectrometer (USB2500+, Ocean Optics, US), constant temperature and humidity incubator (WS-01, Hengfeng Instrument, Huangshi). Methylanthranilate (MA), methyl 4-aminobenzoate and methyl 4-hydroxybenzoate were all purchased from Macklin Biochemical Co. (Shanghai, China). Anthranilic acid and methacrylic acid were purchased from Aladdin Reagent Co. (Shanghai, China). 2,2-Azobisisobutyronitrile was received from Damao Chemical Reagent Co. (Tianjin, China). Ethylene glycol dimethacrylate was obtained from Bailingwei Technology Co. (Beijing, China). Hydrofluoric acid (45%)

A mixture of MA, MAA and EGDMA were dissolved in appropriate amount of methanol for pre-polymerization in the dark to form hydrogen bonding between the template molecules (MA) and the functional monomers (MAA). Then, 5 mg of AIBN was added into above mixture as an initiator. After purging with nitrogen for 5 min, two pieces of PMMA were covered on the surface of the photonic crystal template and clamped, which was similar to a “sandwich” structure. The pre-polymerization solution was slowly injected into the gap of above "sandwich" structure and polymerized at 60 °C for 4 h. Next, the template was soaked in 1% HF solution and 4% HF solution successively to remove SiO2 microspheres and then soaked in a volumetric ratio of methanol-water-acetic acid solution to remove template molecules. Hence, an inverse opal structural MA molecularly imprinted photonic hydrogels (MA-MIPHs) membrane was obtained. The synthetic procedure of MIPHs membrane is shown in Fig.1. Finally, the non-imprinted photonic hydrogels (NIPHs) membrane was

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Fig.1

Schematic diagram of preparation of MIPHs membrane

also prepared through the same procedure but without addition of the template molecules. 2.2.3

Investigation of recognition properties of MIPHs membrane

MIPHs membrane was immersed into 20% methanol solution containing a series of concentrations of MA. After adsorbing completely, the Bragg diffraction peaks of MIPHs membrane were recorded using a USB2500+ fiber optic spectrometer. Based on the change of Bragg diffraction peaks, the recognition properties of MIPHs membrane to template molecules were investigated.

3 Results and discussion 3.1

Characterization of SiO2 microspheres and photonic crystal templates

The optical performance of photonic crystal templates largely depends on the particle size, uniformity, dispersibility of SiO2 microspheres. In this work, the SiO2 microspheres were synthesized by the modified Stöber method. As can be seen from Fig.2A, the silica microspheres had good monodispersity and uniform particle size. As shown in Fig.2B, the SiO2 microspheres arrangement was observed clearly and the photonic crystal templates prepared by vertical deposition self-assembly method were arranged neatly and appeared an opal structure with a close-packed face-centered cubic lattice. 3.2

Optimization of preparation conditions of MIPHs membrane

Fig.2

The molecular recognition principle of MIPHs membrane is based on the cavity which can selectively recognize the template molecules. In the presence of a suitable solvent, the template molecules (MA) formed a complex with the functional monomers via appropriate interactions, such as hydrogen bonding or electrostatic interaction. Then the complex forms polymerized with crosslinker by thermal or photo initiation to produce polymeric membrane. After removing the template molecules from the polymer membrane, the specific binding sites were leaved in the MIPHs membrane for recognition of target molecules. Thus, it is important to choose appropriate solvent, functional monomer and crosslinker during the preparation of MIPHs membrane. Firstly, the polarity and amount of solvent were considered because the solvent was porogen, which determined the size of the pore in membrane. An extremely high amount of solvent would affect the recognition properties of MIPHs membrane. In contrast, too low amount of solvent would reduce the number of recognition sites. Besides, the polarity of solvent also played an important role in polymerization because it influenced the hydrogen bonding between template molecules and functional monomers. Therefore, the amount and the type of solvent were also optimized in detail. As shown in Fig.3A, the recognition properties of the MIPHs membrane had the largest shift of Bragg diffraction peak (), when the dosage of solvent was 0.2 mL of methanol. The recognition of MIPHs membrane towards template molecules could be converted into readable optical signals, which depended on the reversible swelling and shrinking state of the membrane. Moreover, this property was related to crosslinker. Excessive amount of crosslinker enhanced the rigidity of the membrane and led to the difficulty of swelling and shrinking.

Scanning electron microscopy (SEM) images of SiO2 microspheres (A) and opal photonic crystal template (B)

WU Wei-Zhen et al. / Chinese Journal of Analytical Chemistry, 2019, 47(9): 1330–1336

Fig.3

Effect of the amount and type of solvent (A), molar ratio of template molecules to crosslinker (B) and functional monomer (C) on response properties of MIPHs membrane; Scanning electron microscopy (SEM) images of MIPHs membrane (D)

Conversely, too little amount of crosslinker would enhance the flexibility of MIPHs membrane. Therefore it was difficult to obtain stable three-dimensional pore structures, and resulted in losing optical signals. Based on this, the dosage of the crosslinker was investigated in detail. As shown in Fig.3B, the recognition properties of the MIPHs membrane was optimal when the molar ratio of template molecule and crosslinker was 1:1. In addition, the specific adsorption of molecular imprinted polymers was achieved by the means of hydrogen bonding or electrostatic interaction between functional monomers and template molecules, so the selection of a suitable functional monomer was critical for preparation of MIPHs membrane. In this work, several functional monomers such as MAA, AM and AA were also investigated. As shown in Fig.3C, the maximum diffraction peak shift (∆λ) was achieved when the MIPHs membrane was fabricated with 0.5 mmol MAA. Under above optimal conditions, the obtained MIPHs membrane showed highly ordered inverse opal structure (Fig.3D). 3.3

membrane. It would be swollen or shrunk and finally reach its equilibrium at an appropriate time in the process of molecular recognition of MIPHs membrane. Based on this, the response time of MIPHs membrane to MA was optimized. The MIPHs membrane was soaked in 20% methanol solution containing 10 mM MA. It was found that the diffraction peak shift () of MIPHs membrane increased with the increase of the soaked time firstly and then remained roughly constant after 6 min, which indicated the absorption equilibrium of MIPHs membrane towards MA was achieved (Fig.4). 3.4

Sensing properties of MIPHs membrane to MA

Response time of MIPHs membrane to MA

There were both three-dimensional high-ordered porous structure and rich specific recognition sites in the MIPHs

Fig.4

Response time of MIPHs membrane to MA

WU Wei-Zhen et al. / Chinese Journal of Analytical Chemistry, 2019, 47(9): 1330–1336

To study the sensing properties of MIPHs membrane to MA, MIPHs membrane was immersed into 20% methanol solution containing MA with different concentrations. After adsorbing completely, the Bragg diffraction peaks of MIPHs membrane were recorded using a fiber optic spectrometer. The results indicated that the Bragg diffraction peak appeared red-shift with the increase of MA concentration (Fig.5A). This was because the binding sites in the MIPHs membrane could recognize MA and selectively adsorb MA into the MIPHs membrane to increase the ionic strength to a certain extent. Consequently, the lattice structure of the MIPHs membrane expanded and eventually led to a red-shift of the Bragg diffraction peak. As shown in Fig.5B, a linear relationship was obtained between the ∆λ and the concentration of MA in the range of 0.1‒10 mM with a detection limit of 31 μM. And it could be observed by naked eyes via the color change of MIPHs membrane in this process (Fig.5C). The smart MIPHs membrane was successfully applied to rapid and visual detection of MA. Under the same conditions, the Bragg diffraction peak shift of NIPHs membrane was not obvious, due to the absence of specific recognition sites (Fig.5D). All the results indicated that the MIPHs membrane possessed specific recognition sites for target MA. 3.5

Selectivity of MIPHs membrane towards MA

To investigate the selectivity of MIPHs membrane towards

Fig.5

MA, the analogues with similar structure of MA such as anthranilic acid, methyl 4-aminobenzoate and methyl 4-hydroxybenzoate were investigated as comparison. As shown in Fig.6, there was markedly shift of Bragg diffraction peak between MIPHs membrane and MA (Fig.6, black square dots) due to the specific recognition sites while a slight shift was observed between MIPHs membrane and other similar structure analogues of MA under the same measurement conditions. The corresponding results clearly indicated that MIPHs membrane had high selectivity to MA. 3.6

Reusability of MIPHs membrane

The reusability of MIPHs membrane for determination of MA was further investigated. Firstly, MIPHs membrane was immersed into 20% methanol solution containing 10 mM MA. After adsorbing completely, the Bragg diffraction peak of MIPHs membrane rebound MA was recorded by an optic fiber spectrometry. Then the MIPHs membrane bound MA was washed with methanol-water-acetic acid solution and the Bragg diffraction peak of MIPHs membrane was detected again. The above operation was repeated circularly. As shown in Fig.7, the Bragg diffraction peak of MIPHs membrane was basically remain unchanged after 10 times cycle of adsorption and desorption, which exhibited good repeatability and easily achieved recycle use. Thus, MIPHs membrane suggested excellent recognition properties to MA.

Optical response of MIPHs membrane to different concentration of MA (A); Linear relationship between the diffraction peak shift (∆λ) of MIPHs membrane and the concentration of MA (B); Change of color of MIPHs membrane toward different concentrations of MA (C); Diffraction peak shift of MIPHs membrane and NIPHs membrane to different concentration of MA (D)

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which indicated that this proposal method had potential practicality in wine quality control.

4

Fig.6

Diffraction peak shift of MIPHs membrane to different concentration of MA, anthranilic acid, methyl 4-aminobenzoate and methyl 4-hydroxybenzoate

Conclusions

In this study, an inverse opal structural molecularly imprinted photonic hydrogels (MIPHs) membrane was successfully fabricated based on the combination of photonic crystal techniques and molecular imprinting techniques under the optimal preparation conditions. The MIPHs membrane not only exhibited excellent selectivity to target molecule MA, but also possessed good repeatability. The method proposed here realized the visual semi-quantitative detection of target molecules and highlighted the advantages such as rapidity, simplicity and efficient in on-site detection. Therefore, the established method has potential application in wine quality control.

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