Preparation of Molecularly Imprinted Microspheres as

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Jul 16, 2018 - molecularly imprinted microspheres (MIMS) could achieve high yields ... stabilizing SiO2 nanoparticles (30 mg), mixed with a triton X-100 water ...
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Preparation of Molecularly Imprinted Microspheres as Biomimetic Recognition Material for In Situ Adsorption and Selective Chemiluminescence Determination of Bisphenol A Yan Xiong 1,2, * 1 2 3

*

ID

, Qing Wang 1 , Ming Duan 1,2, *, Jing Xu 3 , Jie Chen 1 and Shenwen Fang 1,2

School of Chemistry and Chemical Engineering, Southwest Petroleum University, Chengdu 610500, China; [email protected] (Q.W.); [email protected] (J.C.); [email protected] (S.F.) Oil and Gas Field Applied Chemistry Key Laboratory of Sichuan Province, Southwest Petroleum University, Chengdu 610500, China Liaoning Entry-Exit Inspection and Quarantine Bureau, Dalian 116001, China; [email protected] Correspondence: [email protected] (Y.X.); [email protected] (M.D.); Tel.: +86-28-83037346 (Y.X.)

Received: 22 May 2018; Accepted: 13 July 2018; Published: 16 July 2018

 

Abstract: Bisphenol A (BPA) is an endocrine disrupter in environments which can induce abnormal differentiation of reproductive organs by interfering with the action of endogenous gonadal steroid hormones. In this work, the bisphenol A (BPA) molecularly-imprinted microspheres (MIMS) were prepared and used as biomimetic recognition material for in situ adsorption and selective chemiluminescence (CL) determination of BPA. Through non-covalent interaction, the BPA-MIMS was successfully prepared by Pickering emulsion polymerization using a BPA template, 4-vinylpyridine (4-VP) monomer, ethylene glycol dimethacrylate (EGDMA) cross-linker, and a SiO2 dispersion agent. The characterization of scanning electron microscopy (SEM) and energy-disperse spectroscopy (EDS) showed that the obtained MIMS possessed a regular spherical shape and narrow diameter distribution (25–30 µm). The binding experiment indicated BPA could be adsorbed in situ on the MIMS-packing cell with an apparent maximum amount Qmax of 677.3 µg g−1 . Then BPA could be selectively detected by its sensitive inhibition effect on the CL reaction between luminol and periodate (KIO4 ), and the inhibition mechanism was discussed to reveal the CL reaction process. The CL intensity was linear to BPA concentrations in two ranges, respectively from 0.5 to 1.5 µg mL−1 with a detection limit of 8.0 ng mL−1 (3σ), and from 1.5 to 15 µg mL−1 with a limit of detection (LOD) of 80 ng mL−1 (3σ). The BPA-MIPMS showed excellent selectivity for BPA adsorption and the proposed CL method has been successfully applied to BPA determination in environmental water samples. Keywords: molecularly imprinted microsphere (MIMS); bisphenol A; in situ adsorption; chemiluminescence determination; selectivity

1. Introduction Bisphenol A (BPA) is the most widely used bisphenol (BP), commonly used as the chemical building block in the manufacture of polycarbonate plastics [1] and generally as the weakly acidic color developer in the production of thermal papers [2]. However, the leakage of BPA from receipt papers [3] and drinking bottles [4,5] into food and water will result in potential risks on public health. Many studies have shown that BPA is an endocrine disrupter in the environment, which can induce abnormal differentiation of reproductive organs by interfering with the action of endogenous gonadal steroid hormones [6]. As a result, several states in the United States have prohibited BPA from manufacture, sale, or distribution in some consumer products since 2009 [7]. The European Polymers 2018, 10, 780; doi:10.3390/polym10070780

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Commission [8] and China government restricted the use of BPA in plastic infant feeding bottles in January 2011 and June 2012. The U.S. Food and Drug Administration (FDA) also banned the use of BPA in baby bottles and children’s drinking cups since 2012 [9]. Hence, there is a great need to develop reliable analytical methods for sensitive detection of trace BPA in the fields of environmental monitoring, food safety, and toxicity assessment. Up to now, a large number of analytical methods have been reported for the determination of BPA, including electrochemistry [10–12], chromatography [13–16], mass spectroscopy [17], chemiluminescence (CL) [18,19], and fluorescence [20,21]. However, these methods for BPA measurement still have some disadvantages, as reported in the literature [22]. For the mentioned methods, chemiluminescence combined with flow-injection analysis shows unique merits of high sensitivity, short analysis time, and simple instrumentation manipulation. However, the poor selectivity of the CL technique means it cannot be used for direct analysis of complicated samples [23]. Molecular imprinting technology (MIT) is an attractive technique which has been widely utilized to fabricate artificial tailor-made receptors [23]. With excellent memory of size, shape, and functional groups to template molecules, the resultant molecularly-imprinted polymers (MIPs) show structure predictability, recognition specificity and application universality [24]. The above-mentioned features result in MIPs being employed as biomimetic molecular recognition receptors for a given molecular structure [25]. Among the polymerization methods, traditional bulk polymerization is widely employed for MIPs preparation due to the advantages of rapidity and simplicity [26]. However, the post-treatment of crushing, grinding, and sieving is generally needed for the obtained monolithic polymer by bulk polymerization. This post-treatment procedure is not only time-consuming, but also causes irregular particles and low yields of polymer. To overcome these disadvantages, suspension polymerization [27,28] and precipitation polymerization [29,30] methods were developed to prepare MIPs. However, the MIP microspheres obtained by suspension polymerization have polydisperse particle sizes with large variability, which is not beneficial for the application [31]. Meanwhile, precipitation polymerization uses a large amount of organic solvent and needs very strict reaction conditions with respect to polymerization temperature and stirring speed [32]. A new molecular imprinting method based on Pickering emulsion polymerization (PEP) was developed by Shen et al. [33]. The phenomenon of Pickering emulsion was firstly described by Pickering in 1907 [34], in which solid particles could be employed to stabilize emulsion droplets either in oil-in-water (O/W) or water-in-oil (W/O). By locating the stabilizing particles at the interface between the two immiscible liquids, the coalescence was successfully prevented and the droplets were stabilized [35,36]. By combining Pickering emulsion polymerization (PEP) with MIT, the resulted molecularly imprinted microspheres (MIMS) could achieve high yields of polymer and good control of particle sizes. Meanwhile, with an optimal combination of hydrophobic and electrostatic interactions, this PEP-MIT method can lead to successful formation of molecularly imprinted sites in a continuous water phase [37]. The PEP-MIT based MIMS have been successfully applied as λ-cyhalothrin recognition element [38], peptides catalyst [39], drugs β-receptor blocker [40], solid phase extraction material [41], etc. This work was aimed to develop an improved CL method for in situ and selective determination of BPA using MIMS as biomimetic recognition material. The BPA-MIMS was fabricated by Pickering emulsion polymerization through non-covalent interaction with the BPA template, 4-vinylpyridine (4-VP) monomer, ethylene glycol dimethacrylate (EGDMA) cross-linker, and the SiO2 dispersion agent. Particles of BPA-MIMS were packed into the CL detection cell and used to adsorb BPA on-line. Then the adsorbed BPA can be detected in situ by producing a sensitive inhibition effect on the CL reaction between luminol and periodate (KIO4 ). After the CL reaction, the absorbed BPA was destroyed and taken away by the flow solution with the cavities left on the MIMS for the adsorption and detection of the next sample. The BPA-MIMS showed excellent selectivity for BPA adsorption and has been successfully applied to CL determination of BPA in water samples.

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2. Materials and Methods 2.1. Reagents and Materials 4-Vinylpyridine (4-VP) and luminol were obtained from Aladdin Biochemical Technology Inc., Ltd. (Shanghai, China), ethylene glycol dimethacrylate (EGDMA) was purchased from Letai Chemical Reagent Company (Tianjin, China), and the other reagents were obtained from Kelong Chemical Reagent Company (Chengdu, China). All the reagents were of analytical grade and used as received except EGDMA. EGDMA was distilled to eliminate radical inhibitors. Wahaha® purified water (Wahaha, Hangzhou, China) was used for the preparation of solutions and throughout the experiments. A total of 100 µg mL−1 BPA stock solution was prepared by dissolving 0.05 g BPA in 500 mL of water. A total of 1.0 × 10−3 mol L−1 luminol stock solution was prepared by dissolving 0.0886 g of luminol in 10 mL of NaOH (1.0 mol L−1 ) and diluted to 500 mL. A total of 1.0 × 10−3 mol L−1 potassium periodate (KIO4 ) stock solution was prepared by dissolving 0.1150 g of KIO4 in 500 mL of water. All the solutions were stored in a refrigerator and protected from lighting. 2.2. Preparation of BPA-MIMS Specifically Targeted BPA The BPA-MIMS molecularly imprinted polymers applied for solid phase extraction (SPE) procedure were prepared based on Pickering emulsion polymerization according to [41]. Traditionally, the preparation of BPA-MIMS mainly includes the following steps: (i) preparation of the Pickering emulsion; (ii) free-radical polymerization; and (iii) solvent extraction. Specifically, the water phase used for the emulsion preparation mainly was shaped by solid stabilizing SiO2 nanoparticles (30 mg), mixed with a triton X-100 water solution (0.2%, 10 mL) in a 20 mL thick walled glass tube and sonicated for 10 min. On the other hand, the oil phase was composed of the template BPA (0.228 g, 1 mmol), the functional monomer 4-VP (0.42 mL, 4 mmol), the cross-linking monomer EGDMA (20 mmol, 3.8 mL), and initiator AIBN (40 mg), and then these reagents were dissolved in 3.6 mL of the pore-forming agent toluene in a 10 mL thick-walled glass tube to prepare the pre-polymerization solution, and following sonication for 1 min was carried out to homogenize the mixture in the oil phase. After the addition of 4 mL of pre-polymerization solution to the water phase, the mixture was shaken by intense agitation, a stable Pickering emulsion was obtained when no coalescence of the oil droplets could be observed in 2 h. The free radical polymerization of the monomer 4-VP in the Pickering emulsion was conducted in a water bath at 70 ◦ C for 16 h, polymer–silica microsphere composite was obtained. The mixture was cooled to room temperature, and the supernatant in the mixture was removed. The sinking microspheres, evenly coated with the polymer-silica microsphere powders, were isolated by filtration, were washed with water, ethanol, and acetone in sequence, and dried overnight under vacuum. Then the polymer-silica composite was cleaned by dipping into a hydrofluoric acid (40%) at room temperature for 12 h to remove the silica particles from the surface. The final solvent extraction of the BPA template molecules was achieved using a Soxhlet extractor in a methanol solution containing 10% (vol %) acetic acid as the extraction solvent for 24 h. The overall scheme of BPA-MIMS fabrication process was summarized in Scheme 1a,b. For the preparation of non-imprinted microsphere (NIMS), the similar procedure was adopted except that the pre-polymerization solution without BPA was used and the Soxhlet extraction step was omitted.

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CH3

(b) H2C

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Template remove

poly(EGDMA)

poly(EGDMA)

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poly(EGDMA)

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poly(EGDMA)

Scheme 1. (a) Schematic illustration of the synthesis of molecularly imprinted microspheres with selfScheme 1. (a) Schematic illustration of the synthesis of molecularly imprinted microspheres with assembly at Oil/Water interfaces in a SiO2 NP-stabilized Pickering emulsion; and (b) the overall self-assembly at Oil/Water interfaces in a SiO2 NP-stabilized Pickering emulsion; and (b) the overall scheme of the BPA-MIMS fabrication process. scheme of the BPA-MIMS fabrication process.

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2.3. Characterization The fluorescence spectra for binding studies were measured by a fluorescence spectrometer (LS55, Perkin Elmer, Connecticut, CT, USA). Solutions were placed in a quartz cuvette with 1 cm path length. The fluorescence emission spectra were measured by setting excitation wavelength at 279 nm. The scan speed was chosen to be 500 nm min−1 and the widths of the excitation and the emission slits were both set to 10 nm. Scanning electron microscopy (SEM) images were recorded using a field-emission scanning electron microscope (FESEM, EV0 MA15, Carl Zeiss, OB Cohen, Germany) at an acceleration voltage of 20 kV. All samples were sputter-coated with gold using an E1045 Pt-coater (Carl Zeiss, Germany) before SEM observation. Elemental analysis was conducted with an energy dispersive X-ray spectrometer (EDS) equipped in the EV0 MA15 FESEM at an accelerating voltage of 20 kV. The optical microscope images of the Pickering emulsion were taken using a laser scanning confocal microscope (LSCM, Eclipse Ti, Nikon, Tokyo, Japan) with an excitation wavelength of 488 nm. The zeta potential and dynamic light scattering (DLS) measurements were performed on a NANO ZS apparatus equipped with the DTS Ver. 4.10 software package (Malvern Instruments Ltd., Worcestershire, Malvern, UK). Fourier transform infrared spectroscopy (FTIR, Nicolet 6700, Thermo Scientific, Massachusetts, MA, USA) was used to analyze the chemical structures and compositions of the MIMS. 2.4. Chemiluminescence Measurements Chemiluminescence measurements were carried out using the flow system shown in Figure S1. Solutions were delivered by two peristaltic pumps (HL-2B, Shanghai Huxi, Shanghai, China). PTFE tubing (0.8 mm i.d.) was used to connect the components. A six-way injection valve (XP-206, Shanghai Kincaid Analytical Instrument Co., Ltd., Shanghai, China) was used for solution sampling. An ultra-weak chemiluminescence measuring instrument (RFL-1, Xi’an Remex Analysis Instrument Co. Ltd., China) equipped with an lp21 photomultiplier tube (PMT, Hamamatsu, Japan) was used to detect the CL signal and the data was processed with computerized data processing software. Fifty milligram particles of BPA-MIMS were packed into a glass tube (2 mm inner diameter (i.d.) × 2 cm length) used as the CL detection cell and positioned in front of the PMT detector. The procedure for chemiluminescence determination of BPA was summarized as follows: Step 1 (For BPA on-line adsorption): pump 1 is stopped and BPA solution was delivered to flow through the BPA-MPMS cell by pump 2. As a result, BPA was selectively adsorbed in situ on the MIMS. Step 2 (For BPA in situ CL determination): after a proper adsorption time, the BPA was washed with NaOH solution by using the six-way injection valve. At the same time, the emerging stream of KIO4 -luminol CL reagents was delivered by pump1 and flowed through the BPA-MPMS cell. As a result, BPA could react with the CL reagents under NaOH conditions, producing an inhibition effect on CL emissions and be sensitively detected. Step 3 (For BPA-MIMS cleaning): when the CL intensity of the reaction came to be blank, pump 1 was stopped immediately and the water carrier delivered by pump 2 flowed through the cell to clean the MPMS cavities for the next BPA adsorption and determination. 2.5. Binding Measurements The binding properties of MPMS to BPA were studied by the batch method and the dynamic method. The amount of BPA left in the solution after rebinding was detected through fluorescence measurement. In a typical rebinding experiment for batch method, six portions of 20.0 mL of different concentration BPA solutions (10, 20, 30, 40, 50, and 60 µg mL−1 ) were added to six pieces of 10 mg washed and dried MIMS and placed in the tubes, respectively. Then the suspensions were sealed and were oscillated for 2 h at room temperature to ensure equilibration. After centrifuging at 3000 rpm for 15 min, the supernatants were respectively taken out and put into the quartz cell to detect the

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concentration of free BPA by measuring the fluorescence. The amount of BPA bound to the MIMS polymer was calculated by subtracting the concentration of free BPA from the initial 6BPA Polymers 2018, 10, 780 of 17 concentration. The data obtained was used to draw the adsorption isotherm for Scatchard analysis according to the Equations (1) and (2) [42,43]: concentration of free BPA by measuring the fluorescence. The amount of BPA bound to the MIMS (𝐶 − 𝐶 )𝑣 polymer was calculated by subtracting the concentration of free BPA from the initial BPA concentration. (1) 𝑄 = m The data obtained was used to draw the adsorption isotherm for Scatchard analysis according to the 𝑄 𝑄 𝑄 Equations (1) and (2) [42,43]: = − + (2) 𝐶 (C0𝐾− CR𝐾 )v Q = (1) m where C0 and CR are the initial and final BPA concentrations, v is the sample volume, m is the mass Q of BPA Q Qmax to MIMS at equilibrium, Qmax is the of the used BPA-MIMS, Q is the amount = − adsorbed + (2) C K Kd and Kd is the equilibrium dissociation apparent maximum amount of BPA adsorbed byd MIMS, R constant. where C0 and CR are the initial and final BPA concentrations, v is the sample volume, m is the mass of For BPA-MIMS, the dynamicQmethod, seven portions of 20.0 mL BPA solution with theQsame the used is the amount of BPA adsorbed to MIMS at equilibrium, the apparent max isconcentration −1) were added to seven pieces of 10 mg washed and dried MIMS, respectively. The (10 μg mLamount maximum of BPA adsorbed by MIMS, and Kd is the equilibrium dissociation constant. suspensions were sealed and were oscillated temperature to ensure equilibration. Then one For the dynamic method, seven portionsatofroom 20.0 mL BPA solution with the same concentration − 1 sample was taken at a regular interval to detect concentration of free BPA in the respectively. supernatant (10 µg mL ) wereoutadded to seven pieces of 10the mg washed and dried MIMS, by measuring fluorescence. The data obtained was used to draw the kinetic adsorption for The suspensions were sealed and were oscillated at room temperature to ensure equilibration. curve Then one dynamic analysis. sample was taken out at a regular interval to detect the concentration of free BPA in the supernatant

by measuring fluorescence. The data obtained was used to draw the kinetic adsorption curve for 2.6. Analytical Evaluation of the Proposed Method dynamic analysis. In order to evaluate the proposed method, serials of analytical performances, including 2.6. Analytical Evaluation of the Proposed Method analytical linear range and limit of detection (LOD), repeatability and response time, interference In and order to evaluate the were proposed method,for serials analytical performances, including analytical study, recovery testing investigated BPAofanalysis in detail. Meanwhile, the possible CL linear range and limit detectionand (LOD), repeatability and response interference study, reaction mechanism wasofdiscussed this method was applied to BPA time, determination in several and recovery were investigated for BPA analysis in detail. Meanwhile, the possible CL reaction different real testing water samples. mechanism was discussed and this method was applied to BPA determination in several different real 3. Results water samples.

3. Results 3.1. BPA-MIMS Binding Assays For bindingBinding assays,Assays the concentration of free BPA in the supernatant was detected by measuring 3.1. BPA-MIMS BPA fluorescence emission at λem = 309 nm with λex = 279 nm. The typical fluorescence responses to For binding assays, the concentration free BPA different BPA concentrations are shown inof Figure 1. in the supernatant was detected by measuring BPA fluorescence emission at λem = 309 nm with λex = 279 nm. The typical fluorescence responses to different BPA concentrations are shown in Figure 1. 700 40mg/L 20mg/L 15mg/L 10mg/L 5mg/L 2mg/L 1mg/L 0.5mg/L 0.2mg/L 0.1mg/L

Fluorescence intensity

600 500 400 300 200 100 0 275

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Wavelength (nm) Figure 1. The typical fluorescence responses to different BPA concentrations. Figure 1. The typical fluorescence responses to different BPA concentrations.

The data obtained from batch-type method were plotted according to the Scatchard equation and shown in Figure 2a,b. The equilibration time of the suspensions for the batch-type experiment

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The data obtained from batch-type method were plotted according to the Scatchard equation and shown in Figure 2a,b. the suspensions for the batch-type experiment was 2 h, as illustrated aboveThe in equilibration Section 2.5. time Thisofresult was also confirmed by the dynamic was 2 h, asasillustrated Section This result also confirmed by the dynamic experiment, experiment, shown inabove Figurein2c, which2.5. indicated thewas adsorption and equilibrium time was about as shown in Figure 2c, which indicated the adsorption and equilibrium time was about 50 min. From 50 min. From the Scatchard analysis of Figure 2b, the data plotting showed two linear fittings the Scatchard analysis of Figure 2b, the data plotting showed two linear fittings during the analysis during the analysis BPA concentration range. The first linear regression equation for the straight Theand first equation for theforstraight line line is is = lineBPA is Q1concentration = −0.0367Q1range. + 22.976 the linear secondregression linear regression equation the straight Q2 CR

CR

= −0.0195Q indicated are two types offor binding sites on line MIMS −0.0367𝑄 + 22.976 andThis theresult second linear there regression equation the straight is for = 2 + 17.138. BPA. Then, according Equation the equilibrium Kd and apparent −0.0195𝑄 + 17.138.to This result(2), indicated there are dissociation two types ofconstant binding sites on the MIMS for BPA. −1 and Q maximum amount Q were estimated to be K = 119.36 µmol L = 12.01 µmol/g for max1the apparent maximum d1 dissociation constant Kd and Then, according tomax Equation (2), the equilibrium − 1 theamount lower affinity binding sites to and = 224.63 µmol L and Qmax2 = 16.83 µmol/g for lower the higher Qmax were estimated beKKd2 d1 = 119.36 μmol L−1 and Qmax1 = 12.01 μmol/g for the affinity affinity binding sites. −1 binding sites and Kd2 = 224.63 μmol L and Qmax2 = 16.83 μmol/g for the higher affinity binding sites. 450 400

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Figure 2. (a) The Langmuir adsorption isotherm curve of MIMS with equilibration time of 2 h; (b) Figure 2. (a) The Langmuir adsorption isotherm curve of MIMS with equilibration time of 2 h; (b) Scatchard Scatchard plot to estimate the binding nature of MIMS with equilibration time of 2 h; (c) kinetic plot to estimate the binding nature of MIMS with equilibration time of 2 h; (c) kinetic adsorption curve for adsorption curve for dynamic analysis of 10 μg mL−1; and (d) adsorption rate of MIMS with time dynamic analysis of 10 µg mL−1 ; and (d) adsorption rate of MIMS with time changing. changing.

TheThe different binding characteristics ofof MIMS different binding characteristics MIMSwere weremainly mainlycaused causedby bythe themolecular molecular structure structure of of BPA. which can react with MAA. BPA. As As shown shownin inScheme Scheme1,1,BPA BPAhas hastwo twophenolic phenolichydroxyl hydroxylgroups groups which can react with MAA. Hence, there are two binding sites produced on the MIMS after the extraction of BPA. When the BPA Hence, there are two binding sites produced on the MIMS after the extraction of BPA. When the BPA concentration is low, the subsequent equilibrium constant and apparent maximum amount of BPA concentration is low, the subsequent equilibrium constant and apparent maximum amount of BPA adsorption areare smaller because fewer BPA molecules exist in the low BPA concentration. However, adsorption smaller because fewer BPA molecules exist in the low BPA concentration. However, when BPA concentration is high, the subsequent equilibrium constant and apparent maximum amount when BPA concentration is high, the subsequent equilibrium constant and apparent maximum of BPA adsorption higher because a much greater amount of BPA molecules in the solution. amount of BPA are adsorption are higher because a much greater amount of BPAexist molecules exist in the For the dynamic method, the concentration of free BPA in the supernatant was detected by solution. measuring to evaluate adsorption rate andBPA adsorption The datawas obtained was by Forthe thefluorescence dynamic method, the the concentration of free in the time. supernatant detected used to draw the adsorption curve for dynamic analysis. As shown in Figure 2c,The the data adsorption measuring thekinetic fluorescence to evaluate the adsorption rate and adsorption time. obtained

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was used to draw the kinetic adsorption curve for dynamic analysis. As shown in Figure 2c, the and equilibrium time for 10 µg mL−1 was about−1 50 min. The dynamic kinetic process can be evaluated adsorption and equilibrium time for 10 μg mL was about 50 min. The dynamic kinetic process can by an asymptotic fitting model according to the following equation with R2 = 0.98: be evaluated by an asymptotic fitting model according to the following equation with R2 = 0.98:

yy== 82.923 11.032 ××0.947 0.947t t 82.923 − − 11.032

(3)(3)

where theabsorption absorptionamount amountand andttisisthe theabsorption absorptiontime. time.As Asaaresult, result,the thekinetic kineticrate rateconstant constantk where 𝑦 y isisthe −1 from Equation (3). Meanwhile, as shown in Figure 2d, the − 1 kcan canbebe obtained to be 0.947 min obtained to be 0.947 min from Equation (3). Meanwhile, as shown in Figure 2d, the adsorption adsorption rate waswith decreased with the time. adsorption time. During the first there is a rapid rate was decreased the adsorption During the first 30 min, there30 is min, a rapid adsorption of adsorption of BPA on the MIMS. Then the adsorption slowed down after 30 min. BPA on the MIMS. Then the adsorption slowed down after 30 min. 3.2. 3.2.Synthesis Synthesisand andCharacterization CharacterizationofofBPA-MIMS BPA-MIMS In Inthis thiswork, work,the the BPA-MIMS BPA-MIMS was was prepared prepared with with non-covalent non-covalent interactions interactionsthrough throughthe the typical typical Pickering was used as as a Pickeringemulsion emulsionpolymerization polymerizationand andthe theprocess processwas wasshown shownininScheme Scheme1a.1a.BPA BPA was used template, 4-VP was used asas monomer, a template, 4-VP was used monomer,and andEGDMA EGDMAwas wasused usedasasaacross-linker cross-linkerfor for BPA-MIMS BPA-MIMS polymerization interactions within thethe organic core.core. SiO2 SiO nanoparticles (NPs) (NPs) with polymerizationwith withnon-covalent non-covalent interactions within organic 2 nanoparticles proper hydrophilic-hydrophobic properties were adopted to to stabilize with proper hydrophilic-hydrophobic properties were adopted stabilizethe theemulsion emulsionat at oil/water oil/water interfaces. scattering (DLS) (DLS)indicated indicatedthe theSiO SiO2 2NPs NPshad hadan anaverage averagezeta zetapotential potential interfaces. The The dynamic dynamic light light scattering of of approximately −20.36mV mVwith with excellent stability and average diameter of approximately approximately −20.36 excellent stability and anan average diameter of approximately 400400 nm nm the polydispersity (PDI) ofindicative 0.296, indicative a good dispersibility. optical withwith the polydispersity indexindex (PDI) of 0.296, of a goodofdispersibility. The opticalThe microscope microscope of thewas SiOshown 2 NPs was shown Figure 3a and the diameter distribution was shown image of theimage SiO2 NPs in Figure 3ainand the diameter distribution was shown as Figure 3b. as Figure 3b. The resultinofthis thework DLS in this work is consistent with the literature [44], which The PDI result of PDI the DLS is consistent with the literature [44], which reported the reported the interfacial polymerization inemulsion a Pickering the use of SiO2 NPs. interfacial polymerization of dopamineof indopamine a Pickering by emulsion the use ofby SiO 2 NPs.

Figure Figure3.3.(a) (a)Optical Opticalimage imageof ofSiO SiO22NPs; NPs;(b) (b)DLS DLSmeasurement measurementfor forsize sizedistribution; distribution;and and(c) (c)an anoptical optical image of emulsion droplets after locating the silica NPs at the interface. image of emulsion droplets after locating the silica NPs at the interface.

By locating the stabilizing silica NPs at the interface, the coalescence was successfully prevented By locating the stabilizing silica NPs at the interface, the coalescence was successfully prevented and the droplets were stabilized. As shown in Figure 3c, most of the emulsion droplets were spherical and the droplets were stabilized. As shown in Figure 3c, most of the emulsion droplets were spherical in shape with a diameter range of 20−30 μm. In addition, there was no observation of the DLS signal in shape with a diameter range of 20−30 µm. In addition, there was no observation of the DLS signal for the water phase above the emulsion, which indicated that there was no free SiO2 NPs in the water for the water phase above the emulsion, which indicated that there was no free SiO2 NPs in the water phase. This was because nearly all of the silica particles took part in the emulsion process and were phase. This was because nearly all of the silica particles took part in the emulsion process and were adsorbed by the droplet interfaces. adsorbed by the droplet interfaces. The surface morphologies of the dried BPA-MIMS were examined using FESEM and LSCM, The surface morphologies of the dried BPA-MIMS were examined using FESEM and LSCM, which are shown in Figure 4a,b, respectively. It could be observed that both FESEM and LSCM which are shown in Figure 4a,b, respectively. It could be observed that both FESEM and LSCM indicated that all the MIMS were spherical with an average diameter of approximately 25 μm before indicated that all the MIMS were spherical with an average diameter of approximately 25 µm SiO2 nanoparticle removal. These MIMS showed a good dispersibility without obvious aggregations. before SiO nanoparticle removal. These MIMS showed a good dispersibility without obvious Meanwhile,2the detached SiO2 NPs, which should be derived from the MIMS, were also observed by aggregations. Meanwhile, the detached SiO2 NPs, which should be derived from the MIMS, were also SEM. observed by SEM.

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Figure 4. (a) The surface morphologies of the dried MIMS examined using FESEM; (b) the surface

Figure 4. (a) The surface morphologies of the dried MIMS examined using FESEM; (b) the surface morphologies of the MIMS examined using LSCM; and (c) FTIR spectra of the synthesized MIMS. morphologies of the MIMS examined using LSCM; and (c) FTIR spectra of the synthesized MIMS. The structural characteristics of the presented MIMS before SiO2 removal have been investigated by Fourier transform infrared spectroscopy (FTIR) and spectra were analyzed in(b)detail. Figure 4c Figure 4. (a)characteristics The surface morphologies of the dried MIMS examined FESEM;have the surface The structural of the presented MIMS before SiOusing been investigated 2 removal presented the FTIR spectra of the MIMS after removing the silica nanoparticles and BPA template. morphologies of the MIMS examined using LSCM; and (c) FTIR spectra of the synthesized MIMS. by Fourier transform infrared spectroscopy (FTIR) and spectra were analyzed in detail. Figure 4c Typically, strong peaks at (1) around 3121 and 756 cm–1 corresponding to stretching vibration and presented the FTIR spectra of the MIMS after removing the silica nanoparticles and BPA template. The structural characteristics the presented before SiO2 and removal investigated out-of-plane bending vibration ofofC–H bond for MIMS C=C–H; (2) 2998 2329have cm–1been corresponding to Typically, strong peaks at (1) around 3121 and 756 cm–1 corresponding to stretching vibration and –1 corresponding by Fourier transform infrared spectroscopy (FTIR) and spectra were(3) analyzed detail. Figure 4c asymmetric and symmetric stretching vibration of C–H bond for alkyl; 1724 cmin to –1 corresponding to out-of-plane bending ofcarboxyl; C–H bond for C=C–H; 2998 and 2329 cm presented FTIRvibration spectra MIMS after removing the(2) silica nanoparticles and BPA template. stretching the vibration of C=Oofforthe (4) 1612 and 1397 cm–1 corresponding to asymmetric and asymmetric and symmetric stretching of756 C–H for alkyl; (3) 1724 cm–1vibration corresponding to –1 corresponding Typically, strong peaksvibration at (1) around 3121for and cmbond to stretching and symmetric stretching of vibration C=C pyridine; (5) 1265 cm−1 corresponding to stretching –1 corresponding –1 −1 stretching vibration of C=O for carboxyl; (4) 1612 and 1397 cm to asymmetric and out-of-plane bending vibration(6)of1479 C–H for C=C–H; to (2)bending 2998 and 2329 cm corresponding to vibration of C–O for carboxyl; cmbond corresponding vibration of C–H for carboxyl; 1 corresponding –1to −1 C=C asymmetric stretching vibration ofin-plane C–H bond for−alkyl; (3) 1724ofcm corresponding to symmetric vibration of for pyridine; (5) 1265 cm stretching vibration and (7)stretching 875,and 885,symmetric and 952 cm corresponding to bending vibration pyridine ring. These –1 corresponding to asymmetric and −1carboxyl; stretching vibration C=Ocm for (4) 1612 to and 1397 cm results indicated the EGDMA have been successfully reacted with 4-VP. Meanwhile, there are strong of C–O for carboxyl; (6)of 1479 corresponding bending vibration of C–H for carboxyl; and (7) −1 corresponding to stretching– –1 − 1 symmetric stretching vibration of C=C for pyridine; (5) 1265 cm peaks at (1) 1123 cm corresponding to asymmetric stretching vibration of Si–O–Si bond; (2) 817 cm 875, 885, and 952 cm corresponding to in-plane bending vibration of pyridine ring. These results 1 and around –1 corresponding vibration C–O carboxyl; 1479 cm corresponding to 4-VP. bending vibration ofthere C–H bond, for carboxyl; 486forcm to−1symmetric vibration of Si–O–Si whichpeaks indicated the of EGDMA have been(6)successfully reactedstretching with Meanwhile, are strong −1 and (7) 875, 885, and of 952SiO cm2 nanoparticles corresponding to in-plane indicate the presence in the MIMS. bending vibration of pyridine ring. These –1 at (1) 1123 cm corresponding to asymmetric stretching vibration of Si–O–Si bond; (2) 817 cm–1 and results indicated the EGDMA have been reacted 4-VP. indicating Meanwhile, there are strong The Si apparent concentration was successfully 0.49 before SiO 2 NPswith removal, the presence of Si around 486 cm–1 corresponding to symmetric stretching vibration of Si–O–Si bond, which indicate the peaks cm–1 corresponding to asymmetric vibration ofin Si–O–Si bond; (2) 817 This cm– atoms at in(1) the1123 polymer layer. The results showed thestretching presence of Si atoms the polymer layer. presence SiO nanoparticles in the MIMS. 1 andofaround –1 2 accord 486 cm corresponding to symmetric stretching vibration Si–O–Si which result was in with the result in Figure 4a, in which the detached SiOof 2 NPs werebond, observed. In The before SiO indicatinginto theapresence 2 NPs removal, indicate the presence of SiO 2 nanoparticles in the MIMS. orderSitoapparent remove theconcentration silica NPs, the was dried0.49 polymer-silica microspheres were transferred plastic of Si atoms in the polymer layer.atThe the presence of indicating Si atoms the polymer Si apparent was 0.49showed before 2h. NPs the presence of Silayer. tube The and stirred in HFconcentration (40%) roomresults temperature forSiO 12 Theremoval, obtained MIMS wasin filtrated, washed atoms in theto layer. The results the ofthe Si atoms in the polymer layer. This This result was inpolymer accord with result inshowed Figure 4a,presence inThe which detached SiO NPs were observed. with water be neutral andthe then rinsed with ethanol. EDS and SEM results result for the MIMS 2 result was in accord with the result in Figure 4a, in which the detached SiO 2 NPs were observed. after SiO 2 NPs removal was shown in Figure 5. The EDS result indicated the Si apparent concentration In order to remove the silica NPs, the dried polymer-silica microspheres were transferred into aIn plastic to remove the silica the dried polymer-silica transferred into a plastic changed to be after SiONPs, 2 NPs removing, suggesting that almost allwere of MIMS the silica NPs have been tube order and stirred in 0HF (40%) at room temperature for 12microspheres h. The obtained was filtrated, washed and in BPA-MIMS HFand (40%) atinterfaces room temperature for 12The h. The obtained MIMS was washed removed from the after ethanol. the treatment. Meanwhile, the SEM infiltrated, Figure showed with tube water tostirred be neutral then rinsed with EDS and SEM results result 5for the MIMS with wateroftothe be MIMS neutraldid andnot then rinsed with ethanol. The EDS result for the for MIMS the shape change obviously, indicating thatand theSEM silicaresults NPs were effective the after SiO2 NPs removal was shown in Figure 5. The EDS result indicated the Si apparent concentration after SiO2 NPs removal was shown in Figure 5. The EDS result indicated the Si apparent concentration BPA-MIMS formation. changed to be 0 after SiO2 NPs removing, suggesting that almost all of the silica NPs have been changed to be 0 after SiO 2 NPs removing, suggesting that almost all of the silica NPs have been removed from the BPA-MIMS interfaces Meanwhile, SEM in Figure 5 showed removed from the BPA-MIMS interfacesafter afterthe the treatment. treatment. Meanwhile, thethe SEM in Figure 5 showed the shape of the MIMS diddid not change thatthe the silica NPs were effective for the the shape of the MIMS not changeobviously, obviously, indicating indicating that silica NPs were effective for the BPA-MIMS formation. BPA-MIMS formation.

Figure 5. The EDS results of the MIMS after SiO2 removing.

Figure 5. The EDS results of the MIMS after SiO2 removing.

Figure 5. The EDS results of the MIMS after SiO2 removing.

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3.3. CL Instrument Condition Choice and CL Measurement Condition Optimization 3.3.1. Condition Choice of the CL Instrument First, the negative high voltage of the PMT equipped on the CL instrument was investigated. Generally speaking, the detection sensitivity would increase with the increasing of the PMT’s negative high voltage. However, the noise would also increase at the same time. As a result, a negative high voltage of 600 V was chosen for the PMT for the following determination with a high signal-to-noise ratio. Then the flow speed of the peristaltic pump was also studied. The results showed that the pump speed would have effects on the signal intensity and peak shape. Finally, taking into account the reagent usage and manipulating time together with signal intensity and peak shape, 1.2 and 0.9 mL min−1 were chosen as the pump speed for peristaltic pump 1 (P1) and peristaltic pump 2 (P2), respectively. 3.3.2. Conditions Optimization for CL Measurement In this work, the CL measurement was based on the CL reaction between KIO4 and luminol under a NaOH medium. To obtain a low background emission, luminol and KIO4 solutions were mixed together in a proper distance before they flowed through the detection cell. A prohibition CL emission was recorded when BPA reacted with the mixed solution in the flow detection cell and the CL response was proportional to the BPA concentration. To obtain the most sensitive analysis performance for BPA, the conditions for the luminol-KIO4 -NaOH CL reaction were optimized by measuring the ratio of the CL signal-to-noise (S/N) at different reagent concentrations. First, KIO4 was used as the oxidant for the CL system and its concentration would greatly influence the CL intensity. By fixing the luminol concentration at 2 × 10−5 mol L−1 and NaOH concentration at 0.4 mol L−1 , the effect of KIO4 concentration was examined from 1 × 10−5 to 4 × 10−4 mol L−1 . As shown in Figure S2a, the CL intensity reached a maximum with a better S/N ratio when KIO4 was 5 × 10−5 mol L−1 . Second, luminol acted as the luminescent reagent for the CL system and its concentration was examined from 5 × 10−7 to 4 × 10−5 mol L−1 . As shown in Figure S2b, the CL intensity increased greatly with increasing the concentration of luminol up to 4 × 10−5 mol L−1 . Then the increase of CL intensity is not obvious with further luminol concentration increasing. In order to obtain a preferable CL intensity with a proper luminol concentration, luminol concentration was chosen to be 2 × 10−5 mol L−1 . Third, the experiment indicated that luminol would produce CL emission just under strong basic conditions, so the NaOH concentration was a key factor for the CL reaction. The effect of NaOH concentration as a medium of luminol was examined over a 0.025–0.8 mol L−1 range. As shown in Figure S2c, the CL intensity reached a maximum value when 0.4 mol L−1 NaOH was used. A higher concentration of NaOH would result in lower CL intensity. In conclusion, the optimal conditions for the CL system were 5 × 10−5 mol L−1 KIO4, 2 × 10−5 mol L−1 luminol and 0.4 mol L−1 NaOH. 3.3.3. Optimum of BPA Adsorption Time A proper adsorption time was needed for the adsorption of BPA on the MIMS because too short a time may be insufficient for BPA adsorption but too long time would result in time waste and MIMS saturation. By fixing the CL condition of 5 × 10−5 mol L−1 KIO4 , 2 × 10−5 mol L−1 luminol and 0.4 mol L−1 NaOH, the adsorption time was investigated from 0 to 7 min. The results in Figure S2d indicated that BPA adsorption amount would increase with the increase of adsorption time and produced a higher prohibition effect on the CL reaction. As a result, as shown, it was observed that the CL intensity decreased with the adsorption time. However, the MIMS adsorption for BPA would come to saturation after a period of time. As shown in Figure S2d, the CL intensity reached constant within

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Polymers 2018, 10, x FOR PEER REVIEW Polymers 2018, 10, x FOR PEER REVIEW −11BPA. As a result, 2 min μg mL mL− 4 min min for for 22 µg BPA. As a result, 2 min

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was chosen chosen as as the the optimal optimaltime timeto toobtain obtainthe theproper properBPA BPA was −1 min for 2 μg and mL−1avoid BPA. As adsorption a result, 2 min was chosen as the optimal time to obtain the proper BPA adsorption the saturation. adsorption the min for 2 μgand mLavoid BPA. Asadsorption a result, 2 saturation. min was chosen as the optimal time to obtain the proper BPA adsorption and avoid the adsorption saturation. adsorption and avoid the adsorption saturation. 3.4. Measurements 3.4.The TheAnalytical AnalyticalPerformance PerformanceforforBPA BPA Measurements 3.4. The Analytical Performance for BPA Measurements 3.4. The Analyticalof Performance for BPA Measurements 3.4.1. 3.4.1.Discussion Discussion ofthe thePossible PossibleCL CLReaction ReactionMechanism Mechanism 3.4.1.At Discussion of the Possible CL Reaction Mechanism selected conditions, comparison of theof CLthe spectra and fluorescence spectra inspectra the presence 3.4.1. Discussion of the PossibleaCL Mechanism Atthethe selected conditions, aReaction comparison CL spectra and fluorescence in the and absence of BPA was shown in Figure 6a,b, respectively. As indicating in Figure 6a, the CL intensity At the selected conditions, a comparison of the CL spectra and As fluorescence spectra in6a,the presence absence of BPA was shown in Figure 6a,b, indicating in Figurein the At theand selected conditions, a comparison of the CLrespectively. spectra and fluorescence spectra the ofCL KIO system withdecreased the ofthe BPA. This result confirmed that BPA showed presence and absence of decreased BPA was shown in addition Figure 6a,b, respectively. As indicating in Figure 6a, the 4 -luminol intensity of KIO 4-luminol system with addition of BPA. This result confirmed that presence and absence of BPA was shown in Figure 6a,b, respectively. As indicating in Figure 6a, the a BPA strong CL inhibition effect on the luminol CL the emission. CL intensity of KIO 4-luminol system decreased with the addition of BPA. This result confirmed that showed strong CL inhibition effect on luminol CL emission. CL intensity of aKIO 4-luminol system decreased with the addition of BPA. This result confirmed that BPA showed a strong CL inhibition effect on the luminol CL emission. BPA showed a strong CL inhibition effect on the luminol CL emission. 800 peak 1: without BPA peak 2: with PBA peak 1: without BPA peak BPA peak 1: 2: without with PBA 2 with PBA peak 2:

1 1

500 500400

CL CLintensity intensity CL intensity

400 400300

2 2

300 300200 200 200100 100 100 0 0 0

0

100

0 0

100 100

200

300

Time(s) 300 300 Time(s) (a) Time(s)

200 200

400 400 400

500

spectrum 1: BPA

spectrum 2: BPA+NaOH 800700 spectrum 3: KIO4+Luminol+NaOH spectrum 1: BPA 800 spectrum 1: spectrum 2: BPA BPA+NaOH spectrum 4: KIO4+Luminol 700600 spectrum 2: BPA+NaOH spectrum 3: KIO +Luminol+NaOH 4 700 spectrum 3: KIO +Luminol+NaOH spectrum 4: KIO 4 +Luminol 4 600500 spectrum 4: KIO4+Luminol 600 500400 500 400300 3 400 300200 1 3 300 3 2001001 4 200 1 2 100 0 2 4 100 2300 350 400 450 4 500 550 600 650 0 wavelength 550 600 650 0 300 350 400 450 500 (nm) 300 350 400 450 500 550 600 650

Fluorescence Fluorescenceintensity intensity Fluorescence intensity

1

500

500 500

wavelength (nm) (b) (nm) wavelength (a) (b) (a) (b) and (b) fluorescence Figure 6. Spectra comparison in the presence and absence of BPA. (a) CL spectra; Figure 6. Spectra comparison in the presence and absence of BPA. (a) CL spectra; and (b) fluorescence spectra. Figure 6. Spectra comparison in the presence and absence of BPA. (a) CL spectra; and (b) fluorescence spectra. Figure 6. Spectra comparison in the presence and absence of BPA. (a) CL spectra; and (b) fluorescence spectra. spectra. The fluorescence result in Figure 6b revealed that a maximum emission wavelength was found

The fluorescence result in Figure 6b revealed that a maximum emission wavelength was found atat419 nm, which that the emission the 3-aminophthalate The fluorescence result in Figure 6b revealed that a species maximum emission wavelength was found 419 nm, whichsuggested suggested thepossible emission specieswas was theoxidated oxidated 3-aminophthalate fluorescence result in that Figure 6bpossible revealed that a maximum emission wavelength was found 2The −2−* * ) and 2− (AP the emitter was 3-aminophthalate (AP ) [45,46]. Since the light emission spectrum 2− at 419 nm, which suggested that the possible emission species was the oxidated 3-aminophthalate ) and the emitter wasthat 3-aminophthalate (AP ) [45,46]. light emission spectrumwas was at(AP 419 nm, which suggested the possible emission speciesSince was the oxidated 3-aminophthalate 2−* 2− independent of the inhibitor, this result also indicated the prohibition process by BPA was dynamic (AP ) and the of emitter was 3-aminophthalate (AP2− ) [45,46]. Since the light emission spectrum was independent the inhibitor, this result also indicated the prohibition process by BPA was dynamic 2−* (AP ) and the emitter was 3-aminophthalate (AP ) [45,46]. Since the light emission spectrum was quenching Meanwhile, showed that BPA had andynamic obvious independent of the inhibitor, the this result also indicated the prohibition processofby BPA was quenching[47]. [47]. theresults resultsalso showed thatthe theintensity intensityresponse response obvious independent of theMeanwhile, inhibitor, this result indicated the prohibition process of byBPA BPA had wasan dynamic change with CL reaction, which suggests that BPA has taken part in the CL reaction and interacted quenching [47]. Meanwhile, the results showed that the intensity response of BPA had an obvious change with reaction, which suggests that BPA has intensity taken part in the CL quenching [47].CL Meanwhile, the results showed that the response of reaction BPA hadand an interacted obvious with the intermediate radicals produced the CL reaction. Consequently, the possible light-emitting change with CL reaction, which suggestsbyby that BPA has taken part in the CL reaction and interacted with the intermediate radicals produced the CL reaction. Consequently, the possible light-emitting change with CL reaction, which suggests that BPA has taken part in the CL reaction and interacted pathways can and prohibited BPA follows: with the intermediate radicals produced byby the CLas reaction. Consequently, the possible light-emitting pathways canbebechanged changed and prohibited by BPA as follows: with the intermediate radicals produced by the CL reaction. Consequently, the possible light-emitting pathways can be changed and prohibited by BPA as follows: −BPA − pathways can be changed and prohibited as follows: IO+ OH +by OH IO + O − + H O, (4) IO4 − →→IO (4) 3 + O 2 + H2 O , IO + OH → IO + O + H O, (4) IO + OH + O + H O, (4)(5) LH →+IO OH − → LH− , LH LH , , (5) LH2 ++OH OH → → LH (5) LH + +OOH →→LLH+ HO , (5)(6) LH − − − LH HO2 − (6) (6) LH ++OO2 → →L L ++HO (6)(7) LH + O+ O→ L→ + HO L LO − LL ++OO2 − → LO2 2− (7) (7) → LO CH3 CH3 (7) L + O → LO HO HO HO

-O -O -O

CH CH33 C C C CH3 CH3 CHCH 3 3 CH3 CH3 C C C CH3 CH3 CH3

OH + OHOH + OHOH + OH-

O- + O2+O O + O22O--

-

O

-O

O

O O O O O O

CH3 CH3 C C C CH3 CH3 CH3 CH3 CH3 CHC3 C C CH3 CH3 CH3

O- +H2O +H2O O +H2O O--

(8) (8) (8)

(8) O- + HO2HO2-O ++ HO 2 O--

(9) (9) (9)

(9)

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O O O O

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CH3 CH3 C C CH3 CH3

O-- + O2O + O2-

CH3 CH3 C C CH3 CH3

O O O O

-

O + HO2 O + HO2

(10) (10)

O O

(10) LO 2− → N (11) LO (11) LO2 → →N N 2 + AP , (11) ∗ ∗ (12) AP2−∗∗ → AP 2−∗ + hv. AP AP ∗ ++hv. hv. (12) (12) AP → → AP 2− The symbols LH2 and AP 2− * in the above equations refer to luminol and the excited state The symbols symbols LH LH22 and and AP AP2−* *ininthe theabove above equations refer refer to to luminol luminol and and the the excited excited state state The equations − and LO22− represent the intermediates in the reactions. aminophthalate dianion, respectively. LH−−, L− 2 − − − 2− aminophthalate dianion, andLO LO2 represent representthe theintermediates intermediatesininthe thereactions. reactions. aminophthalate dianion, respectively. respectively.LH LH ,, L L and The above CL and fluorescence results suggested that 2when BPA was injected into the system, BPA The above aboveCL CLand andfluorescence fluorescence results suggested when injected intosystem, the system, The results suggested that−that when BPABPA waswas injected into the BPA − are was oxidized into an intermediate quinones by O2 -induced radical oxidation. Then the O2− − − BPA oxidized was oxidized intermediate quinonesbybyOO2 -induced -inducedradical radicaloxidation. oxidation. Then Then the are was intointo an an intermediate quinones the OO22− are competitively consumed by BPA, less L−− and LO22−2−are2 produced, and the CL was greatly inhibited. − 2− competitively consumed consumed by and LO LO22 are areproduced, produced,and andthe theCL CLwas wasgreatly greatlyinhibited. inhibited. competitively by BPA, BPA, less less LL and Consequently, BPA shows a sensitive inhibition effect on the CL reaction and can be detected based Consequently, BPA CL reaction and cancan be be detected based on Consequently, BPAshows showsaasensitive sensitiveinhibition inhibitioneffect effectononthe the CL reaction and detected based on this inhibition mechanism. This result was in accord with the recorded reference [48]. thisthis inhibition mechanism. This result waswas in accord with thethe recorded reference [48].[48]. on inhibition mechanism. This result in accord with recorded reference ∗ + AP 2−∗ , + AP ∗ ,

3.4.2. Analytical Calibration Curve and Limit of Detection (LOD) 3.4.2. Analytical Analytical Calibration Calibration Curve Curve and and Limit Limit of of Detection Detection (LOD) (LOD) 3.4.2. Under the above optimal conditions, the analytical performance was carried out with 50.0 mg Under the the above above optimal optimal conditions, conditions, the the analytical analytical performance performance was was carried carried out out with with 50.0 50.0 mg mg Under MIP packed in the flow cell. As discussed in Section 3.1, there are two types of binding sites on MIMS MIP packed packed in in the the flow flow cell. cell. As As discussed discussed in in Section Section 3.1, 3.1, there there are are two two types types of of binding binding sites sites on on MIMS MIMS MIP for BPA, with lower affinity binding sites and higher affinity binding sites. As a result, the two for BPA, with lower affinity binding sites and higher affinity binding sites. As a result, the two binding for BPA, with lower affinity binding sites and higher affinity binding sites. As a result, the two binding sites brought two linear regions. As shown in Figure 7, the CL intensity showed two linear sites brought two linear As shown Figure the CL showedshowed two linear binding sites brought tworegions. linear regions. As in shown in 7, Figure 7, intensity the CL intensity tworanges linear ranges with the BPA concentrations. The first linear range was from−150 to 15 ng mL−1 BPA −1 with the BPA concentrations. The first linear range was from 50 to 15 ng mL BPA concentrations and ranges with the BPA concentrations. The first linear range was from 50 to 15 ng mL BPA concentrations and the detection−limit was 8 ng mL−1 (3σ). The regression equation was I = −179.55C 1 (3σ). the detection limit was 8 ng mL The regression equation was I = − 179.55C + 516.58 (C being −1 concentrations and the detection limit was 8 ng−1mL (3σ). The regression equation was I = −179.55C + 516.58 (C being the BPA concentration (μg mL )) with a correlation coefficient of 0.9946. The second BPA(C concentration (µg mL−1 )) with(μg a correlation of 0.9946. Theofsecond range +the 516.58 being the BPA concentration mL−1)) withcoefficient a correlation coefficient 0.9946.linear The second −1 linear range was from 15 to−1150 ng mL BPA concentrations and the detection limit was−80 ng mL−1 1 (3σ). was from 15 to 150 ng mL BPA concentrations and the detection limit was 80 ng mL The −1 −1 linear range was from 15 to 150 ng mL BPA concentrations and the detection limit was 80 ng mL (3σ). The regression equation was I = −9.0192C + 260.65 (C being the BPA concentration (μg mL−1)) −1 )) −1 regression equation was I = − 9.0192C + 260.65 (C being the BPA concentration (µg mL with (3σ). The regression equation was I = −9.0192C + 260.65 (C being the BPA concentration (μg mL )) with a correlation coefficient of 0.9964. a correlation coefficient of 0.9964. with a correlation coefficient of 0.9964.

intensity CLCL intensity

500 500

y = -179.55x + 516.58 y2= -179.55x + 516.58 R = 0.9946 2 R 0.9946 + 260.65 y ==-9.0192x y 2= -9.0192x + 260.65 R = 0.9964 2 R = 0.9964

400 400 300 300 200 200 100 100 0 0

2 2

4 4

6 6

8 8

10 10

12 12

14 -114 -1

BPA concentration(g mL ) BPA concentration(g mL )

16 16

Figure 7. The linear response for different BPA concentration. Figure 7. The linear response for different BPA concentration. Figure 7. The linear response for different BPA concentration.

3.4.3. Repeatability and Response Time 3.4.3. Repeatability and Response Time 3.4.3.By Repeatability and Response Time six times, the method repeatability was measured and the testing a standard BPA solution By testing a standard BPA solution six times, the method repeatability was measured and the relative (R.S.D.) obtained was 2.6%. By standard testing a deviation standard BPA solution six times, the method repeatability was measured and the relative standard deviation (R.S.D.) obtained was 2.6%. Thestandard short response time(R.S.D.) is an analytical relative deviation obtainedparameter was 2.6%.which is commonly desirable for applications. The short response time is an analytical parameter which is commonly desirable for applications. Generally speaking, thetime timeisrequired for 90% changewhich in the equilibrium value was The short response an analytical parameter is response commonlyofdesirable for applications. Generally speaking, the time required for 90% change in the response of equilibrium value was defined as the response time and called t 90 . Experimental results indicated that t 90 was 45 s underwas the Generally speaking, the time required for 90% change in the response of equilibrium value defined as the response time and called t90. Experimental results indicated that t90 was 45 s under the selected conditions. Such a short response made theresults proposed method the BPA defined as the response time and called t90 . time Experimental indicated thatpreferable t90 was 45for s under the selected conditions. Such a short response time made the proposed method preferable for the BPA determination compared with the other traditional time-needed methods. determination compared with the other traditional time-needed methods.

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selected conditions. Such a short response time made the proposed method preferable for the BPA determination compared with the other traditional time-needed methods. 3.4.4. Interference Study and Recovery Test In order to evaluate the overall selectivity of the proposed method for BPA determination, influences of some ions commonly existing in water were investigated according to the recommended procedure. A 50 ng mL−1 BPA solution was analyzed by being added with interfering species. The tolerable limit of interfering ions (Table 1) was taken as a relative error less than 5%. As could be seen, the tolerable limit of CL detection by adopting MIMS was much higher than that without MIMS for the common cations and anions. Most of ions tested did not interfere with determination, except Fe3+ , which could be easily eliminated using EDTA. These results showed that MIMS can be used as biomimetic recognition material in the CL analysis and improve the selectivity of the CL method. However, because no porogen was used for the preparation of MIMS, the MIMS showed indiscriminate adsorption with some structural analogues of BPA, especially for BPF. Thus, porogen should be used in the subsequent work in MIP preparation. Table 1. Tolerable ratio of structural analogues and some interfering species to BPA with and without MIP. Interfering Substances 2−

SO4 CO3 2− Cl− PO4 3− Na+ Ca2+ Mg2+ NH4 + Fe3+ citric acid 4,40 -Dihydroxybiphenyl 4,40 -Cyclohexylidenebisphenol Bisphenol A bis(chloroformate) Bisphenol A acetate propionate 2,2-Bis(4-hydroxy-3-methylphenyl)propane Bis(2-hydroxyphenyl)methane Bis(4-hydroxyphenyl)methane (BPF) tyrosine fulvic acid urea

Without MIMS

With MIMS

100 10 200 10 200 10 20 10 10 10 5 6 5 8 5 5 2 8 10 10

200 200 400 200 400 200 200 200 10 100 20 20 20 15 10 10 3 20 50 100

3.4.5. Analytical Application of the Proposed Method for BPA Determination The proposed method was used to detect BPA in water samples. Four real water samples (including commercial drinking water, boiling water, tap water and river water) were detected by the present CL method based on the MIMS recognition. The water samples were first pretreated by filtrating with 0.45 µm Millipore filters and then a known amount of BPA was added to water samples. The recoveries were calculated from the typical standard calibration graph in triplicate and listed in Table 2. As it can be seen, the recoveries were in the range between 96–110%, indicating that the proposed method had excellent accuracy.

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Table 2. Results of recovery tests on water samples. Water Sample

BPA Added (µg mL−1 )

BPA Founded (µg mL−1 )

Recovery (%)

River water

0 0.5 1

0.03 0.58 1.09

110 106

Tap water

0 0.5 1

0 0.51 1.03

102 103

Boiled water

0 0.5 1

0 0.52 0.96

104 96

Drinking water

0 0.5 1

0 0.49 0.98

98 98

4. Conclusions In this work, the BPA-MIMS were fabricated and used as a biomimetic recognition material for BPA determination. Due to the special binding sites on the MIMS, the BPA could be absorbed in situ on the MIMS and determined by the CL analysis with improved selectivity and sensitivity. The distinguished advantages of the proposed CL method for BPA determination based on MIMS recognition are shown below. First, Pickering emulsion was combined with MIT for the preparation of BPA-MIMS. By locating the stabilizing SiO2 NPs with proper hydrophilic–hydrophobic properties at oil/water interfaces, the coalescence was successfully prevented and the droplets were stabilized. As a result, the synthesized MIMS could achieve high yields of polymer and good control of particle sizes compared with traditional bulk polymerization. Second, the structure of the BPA templates adsorbed on the MIMS would be changed after having a CL reaction with the KIO4 -luminol system. Then the binding sites between the BPA template and the MIMS would be destroyed and the reacted templates could be easily taken away from the MIMS. During this process, the CL reagents were not only used as detection and sensing reagents, but also as extraction eluents. Organic reagents and buffer solutions, which generally would greatly affect CL reaction, were successfully avoided from being used as the eluents to extract the BPA template from MIMS. Third, by packing the MIMS into the CL detection cell, the BPA could be selectively adsorbed on the MIMS and determined in situ through the CL prohibition. Then elements of BPA adsorption and extraction, together with recognition and sensing, were all integrated on the MIMS at the same time. As a result, extra steps were avoided and the analysis procedures were greatly simplified. Finally, when MIMS were used as the biomimetic recognition element, the selectivity of the CL analysis was obviously improved due to the recognition specificity of MIMS and the sensitivity was greatly enhanced due to the enrichment effect of MIMS. As a result, an improved CL method based on MIMS recognition was successfully proposed in this work and could be applied to real sample determination with excellent selectivity and sensitivity. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4360/10/7/780/s1, Figure S1: Chemiluminescence measurements flow system. a: Luminol; b: KIO4 ; c: NaOH; d: BPA/H2O; Figure S2: Conditions optimization for CL measurement. (a) KIO4 concentration; (b) luminol concentration; (c) NaOH concentration; (d) BPA adsorption time. Author Contributions: Y.X. and Q.W. contributed equally to this article. Q.W., J.X., J.C., M.D., and S.F. designed the experiments; Q.W. and J.C. performed the experiments; Y.X. and Q.W. analyzed the data; M.D. contributed reagents, materials, and analysis tools; Y.X. and Q.W. wrote the paper; and all authors reviewed, edited, and approved the manuscript.

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Acknowledgments: This work was supported by the National Natural Science Foundation of China (grant no. 51404203), the Foundation of Youth Science and Technology Innovation Team of Sichuan Province (grant no. 2015TD0007), and the China Postdoctoral Science Foundation Funded Project (grant no. 2017M612993). Conflicts of Interest: The authors declare no conflict of interest.

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