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A microflow chemiluminescence sensor for indirect determination of dibutyl phthalate by hydrolyzing based on biological recognition materials
Journal of Pharmaceutical and Biomedical Analysis 75 (2013) 123–129
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A microflow chemiluminescence sensor for indirect determination of dibutyl phthalate by hydrolyzing based on biological recognition materials Huamin Qiu a,b , Lulu Fan a,b , Xiangjun Li a,b , Leilei Li a,b , Min Sun a,b , Chuannan Luo a,b,∗ a b
School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong (University of Jinan), China
a r t i c l e
i n f o
Article history: Received 29 September 2012 Received in revised form 7 November 2012 Accepted 7 November 2012 Available online 26 November 2012 Keywords: Microflow chemiluminescence Dibutyl phthalate Magnetic molecularly imprinted polymer Hydrolyzing
a b s t r a c t A microflow chemiluminescence (CL) sensor for determination of dibutyl phthalate (DBP) based on magnetic molecularly imprinted polymer (MMIP) as recognition element was fabricated. Briefly, a hydrophilic molecularly imprinted polymer layer was produced at the surface of Fe3 O4 @SiO2 magnetic nanoparticles (MNPs) via combination of molecular imprinting and reversible stimuli responsive hydrogel. In this protocol, the initial step involved co-precipitation of Fe2+ and Fe3+ in an ammonia solution. Silica was then coated on the Fe3 O4 nanoparticles using a sol–gel method to obtain silica shell magnetic nanoparticles. The MMIP was synthesized using methacrylic acid (MAA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linker and 2, 2-azobisisobutyronitrile (AIBN) as initiator in chloroform. Then the synthesized MMIP and magnetic non-molecular imprinted polymers (MNIP) were employed as recognition by packing into lab-made straight shape tubes, connected in CL analyzer for establishing the novel sensor with a single channel syringe pump. And a mixer for hydrolyzing of DBP was followed. Based on this experiment principle, DBP was determined indirectly. And the MMIP showed satisfactory recognition capacity to DBP, resulting to the wide linear range of 3.84 × 10−8 to 2.08 × 10−5 M and the low detection limit of 2.09 × 10−9 M (3) for DBP. The relative standard deviation (RSD) for DBP (3.20 × 10−6 M) was 1.40% (n = 11). Besides improving sensitivity and selectivity, the sensor was reusable. The proposed DBP–MMIP–CL sensor has been successfully applied to determine DBP in drink samples. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Phthalates and other related compounds used in industry include dibutyl phthalate (DBP), dimethyl phthalate (DMP), diethyl phthalate (DEP), diamyl phthalate (DAP) and dioctyl phthalate (DNOP) [1]. Phthalate esters are widely used as additives in the manufacturing of poly (vinyl chloride) (PVC) plastics to make them flexible [2]. Extensive use of these chemicals resulted in their presence in various environmental matrices such as personal care products (e.g., perfumes, lotions, and cosmetics) [3], paints [4], industrial plastics [5], and certain medical devices [6] and pharmaceuticals [7], including drinking water [8] and other environmental samples [9]. Due to the wide spread use in industry, they are considered as ubiquitous environmental pollutants [10]. They have adverse effects on human health, regarding as endocrine disrupting compounds by means of their carcinogenic action [11,12]. According to the previous report, these methods were used for determination of DBP such as solid-phase extraction [1], liquid
∗ Corresponding author at: School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China. Tel.: +86 531 89736065. E-mail address: chm yfl[email protected] (C. Luo). 0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2012.11.010
chromatography [13,14]. Pre-concentration technique was important before determination such as solid-phase extraction (SPE) [15] and solid-phase micro-extraction (SPME) [16]. However, those methods always lead to high blank values and lower limit detection due to complex matrixs in sample preparation procedures. Therefore, it is practically necessary to establish a new method with high selectivity and sensitivity to determine phthalates and related compound in the complex samples. Chemiluminescence (CL) has caused considerable attention for its rapid determination and accuracy. According to the linear relationship between CL intensity and concentration of solution [17], CL was widely used in determination as an analysis method with high sensitivity, wide linear range, and simple equipment. Molecularly imprinted technology (MIT) was introduced into CL for improving selectivity which has high specificity recognition [18]. Magnetite (Fe3 O4 ) nanoparticles (NPs) have drawn considerable attention because of the fundamental scientific interest [19]. It has widely promising applications in magnetic fluids [20], catalysis [21], sensors [22], biomedicine [23], spintronics [24], magnetic recording devices [25] and environmental remediation [26]. Additionally, Fe3 O4 NPs also show advantages such as low toxicity, low cost and eco-friendliness which serve as a stabilizer. Molecularly Imprinted Polymer (MIP) are stable synthetic polymers possessing
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selective molecular cognition sites, which were obtained by using high amounts of cross linking monomers in the presence of template molecule. The advantages of MIP, such as stability at extreme of pH and temperature, ease of preparation, low cost, and reusability. The combination with Fe3 O4 and MIT was widely used for easy separating and high selectivity. In this paper, a novel method for adsorption and determination of DBP using Magnetic Molecularly Imprinted Polymer (MMIP) as recognition element is developed. MMIP was prepared using Fe3 O4 @SiO2 as support, in which methacrylic acid (MAA, functional monomer) and ethylene glycol dimethacrylate (EGDMA, crosslinker) are in the presence of DBP molecules. Particles of the MMIP and Magnetic Non-Molecularly Imprinted Polymer (MNIP) were packed into straight shape glass tubes which served as flow cell, the flow cells were positioned in front of a mixer which for hydrolyzing. The DBP which cannot adsorb by MNIP was hydrolysed in the mixer, the product reacting with the alkaline luminol-H2 O2 to produce strong chemiluminescence (CL). The DBP is selectively adsorbed on the MMIP on-line, no CL produced after the same process. According to the difference of CL, the concentration of hydrolyse product was obtained, and the concentration of DBP was obtained too. After the CL reaction, the absorbed DBP was destroyed and removed by the flow eluent with cavities left on the MMIP ready for the next adsorption assay. The reusable sensor was successfully used in determining DBP of real samples.
(TEOS) [28]. The mixtures were reacted for 12 h at the room temperature with stirring (400 rpm). The products were collected by magnetic separation, washed with diluted hydrochloric acid and ultrapure water, and dried in the vacuum. Fe3 O4 @SiO2 NPs were modified by MPS. Briefly, 250 mg of the Fe3 O4 @SiO2 NPs was dispersed in 50 mL of anhydrous toluene containing 5 mL of MPS, and the mixture was allowed to react at 70 ◦ C for 12 h under dry nitrogen. After magnetic separation, washing by water and drying in vacuum, the products (Fe3 O4 @SiO2 C C) were obtained. The preparation of MMIP is as follows. DBP (0.2 mmol) and MAA (0.8 mmol) were dissolved in chloroform (50 mL). The mixture was shaked in a water bath at 25 ◦ C for 12 h. And then, 200 mg Fe3 O4 @SiO2 C C were added into the system with shaking for 2 h. Subsequently, 4.0 mmol EGDMA and 0.05 mmol AIBN were added into the system and the mixture was sonicated in a water bath. After sparged with nitrogen gas for 5 min to remove oxygen, system was reacted at 60 ◦ C under nitrogen gas for 24 h. After polymerization, the template molecule was removed by washing the polymers with ultrapure water and methanol-acetic acid (9:1, v/v) in turn until no DBP was detected by an UV–vis spectrophotometer at 266 nm. The obtained products (MMIP) were dried at 40 ◦ C under vacuum. For comparison, MNIP were prepared under the same conditions without DBP template. 2.4. MMIP and MNIP rebinding performance
2. Experimental 2.1. Materials and reagents DBP, MAA, EGDMA and AIBN were purchased from Sigma Chemical Company; The EGDMA and MAA were distilled to remove inhibitors. AIBN was re-crystallized prior to its use. The DBP stock solution (1.0 × 10−2 M) was prepared by ethanol, luminol stock solution (1.0 × 10−2 M) was prepared in the alkaline solution. And the stock solutions were stored in refrigerator, respectively. The methanol, acetone, acetic acid, sodium hydroxide, and all the other chemicals used were of analytical reagent grade obtained from Tianjin Chemical Co., Ltd. (China). Doubly distilled water was used throughout the work.
MMIP and MNIP were obtained from the above methods. MMIP and MNIP (20 mg) were soaked in a 10 mL alkaline EtOH of DBP at various concentrations (5 × 10−6 to 5 × 10−4 M), respectively. The solution was performed in a water bath at 30 ◦ C with oscillation for 3 h. Then, MMIP and MNIP were isolated by an external magnetic field, and the remained DBP in solution was measured by CL. The experimental data was presented as the adsorption capacity (Q) per unit mass (mg) of the nanoparticles, and calculated from Eq. (1): (co − ce ) × V m
Q =
(1)
In Eq. (1), co (mg mL−1 ) is the initial concentration of DBP solution, ce (mg mL−1 ) is the DBP concentration of the supernatant solution, V (mL) is the volume of the initial solution and m (mg) is the mass of MMIP or MNIP.
2.2. Apparatus
2.5. Study of molecular recognition and selectivity
The IFFM-E flow injection CL analyzer (Xi’an Remex Electronic instrument High-Tech Ltd., China) was equipped with an automatic injection system and a detection system. PTFE tube (0.8 mm i.d.) was used to connect all components in the flow system. The CL signal was analyzed with a computer. LSP04-1A Single Channel Syringe Pump (Baoding Longer Precision Pump Co., Ltd.)
Diethylhexyl Phthalate (DEHP), l-Tryptophan (l-Try), Sulfamethoxazole (SMZ) and Chloramphenicol (CAP) were chosen for the study molecular recognition and selectivity. The same concentration of solution was prepared for fully adsorption by MMIP. And according to the part 2.4, the Q and ce were obtained. Static adsorption distribution coefficient Kd was obtained as Eq. (2), separation factor ˛ was obtained according to Eq. (3).
2.3. Preparation of MMIP and MNIP
Kd =
Fe3 O4 NPs were prepared by a modified coprecipitation method [27] as follows: The Fe3 O4 NPs were prepared by coprecipitation method. Briefly, FeCl2 ·4H2 O (0.01 mol) and FeCl3 ·6H2 O (0.02 mol) were dissolved in 80 mL water with vigorous stirring (800 rpm) under nitrogen. 10 mL of NH3 ·H2 O (28%, wt.) was added in system dropwise, and the reaction was maintained at 80 ◦ C for 30 min. The black precipitation (Fe3 O4 NPs) was separated with a permanent magnet, and washed. 300 mg Fe3 O4 NPs were dispersed in 40 mL EtOH and 4 mL of ultrapure water by ultrasonication for 15 min, followed by the addition of 5 mL NH3 ·H2 O (28%, wt.) and 2 mL tetraethyl orthosilicate
˛=
Q ce
(2)
Kdi Kdj
(3)
Kdi was the static adsorption distribution coefficient of DBP, and the Kdj was the static adsorption distribution coefficient of other substances. Adsorption ability was higher follows the increase of Kd , selectivity was lower follows the increase of ˛. 2.6. Preparation of the MMIP column As shown in Fig. 1, 40.0 mg of MMIP and MNIP were dispersed in the solution, which was delivered to flow through and MMIP and
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Fig. 1. The schematic diagram of flow system.
MNIP were fastened in the column 1 and 2 by the additional magnetic field. The obtained MMIP and MNIP columns were connected into the flow system as recognition element. 2.7. Procedure for the determination of DBP The schematic diagram of MMIP-CL sensor was shown in Fig. 1 and the determination could be summarized as five steps: 2.7.1. Transit and hydrolyzing of DBP In this step, switch valve (S) was in connection with 2, standard solution (c0 ) was delivered to flow through the MNIP-cell and DBP molecules in the standard solution was NOT selectively adsorbed on the polymer, and delivered into the mixer for hydrolyzing. 2.7.2. Chemiluminescence detection the product of hydrolyzing In this step, switch valve was in connection with 2. The merged stream of luminol, hydrogen peroxide and sodium hydroxide flowed to react the solution with product of hydrolyzing to produce CL1 in the flow cell. 2.7.3. Recognition and adsorption of DBP In this step, S was in connection with 1, standard solution was delivered to flow through the MMIP-cell and DBP molecules in the standard solution were selectively adsorbed on the polymer. None of DBP was flowed into the mixer. The merged stream of luminol, hydrogen peroxide and sodium hydroxide flowed to react with standard solution without product of hydrolyzing to produce CL2 . 2.7.4. Cleaning the MMIP-cell and MNIP-cell for reusable In this step, S was in connection with 1 and 2, eluent and ultrapure water was instead and flowed through the MMIP-cell and MNIP-call to remove DBP for sample solution determination by same processing method. 2.7.5. Calculation the concentration of DBP I = CL2 − CL1 , according to regression equation of CH3 CH2 CH2 CH2 OH, the concentration (c1 ) of product of hydrolyzing (CH3 CH2 CH2 CH2 OH) of standard solution and the concentration (c2 ) of product of hydrolyzing of sample solution were obtained, and the concentration of DBP which hydrolyzed in the standard solution and sample solution were obtained according to the Eqs. (4)–(6).
As shown in Fig. 1, the solution was delivered by single channel syringe pump to the part of “M” for hydrolyzing, the time of deliver was 20 s, and the time of hydrolyzing was 30 s. And the time of determination using MMIP was 30 s, which was the appropriate time for stable CL, and cleaning the MMIP-cell needed 20 s, and adding the time of using MNIP, the time of determination was 80 s, as a result, the time of one analysis was 130 s ideally. 3. Results and discussion 3.1. Preparation of MMIP The synthesis of the MMIP is a multistep procedure, which involved synthesis of Fe3 O4 NPs, silica-shell deposition (Fe3 O4 @SiO2 ), modification of Fe3 O4 @SiO2 with MMIP and removal of DBP template. At first, Fe3 O4 NPs were prepared by the coprecipitation method. Secondly, the surface of Fe3 O4 NPs was coated with silica by TEOS in a sol–gel process. SiO2 shell provided a biocompatible and hydrophilic surface, and prevented oxidation of Fe3 O4 . Furthermore, silanol groups were beneficial to chemical modification on the surface of Fe3O4@SiO2. Thus, double bonds were introduced on Fe3 O4 @SiO2 using MPS to ensure tight growth of imprinted layer. Finally, a hydrophilic magnetic molecularly imprinted layer on Fe3 O4 @SiO2 was produced via a combination of molecular imprinting and reversible stimuli responsive hydrogel [29] which performs reversibility response stimulated by external environment changes. The molecular recognition capability of MMIP was affected by many factors, such as the amount of the template molecule, the type and amount of monomer and cross linking agent. As a porogenic agent and dissolvent, solvent plays an important role in the preparation of the non-covalent type molecularly imprinted polymer. The weak polarity solvent is usually selected as the optimal condition because of non-covalent molecular recognition. In consideration of the weak polarity and excellent dissolving capacity, chloroform was selected as the porogen. MAA, EGDMA and AIBN were used as functional monomers, cross-linker and initiator, respectively. The high cross-linker ratio was generally preferred in order to access permanently porous materials and to generate adequate mechanical stability material, polymers with cross-linker ratio in excess of 80% had been often used. According to reported work, the optimum ratio of the molar amounts of the
(4) c1 Hydrolysis efficiency : w = 2c0 The concentration of DBP in the sample solution cx =
(5) c2 c0 c1
(6)
template molecule, functional monomer, cross-linker and initiator was 1:4:20:0.25. The influences of polymerization time, temperature and hunting speed were also investigated and were confirmed as 24 h, at 60 ◦ C and with 150 rpm.
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Fig. 2. TEM and size statistics of MMIP.
Fig. 3. FT-IR spectra and XRD of MMIP.
3.2. Characterization of MMIP As can be observed in Fig. 2, the diameters of MMIP are approximately 300 nm and 400 nm. So, the thickness of imprinting layer was estimated to be about 50 nm. The TEM images suggested that core–shell nanoparticles with more regular morphological features were prepared. Because of the nano sized particles and thin imprinting layer, the MMIP had much more specific surface area compared with traditional micron-sized MMIP. Size of MMIP was around at 300–400 nm obtained from size statistics. Based on this characteristic, the adsorption capacity of MMIP was obviously increased (Fig. 3). In the spectrum of (A), the peak at 3450, 1760 cm−1 is the –COO bending band, four bands at 2935, 2495, 1622, 1385 cm−1 are the characteristics of benzene ring, 544 cm−1 is the characteristics of Fe3 O4 which gives evidence of the successful preparation of the MMIP. 1390 cm−1 (the characteristics of –CH3 ), 1496 cm−1 (the characteristics of –CH2 ) was able to justify the preparation of the MMIP. X-ray diffraction (XRD) measurements were employed to investigate the phase and structure. As shown in Fig. 4B, the peaks at 2 values of 30.0◦ , 35.3◦ , 42.9◦ , 53.4◦ , 56.9◦ , and 62.5◦ are consistent
with the standard XRD data of Fe3 O4 indicating the coexistence of Fe3 O4 in the recognition materials. 3.3. The selectivity adsorption of MMIP In order to examine the selectivity adsorption, the molecular recognition experiment was also carried out. And the results were shown in Table 1. According to principle as follows, the adsorption ability was higher follows the increase of Kd , selectivity was higher when ˛ was closed to 1, Kd was larger, the adsorption ability was higher, in the results, the Kd of DBP was 30.4, and the others were 6.33, 6.08,
Table 1 the results of selectivity adsorption. Substrate
Q(10−5 mol/g)
ce (10−5 M)
Kd
˛
DBP Bisphenol A l-tryptophan Epinephrine Sulfamethoxazole
30.58 0.23 0.19 0.38 0.09
1.006 0.0363 0.0313 0.0613 0.0154
30.40 6.33 6.08 6.20 5.85
1.00 4.80 5.00 4.90 5.19
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Fig. 4. The possible chemiluminescence mechanism of CL reaction.
6.10, 5.85, respectively. And the ˛ of DBP was 1, the others was higher than DBP which proved the selectivity of MMIP was very well. 3.4. Optimization of CL conditions The reaction between luminol and H2 O2 shows weak CL emission and a strong CL emission was recorded when the product of hydrolyzing was injected into the above mixed solution. The peak heights of the CL emission were proportional to the concentration of product of hydrolyzing. The optimization experiment was carried out to get a better knowledge of the CL reaction of luminol – H2 O2 –NaOH through the schematic diagram shown in Fig. 1. With the luminol concentration in the range from 1.0 × 10−6 to 1.0 × 10−4 M, the CL intensity increased with raising the concentration of luminol up to 1.0 × 10−5 M. Above 1.0 × 10−5 M, the CL intensity decreased, thus, the 1.0 × 10−5 M luminol was used for further work. The effect of H2 O2 concentration was examined from 1.0 × 10−3 to 4.0 × 10−2 M, the CL intensity reached maximum when H2 O2 was 1.0 × 10−2 M. The effect of NaOH concentration as medium of luminol was examined over 1.0 × 10−3 to 0.1 M range, and the CL intensity reached up to maximum value when 0.01 M NaOH was used. But higher concentration of NaOH lowered the CL intensity of this system. The adsorption time which depends on peristaltic pumps speed was an important parameter for the amount of DBP absorbed on the MMIP. It was observed that the adsorption reached maximum at 10 mL min−1 . Simultaneously, according to experiment results, the other substances in the samples would be adsorbed by MMIP when the pumps speed was too small. DBP molecules cannot be adsorbed by MMIP completely under too large pumps speed. So the 10 mL min−1 pumps speed was chosen for the determination of DBP with good results. As a result, the optimal conditions for the CL system is 1.0 × 10−5 M luminol, 1 × 10−2 M H2 O2 , and 1.0 × 10−2 M NaOH, respectively.
BDP was obtained which was wider and lower than the common methods as shown in Table 2. The relative standard deviation (RSD) was 1.40% (n = 11) for BDP of 3.20 × 10−6 M. The regression equation was ICL = 6594 + 1701c (c being the DBP concentration (M)) with R = 0.9998. According to the results, the higher selectivity and more sensitivity of MMIP–CL sensor were obtained, the MMIP–CL sensor has wider linear range and lower detection limit, correlation coefficient was more closed to 1. Molecularly imprinted polymer and chemiluminescence were used at the same time for determination. According to the results, MMIP–CL sensor was more suitable for applying to quantitative analysis. 3.7. Application of MMIP–CL sensor As shown by the experiments, the optimal conditions for the CL system is 1.0 × 10−5 M luminol, 1 × 10−2 M H2 O2 , and 1.0 × 10−2 M NaOH, respectively. Under optimal conditions, the wide linear range (3.84 × 10−8 2.08 × 10−5 M), and the low detection limit (2.09 × 10−9 M) was obtained. The regression equation was ICL = 6594 + 1701c (c being the DBP concentration (M)) with R = 0.9998. In order to evaluate the applicability and reliability of the proposed method, it was applied to determine drink samples. Drink samples (10.0 mL) were obtained from a brand juice drinks. The samples were centrifuged at 5000 rpm for 20 min. The supernatant was transferred into a flask and diluted with ultrapure water for analysis. The blank experiment was carried out at the same time. The results obtained are shown in Tables 3 and 4. 3.8. Reusability The reusable of the sensor was evaluated by comparing the adsorption of MMIP. According to the experiments (part 2.4 and 2.7), the MMIP was extracted with methanol/acetic acid (9/1, v/v) overnight after used, and then for adsorption to get the adsorption
3.5. The study of possible mechanism of CL reaction The possible chemiluminescence mechanism of the luminol – H2 O2 –NaOH and the product of hydrolyzing (CH3 CH2 CH2 CH2 OH) are showed in Fig. 4. After CH3 CH2 CH2 CH2 OH was oxidated by H2 O2 , a kind of intermediate (A) was producing at electronic state, then, ‘hv’ was producing after excitation energy released which makes the CL emission was made stronger. 3.6. The analytical performance of MMIP-CL sensor Under optimal conditions, a wide linear range from 3.84 × 10−8 to 2.08 × 10−5 M and a low detection limit of 2.09 × 10−9 M (3) for
Table 2 The comparison of our work and the common methods. Works
Method
Linear range (M)
Detection limit (M)
Our work
Microflow chemiluminescence sensor Chromatographic Gas chromatography–mass spectrometry Selective solid-phase extraction
3.84 × 10−8 to 2.08 × 10−5
2.09 × 10−9
1.26 × 10−7 to 1.08 × 10−5 6.47 × 10−8 to 1.80 × 10−5
3.95 × 10−7 6.47 × 10−8
–
4.67 × 10−8
[30] [31]
[1]
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Table 3 Analysis of the standard solution (units of c measurement was M). Standard solution
1
c0 I c1
2
3
4
5
6
Average
6605 0.0068
6606 0.0071
6606 0.0073
0.018 6606 0.0070
0.018 6605 0.0067
6609 0.0070
6606 0.0069
Table 4 Analysis of the sample solution (n = 6, units of c measurement was M). Samples
c2
cDBP-hydrolyzing
cx
RSD
Adding
Founding
1 2 3
0.0400 0.0786 0.10607
0.0200 0.0393 0.0803
0.1030 0.2020 0.4130
3.87% 2.98% 3.08%
0.05 0.05 0.05
0.051 0.052 0.052
Recovery 102% 104% 104%
RSD 2.48% 2.54% 2.69%
References
Fig. 5. The analysis of reusability.
capacity. The results are shown in Fig. 5, the difference between ten times was 0.14 × 10−5 mol/g which is tolerable. The sensor could be used more than hundreds of times before the adsorption began to decrease. This was possibly due to loss of binding sites, however it was easy to replace the MMIP-cell and MNIP-cell in the channel.
4. Conclusions In this paper, a novel CL method for adsorption and determination of DBP using MMIP as recognition element is developed. MMIP was prepared using Fe3 O4 as support, in which methacrylic acid (MAA, functional monomer) and ethylene glycol dimethacrylate (EGDMA, cross-linker) are in the presence of DBP molecules. Flow cells were positioned in front of a mixer which for hydrolyzing. The MMIP showed satisfactory recognition capacity to DBP, resulting to the wide linear range of 3.84 × 10−8 to 2.08 × 10−5 M and the low detection limit of 2.09 × 10−9 M (3) for DBP. The relative standard deviation (RSD) for DBP (3.20 × 10−6 M) was 1.40% (n = 11) via the novel sensor. Besides improving sensitivity and selectivity, the sensor was reusable. The proposed DBP–MMIP–CL sensor has been successfully applied to determine DBP in juice drinks samples.
Acknowledgements This work was supported by the Shandong Provincial Natural Science Foundation of China (Nos. ZR2012BM020, ZR2012BQ018) and the Scientific and Technological Development Plan Item of Jinan City in China (No. 201202088).
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
A microflow chemiluminescence sensor for indirect determination of dibutyl phthalate by hydrolyzing based on biological recognition materials Huamin Qiu a,b , Lulu Fan a,b , Xiangjun Li a,b , Leilei Li a,b , Min Sun a,b , Chuannan Luo a,b,∗ a b
School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong (University of Jinan), China
a r t i c l e
i n f o
Article history: Received 29 September 2012 Received in revised form 7 November 2012 Accepted 7 November 2012 Available online 26 November 2012 Keywords: Microflow chemiluminescence Dibutyl phthalate Magnetic molecularly imprinted polymer Hydrolyzing
a b s t r a c t A microflow chemiluminescence (CL) sensor for determination of dibutyl phthalate (DBP) based on magnetic molecularly imprinted polymer (MMIP) as recognition element was fabricated. Briefly, a hydrophilic molecularly imprinted polymer layer was produced at the surface of Fe3 O4 @SiO2 magnetic nanoparticles (MNPs) via combination of molecular imprinting and reversible stimuli responsive hydrogel. In this protocol, the initial step involved co-precipitation of Fe2+ and Fe3+ in an ammonia solution. Silica was then coated on the Fe3 O4 nanoparticles using a sol–gel method to obtain silica shell magnetic nanoparticles. The MMIP was synthesized using methacrylic acid (MAA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linker and 2, 2-azobisisobutyronitrile (AIBN) as initiator in chloroform. Then the synthesized MMIP and magnetic non-molecular imprinted polymers (MNIP) were employed as recognition by packing into lab-made straight shape tubes, connected in CL analyzer for establishing the novel sensor with a single channel syringe pump. And a mixer for hydrolyzing of DBP was followed. Based on this experiment principle, DBP was determined indirectly. And the MMIP showed satisfactory recognition capacity to DBP, resulting to the wide linear range of 3.84 × 10−8 to 2.08 × 10−5 M and the low detection limit of 2.09 × 10−9 M (3) for DBP. The relative standard deviation (RSD) for DBP (3.20 × 10−6 M) was 1.40% (n = 11). Besides improving sensitivity and selectivity, the sensor was reusable. The proposed DBP–MMIP–CL sensor has been successfully applied to determine DBP in drink samples. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Phthalates and other related compounds used in industry include dibutyl phthalate (DBP), dimethyl phthalate (DMP), diethyl phthalate (DEP), diamyl phthalate (DAP) and dioctyl phthalate (DNOP) [1]. Phthalate esters are widely used as additives in the manufacturing of poly (vinyl chloride) (PVC) plastics to make them flexible [2]. Extensive use of these chemicals resulted in their presence in various environmental matrices such as personal care products (e.g., perfumes, lotions, and cosmetics) [3], paints [4], industrial plastics [5], and certain medical devices [6] and pharmaceuticals [7], including drinking water [8] and other environmental samples [9]. Due to the wide spread use in industry, they are considered as ubiquitous environmental pollutants [10]. They have adverse effects on human health, regarding as endocrine disrupting compounds by means of their carcinogenic action [11,12]. According to the previous report, these methods were used for determination of DBP such as solid-phase extraction [1], liquid
∗ Corresponding author at: School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China. Tel.: +86 531 89736065. E-mail address: chm yfl[email protected] (C. Luo). 0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2012.11.010
chromatography [13,14]. Pre-concentration technique was important before determination such as solid-phase extraction (SPE) [15] and solid-phase micro-extraction (SPME) [16]. However, those methods always lead to high blank values and lower limit detection due to complex matrixs in sample preparation procedures. Therefore, it is practically necessary to establish a new method with high selectivity and sensitivity to determine phthalates and related compound in the complex samples. Chemiluminescence (CL) has caused considerable attention for its rapid determination and accuracy. According to the linear relationship between CL intensity and concentration of solution [17], CL was widely used in determination as an analysis method with high sensitivity, wide linear range, and simple equipment. Molecularly imprinted technology (MIT) was introduced into CL for improving selectivity which has high specificity recognition [18]. Magnetite (Fe3 O4 ) nanoparticles (NPs) have drawn considerable attention because of the fundamental scientific interest [19]. It has widely promising applications in magnetic fluids [20], catalysis [21], sensors [22], biomedicine [23], spintronics [24], magnetic recording devices [25] and environmental remediation [26]. Additionally, Fe3 O4 NPs also show advantages such as low toxicity, low cost and eco-friendliness which serve as a stabilizer. Molecularly Imprinted Polymer (MIP) are stable synthetic polymers possessing
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selective molecular cognition sites, which were obtained by using high amounts of cross linking monomers in the presence of template molecule. The advantages of MIP, such as stability at extreme of pH and temperature, ease of preparation, low cost, and reusability. The combination with Fe3 O4 and MIT was widely used for easy separating and high selectivity. In this paper, a novel method for adsorption and determination of DBP using Magnetic Molecularly Imprinted Polymer (MMIP) as recognition element is developed. MMIP was prepared using Fe3 O4 @SiO2 as support, in which methacrylic acid (MAA, functional monomer) and ethylene glycol dimethacrylate (EGDMA, crosslinker) are in the presence of DBP molecules. Particles of the MMIP and Magnetic Non-Molecularly Imprinted Polymer (MNIP) were packed into straight shape glass tubes which served as flow cell, the flow cells were positioned in front of a mixer which for hydrolyzing. The DBP which cannot adsorb by MNIP was hydrolysed in the mixer, the product reacting with the alkaline luminol-H2 O2 to produce strong chemiluminescence (CL). The DBP is selectively adsorbed on the MMIP on-line, no CL produced after the same process. According to the difference of CL, the concentration of hydrolyse product was obtained, and the concentration of DBP was obtained too. After the CL reaction, the absorbed DBP was destroyed and removed by the flow eluent with cavities left on the MMIP ready for the next adsorption assay. The reusable sensor was successfully used in determining DBP of real samples.
(TEOS) [28]. The mixtures were reacted for 12 h at the room temperature with stirring (400 rpm). The products were collected by magnetic separation, washed with diluted hydrochloric acid and ultrapure water, and dried in the vacuum. Fe3 O4 @SiO2 NPs were modified by MPS. Briefly, 250 mg of the Fe3 O4 @SiO2 NPs was dispersed in 50 mL of anhydrous toluene containing 5 mL of MPS, and the mixture was allowed to react at 70 ◦ C for 12 h under dry nitrogen. After magnetic separation, washing by water and drying in vacuum, the products (Fe3 O4 @SiO2 C C) were obtained. The preparation of MMIP is as follows. DBP (0.2 mmol) and MAA (0.8 mmol) were dissolved in chloroform (50 mL). The mixture was shaked in a water bath at 25 ◦ C for 12 h. And then, 200 mg Fe3 O4 @SiO2 C C were added into the system with shaking for 2 h. Subsequently, 4.0 mmol EGDMA and 0.05 mmol AIBN were added into the system and the mixture was sonicated in a water bath. After sparged with nitrogen gas for 5 min to remove oxygen, system was reacted at 60 ◦ C under nitrogen gas for 24 h. After polymerization, the template molecule was removed by washing the polymers with ultrapure water and methanol-acetic acid (9:1, v/v) in turn until no DBP was detected by an UV–vis spectrophotometer at 266 nm. The obtained products (MMIP) were dried at 40 ◦ C under vacuum. For comparison, MNIP were prepared under the same conditions without DBP template. 2.4. MMIP and MNIP rebinding performance
2. Experimental 2.1. Materials and reagents DBP, MAA, EGDMA and AIBN were purchased from Sigma Chemical Company; The EGDMA and MAA were distilled to remove inhibitors. AIBN was re-crystallized prior to its use. The DBP stock solution (1.0 × 10−2 M) was prepared by ethanol, luminol stock solution (1.0 × 10−2 M) was prepared in the alkaline solution. And the stock solutions were stored in refrigerator, respectively. The methanol, acetone, acetic acid, sodium hydroxide, and all the other chemicals used were of analytical reagent grade obtained from Tianjin Chemical Co., Ltd. (China). Doubly distilled water was used throughout the work.
MMIP and MNIP were obtained from the above methods. MMIP and MNIP (20 mg) were soaked in a 10 mL alkaline EtOH of DBP at various concentrations (5 × 10−6 to 5 × 10−4 M), respectively. The solution was performed in a water bath at 30 ◦ C with oscillation for 3 h. Then, MMIP and MNIP were isolated by an external magnetic field, and the remained DBP in solution was measured by CL. The experimental data was presented as the adsorption capacity (Q) per unit mass (mg) of the nanoparticles, and calculated from Eq. (1): (co − ce ) × V m
Q =
(1)
In Eq. (1), co (mg mL−1 ) is the initial concentration of DBP solution, ce (mg mL−1 ) is the DBP concentration of the supernatant solution, V (mL) is the volume of the initial solution and m (mg) is the mass of MMIP or MNIP.
2.2. Apparatus
2.5. Study of molecular recognition and selectivity
The IFFM-E flow injection CL analyzer (Xi’an Remex Electronic instrument High-Tech Ltd., China) was equipped with an automatic injection system and a detection system. PTFE tube (0.8 mm i.d.) was used to connect all components in the flow system. The CL signal was analyzed with a computer. LSP04-1A Single Channel Syringe Pump (Baoding Longer Precision Pump Co., Ltd.)
Diethylhexyl Phthalate (DEHP), l-Tryptophan (l-Try), Sulfamethoxazole (SMZ) and Chloramphenicol (CAP) were chosen for the study molecular recognition and selectivity. The same concentration of solution was prepared for fully adsorption by MMIP. And according to the part 2.4, the Q and ce were obtained. Static adsorption distribution coefficient Kd was obtained as Eq. (2), separation factor ˛ was obtained according to Eq. (3).
2.3. Preparation of MMIP and MNIP
Kd =
Fe3 O4 NPs were prepared by a modified coprecipitation method [27] as follows: The Fe3 O4 NPs were prepared by coprecipitation method. Briefly, FeCl2 ·4H2 O (0.01 mol) and FeCl3 ·6H2 O (0.02 mol) were dissolved in 80 mL water with vigorous stirring (800 rpm) under nitrogen. 10 mL of NH3 ·H2 O (28%, wt.) was added in system dropwise, and the reaction was maintained at 80 ◦ C for 30 min. The black precipitation (Fe3 O4 NPs) was separated with a permanent magnet, and washed. 300 mg Fe3 O4 NPs were dispersed in 40 mL EtOH and 4 mL of ultrapure water by ultrasonication for 15 min, followed by the addition of 5 mL NH3 ·H2 O (28%, wt.) and 2 mL tetraethyl orthosilicate
˛=
Q ce
(2)
Kdi Kdj
(3)
Kdi was the static adsorption distribution coefficient of DBP, and the Kdj was the static adsorption distribution coefficient of other substances. Adsorption ability was higher follows the increase of Kd , selectivity was lower follows the increase of ˛. 2.6. Preparation of the MMIP column As shown in Fig. 1, 40.0 mg of MMIP and MNIP were dispersed in the solution, which was delivered to flow through and MMIP and
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Fig. 1. The schematic diagram of flow system.
MNIP were fastened in the column 1 and 2 by the additional magnetic field. The obtained MMIP and MNIP columns were connected into the flow system as recognition element. 2.7. Procedure for the determination of DBP The schematic diagram of MMIP-CL sensor was shown in Fig. 1 and the determination could be summarized as five steps: 2.7.1. Transit and hydrolyzing of DBP In this step, switch valve (S) was in connection with 2, standard solution (c0 ) was delivered to flow through the MNIP-cell and DBP molecules in the standard solution was NOT selectively adsorbed on the polymer, and delivered into the mixer for hydrolyzing. 2.7.2. Chemiluminescence detection the product of hydrolyzing In this step, switch valve was in connection with 2. The merged stream of luminol, hydrogen peroxide and sodium hydroxide flowed to react the solution with product of hydrolyzing to produce CL1 in the flow cell. 2.7.3. Recognition and adsorption of DBP In this step, S was in connection with 1, standard solution was delivered to flow through the MMIP-cell and DBP molecules in the standard solution were selectively adsorbed on the polymer. None of DBP was flowed into the mixer. The merged stream of luminol, hydrogen peroxide and sodium hydroxide flowed to react with standard solution without product of hydrolyzing to produce CL2 . 2.7.4. Cleaning the MMIP-cell and MNIP-cell for reusable In this step, S was in connection with 1 and 2, eluent and ultrapure water was instead and flowed through the MMIP-cell and MNIP-call to remove DBP for sample solution determination by same processing method. 2.7.5. Calculation the concentration of DBP I = CL2 − CL1 , according to regression equation of CH3 CH2 CH2 CH2 OH, the concentration (c1 ) of product of hydrolyzing (CH3 CH2 CH2 CH2 OH) of standard solution and the concentration (c2 ) of product of hydrolyzing of sample solution were obtained, and the concentration of DBP which hydrolyzed in the standard solution and sample solution were obtained according to the Eqs. (4)–(6).
As shown in Fig. 1, the solution was delivered by single channel syringe pump to the part of “M” for hydrolyzing, the time of deliver was 20 s, and the time of hydrolyzing was 30 s. And the time of determination using MMIP was 30 s, which was the appropriate time for stable CL, and cleaning the MMIP-cell needed 20 s, and adding the time of using MNIP, the time of determination was 80 s, as a result, the time of one analysis was 130 s ideally. 3. Results and discussion 3.1. Preparation of MMIP The synthesis of the MMIP is a multistep procedure, which involved synthesis of Fe3 O4 NPs, silica-shell deposition (Fe3 O4 @SiO2 ), modification of Fe3 O4 @SiO2 with MMIP and removal of DBP template. At first, Fe3 O4 NPs were prepared by the coprecipitation method. Secondly, the surface of Fe3 O4 NPs was coated with silica by TEOS in a sol–gel process. SiO2 shell provided a biocompatible and hydrophilic surface, and prevented oxidation of Fe3 O4 . Furthermore, silanol groups were beneficial to chemical modification on the surface of Fe3O4@SiO2. Thus, double bonds were introduced on Fe3 O4 @SiO2 using MPS to ensure tight growth of imprinted layer. Finally, a hydrophilic magnetic molecularly imprinted layer on Fe3 O4 @SiO2 was produced via a combination of molecular imprinting and reversible stimuli responsive hydrogel [29] which performs reversibility response stimulated by external environment changes. The molecular recognition capability of MMIP was affected by many factors, such as the amount of the template molecule, the type and amount of monomer and cross linking agent. As a porogenic agent and dissolvent, solvent plays an important role in the preparation of the non-covalent type molecularly imprinted polymer. The weak polarity solvent is usually selected as the optimal condition because of non-covalent molecular recognition. In consideration of the weak polarity and excellent dissolving capacity, chloroform was selected as the porogen. MAA, EGDMA and AIBN were used as functional monomers, cross-linker and initiator, respectively. The high cross-linker ratio was generally preferred in order to access permanently porous materials and to generate adequate mechanical stability material, polymers with cross-linker ratio in excess of 80% had been often used. According to reported work, the optimum ratio of the molar amounts of the
(4) c1 Hydrolysis efficiency : w = 2c0 The concentration of DBP in the sample solution cx =
(5) c2 c0 c1
(6)
template molecule, functional monomer, cross-linker and initiator was 1:4:20:0.25. The influences of polymerization time, temperature and hunting speed were also investigated and were confirmed as 24 h, at 60 ◦ C and with 150 rpm.
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Fig. 2. TEM and size statistics of MMIP.
Fig. 3. FT-IR spectra and XRD of MMIP.
3.2. Characterization of MMIP As can be observed in Fig. 2, the diameters of MMIP are approximately 300 nm and 400 nm. So, the thickness of imprinting layer was estimated to be about 50 nm. The TEM images suggested that core–shell nanoparticles with more regular morphological features were prepared. Because of the nano sized particles and thin imprinting layer, the MMIP had much more specific surface area compared with traditional micron-sized MMIP. Size of MMIP was around at 300–400 nm obtained from size statistics. Based on this characteristic, the adsorption capacity of MMIP was obviously increased (Fig. 3). In the spectrum of (A), the peak at 3450, 1760 cm−1 is the –COO bending band, four bands at 2935, 2495, 1622, 1385 cm−1 are the characteristics of benzene ring, 544 cm−1 is the characteristics of Fe3 O4 which gives evidence of the successful preparation of the MMIP. 1390 cm−1 (the characteristics of –CH3 ), 1496 cm−1 (the characteristics of –CH2 ) was able to justify the preparation of the MMIP. X-ray diffraction (XRD) measurements were employed to investigate the phase and structure. As shown in Fig. 4B, the peaks at 2 values of 30.0◦ , 35.3◦ , 42.9◦ , 53.4◦ , 56.9◦ , and 62.5◦ are consistent
with the standard XRD data of Fe3 O4 indicating the coexistence of Fe3 O4 in the recognition materials. 3.3. The selectivity adsorption of MMIP In order to examine the selectivity adsorption, the molecular recognition experiment was also carried out. And the results were shown in Table 1. According to principle as follows, the adsorption ability was higher follows the increase of Kd , selectivity was higher when ˛ was closed to 1, Kd was larger, the adsorption ability was higher, in the results, the Kd of DBP was 30.4, and the others were 6.33, 6.08,
Table 1 the results of selectivity adsorption. Substrate
Q(10−5 mol/g)
ce (10−5 M)
Kd
˛
DBP Bisphenol A l-tryptophan Epinephrine Sulfamethoxazole
30.58 0.23 0.19 0.38 0.09
1.006 0.0363 0.0313 0.0613 0.0154
30.40 6.33 6.08 6.20 5.85
1.00 4.80 5.00 4.90 5.19
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Fig. 4. The possible chemiluminescence mechanism of CL reaction.
6.10, 5.85, respectively. And the ˛ of DBP was 1, the others was higher than DBP which proved the selectivity of MMIP was very well. 3.4. Optimization of CL conditions The reaction between luminol and H2 O2 shows weak CL emission and a strong CL emission was recorded when the product of hydrolyzing was injected into the above mixed solution. The peak heights of the CL emission were proportional to the concentration of product of hydrolyzing. The optimization experiment was carried out to get a better knowledge of the CL reaction of luminol – H2 O2 –NaOH through the schematic diagram shown in Fig. 1. With the luminol concentration in the range from 1.0 × 10−6 to 1.0 × 10−4 M, the CL intensity increased with raising the concentration of luminol up to 1.0 × 10−5 M. Above 1.0 × 10−5 M, the CL intensity decreased, thus, the 1.0 × 10−5 M luminol was used for further work. The effect of H2 O2 concentration was examined from 1.0 × 10−3 to 4.0 × 10−2 M, the CL intensity reached maximum when H2 O2 was 1.0 × 10−2 M. The effect of NaOH concentration as medium of luminol was examined over 1.0 × 10−3 to 0.1 M range, and the CL intensity reached up to maximum value when 0.01 M NaOH was used. But higher concentration of NaOH lowered the CL intensity of this system. The adsorption time which depends on peristaltic pumps speed was an important parameter for the amount of DBP absorbed on the MMIP. It was observed that the adsorption reached maximum at 10 mL min−1 . Simultaneously, according to experiment results, the other substances in the samples would be adsorbed by MMIP when the pumps speed was too small. DBP molecules cannot be adsorbed by MMIP completely under too large pumps speed. So the 10 mL min−1 pumps speed was chosen for the determination of DBP with good results. As a result, the optimal conditions for the CL system is 1.0 × 10−5 M luminol, 1 × 10−2 M H2 O2 , and 1.0 × 10−2 M NaOH, respectively.
BDP was obtained which was wider and lower than the common methods as shown in Table 2. The relative standard deviation (RSD) was 1.40% (n = 11) for BDP of 3.20 × 10−6 M. The regression equation was ICL = 6594 + 1701c (c being the DBP concentration (M)) with R = 0.9998. According to the results, the higher selectivity and more sensitivity of MMIP–CL sensor were obtained, the MMIP–CL sensor has wider linear range and lower detection limit, correlation coefficient was more closed to 1. Molecularly imprinted polymer and chemiluminescence were used at the same time for determination. According to the results, MMIP–CL sensor was more suitable for applying to quantitative analysis. 3.7. Application of MMIP–CL sensor As shown by the experiments, the optimal conditions for the CL system is 1.0 × 10−5 M luminol, 1 × 10−2 M H2 O2 , and 1.0 × 10−2 M NaOH, respectively. Under optimal conditions, the wide linear range (3.84 × 10−8 2.08 × 10−5 M), and the low detection limit (2.09 × 10−9 M) was obtained. The regression equation was ICL = 6594 + 1701c (c being the DBP concentration (M)) with R = 0.9998. In order to evaluate the applicability and reliability of the proposed method, it was applied to determine drink samples. Drink samples (10.0 mL) were obtained from a brand juice drinks. The samples were centrifuged at 5000 rpm for 20 min. The supernatant was transferred into a flask and diluted with ultrapure water for analysis. The blank experiment was carried out at the same time. The results obtained are shown in Tables 3 and 4. 3.8. Reusability The reusable of the sensor was evaluated by comparing the adsorption of MMIP. According to the experiments (part 2.4 and 2.7), the MMIP was extracted with methanol/acetic acid (9/1, v/v) overnight after used, and then for adsorption to get the adsorption
3.5. The study of possible mechanism of CL reaction The possible chemiluminescence mechanism of the luminol – H2 O2 –NaOH and the product of hydrolyzing (CH3 CH2 CH2 CH2 OH) are showed in Fig. 4. After CH3 CH2 CH2 CH2 OH was oxidated by H2 O2 , a kind of intermediate (A) was producing at electronic state, then, ‘hv’ was producing after excitation energy released which makes the CL emission was made stronger. 3.6. The analytical performance of MMIP-CL sensor Under optimal conditions, a wide linear range from 3.84 × 10−8 to 2.08 × 10−5 M and a low detection limit of 2.09 × 10−9 M (3) for
Table 2 The comparison of our work and the common methods. Works
Method
Linear range (M)
Detection limit (M)
Our work
Microflow chemiluminescence sensor Chromatographic Gas chromatography–mass spectrometry Selective solid-phase extraction
3.84 × 10−8 to 2.08 × 10−5
2.09 × 10−9
1.26 × 10−7 to 1.08 × 10−5 6.47 × 10−8 to 1.80 × 10−5
3.95 × 10−7 6.47 × 10−8
–
4.67 × 10−8
[30] [31]
[1]
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Table 3 Analysis of the standard solution (units of c measurement was M). Standard solution
1
c0 I c1
2
3
4
5
6
Average
6605 0.0068
6606 0.0071
6606 0.0073
0.018 6606 0.0070
0.018 6605 0.0067
6609 0.0070
6606 0.0069
Table 4 Analysis of the sample solution (n = 6, units of c measurement was M). Samples
c2
cDBP-hydrolyzing
cx
RSD
Adding
Founding
1 2 3
0.0400 0.0786 0.10607
0.0200 0.0393 0.0803
0.1030 0.2020 0.4130
3.87% 2.98% 3.08%
0.05 0.05 0.05
0.051 0.052 0.052
Recovery 102% 104% 104%
RSD 2.48% 2.54% 2.69%
References
Fig. 5. The analysis of reusability.
capacity. The results are shown in Fig. 5, the difference between ten times was 0.14 × 10−5 mol/g which is tolerable. The sensor could be used more than hundreds of times before the adsorption began to decrease. This was possibly due to loss of binding sites, however it was easy to replace the MMIP-cell and MNIP-cell in the channel.
4. Conclusions In this paper, a novel CL method for adsorption and determination of DBP using MMIP as recognition element is developed. MMIP was prepared using Fe3 O4 as support, in which methacrylic acid (MAA, functional monomer) and ethylene glycol dimethacrylate (EGDMA, cross-linker) are in the presence of DBP molecules. Flow cells were positioned in front of a mixer which for hydrolyzing. The MMIP showed satisfactory recognition capacity to DBP, resulting to the wide linear range of 3.84 × 10−8 to 2.08 × 10−5 M and the low detection limit of 2.09 × 10−9 M (3) for DBP. The relative standard deviation (RSD) for DBP (3.20 × 10−6 M) was 1.40% (n = 11) via the novel sensor. Besides improving sensitivity and selectivity, the sensor was reusable. The proposed DBP–MMIP–CL sensor has been successfully applied to determine DBP in juice drinks samples.
Acknowledgements This work was supported by the Shandong Provincial Natural Science Foundation of China (Nos. ZR2012BM020, ZR2012BQ018) and the Scientific and Technological Development Plan Item of Jinan City in China (No. 201202088).
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