Biosensors and Bioelectronics 64 (2015) 277–284

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2,6-Diaminopyridine-imprinted polymer and its potency to hair-dye assay using graphene/ionic liquid electrochemical sensor Peini Zhao, Jingcheng Hao n Key Laboratory of Colloid and Interface Chemistry & Key Laboratory of Special Aggregated Materials, Shandong University, Ministry of Education, Jinan 250100, China

art ic l e i nf o

a b s t r a c t

Article history: Received 1 July 2014 Received in revised form 31 August 2014 Accepted 4 September 2014 Available online 8 September 2014

A new analytical approach for detecting diaminopyridine derivatives has been constructed using a molecular imprinting-electrochemical sensor. Opposed to the conventional strategy of employing diaminopyridine as the functional monomer and uracil derivatives as the target analyte, in the current study, the 2,6-Diaminopyridine-imprinted core–shell nanoparticles were synthesized with 2,6-Diaminopyridine as the template molecule and 6-aminouracil as the functional monomer. Graphene and ionic liquid which can assist 2,6-Diaminopyridine-imprinted core–shell nanoparticles in electrochemical reaction kinetics by increasing conductivity have been introduced to form one of the electrode modified layers. The proposed analytical method has been applied in 2,6-Diaminopyridine detection in hair-dyes and demonstrated appropriate sensitivity and selectivity, with a linear range of 0.0500–35.0 mg kg  1 and a detection limit as low as 0.0275 mg kg  1. & 2014 Elsevier B.V. All rights reserved.

Keywords: Molecular imprinting-electrochemical sensor Graphene Ionic liquid Diaminopyridine derivatives detection Multiple hydrogen bonding Hair dyes

1. Introduction For fashion pursuit or practical purpose, more and more people choose to change their hair color or cover their gray hair. Hair dyes with many variations in color tone and brightness become popular among these people. However, hair dye products containing dye intermediates are known to be mutagenic and carcinogenic to animals (Ames et al., 1975; Watanabe et al., 1990). Because of the toxicity of their components, the coloring paste composition is under control of the European Council Directive (76/768/EEC) (Gioia et al., 2005). 2,6-Diaminopyridine (2,6-DAP), based on its good dyeing capacity, is also used as a coupler agent for the hair dye synthesis (Xue et al., 2009). Since the mutagenicity and carcinogenic properties of the components in hair dye is still complicated, the determination of 2,6-DAP, one of the components of hair dye is of significant for human health. Many analytical methodologies such as high-performance liquid chromatography (HPLC) (Wang and Huang, 2005; Zhou et al., 2004), gas chromatography–mass spectrometer (GC/MS) (Tanada et al., 1991; Tanada et al., 1994) and micellar electro kinetic capillary chromatography (MEKC) (Lin et al., 1999) have been introduced to monitor dye intermediates in hair dyes. However, these methods usually require expensive equipments, laborious and time expensive n

Corresponding author. Tel.: þ 86 531 88366074. E-mail address: [email protected] (J. Hao).

http://dx.doi.org/10.1016/j.bios.2014.09.016 0956-5663/& 2014 Elsevier B.V. All rights reserved.

extractions of hair dye components, which make them unsuitable for routine analysis. Molecularly imprinted materials have attracted considerable research interests due to their ability of selectively recognizing a chemical species through specific binding between a functional monomer and a target molecule. The integration of molecularly imprinted materials into chemical/biological sensors and biomedical materials is considered as a promising strategy (Haupt and Mosbach, 2000; Stephenson and Shimizu, 2007; Alexander et al., 2006; Takeuchi et al., 2000; Shi et al., 1999; Hayden et al., 2006; Hayden and Dickert, 2001). Multi-hydrogen bonding between templates and functional monomers is a valuable tool to stabilize the template-functional monomer complexes during polymerization. Numerous researches about the synthesis and application of molecularly imprinted polymers (MIPs) based on multi-hydrogen bonds have been reported (Kugimiya et al., 2001; Li et al., 2005, 2006; Manesiotis et al., 2005; Tanabe et al., 1995; Yano et al., 1998). Those studies provide us with an inspiration of developing a different molecularly imprinted polymer, acting in a diametrically opposite way, a new molecularly imprinted polymer in which uracil derivative was used as the functional monomer and 2,6-DAP as the template molecule for recognition of diaminopyridine derivatives has been established. Due to advantages such as high sensitivity, rapid response, ease of control and can realize real-time detection, the electrochemical sensor was chosen to be the analytical method in the proposed paper (Ratautaite et al., 2014; Li et al., 2012a). Since MIPs are

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generally nonconductive, which limits their application in sensoring, it is necessary to introduce conductive materials to improve the conductivity (Zhang et al., 2012; Matsui et al., 2004; Lakshmi et al., 2009; Li et al., 2012b). Graphene, a single layer of sp2 hybrid carbon atoms tightly packed into a two-dimensional (2D) lattice (Zhou et al., 2010a) has many superior properties including outstanding electric conductivity, large specific surface area, good mechanical strength and high mobility of charge carriers (Park and Ruoff, 2009; Geim and Novoselov, 2007; Li et al., 2008) and graphene modified electrodes have great potential in designing new electrochemical sensors (Sun et al., 2013). Meanwhile, ionic liquids (ILs), a new class of solvent made of molten organic cations and various anions, due to its unique physical properties of wide electrochemical windows, commendable chemical and thermal stability, high ionic conductivity and low toxicity (Pandey, 2006), can be used not only as the supporting electrolyte but also as the modifier in chemically modified electrode (Sun et al., 2012). Fluids formed from graphene sheets and ILs which increases the applicability of these nanomaterials will open up possibilities for new applications in different fields. For example, The gels formed by ILs and graphene have been studied as potential electrolytes for dye sensitized solar cells by Ahmad et al. (2011). The method proposed by Han and co-workers proved that the dispersion of a small amount of graphene sheets in [bmim][PF6] could enhance the conductivity of the IL considerably (Zhou et al., 2010b). Zhao and Hu demonstrated that collective Van der Waals forces between ionic liquids and graphene are able to describe both the shortranged cation-π interaction and the long-ranged dispersion interaction, through a combination of a quantum mechanical calculation on the level of density functional theory (Zhao and Hu, 2013). More recently, it was found that ILs could improve the dispersion of graphene by shielding the π–π stacking interaction among graphene sheets. In this paper, 2,6-DAP-imprinted core–shell nanoparticles (DICSNs) has been synthesized. A molecular imprinting-electrochemical sensor based on graphene–IL composite has been constructed and applied to the 2,6-DAP detection in hair dyes.

Compared with HPLC method, the present sensor showed advantages such as high sensitivity and wide linearity range.

2. Experimental section 2.1. Reagents and materials 2,6-Diaminopyridine (2,6-DAP), 2-Aminopyridine (2APY), 3,4Diaminopyridine (3,4-DAP), and 3-Aminopropyltriethoxysilane (APTES), were supplied by J&K Scientific Ltd. The ILs, 1-butyl-3methylimidazolium tetrafluoroborate ([bmim]BF4), 1-butyl-3methylimidazolium hexafluorophosphate ([bmim]PF6), 1-propionitrile-3-methyl-imidazolium tetrafluoroborate ([pmim]BF4), Z99%, were offered by the Center for Green Chemistry and Catalysis (Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences). 6-aminouracil (6AU), ethylene glycol dimethacrylate (EGDMA), and potassium ferricyanide (K3[Fe(CN)6]), were obtained from Aladdin Chemistry Co., Ltd. Hydrazine solution (50 wt%) was obtained from Tianjin Guang-cheng Chemical Reagent Factory (Tianjin, China) and glutaraldehyde (GA, 50 wt%) from Damao Chemical Reagent Factory (Tianjin, China). All other chemicals were analytical reagent grade. Ultrapure water, with a resistivity of 18.25 MΩ cm, was obtained from a UPH-IV ultrapure water purifier (Chengdu Ultrapure Technology Co., Ltd. China). Three hair dyes (L'Oréal, HUYO and Zhanghua) for real sample determination were bought from supermarket. 2.2. Synthesis of 2,6-DAP-imprinted core–shell Nanoparticles (DICSNs) The detailed preparation procedure of DICSNs is illustrated in Scheme 1. The monodispersed silica spheres (SiO2) were prepared by hydrolysis of TEOS with aqueous ammonia referring to the modified Stöber method reported in our previous work (Zhao and Hao, 2013). The synthesized monodispersed SiO2 (0.30 g) was mixed with APTES (7.0 mL) and anhydrous toluene (50 mL) in a flask. The mixture was refluxed for 10 h under dry nitrogen. The

Scheme 1. Illustration of the construction of the 2,6-DAP-imprinted core–shell nanoparticles (DICSNs).

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resulting SiO2@APTES was separated by centrifugation, washed by ethanol for three times, and redispersed in ethanol (50 mL). 0.5 mL of GA (50 wt%) was added to the mixture mentioned above and was vigorously stirred for 12 h and the SiO2@APTES/GA was obtained. SiO2@APTES/GA were collected and washed three times with methanol and then dispersed in 30 mL of methanol. 6-aminouracil (0.168 g, dissolved in 20 mL of DMSO) was added to the mixture, and the solution was stirred for half an hour. A spoon of NaBH4 was added to the system as the catalyst and the mixture was stirred for another 12 h until the SiO2@APTES/GA/6 AU was formed. The DICSNs were synthesized according to a two-step-temperature polymerization method (Zhao et al., 2012). The prepared SiO2@APTES/GA/6AU was transferred into acetonitrile (50 mL). 2,6-DAP (30 mg), MAA (0.4 mL), EGDMA (3.5 mL) and AIBN (20 mg) were added to the dispersion. The mixture was purged with nitrogen for 10 min while cooled in ice bath and then vacuumed. The mixture was first heated to 50 °C for 6 h, and then the temperature was kept at 60 °C for 24 h with stirring. Finally, the mixture was aged at 85 °C for 6 h to get the DICSNs. The target molecule was extracted by a DMSO-acetic acid (4:1, v/v) mixture and the complete extraction was confirmed by cyclic voltammetry (CV). Non-imprinted core–shell nanoparticles (NICSNs) were also prepared as comparison. Both the DICSNs and NICSNs were dried under vacuum for the following studies.

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2.5. Measurement section DICSN–graphene–IL–sensor was immersed in a 2,6-DAP standard solution (3.00 mg kg  1) and incubated for 30 min. During the period, 2,6-DAP molecules were specific adsorbed onto the DICSNs. The sensor was then rinsed with ultrapure water to remove excess, non-specific adsorbed 2,6-DAP. Subsequently, the sensor was placed in electrolytic cell containing nitrogen treated PBS solution (pH ¼6.22). The cyclic voltammogram was carried out under the potential range from  1.0 V to þ 1.0 V at a scan rate of 100 mV s  1. 2.6. Real sample preparation Three hair dyes bought in super market were chosen as real samples. Their brands were Zhanghua (manufactured in Shanghai), HUYO (Jiangsu) and L'Oréal (Suzhou). All of the three hair dyes had two reagents: hair color cream and color reagent. The two parts were mixed together according to their instruction books and the three mixed cream (0.20 g) were transferred to 3 colorimetric cylinders (10 mL). Ethanol (5.0 mL) was added to every cylinder and the mixture in colorimetric cylinders was eddied by the vortex oscillator (IKA Vortex Genius 3, Germany) to make the sample and solvent mixed thoroughly. After about 12 h of the extraction, the sample solution was obtained.

3. Results and discussion 2.3. Preparation of graphene oxide (GO) and graphene reduced from GO Graphene oxide (GO) was obtained from oxidation of natural graphite by a modified Hummers method (Xu et al., 2008; Hummers and Offeman, 1958; Lee et al., 2011). To prepare graphene, GO (50 mg) was firstly dispersed in ultrapure water (100 mL) to make a suspension (0.500 mg mL  1). Polyvinylpyrrolidone (PVP, 40 mg) was added to 50 mL of the suspension, which is with vigorously stirred for 12 h under 50 °C. After the temperature came down, hydrazine (3.5 μL) and aqueous ammonia (28– 30 wt%, 40 μL) were dropped into the suspension which was kept for 1 h at 95 °C. Gradually, the transparent brown GO colloid became an opaque black colloidal dispersion, indicating that graphene was formed (Li et al., 2011). The product was filtered, washed with ultrapure water and ethanol for several times, and then dried under vacuum. 2.4. Construction of the molecular imprinting-electrochemical sensor Two kinds of coating suspensions were prepared. Suspension I: The synthesized graphene (1.0 mg) was dispersed in ultrapure water (1.0 mL) to form graphene suspension. Subsequently, the graphene suspension (0.5 mL) was mixed with 0.5 mL of IL ([bmim]BF4, [bmim]PF6, or [pmim]BF4) solution (5.00 μL mL  1 in N,N-dimethylform-amide (DMF)). Suspension II: DICSNs or NICSNs (2.0 mg) was dispersed in DMF (1.0 mL) by ultra-sonication for about 1 h. Prior to the electrode modification, the bare glassy carbon electrode (GCE) was polished to a mirror-like surface with 0.03–0.05 μm of alumina aqueous slurry. Subsequently, 4.0 μL of suspension I was transferred on the pretreated GCE, when the solvent evaporated, 6.0 μL of suspension II was dropped onto the resulted graphene–IL–sensor. After the solvent evaporated, the DICSN–graphene–IL–sensor was obtained. The NICSN– graphene–IL–sensor, DICSN–GO–IL–sensor and other modified sensors were prepared in a similar way.

3.1. Morphology of the products during the synthetic process The shape and dimension of the prepared materials of SiO2 nanoparticles, DICSNs, GO and graphene were determined by the JEM-1400 transmission electron microscope (TEM, JEOL, Japan) recorded on Gatan 831 CCD and QUANTAFEG250 field emission scanning electron microscope (SEM, QUANTAFEG250, FEI, America). As illustrated in Fig. 1a (TEM image) and Fig. 1b (SEM image), the synthesized SiO2 nanoparticles were monodispersed with a uniformed size of ca. 100 nm. Fig. 1c shows the morphology of the DICSNs. Compared with Fig. 1b, it is clear that in Fig. 1c the size of the SiO2 nanoparticles became bigger and their external surface became rough, indicating a layer of polymer was successfully coated on the surface. Fig. 1(d–f) are the TEM images of DICSNs. Obviously, it can be seen that SiO2 nanoparticles have been encapsulated into the molecularly imprinted polymer shell to obtain the core–shell nanostructures. Compared with the polymer shell, the SiO2 nanoparticles core has a darker color which demonstrates a clear core–shell structure of the molecularly imprinted polymer. From the TEM and SEM images, it can be concluded that the core–shell structure has been constructed successfully. The special core–shell structure of MIP has various advantages including high specific surface area, high density of recognition sites and accessibility to the target species, which will be important to the selectivity of the proposed sensor. The morphology and structure of the GO were observed by TEM and SEM analysis. Fig. 1g exhibits an amorphous and disordered paper-like morphology of the produced GO sheets with some wrinkle on its basal planes and the sheet edges. Fig. 1h shows the multilayer structure of GO platelets, further illustrating their flake-like shapes together with the size around several square micrometers. The obtained graphene sheets were fully analyzed by SEM observation. The size of the obtain graphene sheets was smaller than their relevant GO sheets and the membrane is more corrugated and scrolled after the reduction (Fig. 1i). As reported previously (Meyer et al., 2007), the resemblance of crumpled silk veil waves is intrinsic quality to graphene sheets

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Fig. 1. TEM and SEM images of the prepared SiO2 nanoparticles (a and b), DICSNs (c–f), GO (g and h) and graphene (i).

(Yang et al., 2013). The special structure brings the material many specialties: high electronic and thermal conductivities and exceptional mechanical strength (Geim and Novoselov, 2007; Geim, 2009). Therefore, these have exploited the new application field of graphene sheets in chemical sensors. 3.2. Spectroscopies of FT-IR, Raman and energy dispersive X-RAY The FT-IR spectra of the products were identified by VERTEX-70 infrared spectrometer (Bruker optics Corporation, Germany) (Fig. 2A, B and Fig. S1), and the energy dispersive X-ray spectroscopy (EDS) data was recorded on an Oxford INCA sight X instrument (England) (Fig. 2C). Raman scattering spectrum was recorded with an Ocean Optics QE65000 spectrometer (Fig. 2D). As shown in Fig. 2A, curve a, the peak at 1101 cm  1 ascribes to Si–O–Si stretching vibrations, and the band around 3200 cm  1 shows the presence of the Si–OH. Curve b, the peaks at 3448 cm  1 and 3366 cm  1 attribute to the N–H anti-symmetric stretching vibration of primary amine as well as band at 3291 cm  1 to the N–

H symmetric stretching vibration confirm that APTES has been successful modified on the surface of SiO2. Curve c, the peak at 1640 cm  1 is attributed to CQN stretching vibration of imine combined with the disappearance of the peaks at 3448, 3366 and 3291 cm  1 (compared with curve b) shows that aldehyde groups (–CHO–) of GA have been reacted with the amino groups (–NH2) to form imine, demonstrating GA has been successful grafted on the SiO2@APTES. Curve d, the adsorption at 1639 cm  1 is attributed to the CQN stretching vibration of imine generated between the – NH2 of 6 AU and the –CHO– of the SiO2@APTES/GA. Meanwhile, the band at 3425 cm  1 is due to the N–H symmetric stretching vibration of primary amine. Based on these results, it can be seen the 6 AU has been jointed on the SiO2@APTES/GA. In curve f, the shoulders at 2513 cm  1 and 1732 cm  1 can be assigned to the stretching vibration of tertiary ammonium salt and CQO stretching vibration, respectively. Meanwhile, the band at 1639 cm  1 in curve d has disappeared. By this time, the DICSNs have been perfectly achieved. Compared with DICSNs, the disappearance of the peak at 2513 cm  1, the preservation of the band at

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Fig. 2. FT-IR spectra of the products present in the synthesized process (A): (a) silica spheres (SiO2), (b) SiO2@APTES, (c) SiO2@APTES/GA, (d) SiO2@APTES/GA/ 6 AU; (B): (e) NICSNs, (f) DICSNs and (g) DICSNs after removal of template molecule. EDS results of the products (C): (a) SiO2, (b) SiO2@APTES, (c) SiO2@APTES/GA, (d) SiO2@APTES/GA/6 AU, (e) DICSNs, (f) NICSNs. Raman spectrum of DICSNs–graphene–IL–sensor (D).

1732 cm  1 and the return of the band at 1637 cm  1 in curve e demonstrate the difference between imprinting and non-imprinting of the template molecule in the polymer. The peaks in curve g are generally consistent with those in curve e, showing the complete extraction of template molecules and then the specific binding sites have been formed. EDS results were the assisted proofs for the construction of these products and detailed analysis was listed in Electronic Supporting Information. In Fig. 2D, the band at 1595 cm  1 is the G band which is the dominant Raman signatures of sp2 carbon materials. The band at 1324 cm  1 is assigned to the D band appearing for graphite with defects (or

disordered structure) (Zhong et al., 2013). Fig. 2D shows the Raman spectrum of DICSNs–graphene–IL–sensor and the existence of G band and D band indicates the presence of graphene in the DICSNs–graphene–IL–sensor. 3.3. Investigation of the best construction of the molecular imprinting-electrochemical sensor To compare the binding affinity of DICSNs and NICSNs to the template molecule and study the electrical conductivity of GO, graphene and their respective compounds modified layer with ILs,

Fig. 3. Molecular imprinting-electrochemical sensors with different constructions (A): (a) DICSN–graphene—IL–sensor, (b) NICSN–graphene–IL–sensor, (c) DICSNs–GO–IL– sensor, (d) DICSNs–graphene–sensor, and (e) bare electrode. Effect of different ILs on the molecular imprinting-electrochemical sensors to 2,6-DAP (B). ILs: (a) [bmim]BF4, (b) [bmim]PF6, and (c) [pmim]BF4.

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the cyclic voltammograms of 2,6-DAP on different constructed sensors have been recorded. The cyclic voltammograms were examined on a CHI 600b electrochemical workstation with a three-electrode system (Shanghai Chenhua Instruments Co., Shanghai, China). The sensors were prepared with the coating suspensions slightly different and represented as DICSN–graphene–IL–sensor, NICSN–graphene–IL– sensor, DICSNs–GO–IL–sensor and DICSNs–graphene–sensor. The electrochemical measurements of these electrodes were carried out according to the description in Section 2.5: sensors were immersed in a 2,6-DAP standard solution (3.00 mg kg  1) and incubated for 30 min. The sensors were then rinsed with ultrapure water to remove excess, non-specific adsorbed 2,6DAP. Subsequently, the sensors were placed in electrolytic cell containing nitrogen treated PBS solution (pH ¼ 6.22). The cyclic voltammograms were carried out under the potential range from  1.0 V to þ1.0 V at a scan rate of 100 mV s  1. It is clearly observed from Fig. 3A that there is no redox peak present at the bare electrode (curve e) while quasi-reversible redox peaks present in all of the other electrodes (curve a, b, c, and d). From the comparison between curve (a) and (b), it can be concluded that both the DICSNs and NICSNs have adsorbed 2,6DAP, but the adsorption capacity is significantly different. The peak of DICSNs is higher than that of NICSNs. A reasonable explanation is during the infusion process, 2,6-DAP molecules were probably adsorbed on the surface of NICSNs through physical adsorption while adsorbed on DICSNs through both specific binding and physical adsorption. Thus the specific recognition interaction must play a decisive role for DICSNs in rebinding 2,6-DAP molecules to the specific binding sites distributed on the surface of DICSNs. The comparison of curves (a) and (c) coupled with comparing curve (a) and (d) shows a good electro-conductive effect and electron-transfer capacity due to the combined action of graphene and ILs. From the curve (a) and (c), since GO is a nonconductive carbon material (Marcano et al., 2010) and both of the two modified electrodes contain ILs, the peak current of curve c significantly smaller than curve a, which could attribute to the good electric conductivity of graphene or the synergic effect of graphene and IL in enhancing the electric conductivity and the electron-transfer rate. Meanwhile, it can be seen from the curves (a) and (d), when graphene and IL were used together, the property of the sensor is more predominant than that graphene used alone. There are two possible reasons (Wu et al., 2013): (i) Due to the existence of strong π–π stacking and Van der Waals interaction between graphene sheets, they tend to form irreversible agglomerates or even restack to form graphite which limited their applications (Shiddiky and Torriero, 2011). When the electrode was modified by ILs mixed with graphene, IL may form a functionalized layer on graphene which causes electrostatic repulsion between graphene sheets. This sequentially suppressed the restacking of graphene and then the high surface area of graphene can be fully exploited. (ii) Since graphene is a 2D hexagonal lattice of sp2-hybridized carbon atoms bonded along two plane directions (Liu et al., 2010), the conductivity along the z direction is low, even though its planar electronic transport is outstanding. The introduction of the conductive ILs combining with graphene overcomes this z-direction conduction limitation and facilitates electrochemical signal transmission in the sensing electrodes.

[bmim]PF6 and [pmim]BF4, in the coating suspensions. The cyclic voltammograms of the three different modified electrodes were recorded in Fig. 3B. As shown in Fig. 3B, the peak current of DICSN–graphene– [bmim]BF4–sensor has the biggest value, larger than those of DICSN– graphene–[bmim]PF6–sensor and DICSN–graphene–[pmim]BF4–sensor, but there is not obvious difference between DICSN–graphene– [bmim]PF6–sensor and DICSN–graphene–[pmim]BF4–sensor. It can be seen in Fig. S2 that the cations of [bmim]BF4 and [bmim]PF6 are the same while the anionic components are different, the different performances between DICSN–graphene–[bmim]BF4– sensor and DICSN–graphene–[bmim]PF6–sensor should attribute to the property of anions. This is supported by the fact that the [bmim]BF4 and [pmim]BF4 have the same anions and different cations and there is not obvious difference between them. This may because the branched chains of cations are different. The ability of attracting electron of the propanenitrile is stronger than butyl, which has weaken the interaction among [pmim]BF4, graphene and DICSNs. Hence, three ILs, [bmim]BF4 shows best capability and was chosen through the whole experiment. 3.5. Electrochemical impedance spectroscopy (EIS) For further discussion of the effect of graphene and ILs for the sensor and the possible electron transfer process among DICSNs, graphene and ILs on the electrode surface, electrochemical impedance spectra (EIS) of different sensors were collected (Chen et al., 2008). The impedance spectra are composed of a semicircle and a diagonal straight line. The semicircle portion at higher frequencies corresponds to the electron transfer-controlled process. The semicircle diameter equals the electron transfer resistance, Ret, produced by redox reactions of electrolyte solution at the interface with the electrode (Zhao and Hao, 2013). The linear portion at lower frequencies represents the diffusion-controlled process and the diagonal straight line corresponds to the impedance of the current produced by the diffusion from the bulk solution to the interface solution layer. The impedance spectra of different modified electrochemical sensors have been investigated and the spectra were shown in Fig. 4. From the comparison of the impedance spectra, the feature of each modified layer and the interactions between them during the electrochemical reaction process can be discussed.

3.4. Optimization of Ionic Liquids In order to investigate the influence of different ILs on the molecular imprinting-electrochemical sensors and make the IL play full functions, the capability of the three ILs has been compared. The electrode was modified using [bmim]BF4,

Fig. 4. The electrochemical impedance spectra of electrochemical sensors with different surface conditions recorded in 5.00  10  3 mol L  1 K3[Fe(CN)6] solution containing 0.200 mol L  1 KCl: (a) bare GCE; (b) DICSN–graphene–sensor; (c) DICSN–IL–sensor; (d) DICSN–graphene–IL–sensor; (e) DICSN–graphene–IL– sensor adsorbed 2,6-DAP of sample solution for 30 min.

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As shown in Fig. 4, the impedance spectrum of bare GCE (curve a) exhibits an almost straight line, implying a high electron transfer rate. Therefore, for bare GCE, mass diffusion was the rate limiting step of the electron-transfer process. In contrast to bare GCE, curve (b), (c), (d), and (e) exhibit semicircle in high frequency region and straight line in low frequency region, coinciding with the electron transfer process and the diffuse process, respectively. The semicircle diameter of DICSN–graphene–sensor is the smallest among these sensors indicating the effective electron transfer performance of graphene which further proves its good electrical conductivity and the important role of graphene in the proposed sensor. The semicircle diameter of DICSN–IL–sensor is bigger than others demonstrating that when IL was used alone, the electron transfer performance of IL for the sensor was general, however, the semicircle diameters of curve (d) and (e) present a decreasing tendency. This indicates that when graphene and IL were used together, the capacity of electron transport has been effectively promoted, which may because graphene sheets in IL could enhance the conductivity of the IL considerably. (It should be explained here that combined with Fig. 3A, it cannot say the DICSN–graphene–sensor was the best sensor construction though it has the smallest semicircle diameter in Fig. 4. The DICSN– graphene–IL–sensor has the highest signal in Fig. 3A. The results reflect the different aspects of the sensor′s properties.) The adsorption of 2,6-DAP made the semicircle diameter of curve (e) become smaller than that of curve (d). This may due to that some surface effects and 2,6-DAP molecules binding on the DICSNs by specific recognition may experience a degree of conformational effects (surface reorganisation) (Sallacan et al., 2002) or swelling (Fick et al., 2004) to increase the electron-transfer rate and facilitate the interaction between redox probe and the electrode surface (Apodaca et al., 2011). 3.6. Analytical performance After the material characterization and optimal condition filtration, the linear regression curve is made. Under the optimal conditions, the peak current of 2,6-DAP at different concentrations was performed. The current response was linear to 2,6-DAP concentration varying from 0.0500 to 35.0 mg kg  1, as shown in Fig. S3. The regression equation was I (10  6 A)¼3.81 þ 0.0995c (mg kg  1) with the correlation coefficient r ¼ 0.999. For 0.0995, the confidence interval was (0.0976, 0.101) and for 3.81, the confidence interval was (3.78, 3.83) with 1  α ¼95% and t0.05/2 (9)¼ 2.26. The detection limit of 2,6-DAP was found to be 0.0275 mg kg  1 according to 11 parallel determinations of the blank solution.

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substances can be ignored and the new molecular imprintingelectrochemical sensor possesses the qualification in practical applications. 3.8. Reproducibility, repeatability and stability of the sensor The reproducibility experiment of the method has been measured among 6 paralleled DICSN–graphene–IL–sensors to 2,6-DAP sample solution (3.00 mg kg  1) independently, and the interelectrode relative standard deviation (RSD) was found to be 0.58%, indicating appropriate precision of the proposed method. Five repeated detection procedures of one DICSN–graphene–IL– sensor have been performed to investigate the repeatability of the sensor. The electrode was immersed in the 2,6-DAP standard solution (3.00 mg kg  1) for 30 min to determine the response, and then, the adsorbed template was eluted for the next repeated adsorption-detection trials. Such circulation was repeated for five times and the calculated RSD were about 6.0% (n ¼5), showing excellent repeatability. The stability of the sensor was studied by investigating the CV response change between the fresh DICSN– graphene–IL–sensor and the sensor stored at 4 °C for one week toward 2,6-DAP solution. The signal remained about 90% of its initial current shows the good stability of sensor. 3.9. Applications of DICSN–graphene–IL–sensor in monitoring 2,6DAP in real samples The property of the sensor in practical application was the purpose for this work. Consequently, after all the groundwork mentioned above has been completed, the efficiency of such sensor in practical application was researched. Three hair dyes were chosen as real samples and the concentration of 2,6-DAP in the hair dyes, L'Oréal, HUYO, and Zhanghua, was measured using the molecular imprinting-electrochemical sensor. The recovery test of the detection method has been also investigated by the standard addition method. The determination results were listed in Table 1. According to the ‘32 kinds of cosmetics ingredients Restriction dye detection method’ promulgated by the China Food and Drug Administration, the limit of 2,6-DAP was 200 mg kg  1, the results of real samples showed that the concentration of 2,6-DAP in hair dyes was not exceed the limit. Meanwhile, from the data analysis of the third column in Table 1, the RSD which related to precision of the proposed method was very good as well as the accuracy examined by the recovery test. Therefore, the established molecular imprinting-electrochemical sensor for monitoring of 2,6-DAP demonstrated prominent property.

3.7. Selectivity assay Selectivity of the sensor is one critical index embodying the superior specific recognition ability of the electrochemical sensor. Several interfering substances were introduced to investigate the selectivity of the sensor. The structures of the interfering substances are similar with 2,6-DAP and their concentration is higher than that of 2,6-DAP standard solution. The selectivity of the DICSN–graphene–IL–sensor to 2,6-DAP was evaluated by the peak current ratio (Is/I0), where Is and I0 were reduction peak currents of 2,6-DAP when the interfering substances (2APY and 3,4-DAP) were present and absent, respectively. The result in Fig. S4 shows that the 2-, 10- and 20-fold excess of 2APY and 3,4-DAP over 2,6-DAP hardly causes significant interference on the detection of 2,6-DAP, in which the peak current ratio only varied from 0.78 to 0.98. The results represent that the DICSN–graphene–IL–sensor has good selectivity and it has strong specificity toward 2,6-DAP. The influence of common interfering

4. Conclusions In this work, we presented a new molecular imprintingelectrochemical sensor for detecting 2,6-DAP by reversing the role of the functional monomer and template molecules in traditional molecularly imprinted polymer. Electrochemical sensor based on Table 1 Determination results of 2,6-DAP in real samples. Real sample Founda 7SD mg kg  1

RSD (%) Added mg kg  1

Recovered mg kg  1

Recovery (%)

HUYO L′Oréal Zhanghua

0.40 1.51 1.05

9.84 10.12 10.38

98.40 101.20 103.80

a

4.277 0.02 6.40 7 0.10 6.137 0.06

Average of six determinations.

10.00 10.00 10.00

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P. Zhao, J. Hao / Biosensors and Bioelectronics 64 (2015) 277–284

the MIP and the two popular versatile materials of grapheme and ILs for 2,6-DAP monitoring in hair dyes have been successfully established. The proposed molecular imprinting-electrochemical sensor provides one new detection approach for diaminopyridine derivatives and shows high selectivity, appropriate precision, and accuracy. The proposed method expands the application scope of molecularly imprinted materials based on multiple hydrogen bond interactions as well as the detection approach of diaminopyridine derivatives which can demonstrate important practical significance.

Acknowledgments This work is financially supported by the National Natural Science Foundation of China (Grant nos. 21033005 and 21273134).

Appendix A. Supplementary Information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.09.016.

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ionic liquid electrochemical sensor.

A new analytical approach for detecting diaminopyridine derivatives has been constructed using a molecular imprinting-electrochemical sensor. Opposed ...
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