Biosensors and Bioelectronics 57 (2014) 310–316
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Selective optosensing of clenbuterol and melamine using molecularly imprinted polymer-capped CdTe quantum dots Bui The Huy a,b, Min-Ho Seo a, Xinfeng Zhang a,c, Yong-Ill Lee a,n a
Department of Chemistry, Changwon National University, Changwon 641-773, South Korea Nhatrang Institute of Technology Research and Application, Vietnam Academy of Science and Technology (VAST), 2 Hung Vuong, Nhatrang, Vietnam c Mineral Resources Chemistry Key Laboratory of Sichuan Higher Education Institutions, College of Material and Chemistry & Chemical Engineering, Chengdu University of Technology, Chengdu 610059, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 12 November 2013 Received in revised form 28 January 2014 Accepted 10 February 2014 Available online 25 February 2014
A novel procedure for the optosensing of clenbuterol and melamine was developed using molecularly imprinted polymer-capped CdTe quantum dots (MIP-CdTe QDs). The MIP-CdTe QDs were synthesized by a radical polymerization process among CdTe QDs, a template, 3-aminopropyltriethoxysilane (APTES) and tetraethoxysilane (TEOS). The sizes of the MIP-CdTe particles were controlled by the speed of polymerization, concentration of the template, concentration of the quantum dots, and the ratio of template, monomer and cross-linker. Excellent selectivity and high sensitivity of MIP-CdTe QDs toward clenbuterol/melamine molecules were observed based on the fluorescence quenching of QDs. Experimental results showed that the optimum molar ratios of template, monomer, and cross-linker were 1:8:20 and 1:4:20 for analyzing clenbuterol and melamine, respectively. Under optimum conditions, these MIP-CdTe QDs showed a limit of detection of 0.4 μM (120 ng/mL) for clenbuterol and 0.6 μM (75 ng/mL) for melamine. The feasibility of the developed method in real samples was successfully evaluated through the analysis of clenbuterol and melamine in milk and liver samples with satisfactory recoveries of 92–97%. The MIP-CdTe QDs could be easily regenerated for subsequent sample analysis with water. & 2014 Elsevier B.V. All rights reserved.
Keywords: CdTe Molecularly imprinted polymer Fluorescence quenching Clenbuterol Melamine
1. Introduction Molecular imprinting is a technique to create selective binding sites for a specific molecule. Molecularly imprinted polymers (MIPs) are synthetic polymeric materials with specific recognition sites complementary in shape, size, and functional groups to template molecules. The templates can be imprinted during polymerization and later extracted, creating three-dimensional template binding sites for the purpose of retaining the memory of a template molecule. The general synthetic procedures of MIPs involve complexation in a solution of a template molecule with functional monomers through non-covalent bonds, followed by polymerization of these monomers around the template with the help of a cross-linker in the presence of an initiator. After completing the polymerization process, template molecules are removed by extensive washing steps to disrupt the interactions between the template and the monomer, thus freeing the binding sites. The template extraction directly influences the limit of detection and precision. In general, efficient extraction of the
n
Corresponding authors: Tel.: þ82 55 213 3436; fax: þ 82 55 213 3439. E-mail address: [email protected] (Y.-I. Lee).
http://dx.doi.org/10.1016/j.bios.2014.02.041 0956-5663 & 2014 Elsevier B.V. All rights reserved.
original templates located at the interior area is not easy because of extremely low analyte diffusion rates in highly cross-linked MIPs. Furthermore, the rigid polymeric matrix restrains target species into those deep imprinted and thus reduces compatible recognition (Gao et al., 2007; Peng-Ju et al., 2007). Recently, several approaches have been explored to overcome these problems through the use of surface imprinting with nanospheres for detecting salivary proteins, pyrethroids, atrazine (Lee et al., 2010; Li et al., 2010; Liu, R. et al., 2011) or core–shell imprinted nanoparticles for selective detection of uracil, melamine and lysozyme (Lin et al., 2004; Zhang et al., 2010; Chen et al., 2014). Example, the best imprinting effect was improved by the rebinding kinetic and rebinding affinity (Chen et al., 2014). During the last several decades, quantum dots (QDs) have attracted considerable interest due to their size-dependent optical properties, large surface-to-volume ratios, high quantum effects, and narrow spectral line widths. We know that optical properties of quantum dots are related to its surface and it also have an ability to form coupling complexes with organic compounds (Gerhards et al., 2008; Zhang et al., 2011). These characteristics along with high stability against photobleaching have been shown to be useful in a number of compelling applications such as chemical sensors for bio-macromolecules (Yuan et al., 2009; Zhang et al.,
B. The Huy et al. / Biosensors and Bioelectronics 57 (2014) 310–316
2012), fluorescent probes (Costa-Fernandez, 2006; Liu, X.H. et al., 2011), and influences in bio-environments (Molnár et al., 2011). MIPs with high stability, easy preparation, and low cost present opportunities for analyzing various target analytes including tramadol in human urine (Afkhami et al., 2013), 2,4,6-trinitrotoluene (TNT) (Gao et al., 2007; Wang and Zou, 2011), pyrethroids (Li et al., 2010), and cytochrome (Zhang et al. 2011). Therefore, the use of MIPs-QDs for direct sensing of molecules is a challenge to achieving the tailored selectivity of analytics (Li et al., 2010; Zhang et al. 2011, 2012a, 2012b). Clenbuterol is a well-known sympathomimetic agent and “lean meat powder” because it is fed to stock animals to ensure lean muscle mass. It also causes them to grow quicker by speeding up fat burning and muscle building. Also, clenbuterol is a recently popular drug used by athletes in diverse sports for its purported anabolic effects and reduction of subcutaneous fat. Melamine is a synthetic compound that is widely used as an industrial chemical for the production of plastics, amino resins and flame retardants. Because of its high nitrogen content, the use of melamine can increase the total nitrogen level of poor quality products, seemingly increasing the high protein content. For instance, melamine was mixed in powdered milk to make an artificial protein into a total protein. The toxicity of melamine caught the attention of physicians as a result of a recent spate of renal injury after exposure to melamine-tainted milk in China (Hau et al., 2009). The maximum allowed concentrations by International Legislation from the World Health Organization of melamine in powdered infant food and other food are 1 mg/kg and 2.5 mg/kg, respectively (Sari and Christopher, 2010). A maximum residue limit of 0.5 μg/kg for clenbuterol in the liver of cattle and horses is proposed by European legislation (De Wasch et al., 1998). Recently, clenbuterol and melamine have been found in many kinds of food or food-related products, resulting in many serious human health problems. Various analytical techniques have been suggested to analyze clenbuterol and melamine, such as gas chromatography/mass spectrometry (GC/MS2) (Van et al., 2002), liquid chromatography/mass spectrometry (LC/MS) (Yuen et al., 2005; Yang et al., 2009), solid phase extraction coupled with highperformance liquid chromatography analysis (SPE–HPLC) (He et al., 2009; Yang et al., 2009), mid infrared (MIR) and Raman spectroscopy (Meza-Marquez et al., 2011), surface desorption atmospheric pressure chemical ionization mass spectrometry (SDAPCI-MS) (Jia et al., 2011), combination of magnetic MIP and liquid chromatography–tandem mass spectrometry (He et al. 2014), colorimetric assay using gold nanoparticles (He et al., 2011), potentiometric sensors based on MIP (Liang et al., 2009), electrochemical impedance spectroscopy (EIS) (Wu et al., 2012), immunogold chromatography (IGCA) (Li et al., 2011), and surfaceenhanced Raman scattering (SERS) (Izquierdo-Lorenzo et al., 2010). Although these techniques show promising results for sensitive detection of clenbuterol and melamine, there are still some hindrances including sophisticated instrumentation, extensive sample preparation, and the need for highly skilled personnel. Furthermore, quantitative analysis of clenbuterol is often performed after chemical derivatization, but the derivative procedure may introduce large variability or sample loss (Lee et al., 1998; Crescenzi et al., 2001). Combining the high selectivity of MIP and the efficient fluorescence (FL) properties of QDs could develop a new method for selective recognition and quantitative determination of target analytes. MIP-capped QDs for analytical applications are still in an early stage of development. Due to the abovementioned reasons, the development of an inexpensive, simple approach for the direct detection of clenbuterol and melamine, and as far as we know MIP-capped QDs for the quantitative analysis of clenbuterol and melamine, has not been reported. In the present work, we report the facile synthesis of MIP-capped
311
CdTe quantum dots (MIP-CdTe QDs) in an aqueous phase and their application for the direct detection of clenbuterol and melamine in food. In this study, clenbuterol and melamine were selected as targets because, in addition to the above-mentioned reasons, MIP-CdTe for both cases were produced from the same monomer and cross-linker, resulting in favorable conditions in the process MIP preparation. The sensing of clenbuterol and melamine is based on fluorescence quenching when the analytes are selectively recognized and combined with MIP-capped quantum dots. The sizes of the MIPs particles were controlled by the speed of polymerization, amount of quantum dots, initial concentration of the template, and the molar ratio (T:M:C) of the template (T), monomer (M), and cross-linker (C). Mercaptoacetic acid (MAA)capped CdTe QDs were bound to the analytes (clenbuterol or melamine) and a functional monomer (3-aminopropyltriethoxysilane, APTES) by hydrogen bonding. Experimental results demonstrated that the developed MIP-CdTe QDs method has good potential for quantitative analysis of trace amounts of clenbuterol and melamine with high selectivity.
2. Experiment 2.1. Materials Ammonium hydroxide (NH3H2O), APTES, cadmium chloride (CdCl2), MAA, sodium borohydride (NaBH4), sodium hydroxide (NaOH), tellurium (Te powder), tetraethyl orthosilicate (TEOS), clenbuterol, and melamine were purchased from Sigma-Aldrich. Deionized water with a resistivity of 18.2 MΩ cm was used. All chemicals were used as received without any further purification. The potassium hydrophtalate (0.1 M, pH 2–4), phosphate buffer solution (0.1 M, pH 5–10) were used in the experiments. 2.2. Synthesis of CdTe quantum dots The CdTe QDs in an aqueous phase were synthesized based on the procedure described in the literature with some modifications (Qian et al., 2006; Ying et al., 2014). Briefly, 0.183 g of CdCl2, 0.140 mL of MAA, and 100 mL of water were mixed in a three-neck flask to form a cadmium precursor. The mixture was adjusted to pH 8 with 1.0 N NaOH. Separately, 32 mg of tellurium powder, 20 mg of NaBH4 and 0.5 mL of ultrapure water were added to a one-neck flask. The one-neck flask was sealed and heated at 50 1C for 30 min to produce a deep red clear solution. Then, the obtained NaHTe solution in the one-neck flask was diluted to 40 mL with water and injected into the reaction system under vigorous stirring. Subsequently, the reaction mixture was refluxed for 3 h and then cooled to room temperature. Isopropanol was added to the prepared CdTe solution at the volume ratio of 1:1 (isopropanol:water) to precipitate CdTe particles. 2.3. Synthesis of MIP-capped CdTe QDs (MIP-CdTeQDs) 0.2 mg of CdTe QDs, 5 mL of water, and 25 mmol of template (clenbuterol or melamine) were placed in a 50 mL flask and stirred for 30 min to bind the template to the surface of the CdTe QDs by MAA. Then, 50–300 mmol of APTES as a functional monomer was added and stirred for 30 min. Finally, 0.25–1 mmol of TEOS as a cross-linker and 100 μL of ammonium hydroxide as a catalyst were injected together using the syringe pump, and the mixture was stirred for 12 h. The non-imprinted polymers CdTe QDs (NIP-CdTe QDs) were synthesized using the same procedure without adding the template in order to compare the molecular recognition performances with MIP-CdTe QDs. The resulting MIP-CdTe QDs
312
B. The Huy et al. / Biosensors and Bioelectronics 57 (2014) 310–316
and NIP-CdTe QDs were sonicated, centrifuged, and washed several times with water to extract the template and to remove any free monomer and cross-linker. The products were considered adequately washed when similar FL intensities of both MIP-CdTe QDs and NIP-CdTe QDs were achieved. 2.4. Characterization The morphologies of MIP-CdTe QDs and NIP-CdTe QDs were characterized using a scanning electron microscope (SEM) (FESEM MIRA II LMH, Tescan, USA). The samples for SEM were prepared by drying the QDs in a vacuum. All FL measurements were performed on a FP-6500 spectrofluorometer (JASCO, Tokyo, Japan) with an excitation wavelength at 355 nm. The slit widths for both excitation and emission were set at 3 nm. The UV spectra of the QDs in KBr were recorded on a FT/IR-6300 Fourier transform infrared spectrometer (JASCO, Tokyo, Japan). Transmission electron microscopy (TEM) measurements were carried out using a JEM-2100F transmission electron microscope (JEOL, Tokyo, Japan) operating at 200 kV. TEM samples were prepared by dropping an aqueous solution of the nanoparticles onto amorphous carboncoated copper grids with the excessive solvent evaporated. 2.5. Sample preparation Analytical samples were prepared on the basis of a procedure described elsewhere in the literature (Limian and Joan, 2010; Li et al., 2011). Milk samples were diluted ten-fold using water prior to the analysis but did not undergo any special treatments. The liver samples were washed, chopped into small pieces and homogenized thoroughly with a food grinder. Fifty-gram amounts of homogeneous sample were mixed with 10 mL acetonitrile/ water (1:1, v/v) and then placed in a 50 mL beaker. Extraction was performed under stirring at 300 rpm for 12 h; acetonitrile precipitated the proteins in this process. Subsequently, the mixture was centrifuged for 3 min to remove the precipitated proteins. The supernatant solution was stored for analysis.
3. Results and discussion 3.1. Preparation and characterization of MIP- and NIP-CdTe QDs in the aqueous phase The preparation of NaHTe was facilitated by heating the mixture of NaBH4 and Te powder to 50 1C for 30 min. The highly luminescent CdTe QDs were synthesized in an aqueous phase through the reaction of tellurium and cadmium precursors in the presence of a thiolated agent, MAA. Mercaptoacetic acid can
further cap the surface defects because of its short length, so it can increase the surface tension of CdTe QDs and hinder the growth of CdTe QDs. Larger QD particles were obtained from a lengthy reaction time, leading to the red shift of FL emission of the CdTe QDs ranging in λmax from 520 nm to 580 nm, as illustrated in Fig. S1 (see Supporting information). This is an effect of the Ostwald ripening mechanism as smaller particles dissolve in the process of producing larger particles. The mean particle sizes of the CdTe QDs were determined by TEM on the 200 particles and ranged from 7 nm (reaction time: 3 h) for green and 9 nm (reaction time: 7 h) for yellow. The mean particle size according to TEM was larger than that of the real size because the sizes of water-soluble CdTe nanoparticles were difficult to characterize by TEM due to the aggregation of CdTe nanoparticles during drying on a Cu grid. The inset in Fig. S1 shows a photograph of the watersoluble CdTe QDs. Strong FL emissions with different colors were observed with the naked eye under the illumination of an ultraviolet lamp. Size-selective precipitation of aqueous nanoparticles was achieved by adjusting the amount of isopropanol (Wang et al., 2009a). Under vigorous stirring at room temperature, isopropanol was dropped into the prepared CdTe solution until the solution became slightly turbid, and then the mixture was centrifuged at 4000 rpm for 5 min to obtain the CdTe particles. The precipitation mechanism may be related to the decreased relative dielectric constant of the mixture solution and the reduced interparticle electrostatic repulsion (Wang et al., 2009b). The synthesis of MIP-CdTe QDs involves the complexation of CdTe QDs with the template and functional monomers molecules (APTES) through non-covalent bonds in aqueous solution, followed by the polymerization of these monomers around the template (clenbuterol or melamine) with a cross-linker (TEOS). Fig. 1 illustrates the mechanism of molecularly imprinted polymers with CdTe QDs in the aqueous phase through hydrogen bonding interactions. The amino groups (–NH2) in the molecule of APTES interact with the functional groups in the template molecules to form a complex through hydrogen bonding, which mainly caused the FL quenching (Liu et al., 2010). The role of MIP thickness is very important because it influences the removal of the template and FL intensity. Also, it influences the signal to noise ratio, so MIP thickness should be controlled for applications of MIP-CdTe QDs. The size of the MIP-CdTe QDs particles was affected by the speed of the polymerization process. Therefore, the injection speed of the crosslinker (TEOS) to the reactor is one of the most prevailing parameters for controlling the size of MIP-CdTe QDs. Fig. 2a and b shows the comparison of the differences in particle size influenced by the injection speed of cross-linker with melamine as the template. The average size of the particles was found to be 400 750 nm at high speed injection (200 μL/min), which was
Fig. 1. Schematic illustration for the molecular imprinting mechanism on CdTe QDs through hydrogen bonding interactions.
B. The Huy et al. / Biosensors and Bioelectronics 57 (2014) 310–316
5 nm
313
20 nm
Fig. 2. SEM image of MIP-CdTe QDs with (a) high (200 μL/min) and (b) low (10 μL/min) speed injection of the cross-linker at a T:M:C ratio of 1:4:20. (c) TEM image of MIPCdTe QDs produced with 3 mg CdTe QDs at a T:M:C ratio of 1:4:20. (d) TEM image of CdTe with FL emission wavelength at 580 nm.
ten times larger than that of the particles at low speed injection (10 μL/min), at a T:M:C ratio of 1:4:20. The thickness of MIP polymers on CdTe QDs is also governed by the structures and the amounts of template, monomer, cross-linker, and CdTe used. The thicknesses of MIP polymer on CdTe QDs were 15, 30, and 100 nm for T:M:C ratios of 1:2:10, 1:4:10 and 1:4:20, respectively. Interestingly, the initial amount of added CdTe QDs influenced not only the FL intensity but also the size of the resulting MIP-CdTe QDs when the amounts of template, monomer, and cross-linker were held constant. The TEM image of MIP-CdTe QDs formed using a T:M:C ratio of 1:4:20 with 3 mg CdTe is illustrated in Fig. 2c. In this case, the size of MIP-CdTe was about 380 nm with 60 nm of MIP shell thickness; whereas the size of MIP-CdTe decreased to about 250 nm with 100 nm of MIP shell thickness when 0.2 mg of CdTe QDs was used. A possible cause for this could be associated with decreased inter-particle spacing by increasing the initial amount of CdTe, leading to the agglomeration of CdTe QDs before polymerization. Based on above results, the MIP-CdTe and NIPCdTe were prepared at T:M:C ratios of 1:4:10 from 0.2 mg of CdTe QDs. Fig. 2d shows the TEM image of CdTe QDs with FL emission wavelength at 580 nm, which clearly display narrow size distribution and spherical morphology with an average diameter about 7 nm. The removal of templates bound to MIP-QDs was accomplished via the combination of ultrasonication and centrifugation. MIP-CdTe QDs were dispersed in 10 mL of water followed by 30 min of ultrasonication and 5 min of centrifugation at 4000 rpm. This process was repeated until establishing similar FL intensities of both MIP-CdTe QDs and NIP-CdTe QDs. The efficient removal process of the templates was completed after running five cycles, confirmed by recovering FL intensity up to 93–95% of NIP-CdTe. As seen in Fig. 3, where the template was melamine, a distinct difference was apparent in the FL spectra of NIP-CdTe QDs and MIP-CdTe QDs removed/re-bound templates. The FL intensity of MIP-CdTe QDs was recovered with high reproducibility by removing the template and then quenched again by re-binding the template.
Fig. 3. FL spectra of (a) NIP-CdTe QDs and (b) MIP-CdTe QDs after removing a template (melamine) and (c) MIP-CdTe QDs-bound template (melamine).
3.2. Effects of concentrations of monomer and cross-linker The molar ratio (T:M:C) of template (T), monomer (M), and cross-linker (C) was investigated to evaluate the effects of monomer and cross-linker in the FL intensity of MIP-CdTe QDs. Monomer concentration was varied from 10 mM (T:M:C ¼1:2:20) to 60 mM (T:M:C ¼1:12:20) while the template and the cross-linker were kept constant at 5 mM and at 0.1 M, respectively. The FL intensity of MIP-CdTe QDs increased with increasing concentration of monomer, APTES, and then reached a maximum value at 40 mM of monomer (T:M:C ratio¼ 1:8:20) in the case of clenbuterol and at 20 mM of monomer (T:M:C ratio¼1:4:20) in the case of melamine, as shown in Fig. S2a (see Supporting information). It is well known that the surface coating of QDs strongly affect the physicochemical and photophysical stability of QDs (Hezinger et al., 2008). The proper amount of monomer can make the better packing resulted from the interaction between monomer and QDs, affording to the enhancement of FL intensity by protecting
314
B. The Huy et al. / Biosensors and Bioelectronics 57 (2014) 310–316
the surface against defects and oxidation. The lower concentration of monomer would produce the incomplete coverage of QDs surface and result in more defects on the surface. The thickness of the MIP shell layer depends on the total amount of polymerization precursors such as monomer and crosslinking agent. At higher concentrations of the monomer, a thicker MIP shell layer was observed. However, the FL emission of MIPQDs was obscured when the MIP shell layer was too thick at greater than 500 nm. The thickness of the MIP was tuned at about 250 nm by controlling the ratio of T, M, and C to 1:8:20 during the polymerization process. The effect of cross-linker concentration on the FL emission of MIP-CdTe QDs was also investigated within the range of 50– 200 mM at different molar ratios of T:M from 1:2 to 1:8 (in case clenbuterol was a template). It was generally noticed that the FL intensity of MIP-CdTe QDs changed with increasing concentration of cross-linker, as shown in Fig. S2b and c (see Supporting information). We found that at low concentrations (o50 mM) of cross-linker, TEOS, the MIP-CdTe structure was so weak that CdTe QDs were easily disconnected during the template extraction process. The maximum FL emission was observed when 100 mM of cross-linking agent was used for the MIP capping process. Further increases in the concentration of cross-linking agent decreased the FL emission of MIP-QDs. In case of melamine as a template, the results were similar with those of clenbuterol. The FT-IR spectra of MIP-CdTe-captured melamine and clenbuterol were compared with that of NIP-CdTe to ensure proper MIP coating on the template molecules, as illustrated in Fig. S3 (see Supporting information). In these experiments, the T:M:C ratios were 1:8:20 and 1:4:20 for clenbuterol and melamine, respectively. The strong peaks around 1000–1145 cm 1 indicate the Si–O–Si and Si–OH stretching vibrations. The expanse of this band may be caused by the MIP being modified onto the QDs (Wang et al., 2009a, 2009b; Wang, H.-F. et al., 2009; Li et al., 2010). Other observed bands at 459 and 781 cm 1 also show the Si–O vibrations (shown by vertical arrows). The presence of bands around 2940 cm 1 (aliphatic C–H stretching band) and 3460 and 1541 cm 1 (N–H band) suggests the existence of an aminopropyl group (Wang, H.-F. et al., 2009). The change in absorption peaks at around 1632 cm 1 (N–H band) and 2940 cm 1(C–H band) may be evidence that APTES and TEOS were bound to template molecules. The band at about 3500–3200 cm 1 represents a hydrogen-bonded O–H band. This band expanded and shifted when MIP captured the templates (melamine or clenbuterol) in comparison with NIP because the templates were hydrogen bonded to MAA-capped CdTe QDs. The peaks of the bands, marked by horizontal arrows at both ends, suggest that the template was successfully imprinted into the MIP-CdTe.
3.3. Analytical performance characteristics of MIP-CdTe QDs The influence of pH values between 2 and 10 on the FL intensity was examined, and it was found that FL intensity was considerably stable in the pH interval between 6 and 9, as shown in Fig. S4 (see Supporting information). Further experiments were carried out at pH 7.1. The effects of potential interfering ion species on adsorption and extraction of analytes, clenbuterol or melamine, were studied by addition of higher amounts of foreign ions (1.0 mM) to the aqueous sample solutions. The interference of ions such as Na þ , K þ , Mg2 þ , Ca2 þ , Zn2 þ , Cu2 þ , and Fe2 þ have basically no effect on FL intensity of MIP-CdTe. The results showed that these ions affected only about 1–4% of the FL intensity of MIP-CdTe. Meanwhile, the FL intensity of MIP-CdTe has been off up to 15–18% at 1.0 mM clenbuterol or melamine, as shown in Fig. S4 (see Supporting information). Two series of sample solutions with different concentrations of clenbuterol and melamine were prepared to develop calibration curves based on the relationship between FL intensity of MIP-CdTe and the concentration of templates. Collisional quenching occurs when the excited-state QD is deactivated by contact with analytes (clenbuterol or melamine) in solution as quenchers. For collisional quenching, the decrease in FL intensity is described by the ratio of the fluorescence in the absence of quenching to the presence of quencher by the Stern–Volmer equation (Li et al., 2010). Fig. 4 shows the relationship between the concentration of quencher and FL quenching, (I0/I) 1, where I0 and I are the FL intensities in the absence and presence of a quencher (clenbuterol (a) or melamine (b)), respectively. The FL intensity was proportionally quenched by increasing the clenbuterol or melamine concentration in the MIP-CdTe QDs. Under optimum conditions, the linear calibration curves have been constructed in the range of 2.5–22.5 mM with a correlation coefficient of 0.9975 for clenbuterol and 2.0–35 mM with a correlation coefficient of 0.9944 for melamine. Precision for five replicate measurements of 20 mM was 2.9% RSD and 3.2% RSD for clenbuterol and melamine, respectively. The Stern–Volmer quenching constants, KSV-MIP, for clenbuterol and melamine were calculated to be 5438 M 1 and 6716 M 1, respectively. The KSV-NIP value for NIP-CdTe was 2447 M 1 (clenbuterol) and 2450 M 1 (melamine). The ratio of KSV-MIP and KSV-NIP was defined as the imprinting factor (IF) to evaluate selectivity of the materials (Wang et al., 2009a, 2009b; Wang, H.-F. et al., 2009). The imprinting factors in the MIP-CdTe QDs were 2.22 for clenbuterol and 2.74 for melamine. These results indicate that clenbuterol and melamine can be detected quantitatively using the FL quenching of MIP-CdTe QDs. The limit of detections (LOD) based on 3s were 0.4 μM and 0.6 μM for clenbuterol and melamine, respectively.
Fig. 4. The relationship between (I0/I) 1 and the concentration of (a) clenbuterol and (b) melamine.
B. The Huy et al. / Biosensors and Bioelectronics 57 (2014) 310–316
Several compounds structurally related to clenbuterol (namely, salbutamol, isoproterenol) or melamine (namely, cyanuric and ammeline) were involved to evaluate the selectivity of the MIPCdTe QDS. The KSV values of NIP and MIP are presented in Fig. 5. The relative standard deviation (%RSD) in FL measurements was in the range of 2.93% and 3.25% based on three replicates. For NIP, the KSV values in all cases were nearly identical. The different KSV values of MIP could be due to the functional group variation from the molecular imprinted cavities. The results show that the imprinting factors were 1.18, 1.13, 1.08 and 1.32 for salbutamol, isoproterenol, cyanuric and ammeline, respectively. The IF value of clenbuterol was higher than that of salbutamol and isoproterenol; and the IF value of melamine was higher than that of cyanuric and ammeline. These results indicate the efficient imprinting effect for the high selectivity of MIP-CdTe toward clenbuterol or melamine. It appears that the nonspecific binding does not affect the fluorescence of CdTe QDs to a great extent, as can be seen in Fig. 5. The FL quenching behavior of MIP-CdTe QDs to the template, clenbuterol/melamine, suggests that it could be associated with the interaction of the templates with a limited number of specific binding sites rather than to nonspecific sites on the polymer. In the polymerization process, the functional groups in the template molecule can interact with the amino groups (–NH2) in APTES to form a complex through hydrogen bondings. When there is no template in MIP-CdTe QDs, an emission is generated by accepting the excited energy of CdTe QDs. After re-binding the template, there will be a strong interaction between the template molecule and amino groups, resulted in quenching of the FL of the MIP-CdTe QDs. We suggest that a quenching mechanism is the charge transfer between the template and CdTe QDs, as evidenced by no overlap bands between the absorption spectrum of the template molecule (not shown here and agreed to other report (Izquierdo-Lorenzo et al., 2010)) and the emission spectrum of the CdTe QDs. The charges of the conductive band of the CdTe QDs can transfer to the lowest unoccupied molecular orbital (LUMO) of the UV band of template molecules.
315
Then the excited charges tend to go back and a quenching is generated (Wang et al., 2009a, 2009b; Wang, H.-F. et al., 2009). Without expensive or sophisticated instrumentation, extensive sample preparation, or the need for highly skilled personnel, the LOD values obtained by the present method are on same order as those of a colorimetric assays method for the detection of clenbuterol (He et al., 2011) and a potentiometric method for the detection of melamine (Liang et al., 2009), but much lower than those of MIR-Raman method (Meza-Marquez et al., 2011) and IGCA (Li et al., 2011) for clenbuterol (see Table 1). Although the lower detection (LOD: 1.2 ng/mL) for clenbuterol in horse urine was achieved by using GC–MS2 method (Van et al., 2002), it needs elaborated and time consuming procedures for liquid-liquid extraction and derivatization. Our proposed method is sensitive enough and highly selective for practical uses based on the maximum concentrations allowed by International Legislation from the World Health Organization (De Wasch et al., 1998) to determine clenbuterol and melamine in various matrices. The developed method requires no sample pretreatments including extraction and derivatization and would be a promising alternative for analyzing clenbuterol and melamine quantitatively in food sample. 3.4. Application to real sample analysis The developed optosensing method using MIP-CdTe QDs was adapted to determine melamine concentration in milk drinks and clenbuterol in beef liver samples. These samples were purchased from a local market in Changwon, Korea. Samples were prepared as described in Section 2.5. Recovery tests were performed to evaluate the physical performance of MIP-CdTe QDs. A summary of the analytical results for each spiked sample is shown in Table 2. Recoveries were calculated by comparing the peak areas of the spiked sample with those of standard solutions following the same procedure. Owing to the high selectivity of MIP-CdTe, good recoveries from 92–97% were obtained. These results showed that
Fig. 5. Quenching constants of analyte-imprinted MIP-CdTe and NIP-CdTe for compounds of similar structure to clenbuterol (a) and melamine (b).
Table 1 Comparison of LODs between MIP-CdTe and other methods. Method
Clenbuterol (ng/mL)
Colorimetric GC/MS SERS MIR-Raman IGCA Potentiometric SPE–HPLC Optosensing MIP-CdTe
112 1.2 35 6000
120
Melamine (ng/mL)
2000 750 83 75
Matrix
Reference
Aqueous solution Horse urine Drug Meat River water Milk products Milk powder Milk drink Beef liver
He et al. (2011) Van et al. (2002) Izquierdo-Lorenzo et al. (2010) Meza-Marquez et al. (2011) Li et al. (2011) Liang et al. (2009) Yang et al. (2009) This work
316
B. The Huy et al. / Biosensors and Bioelectronics 57 (2014) 310–316
Table 2 Application of MIP-CdTe to determine melamine concentration in milk drinks and clenbuterol in beef liver. Analyte
Sample
Concentration of template (μg/L) Amount added
Amount found*
Recovery (%)
Melamine
Milk-A Milk-B Milk-C
88.3 189.2 630.6
86.17 3.3 181.17 10.9 580.9 7 31.5
97.5 95.7 92.1
Clenbuterol
Liver-1 Liver-2 Liver-3
156.0 376.3 627.3
144.3 7 15.6 359.4 7 11.9 594.6 7 19.7
92.5 95.5 95.0
n
Average value of three determinations 7 confidence level (95%)
the present approach provides accurate measurements for analyzing clenbuterol and melamine in real samples. 4. Conclusions A novel and simple technique for the optosensing of clenbuterol and melamine was developed based on the FL quenching of MIP-capped CdTe QDs with good recovery, precision, and accuracy. The spatial distribution of MIP-CdTe cavities can be manipulated by controlling the initial amount of template and the ratio of T:M:C in the polymerization process. FL quenching of MIP-CdTe allows the determination of the concentration of clenbuterol in beef liver and melamine in milk drink samples at 0.4 μM (120 ng/mL) and 0.6 μM (75 ng/mL), respectively. The proposed optosensing method with MIP-CdTe QDs will provide an alternative to current practice methods for detecting clenbuterol and melamine. In view of the sensitivity and the selectivity, this method affords a sensitive detection for clenbuterol and melamine. Acknowledgments This work was supported by the Basic Science Research Program (NRF 2011–0010155) and the Priority Research Centers Program (NRF 2010–0029634), through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.02.041. References Afkhami, A., Ghaedi, H., Madrakian, T., Ahmadi, M., Mahmood-Kashani, H., 2013. Biosens. Bioelectron. 44, 34–40. Chen, H., Kong, J., Yuan, D., Fu, G., 2014. Biosens. Bioelectron. 53, 5–11. Costa-Fernandez, J., 2006. Anal. Bioanal. Chem. 384, 37–40.
Crescenzi, C., Bayoudh, S., Cormack, P.A.G., Klein, T., Ensing, K., 2001. Anal. Chem. 73, 2171–2177. De Wasch, K., De Brabander, H., Courtheyn, D., 1998. Analyst 123, 2701–2705. Gao, D., Zhang, Z., Wu, M., Xie, C., Guan, G., Wang, D., 2007. J. Am. Chem. Soc. 129, 7859–7866. Gerhards, C., Schulz-Drost, C., Sgobba, V., Guldi, D.M., 2008. J. Phys. Chem. B 112, 14482–14491. Hau, A.K., Kwan, T.H., Li, P.K, 2009. J. Am. Soc. Nephrol. 20, 245–250. He, D., Zhang, X., Gao, B., Wang, L., Zhao, Q., Chen, H., Wang, H., Zhao, C., 2014. Food Control 36, 36–41. He, L., Su, Y., Shen, X., Zheng, Y., Guo, H., Zeng, Z., 2009. J. Sep. Sci. 32, 3310–3318. He, P., Shen, L., Liu, R., Luo, Z., Li, Z., 2011. Anal. Chem. 83, 6988–6995. Hezinger, A.F.E., Teßmar, J., Göpferich, A., 2008. Eur. J. Pharm. Biopharm. 68, 138–152. Izquierdo-Lorenzo, I., Sanchez-Cortes, S., Garcia-Ramos, J.V., 2010. Langmuir 26, 14663–14670. Jia, B., Ouyang, Y., Sodhi, R.N.S., Hu, B., Zhang, T., Li, J., Chen, H., 2011. J. Mass Spectrom. 46, 313–319. Lee, J., Lho, D.-s., Kim, M., Lee, W., Kim, Y., 1998. Rapid Commun. Mass Spectrom. 12, 1366–1372. Lee, M.-H., Chen, Y.-C., Ho, M.-H., Lin, H.-Y., 2010. Anal. Bioanal. Chem. 397, 1457–1466. Li, H., Li, Y., Cheng, J., 2010. Chem. Mater. 22, 2451–2457. Li, X., Luo, P., Tang, S., Beier, R.C., Wu, X., Yang, L., Li, Y., Xiao, X., 2011. J. Agric. Food Chem. 59, 6064–6070. Liang, R., Zhang, R., Qin, W., 2009. Sens. Actuators. B 141, 544–550. Limian, Z., Joan, S., 2010. LCGC N. Am., 1–8. Lin, C.I., Joseph, A.K., Chang, C.K., Lee, Y.D., 2004. Biosens. Bioelectron. 20, 127–131. Liu, J., Chen, H., Lin, Z., Lin, J.-M., 2010. Anal. Chem. 82, 7380–7386. Liu, R., Guan, G., Wang, S., Zhang, Z., 2011. Analyst 136, 184–190. Liu, X.H., Luo, J.P., Tian, Q., Liu, C.X., Cai, X.X., 2011. Chin. J. Anal. Chem 39, 163–167. Meza-Marquez, O.G., Gallardo-Velazquez, T., Dorantes-Alvarez, L., Osorio-Revilla, G., de la Rosa Arana, J.L., 2011. Analyst 136, 3355–3365. Molnár, M., Ning, Z.J., Chen, Y., Friberg, P., Gan, L.M., Fu, Y., 2011. Sens. Actuators. B 155, 823–830. Peng-Ju, W., Jun, Y., Qing-De, S., Yun, G., Xiao-Lan, Z., Ji-Bao, C., 2007. Chin. J. Anal. Chem. 35, 484–488. Qian, H., Dong, C., Weng, J., Ren, J., 2006. Small 2, 747–751. Sari, S., Christopher, M., 2010. International experts limit melamine levels in food. 〈http://www.who.int/mediacentre/news/releases/2010/melamine_food_20100706/ en/index.html〉. Van, E.P., Delbeke, F.T., Deprez, P., 2002. Biomed. Chromatogr. 16, 475–481. Wang, C.L., Fang, M., Xu, S.H., Cui, Y.P., 2009a. Langmuir 26, 633–638. Wang, C., Fang, M., Han, J., Zhang, H., Cui, Y., Yang, B., 2009b. J. Phys. Chem. C 113, 19445–19451. Wang, H.-F., He, Y., Ji, T.-R., Yan, X.-P., 2009. Anal. Chem. 81, 1615–1621. Wang, Y.-q., Zou, W.-s., 2011. Talanta 85, 469–475. Wu, B., Wang, Z., Zhao, D., Lu, X., 2012. Talanta 101, 374–381. Yang, F., Mao, Y., Zhang, X., Ma, Z., Zhang, X., 2009. J. Sep. Sci. 32, 2974–2978. Yang, H.-H., Zhou, W.-H., Guo, X.-C., Chen, F.-R., Zhao, H.-Q., Lin, L.-M., Wang, X.-R., 2009. Talanta 80, 821–825. Ying, S., Cui, S., Wang, W., Feng, J., Chen, J., 2014. J. Lumin. 145, 575–581. Yuan, J., Guo, W., Yin, J., Wang, E., 2009. Talanta 77, 1858–1863. Yuen, A.H.Y., Yacoub, M.H., Smolenski, R.T., 2005. Rapid Commun. Mass Spectrom. 19, 3603–3606. Zhang, W., He, X.-W., Chen, Y., Li, W.-Y., Zhang, Y.-K., 2011. Biosens. Bioelectron. 26, 2553–2558. Zhang, W., He, X.-W., Chen, Y., Li, W.-Y., Zhang, Y.-K., 2012a. Biosens. Bioelectron. 31, 84–89. Zhang, W., He, X.-W., Li, W.-Y., Zhang, Y.-K., 2012b. Chem. Commun. (Cambridge, UK) 48, 1757–1759. Zhang, Z., Zhang, M., Luo, L., Yang, X., Hu, Y., Zhang, H., Yao, S., 2010. J. Sep. Sci. 33, 2854–2861.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Selective optosensing of clenbuterol and melamine using molecularly imprinted polymer-capped CdTe quantum dots Bui The Huy a,b, Min-Ho Seo a, Xinfeng Zhang a,c, Yong-Ill Lee a,n a
Department of Chemistry, Changwon National University, Changwon 641-773, South Korea Nhatrang Institute of Technology Research and Application, Vietnam Academy of Science and Technology (VAST), 2 Hung Vuong, Nhatrang, Vietnam c Mineral Resources Chemistry Key Laboratory of Sichuan Higher Education Institutions, College of Material and Chemistry & Chemical Engineering, Chengdu University of Technology, Chengdu 610059, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 12 November 2013 Received in revised form 28 January 2014 Accepted 10 February 2014 Available online 25 February 2014
A novel procedure for the optosensing of clenbuterol and melamine was developed using molecularly imprinted polymer-capped CdTe quantum dots (MIP-CdTe QDs). The MIP-CdTe QDs were synthesized by a radical polymerization process among CdTe QDs, a template, 3-aminopropyltriethoxysilane (APTES) and tetraethoxysilane (TEOS). The sizes of the MIP-CdTe particles were controlled by the speed of polymerization, concentration of the template, concentration of the quantum dots, and the ratio of template, monomer and cross-linker. Excellent selectivity and high sensitivity of MIP-CdTe QDs toward clenbuterol/melamine molecules were observed based on the fluorescence quenching of QDs. Experimental results showed that the optimum molar ratios of template, monomer, and cross-linker were 1:8:20 and 1:4:20 for analyzing clenbuterol and melamine, respectively. Under optimum conditions, these MIP-CdTe QDs showed a limit of detection of 0.4 μM (120 ng/mL) for clenbuterol and 0.6 μM (75 ng/mL) for melamine. The feasibility of the developed method in real samples was successfully evaluated through the analysis of clenbuterol and melamine in milk and liver samples with satisfactory recoveries of 92–97%. The MIP-CdTe QDs could be easily regenerated for subsequent sample analysis with water. & 2014 Elsevier B.V. All rights reserved.
Keywords: CdTe Molecularly imprinted polymer Fluorescence quenching Clenbuterol Melamine
1. Introduction Molecular imprinting is a technique to create selective binding sites for a specific molecule. Molecularly imprinted polymers (MIPs) are synthetic polymeric materials with specific recognition sites complementary in shape, size, and functional groups to template molecules. The templates can be imprinted during polymerization and later extracted, creating three-dimensional template binding sites for the purpose of retaining the memory of a template molecule. The general synthetic procedures of MIPs involve complexation in a solution of a template molecule with functional monomers through non-covalent bonds, followed by polymerization of these monomers around the template with the help of a cross-linker in the presence of an initiator. After completing the polymerization process, template molecules are removed by extensive washing steps to disrupt the interactions between the template and the monomer, thus freeing the binding sites. The template extraction directly influences the limit of detection and precision. In general, efficient extraction of the
n
Corresponding authors: Tel.: þ82 55 213 3436; fax: þ 82 55 213 3439. E-mail address: [email protected] (Y.-I. Lee).
http://dx.doi.org/10.1016/j.bios.2014.02.041 0956-5663 & 2014 Elsevier B.V. All rights reserved.
original templates located at the interior area is not easy because of extremely low analyte diffusion rates in highly cross-linked MIPs. Furthermore, the rigid polymeric matrix restrains target species into those deep imprinted and thus reduces compatible recognition (Gao et al., 2007; Peng-Ju et al., 2007). Recently, several approaches have been explored to overcome these problems through the use of surface imprinting with nanospheres for detecting salivary proteins, pyrethroids, atrazine (Lee et al., 2010; Li et al., 2010; Liu, R. et al., 2011) or core–shell imprinted nanoparticles for selective detection of uracil, melamine and lysozyme (Lin et al., 2004; Zhang et al., 2010; Chen et al., 2014). Example, the best imprinting effect was improved by the rebinding kinetic and rebinding affinity (Chen et al., 2014). During the last several decades, quantum dots (QDs) have attracted considerable interest due to their size-dependent optical properties, large surface-to-volume ratios, high quantum effects, and narrow spectral line widths. We know that optical properties of quantum dots are related to its surface and it also have an ability to form coupling complexes with organic compounds (Gerhards et al., 2008; Zhang et al., 2011). These characteristics along with high stability against photobleaching have been shown to be useful in a number of compelling applications such as chemical sensors for bio-macromolecules (Yuan et al., 2009; Zhang et al.,
B. The Huy et al. / Biosensors and Bioelectronics 57 (2014) 310–316
2012), fluorescent probes (Costa-Fernandez, 2006; Liu, X.H. et al., 2011), and influences in bio-environments (Molnár et al., 2011). MIPs with high stability, easy preparation, and low cost present opportunities for analyzing various target analytes including tramadol in human urine (Afkhami et al., 2013), 2,4,6-trinitrotoluene (TNT) (Gao et al., 2007; Wang and Zou, 2011), pyrethroids (Li et al., 2010), and cytochrome (Zhang et al. 2011). Therefore, the use of MIPs-QDs for direct sensing of molecules is a challenge to achieving the tailored selectivity of analytics (Li et al., 2010; Zhang et al. 2011, 2012a, 2012b). Clenbuterol is a well-known sympathomimetic agent and “lean meat powder” because it is fed to stock animals to ensure lean muscle mass. It also causes them to grow quicker by speeding up fat burning and muscle building. Also, clenbuterol is a recently popular drug used by athletes in diverse sports for its purported anabolic effects and reduction of subcutaneous fat. Melamine is a synthetic compound that is widely used as an industrial chemical for the production of plastics, amino resins and flame retardants. Because of its high nitrogen content, the use of melamine can increase the total nitrogen level of poor quality products, seemingly increasing the high protein content. For instance, melamine was mixed in powdered milk to make an artificial protein into a total protein. The toxicity of melamine caught the attention of physicians as a result of a recent spate of renal injury after exposure to melamine-tainted milk in China (Hau et al., 2009). The maximum allowed concentrations by International Legislation from the World Health Organization of melamine in powdered infant food and other food are 1 mg/kg and 2.5 mg/kg, respectively (Sari and Christopher, 2010). A maximum residue limit of 0.5 μg/kg for clenbuterol in the liver of cattle and horses is proposed by European legislation (De Wasch et al., 1998). Recently, clenbuterol and melamine have been found in many kinds of food or food-related products, resulting in many serious human health problems. Various analytical techniques have been suggested to analyze clenbuterol and melamine, such as gas chromatography/mass spectrometry (GC/MS2) (Van et al., 2002), liquid chromatography/mass spectrometry (LC/MS) (Yuen et al., 2005; Yang et al., 2009), solid phase extraction coupled with highperformance liquid chromatography analysis (SPE–HPLC) (He et al., 2009; Yang et al., 2009), mid infrared (MIR) and Raman spectroscopy (Meza-Marquez et al., 2011), surface desorption atmospheric pressure chemical ionization mass spectrometry (SDAPCI-MS) (Jia et al., 2011), combination of magnetic MIP and liquid chromatography–tandem mass spectrometry (He et al. 2014), colorimetric assay using gold nanoparticles (He et al., 2011), potentiometric sensors based on MIP (Liang et al., 2009), electrochemical impedance spectroscopy (EIS) (Wu et al., 2012), immunogold chromatography (IGCA) (Li et al., 2011), and surfaceenhanced Raman scattering (SERS) (Izquierdo-Lorenzo et al., 2010). Although these techniques show promising results for sensitive detection of clenbuterol and melamine, there are still some hindrances including sophisticated instrumentation, extensive sample preparation, and the need for highly skilled personnel. Furthermore, quantitative analysis of clenbuterol is often performed after chemical derivatization, but the derivative procedure may introduce large variability or sample loss (Lee et al., 1998; Crescenzi et al., 2001). Combining the high selectivity of MIP and the efficient fluorescence (FL) properties of QDs could develop a new method for selective recognition and quantitative determination of target analytes. MIP-capped QDs for analytical applications are still in an early stage of development. Due to the abovementioned reasons, the development of an inexpensive, simple approach for the direct detection of clenbuterol and melamine, and as far as we know MIP-capped QDs for the quantitative analysis of clenbuterol and melamine, has not been reported. In the present work, we report the facile synthesis of MIP-capped
311
CdTe quantum dots (MIP-CdTe QDs) in an aqueous phase and their application for the direct detection of clenbuterol and melamine in food. In this study, clenbuterol and melamine were selected as targets because, in addition to the above-mentioned reasons, MIP-CdTe for both cases were produced from the same monomer and cross-linker, resulting in favorable conditions in the process MIP preparation. The sensing of clenbuterol and melamine is based on fluorescence quenching when the analytes are selectively recognized and combined with MIP-capped quantum dots. The sizes of the MIPs particles were controlled by the speed of polymerization, amount of quantum dots, initial concentration of the template, and the molar ratio (T:M:C) of the template (T), monomer (M), and cross-linker (C). Mercaptoacetic acid (MAA)capped CdTe QDs were bound to the analytes (clenbuterol or melamine) and a functional monomer (3-aminopropyltriethoxysilane, APTES) by hydrogen bonding. Experimental results demonstrated that the developed MIP-CdTe QDs method has good potential for quantitative analysis of trace amounts of clenbuterol and melamine with high selectivity.
2. Experiment 2.1. Materials Ammonium hydroxide (NH3H2O), APTES, cadmium chloride (CdCl2), MAA, sodium borohydride (NaBH4), sodium hydroxide (NaOH), tellurium (Te powder), tetraethyl orthosilicate (TEOS), clenbuterol, and melamine were purchased from Sigma-Aldrich. Deionized water with a resistivity of 18.2 MΩ cm was used. All chemicals were used as received without any further purification. The potassium hydrophtalate (0.1 M, pH 2–4), phosphate buffer solution (0.1 M, pH 5–10) were used in the experiments. 2.2. Synthesis of CdTe quantum dots The CdTe QDs in an aqueous phase were synthesized based on the procedure described in the literature with some modifications (Qian et al., 2006; Ying et al., 2014). Briefly, 0.183 g of CdCl2, 0.140 mL of MAA, and 100 mL of water were mixed in a three-neck flask to form a cadmium precursor. The mixture was adjusted to pH 8 with 1.0 N NaOH. Separately, 32 mg of tellurium powder, 20 mg of NaBH4 and 0.5 mL of ultrapure water were added to a one-neck flask. The one-neck flask was sealed and heated at 50 1C for 30 min to produce a deep red clear solution. Then, the obtained NaHTe solution in the one-neck flask was diluted to 40 mL with water and injected into the reaction system under vigorous stirring. Subsequently, the reaction mixture was refluxed for 3 h and then cooled to room temperature. Isopropanol was added to the prepared CdTe solution at the volume ratio of 1:1 (isopropanol:water) to precipitate CdTe particles. 2.3. Synthesis of MIP-capped CdTe QDs (MIP-CdTeQDs) 0.2 mg of CdTe QDs, 5 mL of water, and 25 mmol of template (clenbuterol or melamine) were placed in a 50 mL flask and stirred for 30 min to bind the template to the surface of the CdTe QDs by MAA. Then, 50–300 mmol of APTES as a functional monomer was added and stirred for 30 min. Finally, 0.25–1 mmol of TEOS as a cross-linker and 100 μL of ammonium hydroxide as a catalyst were injected together using the syringe pump, and the mixture was stirred for 12 h. The non-imprinted polymers CdTe QDs (NIP-CdTe QDs) were synthesized using the same procedure without adding the template in order to compare the molecular recognition performances with MIP-CdTe QDs. The resulting MIP-CdTe QDs
312
B. The Huy et al. / Biosensors and Bioelectronics 57 (2014) 310–316
and NIP-CdTe QDs were sonicated, centrifuged, and washed several times with water to extract the template and to remove any free monomer and cross-linker. The products were considered adequately washed when similar FL intensities of both MIP-CdTe QDs and NIP-CdTe QDs were achieved. 2.4. Characterization The morphologies of MIP-CdTe QDs and NIP-CdTe QDs were characterized using a scanning electron microscope (SEM) (FESEM MIRA II LMH, Tescan, USA). The samples for SEM were prepared by drying the QDs in a vacuum. All FL measurements were performed on a FP-6500 spectrofluorometer (JASCO, Tokyo, Japan) with an excitation wavelength at 355 nm. The slit widths for both excitation and emission were set at 3 nm. The UV spectra of the QDs in KBr were recorded on a FT/IR-6300 Fourier transform infrared spectrometer (JASCO, Tokyo, Japan). Transmission electron microscopy (TEM) measurements were carried out using a JEM-2100F transmission electron microscope (JEOL, Tokyo, Japan) operating at 200 kV. TEM samples were prepared by dropping an aqueous solution of the nanoparticles onto amorphous carboncoated copper grids with the excessive solvent evaporated. 2.5. Sample preparation Analytical samples were prepared on the basis of a procedure described elsewhere in the literature (Limian and Joan, 2010; Li et al., 2011). Milk samples were diluted ten-fold using water prior to the analysis but did not undergo any special treatments. The liver samples were washed, chopped into small pieces and homogenized thoroughly with a food grinder. Fifty-gram amounts of homogeneous sample were mixed with 10 mL acetonitrile/ water (1:1, v/v) and then placed in a 50 mL beaker. Extraction was performed under stirring at 300 rpm for 12 h; acetonitrile precipitated the proteins in this process. Subsequently, the mixture was centrifuged for 3 min to remove the precipitated proteins. The supernatant solution was stored for analysis.
3. Results and discussion 3.1. Preparation and characterization of MIP- and NIP-CdTe QDs in the aqueous phase The preparation of NaHTe was facilitated by heating the mixture of NaBH4 and Te powder to 50 1C for 30 min. The highly luminescent CdTe QDs were synthesized in an aqueous phase through the reaction of tellurium and cadmium precursors in the presence of a thiolated agent, MAA. Mercaptoacetic acid can
further cap the surface defects because of its short length, so it can increase the surface tension of CdTe QDs and hinder the growth of CdTe QDs. Larger QD particles were obtained from a lengthy reaction time, leading to the red shift of FL emission of the CdTe QDs ranging in λmax from 520 nm to 580 nm, as illustrated in Fig. S1 (see Supporting information). This is an effect of the Ostwald ripening mechanism as smaller particles dissolve in the process of producing larger particles. The mean particle sizes of the CdTe QDs were determined by TEM on the 200 particles and ranged from 7 nm (reaction time: 3 h) for green and 9 nm (reaction time: 7 h) for yellow. The mean particle size according to TEM was larger than that of the real size because the sizes of water-soluble CdTe nanoparticles were difficult to characterize by TEM due to the aggregation of CdTe nanoparticles during drying on a Cu grid. The inset in Fig. S1 shows a photograph of the watersoluble CdTe QDs. Strong FL emissions with different colors were observed with the naked eye under the illumination of an ultraviolet lamp. Size-selective precipitation of aqueous nanoparticles was achieved by adjusting the amount of isopropanol (Wang et al., 2009a). Under vigorous stirring at room temperature, isopropanol was dropped into the prepared CdTe solution until the solution became slightly turbid, and then the mixture was centrifuged at 4000 rpm for 5 min to obtain the CdTe particles. The precipitation mechanism may be related to the decreased relative dielectric constant of the mixture solution and the reduced interparticle electrostatic repulsion (Wang et al., 2009b). The synthesis of MIP-CdTe QDs involves the complexation of CdTe QDs with the template and functional monomers molecules (APTES) through non-covalent bonds in aqueous solution, followed by the polymerization of these monomers around the template (clenbuterol or melamine) with a cross-linker (TEOS). Fig. 1 illustrates the mechanism of molecularly imprinted polymers with CdTe QDs in the aqueous phase through hydrogen bonding interactions. The amino groups (–NH2) in the molecule of APTES interact with the functional groups in the template molecules to form a complex through hydrogen bonding, which mainly caused the FL quenching (Liu et al., 2010). The role of MIP thickness is very important because it influences the removal of the template and FL intensity. Also, it influences the signal to noise ratio, so MIP thickness should be controlled for applications of MIP-CdTe QDs. The size of the MIP-CdTe QDs particles was affected by the speed of the polymerization process. Therefore, the injection speed of the crosslinker (TEOS) to the reactor is one of the most prevailing parameters for controlling the size of MIP-CdTe QDs. Fig. 2a and b shows the comparison of the differences in particle size influenced by the injection speed of cross-linker with melamine as the template. The average size of the particles was found to be 400 750 nm at high speed injection (200 μL/min), which was
Fig. 1. Schematic illustration for the molecular imprinting mechanism on CdTe QDs through hydrogen bonding interactions.
B. The Huy et al. / Biosensors and Bioelectronics 57 (2014) 310–316
5 nm
313
20 nm
Fig. 2. SEM image of MIP-CdTe QDs with (a) high (200 μL/min) and (b) low (10 μL/min) speed injection of the cross-linker at a T:M:C ratio of 1:4:20. (c) TEM image of MIPCdTe QDs produced with 3 mg CdTe QDs at a T:M:C ratio of 1:4:20. (d) TEM image of CdTe with FL emission wavelength at 580 nm.
ten times larger than that of the particles at low speed injection (10 μL/min), at a T:M:C ratio of 1:4:20. The thickness of MIP polymers on CdTe QDs is also governed by the structures and the amounts of template, monomer, cross-linker, and CdTe used. The thicknesses of MIP polymer on CdTe QDs were 15, 30, and 100 nm for T:M:C ratios of 1:2:10, 1:4:10 and 1:4:20, respectively. Interestingly, the initial amount of added CdTe QDs influenced not only the FL intensity but also the size of the resulting MIP-CdTe QDs when the amounts of template, monomer, and cross-linker were held constant. The TEM image of MIP-CdTe QDs formed using a T:M:C ratio of 1:4:20 with 3 mg CdTe is illustrated in Fig. 2c. In this case, the size of MIP-CdTe was about 380 nm with 60 nm of MIP shell thickness; whereas the size of MIP-CdTe decreased to about 250 nm with 100 nm of MIP shell thickness when 0.2 mg of CdTe QDs was used. A possible cause for this could be associated with decreased inter-particle spacing by increasing the initial amount of CdTe, leading to the agglomeration of CdTe QDs before polymerization. Based on above results, the MIP-CdTe and NIPCdTe were prepared at T:M:C ratios of 1:4:10 from 0.2 mg of CdTe QDs. Fig. 2d shows the TEM image of CdTe QDs with FL emission wavelength at 580 nm, which clearly display narrow size distribution and spherical morphology with an average diameter about 7 nm. The removal of templates bound to MIP-QDs was accomplished via the combination of ultrasonication and centrifugation. MIP-CdTe QDs were dispersed in 10 mL of water followed by 30 min of ultrasonication and 5 min of centrifugation at 4000 rpm. This process was repeated until establishing similar FL intensities of both MIP-CdTe QDs and NIP-CdTe QDs. The efficient removal process of the templates was completed after running five cycles, confirmed by recovering FL intensity up to 93–95% of NIP-CdTe. As seen in Fig. 3, where the template was melamine, a distinct difference was apparent in the FL spectra of NIP-CdTe QDs and MIP-CdTe QDs removed/re-bound templates. The FL intensity of MIP-CdTe QDs was recovered with high reproducibility by removing the template and then quenched again by re-binding the template.
Fig. 3. FL spectra of (a) NIP-CdTe QDs and (b) MIP-CdTe QDs after removing a template (melamine) and (c) MIP-CdTe QDs-bound template (melamine).
3.2. Effects of concentrations of monomer and cross-linker The molar ratio (T:M:C) of template (T), monomer (M), and cross-linker (C) was investigated to evaluate the effects of monomer and cross-linker in the FL intensity of MIP-CdTe QDs. Monomer concentration was varied from 10 mM (T:M:C ¼1:2:20) to 60 mM (T:M:C ¼1:12:20) while the template and the cross-linker were kept constant at 5 mM and at 0.1 M, respectively. The FL intensity of MIP-CdTe QDs increased with increasing concentration of monomer, APTES, and then reached a maximum value at 40 mM of monomer (T:M:C ratio¼ 1:8:20) in the case of clenbuterol and at 20 mM of monomer (T:M:C ratio¼1:4:20) in the case of melamine, as shown in Fig. S2a (see Supporting information). It is well known that the surface coating of QDs strongly affect the physicochemical and photophysical stability of QDs (Hezinger et al., 2008). The proper amount of monomer can make the better packing resulted from the interaction between monomer and QDs, affording to the enhancement of FL intensity by protecting
314
B. The Huy et al. / Biosensors and Bioelectronics 57 (2014) 310–316
the surface against defects and oxidation. The lower concentration of monomer would produce the incomplete coverage of QDs surface and result in more defects on the surface. The thickness of the MIP shell layer depends on the total amount of polymerization precursors such as monomer and crosslinking agent. At higher concentrations of the monomer, a thicker MIP shell layer was observed. However, the FL emission of MIPQDs was obscured when the MIP shell layer was too thick at greater than 500 nm. The thickness of the MIP was tuned at about 250 nm by controlling the ratio of T, M, and C to 1:8:20 during the polymerization process. The effect of cross-linker concentration on the FL emission of MIP-CdTe QDs was also investigated within the range of 50– 200 mM at different molar ratios of T:M from 1:2 to 1:8 (in case clenbuterol was a template). It was generally noticed that the FL intensity of MIP-CdTe QDs changed with increasing concentration of cross-linker, as shown in Fig. S2b and c (see Supporting information). We found that at low concentrations (o50 mM) of cross-linker, TEOS, the MIP-CdTe structure was so weak that CdTe QDs were easily disconnected during the template extraction process. The maximum FL emission was observed when 100 mM of cross-linking agent was used for the MIP capping process. Further increases in the concentration of cross-linking agent decreased the FL emission of MIP-QDs. In case of melamine as a template, the results were similar with those of clenbuterol. The FT-IR spectra of MIP-CdTe-captured melamine and clenbuterol were compared with that of NIP-CdTe to ensure proper MIP coating on the template molecules, as illustrated in Fig. S3 (see Supporting information). In these experiments, the T:M:C ratios were 1:8:20 and 1:4:20 for clenbuterol and melamine, respectively. The strong peaks around 1000–1145 cm 1 indicate the Si–O–Si and Si–OH stretching vibrations. The expanse of this band may be caused by the MIP being modified onto the QDs (Wang et al., 2009a, 2009b; Wang, H.-F. et al., 2009; Li et al., 2010). Other observed bands at 459 and 781 cm 1 also show the Si–O vibrations (shown by vertical arrows). The presence of bands around 2940 cm 1 (aliphatic C–H stretching band) and 3460 and 1541 cm 1 (N–H band) suggests the existence of an aminopropyl group (Wang, H.-F. et al., 2009). The change in absorption peaks at around 1632 cm 1 (N–H band) and 2940 cm 1(C–H band) may be evidence that APTES and TEOS were bound to template molecules. The band at about 3500–3200 cm 1 represents a hydrogen-bonded O–H band. This band expanded and shifted when MIP captured the templates (melamine or clenbuterol) in comparison with NIP because the templates were hydrogen bonded to MAA-capped CdTe QDs. The peaks of the bands, marked by horizontal arrows at both ends, suggest that the template was successfully imprinted into the MIP-CdTe.
3.3. Analytical performance characteristics of MIP-CdTe QDs The influence of pH values between 2 and 10 on the FL intensity was examined, and it was found that FL intensity was considerably stable in the pH interval between 6 and 9, as shown in Fig. S4 (see Supporting information). Further experiments were carried out at pH 7.1. The effects of potential interfering ion species on adsorption and extraction of analytes, clenbuterol or melamine, were studied by addition of higher amounts of foreign ions (1.0 mM) to the aqueous sample solutions. The interference of ions such as Na þ , K þ , Mg2 þ , Ca2 þ , Zn2 þ , Cu2 þ , and Fe2 þ have basically no effect on FL intensity of MIP-CdTe. The results showed that these ions affected only about 1–4% of the FL intensity of MIP-CdTe. Meanwhile, the FL intensity of MIP-CdTe has been off up to 15–18% at 1.0 mM clenbuterol or melamine, as shown in Fig. S4 (see Supporting information). Two series of sample solutions with different concentrations of clenbuterol and melamine were prepared to develop calibration curves based on the relationship between FL intensity of MIP-CdTe and the concentration of templates. Collisional quenching occurs when the excited-state QD is deactivated by contact with analytes (clenbuterol or melamine) in solution as quenchers. For collisional quenching, the decrease in FL intensity is described by the ratio of the fluorescence in the absence of quenching to the presence of quencher by the Stern–Volmer equation (Li et al., 2010). Fig. 4 shows the relationship between the concentration of quencher and FL quenching, (I0/I) 1, where I0 and I are the FL intensities in the absence and presence of a quencher (clenbuterol (a) or melamine (b)), respectively. The FL intensity was proportionally quenched by increasing the clenbuterol or melamine concentration in the MIP-CdTe QDs. Under optimum conditions, the linear calibration curves have been constructed in the range of 2.5–22.5 mM with a correlation coefficient of 0.9975 for clenbuterol and 2.0–35 mM with a correlation coefficient of 0.9944 for melamine. Precision for five replicate measurements of 20 mM was 2.9% RSD and 3.2% RSD for clenbuterol and melamine, respectively. The Stern–Volmer quenching constants, KSV-MIP, for clenbuterol and melamine were calculated to be 5438 M 1 and 6716 M 1, respectively. The KSV-NIP value for NIP-CdTe was 2447 M 1 (clenbuterol) and 2450 M 1 (melamine). The ratio of KSV-MIP and KSV-NIP was defined as the imprinting factor (IF) to evaluate selectivity of the materials (Wang et al., 2009a, 2009b; Wang, H.-F. et al., 2009). The imprinting factors in the MIP-CdTe QDs were 2.22 for clenbuterol and 2.74 for melamine. These results indicate that clenbuterol and melamine can be detected quantitatively using the FL quenching of MIP-CdTe QDs. The limit of detections (LOD) based on 3s were 0.4 μM and 0.6 μM for clenbuterol and melamine, respectively.
Fig. 4. The relationship between (I0/I) 1 and the concentration of (a) clenbuterol and (b) melamine.
B. The Huy et al. / Biosensors and Bioelectronics 57 (2014) 310–316
Several compounds structurally related to clenbuterol (namely, salbutamol, isoproterenol) or melamine (namely, cyanuric and ammeline) were involved to evaluate the selectivity of the MIPCdTe QDS. The KSV values of NIP and MIP are presented in Fig. 5. The relative standard deviation (%RSD) in FL measurements was in the range of 2.93% and 3.25% based on three replicates. For NIP, the KSV values in all cases were nearly identical. The different KSV values of MIP could be due to the functional group variation from the molecular imprinted cavities. The results show that the imprinting factors were 1.18, 1.13, 1.08 and 1.32 for salbutamol, isoproterenol, cyanuric and ammeline, respectively. The IF value of clenbuterol was higher than that of salbutamol and isoproterenol; and the IF value of melamine was higher than that of cyanuric and ammeline. These results indicate the efficient imprinting effect for the high selectivity of MIP-CdTe toward clenbuterol or melamine. It appears that the nonspecific binding does not affect the fluorescence of CdTe QDs to a great extent, as can be seen in Fig. 5. The FL quenching behavior of MIP-CdTe QDs to the template, clenbuterol/melamine, suggests that it could be associated with the interaction of the templates with a limited number of specific binding sites rather than to nonspecific sites on the polymer. In the polymerization process, the functional groups in the template molecule can interact with the amino groups (–NH2) in APTES to form a complex through hydrogen bondings. When there is no template in MIP-CdTe QDs, an emission is generated by accepting the excited energy of CdTe QDs. After re-binding the template, there will be a strong interaction between the template molecule and amino groups, resulted in quenching of the FL of the MIP-CdTe QDs. We suggest that a quenching mechanism is the charge transfer between the template and CdTe QDs, as evidenced by no overlap bands between the absorption spectrum of the template molecule (not shown here and agreed to other report (Izquierdo-Lorenzo et al., 2010)) and the emission spectrum of the CdTe QDs. The charges of the conductive band of the CdTe QDs can transfer to the lowest unoccupied molecular orbital (LUMO) of the UV band of template molecules.
315
Then the excited charges tend to go back and a quenching is generated (Wang et al., 2009a, 2009b; Wang, H.-F. et al., 2009). Without expensive or sophisticated instrumentation, extensive sample preparation, or the need for highly skilled personnel, the LOD values obtained by the present method are on same order as those of a colorimetric assays method for the detection of clenbuterol (He et al., 2011) and a potentiometric method for the detection of melamine (Liang et al., 2009), but much lower than those of MIR-Raman method (Meza-Marquez et al., 2011) and IGCA (Li et al., 2011) for clenbuterol (see Table 1). Although the lower detection (LOD: 1.2 ng/mL) for clenbuterol in horse urine was achieved by using GC–MS2 method (Van et al., 2002), it needs elaborated and time consuming procedures for liquid-liquid extraction and derivatization. Our proposed method is sensitive enough and highly selective for practical uses based on the maximum concentrations allowed by International Legislation from the World Health Organization (De Wasch et al., 1998) to determine clenbuterol and melamine in various matrices. The developed method requires no sample pretreatments including extraction and derivatization and would be a promising alternative for analyzing clenbuterol and melamine quantitatively in food sample. 3.4. Application to real sample analysis The developed optosensing method using MIP-CdTe QDs was adapted to determine melamine concentration in milk drinks and clenbuterol in beef liver samples. These samples were purchased from a local market in Changwon, Korea. Samples were prepared as described in Section 2.5. Recovery tests were performed to evaluate the physical performance of MIP-CdTe QDs. A summary of the analytical results for each spiked sample is shown in Table 2. Recoveries were calculated by comparing the peak areas of the spiked sample with those of standard solutions following the same procedure. Owing to the high selectivity of MIP-CdTe, good recoveries from 92–97% were obtained. These results showed that
Fig. 5. Quenching constants of analyte-imprinted MIP-CdTe and NIP-CdTe for compounds of similar structure to clenbuterol (a) and melamine (b).
Table 1 Comparison of LODs between MIP-CdTe and other methods. Method
Clenbuterol (ng/mL)
Colorimetric GC/MS SERS MIR-Raman IGCA Potentiometric SPE–HPLC Optosensing MIP-CdTe
112 1.2 35 6000
120
Melamine (ng/mL)
2000 750 83 75
Matrix
Reference
Aqueous solution Horse urine Drug Meat River water Milk products Milk powder Milk drink Beef liver
He et al. (2011) Van et al. (2002) Izquierdo-Lorenzo et al. (2010) Meza-Marquez et al. (2011) Li et al. (2011) Liang et al. (2009) Yang et al. (2009) This work
316
B. The Huy et al. / Biosensors and Bioelectronics 57 (2014) 310–316
Table 2 Application of MIP-CdTe to determine melamine concentration in milk drinks and clenbuterol in beef liver. Analyte
Sample
Concentration of template (μg/L) Amount added
Amount found*
Recovery (%)
Melamine
Milk-A Milk-B Milk-C
88.3 189.2 630.6
86.17 3.3 181.17 10.9 580.9 7 31.5
97.5 95.7 92.1
Clenbuterol
Liver-1 Liver-2 Liver-3
156.0 376.3 627.3
144.3 7 15.6 359.4 7 11.9 594.6 7 19.7
92.5 95.5 95.0
n
Average value of three determinations 7 confidence level (95%)
the present approach provides accurate measurements for analyzing clenbuterol and melamine in real samples. 4. Conclusions A novel and simple technique for the optosensing of clenbuterol and melamine was developed based on the FL quenching of MIP-capped CdTe QDs with good recovery, precision, and accuracy. The spatial distribution of MIP-CdTe cavities can be manipulated by controlling the initial amount of template and the ratio of T:M:C in the polymerization process. FL quenching of MIP-CdTe allows the determination of the concentration of clenbuterol in beef liver and melamine in milk drink samples at 0.4 μM (120 ng/mL) and 0.6 μM (75 ng/mL), respectively. The proposed optosensing method with MIP-CdTe QDs will provide an alternative to current practice methods for detecting clenbuterol and melamine. In view of the sensitivity and the selectivity, this method affords a sensitive detection for clenbuterol and melamine. Acknowledgments This work was supported by the Basic Science Research Program (NRF 2011–0010155) and the Priority Research Centers Program (NRF 2010–0029634), through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.02.041. References Afkhami, A., Ghaedi, H., Madrakian, T., Ahmadi, M., Mahmood-Kashani, H., 2013. Biosens. Bioelectron. 44, 34–40. Chen, H., Kong, J., Yuan, D., Fu, G., 2014. Biosens. Bioelectron. 53, 5–11. Costa-Fernandez, J., 2006. Anal. Bioanal. Chem. 384, 37–40.
Crescenzi, C., Bayoudh, S., Cormack, P.A.G., Klein, T., Ensing, K., 2001. Anal. Chem. 73, 2171–2177. De Wasch, K., De Brabander, H., Courtheyn, D., 1998. Analyst 123, 2701–2705. Gao, D., Zhang, Z., Wu, M., Xie, C., Guan, G., Wang, D., 2007. J. Am. Chem. Soc. 129, 7859–7866. Gerhards, C., Schulz-Drost, C., Sgobba, V., Guldi, D.M., 2008. J. Phys. Chem. B 112, 14482–14491. Hau, A.K., Kwan, T.H., Li, P.K, 2009. J. Am. Soc. Nephrol. 20, 245–250. He, D., Zhang, X., Gao, B., Wang, L., Zhao, Q., Chen, H., Wang, H., Zhao, C., 2014. Food Control 36, 36–41. He, L., Su, Y., Shen, X., Zheng, Y., Guo, H., Zeng, Z., 2009. J. Sep. Sci. 32, 3310–3318. He, P., Shen, L., Liu, R., Luo, Z., Li, Z., 2011. Anal. Chem. 83, 6988–6995. Hezinger, A.F.E., Teßmar, J., Göpferich, A., 2008. Eur. J. Pharm. Biopharm. 68, 138–152. Izquierdo-Lorenzo, I., Sanchez-Cortes, S., Garcia-Ramos, J.V., 2010. Langmuir 26, 14663–14670. Jia, B., Ouyang, Y., Sodhi, R.N.S., Hu, B., Zhang, T., Li, J., Chen, H., 2011. J. Mass Spectrom. 46, 313–319. Lee, J., Lho, D.-s., Kim, M., Lee, W., Kim, Y., 1998. Rapid Commun. Mass Spectrom. 12, 1366–1372. Lee, M.-H., Chen, Y.-C., Ho, M.-H., Lin, H.-Y., 2010. Anal. Bioanal. Chem. 397, 1457–1466. Li, H., Li, Y., Cheng, J., 2010. Chem. Mater. 22, 2451–2457. Li, X., Luo, P., Tang, S., Beier, R.C., Wu, X., Yang, L., Li, Y., Xiao, X., 2011. J. Agric. Food Chem. 59, 6064–6070. Liang, R., Zhang, R., Qin, W., 2009. Sens. Actuators. B 141, 544–550. Limian, Z., Joan, S., 2010. LCGC N. Am., 1–8. Lin, C.I., Joseph, A.K., Chang, C.K., Lee, Y.D., 2004. Biosens. Bioelectron. 20, 127–131. Liu, J., Chen, H., Lin, Z., Lin, J.-M., 2010. Anal. Chem. 82, 7380–7386. Liu, R., Guan, G., Wang, S., Zhang, Z., 2011. Analyst 136, 184–190. Liu, X.H., Luo, J.P., Tian, Q., Liu, C.X., Cai, X.X., 2011. Chin. J. Anal. Chem 39, 163–167. Meza-Marquez, O.G., Gallardo-Velazquez, T., Dorantes-Alvarez, L., Osorio-Revilla, G., de la Rosa Arana, J.L., 2011. Analyst 136, 3355–3365. Molnár, M., Ning, Z.J., Chen, Y., Friberg, P., Gan, L.M., Fu, Y., 2011. Sens. Actuators. B 155, 823–830. Peng-Ju, W., Jun, Y., Qing-De, S., Yun, G., Xiao-Lan, Z., Ji-Bao, C., 2007. Chin. J. Anal. Chem. 35, 484–488. Qian, H., Dong, C., Weng, J., Ren, J., 2006. Small 2, 747–751. Sari, S., Christopher, M., 2010. International experts limit melamine levels in food. 〈http://www.who.int/mediacentre/news/releases/2010/melamine_food_20100706/ en/index.html〉. Van, E.P., Delbeke, F.T., Deprez, P., 2002. Biomed. Chromatogr. 16, 475–481. Wang, C.L., Fang, M., Xu, S.H., Cui, Y.P., 2009a. Langmuir 26, 633–638. Wang, C., Fang, M., Han, J., Zhang, H., Cui, Y., Yang, B., 2009b. J. Phys. Chem. C 113, 19445–19451. Wang, H.-F., He, Y., Ji, T.-R., Yan, X.-P., 2009. Anal. Chem. 81, 1615–1621. Wang, Y.-q., Zou, W.-s., 2011. Talanta 85, 469–475. Wu, B., Wang, Z., Zhao, D., Lu, X., 2012. Talanta 101, 374–381. Yang, F., Mao, Y., Zhang, X., Ma, Z., Zhang, X., 2009. J. Sep. Sci. 32, 2974–2978. Yang, H.-H., Zhou, W.-H., Guo, X.-C., Chen, F.-R., Zhao, H.-Q., Lin, L.-M., Wang, X.-R., 2009. Talanta 80, 821–825. Ying, S., Cui, S., Wang, W., Feng, J., Chen, J., 2014. J. Lumin. 145, 575–581. Yuan, J., Guo, W., Yin, J., Wang, E., 2009. Talanta 77, 1858–1863. Yuen, A.H.Y., Yacoub, M.H., Smolenski, R.T., 2005. Rapid Commun. Mass Spectrom. 19, 3603–3606. Zhang, W., He, X.-W., Chen, Y., Li, W.-Y., Zhang, Y.-K., 2011. Biosens. Bioelectron. 26, 2553–2558. Zhang, W., He, X.-W., Chen, Y., Li, W.-Y., Zhang, Y.-K., 2012a. Biosens. Bioelectron. 31, 84–89. Zhang, W., He, X.-W., Li, W.-Y., Zhang, Y.-K., 2012b. Chem. Commun. (Cambridge, UK) 48, 1757–1759. Zhang, Z., Zhang, M., Luo, L., Yang, X., Hu, Y., Zhang, H., Yao, S., 2010. J. Sep. Sci. 33, 2854–2861.
