Upconversion particles coated with molecularly imprinted polymers as fluorescence probe for detection of clenbuterol

Biosensors and Bioelectronics 71 (2015) 44–50

Contents lists available at ScienceDirect

Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios

Upconversion particles coated with molecularly imprinted polymers as fluorescence probe for detection of clenbuterol Yiwei Tang a, Ziyuan Gao a, Shuo Wang b, Xue Gao a, Jingwen Gao a, Yong Ma a, Xiuying Liu a, Jianrong Li a,n a Food Science Research Institute of Bohai University, Food Safety Key Lab of Liaoning Province, College of Chemistry, Chemical Engineering and Food Safety, Bohai University, Jinzhou 121013, China b Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science & Technology, Tianjin 300457, China

art ic l e i nf o

a b s t r a c t

Article history: Received 28 January 2015 Received in revised form 26 March 2015 Accepted 5 April 2015 Available online 7 April 2015

A novel fluorescence probe based on upconversion particles, YF3:Yb3 þ , Er3 þ , coating with molecularly imprinted polymers (MIPs@UCPs) has been synthesized for selective recognition of the analyte clenbuterol (CLB), which was characterized by scan electron microscope and X-ray powder diffraction. The fluorescence of the MIPs@UCPs probe is quenched specifically by CLB, and the effect is much stronger than the NIPs@UCPs (non-imprinting polymers, NIPs). Good linear correlation was obtained for CLB over the concentration range of 5.0–100.0 μg L  1 with a detection limit of 0.12 μg L  1 (S/N ¼3). The developed method was also used in the determination of CLB in water and pork samples, and the recoveries ranged from 81.66% to 102.46% were obtained with relative standard deviation of 2.96–4.98% (n ¼ 3). The present study provides a new and general tactics to synthesize MIPs@UCPs fluorescence probe with highly selective recognition ability to the CLB and is desirable for application widely in the near future. & 2015 Elsevier B.V. All rights reserved.

Keywords: Upconversion particles Molecularly imprinted polymer Fluorescence probe Clenbuter

1. Introduction Fluorophores have been widely used in fluorescent sensors, probes and tags for various applications (Ren and Chen, 2015; Li et al., 2015a, 2015b) owing to its fast, sensitive and reproducible signal. Generally, most of fluorophores obey the law of Stocks, converting short wavelength light to longer ones (downconversion) (Dou et al., 2014). A case in point is quantum dots, which seem to be a good candidate for analytical assays because they exhibit a Stern–Volmer quenching behavior (Ren and Chen, 2015; Durán-Toro et al., 2014). Sadly, the quantum dots have many drawbacks in application process. Firstly, the intrinsic toxicity of the quantum dots composing with Cd, Se etc. poses risks to environment and health. Secondly, the autofluorescence from the analyte may decrease the sensitivity of the detection. So, it is a topic of great interest that a new fluorescence probe is prepared to apply in assay. Upconverting particles (UCPs) doped with lanthanide have gained widely attention due to their specific character, termed anti-Stocks, converting low-energy light such as near-infrared (NIR) or infrared (IR) to high-energy light (UV or visible) through multiple photon absorptions or energy transfers (Auzel, 2004). n

Corresponding author. Fax: þ86 416 3400013. E-mail address: [email protected] (J. Li).

http://dx.doi.org/10.1016/j.bios.2015.04.005 0956-5663/& 2015 Elsevier B.V. All rights reserved.

Moreover, UCPs owning low cytotoxicity, narrow emission, long lifetime, and high photostability make them ideal as luminescent labels (Wang et al., 2010). Now UCPs are mainly used in medical field (Xia et al., 2014; Chen et al. 2013; Ma et al., 2014; Ang et al., 2011), pesticide detection (Qian et al., 2013) and biological sensing (Zhang et al., 2011). Molecularly imprinted polymers (MIPs) as a new molecular recognition material, one kind of bionic antibodies, have attracted considerable interest due to their predetermination, specific recognition and practicability. Currently, MIPs have been used in solid-phase microextraction (Zhao et al., 2015), sensor (Ding et al., 2015; Chen et al., 2015; Li et al., 2015a, 2015b), membrane (Takeda et al., 2009) and artificial antibody (Tang et al., 2013). Evidently, MIPs will be a powerful molecular receptor to improve the selectivity of the recognition material. Clenbuterol (CLB) that belongs to β-agonizts was not only widely used as a bronchodilator and a tocolytic agent in clinical management, but also used as a growth promoter in animal production to promote animal growth and increase the muscular mass (Qiao and Du, 2013; Zhang et al., 2012; McCaughey et al., 1990). However, it had been led to serious side effects, such as muscular tremors, headache and palpitations (Song et al., 2013; Brambilla et al., 2000), and acute poisoning because of the abuse of CLB. Therefore, the law forbids using CLB as a feed additive to meat producing animals in Europe Union and China (Geng et al., 2011).

Y. Tang et al. / Biosensors and Bioelectronics 71 (2015) 44–50

At present, many methods, including enzyme-linked immunosorbent assay (ELISA) (Prezelj et al., 2003) and instrument detected assay such as high performance liquid chromatography (HPLC) (Du et al., 2013; Morales-Trejo et al., 2013; Chang et al., 2005; Botterblom et al., 1993), gas chromatography–mass spectrometry (GC–MS) (Amendola et al., 2002; Hooijerink et al., 1994; Abukhalaf et al., 2000; Ramos et al., 2003), liquid chromatography–mass spectrometry (Li et al., 2013a, 2013b), capillary zone electrophoresis method (CE) (Li et al., 2013a, 2013b) and electrochemical method (Zhao et al., 2011), to detect CLB have been reported. ELISAs are fast, simple, and sensitive, but the high expense of producing antibodies, poor reagent stability, and the laboratory animals needed are recognized problems. Instrument detected assay also involved deficiencies, such as time consuming, expensive instruments and requirement of skilled workers. Hence, to prepare a rapid and effective method to detect CLB is very important. To design and prepare an effective fluorescent probe is of paramount interest in sensor applications for detection of analyte. In this paper, a novel fluorescence probe (MIPs coated UCPs, MIPs@UCPs) combined high selectivity of MIPs and high sensitivity of the UCPs based on YF3:Yb3 þ , Er3 þ to detect CLB in pork and water samples was prepared, which was characterized by scan electron microscope (SEM), X-ray powder diffraction (XRD) and adsorption properties. The fluorescence quenching relation between the MIPs@UCPs probe and CLB was also studied. The present research proposed a facile strategy for synthesizing the fluorescent probe exhibiting the characteristics of stability, selectivity, sensitivity, eco-friendliness, operational simplicity, which is desirable for application widely in the near future.

45

was used to characterize the CLB derivative, and the chemical shifts were expressed in parts per million (δ scale) using the tetramethylsilane as an internal standard. Mass spectra were obtained using an Advantage HPLC-MS (ThermoFinnigan, San Jose, USA) equipped with electrospray ionization (ESI) in positive mode. 2.3. Synthesis of clenbuterol derivative The CLB derivative, 2-(tert-butylamino)-1-(3, 5-dichloro-4methacrylamidophenyl) ethyl methacrylate (TBDMEM), was synthesized as following: 10.0 mmol of CLB was dissolved in 50.0 mL of dry dichloromethane containing 25.0 mmol of triethylamine. Then 22.0 mmol of methacryloyl chloride was added dropwisely into the mixed solution with stirring. The chemical reaction was carried out for 24 h at room temperature. After reaction, a saturated solution of sodium hydrocarbonate was added, and the organic phase was then separated and collected using a separatory funnel. The aqueous phase was washed twice with dichloromethane. Then, all the organic phases were combined and dried using anhydrous sodium sulfate. After that, the organic solvent was removed using a rotary evaporator. The residual was purified on a silica gel column, using the mixture solvent of petroleum benzin and ethyl acetate (3:1, v/v) as the eluant. The eluant was evaporated to dryness under reduced pressure to obtain the pure compound TBDMEM (2.34 g) with a total yield of 46.1%. It was characterized by 1H NMR and mass spectrometry. 1 H NMR (400 MHz, CDCl3), δ 7.13 (s, 2H), 6.19–6.09 (m, 1H), 5.80 (dd, J ¼9.9, 3.9 Hz, 1H), 5.64–5.66 (m, 1H), 5.28–5.15 (m, 1H), 5.13–4.93 (m, 1H), 4.52 (s, 2H), 4.11–3.91 (m, 1H), 3.63 (dd, J ¼15.7, 3.9 Hz, 1H), 2.02–1.89 (m, 6H), 1.51 (s, 9H). Mass spectrometry (ESI, positive), m/z: 436.1 [Mþ Na], 849.2 [2M þNa].

2. Experimental 2.4. Preparation of MIPs@UCPs probe 2.1. Reagents CLB, isoproterenol (ISOP), terbutaline (TER), salbutamol (SAL), ambroxol (AMB) were obtained from Sigma (Sigma-Aldrich, USA), and used for adsorption test. Yttrium oxide (Y2O3, 99.99%), ytterbium oxide (Yb2O3, 99.99%), erbium oxide (Er2O3, 99.99%), sodium fluoride (NaF), sodium hydroxide (NaOH), concentrated nitric acid (HNO3), ethylenediamine tetra-acetic acid (EDTA), methyl acrylic acid (MAA), azobis (isobutryonitrile) (AIBN) and methacryloyl chloride were obtained from Aladdin (Shanghai, China). Ethylene glycol dimethacrylate (EGDMA) was got from Sigma (Sigma-Aldrich, USA). Other reagents were purchased from Tianjin Chemical Reagent Factory (Tianjin, China). EGDMA and MAA were distilled before using. All chemicals employed in this study were of the highest available purity and at least of analytical grade. 2.2. Apparatus An Agilent 1260 HPLC system with ultraviolet detector (detection wavelength 246 nm) and a reversed-phase C18 analytical column (4.6 mm  250 mm, 5 mm, Agilent, USA) was employed to analysis CLB. The mobile phase was 50.0 mM NaH2PO4 and methanol (65/35, V/V) and the flow rate was maintained at 1.0 mL min  1 (column temperature, 25 °C). Scanning electron microscope coupled with energy dispersive X-ray (Hitachi S-4800, Japan) was used to observe the surface morphology and elemental composition of the prepared samples. Fluorescence intensity studies were carried out by using an F-7000 fluorescence spectrophotometer (Hitachi, Japan) at room temperature. The X-ray diffraction (XRD) spectrum was obtained using an X'pert PRO diffractometer (NOV Crop., Japan). 1H NMR spectra recorded by Bruker AV-400 spectrometer (Rheinstetten, Germany)

The preparative procedure of the MIPs@UCPs probe involves two major steps: the first step is the preparation of UCPs, and the second one is the coating MIPs onto UCPs. The YF3:Yb3 þ , Er3 þ particle was synthesized using a facile hydrothermal approach as follows. At first, Y2O3, Yb2O3 and Er2O3 was used to synthesize nitrate, and then the nitrate was utilized to configure water solution consisting of 8.25 mL of Y(NO3)3 (0.8 mol L  1), 2.686 mL of Yb(NO3)3 (0.63 mol L  1), 0.423 mL of Er (NO3)3 (0.4 mol L  1), and 33.0 mL of EDTA (0.2 mol L  1), which were well mixed at room temperature to form a white viscous solution. Then, the mixture was mixed uniformly by a magnetic stirrer. Subsequently, 38.0 mL of NaF (46.0 mmol) solution was added into the mixture with stirring constantly. Five minutes later, the pH value of the solution was adjusted to 3.0 with HNO3 or NaOH solution. Then the mixture was transferred to a 100.0 mL Teflon-lined stainless-steel autoclave, which were annealed at 180 °C for 24 h. After reaction, the UCPs was obtained after centrifugation and then be washed with ethanol and water three times and dried in oven. The MIPs@UCPs was prepared as follows. Clenbuterol derivative (1 mmol), UCPs (0.25 g), cross-linkers EGDMA (3.0 mmol) and free-radical initiator AIBN (30.0 mg) were dissolved in a mixed solvent of ethyl acetate (2.0 mL) and methanol (3.0 mL) in a flask. Then the solution was purged with a gentle flow of nitrogen for 5 min. Subsequently, the flask was sealed and placed in an oil bath at 70 °C for 48 h. After polymerization, the MIPs@UCPs was obtained. In order to extract CLB from the fluorescence probe completely, the MIPs@UCPs was washed successively with 3.0 mol L  1 of NaOH solution for 48 h, the mixed solution of methanol/acetic acid (9:1, v/v) for 72 h, and methanol until no CLB can be observed. The resulting fluorescence probe was dried at 50 °C.

46

Y. Tang et al. / Biosensors and Bioelectronics 71 (2015) 44–50

For comparison, the NIPs@UCPs (non-imprinted polymers, NIPs) was synthesized and treated using 1 mmol of methacrylic acid as the monomer, 3.0 mmol of EGDMA as the cross-linker, and 30.0 mg of AIBN as the initiator under the same polymerization conditions without adding a template. 2.5. Fluorescence analysis Fluorescence analysis was performed using an F-7000 fluorescence spectrometer. The MIPs@UCPs (15.0 mg) and different concentrations of CLB methanol solution (4.0 mL) were incubated at room temperature for 5 min, followed by recording the fluorescence spectrum of each solution. The spectra were recorded in the wavelength range of 580–610 nm upon excitation at 890 nm. Slit widths (5 nm), scan speed (240 nm min  1), quartz cell (1 cm path length) and the excitation voltage (700 V) were kept constantly within each data set. The average data was obtained with three replicates. 2.6. Preparation of samples Water and pork were selected to evaluate the performance of the developed fluorescent detection method. All samples were bought from local markets and verified to not contain CLB by HPLC. For the spiking study of samples, water samples (5.0 mL) were spiked with 600.0, 800.0, and 1000.0 μL of the CLB methanol solution (1.0 mg L  1), separately. After the mixture was shaken on a shaker for 3 min, it was dried using a rotary evaporator. Then the residues were dissolved in methanol for analysis. Smashed pork (5.0 g) was placed in centrifuge tube and spiked with 600.0, 800.0, and 1000.0 μL of CLB methanol solution (1.0 mg L  1), separately. Then the spiked sample was homogenized on the rotary shaker for 3 min and kept at 4 °C for 12 h. Subsequently, the spiked sample was extracted with 20.0 mL of acetonitrile by ultrasonic agitation for 30 min and then centrifuged at 3800 rmp for 10 min at 4 °C. The extraction procedure was repeated twice. The two-part supernatants were combined and mixed with 20.0 mL of hexane saturated with acetonitrile to remove any fat. After being shaken for 2 min, the acetonitrile was separated and dried by a rotary evaporator at 45 °C. Then the residues were dissolved in methanol for analysis.

3. Results and discussion The fluorescence probe (Scheme 1) consists of the core, upconversion particles used for recognition signal amplification and optical readout, and the shell, MIPs used for molecular recognition and avoiding interferences contacting with the UCPs. Upconversion particles doped with lanthanide, YF3:Yb3 þ , Er3 þ , owns novel upconversion emission properties, including narrow and symmetric emission and wide excitation. To obtain a highly selectivity probe, covalent imprinting method was employed in this study. At first, the covalent linkage between CLB and methacryloyl chloride was first synthesized, which can increase the structural stability in the prepolymerization process. After polymerization, a three-dimensional network complemented with CLB was formed in polymers. The CLB molecule was removed from the MIPs by hydrolysis, and the complementary binding sites having two carboxyl groups in the cavities were left, which can rebind CLB through hydrogen bonding. To prepare the MIPs with covalent imprinting method is an effective way to improve the specificity of the polymers. The fluorescence quenching of the MIPs@UCPs occurs when the CLB is bound to the fluorescence probe. First, we ruled out energy transfer as a possible mechanism for the fluorescence quenching due to not having spectral overlap between the adsorption spectrum of the CLB and the emission spectrum of the MIPs@UCPs. So, the mechanism for the luminescence quenching of the MIPs@UCPs was probably the photo-induced electron transfer due to the amine and hydroxyl groups of CLB interacting with carboxyl groups in the MIPs@UCPs, the lone pair of electrons in the oxygencontaining groups is available for photo-induced electron transfer, leading to a decrease of the emission (Zhou et al., 2015). Such charge transfer mechanism has also been reported in the paper (Wang et al., 2009). 3.1. Optimization of cross-linker In order to investigate the effect of the amount of cross-linker on binding ability of the MIPs@UCPs probe for CLB, four kinds of probes were synthesized based on different molar ratios between CLB derivative and cross-linker, 1:2 (P1), 1:3 (P2), 1:4 (P3) and 1:5 (P4). To obtain the optimal MIPs@UCPs, 30.0 mg of the imprinted polymer probe and non-imprinted polymer probe was mixed with 3.0 mL of CLB methanol solution, respectively. The mixture was then oscillated horizontally for 12 h. After that, the filtrate was got

Scheme 1. The preparation process of MIPs@UCPs fluorescence probe.

Y. Tang et al. / Biosensors and Bioelectronics 71 (2015) 44–50

47

Fig. 1. SEM images of UCPs (A1), MIPs@UCPs (A2) and powder X-ray diffraction patterns for UCPs and MIPs@UCPs (B).

using a membrane filter (pore size, 0.22 μm) and was measured for the unextracted CLB by HPLC. Fig. S1 (see the Supplementary materials) showed the adsorption capacity of the MIPs@UCPs and NIPs@UCPs, indicating that the adsorption capacity of the MIP2@UCPs (1.13 mg g  1) was more than 2 times that of the corresponding NIPs@UCPs. The results suggested that the amount of cross linker was an important factor in polymerization process, which can fix the template-binding site firmly. However, excessive amount of crosslinker also resulted in removing the template, very difficult, from the polymer matrix owing to steric hindrance (Tang et al., 2011). So the best molar ratio of TBDMEM and cross-linker, 1:3, was chosen to synthesize the fluorescence probe. 3.2. Characterization of the MIPs@UCPs The SEM images as shown in Fig. 1A showed the morphologies of the UCPs (Fig. 1A (1)) and the MIPs@UCPs (Fig. 1A (2)) probe. Fig. 1A (1) revealed that the developed UCPs were regular truncated octahedrons with uniformity, and the surface is very smooth. Fig. 1A (2) showed that the UCPs were encapsulated fully by the MIPs, indicating that the fluorescence probe was prepared

successfully. Fig. 1B showed XRD results of the synthesized UCPs and MIPs@UCPs probe. The positions and intensities of all peaks can be well indexed to pure orthorhombic YF3, which were in good agreement with the literature values (JCPDS Card No. 74-0911). The XRD spectrum of the MIPs@UCPs had a similar XRD pattern to the UCPs, which confirmed the crystal type of the UCPs was not changed in the MIPs@UCPs. It was noteworthy that the spectrum of the MIPs@UCPs had a lower intensity because of the effect of the coating MIPs shell. This result also showed that the MIPs shell was successfully coated on the UCPs. Energy dispersive X-ray spectroscopy (Fig. S2, see the Supplementary materials) revealed the presence of Y, F, Er, Yb, C, O and N elements in the MIPs@UCPs, which indicated that the UCPs crystals were composed of the elements Y, F, Er and Yb, and other elements were existing in the shell, MIPs. These results demonstrated the successful coating procedure. 3.3. Kinetic uptake The kinetic adsorption process of the developed fluorescence probe to CLB was studied by employing 30.0 mg of the MIPs@UCPs

48

Y. Tang et al. / Biosensors and Bioelectronics 71 (2015) 44–50

Fig. 2. Uptake kinetic plot of CLB onto the MIPs@UCPs (A) and pseudo-second-order kinetic model for the adsorption of CLB onto the MIPs@UCPs (B).

incubated with CLB methanol solution (3.0 mL, 20.0 mg L  1) for different time. The results as shown in Fig. 2A indicated that the MIPs@UCPs had rapid binding rate for the target molecule, reaching adsorption equilibrium within 20 min at the experimental concentration. It implied that the MIPs@UCPs exhibited fast mass transfer rate compared with the traditional imprinted method, which might be attributed to the surface imprinting method (Du et al., 2014). Moreover, the lower the concentration of CLB is, the shorter the adsorption equilibrium time will become. The good mass transport of the MIPs@UCPs was an advantage for reducing the test time. The pseudo-second-order kinetic model, t /qt = 1/kq2e + t /qe , where k is the rate constant of second-order sorption (μg g  1 s  – 1 ), qt is the adsorption capacity at any time (μg g  1), qe is the adsorption capacity at equilibrium (μg g  1), was used to describe the adsorption process. The fitting curve was obtained by t/qt versus t (as shown in Fig. 2B), and the correlation coefficient (R2) was 0.9954. The calculated qe value (2.27 mg g  1) depending on the pseudo-second-order kinetic model was close to the qe value (2.18 mg g  1) obtained from the experiment. These results suggested that the adsorption process of the MIPs@UCPs toward the target molecular (CLB) followed well the pseudo-second-order kinetic model, and the rate-limiting step of the adsorption process might be chemical adsorption (Xu et al., 2011). 3.4. Adsorption isotherm Isothermal adsorption capacity of the probe was carried out by employing 30.0 mg of MIPs@UCPs and NIPs@UCPs equilibrating with 3.0 mL of different concentration of CLB methanol solution (20.0–160.0 mg L  1), respectively. The results (Fig. S3, see the Supplementary materials) showed that the adsorption capacity of the MIPs@UCPs and NIPs@UCPs increased with the increase of the concentration of CLB solution. When the concentration of CLB solution was 160.0 mg L  1, the adsorption capacity of the MIPs@UCPs (3.2029 mg g  1) was 1.73 times more than the NIPs@UCPs (2.5802 mg g  1), which was higher than the reported work (Du et al., 2014). It showed that the imprinted polymer probe had strong adsorption effect and better selectivity to CLB.

for 5 min between the probe and CLB was adopted in our experiments. 3.6. Selectivity of the MIPs@UCPs When the CLB was removed via hydrolysis, imprinted binding sites were left in the composites that selective bound the target CLB molecules. To investigate the specificity of the MIPs@UCPs probe for the CLB, four kinds of competitors, terbutaline, isoproterenol, salbutamol and ambroxol (see Fig. S5 for structures, Supplementary materials), with similar structures to CLB were selected to quench the probe at the same concentration. Results were shown in Fig. S6 (see the Supplementary materials), indicating that only CLB can quench strongly the MIPs@UCPs probe, and the analoges had little influence on the fluorescence quenching. The changes in fluorescence intensity of NIPs@UCPs were similar for CLB and its analogs, suggesting that there were no selective recognition sites in the NIPs. These results confirmed that the MIPs@UCPs probe owned highly selective recognition ability to the analyte, which mainly attributed the MIPs synthesized by covalent imprinting method. 3.7. MIPs@UCPs and NIPs@UCPs with template molecule of different concentrations The relationship between the fluorescent intensity of the fluorescence probe and the concentration of CLB methanol solution was determined by recording the fluorescent intensity of the MIPs@UCPs and NIPs@UCPs (15.0 mg) incubated with 4.0 mL of different concentrations of CLB methanol solution (0, 5.0, 10.0 20.0, 40.0, 60.0, 80.0, 100.0 μg L  1) for 5 min. The results as shown in Fig. 3 indicated the decrease of fluorescence intensity of the MIPs@UCPs was much larger than that of the NIPs@UCPs by the CLB molecule at the same concentration. This result revealed that the fluorescence quenching depended crucially on the binding ability of the MIPs@UCPs with the target. So the MIPs with good specificity was a crucial factor to synthesize this fluorescence probe. 3.8. Fluorescence quenching analysis

3.5. Influence of incubation time A suitable incubation time between the MIPs@UCPs probe and CLB was obtained by recording the fluorescence intensity at different time scales. The results (Fig. S4, see the Supplementary materials) indicated that the stable fluorescence intensity can be found after 5 min, and can remain for at least 1.0 h. So, to incubate

The MIPs@UCPs probe showed typical fluorescence quenching at the concentration of CLB from 5.0 μg L  1 to 100.0 μg L  1, suggesting that it was very suitable and effective for practical application. Quantified analysis can be carried out by the Stern–Volmer equation, F0/F = 1 + KsvCq , where F0 is the initial fluorescence intensity in the absence of quencher, F is the fluorescence intensity

Y. Tang et al. / Biosensors and Bioelectronics 71 (2015) 44–50

49

Fig. 3. Fluorescence spectra of the MIPs@UCPs (A) and NIPs@UCPs (B) in the presence of varying concentrations of CLB, a–h: 0, 5.0, 10.0, 20.0, 40.0, 60.0, 80.0, 100.0 μg L  1. The inset shows the relationship between fluorescence intensity ratio F0/F and the CLB concentration.

in the presence of analyte, Ksv is the quenching constant of the quencher, and Cq is the concentration of the analyte. The Stern– Volmer plot of the MIPs@UCPs with different concentration of CLB was shown in Fig. 3A. The regression equation, F0/F = 0.0043Cq + 1.0251, was obtained, with a correlation coefficient of 0.9940. The linear range of the calibration curve was determined from 5 μg L  1 to 100 μg L  1 with a detection limit (S/ N ¼3) of 0.12 μg L  1. Compared with the results obtained from electrophoresis and chromatography methods for analysis of CLB (Table 1), this method is simple, time-saving and without complicated separation process. Similar, fluorescence analysis owned low detection limit and good renewability compared with immunosensor assay. Most of all, fluorescence analysis based on MIPs@UCPs probe owned better selectivity with using the MIPs coated UCPs. 3.9. Analytical performance of the MIPs@UCPs probe for CLB To assess the analytical performance of the MIPs@UCPs probe, water and pork samples were spiked with the target analyte. Each spiked sample was evaluated in three replicates to repeatability of the developed method and HPLC. The results as shown in Table 2 indicated that the recoveries of these spiked samples obtained from fluorescence detection were 81.66–102.46%, and the relative standard deviation (RSD) ranges were all less than 5.0%. The accuracy of this fluorescence detection method was validated by comparative analysis of the spiked samples with HPLC (Table 2); there was no significant difference between the results analyzed by both methods. These results demonstrated that the

Table 2 Determination of CLB in water and pork samples by fluorescence detection method based on the MIPs@UCPs probe and HPLC (n ¼3). Sample Spiking levels

Water

Pork

Fluorescence detection

HPLC

(μg mL  1 or μg g  1)

Recovery (%)

RSD (%)

Recovery (%) RSD (%)

0.12 0.16 0.20 0.12 0.16 0.20

102.46 94.40 97.45 89.18 90.50 81.66

4.98 4.67 3.91 4.82 3.46 2.96

105.44 97.06 95.40 91.57 93.41 80.57

5.78 3.34 2.11 5.30 4.49 3.76

proposed method based on the MIPs@UCPs fluorescence probe is an alternative and practical approach for CLB detection in water and pork samples.

4. Conclusion In summary, a facile strategy was developed successfully to prepare the novel MIPs@UCPs fluorescence probe that combined the selectivity of the MIPs and high sensitivity of the fluorescence emitted from the UCPs. A simple and rapid analysis method for determination of CLB was established based on the fluorescence probe. All studied results indicated that the MIPs@UCPs has high selectivity and adsorption capacity for CLB, which will be a great potential fluorescence probe for its wide application in the near

Table 1 Comparison of this method with other methods used in the literatures. Methods

Linear range (μg L  1)

LODs (μg L  1)

Recovery (%) Detection time (min)

RSD (%)

Renewable Antiinterference

Operation Ref.

Capillary electrophoresis

0.63–37.64

0.63

–

11

o 5.7

–

Medium

Complex

UHPLC-MS

0.005–0.3

–

90.0–109.0

–

o 15

–

Medium

Complex

SMIPs-MEPS-HPLC Electrochemical immunosensor Fluorescent probe

0.02–50.0 0.5–200.0

0.009 0.22

86.5–91.2 94.0–102.0

15 15

r 6.3 1.8–3.5

Yes No

Medium Medium

Complex Complex

Li et al., 2013a, 2013b Nicoli et al., 2013 Du et al., 2014 Yang et al., 2014

5.0–100.0

0.12

81.6–102.4

5

2.96–4.98 Yes

Good

Simple

This work

50

Y. Tang et al. / Biosensors and Bioelectronics 71 (2015) 44–50

future.

Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant nos. 31201370 and 31471639), Scientific Research Foundation for Doctor of Science and Technology Department of Liaoning Province (Grant no. 20121080), National Natural Science Foundation for Distinguished Young Scholars of China (Grant no. 31225021) and the National Key Technologies R&D Program of China during the 12th Five-Year Period (2012BAD29B06).

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.04.005.

References Abukhalaf, I.K., Deutsch, D.A., Parks, B.A., Wineski, L., Paulsen, D., Aboul-Enein, H.Y., Potter, D.E., 2000. Biomed. Chromatogr. 14, 99–105. Amendola, L., Colamonici, C., Rossi, F., Botre, B., 2002. J. Chromatogr. B 773, 7–16. Ang, L.Y., Lim, M.E., Ong, L.C., Zhang, Y., 2011. Nanomedicine 6, 1273–1289. Auzel, F., 2004. Chem. Rev. 104, 139–173. Botterblom, M.H., Feenstra, M.G., Erdtsieck-Ernste, E.B., 1993. J. Chromatogr. A 613, 121–126. Brambilla, G., Cenci, T., Franconi, F., Galarini, R., Macr, A., Rondoni, F., Strozzi, M., Loizzo, A., 2000. Toxicol. Lett. 114, 47–53. Chang, L.Y., Chou, S.S., Hwang, D.F., 2005. J. Food Drug Anal. 13, 163–167. Chen, F., Bu, W.B., Cai, W.B., Shi, J.L., 2013. Engineering upconversion nanoparticles for biomedical imaging and therapy In: Cai, W.B. (Ed.), Engineering in Translational Medicine. Springer, London, pp. 585–609. Chen, J.R., Huang, H., Zeng, Y.B., Tang, H., Li, L., 2015. Biosens. Bioelectron. 65, 366–374. Ding, Z.Q., Annie-Bligh, S.W., Tao, L., Quan, J., Nie, H.L., Zhu, L.M., Gong, X., 2015. Mat. Sci. Eng. C: Mater. 48, 469–479. Dou, Q.Q., Guo, H.C., Ye, E.Y., 2014. Mat. Sci. Eng. C: Mater. 45, 635–643. Durán-Toro, V., Gran-Scheuch, A., Órdenes-Aenishanslins, N., Monrás, J.P., Saona, L. A., Venegas, F.A., Chasteen, T.G., Bravo, D., Pérez-Donoso, J.M., 2014. Anal. Biochem. 450, 30–36.

Du, W., Lei, C.M., Zhang, S.R., Bai, G., Zhou, H.Y., Sun, M., Fua, Q., Chang, C., 2014. J. Pharm. Biomed. Anal. 91, 160–168. Du, W., Zhang, S.R., Fu, Q., Zhao, G., Chang, C., 2013. Biomed. Chromatogr. 27, 1775–1781. Geng, Y., Zhang, M., Yuan, W., Xiang, B., 2011. Food Addit. Contam. A 28, 1006–1012. Hooijerink, H., Schilt, R., van Bennekom, E.O., Huf, F.A., 1994. J. Chromatogr. B 660, 303–313. Li, K.J., Wang, Y.J., Zhang, L.L., Qin, F., Guo, X.J., Li, F.M., 2013a. J. Chromatogr. B 934, 89–96. Li, L.B., Du, H.W., Yu, H., Xu, L., You, Y.T.Y., 2013b. Electrophoresis 34, 277–283. Li, S.H., Luo, J.H., Yin, G.H., Xu, Z., Le, Y., Wu, X.F., Wu, N.C., Zhang, Q., 2015a. Sens. Actuators B: Chem. 206, 14–21. Li, Z., Wang, Y., Ni, Y.N., Kokot, S., 2015b. Spectrochim. Acta A 137, 1213–1221. Ma, C., Yang, R.H., Li, J.S., Li, Y.H., Yang, J.F., Zhang, T.R., Liu, C.H., Yang, S., Tan, W.H., Bian, T., 2014. Anal. Chem. 86, 6508–6515. McCaughey, W.J., Elliott, C.T., Crooles, S.R.H., 1990. Vet. Rec. 126, 351–354. Morales-Trejo, F., Vega-y León, S., Escobar-Medina, A., Gutidrrez-Tolentino, R., 2013. J. Food Drug Anal. 21, 414–420. Nicoli, R., Petrou, M., Badoud, F., Dvorak, J., Saugy, M., Baume, N., 2013. J. Chromatogr. A 1292, 142–150. Prezelj, A., Obreza, A., Pecar, S., 2003. Curr. Med. Chem. 10, 281–290. Qian, K., Fang, G.Z., Wang, S., 2013. RSC Adv. 3, 3825–3828. Qiao, F.X., Du, J.J., 2013. J. Chromatogr. B 923-924, 136–140. Ramos, F., Cristino, A.V., Carrola, P., Eloy, T., Silva, J.M., Castiho, M.C., Silveira, M.I.N., 2003. Anal. Chim. Acta 483, 207–213. Ren, X.H., Chen, L.G., 2015. Biosens. Bioelectron. 64, 182–188. Song, C.M., Zhi, A.M., Liu, Q.T., Yang, J.F., Jia, G.C., Shervin, J., Tang, L., Hu, X.F., Deng, R.G., Xu, C.L., Zhang, G.P., 2013. Biosens. Bioelectron. 50, 62–65. Takeda, K., Ohashi, A., Hibiya, M., Sugiyama, S., Kobayashi, T., 2009. Ther. Apher. Dial. 13, 19–26. Tang, Y.W., Fang, G.Z., Wang, S., Li, J.L., 2011. Anal. Bioanal. Chem. 401 2275-2282. Tang, Y.W., Fang, G.Z., Wang, S., Sun, J.W., Qian, K., 2013. J. AOAC Int. 96, 453–458. Wang, H.F., He, Y., Ji, T.R., Yan, X.P., 2009. Anal. Chem. 81, 1615–1621. Wang, F., Han, Y., Lim, C.S., Lu, Y., Wang, J., Xu, J., Chen, H., Zhang, C., Hong, M., Liu, X., 2010. Nature 463, 1061–1065. Xia, A., Zhang, X.F., Zhang, J., Deng, Y.Y., Chen, Q., Wu, S.S., Huang, X.H., Shen, J., 2014. Biomaterials 35, 9167–9176. Xu, W.Z., Zhou, W., Bian, L.H., Huang, W.H., Wu, X.Y., 2011. J. Sep. Sci. 34, 1746–1753. Yang, X., Wu, F., Chen, D.Z., Lin, H.W., 2014. Sens. Actuators B: Chem. 192, 529–535. Zhao, C., Jin, G.P., Chen, L.L., Li, Y., Yu, B., 2011. Food Chem. 129, 595–600. Zhao, T., Guan, X.J., Tang, W.J., Ma, Y., Zhang, H.X., 2015. Anal. Chim. Acta 853, 668–675. Zhang, H.C., Liu, C.Y., Liu, G.Y., Chen, X.L., Ye, Y.D., Wang, Y.R., Chai, C.Y., 2012. Chin. J. Anal. Chem. 40, 852–856. Zhang, J., Li, C.Y., Zhao, W.Z., Liu, B.C., Liu, Y.X., Gaole Aletan, G.L., 2011. Biosensing based on luminescent semiconductor quantum dots and rare earth up-conversion nanoparticles In: Serra, P.A. (Ed.), New Perspectives in Biosensors Technology and Applications. Intech, Rijeka, pp. 127–148. Zhou, X., Ma, P.P., Wang, A.Q., Yu, C.F., Qian, T., Wu, S.S., Shen, J., 2015. Biosens. Bioelectron. 64, 404–410.