Biosensors and Bioelectronics 71 (2015) 342–347

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Selective detection of fenaminosulf via a molecularly imprinted fluorescence switch and silver nano-film amplification Shuhuai Li a,b,c,n, Guihao Yin a,b,c, Qun Zhang a,b,c, Chunli Li a,b,c, Jinhui Luo a,b,c, Zhi Xu a,b,c, Anli Qin a,b,c a

Analysis and Test Center of Chinese Academy of Tropical Agricultural Sciences, Haikou, 571101, China Laboratory of Quality and Safety Risk Assessment for Tropical Products (Haikou), Ministry of Agriculture, Haikou 571101, China c Hainan Provincial Key Laboratory of Quality and Safety for Tropical Fruits and Vegetables, Haikou, 571101, China b

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 12 April 2015 Accepted 20 April 2015 Available online 21 April 2015

A novel fluorescence switch sensor was constructed for detecting the fungicide fenaminosulf (FM), based on a dye-doped molecularly imprinted polymer (MIP) and silver nanofilm amplification. The MIP was prepared by electropolymerization of hydroquinone doped with neutral red on the silver nanofilm modified electrode. A fluorescence signal was produced by the neutral red and the fluorescence intensity was diminished by the ion pair that formed via electrostatic forces between FM and the dye. Therefore, elution and adsorption of FM by the MIP acted as a switch to control the fluorescence intensity, which was effectively amplified by the silver nanofilm. The decrease in fluorescence intensity was linear with the FM concentration, establishing a new method for FM detection. Under optimal conditions, good linear correlation was obtained for FM concentrations over the range from 2.0
10  10 to 4.0
10  8 mol/L, with a detection limit of 1.6
10  11 mol/L. This method was utilized to determine residual FM in vegetable samples, and recoveries ranging from 92.0% to 110% were obtained. & 2015 Elsevier B.V. All rights reserved.

Keywords: Molecular imprinting Switch Silver nanofilm Fenaminosulf

1. Introduction Fenaminosulf (FM, p-dimethylaminobenzenediazosodium sulfonate) (Pai, 1983) is a fungicide and microbiocide that improves agriculture crop yields and a moderately toxic pesticide. Given the increased use of FM in agricultural production, much research on its impact on human and environmental safety has been conducted recently. For example, FM has been shown to be mutagenic (Liman et al., 2011). FM can be detected and analyzed with liquid chromatography–tandem mass spectrometry (Pang et al., 2006) and gas chromatography–mass spectrometry (Yang et al., 2010). However, because these methods have high costs and long analysis times, it is desirable to develop a simple, low-cost, highly sensitive method to detect FM in a controllable way with a very low detection limit. Molecularly imprinted polymers (MIPs) (Yang et al., 2010) can be used to separate and detect specific molecules quite effectively, with high selectivity and sensitivity. MIPs have been widely used as biological sensors, because of their high cross-linked structure, discrimination, stability, long service life, and low cost (Greene and n Corresponding author at: Analysis and Test Center of Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China. E-mail address: [email protected] (S. Li).

http://dx.doi.org/10.1016/j.bios.2015.04.066 0956-5663/& 2015 Elsevier B.V. All rights reserved.

Shimizu, 2005; Haupt and Mosbach, 2000). The most common MIP sensors are electrochemical, such as potentiometric sensors (Liang et al., 2009), conductometric sensors (Sergeyeva et al., 1999), and amperometric sensors (Yang and Zhao, 2015). However, it is difficult to detect templates without electroactivity; hence, indirect methods must be used (Li et al., 2012). Furthermore, as the complexity of samples increases, the analysis efficiency decreases. “Gate-controlled” MIP sensors have been reported (Li et al., 2013), in which the process of template elution and rebinding acts as a gate to control the flux of probes, thereby amplifying the electrochemical signals. However, the electrochemical stability needs to be improved. In contrast, fluorescence-based MIP sensors (Ton et al., 2015; Ton et al., 2013) are easy to develop and can yield highly reproducible results. Moreover, they exhibit high sensitivity and selectivity, and require only basic equipment. Nanoscale materials are frequently used as carriers (Feng et al., 2014; Zhou et al., 2014) or tags (Costa-Fernández et al., 2006; Zhang et al., 2012) to increase the sensitivity of fluorescent MIP sensors. Silver nanoparticles (Sun and Xia, 2002) 1  100 nm in size have unique optical, electrical, and thermal properties, and have been extensively used for sensors (Han et al., 2014; McFarland and van Duyne, 2003). Here, we report the first application of silver nanofilm amplification of a molecularly imprinted fluorescent switch (MIP– FL–SW).

S. Li et al. / Biosensors and Bioelectronics 71 (2015) 342–347

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Fig. 1. Procedure for MIP-FL-SW-sensor fabrication for the detection of fenaminosulf (FM).

An MIP–FL–SW sensor was fabricated for selective detection of FM. First, a two-dimensional silver nanofilm was electro-deposited on an indium tin oxide (ITO) electrode. Then, MIP was electropolymerized onto the silver nano-film. The MIP monomer was neutral red (NR)-doped hydroquinone, and FM was the template. After removal of the template molecules, the MIP sensors had stereo cavities (Li et al., 2012) in the imprinted membrane for detecting (binding) FM. When the cavity was empty, the bright fluorescence of NR in the MIP was detected; in this mode, the switch was “ON”. When was FM adsorbed on the MIP, the NR fluorescence intensity decreased because of the ion pair that formed via electrostatic forces between the FM and the NR (Snyder, 2010); in this mode, the switch was “OFF”. Thus, the switch controlled the NR fluorescence intensity in the MIP by eluting and adsorbing FM, as shown in Fig. 1. The decrease in fluorescence intensity was linear with the Fenaminosulf concentration, establishing a new method for FM detection. Furthermore, because the silver nanofilm significantly enhanced the NR redox activity, the fluorescence was effectively amplified. Therefore, the fluorescent switch sensor not only had high linearity and stability, but also high sensitivity and resolution.

standard three-electrode workstation (CHI660E, Shanghai Chenhua Instrument Co. Ltd., Shanghai, China). A KCl-saturated Ag/AgCl electrode was the reference, a platinum wire was the auxiliary electrode, and the MIP-modified ITO was the working electrode. Fluorescence spectra and time dependences were recorded with a F-2500 spectrophotometer (Hitachi, Tokyo, Japan). Fourier transform infrared spectra were recorded with a FT-IR-8400 spectrometer (Shimadzu, Tokyo, Japan). Scanning electron microscopy images were acquired with an Axio Imager instrument (Carl Zeiss AG, Oberkochen, Germany). 2.2. Preparation of silver-nano-film-modified electrodes The ITO electrodes were dipped into acetone and ethanol, and cleaned ultrasonically for 5 min. Silver nanofilms were applied to prepared on the ITO electrode as reported previously (Yin et al., 2003). Briefly, the ITO electrode, in 0.2 mol/L KNO3 and 5.0 mmol/L AgNO3, was scanned over 0.1  1.0 V using cyclic voltammetry (CV) at 50 mV/s for 10 cycles to form the silver nano-films. This resulted in silver nanofilms being electro-deposited onto the electrodes. 2.3. Preparation of MIP and non-molecular imprinted polymer (nMIP) modified electrodes

2. Materials and methods 2.1. Chemicals, reagents, and electrochemistry Fenaminosulf (FM) standards were obtained from Dr. Ehrenstorfe GmbH (Augsburg, Germany). Kelthane, Cypermethrin, Dursban, Methamidophos, Paraquat, Abamectin, Carbofuran, and Methomyl were obtained from the Agricultural Environmental Quality Supervision and Testing Center of China. NR and hydroquinone were obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). All the reagents used in the experiment were of analytical grade. All aqueous solutions were prepared with doubly distilled water. Electrochemical measurements were performed with a

The newly silvered electrodes were then washed with water, alcohol, and HNO3 (50% by volume). Then, MIPs and nMIPs were applied to the modified ITO electrodes via electropolymerization, as follows. First, hydroquinone was dissolved in 0.1 mol/L of acetate-buffered polymer solution, and the pH was adjusted to 3.6. Then, 2.5
10  4 mol/L of FM solution and 8.0
10  5 mol/L of NR were added and mixed. Then, the resulting electrolyte and the three silvered electrodes were installed in a CV test system. The system was configured for a potential range of the CV was 0 to þ1.0 V, with 20 cycles. Free radicals and negative ions were generated during the processes, which initiated polymerization of the hydroquinone on the electrodes. Then, the electrodes were washed with methanol for 6 min to remove the template molecules.

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The nMIP was fabricated using the same method, but without the FM.

3. Results and discussion 3.1. Electropolymerization of MIP

2.4. Electrochemical and fluorescence measurements The electrochemical experiments were performed in a 3
10  4 mol/L solution of K3[Fe(CN)6] /K4[Fe(CN)6] solution containing 0.5 mol/L of KCl. CV was performed at a scan rate of 50 mV/s, where differential pulse voltammetry measurements were taken over the potential range of  0.2 to þ0.8 V, at a scan rate of 50 mV/s and a pulse amplitude of 50 mV. Alternating current (AC) impedance was measured at a potential of 0.19 V over the frequency range 100 mHz to 100 kHz with an alternating voltage of 5 mV. Fluorescence spectra were acquired over 500–750 nm with 510 nm excitation. The excitation and emission slits were 5 nm.

2.5. Sample preparation 25 mL of acetone and 10 mL of dichloromethane were added to 20 g of vegetable samples and 5.0 g of sodium chloride in a 100 mL beaker. The mixture was homogenized in a blender until smooth. After allowing the solution to stand, the supernatant was collected in conical flask, and 5 g of anhydrous sodium sulfate was added to it. After stirring and filtration, the filtrate was dried by rotary evaporation. The final residue was then dissolved in 5 mL of ultrapure water.

The electropolymerization of MIP was by cyclic voltammograms (Supplementary material Fig. S1). Templates bonded to functional monomers with hydrogen bonds. In the electropolymerization processes, radical anions were generated in the presence of FM, which initiated the polymerization of hydroquinone. The irreversible polymerization had a distinct oxidation peak at 0.90 V. As the number of cycles increased, the peak current of hydroquinone oxidation continuously decreased because of the compacted polymer film that uniformly formed on the electrode. When the number of cycles reached 20, the MIP fully covered the modified ITO electrode. Further experiments suggest that, the number of excitement cycles can significantly affect the thickness of the MIP layer, directly affecting the sensor performance. If the number of cycles is too low, the MIP may become thinner, making it hard for template molecules to imprint the MIP. If the number of cycles is too high, the MIP may become too thick, which is likely to cause template molecules to become embedded in the MIP, making template removal difficult. Therefore, an appropriate number of excitement cycles are imperative. Twenty was chosen as the best number of cycles to ensure the MIP thickness is moderate. There is no peak current of FM or NR in electropolymerization processes. This implies that the molecular structure of FM and NR is not changed by the process of imprinting, even when FM and NR are entrapped in the MIP.

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(220)

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2θ (degress) Fig. 2. Silver nano-film characterization: (A) SEM image; (B) particle size distribution; (C) XRD spectra; and (D) FAM image.

S. Li et al. / Biosensors and Bioelectronics 71 (2015) 342–347

3.2. Characterization of silver nano-films The optical properties of silver nanofilms depend on particle size. The morphology of a silver nanofilm was characterized using scanning electron microscope (SEM). As shown in Fig. 2A, it exhibited a regular structure and was mono-dispersed, with an average diameter of 32 nm. Fig. 2B indicated that the particle size distribution is between 0 and 45 nm. The X-ray diffraction (XRD) pattern for the film clearly has well-defined peaks corresponding to highly crystalline nanoparticles (Fig. 2C). The three major reflections at 38.50, 44.00, and 65.02 can be assigned to the (111), (200), and (220) crystal planes of silver, respectively. The structure and morphology of the resulting silver nanofilms were characterized using atomic force microscopy (AFM). Fig. 2D indicated that most of the nanoparticles were gathered together and distributed uniformly, and only a few particles were scattered outside the film. The edges and crumpled silk waves of the silver nanofilms lead us to believe that these nanoparticles were indeed deposited on the film. From Fig. 2D, we could see that the silver nanofilms are well covered by the silver nanoparticles and the average size of the silver nanoparticles was roughly consistent with the TEM results. 3.3. CV and AC characterization of MIP

400

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used as the redox probe between the imprinted electrodes and the substrate solutions during the CV and AC characterization of the MIP film formation. Cavities in the MIP can act as channels for electron transport; thus binding of the template in the cavities can result in a corresponding change in redox current. As shown in Fig. 3A, the peak current of the probe was lower for the silver-nanofilm-modified ITO electrode (curve b) relative to that for the bare ITO electrode (curve a), this is because the silver nanofilm hindered electron transport. When the compact water-insoluble polymer membrane was formed, it was difficult for the redox probe to react on the ITO electrode. Thus, the peak current of the probe decreased (curve d). After template removal, the peak current of the probe increased (curve c), but it was still less than that of curve a, because only the cavities in the MIP acted as channels for electron transport. When FM re-adsorbed onto the MIP electrode, the cavities were blocked again, decreasing the peak probe current (curve e). For the control nMIP, there was almost no change in the peak currents following template removal because there were no template-binding cavities in the polymer (curves f and g). AC impedances data indicate similar changes before and after FM adsorption and re-adsorption in the cavities (Fig. 3B). The resistance was affected by surface passivation of the electrode. When the silver nanofilm was deposited on the ITO electrode, the resistance increased (curves a and b). When the MIP covered the ITO electrode, passivation and resistance increased (curve c). When the FM template was removed, the resistance decreased (curve d).

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Because no FM redox peaks were observed on the ITO electrode over the range of  0.2 V to þ 0.6 V, K3[Fe(CN)6]/K4[Fe(CN)6] was

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Fig. 3. (A) CVs of MIP and nMIP sensors under different conditions: (a) Bare ITO electrode; (b) Silver-nano-film-modified ITO electrode; (c) MIP-modified ITO electrode; (d) MIP electrode after template removal; (e) MIP electrode after re-adsorption of FM (6
10  9 mol/L FM); (f) nMIP-modified ITO electrode; and (g) nMIP electrode after template removal. (B) AC impedance characterization of MIP films. (a) Bare ITO electrode; (b) Silver-nano-film- modified ITO electrode; (c) MIP-modified ITO electrode; (d) MIP electrode after template removal; and (e) MIP electrode after re-adsorption of FM (6
10  9 mol/L FM). (C) XPS of MIP: (a) MIP with bound template; and (b) MIP after template removal. (D) FT-IR of MIP: (a) MIP with bound template; and (b) MIP after template removal.

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When FM was re-introduced, the resistance increased (curve e), demonstrating again that MIP binds FM. As shown in Fig. 3 C, X-ray photoelectron spectroscopy (XPS) of MIP reveals intense peaks corresponding to S (2p) at 181 eV, C (1s) at 285 eV, Ag (3d5/2) at 367.4 eV, Ag (3d3/2) at 374.5 eV, N (1 s) at 399 eV, and O (1s) at 533 eV (curve a). These results indicate that MIP was successfully deposited on the silver-nanofilm-modified ITO electrode. After the FM template molecules were removed, the S peaks almost disappeared, while the C, O, and N peaks became slightly weaker (curves a and b). The fourier translation infrared spectrum (FT-IR) of the MIP are shown in Fig. 3D. As shown, the peaks near 3500 cm  1, 3000 cm  1 and 1500 cm  1 are separately attributed to the stretching vibration of –OH, C–H, and C ¼C, respectively, in hydroquinone (curve a). The peaks near 2920 cm  1 and 1205–1500 cm  1 are separately attributed to the stretching vibration of C–H and the aromatic ring in NR. The peaks near 1068 cm  1, 1010 cm  1, 620 cm  1, and 530 cm  1 are attributed to the sulfonic group in FM. This shows that a composite MIP was successfully prepared on the ITO electrode. When the FM was removed from the MIP, the peaks of FM disappeared (curve b). 3.4. Amplifier effect of silver nano-films Fluorescence signals were studied under different conditions, as shown in Fig.4. A fluorescence emission of NR in solution peaked at about 625 nm, when a 510 nm laser was used as an exciting source (curve a). A bright fluorescence signal of NR was obtained after polymerizationon the surface of ITO electrode without silver nanofilms, because NR was concentrated and its stability was greatly improved (curve b). When FM re-adsorbed on the MIP electrode, the NR fluorescence intensity decreased because of the ion pairs formed (curve c). By using silver nanofilms for measurement, the fluorescence intensity was not only stronger but also more stable (curve d). When FM re-adsorbed on the MIP electrode, the reduction of NR fluorescence intensity was more significant in the presence of silver nano-films (curve e). It indicates that silver nano-films have a tremendous amplifier effect in MIP. The mechanism responsible for the amplification effect was probably associated with an enhanced static magnetic field (Geddes et al., 2003; Lukomska et al., 2004). The local field enhancement provides a higher excitation rate but does not alter the lifetime of the fluorophore. Fluorescence is mainly modulated by two parameters: excitation rate and quantum yield. The

500

3.5. Optimization of experimental conditions The experimental conditions were optimized for FM detection, including the doping amount of NR (Supplementary material Fig. S2), and times for template removal and re-adsorption (Supplementary material Fig. S3). The doping amount of NR has a direct influence on fluorescence signals. A very high doping level will destroy the smoothness of film surfaces, resulting in the fluorescence quenching effect. However, if the doping level is low, it will cause a decreased signal and low sensitivity. The experimental results show that 8.0
10  5 mol/L of NR doped in the MIP is optimal. FM template removal was performed in 10 mL of carbinol. The fluorescence intensity was measured every 2 min; it increased gradually and then remained constant after 8 min. Thus, the optimum time for FM removal was 8 min. The re-adsorption of the templates was performed with 6.0
10  9 mol/L FM. In this case, the fluorescence intensity decreased gradually and then remained constant after 12 min. Thus, 12 min was the optimum FM re-adsorption time in all the following assays. 3.6. Fluorescence response to FM Under optimized conditions, the fluorescence intensity was measured after FM re-adsorption on the sensor in different FM concentrations (Fig. 5). The decrease in fluorescence intensityIwas linear with respect to FM concentration c over the range of 2.0
10  10 mol/L to 4.0
10  8 mol/L. The linear regression fit was ΔI ¼4.53c (10  10 mol/L) þ 0.703, with a coefficient of correlation r ¼0.999. The detection limit DL was 1.6
10  11 mol/L (DL ¼KSb /a, K ¼3). 3.7. Selectivity To test the selectivity of the MIP sensor, the re-adsorption procedure was performed as described above using other commercial pesticides such as Kelthane, Cypermethrin, Dursban,

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excitation rate is directly proportional to the square of the electric field. The higher the square of the electric field region is restricted on the metal nanohole array region, which forecasts stronger excitation of fluorescence (Guo et al., 2009; Kannadorai et al., 2012). When plasmon resonance occurs between the incident light and silver nano-particles, a strong electric field is generated. This field may cause widening the density of state and enhanced excitation efficiency of NR.

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10  5 mol/L NR solution; (b) MIP sensor after removing FM without silver nano-film; (c) MIP sensor after FM re-adsorption with 4
10  8 mol/L FM without silver nano-film; (d) MIP sensor after removing FM; and (e) MIP sensor after FM re-adsorption with 4
10  8 mol/L FM.

0 500

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10  10 mol/L FM.

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Methamidophos, Paraquat, Abamectin, Carbofuran, and Methomyl in 6.0
10  9 mol/L FM. The resulting fluorescence intensities were studied (Supplementary material Fig. S4) and no change was observed after FM re-adsorption on the MIP sensors in a solution with or without a mixture of the above-mentioned pesticides. The results demonstrate that these pesticides, with similar structures to that of FM, did not bind to the FM-templated cavities. Interferences from several inorganic ions were also studied. In these cases, the fluorescence signals did not change after FM re-adsorption on the MIP sensors in a solution with or without a mixture of 2500-fold (relative to FM) PO4 3− and SO42−, 1800-fold CO32−, 1000-fold Cl  , NO3−, NH4+ , Ca2 þ , Mg2 þ , and SO32−, 500-fold Fe3 þ , Cr3 þ , Cu2 þ , Br-, Ba2 þ , and Fe2 þ , and 200-fold Hg2 þ , Zn2 þ , Co2 þ , and Cd2 þ .

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Acknowledgements The authors gratefully acknowledge the financial support received from the Fundamental Scientific Research Funds for Chinese Academy of Tropical Agricultural Sciences (No. 1630042015011) and the National Key Technology R and D Program of China (No. 2012BAK01B00).

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.2015.04.066.

References 3.8. Reproducibility and stability Sensor reproducibility was examined by measuring the fluorescence intensities of five different sensors fabricated under the same conditions during FM re-adsorption in 6.0
10  9 mol/L FM. A 3.9% relative standard deviation was obtained. Similarly, five separate fluorescence measurements for the same sensor were performed for FM re-adsorption in 6.0
10  9 mol/L FM; the relative standard deviation was 4.2%. Thus, the MIP sensors have excellent reproducibility. To ensure stability, the sensor was kept in the dark when not in use. Periodic testing after 6.0
10  9 mol/L of FM re-adsorption revealed no decrease in sensitivity over the first 15 days. After a month, the sensitivity decreased by 11.5% relative to that measured initially. 3.9. Sample detection The samples detection and the recovery experiments were performed simultaneously. The recoveries of the sensor ranged from 92.0% to 110% (Supplementary material Table S1).

4. Conclusions A sensor based on electrostatic control and silver nanofilm amplification is suitable for fabrication of sensitive, selective, and stable MIP sensors. It provides a new strategy for constructing molecular imprinted sensors, which could be broadly used in medical diagnoses, environmental assessments, and food analyses.

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