A selective molecularly imprinted electrochemical sensor with [email protected] signal amplification for the simultaneous determination of sulfadiazine and acetaminophen

Sensors & Actuators: B. Chemical 300 (2019) 126993

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

A selective molecularly imprinted electrochemical sensor with GO@COF signal amplification for the simultaneous determination of sulfadiazine and acetaminophen

T

Yufeng Suna, Jingbo Hea, Geoffrey I.N. Waterhouseb, Longhua Xua, Hongyan Zhangc, ⁎ Xuguang Qiaoa, Zhixiang Xua, a

Key Laboratory of Food Processing Technology and Quality Control in Shandong Province, College of Food Science and Engineering, Shandong Agricultural University, Tai’an, 271018, PR China School of Chemical Sciences, The University of Auckland, Auckland, 1142, New Zealand c College of Life Science, Shandong Normal University, Jinan, 250014, PR China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Graphene oxide Covalent organic framework Molecular imprinted polymer Electrochemical sensor Sulfadiazine Acetaminophen

In this work, the development of a simple and sensitive electrochemical sensor for the simultaneous determination of sulfadiazine (SDZ) and acetaminophen (AP) is described. The sensor harnessed a molecularly imprinted polymer (MIP) for selective recognition of SDZ and AP, and a graphene oxide@covalent organic framework (GO@COF) nanocomposite for signal amplification. The GO@COF nanocomposite was first immobilized on a glassy carbon electrode (GCE), after which a polypyrrole MIP was electrodeposited on the modified electrode. The resulting electrochemical sensor (denoted as MIP/GO@COF/GCE) showed a strong current response to both SDZ and AP in phosphate buffer at pH 7.0. Under optimal testing conditions, linear calibration curves were obtained over the concentration range 0.5–200 μM for SDZ and 0.05–20 μM for AP, with limits of detection being 0.16 μM and 0.032 μM, respectively. Beef and fodder samples spiked with SDZ and AP were extracted in ethyl acetate, then the SDZ and AP in the extracts quantified using the MIP/GO@COF/GCE sensor. Recoveries were excellent, ranging from 82.0 to 108.0%. The same method was also employed to determine SDZ and AP residues in pork and chicken samples, with the results correlating well with those obtained using high-performance liquid chromatography.

1. Introduction Sulfadiazine (SDZ) is an antibiotic that is commonly used to treat urinary tract infections, ear infections, burns, meningitis, malaria and toxoplasmosis. SDZ inhibits bacteria growth by interfering with folate metabolism [1]. On account of its potency as a broad-spectrum antibiotic and its low cost, SDZ is also widely used to treat diseases in cattle and other animals intended for human consumption. To ensure the safety of animal products and protect consumers from risks related to SDZ residues, European Union regulation sets a safe limit of 100 μg kg−1 for sulfonamides in animal origin food stuffs [2]. Acetaminophen (AP) is widely used as an antipyretic to reduce fever symptoms and an analgesic for pain relief [3]. In many countries, AP is administered as an alternative to aspirin and phenacetin [4]. However, the excessive consumption of AP can result in the accumulation of toxic metabolites that can eventually lead to liver and kidney impairment

⁎

[5]. AP is also used in animal care to treat fever and as a pain reliever. Fast and sensitive methods for SDZ and AP detection in animal tissues and meats for consumption are needed to protect consumers. Currently, a number of methods are used to determine SDZ and AP, including high-performance liquid chromatography (HPLC) [6,7], liquid chromatography-mass spectrometry [8,9], spectrophotometry [10], spectroscopic methods [11,12] and capillary electrophoresis [13,14]. These methods are reliable, yet are not ideal since they use expensive instruments and have long analysis times (due to the lengthy sample preparation needed prior to analysis). Since SDZ and AP are frequently used together to treat cold and fever in cattle and other animals, analytical methods which can simultaneously determine SDZ and AP are demanded. Electrochemical methods offer many advantages over conventional analytical techniques for trace analyte detection, on account of their relatively low capital costs and short analysis times. Most

Corresponding author. E-mail address: [email protected] (Z. Xu).

https://doi.org/10.1016/j.snb.2019.126993 Received 17 June 2019; Received in revised form 28 July 2019; Accepted 16 August 2019 Available online 17 August 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.

Sensors & Actuators: B. Chemical 300 (2019) 126993

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2.2. Instruments and apparatus

electrochemical sensors are based on a glassy carbon electrode (GCE), which is subsequently modified with conductive nanomaterials to improve the sensitivity towards the target analyte(s). Commonly nanomaterials used for GCE modification include carbon black [15–17], graphene [18–20], polypyrrole (PPy) [21,22], and metal nanoparticles [23–26]. Covalent organic frameworks (COFs) are porous organic crystalline materials formed via the ordered covalent assembly of organic building units [27,28]. Due to their high specific surface area, high thermal stability and low density [29], COFs are attractive materials for gas storage, catalysis (as supports), pseudocapacitors and photoconductive devices [30–32]. However, the poor conductivity of COFs generally limits their application in electrochemical sensing. Recently, a highly conductive graphene oxide@covalent organic framework composite (GO@COF) was reported which demonstrated excellent electrochemical performance [33], opening the way for the wider utilization of COFs in electrochemical sensor development. Recognition element is an important component of electrochemical sensors due to their role in the selective recognition of target analytes. A host of recognition elements have been used to improve the sensitivity and selectivity of electrochemical sensors, including antibodies, phages, aptamers and molecularly imprinted polymers [34]. MIPs are usually applied in conjunction with other detection elements to achieve very high selectivity, short analysis times and stable operation performance. Many methods have been used to synthesize MIPs [35], with the direct electrochemical polymerization of an imprinted conducting polymer (such as polypyrrole) on an electrode surface being the most practical approach for electrochemical sensor fabrication. In this work, we targeted the development a selective electrochemical sensor for the simultaneous detection of SDZ and AP. Our approach involved modification of a GCE with a highly conductive GO@COF nanocomposite for electrochemical signal amplification, followed by electrodeposition of a PPy MIP imprinted with SDZ and AP. The electrochemical response of the MIP/GO@COF/GCE for the simultaneous quantification of SDZ and AP was then evaluated using differential pulse voltammetry (DPV) and cyclic voltammetry (CV). Particular emphasis here is placed on the selectivity, stability, reproducibility and accuracy of the MIP/GO@COF/GCE electrode, as well as the viability of the developed sensor for the detection of SDZ and AP in real meat samples.

All electrochemical tests were conducted on an electrochemical workstation (CHI630E, CH Instrument Company, Shanghai, China) using a three-electrode system, comprising a modified GCE as the working electrode, a saturated calomel reference electrode (SCE) and a platinum wire auxiliary electrode. Scanning electron microscopy (SEM) observations were performed on a JSM-6610 microscope operated at an accelerating voltage of 15 kV. X-ray diffraction (XRD) data were collected using a D8-advance diffractometer (Bruker, Germany) equipped with Cu Kα X-ray source. The HPLC system consisted of two LC-10ATVP pumps and a SPD10AVP UV detector (Shimadzu, Kyoto, Japan). All separations were achieved on a C18 reversed-phase column (4.6 × 250 mm, 5 m) at a mobile phase flow rate of 1.0 mL min−1. The mobile phase for SDZ determination was formic acid/acetonitrile with a gradient elution. The mobile phase for AP determination was methyl alcohol/ammonium hydroxide. The UV detector was set at 270 nm for SDZ and 250 nm for AP. 2.3. Preparation of the MIP/GO@COF/GCE The GCE was polished successively with 0.3 μm and 0.05 μm alumina pastes followed by thorough rising with double distilled water (DDW). Next, the polished GCE was ultrasonicated for 3 min in DDW, then 3 min in ethanol and finally 3 min in DDW. After drying in air, 8 μL of a 1.5 mg mL−1 GO@COF suspension was dropped onto the GCE, and the resulting modified electrode (denoted GO@COF/GCE) left to dry at room temperature (Scheme 1). The GO@COF/GCE electrode was then immersed in an acetonitrile solution containing 5 mM SDZ, 5 mM AP, 0.1 M pyrrole and 0.1 M TBAP. High-purity nitrogen was then bubbled through the acetonitrile solution to remove dissolved oxygen. Cyclic voltammetry (CV) was then used to electrochemically polymerize a PPy MIP on the GO@COF/GCE electrode. The polymerization was carried at potentials ranging from −0.6 V to 1.2 V over 5 cycles. Following the polymerization, a polymer overoxidation treatment was used to remove the SDZ and AP molecular templates. The treatment involved scanning the modified electrode from −0.6 V to 1.2 V for fifteen cycles at a scan rate of 50 mV s−1 in 0.1 M sodium hydroxide solution. Finally, MIP/ GO@COF/GCE was rinsed with DDW and allowed to dry at room temperature. A NIP (non-imprinted polymer)/GO@COF/GCE was fabricated under the same conditions of the MIP/GO@COF/GCE, except that SDZ and AP were not added in the polymerization step.

2. Experimental section 2.1. Materials and chemicals

2.4. Electrochemical measurements The pork, chicken, beef and fodder samples were purchased from a local market in Tai’an (Shandong, China). N,N-dimethylformamide (DMF) was obtained from Aladdin Industrial Corporation (Shanghai, China). Pyrrole, sulfamerazine, tetrabutyl ammonium perchlorate (TBAP), sulfacetamide, SDZ and AP were purchased from Shandong Xiya Chemical Industry Co., Ltd. (China). The GO@COF (benzene-1,4-diboronic acid as monomer) was supplied by Baiyin COFs Chemistry Technology Co., Ltd. (Gansu, China). Glucose and sodium hydroxide were purchased from Tianjin Kaitong Chemical Reagent Co., Ltd. (Tianjin, China). Ascorbic acid was obtained from Hualan Biological Engineering Inc. (Henan, China). Glutamic acid was supplied by Solarbio Biotechnology Co., Ltd. (Beijing, China). P-nitrophenol was purchased from Aikeda Chemical Reagent Co., Ltd. (Sichuan, China). A redox probe solution containing 2.0 mM K3Fe(CN)6, 2.0 mM K4Fe(CN)6 and 0.2 M KCl was prepared by dissolving the three compounds in a phosphate buffer (0.2 M, pH 7.0). Phosphate buffer with different pH values were prepared by mixing stock solutions of 0.2 M Na2HPO4 and 0.2 M NaH2PO4 in different ratios.

Differential pulse voltammetry (DPV) and CV were employed to investigate electrochemical behaviors of the different modified electrodes. Cyclic voltammograms were collected from −0.2 V to 0.6 V at a scan rate of 50 mV s−1 in a redox probe solution. DPV was used to record the peak current of the modified electrodes in the presence of SDZ and AP, with data collected in the potential range from 0.25 V to 1.6 V. 2.5. Sample preparation Recovery tests for SDZ and AP in beef and fodder samples were investigated. According to the limit of detection, linear range and maximum residue limit of China, three concentrations of AP (5.0 × 10−7, 5.0 × 10−6 or 2.0 × 10−5 M) and SDZ (5.0 × 10−6, 5.0 × 10−5 or 2.0 × 10−4 M) were selected for recovery test to verify the accuracy of the developed MIP/GO@COF/GCE sensor more comprehensively. Beef and fodder samples were determined to be free of SDZ and AP using HPLC (GB 29694-2013 and GB 29683-2013) and MIP/GO@COF/GCE sensor. Subsequently the samples were spiked using the following protocol: Homogenized sample (5.0 g) was carefully 2

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Scheme 1. Schematic illustration of MIP/GO@COF/GCE sensor fabrication.

are due to the crystalline COF. The XRD is thus consistent with a GO@COF nanocomposite.

weighed into a 50 mL tube and spiked with 1 mL of SDZ and AP standard solution. After incubation for 24 h, 20 mL of ethyl acetate was added and the resulting mixture was vortexed for 2 min, then centrifuged at 6000 rpm for 15 min. The organic extract was transferred into a clean 100-mL round bottom flask. The process was then repeated (i.e. 20 mL of ethyl acetate was added to the sample, followed by vortexing and centrifugation), and the organic extract was again collected. The organic extracts were combined, and then evaporated to dryness under a gentle flow of nitrogen gas. Finally, the residue was dissolved in 5 mL phosphate buffer (0.2 M, pH 7.0), then filtered through 0.22 μm filter membrane and the filtrate analyzed with the MIP/GO@COF/GCE sensor. The MIP/GO@COF/GCE sensor was also used to detect SDZ and AP residue in real pork and chicken samples. The pork and chicken samples were subjected to the same extraction procedure described above for the beef and fodder samples, except that SDZ and AP were not added. The extracts were analyzed using the MIP/GO@COF/GCE sensor and the HPLC method, and the SDZ and AP levels in the pork and chicken samples determined by each method were calculated and compared.

3.2. Electropolymerization of the PPy MIP imprinted with SDZ and AP The electropolymerization of pyrrole on the GO@COF/GCE was performed using CV in an acetonitrile solution containing 0.1 M pyrrole and 0.1 M TBAP at potentials ranging from −0.6 V to + 1.20 V (Fig. S3). During the electrodeposition, pyrrole monomers are oxidized giving a peak at 1.1 V which can be ascribed to the formation of pyrrole radical cations. These radical cations then polymerize to give PPy films. Film growth during the CV cycles is evidenced by the development of a broad anodic peak around 0.1 V and a reverse cathodic peak at −0.2 V (Fig. S3B). In the absence of SDZ and AP during PPy film growth, a nonimprinted polymer (NIP) is obtained (the electrode was denoted as NIP/ GO@COF/GCE). To obtain a molecularly imprinted PPy film on GO@COF/GCE, SDZ and AP were added into the pyrrole monomer solution used for PPy film electrodeposition. Fig. S3A shows CVs for the electrodeposition of PPy in the presence of SDZ and AP. The oxidation peak potential of polypyrrole shifts from 0.1 to 0.3 V (Fig. S3A), suggesting that the template is becoming part of the polymeric chain. These results are consistent with the previous study of PPy MIP formation [37]. Fig. S4A shows a schematic representation of the as-deposited molecularly imprinted PPy film containing SDZ and AP. Hydrogen bonding interactions between the NeH groups of PPy and electron lone pairs on SDZ and AP hold the template molecules firmly in place [3,37]. To remove the embedded molecular templates, the imprinted polymer is oxidized in a 0.1 M sodium hydroxide solution. At such high pH, the cationic charges along the polymeric PPy backbone are eliminated, resulting in the release of the molecular templates (now in an anion form due to the pH change) in order to achieve electrostatic neutrality. The high pH oxidation step could possibly have introduced some additional functional groups on the PPy surface [38], though results below suggest that these additional groups (if formed) do not adverse impact the ability of the MIP to recognize imprinted molecules. After the SDZ and AP removal, a MIP for selective recognition of SDZ and AP based on shape selection and precise positioning of functional groups is created (Fig. S4B).

3. Results and discussion 3.1. Morphological characterization of GO@COF and the PPy MIP before and after SDZ and AP extraction Fig.1 A and B show SEM images of the GO@COF nanocomposite, showing the presence of COF fragments of different sizes immobilized on the graphene oxide sheets (Fig. S1 shows graphene oxide is a folded sheet). Fig. 1C and D show the surface morphologies of the electrodeposited PPy MIP before and after extraction of SDZ and AP, respectively. The surface morphologies of the MIP pre and post extraction are similar, exhibiting a cauliflower-like morphology which is typical of electrodeposited PPy. After extraction of the SDZ and AP templates (Fig. 1D) via the overoxidation treatment, the MIP surface becomes more textured, consistent with the findings of Gürler et al., [36]. Fig. S2 shows the XRD pattern for the GO@COF nanocomposite. The XRD pattern shows an intense feature at 10.2°, which can readily be assigned to the (001) reflection of graphene oxide. All other reflections 3

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Fig. 1. SEM images of (A) GO@COF (5000×), (B) GO@COF (9000×), (C) MIP before removal of SDZ and AP (60000×) and (D) MIP after removal of SDZ and AP (60000×).

oxidation peak current of GO@COF/GCE (curve b) is larger than that of the bare GCE (curve a), though the oxidation potentials of SDZ and AP (curve b) are lower on the GO@COF/GCE than on GCE. Results suggest that GO@COF can catalyze the oxidation of SDZ and AP. The oxidation peak currents for MIP/GO@COF/GCE (curve c) and NIP/GO@COF/ GCE (curve d) are larger than that of GO@COF/GCE. Further, the current response obtained with MIP electrode is higher than that obtained with the NIP electrode. The results clearly demonstrate that the MIP electrode is more sensitive towards SDZ and AP than the NIP electrode. Fig. 2D shows CVs collected at different scan rates (20 mV s−1 to 300 mV s−1) on a MIP/GO@COF/GCE. The same analysis was performed for GCE and GO@COF/GCE (Fig. S5), and the slopes of the respective Ip versus v1/2 plots used to calculate electroactive surface areas from the Randles-Sevcik equation (IP = 2.69 × 105n3/2AD1/2Cv1/ 2 , where IP is the anodic or the cathodic peak current (A), n is the number of transferred electrons, A is the electroactive area (cm2), D is the diffusion coefficient of [Fe(CN)6]3/4− (cm2 s−1), C is the concentration of the [Fe(CN)6]3/4− (mol cm−3), and v1/2 is the square root of scan rate (V s−1). The calculated electroactive surface areas for the bare GCE, GO@COF/GCE and MIP/GO@COF/GCE are 0.083, 0.091 and 0.100 cm2, respectively.

3.3. Electrochemical characteristics of the modified electrodes CV and DPV were applied to investigate electrochemical behaviors of different modified electrodes fabricated in this study. Cyclic voltammograms for the different electrodes in the redox probe solution are shown in Fig. 2A. Compared to GCE (curve a), the peak current of GO@COF/GCE (curve b) greatly increases, which can be attributed to the good electrical conductivity and high surface area of the GO@COF nanocomposite. After electrodeposition of the MIP on the GO@COF/ GCE and removal of the SDZ and AP molecular templates, the peak current increases further (curve c). This increase is likely due to the rough cauliflower-like surface of the PPy MIP which is expected to facilitate electronic transfer between the MIP/GO@COF/GCE and the redox probe. Fig. 2B shows the DPV behavior of MIP/GO@COF/GCE in different solutions. In AP solutions (curve a), an oxidation peak is observed at 0.36 V, which can be attributed to the oxidation of eOH and NH eegroups in the molecule. In SDZ solutions (curve b), an oxidation peak is observed at 0.86 V due to oxidation of the –NH2 group in SDZ. In solutions containing both AP and SDZ (curve c), two distinct oxidation peaks are seen at 0.36 V and 0.86 V, corresponding to the oxidation of AP and SDZ, respectively. This indicates that AP and SDZ are adsorbed independently on the electrode surface (as expected for a MIP and two structurally distinct molecules) and MIP/GO@COF/GCE is suitable for the simultaneous determination of SDZ and AP. DPV data for the different modified electrodes are shown in Fig. 2C. As expected, the

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Fig. 2. (A) CV responses of (a) the GCE, (b) GO@COF/GCE and (c) MIP/GO@COF/GCE (after SDZ and AP removal) in the redox probe solution. The scan rate was 50 mV s−1. (B) DPV response of the MIP/GO@COF/GCE in the presence of (a) 0.01 mM AP in phosphate buffer (pH 7.0), (b) 0.1 mM SDZ in phosphate-buffer (pH 7.0) and (c) 0.01 mM AP and 0.1 mM SDZ in phosphate-buffer (pH 7.0). (C) DPV response of (a) the GCE, (b) GO@COF/GCE, (c) MIP/GO@COF/GCE and (d) NIP/ GO@COF/GCE in the presence of 0.01 mM AP and 0.1 mM SDZ in phosphate-buffer (pH 7.0). (D) CVs for the MIP/GO@COF/GCE at different scan rates from 20 to 300 mV/s in 1.0 mM [Fe(CN)6]3/4− solution.

Fig. S6C shows the effect of pH of the phosphate buffer on the current response of MIP/GO@COF/GCE towards SDZ and AP. The pH range studied is 6.0–8.0. Ip value increases with pH up to 7.0 for both SDZ and AP, then decreases at higher pH value. Accordingly, phosphate buffer of pH 7.0 is used for detection and quantification of SDZ and AP in subsequent experiments.

3.4. Optimization of the experimental testing conditions of the electrochemical sensor In order to maximize the sensitivity of the MIP/GO@COF/GCE sensor for the detection of SDZ and AP, a wide range of experimental parameters need to be considered and optimized. The first parameter that needed to be optimized is the thickness of the MIP, which is controlled by the number of electrochemical polymerization cycles. Fig. S6A shows the effect of the number of electrochemical polymerization cycles on the current response of the MIP/GO@COF/GCE towards SDZ and AP. As the number of cycles increases, the current response towards both SDZ and AP firstly increases and then decreases. The response current reaches maximum value when the number of electrochemical polymerization cycle is five, thus prompting the use of five polymerization cycles for MIP/GO@COF/GCE sensor fabrication. Next, the effect of the incubation time of the MIP/GO@COF/GCE in the SDZ and AP solution on response current was examined. Incubation times are varied from 3 to 18 min. Fig. S6B shows that the current response towards SDZ and AP increases with incubation time and then reaches a plateau after 12 min, indicating that SDZ and AP adsorption on the MIP/GO@COF/GCE has reached saturation. Thus, 12 min is selected as the optimal incubation time for all subsequent experiments.

3.5. Simultaneous determination of SDZ and AP using MIP/GO@COF/GCE DPV experiments were performed under the optimal experimental conditions established above. Fig. 3A shows DPV data collected as the concentrations of SDZ and AP were simultaneously increased. DPV curves are recorded in the range of 0.5–200 μM and 0.05–20 μM, respectively. For both SDZ and AP, strong linear relationships are established between Ip and the analyte concentration. The current value corresponding to each concentration is the average of three measurements. The obtained linear expressions are Ip (−μA) = 0.565 × [AP] (μM) + 0.016 (R2 = 0.994) for acetaminophen, and Ip (−μA) = 0.058 × [SDZ] (μM) + 0.021 (R2 = 0.998) for sulfadiazine (Fig. 3B and C, respectively). The limits of detection of MIP/GO@COF/ GCE for AP and SDZ are 0.032 μM (4.8 μg kg−1) and 0.16 μM (48 μg kg−1), respectively. The agriculture ministry of the People's 5

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Fig. 3. (A) DPV responses of MIP/GO@COF/GCE in phosphate-buffer (pH 7.0) containing different concentrations of SDZ and AP. (B) Linear relationship between the DPV response currents and AP concentration. (C) Linear relationship between the DPV response currents and SDZ concentration.

Republic of China sets a safe limit of 100 μg kg-1 for sulfonamides in animal origin food stuffs and does not set maximum residue limit for AP. Therefore, these limits are compliant with the ones imposed by the health legislations. Table S1 compares the performance of the developed sensor with other sensors reported in literature for SDZ and AP determination. Impressively, the limits of detection of the MIP/ GO@COF/GCE sensor for SDZ and AP are lower than other sensors reported to date [39–44]. Further, the MIP/GO@COF/GCE allows the simultaneous determination of SDZ and AP, highlighting a major advantage of the MIP-based electrochemical sensor.

MIP/GO@COF/GCE towards SDZ and AP are higher than those of the respective structural analogs. The selectivity of the proposed sensor can be further evaluated by calculating the imprinted factor (IF). IF = IMIP/ INIP, where IMIP and INIP are the response currents for each analyte determined with the MIP and NIP sensors, respectively. For SDZ and AP, the IFs are 2.5 and 2.3, respectively. However, the IFs calculated for the analogs (sulfamerazine, sulfacetamide, ascorbic acid and p-nitrophenol) are all around 1, indicating that MIP/GO@COF/GCE can specifically recognize AP and SDZ. In order to investigate the potential interference of common small organic molecules and metal ions on the response current of MIP/GO@COF/GCE for SDZ and AP, interference experiments were carried out. Fig. 4C shows that the presence of glucose, glutamic acid, Fe3+ and Cu2+ has negligible influence on the determination of SDZ and AP. The above results demonstrate that the MIP/GO@COF/GCE sensor has excellent recognition selectivity towards SDZ and AP (as was intended when fabricating the sensor), with the ability to simultaneous determine SDZ and AP being a major further advantage of the developed sensor. To investigate the reproducibility of MIP/GO@COF/GCE sensor, six sensors were fabricated and used to determine SDZ (0.1 mM) and AP (0.01 mM) by DPV (Table S2). Relative standard deviations (RSDs) for the determination of SDZ and AP are 5.5% and 6.7%, respectively,

3.6. Selectivity, reproducibility, repeatability and stability of the electrochemical sensor The ability of MIP/GO@COF/GCE to selectively recognize SDZ and AP is a key feature of the developed sensor. To test the selectivity of MIP/GO@COF/GCE, the response of the sensor towards structural analogs of SDZ (sulfamerazine and sulfacetamide) and AP (ascorbic acid and p-nitrophenol) was examined. Fig. 4A and B compare the response currents of the MIP and NIP sensors towards SDZ (0.1 mM), AP (0.01 mM) and their structural analogs (at a concentration of 0.1 mM in pH 7.0 phosphate buffer). The results show the response currents of 6

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Fig. 4. (A) DPV responses of the MIP and NIP sensors for SDZ (0.1 mM) and structural analogs (sulfamerazine (SMR), sulfacetamide (SMZ)) at a concentration of 0.1 mM. (B) DPV responses of the MIP and NIP sensors for AP (0.01 mM) and structural analogs (p-nitrophenol and ascorbic acid) at a concentration of 0.1 mM. (C) DPV responses of MIP/GO@COF/GCE towards 0.1 mM SDZ and 0.01 mM AP in the absence of interferents and DPV responses in presence of 0.1 mM concentrations of (a) glucose, (b) glutamic acid, (c) Fe2+ ions and (d) Cu2+ ions, respectively.

were made using a single MIP/GO@COF/GCE sensor, with the resulting RSDs for the determination SDZ (0.1 mM) and AP (0.01 mM) calculated to be 2.7% and 5.8%. Again, this is a highly acceptable level of measurement repeatability. The stability of MIP/GO@COF/GCE sensor was investigated subsequently (Table S4). Three MIP/GO@COF/GCE electrodes are stored at 4 °C for a period of 30 days. Every 5 days, the response currents of each MIP/GO@COF/GCE electrode towards SDZ (0.1 mM) and AP (0.01 mM) were measured. After 30 days, the response currents of MIP/GO@COF/GCE sensor for determination SDZ and AP are still high (82.4% and 85.4%, respectively, of the original values), indicating that the MIP/GO@COF/GCE sensor possesses excellent stability.

Table 1 Recovery of SDZ and AP in spiked samples determined using the MIP/ GO@COF/GCE sensor ( ± RSD, n = 3). Samples

Beef

Fodder

Spiked level(10−5 mol L−1)

Found level (×10−5 mol L−1)

Recovery (%)

RSD(%)

SDZ

AP

SDZ

AP

SDZ

AP

SDZ

AP

20 5 0.5 20 5 0.5

2.0 0.5 0.05 2.0 0.5 0.05

20.7 4.4 0.46 21.6 4.5 0.43

1.86 0.44 0.048 2.0 0.44 0.041

103.5 88.0 92.0 108.0 90.0 86.0

93.0 87.0 96.0 100.0 88.0 82.0

2.5 1.3 1.2 2.6 5.8 6.1

3.7 1.6 1.5 2.6 3.4 4.7

3.7. Applicability and accuracy of the MIP/GO@COF/GCE sensor indicating that the MIP/GO@COF/GCE sensor possesses acceptable reproducibility. Measurement repeatability is a further important performance indicator for any type of sensor (Table S3). Six repeat measurements (The determined MIP electrode was immersed in 0.1 M sodium hydroxide solution to remove SDZ and AP through CV and then immersed in the target solution to continue the next measurement)

To evaluate the accuracy of the proposed method, SDZ and AP recovery tests were performed using spiked beef and fodder samples. The results are shown in Table 1. The recoveries range from 82.0% to 108.0% with RSDs below 6.1%, indicating the SDZ and AP extraction protocol with ethyl acetate and their subsequent determination using 7

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the sensor has good accuracy. To demonstrate the potential of the MIP/GO@COF/GCE sensor for SDZ and AP quantification in real samples, we applied the sensor to the determination of SDZ and AP in pork and chicken samples obtained from a local supermarket. The concentrations of SDZ and AP in the pork sample are determined to 1962.0 ± 2.5 μg kg−1 and 67.7 ± 7.0 μg kg−1, respectively. In the chicken sample, the concentrations are 835.2 ± 8.5 μg kg−1 and 29.7 ± 5.2 μg kg−1, respectively. To validate these findings, the SDZ and AP concentrations in the pork and chicken samples were also determined by HPLC (Fig. S7). The concentrations of SDZ and AP determined by HPLC in the pork sample are 1937.8 ± 5.3 μg kg−1 and 68.4 ± 6.5 μg kg−1, respectively, and in the chicken sample 855.5.0 ± 3.9 μg kg−1 and 29.5 ± 1.4 μg kg−1, respectively. The results obtained by these two different analytical methods have no significant difference (P > 0.05), indicating that the proposed sensor has excellent accuracy and is suitable for the selective and simultaneous determination of SDZ and AP in meat products.

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Jingbo He obtained her bachelor’s degree from Shandong Agricultural University, Taian, China in 20167. Her research interests are electrochemical sensor detection methods. Geoffrey I.N. Waterhouse was born in 1972, is now a associate professor in School of Chemical Sciences, The University of Auckland. His research interests focus on the development of low cost semiconductor photocatalysts for solar hydrogen production and carbon dioxide reduction, and also the fabrication of smart platforms for optical sensing and biosensing. Longhua Xu got her Ph. D in Food science in 2016 from Tianjin University of Science & Technology, Tianjin, China and then became a faculty in College of Food Science and Engineering, Shandong Agricultural University. Her research interests are molecularly imprinted polymers design and application. Hongyan Zhang got her Ph. D in Food science in 2007 from Tianjin University of Science & Technology, Tianjin, China and then became a faculty in College of Life Science, Shandong Normal University. Her research interest is development of new food analytical method. Xuguang Qiao was born in 1965, is now a Professor in College of Food Science and Engineering, Shandong Agricultural University. His research interests focus on food quality control. Zhixiang Xu was born in 1973, is now a Professor in College of Food Science and Engineering, Shandong Agricultural University. His research interests focus on tailored (bio) molecular recognition interfaces, molecularly imprinted materials, and electrochemical sensor.

Yufeng Sun will get her master’s in Shandong Agricultural University (2018), Taian, China. Her research interests are electrochemical sensor detection methods.

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