Molecularly Imprinted Poly(thionine)-Based Electrochemical Sensing

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A Molecularly Imprinted Poly(thionine)-Based Electrochemical Sensing Platform for Fast and Selective Ultratrace Determination of Patulin Qingwen Huang, Zhihui Zhao, Dongxia Nie, Keqiu Jiang, Wenbo Guo, Kai Fan, Zhiqi Zhang, Jiajia Meng, Yongjiang Wu, and Zheng Han Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05791 • Publication Date (Web): 22 Feb 2019 Downloaded from http://pubs.acs.org on February 25, 2019

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Analytical Chemistry

A Molecularly Imprinted Poly(thionine)-Based Electrochemical Sensing Platform for Fast and Selective Ultratrace Determination of Patulin

Qingwen Huanga,b,1, Zhihui Zhaoa,1, Dongxia Niea, Keqiu Jianga,b, Wenbo Guoa, Kai Fana, Zhiqi Zhanga, Jiajia Menga, Yongjiang Wub,*, Zheng Hana,*

aInstitute

for Agro-food Standards and Testing Technology, Shanghai Key Laboratory of

Protected Horticultural Technology, Laboratory of Quality and Safety Risk Assessment for Agro-products (Shanghai), Ministry of Agriculture, Shanghai Academy of Agricultural Sciences, 1000 Jingqi Road, Shanghai 201403, China bCollege

of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road,

Hangzhou 310058, China

1These

authors contributed equally to this work.

*Corresponding author Tel.: +86-21-62203612; Fax: +86-21-62203612 E-mail addresses: [email protected] (Zheng Han); [email protected] (Yongjiang Wu)

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ABSTRACT An innovative approach based on a surface functional monomer-directing strategy for the construction of a sensitive and selective molecularly imprinted electrochemical sensor for patulin recognition is described. A patulin imprinted platinum nanoparticle (PtNP)-coated poly(thionine) film was grown on a pre-formed thionine tailed surface of PtNP-nitrogen-doped graphene (NGE) by electropolymerization, which provided high capacity and fast kinetics to uptake patulin molecules. Thionine acted not only as a functional monomer for molecularly imprinted polymer (MIP), but also as a signal indicator. Enhanced sensitivity was obtained by combining the excellent electric conductivity of PtNPs, NGE and thionine with multi-signal amplification. The designed sensor displayed excellent performance for patulin detection over the range of 0.002–2 ng mL–1 (R2 = 0.995) with a detection limit of 0.001 ng mL–1 for patulin. In addition, the resulting sensor showed good stability and high repeatability and selectivity. Furthermore, the feasibility of its applications has also been demonstrated in the analysis of real samples, providing novel tactics for the rational design of MIP-based electrochemical sensors to detect a growing number of deleterious substances.


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Patulin (see Figure 1), produced by Aspergillus, Penicillium and Byssochlamys species,1,2 is frequently found in various fruits, especially in apples and grapes, and their derived products.3,4 It has been proved that patulin has acute, subacute and chronic toxic effects on animal and human health, such as agitation, mutagenicity, carcinogenicity, convulsion, hyperemia, ulceration and epithelial cell degeneration.2,5 Due to its high toxicity and widespread occurrence, a maximum residue limit for patulin has been set at 50 µg L–1 in apple juice in the USA and China (CPG Sec.510.150 and GB 2761-2017). Patulin is frequently determined by chromatographic techniques, such as gas chromatography6 and high performance liquid chromatography (HPLC) with various detectors.7,8 Although showing good reproducibility and accuracy, these methods invariably require time-consuming pretreatments, expensive instruments and skilled operators.9,10 Recently, electrochemical methodologies have drawn significant attention by virtue of their quick and easy operation, low cost, high sensitivity, portability and miniaturization, and have been widely used for the detection of drugs,11 pesticides,12 mycotoxins13 and explosives.14 Molecularly imprinted polymers (MIPs) were proposed as the most efficient recognition elements in electrochemical methods for their high selectivity and chemical stability.15 MIPs, which mainly mimic the biological antibodies model, are synthesized by polymerizing functional monomers in the presence of template molecules and forming specific recognition sites after removal of the templates. Several methods, including in-situ electrochemical polymerization,16 optical17 and thermal technologies,18 have been 3 ACS Paragon Plus Environment


developed for MIP preparation, among which, the first approach has aroused the most considerable attention due to its unique advantages, such as simple and fast preparation, high adherence to the electrode surface, easy control of the film thickness and high reproducibility.19 Development of optimal functional monomers is the primary step for the preparation of MIPs. Thionine, an aromatic redox dye with two amino groups, has proven to be an excellent candidate, serving as a signal molecule with good electron transfer ability and high water solubility.20,21 In contrast, the electropolymerization of thionine can form a poly(thionine) film through the amino group, and could also be a promising MIP functional monomer due to its free –NH2 group, which could conjunct with the –OH group22 and acyl group2 in targeted substances. However, the utilization of thionine for the preparation of a MIP-based electrochemical sensor remains at an early stage. To the best of our knowledge, there have been no reports of the exploration of thionine, not only as a functional monomer of MIPs, but also as a signal indicator for the construction of an electrochemical sensor. To obtain ideal MIP-based electrochemical sensors, it is important to modify the electrode and fabricate the MIPs on the electrode surface to obtain better electrical performance. Many functionalized nanomaterials with a large surface area and good conductivity were utilized to increase the effective binding sites, and improve the selectivity, sensitivity and the stability of the developed electrochemical sensors.23 4 ACS Paragon Plus Environment

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Nitrogen-doped graphene (NGE), a new carbon derived material, has been abundantly used in the field of sensors due to its excellent thermal/mechanical stability, good electrical conductivity and large functional surface area.24 Pt nanoparticles (PtNPs) have also been frequently studied for amplifying the electric signal due to their large specific surface area and good conductivity. By combining the advantages of the two above-mentioned materials, NGE functionalized with PtNPs could be an excellent promising material for the fabrication of electrodes and could provide favorable environmental conditions for the immobilization of functional molecules. In this study, a novel electrochemical sensor was developed for ultratrace patulin determination by fabrication of a molecularly imprinted film on a thionine-PtNP-NGE modified electrode. The MIP/thionine-PtNP-NGE-based sensor was designed as illustrated in Scheme 1 based on four main points: (1) NGE primarily offered an effective platform with large surface area and high electrical conductivity, which facilitated the assembly of PtNPs, and coupled with the large amount of thionine through Pt-NH2 bonding. (2) PtNPs reduced in situ were uniformly incorporated into the polythionine MIPs to enhance the conductivity and stability of the MIP. (3) Thionine was used not only as a functional monomer of MIP but also as an electrochemical probe for the first time for patulin. (4) MIP was electrochemically polymerized based on a surface functional monomer-directing strategy, improving the uptake capacity of patulin molecules and enhancing the adhesion stability of the MIP film to the electrode surface. 5 ACS Paragon Plus Environment


This work is expected to propose a novel and promising strategy for the construction of electrochemical sensors with good performance for patulin detection.

EXPERIMENTAL SECTION

Materials and reagents. Chloroplatinic acid hexahydrate (99.9%, H2PtCl6·6H2O) and N, N-Dimethylformamide (DMF, ≥99.8%) were purchased from Adamas Reagent Co., Ltd. (Shanghai, China). L-(+)-Ascorbic acid (>99%) was purchased from Alfa Aesar (China) Chemicals Co., Ltd. (Shanghai, China). MycoSep®228 AflaPat columns were acquired from Romer labs (Washington, MO, USA). NGE was obtained from Nanjing Xianfeng Nanomaterials Technology Co., Ltd. (Nanjing, China). Phosphate buffer solution (PBS) was supplied by Zrbiorise Inc. (Shanghai, China). Thionine was provided by Acros Organics (Belgium). Deionized triple-distilled water purified by a MING-CHE 24 UV system (Merck Millipore, Billerica, MA, USA) was used throughout the whole experiment. The standards of 5-hydroxymethylfurfural (5-HMF) were purchased from Adamas Reagent Co., Ltd. Aflatoxin B1 (AFB1), alternariol (AOH), citrinin (CIT), ochratoxin A (OTA) and patulin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Apple and grape juice samples were randomly collected from supermarkets in Shanghai and stored at 4 °C in the dark. 6 ACS Paragon Plus Environment

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Apparatus. The synthesized nanomaterials were characterized by a JEM-1010 transmission electron microscope (TEM; JEOL Ltd., Tokyo, Japan) and an S-4800 scanning electron microscope (SEM, Hitachi High-Technologies Co., Japan). Nitrogen adsorption-desorption analysis was performed on a Quantachrome-Autosorb-3D (Florida, USA). UV-vis absorption spectra were carried out on a NanoDrop 2000c spectrophotometer (Thermo Scientific, Waltham, MA, USA). The thermal characteristics of the nanocomposites were analyzed by thermogravimetric analysis and differential scanning calorimetry (TGA/DSC) using TGA/DSC 3+ (Mettler Toledo, Switzerland) with the temperature heating from 25 to 800 C at a rate of 10 K min–1 and air gas at a flow rate of 50 mL min–1. All electrochemical measurements were carried out on a CHI660D electrochemical workstation (Chenhua Instruments Co, China) with a conventional three-electrode system. A glassy carbon electrode (GCE, d = 3 mm), a platinum electrode and a saturated calomel electrode were served as the working electrode, auxiliary electrode and the reference electrode, respectively.

Synthesis of thionine-PtNP-NGE composite. Solid portions (50 mg) of NGE were dissolved in 4.5 mL of a H2PtCl6·6H2O (20 mmol L–1) aqueous solution. Then, 110 μL of hydrochloric acid (6 mol L–1) and 5.0 mL of ascorbic acid (0.1 mol L–1) were added, and 7 ACS Paragon Plus Environment


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the mixture was ultrasonicated for 4 h. After centrifugation at 2000 g for 20 min, the black precipitates (PtNP-NGE) were collected and subsequently washed with acetone and distilled water three times, and then dried at 50 C. Accurately weighed solid portions (5 mg) of PtNP-NGE and 2.5 mg of thionine were added to 5 mL of DMF, and the mixture was ultrasonicated for 4 h. After centrifugation at 2000 g for 3 min, the residues were collected, washed with distilled water three times and then dried at room temperature. Finally, the prepared thionine-PtNP-NGE (3 mg) composites were dispersed in water (600 μL) with the help of ultrasonic treatment to form a homogeneous thionine-PtNP-NGE suspension solution (5 mg mL–1).

Fabrication of thionine-PtNP-NGE composite modified GCE. The GCE was successively polished with 1, 0.3 and 0.05 μm alumina powders, and then washed with water. An aliquot (6 μL) of the thionine-PtNP-NGE suspension was coated onto the electrode and dried at room temperature.

Fabrication

of

MIP/thionine-PtNP-NGE/GCE.

The

prepared

thionine-PtNP-NGE/GCE was immersed in a 2 mL PBS solution containing thionine (3 mmol L–1), patulin (0.5 mmol L–1) and H2PtCl6·6H2O (2 mmol L–1). The electropolymerization cycle was run 15 times with the potential range of –0.4–0.4 V and a scan rate of 50 mV s–1. Afterwards, the polymer modified electrode was soaked in 8 ACS Paragon Plus Environment

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sulfuric acid (H2SO4, 2 mol L–1) for 10 min to remove the template molecules. MIP/thionine-PtNP-NGE/GCE was obtained after the modified electrode was thrice washed with water and dried at room temperature. The non-imprinted polymer (NIP)/thionine-PtNP-NGE/GCE was prepared in the same procedure in the absence of patulin.

Electrochemical measurements. For the detection of patulin, differential pulse voltammetry (DPV) measurements were carried out in 5.0 mL PBS solutions (0.1 mol L–1, pH 6.0) with the experiment parameters as follows: initial potential, 0.2 V; final potential, –0.5 V; pulse amplitude, 0.05 V; pulse width, 0.05 s; sampling width, 0.0167 s. The adsorption time was 180 s. The current difference (ΔI) was calculated according to equation (1): ΔI = I0 – I

(1)

where I0 is the peak current of blank PBS and I represents the peak current of PBS containing patulin. All measurements were carried out at room temperature. Cyclic voltammetry (CV) measurements were performed using [Fe(CN)6]3-/4- as a redox probe in the potential range of –0.2–0.6 V to evaluate the conductivity of the fabricated electrode, while it was recorded in PBS solution (0.1 mol L–1, pH 6.0) with the scan range of –0.2–0.6 V and the scan rate of 50 mV s–1 for the characterization of the other electrochemical performance. 9 ACS Paragon Plus Environment


RESULTS AND DISCUSSION

Characterization of synthesized materials. The morphologies of the NGE, thionine-PtNP-NGE and MIP/thionine-PtNP-NGE were characterized by TEM (Figure 2). The shape of the NGE (Figure 2A) is flake-like with some wrinkles, verifying the high surface area of this material. The thionine-PtNP-NGE was simply prepared with the assistance of ultrasonication. It could be obviously seen that in the synthesized nanomaterial, PtNPs were homogeneously distributed on the surface of the NGE, leading to more roughness and wrinkles, which greatly improved the electroactive surface (Figure 2B). After conjunction with MIP through electropolymerization, the thionine-PtNP-NGE was covered by a dense membrane with flexural nanostrips (Figure 2C), proving the successful synthesis of the poly(thionine) (MIP) film through the –NH– bridge.25 The structure of the NGE, thionine-PtNP-NGE and MIP/thionine-PtNP-NGE were also characterized by SEM (Figure S1, Supporting Information). Interestingly, as shown in Figure S1C (Supporting Information), the structure of MIP is rough and porous, implying that the recognition sites were successfully generated after the removal of the template. UV-vis adsorption spectroscopy was further used for the characterization of the thionine, NGE, PtNP-NGE and thionine-PtNP-NGE. As displayed in Figure 2D, the NGE (curve a) and PtNP-NGE (curve b) showed no obvious UV adsorption. The thionine 10 ACS Paragon Plus Environment

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solution (curve c) exhibited two characteristic adsorption peaks at 285 and 605 nm, which belong to the long wavelength π-π* transitions of the aromatic ring and the n-π* transition of the C=N bond, respectively.26 This is similar to the spectrum of the thionine-PtNP-NGE suspension solution (curve d), but the adsorption peak at 605 nm is blue-shifted to 602 nm, which could be due to the π-π* stacking force and Pt-NH2 bond between thionine and the PtNP-NGE. All the above observations clearly indicate that thionine-PtNP-NGE composites had been successfully synthesized. To determine the functional surface area and prove the suitability of the NGE as the supporting substance, nitrogen adsorption-desorption tests were performed. The isotherms (Figure S2, Supporting Information) showed a typical IV character with a hysteresis loop (0.4–1.0), demonstrating the presence of mesopores in the framework of NGE. The large specific surface area and total pore volume were calculated as 763.8 m2 g–1 and 6.354 cm3 g–1, respectively, verifying it to be an excellent reinforcement material, providing abundant binding sites, and thus is suitable to be used in the present study. TGA was used for analysis of the thermal stability of the thionine-PtNP-NGE. As shown in Figure S3 (Supporting Information), from 25 to 100 C, the weight decreased due to the dislodgement of water. From 100 to 360 C, carbon combustion and labile organic amine decomposition caused the weight lost.27 From 360 to 615 C, the weight of the prepared nanocomposites sharply decreased because of the destruction of the carbon skeleton. From 615 to 800 C, the weight loss was minor again, and finally, the residues 11 ACS Paragon Plus Environment


of the thionine-PtNP-NGE were 2.15%.

Electrochemical behaviors of fabricated electrodes. When the bare GCE was used, there was no peak (curve a) in PBS (0.1 mol L–1, pH 6.0), as shown in Figure 3. When the GCE was modified with thionine-PtNP-NGE nanocomposites (curve b), a typical reduction current peak was observed at –0.2 V in PBS, manifesting that the thionine in the composites could be a favorable indicator for monitoring the reactions on the electrode surface.20 Moreover, when the thionine-PtNP-NGE modified electrode was immersed into the polymerization precursor solution containing thionine (3 mmol L–1), patulin (0.5 mmol L–1) and H2PtCl6·6H2O (2 mmol L–1), thionine layer at the surface of PtNP-NGE can not only direct the selective occurrence of imprinting polymerization by amino group (–NH2), but also drive patulin molecules into the formed polymers through hydrogen bond interaction between the residual –NH2 of thionine and –OH and acyl group of patulin molecules at the same time. After electropolymerization, patulin templates were preserved in the MIP film and enriched on the surface of the electrode. Compared to the thionine-PtNP-NGE-modified GCE (thionine-PtNP-NGE/GCE), the peak current is greatly reduced (curve c) when the MIP/thionine-PtNP-NGE/GCE before template removal was used, due to the poor electron transfer ability of the compact membrane. After elution of the template molecules (curve d), the reduction peak sharply increased, and was even significantly higher than that of curve b, certifying the excellent 12 ACS Paragon Plus Environment

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electrical conductivity of the prepared MIP film. Afterwards, incubation of the MIP/thionine-PtNP-NGE-modified GCE (MIP/thionine-PtNP-NGE/GCE) in patulin solution (1 ng mL–1) caused the reduction of the redox peak (curve e), due to the binding of patulin molecules and the selective cavity blocking the arrival channel of the probe. All these results clearly indicated that patulin-imprinted MIPs have been successfully prepared

with

thionine

as

the

functional

monomer,

and

the

synthesized

MIP/thionine-PtNP-NGE modified electrode was sensitive and selective, and could be used for the detection of patulin in real samples. The fabrication of the MIP-based electrochemical sensor is technically simpler with considerably reduced time and cost compared to the previously reported rapid methods for patulin detection.28,29 The influences of scan rate on the MIP/thionine-PtNP-NGE/GCE performance were investigated by the construction of CV curves in PBS (0.1 mmol L–1, pH 6.0). As shown in Figure 4A, both the anodic peak current (Ipa) and cathodic peak current (Ipc) increased linearly with the square root of the scan rate (v1/2) from 10 to 270 mV s–1. The linear regression equations are Ipa = 42.784 v1/2 – 13.818 (R2 = 0.988) and Ipc = –35.167 v1/2 + 11.873 (R2 = 0.992), respectively, implying that the electrochemical kinetics were a diffusion-controlled process.2 The CV curves for thionine-PtNP-NGE/GCE and bare GCE were constructed at scan rates from 50 to 150 mV s–1 in 0.1 mol L–1 KCl aqueous solution containing 5 mmol L–1 [Fe(CN)6]3-/4-. The linear regression equations were Ipa (μA) = 26.758 v1/2 – 42.094 (R2 = 13 ACS Paragon Plus Environment


0.999), Ipc (μA) = –23.822 v1/2 + 22.908 (R2 = 0.99) for thionine-PtNP-NGE/GCE and Ipa (μA) = 14.354 v1/2 + 2.417 (R2 = 0.999), Ipc (μA) = –13.997 v1/2 – 5.479 (R2 = 0.998) for the bare GCE (data not shown), verifying the excellent electrode-transfer rate. The influences of PtNPs on the performance of the constructed electrochemical sensors were also investigated. For preparation of the supporting substrate, PtNPs could significantly improve the signal responses and efficiently promote a firm conjunction of thionine on the NGE via Pt-NH2 bonding, ensuring the stability and sensitivity of the developed sensor. In contrast, PtNPs were also utilized for the preparation of MIPs. DPV measurements were carried out in PBS (0.1 mmol L–1, pH 6.0) containing 0, 0.05 and 0.5 ng mL−1 patulin using the electrode fabricated with MIP containing PtNPs and one without PtNPs (Figure S4, Supporting Information). The signals (I) were greatly improved with larger current differences (ΔI) when PtNPs were added. All these results clearly proved the importance of PtNPs on sensor construction, and thus should be utilized in the current study.

Optimization of experimental conditions. For preparation of the as-designed electrochemical sensor, several key parameters, including the ratio of PtNP-NGE and thionine, the ratio of template and functional monomer, pH values and the adsorption time for targeted analytes, were all thoroughly investigated to achieve optimal sensitivity, selectivity and accuracy of the established method. 14 ACS Paragon Plus Environment

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Different ratios between the PtNP-NGE and thionine (8/1, 4/1, 2/1, 1/1 and 1/2, m/m) were tested for the preparation of the thionine-PtNP-NGE in PBS (0.1 mol L–1, pH 6.0). As shown in Figure S5 (Supporting Information), the peak current significantly increased with the ratios decreasing from 8/1 to 2/1 because of the excellent conductivity of thionine. However, the increasing amounts of thionine hindered the electron transfer with redundant aggregation of thionine molecules. Therefore, the ratio of the PtNP-NGE and thionine was set as 2/1. The pH value is an important factor that affects the electrochemical behaviors. In the present study, different pH values in the range of 4.0–7.0 were investigated in PBS solutions by CV measurements. From Figure 4B, it could be discovered that the oxidation peak potential values were shifted to negative potentials with the increase of pH values. The formal potential, E0, calculated as the average of Epa and Epc showed a linear relationship with pH value and the regression equation was E0 = 0.021 – 0.038 pH (R2 = 0.991) (Figure S6, Supporting Information), demonstrating that two electrons and one proton were involved in the redox process, which is consistent with previous studies.21,30 The highest peak current was obtained with a pH of 6.0. The molar ratios of template to monomer were carefully optimized to ensure the quantity of the imprinting recognition sites in the MIP and to increase the analytical performance of the developed electrochemical sensor. Different ratios of template to functional monomer (1/2, 1/4, 1/6, 1/8, npatulin/nthionine) were evaluated by DPV 15 ACS Paragon Plus Environment


measurements in PBS solution (0.1 mol L–1, pH 6.0) containing 0.5 mmol L–1 patulin and H2PtCl6·6H2O (2 mmol L–1). The electropolymerization cycles was run 15 times with the potential ranging from –0.4 to 0.4 V and a scan rate of 50 mV s–1. After removal of the template, the prepared MIP with a ratio of 1/6 for the template/functional monomer showed the highest current response (Figure 4C). The lower ratios (1/2 and 1/4) were not enough to prepare the grafted MIP layers with sufficient active cavities, and the high ratio (1/8) might lead to self-polymerization, all decreasing the selective binding sites, resulting in the weakened signals. For the detection of patulin in real samples, the adsorption time was also carefully optimized. With increasing adsorption time from 60 to 180 s (Figure 4D), the peak currents accordingly decreased. When the adsorption time further increased from 180 to 210 s, the signals remained consistent with no significant change observed. The fast adsorption kinetics for patulin may be explained by the fact that most of the imprinted sites are in the proximity surface providing better site accessibility and lower mass transfer. Herein, considering the complete adsorption at the lowest cost and highest efficiency, 180 s was chosen as the optimum adsorption time.

Analytical performance of the MIP/thionine-PtNP-NGE-based sensor. Under the optimal conditions, the oxidation current of thionine decreased with the increased concentrations of patulin (Figure 5A), and a linear relationship between the current 16 ACS Paragon Plus Environment

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decrement (ΔI) and logarithm value of the patulin concentration (LogCpatulin) over the range of 0.002–2 ng mL–1 was found (Figure 5B). The linear regression equation was ΔI (μA) = 5.557 Log(C patulin/pg mL–1) – 1.388, with a correlation coefficient (R2) of 0.995. For comparison, NIP was also prepared, and used for fabrication of the electrode. In contrast to MIP, there was no obvious change with different concentrations of patulin when NIP was used, proving that NIP could not efficiently combine with patulin due to the lack of specific cavity. The sensitivity of the established method was evaluated by determination of the limit of detection (LOD) that is equal to the concentration of the analyte of three times the signal to noise ratio (S/N = 3). After addition of different concentrations of patulin standards into PBS, LOD was finally fixed as 0.001 ng mL–1. Compared to the previous studies, the current established sensor showed a preferable sensitivity with a relatively lower detection limit, allowing the accurate determination of ultratrace amounts of patulin.2,31 The selectivity of the electrochemical sensor was evaluated by determination of patulin and other interfering substances, including the similar chemical compound (5-HMF) and frequently co-occurring mycotoxins (AFB1, AOH, CIT and OTA) in fruit juice. Figure 6 shows the DPV responses of patulin and other interferences at the same concentration (0.05 ng mL–1) in PBS (0.1 mol L–1, pH 6.0). The experiments were performed in triplicate. The results show that the presence of patulin could cause drastic changes in the current peaks, while no significant ΔI values were observed for the others (less than 15%). 17 ACS Paragon Plus Environment


Due to the consideration of one sample containing multiple mycotoxins in real situations, the responses of patulin in standard solutions containing other several interfering substances were further investigated. The DPV responses did not significantly change with ΔI values ranging from 7.3 to 8.9 μA, suggesting that satisfactory selectivity of the established electrochemical sensor was obtained. Since the concentrations of 5-HMF were practically exceeding the level allowed for patulin, higher concentrations of 5-HMF (10 times and 40 times of patulin) were also investigated to accurately evaluate the interference of 5-HMF in real samples. Less than 20% of the interference was observed (Figure S7), suggesting that satisfactory selectivity of the established electrochemical sensor was obtained. It could be attributed to the high affinity of amine ligands to patulin molecules. Moreover, the optimal match of imprinted cavities with the shape of patulin molecules can enhance the affinity of imprinted sites, allowing the detection of patulin from a complex matrix without separations. To evaluate the stability of the modified electrode, the peak currents of 20 successive measurements by CV in PBS containing 1 ng mL–1 patulin were examined. Satisfactory stability of the electrochemical responses was obtained with a slight decrease in current (less than 2%). The storage stability of the established sensor was also determined by comparing the mean current responses using the stored immunosensors (4 C for 10 d) with those of freshly prepared ones. The sensor retained 95% of its initial responses, confirming a long lifetime for the prepared electrode. The repeatability of the MIP sensor 18 ACS Paragon Plus Environment

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was tested by determination of patulin in PBS solution (0.05 ng mL–1) in quintuplicate. The relative standard deviation (RSD) of the measurements was 4.94%, which indicated that the repeatability of the designed sensor is excellent.

Monitoring of patulin in real samples. The developed and validated electrochemical sensor was applied to the determination of patulin in real apple and grape juice samples. Two groups of tests were performed: (1) Recovery: Blank apple and grape juice samples (patulin free) were spiked with low, intermediate and high concentration levels of patulin standard (0.005, 0.05 and 0.5 ng mL–1). The spiked samples were passed through a 0.22-μm nylon filter and a 0.25 mL filtrate was collected and diluted with 4.75 mL PBS (0.1 mol L–1, pH 6.0), and used for analysis. As shown in Table 1, the recoveries are 99.8–113.0% for apple juice and 95.4–104.8% for grape juice samples, affirming a considerable application potential of our sensing platform. (2) Contaminated samples: one apple juice and one grape juice (containing a certain amount of patulin) directly collected from supermarket were determined by the developed electrochemical sensor, and the results were further verified by an in-parallel LC-MS/MS approach previously established in our lab.32 Similar results were obtained for UHPLC-MS/MS and the current method (Table S1, Supporting Information), demonstrating the excellent detection capability of the proposed sensing scheme.



CONCLUSIONS In this study, a molecularly imprinted film fabricated on a thionine-PtNP-NGE modified electrode was developed for ultratrace patulin determination for the first time. Strategically, the composite of thionine-PtNP-NGE not only acts as a reinforcement material with excellent electrical conductivity, but also worked as an internal reference signal of the sensing platform. The double amplification based on the MIP film and thionine-PtNP-NGE has been verified to favor the highly sensitive and selective detection of patulin. By virtue of the optimal conditions, the proposed electrochemical sensor showed a wide linear range, low detection limit, high selectivity and excellent repeatability, and can be applied for the rapid detection of patulin in fruit juice. This simple, sensitive and selective strategy opens a new horizon for the rapid and sensitive recognition of patulin, which also holds promise for future applications to other mycotoxins.

ASSOCIATED CONTENT Supporting Information Additional Figures S1−S6 and Table S1.

ACKNOWLEDGEMENTS


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This work was supported by the National Key R&D Plan [No. 2017YFC1700806], National Natural Science Foundation of China [No. 31671950], Shanghai City Sci-Tech Joint Research Project in Yangtze River Delta of Shanghai Municipal Science and Technology Commission [No. 18395810100] and the Science and Technology Innovation Action Plan Project of Shanghai Municipal Commission of Science and Technology [No. 17391901200].

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For Table of Contents Only


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Figure Caption Scheme 1. Schematic illustration of electrode fabrication, molecular imprinting and the recognition principle of the developed electrochemical sensor for patulin detection. Figure 1. Chemical structure of patulin. Figure

2.

TEM

images

of

NGE

(A),

thionine-PtNP-NGE

(B)

and

MIP/thionine-PtNP-NGE where the inset shows a partial enlargement (C). (D) UV-Vis spectra of NGE (a), PtNP-NGE (b), thionine (c) and thionine-PtNP-NGE (d). Figure 3. DPV measurements in PBS (0.1 mol L–1, pH 6.0) by using GCE (a), thionie-PtNP-NGE/GCE (b), MIP/thionine-PtNP-NGE/GCE before template removal (c) and MIP/thionine-PtNP-NGE/GCE after template removal (d). DPV measurements in PBS solution (0.1 mol L–1, pH 6.0) containing 1 ng mL–1 patulin by using MIP/thionine-PtNP-NGE/GCE (e). Figure 4. (A) CV measurements in PBS (0.1 mol L–1, pH 6.0) with a scan rate of 10–270 mV s–1 and a scan range of –0.5–0.2 V. Insert shows the linearity anodic peak current (Ipa) and cathodic peak current (Ipc) with the square root of the scan rate (v1/2) from 10 to 270 mV s–1. (B) CV measurements in PBS solutions (0.1 mol L–1) with the pH values in the range of 4.0–7.0. (C) DPV measurements in PBS (0.1 mol L–1, pH 6.0) with different ratios of template to monomer (npatulin/nthionine) 1/2 (a), 1/4 (b), 1/6 (c) and 1/8 (d). (D) DPV measurements in PBS (0.1 mol L–1, pH 6.0) solution containing 0.5 ng mL–1 patulin with the adsorption time in the range of 0–210 s. 25 ACS Paragon Plus Environment


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Figure 5. (A) DPV measurements in PBS solution (0.1 mol L–1, pH 6.0) containing different concentrations (0, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1 and 2 ng mL–1) of patulin. (B) Linear relationships of the current difference (ΔI) and logarithm concentration of patulin for the MIP and NIP. Figure 6. Selectivity of established electrochemical platform toward different compounds. The concentration for each mycotoxin is 0.05 ng mL−1.

Table 1. Recoveries of patulin in apple and grape juice samples determined by the established electrochemical sensor (n = 3). Sample

Apple juice

Grape juice

Spiked

Found

Recovery

(ng mL–1)

( ±SD, ng mL–1)

( ±SD, %)

0.005

0.00499±0.00041

99.8±8.2

0.05

0.0547±0.0059

109.4±11.8

0.5

0.565±0.039

113.0±7.8

0.005

0.00507±0.00093

101.4±18.6

0.05

0.0477±0.0036

95.4±7.2

0.5

0.524±0.054

104.8±10.8


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Scheme 1. Schematic illustration of electrode fabrication, molecular imprinting and the recognition principle of the developed electrochemical sensor for patulin detection.

ACS Paragon Plus Environment


Figure 1. Chemical structure of patulin. 39x28mm (300 x 300 DPI)


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Figure 2. TEM images of NGE (A), thionine-PtNP-NGE (B) and MIP/thionine-PtNP-NGE where the inset shows a partial enlargement (C). (D) UV-Vis spectra of NGE (a), PtNP-NGE (b), thionine (c) and thionine-PtNP-NGE (d).



Figure 3. DPV measurements in PBS (0.1 mol L–1, pH 6.0) by using GCE (a), thionie-PtNP-NGE/GCE (b), MIP/thionine-PtNP-NGE/GCE before template removal (c) and MIP/thionine-PtNP-NGE/GCE after template removal (d). DPV measurements in PBS solution (0.1 mol L–1, pH 6.0) containing 1 ng mL–1 patulin by using MIP/thionine-PtNP-NGE/GCE (e). 214x172mm (300 x 300 DPI)


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Figure 4. (A) CV measurements in PBS (0.1 mol L–1, pH 6.0) with a scan rate of 10–270 mV s–1 and a scan range of –0.5–0.2 V. Insert shows the linearity anodic peak current (Ipa) and cathodic peak current (Ipc) with the square root of the scan rate (v1/2) from 10 to 270 mV s–1. (B) CV measurements in PBS solutions (0.1 mol L–1) with the pH values in the range of 4.0–7.0. (C) DPV measurements in PBS (0.1 mol L–1, pH 6.0) with different ratios of template to monomer (npatulin/nthionine) 1/2 (a), 1/4 (b), 1/6 (c) and 1/8 (d). (D) DPV measurements in PBS (0.1 mol L–1, pH 6.0) solution containing 0.5 ng mL–1 patulin with the adsorption time in the range of 0–210 s.



Figure 5. (A) DPV measurements in PBS solution (0.1 mol L–1, pH 6.0) containing different concentrations (0, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1 and 2 ng mL–1) of patulin. (B) Linear relationships of the current difference (ΔI) and logarithm concentration of patulin for the MIP and NIP.


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Figure 6. Selectivity of established electrochemical platform toward different compounds. The concentration for each mycotoxin is 0.05 ng mL−1.


Molecularly Imprinted Poly(thionine)-Based Electrochemical Sensing

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