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A novel nanoenzyme based on Fe3O4 nanoparticles@thionine-imprinted polydopamine for electrochemical biosensing
Sensors and Actuators B 253 (2017) 108–114
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A novel nanoenzyme based on Fe3 O4 nanoparticles@thionine-imprinted polydopamine for electrochemical biosensing Li Wang, Longfei Miao, Han Yang, Jie Yu, Yingzhen Xie, Lijuan Xu, Yonghai Song ∗ Key Laboratory of Functional Small Organic Molecule, Ministry of Education, Key Laboratory of Chemical Biology, Jiangxi Province, College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang 330022, China
a r t i c l e
i n f o
Article history: Received 8 February 2017 Received in revised form 28 May 2017 Accepted 19 June 2017 Available online 21 June 2017 Keywords: Nanoenzyme Fe3 O4 NPs MMIPs H2 O2 Thionine Dopamine Electrochemical biosensors
a b s t r a c t Here, a new nanoenzyme of Fe3 O4 nanoparticles (NPs) magnetic molecularly imprinted polymers (MMIPs) was prepared by polymerizing dopamine on the Fe3 O4 NPs surface in the presence of templated thionine (Thi) for the first time. The results showed that uniform spherical and core-shell structured Fe3 O4 NPs MMIPs which were about 600 nm in diameter were successfully formed and the imprinting sites improved the selectivity of Fe3 O4 NPs MMIPs greatly. The as-prepared Fe3 O4 NPs MMIPs could catalyze the reduction of Thi selectively, which could be enhanced by H2 O2 owing to the peroxidaselike activity of Fe3 O4 NPs. Accordingly, a highly selective and sensitive H2 O2 electrochemical biosensor was proposed based on the Fe3 O4 NPs MMIPs-modified glassy carbon electrode. The electrochemical biosensor based on the Fe3 O4 NPs MMIPs nanoenzyme exhibited low detection limit of 1.58 nM and high selectivity. Since acetylthiocholine chloride (AChl) could be hydrolyzed into choline with the help of acetylcholinesterase (AChE) and simultaneously the choline oxidase (ChOx) could reduce choline into betaine accompanied by the production of H2 O2 , the proposed electrochemical H2 O2 biosensor could be further used to detect AChl, AChE and ChOx. The results also exhibited wide linear range (2.85–160 M for AChl, 0.53–20000 ng mL−1 for AChE and 22.76–400 ng mL−1 for ChOx), low detection limit (0.86 M for AChl, 0.16 ng mL−1 for AChE and 6.83 ng mL−1 for ChOx) and high selectivity. Therefore, the Fe3 O4 NPs MMIPs should be a promising nanoenzyme for electrochemical biosensors. © 2017 Elsevier B.V. All rights reserved.
1. Introduction The biosensors could transform the biological response into the detectable signal. Among various biosensors, the enzyme-based electrochemical biosensors have attracted extensive interests due to their convenient operation, fast signal conversion, low detection limit, and excellent specificity [1–5]. Enzyme is the protein with special catalytic function. Enzymes could be obtained from organisms by complex procedures with high cost [6–10]. Furthermore, natural enzymes are always easy to lose their bioactivity at high temperature, in a low or high pH solution, even when contact with electrode surface [11–16]. Accordingly, it is necessary to develop nanoenzymes instead of natural enzymes for constructing enzyme-based electrochemical biosensors [17–19].
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (Y. Song). http://dx.doi.org/10.1016/j.snb.2017.06.132 0925-4005/© 2017 Elsevier B.V. All rights reserved.
Nanostructures with enzymatic activities, those are called nanoenzymes, have attracted increasing attention for natural enzyme mimics and found wide applications in bioanalysis, bioimaging, and biomedicine [20,21]. Among the known nanoenzymes, magnetic Fe3 O4 nanoparticles (NPs) have received much attention due to their application in biological imaging and separation techniques [22–26]. In 2007, it was reported that Fe3 O4 NPs exhibited intrinsic peroxidase-like activity, just like natural horseradish peroxidase (HRP) [27]. It was well known that peroxidases have the ability to oxidize the organic substrate for reducing their toxicity which was employed for wastewater treatment. For instance, Fe3 O4 NPs could oxide 3,3 ,5,5 -tetramethylbenzidine (TMB) into a blue product instead of HRP in the presence of H2 O2 [28]. In 2014, it was also discovered that the Fe3 O4 NPs could be used as mimetic HRP to electrochemically catalyze the reduction of thionine (Thi) without H2 O2 [29]. Although Fe3 O4 NPs is a good kind of HRP mimic enzyme, its specificity is still far lower than that of natural enzymes. This weakness has hampered the application of Fe3 O4 NPs nanoenzymes in biosensors. Therefore, to develop
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novel nanoenzymes with high specificity is still challenging and fascinating. Molecular imprinting is a facile and well-established method to create recognition cavities in accordance with template-molecule’s shape, size and functional group [30–32]. Currently, molecular imprinting polymers (MIPs) showed wide application for solidphase extraction, catalysis, drug-controlled release, and chemical sensors due to possessing ideal selectivity, thermal stability, easy preparation, etc. [33–36]. Dopamine (DA) could be easily polymerized on a solid surface owing to its catechol and amine groups [37]. In 2012, DA was chosen as the monomer to construct bovine hemoglobin imprinted biosensors [38]. In 2013, Yao designed a magnetic MIPs (MMIPs) by DA self-polymerization on the surface of NPs in the presence of template chlorpyrifos [39]. Inspired by these breakthroughs, we designed a novel nanoenzyme of Thi-imprinted Fe3 O4 NPs MMIPs by polymerizing DA on Fe3 O4 NPs in the presence of template Thi for the first time. The Thi is an important electroactive small molecule which shows a pair of well developed redox peaks and has been extensively used as probe in electrochemical biosensors. The imprinting sites improved the selectivity of Fe3 O4 NPs MMIPs toward Thi greatly. We found the Fe3 O4 NPs MMIPs could catalyze the reduction of Thi selectively with the help of H2 O2 . Accordingly, a highly selective and sensitive electrochemical H2 O2 biosensor was proposed based on the Fe3 O4 NPs MMIPs by using Thi as probe. Since acetylthiocholine chloride (AChl) could be hydrolyzed into choline with the help of acetylcholinesterase (AChE) and simultaneously the choline oxidase (ChOx) could reduce choline into betaine accompanied by the production of H2 O2 [40,41], the proposed electrochemical H2 O2 biosensor could be further used to detect AChl, AChE and ChOx, respectively.
2. Experimental 2.1. Chemicals and solutions Tris (hydroxymethyl) aminomethane (Tris), FeCl3 ·6H2 O, TMB, DA, Thi, AChE (type C3389, 500 U mg−1 ), AChl, and ChOx were obtained from Sigma–Aldrich. NaAc, ethylene glycol, NaH2 PO4 , Na2 HPO4 , H2 O2 (AR, 30 wt.% in H2 O) and other reagents were purchased from Aladdin Reagent Co., Ltd (Shanghai, China). Phosphate buffer solution (PBS, 0.2 M) was prepared by mixing Na2 HPO4 and NaH2 PO4 . Tris-HCl buffer (pH 8.5) was prepared as followed [42]. Briefly, 12.14 g Tris were added into 1000 mL water, and then adjusted the pH to 8.5 with 6 M HCl. The ChOx, AChl and AChE solution were daily prepared by dissolving proper amount of commercial substance in 0.2 M PBS (pH 7.0) and then stored in 4 ◦ C. Ultrapure water (18.2 M cm−1 ) was used.
2.2. Preparation of Fe3 O4 NPs MMIPs The Fe3 O4 NPs were prepared by dissolving 0.30 g FeCl3 and 0.80 g NaAc in 10 mL of ethylene glycol and then heating at 200 ◦ C for 10 h [36]. Then 25 mg Fe3 O4 NPs powder were added into 10 mL of 10 mM Tris-HCl buffer (pH = 8.5) followed by the addition of 2.5 mL of 2.0 mg mL−1 Thi. Then the above suspension was mechanically stirred for 2 h. Next, 25 mg DA was dissolved in the O2 -saturated solution and incubated for 4 h. The resulted Fe3 O4 NPs MMIPs were collected by external magnetic separation. The template molecules were extracted by acetic acid/acetonitrile solution. For comparison, magnetic non-imprinted polymers (MNIPs) were also fabricated in the absence of Thi during the self-polymerization process. The preparation process was illustrated in Scheme 1.
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Scheme 1. Schematic illustration of the fabrication process of Fe3 O4 NPs MMIPs.
2.3. Preparation of Fe3 O4 NPs MMIPs-modified glassy carbon electrode (GCE) 25 mg Fe3 O4 NPs MMIP powder was firstly dispersed in 5 mL of 0.05% nafion solution. Then 7 L of 5.0 mg mL−1 Fe3 O4 NPs MMIPs aqueous suspension was cast on the newly polished GCE with a diameter of 3 mm from Shanghai Chenhua Instrument Co., Ltd. and then dried under N2 atmosphere. The nafion improved the distribution and stability of Fe3 O4 NPs MMIP on GCE surface greatly. To further enhance the stability and repeatability of modified electrode, the Fe3 O4 NPs MMIPs-modified GCE was immersed in 0.2 M PBS (pH 7.0) for 4 h and then washed by ultrapure water. In the immersing process, some loosely bound Fe3 O4 NPs MMIPs could drop off from the Fe3 O4 NPs MMIPs-modified GCE, which enhanced the stability and repeatability of the Fe3 O4 NPs MMIPs-modified GCE. The MNIPs-modified GCE was also prepared by this method. 2.4. Instruments Scanning electron microscopy (SEM) and energy dispersive Xray spectroscopy (EDXS) tests were implemented on a HITACHI S-3400N scanning electron microscope with a Phoenix energy Xray analyzer. X-ray powder diffraction (XRD) data were collected on a D/Max 2500V/PC X–ray powder diffractometer using Cu K␣ radiation ( = 1.54056 Å, 40 kV, 200 mA). UV–vis absorption spectra were collected on a Hitachi U-3900H UV–vis Spectrophotometer. All electrochemical measurements were performed on a CHI 660C electrochemical workstation (Shanghai, China) by conventional three-electrode system including the modified GCE as the working electrode, a platinum wire as the auxiliary electrode and a saturated calomel electrode (SCE, saturated KCl) as the reference electrode. 3. Results and discussion 3.1. Characterization of Fe3 O4 MMIPs The shape and core-shell nanostructure of Fe3 O4 NPs MMIPs were characterized by SEM and the results are shown in Fig. 1. The low resolution SEM image (Fig. 1A) showed a large number of uniform spherical Fe3 O4 NPs. The high resolution SEM image (Fig. 1C) indicated that the surface of Fe3 O4 NPs was very rough, which provided lots of active sites to improve its catalytic activities. The diameter of the spherical Fe3 O4 NPs was about 600 nm which was slightly bigger as compared with other nanomaterials, but similar to some previous results [36]. Although small Fe3 O4 NPs were benefit for their electrochemical performances, it was very difficult to reduce their size owing to the strong magnetism. Here, we tried our best to synthesize uniform and relatively small Fe3 O4 NPs. The good uniformity was also very beneficial to the good reproducibility of electrochemical biosensors based on Fe3 O4 NPs. The low resolution SEM image of the Fe3 O4 NPs MMIPs (Fig. 1B) also showed many uniform spherical particles but their surface seemed more smooth as compared with Fe3 O4 NPs (Fig. 1D). It might be because the DA polymerized on the surface of Fe3 O4 NPs and finally formed ploydopamine (PDA) to cover the Fe3 O4 NPs. The diameter of the Fe3 O4 NPs MMIPs was also about 600 nm, which indicated
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Fig. 1. SEM images of Fe3 O4 NPs (A,C) and Fe3 O4 NPs MMIPs (B,D).
only a thin layer of PDA was covered on the surface of Fe3 O4 NPs. The thickness of PDA layer was a crucial factor for the electrochemical performances of Fe3 O4 NPs MMIPs nanoenzyme. If it was too thick, the electrochemical performances would be hampered. If it was too thin, the selectivity of nanoenzyme might be poor. Thus, only a suitable thickness of PDA was optimized and presented in the experiments. Compared with the SEM images of Fe3 O4 NPs and Fe3 O4 NPs MMIPs, DA was confirmed to be successfully polymerized on the surface of Fe3 O4 NPs. The amount of Thi used to prepare Fe3 O4 NPs MMIPs nanoenzyme was also an important factor for the electrochemical performances of Fe3 O4 NPs MMIPs nanoenzyme. If the amount of Thi was too high, the elution of Thi would be very difficult. If the amount too low, the efficiency of molecular imprinting was poor. It was found that 0.4 mg mL−1 Thi was optimal for preparing Fe3 O4 NPs MMIPs nanoenzyme. The as-prepared Fe3 O4 NPs were ideal matrix materials for the preparation of MMIPs. During the polymerization of DA, the Thi molecules could be entrapped into the PDA layers based on the hydrogen bonds between PDA and Thi. What’s more, the template molecules have heteroatom ring and benzene ring which could combine with the benzene ring in the chain of PDA and then resulted in stronger Van der Waals interaction between templates and polymers. Thi imprinted sites could be easily generated after the Thi molecules were removed out by destroying the Van der Waals interaction and hydrogen bonds with the acetic acid/acetonitrile solution. In order to ensure that Thi molecules were thoroughly removed out, EDX of Fe3 O4 NPs MMIPs-Thi, Fe3 O4 NPs MMIPs and MNIPs were shown in Fig. 2A. Several typical Fe, O, C and S elements appeared in EDX of Fe3 O4 NPs MMIPs-Thi (curve a). The Fe and O should come from the Fe3 O4 NPs, C and O might originate from PDA, and S resulted from Thi. The peak of O was higher than that of Fe because O not only came from Fe3 O4 NPs but also from PDA. The appearance of S indicated that Thi was successfully packaged by PDA. The disappearance of the
S peak disappeared in Fe3 O4 NPs MMIPs (curve b) was due to the removing of Thi template. It was worth noting that the Fe3 O4 NPs MMIPs contained the same elements as the MNIPs (curve c). It further demonstrated that the Fe3 O4 NPs MMIPs were successfully prepared. Fig. 2B shows the XRD patterns of Fe3 O4 NPs (curve a) and the as-prepared Fe3 O4 NPs MMIPs (curve b). Five peaks at 30.2◦ , 35.6◦ , 43.4◦ , 56.9◦ , and 62.7◦ were observed for the two samples from 5◦ to 90◦ . They were assigned to the (220), (311), (400), (511), and (440) crystal facets of Fe3 O4 (JCPDS Card 19-629). The results further confirmed that Fe3 O4 NPs were successfully synthesized. Moreover, the peak positions of Fe3 O4 NPs were not changed after coating with the polymer layers, indicating that the Fe3 O4 NPs MMIPs still maintained the crystalline structure of Fe3 O4 NPs. 3.2. Electrochemical behaviors of Fe3 O4 NPs MMIPs nanoenzyme The electrocatalytic activity of the Fe3 O4 NPs MMIPs nanoenzyme toward 6 M Thi was explored by CVs. As shown in Fig. 3A, a pair of asymmetric redox peaks was observed. Based on the peaks’ potentials, the asymmetric redox peaks could be attributed to the Thi [29]. Obviously, both anodic peak current and cathodic peak current increased as the scan rate varied from 100 to 900 mV s−1 . The plots of both anodic peak current and cathodic peak current versus the square root of scan rates presented linear relationship, suggesting the Thi redox occurred through a diffusion-controlled electrochemical process (Fig. 3B). The CVs results indicated that the thickness of PDA layer was appropriate and Thi molecules could go through the MMIPs to arrive at the surface of Fe3 O4 NPs. The cathodic peak was obviously larger than the anodic peak, indicating a typical catalytic reaction. The possible mechanism of Fe3 O4 NPs MMIPs nanoenzyme towards Thi was presented: Fe3+ + Thi → Fe2+ + Thi+
(1)
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Fig. 2. (A) EDS analysis of Fe3 O4 NPs MMIPs-Thi (curve a), Fe3 O4 NPs MMIPs (curve b) and Fe3 O4 NPs MNIPs (curve c). (B) XRD patterns of Fe3 O4 NPs (curve a) and Fe3 O4 NPs MMIPs (curve b).
Fig. 3. (A) CVs of Fe3 O4 NPs MMIPs-modified GCE in 0.2 M N2 -saturated PBS (pH = 7.0) in the presence of 6 M Thi at different scan rates: (a) 200 mV s− 1 , (b) 300 mV s− 1 , (c) 400 mV s− 1 , (d) 500 mV s− 1 , (e) 600 mV s− 1 , (f) 700 mV s− 1 , (g) 800 mV s− 1 and (h) 900 mV s− 1 . (B) Plot of peak current versus the square root of scan rates.
Thi+ + e → Thi
(2)
Since the as-prepared Fe3 O4 NPs MMIPs nanoenzyme electrochemically responded toward Thi, it could be used to construct electrochemical biosensors in the presence of Thi. Accordingly, the concentration of Thi was optimized for subsequent electrochemical experiments. As shown in Fig. S1 (Supporting information), the optimal concentration of Thi was 20 M. The CVs result also clearly showed that H2 O2 took part in the electrochemical reaction and the Fe3 O4 NPs MMIPs-modified GCE could be used to detect H2 O2 . 3.3. Binding ability, selectivity, stability and reproducibility To explore the rebinding ability of Thi, we compared the molecular imprinting effectiveness of Fe3 O4 NPs MMIPs and Fe3 O4 NPs MNIPs in 0.2 M PBS solution (pH 7.0) containing 20 M Thi and 10 M H2 O2 (Fig. 4A). No obvious peak of Thi appeared on the Fe3 O4 NPs MNIPs-modified GCE electrode. While, the Fe3 O4 NPs MMIPs-modified GCE possessed obvious redox peaks. It clearly indicated that Fe3 O4 NPs MMIPs had a much better rebinding capacity than Fe3 O4 NPs MNIPs under the same conditions. Since Fe3 O4 NPs had peroxidase-like bioactivity, in the presence of H2 O2 and Fe3 O4 NPs, the colorless TMB could be oxidized into blue oxTMB which showed a typical UV–vis absorption peak at 650 nm [42]. Thus, TMB was used as the target molecule to test the selectivity of Fe3 O4 NPs MMIPs. Fig. 4B showed the UV–vis absorption spectra of 100 M TMB solutions containing 500 M H2 O2 after Fe3 O4 NPs
and Fe3 O4 NPs MMIPs were added. It was obvious that the TMB solution in the presence of Fe3 O4 NPs and 500 M H2 O2 (curve a) had a strong absorption while the TMB solution in the presence of Fe3 O4 NPs MMIPs and 500 M H2 O2 (curve b) did not show any absorption at 650 nm. The results indicated that the nanoenzyme based on Fe3 O4 NPs MMIPs showed good selectivity toward Thi. The selectivity of the nanoenzyme based on Fe3 O4 NPs MMIPs toward electrochemical detection of Thi was also evaluated by adding some interferents including glucose, TMB, methylene blue (MB), Na+ , ascorbic acid (AA), dopamine (DA) into 0.2 M PBS in the absence of Thi. As shown in Fig. S2 (Supporting information), the same concentration of the possible interfering substances did not show obvious interference, also indicating good selectivity. The selectivity of Fe3 O4 NPs MMIPs nanoenzyme might result from the imprinted sites of Fe3 O4 NPs MMIPs which retained accurate size of the template, shape memory, and orientation of chemical functionality of the template Thi. The selectivity was necessary for the electrochemical biosensors based on the Fe3 O4 NPs MMIPs. The stability of Fe3 O4 NPs MMIPs-modified GCE was investigated by storing it at 4 ◦ C for different time. After 2 days, no obvious change was found. After 30 days, 97.2% of the original value of peak current was remained. The results demonstrated a good stability which was obviously superior to natural enzyme. Natural enzymes always easily lose their bioactivity at high temperature, in a low or high pH solution, even when contact with electrode surface, which would result in a poor stability. Seven separately prepared Fe3 O4 NPs MMIPs-modified GCE were used to test the
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Fig. 4. (A) CVs of Fe3 O4 NPs MMIPs-modified GCE and Fe3 O4 NPs MNIPs-modified GCE in 0.2 M N2 -saturated PBS (pH = 7.0) containing 20 M Thi and 10 M H2 O2 at scan rate of 0.05 V s−1 . (B) UV–vis absorption spectra of Fe3 O4 NPs (curve a) and Fe3 O4 NPs MMIPs (curve b) in the presence of 500 M H2 O2 and 100 M TMB.
Fig. 5. CVs of Fe3 O4 NPs MMIPs-modified GCE in 0.2 M N2 -saturated PBS (pH = 7.0) with different concentration of H2 O2 (A), AChl (B), AChE (C) and ChOx (D). Insets were the corresponding working curves.
reproducibility under the same conditions. The results showed a good reproducibility with a relative standard deviation (RSD) of 2.45%. 3.4. Electrochemical detection of H2 O2 , AChl, AChE and ChOx by using the Fe3 O4 NPs MMIPs-modified GCE The possible catalytic mechanism of the Fe3 O4 NPs MMIPs nanoenzyme towards H2 O2 in the presence of Thi was explored. As shown in Fig. 3, the reduction of Thi could be catalyzed by the Fe3 O4 NPs MMIPs, and we subsequently found that the Fe3 O4 NPs MMIPs could exhibit better electrocatalytic effect on the reduction
of Thi in the presence of H2 O2 as shown in Fig. S1A (Supporting information). Accordingly, the Fe3 O4 NPs MMIPs-modified GCE could be also used to electrochemically detect H2 O2 in the presence of Thi. As shown in Fig. 5A, the reduction peak of Thi increased gradually as the H2 O2 concentration increased, showing a typical electrocatalytic process. The electrochemical detection of H2 O2 by using the Fe3 O4 NPs MMIPs-modified GCE exhibited good linear relationship in the range of 4.08 nM–9.0 M with a low detection limit of 1.58 nM (R/N = 3) (Inset in Fig. 5A). The result is similar to that obtained by the amperometric technique (Fig. S3, Supporting information). The result was superior to some previous H2 O2 biosensors [43–47] (Table S1, Supporting information). The result
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also proved that the presence of H2 O2 could improve the electrocatalytic ability of Fe3 O4 NPs MMIPs toward the reduction of Thi. The mechanism was presumed: Fe2+ + H2 O2 → Fe3+ + H2 O
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2017.06.132.
(3)
Here, the H2 O2 firstly oxidized the Fe2+ of Fe3 O4 NPs MMIPs into Fe3+ , and then the oxidized Fe3 O4 NPs MMIPs (Fe3+ ) could oxidize the Thi into Thi+ which was reduced by electrochemical reaction. Accordingly, with the increase of H2 O2 , the cathodic peak current of Thi was increased gradually. It was well known that AChl could hydrolyze into choline with the help of AChE and simultaneously the ChOx could reduce choline into betaine accompanied by the production of H2 O2 . Thus the proposed electrochemical H2 O2 biosensor could be further used to detect AChl, AChE, and ChOx. As shown in Fig. 5B, the eletrocatalytic detection of AChl by using the Fe3 O4 NPs MMIPs-modified GCE exhibited well linear relationship in the range of 2.80–160 M with a low detection of 0.86 M (R/N = 3). The linear range was wider than other electrochemical AChl biosensors [48–51] (Table S2, Supporting information), such as ChOx/Silicate/MWCNTs/Pt (5.0–100 M) [48] and AChE/ChO/carbon paste electrode (0.07–10 M) [50]. The linear relationship of AChE and ChOx was 0.53 ng mL−1 − 20 g mL−1 (Fig. 5C) and 22.76 ng mL−1 –0.40 g mL−1 (Fig. 5D), respectively. The detection limit of AChE and ChOx was 0.16 ng mL−1 and 6.83 ng mL−1 , respectively. The selectivity of Fe3 O4 NPs MMIPs-modified GCE toward electrochemical detection of H2 O2 , AChl, AChE and ChOx were also evaluated by adding some possible interfering substances into 0.2 M PBS. As shown in Fig. S4 (Supporting information), the same concentration of the possible interfering substances to electrochemical detection of H2 O2 , AChl, AChE and ChOx were tested, and no obvious interference was observed, indicating good selectivity of the Fe3 O4 NPs MMIPs nanoenzyme.
4. Conclusion In conclusion, a novel Fe3 O4 NPs MMIPs nanoenzyme was successfully constructed by a magnetic surface imprinting technique in which the MMIPs were prepared by a facile approach based on self-polymerization of DA in the presence of template Thi, and then the template Thi was removed out by an extraction procedure with organic solution. The obtained Fe3 O4 NPs MMIPs showed good selectivity toward Thi as well as the excellent catalytic activity, which led to an excellent sensitivity, and good selectivity of the electrochemical nonenzymatic H2 O2 biosensor based on Fe3 O4 NPs MMIPs nanoenzyme. At the same time, the proposed electrochemical biosensor was further used to detect AChl, AChE and ChOx, respectively. The results exhibited wide detection range and low detection limit. Based on this work, the Fe3 O4 NPs MMIPs might provide a new way to construct nanoenzymes. Overall, the proposed preparation method of the Fe3 O4 NPs MMIPs was simple, efficient, cheap and easy to mass production. It might also open up a new way for diverse targets monitoring.
Acknowledgments This work was financially supported by National Natural Science Foundation of China (21465014, 21465015 and 21665012), Natural Science Foundation of Jiangxi Province (20143ACB21016) and the Ground Plan of Science and Technology Projects of Jiangxi Educational Committee (KJLD14023).
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Biographies Li Wang received her Ph.D. in analytical chemistry from the Changchun Institute of Applied Chemistry, Chinese Academy of Science, China. She is currently working as a professor at Jiangxi Normal University. Her current research interest is focused on biosensors. Longfei Miao received his science bachelor in chemistry in 2016 from Jiangxi Normal University, China. He is working for his master’s degree in Jiangxi Normal University, China. His research interests are electrochemical sensor. Han Yang received her science bachelor in chemistry in 2015 from Jiangxi Normal University, China. She is working for her master’s degree in Jiangxi Normal University, China. Her research interests are electrochemical sensor. Jie Yu received her science bachelor in chemistry in 2014 from Jiangxi Science and technology Normal University, China. She is working for her master’s degree in Jiangxi Normal University, China. Her research interests are nano-materials and their applications in sensor. Yingzhen Xie received her science bachelor in chemistry in 2013 from Jingganshan University, China. She is working for her master’s degree in Jiangxi Normal University, China. Her research interests are bioelectrochemical sensor. Lijuan Xu received her science bachelor in chemistry in 2016 from Jiangxi Normal University, China. She is working for her master’s degree in Jiangxi Normal University, China. Her research interests are electrochemical sensor. Yonghai Song received his Ph.D. in analytical chemistry from the Changchun Institute of Applied Chemistry, Chinese Academy of Science, China. He is currently working as a professor at Jiangxi Normal University. His current research interests focus on biosensors.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A novel nanoenzyme based on Fe3 O4 nanoparticles@thionine-imprinted polydopamine for electrochemical biosensing Li Wang, Longfei Miao, Han Yang, Jie Yu, Yingzhen Xie, Lijuan Xu, Yonghai Song ∗ Key Laboratory of Functional Small Organic Molecule, Ministry of Education, Key Laboratory of Chemical Biology, Jiangxi Province, College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang 330022, China
a r t i c l e
i n f o
Article history: Received 8 February 2017 Received in revised form 28 May 2017 Accepted 19 June 2017 Available online 21 June 2017 Keywords: Nanoenzyme Fe3 O4 NPs MMIPs H2 O2 Thionine Dopamine Electrochemical biosensors
a b s t r a c t Here, a new nanoenzyme of Fe3 O4 nanoparticles (NPs) magnetic molecularly imprinted polymers (MMIPs) was prepared by polymerizing dopamine on the Fe3 O4 NPs surface in the presence of templated thionine (Thi) for the first time. The results showed that uniform spherical and core-shell structured Fe3 O4 NPs MMIPs which were about 600 nm in diameter were successfully formed and the imprinting sites improved the selectivity of Fe3 O4 NPs MMIPs greatly. The as-prepared Fe3 O4 NPs MMIPs could catalyze the reduction of Thi selectively, which could be enhanced by H2 O2 owing to the peroxidaselike activity of Fe3 O4 NPs. Accordingly, a highly selective and sensitive H2 O2 electrochemical biosensor was proposed based on the Fe3 O4 NPs MMIPs-modified glassy carbon electrode. The electrochemical biosensor based on the Fe3 O4 NPs MMIPs nanoenzyme exhibited low detection limit of 1.58 nM and high selectivity. Since acetylthiocholine chloride (AChl) could be hydrolyzed into choline with the help of acetylcholinesterase (AChE) and simultaneously the choline oxidase (ChOx) could reduce choline into betaine accompanied by the production of H2 O2 , the proposed electrochemical H2 O2 biosensor could be further used to detect AChl, AChE and ChOx. The results also exhibited wide linear range (2.85–160 M for AChl, 0.53–20000 ng mL−1 for AChE and 22.76–400 ng mL−1 for ChOx), low detection limit (0.86 M for AChl, 0.16 ng mL−1 for AChE and 6.83 ng mL−1 for ChOx) and high selectivity. Therefore, the Fe3 O4 NPs MMIPs should be a promising nanoenzyme for electrochemical biosensors. © 2017 Elsevier B.V. All rights reserved.
1. Introduction The biosensors could transform the biological response into the detectable signal. Among various biosensors, the enzyme-based electrochemical biosensors have attracted extensive interests due to their convenient operation, fast signal conversion, low detection limit, and excellent specificity [1–5]. Enzyme is the protein with special catalytic function. Enzymes could be obtained from organisms by complex procedures with high cost [6–10]. Furthermore, natural enzymes are always easy to lose their bioactivity at high temperature, in a low or high pH solution, even when contact with electrode surface [11–16]. Accordingly, it is necessary to develop nanoenzymes instead of natural enzymes for constructing enzyme-based electrochemical biosensors [17–19].
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (Y. Song). http://dx.doi.org/10.1016/j.snb.2017.06.132 0925-4005/© 2017 Elsevier B.V. All rights reserved.
Nanostructures with enzymatic activities, those are called nanoenzymes, have attracted increasing attention for natural enzyme mimics and found wide applications in bioanalysis, bioimaging, and biomedicine [20,21]. Among the known nanoenzymes, magnetic Fe3 O4 nanoparticles (NPs) have received much attention due to their application in biological imaging and separation techniques [22–26]. In 2007, it was reported that Fe3 O4 NPs exhibited intrinsic peroxidase-like activity, just like natural horseradish peroxidase (HRP) [27]. It was well known that peroxidases have the ability to oxidize the organic substrate for reducing their toxicity which was employed for wastewater treatment. For instance, Fe3 O4 NPs could oxide 3,3 ,5,5 -tetramethylbenzidine (TMB) into a blue product instead of HRP in the presence of H2 O2 [28]. In 2014, it was also discovered that the Fe3 O4 NPs could be used as mimetic HRP to electrochemically catalyze the reduction of thionine (Thi) without H2 O2 [29]. Although Fe3 O4 NPs is a good kind of HRP mimic enzyme, its specificity is still far lower than that of natural enzymes. This weakness has hampered the application of Fe3 O4 NPs nanoenzymes in biosensors. Therefore, to develop
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novel nanoenzymes with high specificity is still challenging and fascinating. Molecular imprinting is a facile and well-established method to create recognition cavities in accordance with template-molecule’s shape, size and functional group [30–32]. Currently, molecular imprinting polymers (MIPs) showed wide application for solidphase extraction, catalysis, drug-controlled release, and chemical sensors due to possessing ideal selectivity, thermal stability, easy preparation, etc. [33–36]. Dopamine (DA) could be easily polymerized on a solid surface owing to its catechol and amine groups [37]. In 2012, DA was chosen as the monomer to construct bovine hemoglobin imprinted biosensors [38]. In 2013, Yao designed a magnetic MIPs (MMIPs) by DA self-polymerization on the surface of NPs in the presence of template chlorpyrifos [39]. Inspired by these breakthroughs, we designed a novel nanoenzyme of Thi-imprinted Fe3 O4 NPs MMIPs by polymerizing DA on Fe3 O4 NPs in the presence of template Thi for the first time. The Thi is an important electroactive small molecule which shows a pair of well developed redox peaks and has been extensively used as probe in electrochemical biosensors. The imprinting sites improved the selectivity of Fe3 O4 NPs MMIPs toward Thi greatly. We found the Fe3 O4 NPs MMIPs could catalyze the reduction of Thi selectively with the help of H2 O2 . Accordingly, a highly selective and sensitive electrochemical H2 O2 biosensor was proposed based on the Fe3 O4 NPs MMIPs by using Thi as probe. Since acetylthiocholine chloride (AChl) could be hydrolyzed into choline with the help of acetylcholinesterase (AChE) and simultaneously the choline oxidase (ChOx) could reduce choline into betaine accompanied by the production of H2 O2 [40,41], the proposed electrochemical H2 O2 biosensor could be further used to detect AChl, AChE and ChOx, respectively.
2. Experimental 2.1. Chemicals and solutions Tris (hydroxymethyl) aminomethane (Tris), FeCl3 ·6H2 O, TMB, DA, Thi, AChE (type C3389, 500 U mg−1 ), AChl, and ChOx were obtained from Sigma–Aldrich. NaAc, ethylene glycol, NaH2 PO4 , Na2 HPO4 , H2 O2 (AR, 30 wt.% in H2 O) and other reagents were purchased from Aladdin Reagent Co., Ltd (Shanghai, China). Phosphate buffer solution (PBS, 0.2 M) was prepared by mixing Na2 HPO4 and NaH2 PO4 . Tris-HCl buffer (pH 8.5) was prepared as followed [42]. Briefly, 12.14 g Tris were added into 1000 mL water, and then adjusted the pH to 8.5 with 6 M HCl. The ChOx, AChl and AChE solution were daily prepared by dissolving proper amount of commercial substance in 0.2 M PBS (pH 7.0) and then stored in 4 ◦ C. Ultrapure water (18.2 M cm−1 ) was used.
2.2. Preparation of Fe3 O4 NPs MMIPs The Fe3 O4 NPs were prepared by dissolving 0.30 g FeCl3 and 0.80 g NaAc in 10 mL of ethylene glycol and then heating at 200 ◦ C for 10 h [36]. Then 25 mg Fe3 O4 NPs powder were added into 10 mL of 10 mM Tris-HCl buffer (pH = 8.5) followed by the addition of 2.5 mL of 2.0 mg mL−1 Thi. Then the above suspension was mechanically stirred for 2 h. Next, 25 mg DA was dissolved in the O2 -saturated solution and incubated for 4 h. The resulted Fe3 O4 NPs MMIPs were collected by external magnetic separation. The template molecules were extracted by acetic acid/acetonitrile solution. For comparison, magnetic non-imprinted polymers (MNIPs) were also fabricated in the absence of Thi during the self-polymerization process. The preparation process was illustrated in Scheme 1.
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Scheme 1. Schematic illustration of the fabrication process of Fe3 O4 NPs MMIPs.
2.3. Preparation of Fe3 O4 NPs MMIPs-modified glassy carbon electrode (GCE) 25 mg Fe3 O4 NPs MMIP powder was firstly dispersed in 5 mL of 0.05% nafion solution. Then 7 L of 5.0 mg mL−1 Fe3 O4 NPs MMIPs aqueous suspension was cast on the newly polished GCE with a diameter of 3 mm from Shanghai Chenhua Instrument Co., Ltd. and then dried under N2 atmosphere. The nafion improved the distribution and stability of Fe3 O4 NPs MMIP on GCE surface greatly. To further enhance the stability and repeatability of modified electrode, the Fe3 O4 NPs MMIPs-modified GCE was immersed in 0.2 M PBS (pH 7.0) for 4 h and then washed by ultrapure water. In the immersing process, some loosely bound Fe3 O4 NPs MMIPs could drop off from the Fe3 O4 NPs MMIPs-modified GCE, which enhanced the stability and repeatability of the Fe3 O4 NPs MMIPs-modified GCE. The MNIPs-modified GCE was also prepared by this method. 2.4. Instruments Scanning electron microscopy (SEM) and energy dispersive Xray spectroscopy (EDXS) tests were implemented on a HITACHI S-3400N scanning electron microscope with a Phoenix energy Xray analyzer. X-ray powder diffraction (XRD) data were collected on a D/Max 2500V/PC X–ray powder diffractometer using Cu K␣ radiation ( = 1.54056 Å, 40 kV, 200 mA). UV–vis absorption spectra were collected on a Hitachi U-3900H UV–vis Spectrophotometer. All electrochemical measurements were performed on a CHI 660C electrochemical workstation (Shanghai, China) by conventional three-electrode system including the modified GCE as the working electrode, a platinum wire as the auxiliary electrode and a saturated calomel electrode (SCE, saturated KCl) as the reference electrode. 3. Results and discussion 3.1. Characterization of Fe3 O4 MMIPs The shape and core-shell nanostructure of Fe3 O4 NPs MMIPs were characterized by SEM and the results are shown in Fig. 1. The low resolution SEM image (Fig. 1A) showed a large number of uniform spherical Fe3 O4 NPs. The high resolution SEM image (Fig. 1C) indicated that the surface of Fe3 O4 NPs was very rough, which provided lots of active sites to improve its catalytic activities. The diameter of the spherical Fe3 O4 NPs was about 600 nm which was slightly bigger as compared with other nanomaterials, but similar to some previous results [36]. Although small Fe3 O4 NPs were benefit for their electrochemical performances, it was very difficult to reduce their size owing to the strong magnetism. Here, we tried our best to synthesize uniform and relatively small Fe3 O4 NPs. The good uniformity was also very beneficial to the good reproducibility of electrochemical biosensors based on Fe3 O4 NPs. The low resolution SEM image of the Fe3 O4 NPs MMIPs (Fig. 1B) also showed many uniform spherical particles but their surface seemed more smooth as compared with Fe3 O4 NPs (Fig. 1D). It might be because the DA polymerized on the surface of Fe3 O4 NPs and finally formed ploydopamine (PDA) to cover the Fe3 O4 NPs. The diameter of the Fe3 O4 NPs MMIPs was also about 600 nm, which indicated
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Fig. 1. SEM images of Fe3 O4 NPs (A,C) and Fe3 O4 NPs MMIPs (B,D).
only a thin layer of PDA was covered on the surface of Fe3 O4 NPs. The thickness of PDA layer was a crucial factor for the electrochemical performances of Fe3 O4 NPs MMIPs nanoenzyme. If it was too thick, the electrochemical performances would be hampered. If it was too thin, the selectivity of nanoenzyme might be poor. Thus, only a suitable thickness of PDA was optimized and presented in the experiments. Compared with the SEM images of Fe3 O4 NPs and Fe3 O4 NPs MMIPs, DA was confirmed to be successfully polymerized on the surface of Fe3 O4 NPs. The amount of Thi used to prepare Fe3 O4 NPs MMIPs nanoenzyme was also an important factor for the electrochemical performances of Fe3 O4 NPs MMIPs nanoenzyme. If the amount of Thi was too high, the elution of Thi would be very difficult. If the amount too low, the efficiency of molecular imprinting was poor. It was found that 0.4 mg mL−1 Thi was optimal for preparing Fe3 O4 NPs MMIPs nanoenzyme. The as-prepared Fe3 O4 NPs were ideal matrix materials for the preparation of MMIPs. During the polymerization of DA, the Thi molecules could be entrapped into the PDA layers based on the hydrogen bonds between PDA and Thi. What’s more, the template molecules have heteroatom ring and benzene ring which could combine with the benzene ring in the chain of PDA and then resulted in stronger Van der Waals interaction between templates and polymers. Thi imprinted sites could be easily generated after the Thi molecules were removed out by destroying the Van der Waals interaction and hydrogen bonds with the acetic acid/acetonitrile solution. In order to ensure that Thi molecules were thoroughly removed out, EDX of Fe3 O4 NPs MMIPs-Thi, Fe3 O4 NPs MMIPs and MNIPs were shown in Fig. 2A. Several typical Fe, O, C and S elements appeared in EDX of Fe3 O4 NPs MMIPs-Thi (curve a). The Fe and O should come from the Fe3 O4 NPs, C and O might originate from PDA, and S resulted from Thi. The peak of O was higher than that of Fe because O not only came from Fe3 O4 NPs but also from PDA. The appearance of S indicated that Thi was successfully packaged by PDA. The disappearance of the
S peak disappeared in Fe3 O4 NPs MMIPs (curve b) was due to the removing of Thi template. It was worth noting that the Fe3 O4 NPs MMIPs contained the same elements as the MNIPs (curve c). It further demonstrated that the Fe3 O4 NPs MMIPs were successfully prepared. Fig. 2B shows the XRD patterns of Fe3 O4 NPs (curve a) and the as-prepared Fe3 O4 NPs MMIPs (curve b). Five peaks at 30.2◦ , 35.6◦ , 43.4◦ , 56.9◦ , and 62.7◦ were observed for the two samples from 5◦ to 90◦ . They were assigned to the (220), (311), (400), (511), and (440) crystal facets of Fe3 O4 (JCPDS Card 19-629). The results further confirmed that Fe3 O4 NPs were successfully synthesized. Moreover, the peak positions of Fe3 O4 NPs were not changed after coating with the polymer layers, indicating that the Fe3 O4 NPs MMIPs still maintained the crystalline structure of Fe3 O4 NPs. 3.2. Electrochemical behaviors of Fe3 O4 NPs MMIPs nanoenzyme The electrocatalytic activity of the Fe3 O4 NPs MMIPs nanoenzyme toward 6 M Thi was explored by CVs. As shown in Fig. 3A, a pair of asymmetric redox peaks was observed. Based on the peaks’ potentials, the asymmetric redox peaks could be attributed to the Thi [29]. Obviously, both anodic peak current and cathodic peak current increased as the scan rate varied from 100 to 900 mV s−1 . The plots of both anodic peak current and cathodic peak current versus the square root of scan rates presented linear relationship, suggesting the Thi redox occurred through a diffusion-controlled electrochemical process (Fig. 3B). The CVs results indicated that the thickness of PDA layer was appropriate and Thi molecules could go through the MMIPs to arrive at the surface of Fe3 O4 NPs. The cathodic peak was obviously larger than the anodic peak, indicating a typical catalytic reaction. The possible mechanism of Fe3 O4 NPs MMIPs nanoenzyme towards Thi was presented: Fe3+ + Thi → Fe2+ + Thi+
(1)
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Fig. 2. (A) EDS analysis of Fe3 O4 NPs MMIPs-Thi (curve a), Fe3 O4 NPs MMIPs (curve b) and Fe3 O4 NPs MNIPs (curve c). (B) XRD patterns of Fe3 O4 NPs (curve a) and Fe3 O4 NPs MMIPs (curve b).
Fig. 3. (A) CVs of Fe3 O4 NPs MMIPs-modified GCE in 0.2 M N2 -saturated PBS (pH = 7.0) in the presence of 6 M Thi at different scan rates: (a) 200 mV s− 1 , (b) 300 mV s− 1 , (c) 400 mV s− 1 , (d) 500 mV s− 1 , (e) 600 mV s− 1 , (f) 700 mV s− 1 , (g) 800 mV s− 1 and (h) 900 mV s− 1 . (B) Plot of peak current versus the square root of scan rates.
Thi+ + e → Thi
(2)
Since the as-prepared Fe3 O4 NPs MMIPs nanoenzyme electrochemically responded toward Thi, it could be used to construct electrochemical biosensors in the presence of Thi. Accordingly, the concentration of Thi was optimized for subsequent electrochemical experiments. As shown in Fig. S1 (Supporting information), the optimal concentration of Thi was 20 M. The CVs result also clearly showed that H2 O2 took part in the electrochemical reaction and the Fe3 O4 NPs MMIPs-modified GCE could be used to detect H2 O2 . 3.3. Binding ability, selectivity, stability and reproducibility To explore the rebinding ability of Thi, we compared the molecular imprinting effectiveness of Fe3 O4 NPs MMIPs and Fe3 O4 NPs MNIPs in 0.2 M PBS solution (pH 7.0) containing 20 M Thi and 10 M H2 O2 (Fig. 4A). No obvious peak of Thi appeared on the Fe3 O4 NPs MNIPs-modified GCE electrode. While, the Fe3 O4 NPs MMIPs-modified GCE possessed obvious redox peaks. It clearly indicated that Fe3 O4 NPs MMIPs had a much better rebinding capacity than Fe3 O4 NPs MNIPs under the same conditions. Since Fe3 O4 NPs had peroxidase-like bioactivity, in the presence of H2 O2 and Fe3 O4 NPs, the colorless TMB could be oxidized into blue oxTMB which showed a typical UV–vis absorption peak at 650 nm [42]. Thus, TMB was used as the target molecule to test the selectivity of Fe3 O4 NPs MMIPs. Fig. 4B showed the UV–vis absorption spectra of 100 M TMB solutions containing 500 M H2 O2 after Fe3 O4 NPs
and Fe3 O4 NPs MMIPs were added. It was obvious that the TMB solution in the presence of Fe3 O4 NPs and 500 M H2 O2 (curve a) had a strong absorption while the TMB solution in the presence of Fe3 O4 NPs MMIPs and 500 M H2 O2 (curve b) did not show any absorption at 650 nm. The results indicated that the nanoenzyme based on Fe3 O4 NPs MMIPs showed good selectivity toward Thi. The selectivity of the nanoenzyme based on Fe3 O4 NPs MMIPs toward electrochemical detection of Thi was also evaluated by adding some interferents including glucose, TMB, methylene blue (MB), Na+ , ascorbic acid (AA), dopamine (DA) into 0.2 M PBS in the absence of Thi. As shown in Fig. S2 (Supporting information), the same concentration of the possible interfering substances did not show obvious interference, also indicating good selectivity. The selectivity of Fe3 O4 NPs MMIPs nanoenzyme might result from the imprinted sites of Fe3 O4 NPs MMIPs which retained accurate size of the template, shape memory, and orientation of chemical functionality of the template Thi. The selectivity was necessary for the electrochemical biosensors based on the Fe3 O4 NPs MMIPs. The stability of Fe3 O4 NPs MMIPs-modified GCE was investigated by storing it at 4 ◦ C for different time. After 2 days, no obvious change was found. After 30 days, 97.2% of the original value of peak current was remained. The results demonstrated a good stability which was obviously superior to natural enzyme. Natural enzymes always easily lose their bioactivity at high temperature, in a low or high pH solution, even when contact with electrode surface, which would result in a poor stability. Seven separately prepared Fe3 O4 NPs MMIPs-modified GCE were used to test the
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Fig. 4. (A) CVs of Fe3 O4 NPs MMIPs-modified GCE and Fe3 O4 NPs MNIPs-modified GCE in 0.2 M N2 -saturated PBS (pH = 7.0) containing 20 M Thi and 10 M H2 O2 at scan rate of 0.05 V s−1 . (B) UV–vis absorption spectra of Fe3 O4 NPs (curve a) and Fe3 O4 NPs MMIPs (curve b) in the presence of 500 M H2 O2 and 100 M TMB.
Fig. 5. CVs of Fe3 O4 NPs MMIPs-modified GCE in 0.2 M N2 -saturated PBS (pH = 7.0) with different concentration of H2 O2 (A), AChl (B), AChE (C) and ChOx (D). Insets were the corresponding working curves.
reproducibility under the same conditions. The results showed a good reproducibility with a relative standard deviation (RSD) of 2.45%. 3.4. Electrochemical detection of H2 O2 , AChl, AChE and ChOx by using the Fe3 O4 NPs MMIPs-modified GCE The possible catalytic mechanism of the Fe3 O4 NPs MMIPs nanoenzyme towards H2 O2 in the presence of Thi was explored. As shown in Fig. 3, the reduction of Thi could be catalyzed by the Fe3 O4 NPs MMIPs, and we subsequently found that the Fe3 O4 NPs MMIPs could exhibit better electrocatalytic effect on the reduction
of Thi in the presence of H2 O2 as shown in Fig. S1A (Supporting information). Accordingly, the Fe3 O4 NPs MMIPs-modified GCE could be also used to electrochemically detect H2 O2 in the presence of Thi. As shown in Fig. 5A, the reduction peak of Thi increased gradually as the H2 O2 concentration increased, showing a typical electrocatalytic process. The electrochemical detection of H2 O2 by using the Fe3 O4 NPs MMIPs-modified GCE exhibited good linear relationship in the range of 4.08 nM–9.0 M with a low detection limit of 1.58 nM (R/N = 3) (Inset in Fig. 5A). The result is similar to that obtained by the amperometric technique (Fig. S3, Supporting information). The result was superior to some previous H2 O2 biosensors [43–47] (Table S1, Supporting information). The result
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also proved that the presence of H2 O2 could improve the electrocatalytic ability of Fe3 O4 NPs MMIPs toward the reduction of Thi. The mechanism was presumed: Fe2+ + H2 O2 → Fe3+ + H2 O
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2017.06.132.
(3)
Here, the H2 O2 firstly oxidized the Fe2+ of Fe3 O4 NPs MMIPs into Fe3+ , and then the oxidized Fe3 O4 NPs MMIPs (Fe3+ ) could oxidize the Thi into Thi+ which was reduced by electrochemical reaction. Accordingly, with the increase of H2 O2 , the cathodic peak current of Thi was increased gradually. It was well known that AChl could hydrolyze into choline with the help of AChE and simultaneously the ChOx could reduce choline into betaine accompanied by the production of H2 O2 . Thus the proposed electrochemical H2 O2 biosensor could be further used to detect AChl, AChE, and ChOx. As shown in Fig. 5B, the eletrocatalytic detection of AChl by using the Fe3 O4 NPs MMIPs-modified GCE exhibited well linear relationship in the range of 2.80–160 M with a low detection of 0.86 M (R/N = 3). The linear range was wider than other electrochemical AChl biosensors [48–51] (Table S2, Supporting information), such as ChOx/Silicate/MWCNTs/Pt (5.0–100 M) [48] and AChE/ChO/carbon paste electrode (0.07–10 M) [50]. The linear relationship of AChE and ChOx was 0.53 ng mL−1 − 20 g mL−1 (Fig. 5C) and 22.76 ng mL−1 –0.40 g mL−1 (Fig. 5D), respectively. The detection limit of AChE and ChOx was 0.16 ng mL−1 and 6.83 ng mL−1 , respectively. The selectivity of Fe3 O4 NPs MMIPs-modified GCE toward electrochemical detection of H2 O2 , AChl, AChE and ChOx were also evaluated by adding some possible interfering substances into 0.2 M PBS. As shown in Fig. S4 (Supporting information), the same concentration of the possible interfering substances to electrochemical detection of H2 O2 , AChl, AChE and ChOx were tested, and no obvious interference was observed, indicating good selectivity of the Fe3 O4 NPs MMIPs nanoenzyme.
4. Conclusion In conclusion, a novel Fe3 O4 NPs MMIPs nanoenzyme was successfully constructed by a magnetic surface imprinting technique in which the MMIPs were prepared by a facile approach based on self-polymerization of DA in the presence of template Thi, and then the template Thi was removed out by an extraction procedure with organic solution. The obtained Fe3 O4 NPs MMIPs showed good selectivity toward Thi as well as the excellent catalytic activity, which led to an excellent sensitivity, and good selectivity of the electrochemical nonenzymatic H2 O2 biosensor based on Fe3 O4 NPs MMIPs nanoenzyme. At the same time, the proposed electrochemical biosensor was further used to detect AChl, AChE and ChOx, respectively. The results exhibited wide detection range and low detection limit. Based on this work, the Fe3 O4 NPs MMIPs might provide a new way to construct nanoenzymes. Overall, the proposed preparation method of the Fe3 O4 NPs MMIPs was simple, efficient, cheap and easy to mass production. It might also open up a new way for diverse targets monitoring.
Acknowledgments This work was financially supported by National Natural Science Foundation of China (21465014, 21465015 and 21665012), Natural Science Foundation of Jiangxi Province (20143ACB21016) and the Ground Plan of Science and Technology Projects of Jiangxi Educational Committee (KJLD14023).
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Biographies Li Wang received her Ph.D. in analytical chemistry from the Changchun Institute of Applied Chemistry, Chinese Academy of Science, China. She is currently working as a professor at Jiangxi Normal University. Her current research interest is focused on biosensors. Longfei Miao received his science bachelor in chemistry in 2016 from Jiangxi Normal University, China. He is working for his master’s degree in Jiangxi Normal University, China. His research interests are electrochemical sensor. Han Yang received her science bachelor in chemistry in 2015 from Jiangxi Normal University, China. She is working for her master’s degree in Jiangxi Normal University, China. Her research interests are electrochemical sensor. Jie Yu received her science bachelor in chemistry in 2014 from Jiangxi Science and technology Normal University, China. She is working for her master’s degree in Jiangxi Normal University, China. Her research interests are nano-materials and their applications in sensor. Yingzhen Xie received her science bachelor in chemistry in 2013 from Jingganshan University, China. She is working for her master’s degree in Jiangxi Normal University, China. Her research interests are bioelectrochemical sensor. Lijuan Xu received her science bachelor in chemistry in 2016 from Jiangxi Normal University, China. She is working for her master’s degree in Jiangxi Normal University, China. Her research interests are electrochemical sensor. Yonghai Song received his Ph.D. in analytical chemistry from the Changchun Institute of Applied Chemistry, Chinese Academy of Science, China. He is currently working as a professor at Jiangxi Normal University. His current research interests focus on biosensors.