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nitrogen-doped graphene nanoribbons
Accepted Manuscript Fabricating photoelectrochemical aptasensor for sensitive detection of aflatoxin B1 with visible-light-driven BiOBr/ nitrogen-doped graphene nanoribbons
Wei Chen, Mingyue Zhu, Qian Liu, Yingshu Guo, Huaisheng Wang, Kun Wang PII: DOI: Reference:
S1572-6657(19)30194-8 https://doi.org/10.1016/j.jelechem.2019.03.033 JEAC 12992
To appear in:
Journal of Electroanalytical Chemistry
Received date: Revised date: Accepted date:
25 December 2018 17 March 2019 17 March 2019
Please cite this article as: W. Chen, M. Zhu, Q. Liu, et al., Fabricating photoelectrochemical aptasensor for sensitive detection of aflatoxin B1 with visible-lightdriven BiOBr/nitrogen-doped graphene nanoribbons, Journal of Electroanalytical Chemistry, https://doi.org/10.1016/j.jelechem.2019.03.033
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ACCEPTED MANUSCRIPT Fabricating photoelectrochemical aptasensor for sensitive detection of aflatoxin B1 with visible-light-driven BiOBr/nitrogen-doped graphene nanoribbons Wei Chen1a, Mingyue Zhu1a, Qian Liua, Yingshu Guob,*, Huaisheng Wangc, Kun
a
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Wanga,* Key Laboratory of Modern Agriculture Equipment and Technology, School of
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Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013, PR
b
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China
Shandong Province Key Laboratory of Detection Technology for Tumor Makers,
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School of Chemistry and Chemical Engineering, Linyi University, Linyi 276005, PR
c
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China
School of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng
These authors contributed equally to this work.
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1
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252059, PR China
*Corresponding author: [email protected] (Y.S. Guo); [email protected] (K.
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Wang).
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ACCEPTED MANUSCRIPT Abstract A selective and sensitive photoelectrochemical (PEC) aptasensor for AFB1 detection in corn samples was fabricated by introducing BiOBr/nitrogen-doped graphene
nanoribbons
(N-GNRs)
as
photoactive
interface.
As
efficient
visible-light-driven photoactive species, the prepared BiOBr/N-GNRs exhibited
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higher photoactivity than pure materials under visible light irradiation. N-GNRs,
UV-vis
diffuse
reflectance
spectroscopy
demonstrated
that
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composites.
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acting as promising supporters for nanomaterials, can improve the performance of
BiOBr/N-GNRs possessed the narrower band gap energy, which could be easily
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irradiated by visible light. In addition, steady-state photoluminescence (PL) spectra also revealed that BiOBr/N-GNRs exhibited the lower recombination rate of
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photogenerated electron−hole pairs. The formation of the aptamer-AFB1 complex increased the resistance of the electrode, restrained the electron transfer, and thus
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quenched the PEC signal. Thereafter, a “signal-off” PEC aptasensor was fabricated
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successfully. The proposed PEC aptasensor demonstrated sensitive detection of AFB1 in a wide range from 5 pg mL–1 to 15 ng mL–1 with a low detection limit of 1.7 pg
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mL–1 and possessed high specificity and good reproducibility. The PEC aptasensor was suitable for corn samples with a good recovery in the range of 98.0–102.0% and
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the relative standard deviation (RSD) of 1.7–2.1% to confirm practical utility. Furthermore, this strategy would be extended to detect different targets as versatile PEC devices by replacing the aptamers with other sequences.
Keywords: Photoelectrochemical aptasensor; Visible light; BiOBr; Nitrogen-doped graphene nanoribbon; Aflatoxin B1
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ACCEPTED MANUSCRIPT 1. Introduction Aflatoxin B1 (AFB1) is one of the most toxic and carcinogenic compounds in aflatoxins and widely distributed in food and agricultural commodities [1]. Foodstuff including corn, peanuts, and cereals, are extremely vulnerable to AFB1 contamination, which leading to significant health and economic problems. In order to alleviate these
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serious threats, regulation limits have been set by many countries. China has set the
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maximum residue limits (MRLs) for AFB1 as 5 μg/kg. In European Union (EU), the
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MRLs for AFB1 was set at 2 μg/kg [2]. Thus, the sensitive detection of AFB1 is needed for food safety control and human health care. Up to date, there are some
ultra-high
performance
liquid
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methods developed for AFB1 detection, such as thin layer chromatography (TLC) [3], chromatography-tandem
mass
spectrometry
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(UPLC-MS/MS) [4], and enzyme-linked of immunosorbent assay (ELISA) [5]. Although these methods could be applied to obtain good sensitivity and selectivity to
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AFB1 detection, these methods are still not suitable for onsite analysis due to
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time-consuming pretreatment, expensive instruments, and required profession skills [6]. Therefore, a simple, rapid, and low-cost method for AFB1 detection is urgently
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anticipated.
Recently, Tang et al. designed a photoelectrochemical (PEC) immunosensing
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system for AFB1 detection in foodstuff, which demonstrated that PEC immunosensor could be employed to monitor AFB1 [7, 8]. However, immunoassay-based methods have limitations associated with high cost, and complex cross-reactivity due to the required antibodies. Thus, it is very important to establish rapid, sensitive, and inexpensive detection techniques for application in real samples. As antibody mimetics with high specificities and affinities to specific targets, aptamer has been regarded as a promising alternative to antibody in analytics [9, 10]. Aptamers have 3
ACCEPTED MANUSCRIPT been used to fabricate high-performance biosensors with high specificities and affinities [11,12]. Furthermore, DNA aptamers possess some obvious merits compared with antibodies, including selection in vitro, smaller sizes, low cost, high stability, and easier to modify in sensor design [13]. Therefore, aptamer-based assays have been coupled with PEC sensing. PEC aptasensor, combining the advantages of PEC
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detection and the character of target-dependent aptamer, provides a promising method
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for sensitive biomolecular detection in many fields [14, 15]. PEC aptasensor exhibits
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the advantages of both optical and electrochemical methods such as easy operation, low cost, and high sensitivity, which has attracted the interest of researchers [16,17].
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The establishment of selective, sensitive, and reproducible PEC aptasensor for identifying and quantifying trace amount of target biomolecules is closely associated
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with the PEC active species-based photoelectrode [18]. Therefore, it is necessary to
efficiency for AFB1 detection.
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employ efficient PEC photoactive materials with superior photo-electric conversion
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The photoelectric conversion efficiency and biocompatibility of photoactive materials need to be considered in the design and construction of PEC aptasensors. It
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is accepted that the UV light is a high-energy excitation light source that easily leading to fatal damage towards biomolecules [19]. Thus, some efforts have
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continuously highlighted some visible-light-driven semiconductors due to their good chemical stability, visible-light absorption capacity, and photochemical performances [20, 21]. Among them, BiOBr have drawn much attention owing to its proper energy band positions and excellent photochemical properties [22]. However, the photochemical performance of pure BiOBr is still limited by its higher recombination rate of the photogenerated electron-hole pairs [23]. Therefore, the rational design of the BiOBr-based hybrids combining other functional component with fast electron 4
ACCEPTED MANUSCRIPT mobility is highly anticipated. Among them, nitrogen-doped graphene nanoribbons (N-GNRs), as novel carbon materials, have been widely employed in oxygen reduction reaction (ORR) and energy conversion/storage devices [24]. Compared with one dimensional carbon materials, N-GNRs demonstrated more chemical active sites, faster electron transfer, and reduced charge-transfer resistance, thus leading to the
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enhanced performance [25]. In addition, N-GNRs possessed more straight edges and
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more ideal surface regularity than random graphene sheets [26]. With good properties,
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N-GNRs can serve as promising supporters for nanomaterials, and apply to improve the performance of composites [27].
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Herein, we fabricated a selective PEC aptasensor based on BiOBr/N-GNRs visible-light-driven photoactive species for AFB1 detection. The proposed PEC
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aptasensing integrated high specific recognition ability of aptamer, demonstrating relative advantages over antibodies about easier synthesis and wider target choices. In
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particular, BiOBr/N-GNRs obtained the optimal PEC performance compared with
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BiOBr, N-GNRs and BiOBr/NG. Therefore, a new PEC aptasensor has been fabricated for sensitive and selective detection of AFB1. AFB1 can be detected from 5
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pg mL–1 to 15 ng mL–1. The limit of detection (LOD) of the PEC aptasensor was significantly lowered to 1.7 pg mL–1. This work provides a feasible approach to
food.
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establish a PEC aptasensor platform in the detection of other hazardous substances in
2. Experimental section 2.1 Materials and reagents Multi-walled carbon nanotubes (MWCNTs) (diameter: 30 ~ 40 nm), potassium permanganate (KMnO4), concentrated sulfuric acid (H2SO4), hydrogen peroxide 5
ACCEPTED MANUSCRIPT (H2O2), concentrated hydrochloric acid (HCl), glycine, bismuth nitrate pentahydrate (Bi(NO3)3·5H2O), the ionic liquid [C16mim]Br (1-hexadecyl-3-methylimidazolium bromide) were all obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) was purchased from Sunshine Biotechnology Co., Ltd. (Nanjing, China). Aflatoxin B1 (AFB1) aptamer with the sequence of
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5’–NH2–GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA
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GGC CC–3’ were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The
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DNA aptamer solution was received by dissolving DNA in the phosphate-buffered saline (PBS, pH 7.0). Standard AFB1 samples with various concentrations were also
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prepared in PBS. The indium tin oxide (ITO) electrode was purchased from Zhuhai Kaivo Optoelectronic Technology Co., Ltd. All other reagents were of analytical
2.2 Preparation of BiOBr/N-GNRs
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reagent grade. Ultrapure water (≥ 18.2 MΩ.cm) was used throughout the study.
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Graphene oxide nanoribbons (GONRs) were longitudinally unzipped from
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MWCNTs by using a pressurized oxidation method according to the literature [28]. Annealing the mixture of GONRs and glycine to obtain N-GNRs at 500 °C for 2 h
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under argon atmosphere. Next, BiOBr/N-GNRs composites was synthesized as follows: 38.7 mg the ionic liquid [C16mim]Br (1-hexadecyl-3-methylimidazolium
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bromide) and 48.5 mg Bi(NO3)3·5H2O was dissolved in 20 mL ultrapure water with continual magnetic stirring for 1.0 h. Subsequently, a certain amount of N-GNRs was added into the above suspension with sonication for 0.5 h. Then the mixture was put into a 25-mL Teflon-lined autoclave, which was heated at a temperature of 140 °C for 24 h [29]. The resulting precipitate was collected and washed several times with ultrapure water and alcohol and then dried at 60 °C for 8 h to get the BiOBr/N-GNRs powders. For comparison, BiOBr were fabricated by the same procedure without 6
ACCEPTED MANUSCRIPT adding N-GNRs. 2.3 Fbrication of PEC aptasensing interface The construction procedure of the PEC aptasensor was displayed in Scheme 1. Firstly, ITO electrodes were cleaned by 1 mol L–1 NaOH solution and ultrasonically washed in double-distilled water and alcohol, respectively. 20 μL of BiOBr/N-GNRs
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dispersion (2 mg mL–1) was dropped onto the ITO surface with a covered area of 0.5
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cm2 and dried under an infrared lamp. Subsequently, 20 µL of 0.05 % chitosan (CHIT) solution as the fixing agent was casted onto the ITO electrode. After drying in air, 20
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μL of 0.05 % glutaraldehyde (GA) was dropped onto the CHIT/BiOBr/N-GNRs/ITO
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electrode surface, and kept for 1 h at room temperature, followed by thoroughly rinsing with ultrapure water for several times. 20 µL of amine-functionalized AFB1
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aptamer (2 µM) was immobilized on the CHIT/BiOBr/N-GNRs/ITO electrode surface and incubated at 37 °C for 4 h. It is mentioned that NH2 group in the aptamer is
glutaraldehyde
(GA)
as
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using
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covalently attached to NH2 group of the immobilized CHIT on the ITO electrode crosslinking
agent
[30].
The
obtained
aptamer/GA/CHIT/BiOBr/N-GNRs/ITO electrode was washed thoroughly with
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ultrapure water to remove unabsorbed aptamers. Then, the electrode was covered with 10 μL of 1×10-3 mol L–1 bull serum albumin (BSA) was put in air ambient for 1 h
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to block nonspecific active sites, followed by rinsing with ultrapure water and ethanol, respectively. Finally, the aptamer/GA/CHIT/BiOBr/N-GNRs/ITO were used for AFB1 detection. 2.4 Real sample preparation Corn samples were obtained from local markets with no AFB1 involved. The AFB1 samples were prepared according to the previous literature [31]. The power of corn samples (1 g) and known amounts of AFB1 were mixed completely in 5 mL of 7
ACCEPTED MANUSCRIPT methanol/water (20:80, v/v). The resulting products was centrifuged thrice at 8000 rpm for 10 min. After collecting supernatant, the extract was passed through a 0.45 µm syringe filter and then adjusted to pH 7.4. Finally, the diluted extracts with various amounts of AFB1 were analyzed by the proposed PEC aptasensor.
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3. Results and discussion
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3.1 Characterization of the samples
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TEM and XRD measurements were utilized to characterize of as-synthesized samples. As displayed in Fig. 1A, BiOBr nanosheets were randomly dispersed on the
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surface of the N-GNRs. And for N-GNRs with graphene-like morphologies exposed abundant edge sites of nanoribbons [25]. As illustrated in Fig. 1B, all the main
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diffraction peaks could be assigned to the phase of BiOBr (JCPDS NO. 09-0393) [29]. However, diffraction peaks of N-GNRs could not be detected by XRD due to less
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content and pretty weak diffraction intensity of N-GNRs [32]. Moreover, no other
as-prepared samples.
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diffraction peaks could be found in BiOBr/N-GNRs, indicating the high quality of
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XPS analysis was employed to elucidate the chemical property of BiOBr/N-GNRs. The survey XPS (Fig. 2A) obviously displayed the existence of Bi,
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Br, O, C and N elements in BiOBr/N-GNRs. As shown in Fig. 2B, two strong peaks at 159.0 eV and 164.3 eV were attributed to Bi 4f7/2 and Bi 4f5/2 originating from BiOBr, respectively [29]. However, when BiOBr combined with N-GNRs, the Bi 4f peaks of BiOBr/N-GNRs composites exhibited a little shift towards the high binding energy. Meanwhile, the high binding energy of Br 3d (Fig. 2C) and O 1s (Fig. 2D) in the BiOBr/N-GNRs composites also displayed a slight shift compared with pure BiOBr. This phenomenon indicated that the interaction between BiOBr and N-GNRs existed 8
ACCEPTED MANUSCRIPT in BiOBr/N-GNRs. Furthermore, the binding energies of 284.6 e, 286.0 eV, 287.6 eV and 288.6 eV associated to C 1s, which were characteristics of C=C, C=N, C–N, C–O bonding in BiOBr/N-GNRs (Fig. 2E) [33]. For the N 1s in Fig. 2F, the binding energies of 398.5 eV, 399.9 eV and 401.8 eV are assigned to pyridine N, pyrrolic N, and graphitic N, respectively [34]. XPS spectrum further confirms that
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BiOBr/N-GNRs are successfully synthesized.
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3.2 PEC properties and photoactivity of the samples
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In order to examine the PEC properties of BiOBr/N-GNRs, a comparative trial have been performed at a potential of 0 V upon photoexcitation with light illumination.
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As shown in Fig. 3A, the PEC signals of N-GNRs (a) and BiOBr (b) were ca. 40 and 125 nA, respectively. After combining N-GNRs with BiOBr, BiOBr/N-GNRs (d)
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displayed ca. 15-fold and 5-fold enhancement compared with the PEC signals of N-GNRs and BiOBr. Moreover, BiOBr/N-GNRs demonstrated an obviously enhanced
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PEC response compared with the BiOBr/NG nanocomposites (c). This fact suggested
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that the introduction of N-GNRs accelerated the separation of the photogenerated charge, and then led to the improvement of PEC performances [35].
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Photoluminescence (PL) analysis was employed to study the charge separation efficiency of as-prepared samples. It’s generally considered that the recombination of
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photogenerated electrons and holes could enhance the PL emission signal [36]. Fig. 3B showed the PL spectra of BiOBr and BiOBr/N-GNRs with an excitation wavelength of 360 nm. The BiOBr/N-GNRs had the decreased fluorescence intensity centered at around 470 nm compared with pure BiOBr. Therefore, the PL results suggested that BiOBr/N-GNRs obtained the improved photogenerated electron-hole pairs separation efficiency. Moreover, UV-visible absorption spectra were conducted to study the light 9
ACCEPTED MANUSCRIPT absorption ability of BiOBr and BiOBr/N-GNRs. As illustrated in Fig. 3C, the optical absorption edge of pure BiOBr was about 440 nm, and BiOBr/N-GNRs showed a slightly red shift in the visible range compared with pristine BiOBr. Morever, there was obviously reduced band gap energy for BiOBr/N-GNRs compared with BiOBr (Fig. 3D). This result implied that BiOBr/N-GNRs could be easier irradiated by
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visible light, leading to the charge effective separation. It is generally accepted that
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the lower recombination rate of the charge would exhibit more excellent PEC
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performances [37]. The UV-vis results suggested that the introduction of N-GNRs played an important role in BiOBr/N-GNRs, leading to enhanced PEC responses
3.3 Fabrication of PEC aptasensor
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compared to pure BiOBr.
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As displayed in Scheme 1, a selective PEC aptasensor was constructed for AFB1 assay on the basis of the stable PEC signal of BiOBr/N-GNRs. Fig. 4A exhibited the
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PEC intensity of the “signal-off” PEC aptasensor fabrication process. Under visible
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light irradiation, BiOBr/N-GNRs/ITO (a) displayed a high photocurrent of 0.60 μA owing to the improved the charge separation efficiency. However, the PEC intensity
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of the electrode modified by AFB1 aptamer (b) decreased owing to the steric hindrance of the aptamer [38]. The PEC response decreased to about 0.32 μA after the
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aptasensor having specific reaction with 5 ng mL–1 AFB1 (c). This phenomenon demonstrated that the change on the electrode surface restrained the transport of photogenerated electrons. EIS analysis was conducted on the ITO electrode to further confirm the successful assembly process of the PEC aptasensor. As illustrated in Fig. 4B, the EIS spectra of different modified electrodes were associated with each fabrication step of this PEC aptasensor. The equivalent circuit applied for fitting the EIS date (Fig. 4B 10
ACCEPTED MANUSCRIPT inset), where Ret, Zw, Rs, and Q represent electron transfer resistance, Warburg impedance, electrolyte solution resistance, and constant phase elements, respectively. After, the aptamer modified on the BiOBr/N-GNRs/ITO electrode (b), the value of charge-transfer resistance (Rct) increased from 25 Ω to 50 Ω. It could be explained that the aptamer would increase the steric hindrance of the electrode interface, and
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then restrained the transfer of the electrons. At the presence of 5 ng mL–1 AFB1, the
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Rct increased dramatically to 115 Ω due to the specific reaction between AFB1 and the
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aptamer (c). The formation of complex further impeded the transfer the electrons, and then resulted in increased Rct [39]. All these results have verified the successful
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construction of the proposed PEC aptasensor.
3.4 Optimization of conditions for PEC aptasensor
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In order to obtain efficient PEC aptasensor for AFB1 detection, some experimental parameters including the amount of N-GNRs in the BiOBr/N-GNRs, the
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concentration of aptamer, and the incubation time of AFB1 with aptamer were
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optimized. As illustrated in Fig. S1, the photocurrent response increased and then decreased gradually with increasing the weight ratio of N-GNRs from 2% to 10%.
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This fact indicated that the increasing N-GNRs overlapped on the surface of BiOBr nanosheets would block the light absorption of BiOBr. Thus, BiOBr/N-GNRs (5%)
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was chosen for construction of the PEC aptasensor in this work. Furthermore, the aptamer concentration and the incubation time had obvious influence on the performances of the fabricated aptasensor. As shown in Fig. S2A, the PEC response decreased gradually with the increase of the aptamer concentration from 5×10-7 to 3.0 ×10-6 mol L-1. This is ascribed that higher aptamer concentration modified on the electrode could impede the electrons transfer. While the aptamer concentration was further increased to over 2 ×10-6 mol L-1, the photocurrent response almost kept 11
ACCEPTED MANUSCRIPT constant. This phenomenon could be explained that the aptamer immobilized on the surface of the electrode had reached plateau. Therefore, 2 ×10-6 mol L-1 aptamer concentration was selected as the optimal aptamer concentration for the PEC aptasensor. As displayed in Fig. S2B, the photocurrent declined with the increase of incubation time. When it reached 40 min, the photocurrent response almost kept
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constant. It indicated that the aptamer on the electrode has reached saturation.
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Therefore, 40 min was chosen as the optimal incubation time.
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3.5 Analytical performances of PEC aptasensor
Under the optimal conditions, an efficient PEC aptasensor for AFB1 detection
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was fabricated. As shown in Fig. 4C, the PEC signal decreased gradually with the
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increase of the AFB1 concentration. And Fig. 4D displayed the corresponding linear calibration curve reporting the PEC response as a function of the AFB1 concentration
(pg mL–1)], with a correlation coefficient R2 =0.9973. In
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0.4268 -0.0186 [C
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ranging from 5 pg mL–1 to 15 ng mL–1. The regression equation is determined as I = AFB1
addition, the limit of detection was calculated to be 1.7 pg mL–1 (defined as S/N = 3).
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The analytical performance of the fabricated aptasensor has been compared to the
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reported methods towards AFB1, as shown in Table 1. The typical instrumental analytical methods, including TLC [3], UPLC-MS/MS [4] and ELISA [5], have been applied to detect AFB1. However, these methods are restricted to time-consuming pretreatment, high-cost instruments, and required profession skills [6]. Compared to other methods on the basis of fluorescence [40] and electrochemistry [41], the proposed aptasensor possessed a wider linear range. Although there were reported methods demonstrating wide linear ranges comparable to the aptasensor [7, 8], the 12
ACCEPTED MANUSCRIPT PEC immunosensor had limitations associated with high cost, and complex cross-reactivity due to the required antibodies. Noticeably, PEC aptasensor exhibited some advantages including low cost, easy operation, and high sensitivity. 3.6 Selectivity and reproducibility of the PEC aptasensor
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In order to investigate the selectivity of the PEC aptasensor, some interfering species ochratoxin A (OTA) and fumonisin B1 (FB1) were studied in the interference
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test. Under the same conditions, the photocurrrent responses of 50 ng mL–1 interfering
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species with 5 ng mL–1 AFB1 were recorded. As displayed in Fig. S3A, there was more significant photocurrent change of the aptasensor toward 5 ng mL–1 AFB1 than
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that of 10-fold concentration of nonspecific molecules. This sugggested that the
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proposed PEC aptasensor possessed good specificity. Furthermore, the reproducibility and stability are necessary factors to valuate PEC sensor. As displayed in Fig. S3B, the photocurrent of prepared aptamer-BiOBr/N-GNRs/ITO towards 5 ng mL–1 AFB1
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was very stable. In addition, the photocurrent had no obvious change after the electrodde was stored for two weeks at 4 °C. This meant the high stability and good reproducibility of as-fabricated PEC aptasensor.
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3.7 Analytical application in real samples
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The established PEC aptasensor was evaluated by corn samples detection. As illustrated in Table S1, a good recovery in the range of 98.0–102.0% with the relative standard deviation (RSD) of 1.7–2.1% was recorded. Thus, the PEC aptamer-based assay method could be employed to AFB1 detection in real samples.
4. Conclusions In summary, this work demonstrated PEC aptasensing with high selective and sensitive by combining the high-efficient PEC technique with specificity aptamer as 13
ACCEPTED MANUSCRIPT well as the improved photoactivity of BiOBr/N-GNRs. The proposed PEC aptasenor exhibited siginificant advantages in selectivity and sensitity owing to the merits of aptamer and BiOBr/N-GNRs photoactive species. Based on a “signal-off” PEC response of the proposed aptasensor, a broad linear range and a low limit of detection for AFB1 was acquired. And good recoveries were achieved when the established PEC
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aptasenor was applied to detect AFB1 in corn samples. This work not only extended to
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determine other aflatoxins in foodstuff but also provided a promising strategy for food
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safety in PEC biosensing.
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Acknowledgments
This research was supported under the National Natural Science Foundation of
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China (21375050, 21675066, 61601204), the Research Foundation of Zhenjiang
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Science and Technology Bureau (No. NY2016011).
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Compliance with Ethical Standards
Conflict of Interest The authors certify that there is no conflict of interest with any
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individual/organization for the present work. Ethical Approval This article does not contain any studies with human participants or
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animals performed by any of the authors. Informed consent Informed consent was obtained from all individual participants included in the study.
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Yi, Small 11 (2015) 1423-1429. [25] X. Meng, C. Yu, X. Song, Y. Liu S. Liang, Z. Liu, C. Hao, J. Qiu, Adv. Energy Mater. 5 (2015). 1-9. [26] A.L. Higginbotham, D.V. Kosynkin, A. Sinitskii, Z. Sun, J.M. ACS Nano 4 (2010) 16
ACCEPTED MANUSCRIPT 2059-2069. [27] N. Li, H. Ma, W. Cao, D. Wu, T. Yan, B. Du, Q. Wei, Biosens. Bioelectron. 74 (2015) 786-791. [28] Q. Liu, M. Cheng, J. Wang, G. Jiang, Chem. Eur. J. 21 (2015) 5594-5599.
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[29] J. Xia, J. Di, H. Li, H. Xu, H. Li, S. Guo, Appl. Catal. B: Environ. 181 (2016)
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[30] S. Khezrian, A. Salimi, H. Teymourian, R. Hallaj, Biosens. Bioelectron. 43 (2013) 218-225.
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[31] W.B. Shim, M.J. Kim, H. Mun, M.G. Kim, Biosens. Bioelectron. 62 (2014)
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288-294.
[32] Y. Yin, Q. Liu, D. Jiang, X. Du, J. Qian, H. Mao, K. Wang, Carbon 96 (2016)
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4180-4187.
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[33] X. Yan, Y. Li, M. Li, Y. Jin, F. Du, G. Chen, Y. Wei, J. Mater. Chem. A 3 (2015)
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[34] M. Liu, Y. Song, S. He, W.W. Tjiu, J. Pan, Y.Y. Xia, T. Liu, ACS Appl. Mater.
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Interfaces 6 (2014) 4214-4222. [35] X. Du, D. Jiang, L. Dai, L. Zhou, N. Hao, J. Qian, B. Qiu, K. Wang, Biosens. Bioelectron. 81 (2016) 242-248. [36] J.W. Tang, Z.G. Zou J.H. Ye, J. Phys. Chem. B 107 (2003) 14265-14269. [37] M. Zhang, X.Bai, D. Liu, J. Wang, Y. Zhu, Appl. Catal. B: Environ. 164 (2015) 77-81. [38] D. Jiang, X. J.Du, D.Y. Chen, L. Zhou, W. Chen, Y.Q. Li, N. Hao, J. Qian, Q. Liu, 17
ACCEPTED MANUSCRIPT K. Wang, Biosens. Bioelectron. 83 (2016) 149-155. [39] D. Jiang, X. J. Du, Q. Liu, L. Zhou, L. M. Dai, J. Qian, K. Wang, Analyst 140 (2015) 6404-6411. [40] J. Qian, C. Ren, C. Wang, W.Chen, X.Lu, , H. Li, Q.Liu, N.Hao, H. Li, K. Wang,
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Anal. Chim. Acta 1019 (2018), 119-127.
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[41] G. Evtugyn, A. Porfireva, V. Stepanova, R. Sitdikov, I. Stoikov, D. Nikolelis, T.
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Hianik, Electroanal. 26(2014), 2100-2109.
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ACCEPTED MANUSCRIPT Figure Captions
Scheme 1. Schematic diagram for the construction of PEC aptasensor for AFB1 detection on the basis of the BiOBr/N-GNRs/ITO electrode.
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Fig. 1. (A) High-magnification TEM image of BiOBr/N-GNRs. (B) XRD patterns of
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BiOBr (a) and BiOBr/N-GNRs (b).
Fig. 2. XPS spectra of BiOBr and BiOBr/N-GNRs samples: (A) survey spectrum, (B)
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Bi 4f, (C) Br 3d, (D) O 1s, (E) C 1s and (F) N 1s.
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Fig. 3. (A) Photocurrent responses 0.1 mol L–1 PBS at bias potential of 0 V of pure N-GNRs (a), BiOBr (b), BiOBr/NG (c) and BiOBr/N-GNRs (d) modified ITO
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electrodes. (B) Steady-state PL spectra of BiOBr (a) and BiOBr/N-GNRs (b); (C)
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UV−vis diffuse reflectance spectra and (D) plot of (Ahʋ)2 versus the energy (hʋ) for
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the band gap energy of BiOBr (a) and BiOBr/N-GNRs (b).
Fig. 4. (A) PEC in 0.1 mol L–1 PBS at bias potential of 0 V and (B) EIS
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characterization of aptasensor fabrication process in 0.1 mol L–1 KCl solution containing
5
mmol
aptamer-BiOBr/N-GNRs/ITO
L–1
[Fe(CN)6]3–/4–: (b),
and
BiOBr/N-GNRs/ITO AFB1
interact
(a), with
aptamer-BiOBr/N-GNRs/ITO (c). (C) Photocurrent response of the PEC aptasensor at different concentrations of AFB1 in 0.1 mol L–1 PBS at bias potential of 0 V: 5 pg mL–1 (a), 0.01 ng mL–1 (b), 0.1 ng mL–1 (c), 0.5 ng mL–1 (d), 1 ng mL–1 (e), 3 ng mL–1 (f), 5 ng mL–1 (g), 10 ng mL–1 (h) and 15 ng mL–1 (i); (D) the corresponding linear 19
ACCEPTED MANUSCRIPT calibration curve.
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Table 1 Comparison of the reported techniques for AFB1 detection.
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Scheme 1
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ACCEPTED MANUSCRIPT Fig. 1
(A)
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20
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N-GNRs
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30
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BiOBr
Intensity (a.u.)
(B)
40
50
JCPDS NO. 09-0393
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2 Theta (degree)
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70
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Intensity (a.u.)
Bi 4p
BiOBr/N-GNRs
159.2 eV
500
750
156
160
164
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(D)
69.2 eV
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BiOBr
O 1s
529.8 eV
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Br 3d
68.2 eV
BiOBr
530.0 eV
BiOBr/N-GNRs 70
BiOBr/N-GNRs
72
528
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68
Binding Energy (eV)
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(E)
531
(F)
C 1s
C-N 287.6 eV C-O 288.6 eV
Intensity (a.u.)
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Intensity (a.u.)
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285
C=N 286.0 eV
537
N 1s Pyrrolic N 399.9 eV
Pyridine N 398.5 eV
Graphitic N 401.8 eV
BiOBr/N-GNRs 288
534
Binding Energy (eV)
C=C 284.6 eV
282
168
Binding Energy (eV)
Intensity (a.u.)
Intensity (a.u.)
68.1 eV 69.1 eV
66
164.5 eV
BiOBr
BiOBr/N-GNRs
Binding Energy (eV)
(C)
Bi 4f
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Bi 4p
BiOBr
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O 1s
Bi 4d5/2 Bi 4d3/2 Bi 4d5/2
N 1s
C 1s
Bi 4f7/2 Bi 4f5/2
Bi 5d Br 3d
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(B) 159.0 eV
Survey
Bi 4d3/2 O 1s
C 1s
Bi 4f5/2
Bi 5d Br 3d
Intensity (a.u.)
(A)
Bi 4f7/2
Fig. 2
BiOBr/N-GNRs
291
396
Binding Energy (eV)
400
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Binding Energy (eV)
23
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(B) d c
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(A) 0.6 0.4
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Photocurrent (A)
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Detection limit (pg mL–1)
References
TLC
16 ~ 115
–
[3]
UPLC-MS/MS
0.5 ~ 69.4
–
[4]
0.1 ~ 10
10
[5]
0.005 ~ 10
1.7
0.0312 ~ 31.2
15.6
PEC immunosensor
0.01 ~ 20
2.1
PEC immunosensor
0.01 ~ 15
3.0
[8]
PEC aptasensor
0.005 ~ 15
1.7
This work
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Electrochemical aptasensor
[40] [41]
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Fluorescent aptasensor
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ELISA
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Linear range (ng mL–1)
Method
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[7]
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Graphical Abstract
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ACCEPTED MANUSCRIPT Highlights
BiOBr/nitrogen-doped graphene nanoribbons as photoactive species were constructed A photoelectrochemical aptasensor was fabricated for aflatoxin B1 detection
The PEC aptasensor exhibited a wider linear range with a lower detection limit
The PEC aptasensor was applied to detect aflatoxin B1 in corn samples
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Wei Chen, Mingyue Zhu, Qian Liu, Yingshu Guo, Huaisheng Wang, Kun Wang PII: DOI: Reference:
S1572-6657(19)30194-8 https://doi.org/10.1016/j.jelechem.2019.03.033 JEAC 12992
To appear in:
Journal of Electroanalytical Chemistry
Received date: Revised date: Accepted date:
25 December 2018 17 March 2019 17 March 2019
Please cite this article as: W. Chen, M. Zhu, Q. Liu, et al., Fabricating photoelectrochemical aptasensor for sensitive detection of aflatoxin B1 with visible-lightdriven BiOBr/nitrogen-doped graphene nanoribbons, Journal of Electroanalytical Chemistry, https://doi.org/10.1016/j.jelechem.2019.03.033
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Fabricating photoelectrochemical aptasensor for sensitive detection of aflatoxin B1 with visible-light-driven BiOBr/nitrogen-doped graphene nanoribbons Wei Chen1a, Mingyue Zhu1a, Qian Liua, Yingshu Guob,*, Huaisheng Wangc, Kun
a
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Wanga,* Key Laboratory of Modern Agriculture Equipment and Technology, School of
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Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013, PR
b
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China
Shandong Province Key Laboratory of Detection Technology for Tumor Makers,
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School of Chemistry and Chemical Engineering, Linyi University, Linyi 276005, PR
c
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China
School of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng
These authors contributed equally to this work.
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252059, PR China
*Corresponding author: [email protected] (Y.S. Guo); [email protected] (K.
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Wang).
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ACCEPTED MANUSCRIPT Abstract A selective and sensitive photoelectrochemical (PEC) aptasensor for AFB1 detection in corn samples was fabricated by introducing BiOBr/nitrogen-doped graphene
nanoribbons
(N-GNRs)
as
photoactive
interface.
As
efficient
visible-light-driven photoactive species, the prepared BiOBr/N-GNRs exhibited
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higher photoactivity than pure materials under visible light irradiation. N-GNRs,
UV-vis
diffuse
reflectance
spectroscopy
demonstrated
that
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composites.
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acting as promising supporters for nanomaterials, can improve the performance of
BiOBr/N-GNRs possessed the narrower band gap energy, which could be easily
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irradiated by visible light. In addition, steady-state photoluminescence (PL) spectra also revealed that BiOBr/N-GNRs exhibited the lower recombination rate of
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photogenerated electron−hole pairs. The formation of the aptamer-AFB1 complex increased the resistance of the electrode, restrained the electron transfer, and thus
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quenched the PEC signal. Thereafter, a “signal-off” PEC aptasensor was fabricated
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successfully. The proposed PEC aptasensor demonstrated sensitive detection of AFB1 in a wide range from 5 pg mL–1 to 15 ng mL–1 with a low detection limit of 1.7 pg
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mL–1 and possessed high specificity and good reproducibility. The PEC aptasensor was suitable for corn samples with a good recovery in the range of 98.0–102.0% and
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the relative standard deviation (RSD) of 1.7–2.1% to confirm practical utility. Furthermore, this strategy would be extended to detect different targets as versatile PEC devices by replacing the aptamers with other sequences.
Keywords: Photoelectrochemical aptasensor; Visible light; BiOBr; Nitrogen-doped graphene nanoribbon; Aflatoxin B1
2
ACCEPTED MANUSCRIPT 1. Introduction Aflatoxin B1 (AFB1) is one of the most toxic and carcinogenic compounds in aflatoxins and widely distributed in food and agricultural commodities [1]. Foodstuff including corn, peanuts, and cereals, are extremely vulnerable to AFB1 contamination, which leading to significant health and economic problems. In order to alleviate these
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serious threats, regulation limits have been set by many countries. China has set the
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maximum residue limits (MRLs) for AFB1 as 5 μg/kg. In European Union (EU), the
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MRLs for AFB1 was set at 2 μg/kg [2]. Thus, the sensitive detection of AFB1 is needed for food safety control and human health care. Up to date, there are some
ultra-high
performance
liquid
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methods developed for AFB1 detection, such as thin layer chromatography (TLC) [3], chromatography-tandem
mass
spectrometry
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(UPLC-MS/MS) [4], and enzyme-linked of immunosorbent assay (ELISA) [5]. Although these methods could be applied to obtain good sensitivity and selectivity to
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AFB1 detection, these methods are still not suitable for onsite analysis due to
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time-consuming pretreatment, expensive instruments, and required profession skills [6]. Therefore, a simple, rapid, and low-cost method for AFB1 detection is urgently
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anticipated.
Recently, Tang et al. designed a photoelectrochemical (PEC) immunosensing
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system for AFB1 detection in foodstuff, which demonstrated that PEC immunosensor could be employed to monitor AFB1 [7, 8]. However, immunoassay-based methods have limitations associated with high cost, and complex cross-reactivity due to the required antibodies. Thus, it is very important to establish rapid, sensitive, and inexpensive detection techniques for application in real samples. As antibody mimetics with high specificities and affinities to specific targets, aptamer has been regarded as a promising alternative to antibody in analytics [9, 10]. Aptamers have 3
ACCEPTED MANUSCRIPT been used to fabricate high-performance biosensors with high specificities and affinities [11,12]. Furthermore, DNA aptamers possess some obvious merits compared with antibodies, including selection in vitro, smaller sizes, low cost, high stability, and easier to modify in sensor design [13]. Therefore, aptamer-based assays have been coupled with PEC sensing. PEC aptasensor, combining the advantages of PEC
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detection and the character of target-dependent aptamer, provides a promising method
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for sensitive biomolecular detection in many fields [14, 15]. PEC aptasensor exhibits
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the advantages of both optical and electrochemical methods such as easy operation, low cost, and high sensitivity, which has attracted the interest of researchers [16,17].
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The establishment of selective, sensitive, and reproducible PEC aptasensor for identifying and quantifying trace amount of target biomolecules is closely associated
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with the PEC active species-based photoelectrode [18]. Therefore, it is necessary to
efficiency for AFB1 detection.
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employ efficient PEC photoactive materials with superior photo-electric conversion
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The photoelectric conversion efficiency and biocompatibility of photoactive materials need to be considered in the design and construction of PEC aptasensors. It
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is accepted that the UV light is a high-energy excitation light source that easily leading to fatal damage towards biomolecules [19]. Thus, some efforts have
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continuously highlighted some visible-light-driven semiconductors due to their good chemical stability, visible-light absorption capacity, and photochemical performances [20, 21]. Among them, BiOBr have drawn much attention owing to its proper energy band positions and excellent photochemical properties [22]. However, the photochemical performance of pure BiOBr is still limited by its higher recombination rate of the photogenerated electron-hole pairs [23]. Therefore, the rational design of the BiOBr-based hybrids combining other functional component with fast electron 4
ACCEPTED MANUSCRIPT mobility is highly anticipated. Among them, nitrogen-doped graphene nanoribbons (N-GNRs), as novel carbon materials, have been widely employed in oxygen reduction reaction (ORR) and energy conversion/storage devices [24]. Compared with one dimensional carbon materials, N-GNRs demonstrated more chemical active sites, faster electron transfer, and reduced charge-transfer resistance, thus leading to the
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enhanced performance [25]. In addition, N-GNRs possessed more straight edges and
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more ideal surface regularity than random graphene sheets [26]. With good properties,
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N-GNRs can serve as promising supporters for nanomaterials, and apply to improve the performance of composites [27].
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Herein, we fabricated a selective PEC aptasensor based on BiOBr/N-GNRs visible-light-driven photoactive species for AFB1 detection. The proposed PEC
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aptasensing integrated high specific recognition ability of aptamer, demonstrating relative advantages over antibodies about easier synthesis and wider target choices. In
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particular, BiOBr/N-GNRs obtained the optimal PEC performance compared with
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BiOBr, N-GNRs and BiOBr/NG. Therefore, a new PEC aptasensor has been fabricated for sensitive and selective detection of AFB1. AFB1 can be detected from 5
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pg mL–1 to 15 ng mL–1. The limit of detection (LOD) of the PEC aptasensor was significantly lowered to 1.7 pg mL–1. This work provides a feasible approach to
food.
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establish a PEC aptasensor platform in the detection of other hazardous substances in
2. Experimental section 2.1 Materials and reagents Multi-walled carbon nanotubes (MWCNTs) (diameter: 30 ~ 40 nm), potassium permanganate (KMnO4), concentrated sulfuric acid (H2SO4), hydrogen peroxide 5
ACCEPTED MANUSCRIPT (H2O2), concentrated hydrochloric acid (HCl), glycine, bismuth nitrate pentahydrate (Bi(NO3)3·5H2O), the ionic liquid [C16mim]Br (1-hexadecyl-3-methylimidazolium bromide) were all obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) was purchased from Sunshine Biotechnology Co., Ltd. (Nanjing, China). Aflatoxin B1 (AFB1) aptamer with the sequence of
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5’–NH2–GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA
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GGC CC–3’ were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The
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DNA aptamer solution was received by dissolving DNA in the phosphate-buffered saline (PBS, pH 7.0). Standard AFB1 samples with various concentrations were also
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prepared in PBS. The indium tin oxide (ITO) electrode was purchased from Zhuhai Kaivo Optoelectronic Technology Co., Ltd. All other reagents were of analytical
2.2 Preparation of BiOBr/N-GNRs
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reagent grade. Ultrapure water (≥ 18.2 MΩ.cm) was used throughout the study.
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Graphene oxide nanoribbons (GONRs) were longitudinally unzipped from
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MWCNTs by using a pressurized oxidation method according to the literature [28]. Annealing the mixture of GONRs and glycine to obtain N-GNRs at 500 °C for 2 h
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under argon atmosphere. Next, BiOBr/N-GNRs composites was synthesized as follows: 38.7 mg the ionic liquid [C16mim]Br (1-hexadecyl-3-methylimidazolium
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bromide) and 48.5 mg Bi(NO3)3·5H2O was dissolved in 20 mL ultrapure water with continual magnetic stirring for 1.0 h. Subsequently, a certain amount of N-GNRs was added into the above suspension with sonication for 0.5 h. Then the mixture was put into a 25-mL Teflon-lined autoclave, which was heated at a temperature of 140 °C for 24 h [29]. The resulting precipitate was collected and washed several times with ultrapure water and alcohol and then dried at 60 °C for 8 h to get the BiOBr/N-GNRs powders. For comparison, BiOBr were fabricated by the same procedure without 6
ACCEPTED MANUSCRIPT adding N-GNRs. 2.3 Fbrication of PEC aptasensing interface The construction procedure of the PEC aptasensor was displayed in Scheme 1. Firstly, ITO electrodes were cleaned by 1 mol L–1 NaOH solution and ultrasonically washed in double-distilled water and alcohol, respectively. 20 μL of BiOBr/N-GNRs
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dispersion (2 mg mL–1) was dropped onto the ITO surface with a covered area of 0.5
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cm2 and dried under an infrared lamp. Subsequently, 20 µL of 0.05 % chitosan (CHIT) solution as the fixing agent was casted onto the ITO electrode. After drying in air, 20
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μL of 0.05 % glutaraldehyde (GA) was dropped onto the CHIT/BiOBr/N-GNRs/ITO
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electrode surface, and kept for 1 h at room temperature, followed by thoroughly rinsing with ultrapure water for several times. 20 µL of amine-functionalized AFB1
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aptamer (2 µM) was immobilized on the CHIT/BiOBr/N-GNRs/ITO electrode surface and incubated at 37 °C for 4 h. It is mentioned that NH2 group in the aptamer is
glutaraldehyde
(GA)
as
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using
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covalently attached to NH2 group of the immobilized CHIT on the ITO electrode crosslinking
agent
[30].
The
obtained
aptamer/GA/CHIT/BiOBr/N-GNRs/ITO electrode was washed thoroughly with
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ultrapure water to remove unabsorbed aptamers. Then, the electrode was covered with 10 μL of 1×10-3 mol L–1 bull serum albumin (BSA) was put in air ambient for 1 h
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to block nonspecific active sites, followed by rinsing with ultrapure water and ethanol, respectively. Finally, the aptamer/GA/CHIT/BiOBr/N-GNRs/ITO were used for AFB1 detection. 2.4 Real sample preparation Corn samples were obtained from local markets with no AFB1 involved. The AFB1 samples were prepared according to the previous literature [31]. The power of corn samples (1 g) and known amounts of AFB1 were mixed completely in 5 mL of 7
ACCEPTED MANUSCRIPT methanol/water (20:80, v/v). The resulting products was centrifuged thrice at 8000 rpm for 10 min. After collecting supernatant, the extract was passed through a 0.45 µm syringe filter and then adjusted to pH 7.4. Finally, the diluted extracts with various amounts of AFB1 were analyzed by the proposed PEC aptasensor.
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3. Results and discussion
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3.1 Characterization of the samples
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TEM and XRD measurements were utilized to characterize of as-synthesized samples. As displayed in Fig. 1A, BiOBr nanosheets were randomly dispersed on the
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surface of the N-GNRs. And for N-GNRs with graphene-like morphologies exposed abundant edge sites of nanoribbons [25]. As illustrated in Fig. 1B, all the main
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diffraction peaks could be assigned to the phase of BiOBr (JCPDS NO. 09-0393) [29]. However, diffraction peaks of N-GNRs could not be detected by XRD due to less
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as-prepared samples.
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diffraction peaks could be found in BiOBr/N-GNRs, indicating the high quality of
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XPS analysis was employed to elucidate the chemical property of BiOBr/N-GNRs. The survey XPS (Fig. 2A) obviously displayed the existence of Bi,
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Br, O, C and N elements in BiOBr/N-GNRs. As shown in Fig. 2B, two strong peaks at 159.0 eV and 164.3 eV were attributed to Bi 4f7/2 and Bi 4f5/2 originating from BiOBr, respectively [29]. However, when BiOBr combined with N-GNRs, the Bi 4f peaks of BiOBr/N-GNRs composites exhibited a little shift towards the high binding energy. Meanwhile, the high binding energy of Br 3d (Fig. 2C) and O 1s (Fig. 2D) in the BiOBr/N-GNRs composites also displayed a slight shift compared with pure BiOBr. This phenomenon indicated that the interaction between BiOBr and N-GNRs existed 8
ACCEPTED MANUSCRIPT in BiOBr/N-GNRs. Furthermore, the binding energies of 284.6 e, 286.0 eV, 287.6 eV and 288.6 eV associated to C 1s, which were characteristics of C=C, C=N, C–N, C–O bonding in BiOBr/N-GNRs (Fig. 2E) [33]. For the N 1s in Fig. 2F, the binding energies of 398.5 eV, 399.9 eV and 401.8 eV are assigned to pyridine N, pyrrolic N, and graphitic N, respectively [34]. XPS spectrum further confirms that
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BiOBr/N-GNRs are successfully synthesized.
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3.2 PEC properties and photoactivity of the samples
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In order to examine the PEC properties of BiOBr/N-GNRs, a comparative trial have been performed at a potential of 0 V upon photoexcitation with light illumination.
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As shown in Fig. 3A, the PEC signals of N-GNRs (a) and BiOBr (b) were ca. 40 and 125 nA, respectively. After combining N-GNRs with BiOBr, BiOBr/N-GNRs (d)
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displayed ca. 15-fold and 5-fold enhancement compared with the PEC signals of N-GNRs and BiOBr. Moreover, BiOBr/N-GNRs demonstrated an obviously enhanced
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PEC response compared with the BiOBr/NG nanocomposites (c). This fact suggested
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that the introduction of N-GNRs accelerated the separation of the photogenerated charge, and then led to the improvement of PEC performances [35].
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Photoluminescence (PL) analysis was employed to study the charge separation efficiency of as-prepared samples. It’s generally considered that the recombination of
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photogenerated electrons and holes could enhance the PL emission signal [36]. Fig. 3B showed the PL spectra of BiOBr and BiOBr/N-GNRs with an excitation wavelength of 360 nm. The BiOBr/N-GNRs had the decreased fluorescence intensity centered at around 470 nm compared with pure BiOBr. Therefore, the PL results suggested that BiOBr/N-GNRs obtained the improved photogenerated electron-hole pairs separation efficiency. Moreover, UV-visible absorption spectra were conducted to study the light 9
ACCEPTED MANUSCRIPT absorption ability of BiOBr and BiOBr/N-GNRs. As illustrated in Fig. 3C, the optical absorption edge of pure BiOBr was about 440 nm, and BiOBr/N-GNRs showed a slightly red shift in the visible range compared with pristine BiOBr. Morever, there was obviously reduced band gap energy for BiOBr/N-GNRs compared with BiOBr (Fig. 3D). This result implied that BiOBr/N-GNRs could be easier irradiated by
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visible light, leading to the charge effective separation. It is generally accepted that
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the lower recombination rate of the charge would exhibit more excellent PEC
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performances [37]. The UV-vis results suggested that the introduction of N-GNRs played an important role in BiOBr/N-GNRs, leading to enhanced PEC responses
3.3 Fabrication of PEC aptasensor
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compared to pure BiOBr.
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As displayed in Scheme 1, a selective PEC aptasensor was constructed for AFB1 assay on the basis of the stable PEC signal of BiOBr/N-GNRs. Fig. 4A exhibited the
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PEC intensity of the “signal-off” PEC aptasensor fabrication process. Under visible
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light irradiation, BiOBr/N-GNRs/ITO (a) displayed a high photocurrent of 0.60 μA owing to the improved the charge separation efficiency. However, the PEC intensity
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of the electrode modified by AFB1 aptamer (b) decreased owing to the steric hindrance of the aptamer [38]. The PEC response decreased to about 0.32 μA after the
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aptasensor having specific reaction with 5 ng mL–1 AFB1 (c). This phenomenon demonstrated that the change on the electrode surface restrained the transport of photogenerated electrons. EIS analysis was conducted on the ITO electrode to further confirm the successful assembly process of the PEC aptasensor. As illustrated in Fig. 4B, the EIS spectra of different modified electrodes were associated with each fabrication step of this PEC aptasensor. The equivalent circuit applied for fitting the EIS date (Fig. 4B 10
ACCEPTED MANUSCRIPT inset), where Ret, Zw, Rs, and Q represent electron transfer resistance, Warburg impedance, electrolyte solution resistance, and constant phase elements, respectively. After, the aptamer modified on the BiOBr/N-GNRs/ITO electrode (b), the value of charge-transfer resistance (Rct) increased from 25 Ω to 50 Ω. It could be explained that the aptamer would increase the steric hindrance of the electrode interface, and
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then restrained the transfer of the electrons. At the presence of 5 ng mL–1 AFB1, the
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Rct increased dramatically to 115 Ω due to the specific reaction between AFB1 and the
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aptamer (c). The formation of complex further impeded the transfer the electrons, and then resulted in increased Rct [39]. All these results have verified the successful
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construction of the proposed PEC aptasensor.
3.4 Optimization of conditions for PEC aptasensor
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In order to obtain efficient PEC aptasensor for AFB1 detection, some experimental parameters including the amount of N-GNRs in the BiOBr/N-GNRs, the
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concentration of aptamer, and the incubation time of AFB1 with aptamer were
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optimized. As illustrated in Fig. S1, the photocurrent response increased and then decreased gradually with increasing the weight ratio of N-GNRs from 2% to 10%.
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This fact indicated that the increasing N-GNRs overlapped on the surface of BiOBr nanosheets would block the light absorption of BiOBr. Thus, BiOBr/N-GNRs (5%)
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was chosen for construction of the PEC aptasensor in this work. Furthermore, the aptamer concentration and the incubation time had obvious influence on the performances of the fabricated aptasensor. As shown in Fig. S2A, the PEC response decreased gradually with the increase of the aptamer concentration from 5×10-7 to 3.0 ×10-6 mol L-1. This is ascribed that higher aptamer concentration modified on the electrode could impede the electrons transfer. While the aptamer concentration was further increased to over 2 ×10-6 mol L-1, the photocurrent response almost kept 11
ACCEPTED MANUSCRIPT constant. This phenomenon could be explained that the aptamer immobilized on the surface of the electrode had reached plateau. Therefore, 2 ×10-6 mol L-1 aptamer concentration was selected as the optimal aptamer concentration for the PEC aptasensor. As displayed in Fig. S2B, the photocurrent declined with the increase of incubation time. When it reached 40 min, the photocurrent response almost kept
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constant. It indicated that the aptamer on the electrode has reached saturation.
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Therefore, 40 min was chosen as the optimal incubation time.
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3.5 Analytical performances of PEC aptasensor
Under the optimal conditions, an efficient PEC aptasensor for AFB1 detection
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was fabricated. As shown in Fig. 4C, the PEC signal decreased gradually with the
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increase of the AFB1 concentration. And Fig. 4D displayed the corresponding linear calibration curve reporting the PEC response as a function of the AFB1 concentration
(pg mL–1)], with a correlation coefficient R2 =0.9973. In
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0.4268 -0.0186 [C
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ranging from 5 pg mL–1 to 15 ng mL–1. The regression equation is determined as I = AFB1
addition, the limit of detection was calculated to be 1.7 pg mL–1 (defined as S/N = 3).
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The analytical performance of the fabricated aptasensor has been compared to the
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reported methods towards AFB1, as shown in Table 1. The typical instrumental analytical methods, including TLC [3], UPLC-MS/MS [4] and ELISA [5], have been applied to detect AFB1. However, these methods are restricted to time-consuming pretreatment, high-cost instruments, and required profession skills [6]. Compared to other methods on the basis of fluorescence [40] and electrochemistry [41], the proposed aptasensor possessed a wider linear range. Although there were reported methods demonstrating wide linear ranges comparable to the aptasensor [7, 8], the 12
ACCEPTED MANUSCRIPT PEC immunosensor had limitations associated with high cost, and complex cross-reactivity due to the required antibodies. Noticeably, PEC aptasensor exhibited some advantages including low cost, easy operation, and high sensitivity. 3.6 Selectivity and reproducibility of the PEC aptasensor
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In order to investigate the selectivity of the PEC aptasensor, some interfering species ochratoxin A (OTA) and fumonisin B1 (FB1) were studied in the interference
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test. Under the same conditions, the photocurrrent responses of 50 ng mL–1 interfering
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species with 5 ng mL–1 AFB1 were recorded. As displayed in Fig. S3A, there was more significant photocurrent change of the aptasensor toward 5 ng mL–1 AFB1 than
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that of 10-fold concentration of nonspecific molecules. This sugggested that the
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proposed PEC aptasensor possessed good specificity. Furthermore, the reproducibility and stability are necessary factors to valuate PEC sensor. As displayed in Fig. S3B, the photocurrent of prepared aptamer-BiOBr/N-GNRs/ITO towards 5 ng mL–1 AFB1
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was very stable. In addition, the photocurrent had no obvious change after the electrodde was stored for two weeks at 4 °C. This meant the high stability and good reproducibility of as-fabricated PEC aptasensor.
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3.7 Analytical application in real samples
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The established PEC aptasensor was evaluated by corn samples detection. As illustrated in Table S1, a good recovery in the range of 98.0–102.0% with the relative standard deviation (RSD) of 1.7–2.1% was recorded. Thus, the PEC aptamer-based assay method could be employed to AFB1 detection in real samples.
4. Conclusions In summary, this work demonstrated PEC aptasensing with high selective and sensitive by combining the high-efficient PEC technique with specificity aptamer as 13
ACCEPTED MANUSCRIPT well as the improved photoactivity of BiOBr/N-GNRs. The proposed PEC aptasenor exhibited siginificant advantages in selectivity and sensitity owing to the merits of aptamer and BiOBr/N-GNRs photoactive species. Based on a “signal-off” PEC response of the proposed aptasensor, a broad linear range and a low limit of detection for AFB1 was acquired. And good recoveries were achieved when the established PEC
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aptasenor was applied to detect AFB1 in corn samples. This work not only extended to
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determine other aflatoxins in foodstuff but also provided a promising strategy for food
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safety in PEC biosensing.
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Acknowledgments
This research was supported under the National Natural Science Foundation of
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China (21375050, 21675066, 61601204), the Research Foundation of Zhenjiang
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Science and Technology Bureau (No. NY2016011).
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Compliance with Ethical Standards
Conflict of Interest The authors certify that there is no conflict of interest with any
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individual/organization for the present work. Ethical Approval This article does not contain any studies with human participants or
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animals performed by any of the authors. Informed consent Informed consent was obtained from all individual participants included in the study.
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[41] G. Evtugyn, A. Porfireva, V. Stepanova, R. Sitdikov, I. Stoikov, D. Nikolelis, T.
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ACCEPTED MANUSCRIPT Figure Captions
Scheme 1. Schematic diagram for the construction of PEC aptasensor for AFB1 detection on the basis of the BiOBr/N-GNRs/ITO electrode.
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Fig. 1. (A) High-magnification TEM image of BiOBr/N-GNRs. (B) XRD patterns of
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BiOBr (a) and BiOBr/N-GNRs (b).
Fig. 2. XPS spectra of BiOBr and BiOBr/N-GNRs samples: (A) survey spectrum, (B)
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Bi 4f, (C) Br 3d, (D) O 1s, (E) C 1s and (F) N 1s.
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Fig. 3. (A) Photocurrent responses 0.1 mol L–1 PBS at bias potential of 0 V of pure N-GNRs (a), BiOBr (b), BiOBr/NG (c) and BiOBr/N-GNRs (d) modified ITO
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electrodes. (B) Steady-state PL spectra of BiOBr (a) and BiOBr/N-GNRs (b); (C)
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UV−vis diffuse reflectance spectra and (D) plot of (Ahʋ)2 versus the energy (hʋ) for
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the band gap energy of BiOBr (a) and BiOBr/N-GNRs (b).
Fig. 4. (A) PEC in 0.1 mol L–1 PBS at bias potential of 0 V and (B) EIS
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characterization of aptasensor fabrication process in 0.1 mol L–1 KCl solution containing
5
mmol
aptamer-BiOBr/N-GNRs/ITO
L–1
[Fe(CN)6]3–/4–: (b),
and
BiOBr/N-GNRs/ITO AFB1
interact
(a), with
aptamer-BiOBr/N-GNRs/ITO (c). (C) Photocurrent response of the PEC aptasensor at different concentrations of AFB1 in 0.1 mol L–1 PBS at bias potential of 0 V: 5 pg mL–1 (a), 0.01 ng mL–1 (b), 0.1 ng mL–1 (c), 0.5 ng mL–1 (d), 1 ng mL–1 (e), 3 ng mL–1 (f), 5 ng mL–1 (g), 10 ng mL–1 (h) and 15 ng mL–1 (i); (D) the corresponding linear 19
ACCEPTED MANUSCRIPT calibration curve.
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Table 1 Comparison of the reported techniques for AFB1 detection.
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Scheme 1
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ACCEPTED MANUSCRIPT Fig. 1
(A)
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N-GNRs
b
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BiOBr
Intensity (a.u.)
(B)
40
50
JCPDS NO. 09-0393
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2 Theta (degree)
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a
70
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Intensity (a.u.)
Bi 4p
BiOBr/N-GNRs
159.2 eV
500
750
156
160
164
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(D)
69.2 eV
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BiOBr
O 1s
529.8 eV
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Br 3d
68.2 eV
BiOBr
530.0 eV
BiOBr/N-GNRs 70
BiOBr/N-GNRs
72
528
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68
Binding Energy (eV)
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(E)
531
(F)
C 1s
C-N 287.6 eV C-O 288.6 eV
Intensity (a.u.)
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Intensity (a.u.)
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285
C=N 286.0 eV
537
N 1s Pyrrolic N 399.9 eV
Pyridine N 398.5 eV
Graphitic N 401.8 eV
BiOBr/N-GNRs 288
534
Binding Energy (eV)
C=C 284.6 eV
282
168
Binding Energy (eV)
Intensity (a.u.)
Intensity (a.u.)
68.1 eV 69.1 eV
66
164.5 eV
BiOBr
BiOBr/N-GNRs
Binding Energy (eV)
(C)
Bi 4f
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Bi 4p
BiOBr
164.3 eV
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O 1s
Bi 4d5/2 Bi 4d3/2 Bi 4d5/2
N 1s
C 1s
Bi 4f7/2 Bi 4f5/2
Bi 5d Br 3d
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(B) 159.0 eV
Survey
Bi 4d3/2 O 1s
C 1s
Bi 4f5/2
Bi 5d Br 3d
Intensity (a.u.)
(A)
Bi 4f7/2
Fig. 2
BiOBr/N-GNRs
291
396
Binding Energy (eV)
400
404
Binding Energy (eV)
23
408
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(B) d c
0.3 b a
a
b
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Photocurrent (A)
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Time (s)
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(A) 0.6 0.4
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-Z'' ()
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Photocurrent (A)
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Detection limit (pg mL–1)
References
TLC
16 ~ 115
–
[3]
UPLC-MS/MS
0.5 ~ 69.4
–
[4]
0.1 ~ 10
10
[5]
0.005 ~ 10
1.7
0.0312 ~ 31.2
15.6
PEC immunosensor
0.01 ~ 20
2.1
PEC immunosensor
0.01 ~ 15
3.0
[8]
PEC aptasensor
0.005 ~ 15
1.7
This work
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Electrochemical aptasensor
[40] [41]
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Fluorescent aptasensor
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ELISA
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Method
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[7]
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Graphical Abstract
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ACCEPTED MANUSCRIPT Highlights
BiOBr/nitrogen-doped graphene nanoribbons as photoactive species were constructed A photoelectrochemical aptasensor was fabricated for aflatoxin B1 detection
The PEC aptasensor exhibited a wider linear range with a lower detection limit
The PEC aptasensor was applied to detect aflatoxin B1 in corn samples
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