Aflatoxin B1 electrochemical aptasensor based on tetrahedral DNA

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Biological and Medical Applications of Materials and Interfaces

Aflatoxin B1 electrochemical aptasensor based on tetrahedral DNA nanostructures functionalized three dimensionally ordered macroporous MoS2-AuNPs film Gang Peng, Xiaoyan Li, Feng Cui, Qianying Qiu, Xiaojun Chen, and He Huang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01693 • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 7, 2018

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ACS Applied Materials & Interfaces

Aflatoxin

B1

tetrahedral

electrochemical

DNA

nanostructures

aptasensor

based

functionalized

on three

dimensionally ordered macroporous MoS2-AuNPs film Gang Peng a, Xiaoyan Li a, b, Feng Cui b, Qianying Qiu b, Xiaojun Chen b, *, He Huang c, d * a

School of Biotechnology and Pharmaceutical Engineering, bCollege of Chemistry and Molecular

Engineering, cSchool of Pharmaceutical Sciences and dJiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University, Nanjing 211816, PR China ABSTRACT: In the food safety evaluation, aflatoxin B1 (AFB1) is an important indicator. In this work, we developed an AFB1 electrochemical aptasensor based on tetrahedral DNA nanostructures (TDNs) immobilized three dimensionally ordered macroporous MoS2-AuNPs hybrid (3DOM MoS2-AuNPs) recognition interface and horseradish peroxidase (HRP) functionalized magnetic signal amplifier. To greatly enhance the recognition efficiency, sensitivity and stability of the aptasensor, the AFB1 aptamer-incorporated TDNs were ingeniously combined with the 3DOM MoS2-AuNPs film for the construction of the sensing interface. The aptamers would release from the electrode surface after they reacted with AFB1, and then the hybridization-free TDNs formed. Thus, the biocomposite of DNA helper strands (H1)/HRP functionalized AuNPs-SiO2@Fe3O4 nanospheres would combine with the hybridization-free TDNs due to the hybridization of H1 and TDNs. The more AFB1 existed in the solution, the more H1/HRP-AuNPs-SiO2@Fe3O4 could be combined onto the 3DOM MoS2-AuNPs surface. The current response coming from HRP-catalyzed reduction of H2O2 using thionine (Thi) as electrochemical probe was proportional with the AFB1 concentration. Upon optimal conditions, the aptasensor showed specificity for AFB1, achieving a good linear range of 0.1 fg/mL-0.1 µg/mL and the detection limit of 0.01 fg/mL. Furthermore, the developed aptasensor was also applied for detecting AFB1 content in rice and wheat powder samples, obtaining good results in conformity with those achieved from the high-performance liquid chromatography tandem mass spectrometry (HPLC-MS) method. KEYWORDS: Three dimensionally ordered macroporous, MoS2-AuNPs film, aptasensor, tetrahedral DNA nanostructures, aflatoxin B1, rice sample ■ INTRODUCTION It is well known that toxigenic fungi can produce agricultural commodity contamination mycotoxins. There is a proverb called the people to food for the day, food to security for the first. If people eat the mycotoxin contaminated food, their body will be harmed. Among the series of mycotoxins, aflatoxin B1 (AFB1) is the most common contaminant in foods1 and possesses the highest toxicity due to its ability of binding DNA and proteins.2,3 In the carcinogens, AFB1 is classfied as the Group 1 by the International Agency for Research on Cancer.4 Therefore, the development of rapid and sensitive strategy for detecting AFB1 is essential for food safety. Currently, chromatography methods and immunoassays have been developed for AFB1 detection, such

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as high-performance liquid chromatography (HPLC),5 liquid chromatography tandem mass spectrometry (LC-MS/MS),6 thin layer chromatography (TLC),7 enzyme-linked immunosorbent assay (ELISA),8 immunosensors9 and colorimetric immunoassay.10 Although these methods provide good sensitivity, but there are still some disadvantages including high cost, well-trained operators and difficulty in the preparation of antibodies for small molecules. In the past ten years, researchers have developed varieties of electrochemical aptasensors for AFB1.1-3,11 Compared with other methods, electrochemical aptasensors exhibit attractive superiorities like rapid response, high sensitivity, simple preparation and low cost.12-14 However, the orientation and density of DNA single strand immobilized on the electrode surface was unable to control, leading to the fact that the binding activity of the single strand DNA with the targets were inhibited,15, 16 and the sensitivity and stability of the aptasensors might be affected. For the sake of improving the capture efficiency of the single strand DNA with the target molecules and the stability of aptasensor, tetrahedral DNA nanostructures (TDNs) were recently introduced into the building of aptasensors.17-19 TDNs are a new kind of DNA structures, which possess excellent properties such as mechanical rigidity, structural stability, accurately controlled recognition units and specific orientation. 15, 20 Simultaneously, TDNs could be easily functionalized using different chemical substances.15 Chen et al. have developed an electrochemical sensor for prostate-specific antigen based on TDNs, with the detection limit of 1 pg/mL.16 Zeng et al. also reported that TDNs could be used for developing an electrochemical miRNAs simultaneous detection system.21 Thus, TDNs will be widely applied in the construction of aptasensor. In this paper, we creatively constructed an ultrasensitive electrochemical aptasensor for AFB1 based on aptamer-incorporated TDNs recognition system, which provided higher accessibility and spatial orientation controllability. A 3D ordered macroporous MoS2-Au (3DOM MoS2-Au) film was fabricated as the sensing platform, which enhanced the immobilization amount of TDNs and facilitated the movement of the electrons between the redox probe and the electrode surface. Horseradish peroxidase (HRP) functionalized magnetic nanobeads were used as biomarker, which simplified the separation process and amplified the catalytic current responses. The working mechanism of the newly-fabricated AFB1 aptasensor was based on the conversion of biochemical signals into electrical ones, and it could be also applied in real rice and wheat powder samples detection. ■ EXPERIMENTAL Materials and reagents. All oligonucleotides including the thiolated TDNs sequences, aptamer, helper strand H1 and thiolated sequence D (ssDNA) were customized and purified by Takara Biotechnology Co., Ltd. (Dalian, China). The sequences information was described in Table S1. Every sequence participated in the TDNs construction contains three functional segments, which would combine each other to fabricate the stable TDNs structures. The ssDNA was synthesized for the comparison with TDNs to verify the stability of our proposed aptasensor. Ammonium tetrathiomolybdate [(NH4)2MoS4)], 6-Mercaptohexanol (MCH) and poly (diallyldimethylammonium chloride) (PDDA, 20 wt. %) were purchased from Sigma Chemical Co., Ltd. (St. Louis, MO, USA). The mono-dispersed silica spheres (SiO2, D=500 nm) and thionin acetate (Thi) were obtained from Alfa Asear. Tetraethyl orthosilicate (TEOS), iron chloride anhydrous (FeCl3), sodium acetate anhydrous (NaAc) and hydrogen peroxide (H2O2)were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Polyethylene glycol (PEG) 10,000 was obtained from Xilong Scientific Co., Ltd. (Shantou, China). Potassium ferricyanide (K3[Fe(CN)6]), potassium chloride (KCl), ethylene glycol (EG) and potassium ferrocyanide (K4[Fe(CN)6]), were all obtained from Shanghai Linfeng Chemical Reagent Co., Ltd. (Shanghai, China). Chloroauric acid hydrated (HAuCl4·4H2O) was purchased from Nanjing Chemical


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Reagent Co., Ltd. (Nanjing, China). HRP was obtained from Shanghai Baoman biological technology Co., Ltd., (Shanghai, China). Aflatoxin B1 (AFB1), aflatoxinM1 (AFM1), zearalenone (ZEN), ochratoxin A (OTA) and aflatoxin B2 (AFB2) were purchased from Pribolab Pte. Ltd. (Singapore). All other chemicals were of analytical grade and used without further purification. Stock solution of 3 % PDDA was prepared in the mixture of Tris and NaCl. 0.01 M pH 7.4 of phosphate buffered solution (PBS) was used as the electrolyte. Double-distilled water (DDW) was used throughout the experiments. Apparatus. Surface morphology of 3DOM MoS2-AuNPs electrode was investigated by scanning electron microscopy (SEM, Hitachi S4800). The elemental composition analysis was performed by energy dispersive X-ray spectroscopy (EDS Falcon 60S, EDAX Inc.). Transmission electron microscopy (TEM) images were obtained from a JEOL JEM-200CX microscope. CV, EIS and amperometric measurements were carried out on a CHI 660D electrochemical workstation (Shanghai Chenhua Instrument Company, China). The typical three-electrode cell system consisting a saturated calomel reference electrode (SCE), a 3DOM MoS2-AuNPs modified Au slice working electrode and a Pt auxiliary electrode was used for all the electrochemical measurements. All electrochemical experiments were purged with high-purity N2 to remove O2. Fabrication of the 3DOM MoS2-AuNPs film. The Au substrates were initially cleaned by ultra-sonication in the acetone and ethanol for 10 min, respectively. According to our previous work,22 the Au substrates modified by 3D silica colloidal crystal were prepared. The apertured insulating tape was used to control the electrode area by the diameter (ϕ) of 4 mm. Then the MoS2-AuNPs film was obtained by co-deposition. Briefly, the Au electrode modified by silica was immersed in a 0.1 M KCl solution containing 0.2 mM HAuCl4·4H2O and 5 mM (NH4)2MoS4 for 5 min, and then electro-deposited MoS2-AuNPs into the template interspaces at constant potential of -1 V and the charge was controlled as 18.7 mC. To eliminate the interference of oxygen, the whole electro-deposition process was protected by N2. After that, 5 % HF was used to dissolve the template in 2 min for the obtaining of 3DOM MoS2-AuNPs film. Formation of TDNs. The construction of TDNs based on Watson-Crick base pairing (A1-B1, A2-D2, A3-C3, B2-C2, B3-D3, C1-D1) was depicted in Scheme S1.The mixture of A, B, C and D at the concentration of 1 µM for each strand were firstly prepared using TE buffer (the mixture of 20 mM Tris and 50 mM MgCl2). Afterwards, the resulting mixture was heated to 95 oC for 10 min and then cooled to 4 oC in 30 min, and the TDNs were conceived to be constructed according to the previous literature.17, 19 Fabrication of the biocomposite of HRP/H1-AuNPs–SiO2@Fe3O4. In accordance with an anteriorly reported study with minor modification, Fe3O4 magnetic nanobeads were prepared.23 Briefly, with the help of ultrasonication, 1.0 g of PEG 10000, 0.811 g of FeCl3 and 3.6 g NaAc were dissolved in 40 mL of EG at room temperature. Then, the resulting mixture was held in a poly tetrafluoroethylene reaction kettle at 200 o

C for 8 h. The obtained black solid products were separated by magnetic decantation, rinsed with DDW and

ethanol alternately several times, and dried under vacuum at 60 oC. Au nanoparticles (AuNPs) were prepared following a previous protocol,24 and stored in dark. AuNPs-SiO2@Fe3O4 magnetic composite was prepared according to our previous work25 and dispersed in PBS to achieve a concentration of 20 mg/mL. Then 75 µL of 2 mg/mL HRP and 50 µL of 10 µM H1 were added and the mixture was shaken overnight at room temperature. The HRP/H1-AuNPs-SiO2@Fe3O4 was collected and rinsed, and then dispersed in 1 mL PBS for further use. To verify the amplification effect coming from the HRP/H1-AuNPs-SiO2@Fe3O4, the HRP/H1-AuNPs without the magnetic beads of



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SiO2@Fe3O4 was also prepared for comparison by the similar procedure. Fabrication of the AFB1 aptasensor. The designed strategy of the AFB1 aptasensor was depicted in Scheme 1. After the 3DOM MoS2-AuNPs film was prepared, it was cleaned in 0.5 M H2SO4 via potential scanning between 0 and 1.6 V until obtaining a reproducible CV curve, and was incubated immediately into a 10 µL TDNs solution overnight at room temperature. The thiolated TDNs were then covalently immobilized on the 3DOM MoS2-AuNPs electrode surface to form TDNs-3DOM MoS2-AuNPs. After that the residual active sites were passivated by 2 mM MCH for 2 h, it was incubated in 10 µM AFB1 aptamer (APT) solution at 37 oC for 2.5 h to let the aptamer hybridize with TDNs. When the fabricated AFB1 aptasensor reacted with AFB1 in the solution at 37 oC for 50 min, part of APT would release from the electrode

surface

due

to

the

recognition

combination

of

APT

and

AFB1.

20

mg/mL

HRP/H1/AuNPs-SiO2@Fe3O4 dispersion was then incubated onto the electrode surface at 37 oC and 100 % relative humidity for 2.5 h, which would combine onto the electrode surface due to the hybridization of H1 with the free TDNs. After the excessive HRP/H1/AuNPs-SiO2@Fe3O4 was washed away, the catalytic current was measured with the aid of 0.01 M pH 7.4 PBS containing 25 µM Thi and 1 mM H2O2.

Scheme 1. The schematic protocol of the electrochemical AFB1 aptasensor. Real samples detection. The rice and wheat powder samples were provided by the National Light Industry Food Quality Supervision and Inspection Station of Nanjing. The rice sample was finely ground into powder. Take 5 g of the rice or wheat powder disperse into a 15 mL extraction solution with 80 % methanol and 4 % NaCl. Shake the obtained mixture for 45 min, followed by centrifuging at 4000 rpm for 5 min. Collect the supernatant and dilute it with DDW by the volume ratio of 1:9, and the dilution solution was used as real samples for AFB1 detection. 26, 27 ■ RESULTS AND DISCUSSION Characterization of 3DOM MoS2-AuNPs modified electrode. 3DOM nanomaterials are good candidates for developing biosensors, owing to their outstanding performances, such as controllable aperture, large surface area and good biocompatibility.28 Recently, an analog of graphene, MoS2 was successfully used in electrochemical sensing field due to its 2D structure and good catalytic activity.29 In this work, a 3DOM


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MoS2-AuNPs film modified electrode was obtained by electrodeposition. As shown in Figure 1, the 3DOM structure was clearly observed, indicating that 3DOM MoS2-AuNPs film was successful synthesized. To obtain the optimal performance, the effect of deposition charge on the 3DOM structure was studied. Seen from Figure 1A-D, the diameter of 3DOM increased along with the deposition charge increasing from 2.58 to 18.7 mC. When the deposition charge is 2.58 mC, the 3DOM structure was initially formed with the pore size of about 355 nm, illustrating the depth of the pores is fairly shallow (Figure 1A). Along with the deposition charge increasing, the pore size enlarged gradually. When the deposition charge reached 18.7 mC, the pore size is averagely 430 nm with the wall thickness of about 70 nm. The sum is similar to the diameter of the template silica sphere (Figure 1D), that meant the depth of pores was just the radius of the silica sphere (~250 nm). However, the average pore size decreased to about 320 nm after increasing the deposition charge to 25.8 mC (Figure 1E), due to the appearance of the spherical shell. Seen from the inset of Figure 1E, we could deduce that more than half surface of silica sphere had been covered by MoS2-AuNPs film. The excessive deposition of MoS2-AuNPs made the 3DOM structure become a little disordered, which might be because it was damaged when the template was removed. In addition, the spherical shell would also decrease the amount of immobilized TDNs and increase the steric hindrance of biorecognition. Thus, the optimal deposition charge of 18.7 mC was selected in this work. Moreover, the EDS spectrum displayed in Figure 1F verified the elemental composition of the 3DOM MoS2-AuNPs film were Mo, S and Au, and the atom ratio of Mo and S was 1:2. The electrochemical characteristics of 3DOM MoS2-AuNPs film was investigated by CV and EIS. Figure S1A showed CV of the 3DOM MoS2-AuNPs modified electrode in 5 mM [Fe(CN)6]3-/4- contained 0.1 M KCl solution (curve d). The CVs of bare Au electrode (curve a), 3DOM MoS2 electrode (curve b) and 3DOM Au electrode (curve c) were also presented for comparison. The current response of 3DOM MoS2 electrode was smaller than that of bare electrode due to the poor conductivity of MoS2.30 The synergy of AuNPs and MoS2 based on the excellent conductivity of AuNPs and large specific surface area of MoS2 film caused the peak current of 3DOM MoS2-AuNPs was the largest among all the modified electrodes. According to the Randled-Sevcik equation:31

Ip=2.69×105n3/2AD1/2ν1/2C

(1)

Where Ip is defined as the peak current, n is the quantity of electrons referring to the redox reaction process (=1), D shows the diffusion coefficient (6.7×10-6 cm2s-1), the scan rate is ν (0.1 V s-1) and the concentration of the probe molecule in the solution is C (5.0×10-6 mol cm-3). By calculating the data, the effective surface area (A) of bare electrode, 3DOM MoS2 electrode, 3DOM Au electrode and 3DOM MoS2-AuNPs electrode was 0.071, 0.055, 0.091 and 0.109 cm2, respectively. Thus, the 3DOM MoS2-AuNPs caused a 1.54-fold increase in the electro-active surface area comparing with the bare electrode. EIS was also applied to study the different electrodes. The basic equivalent circuit model was shown as the inset of Figure S1B, which was composed of four parts, Rs, Cdl, Rct and Zw, representing the electrolyte solution resistance, the double layer capacitance, the interfacial charge-transfer resistance and Warburg impedance, respectively.28 EIS image includes two parts, a linear one and a semicircular one, representing the diffusion process at lower frequencies and the electron transfer-limited process at higher frequencies, respectively. The diameter of semicircular portion is equal to the Rct value.32 The Rct value of bare electrode (curve a), 3DOM MoS2 electrode (curve b), 3DOM Au electrode (curve c) and 3DOM MoS2-AuNPs electrode (curve d) were 474, 851, 167 and 54 Ω, respectively; indicating that the speed of electron transfer of 3DOM MoS2-AuNPs film was the fastest. Thus, the EIS measurements agreed with the above CVs results.



Figure 1. SEM images of 3DOM MoS2-AuNPs films with different deposition charges: (A) 2.58, (B) 5.86, (C) 12.9, (D) 18.7 and (E) 25.8 mC, respectively. Inset: the partial enlarged SEM image of Figure E. (F) The EDS spectra of 3DOM MoS2-AuNPs. Characterization of AuNPs-SiO2@Fe3O4. TEM and EDS were used for the characterization of AuNPs-SiO2@Fe3O4. Shown as Figure S2A, the size of monodispersed Fe3O4 spherical particles was about 245 nm. After the modification of SiO2, the black Fe3 O4 core was surrounded by gray layer with the thickness of about 22 nm, indicating the core-shell structure SiO2@Fe3O4 nanosphere was obtained (Figure S2B). The TEM image of AuNPs was displayed in Figure S2C, showing the average particle size of 13.7 nm. After the SiO2@Fe3 O4 nanosphere was positively charged by PDDA modification, it could electrostatically attract AuNPs as much as possible. Thus, observed from Figure S2D, the AuNPs-SiO2@Fe3O4 gathered together owing to the linkage of numerous AuNPs. Furthermore, the elemental composition of AuNPs-SiO2@Fe3O4 was also clarified by EDS, as seen in the inset of Figure S2D. Electrochemical characterization of the AFB1 aptasensor. CV and EIS techniques were used to study the aptasensor fabrication process. Generally, the interfacial property of the biosensor greatly influenced the amount of immobilized oligonucleotides. In this study, the newly-fabricated 3DOM MoS2-AuNPs film was used as the sensing interface, which may increase the immobilized amount of TDNs, and enhance the biocompatibility and conductivity. During the modification process of the electrode, [Fe(CN)6]3-/4- was selected as the electro-active probe for studying the CV changes. Figure 2A showed the CVs of different modification steps: the 3DOM MoS2-AuNPs electrode (curve a), TDNs/3DOM MoS2-AuNPs (curve b), passivated by MCH (curve c), APT/TDNs/3DOM MoS2-AuNPs (curve d) and after the capture of AFB1 (curve e). For the bare 3DOM MoS2-AuNPs electrode, there could be observed a pair of well-defined redox peaks with the anodic peak (Epa) and the cathodic peak (Epc) at 0.265 and 0.151 V, respectively. It exhibited good conductivity and efficient redox-activity, reflecting the peak potential difference (∆Ep) as low as 0.104 V. Immobilize TDNs onto the electrode surface, the ∆Ep increased obviously and the peak current decreased clearly, owing to that the insulative TDNs hindered the electron transfer between the bulk solution and the electrode surface33 and the negatively charged oligonucleotides phosphate backbone prevented [Fe(CN)6]3-/4- approaching the electrode surface.28 After blocking nonspecific sites and passivating the remaining active sites using MCH, the peak current further reduced. After the


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hybridization with APT, the amount of negatively charged oligonucleotides increased again, leading to the peak current continued to decrease. The peak current increased obviously after incubating with 1 ng/mL AFB1, owing to the part of AFB1-captured APT released from the electrode surface. Meanwhile, the electrode modification process was also studied by EIS, and the result was shown in Figure 2B. For the bare 3DOM MoS2-AuNPs electrode (curve a), the value of Rct was 54 Ω and the curve was almost a straight line, indicating the electrons on the electrode surface transferred rapidly and thus the mass diffusion was considered as the limiting step in the electron-transfer process. The value of Rct was 453 Ω after immobilizing TDNs onto the electrode surface (curve b), attributing to the fact that the negatively charged oligonucleotides electrostatically repelled the negative [Fe(CN)6]3-/4- probe29. Furthermore, the fractional surface coverage (θ) of TDNs immobilized on the electrode was estimated by using the formula of θ=1-(Rct(a)/Rct(b))×100%, where Rct(a) and Rct(b) were the Rct values of bare 3DOM MoS2-AuNPs electrode and TDNs modified electrode, respectively. So, the θ value was calculated as 88.1%, exhibiting a fairly high coverage of TDNs. Since the molecule length of one DNA base is averagely 0.34 nm, the length of TDNs edge was about 6.2 nm.34 According to the equilateral triangle area formula, the bottom area of TDNs (STDNs) was about 16.6 nm2. Therefore, the numbers of TDNs (N) can be calculated by the formula of N=Sθ/STDNs, Where S is the effective electroactive surface area of 3DOM MoS2-AuNPs electrode (0.109 cm2). Then, the N was 5.8×1011, which was considered enough for the immobilization of APT. When the electrode surface was immobilized by MCH (curve c) and APT (curve d), the Rct values further increased to 844 and 1245 Ω, respectively. When the electrode was treated with 1 ng/mL AFB1, the Rct value decreased to 947 Ω, indicating the successful capture of AFB1 by APT. All the EIS results agreed with the aforementioned CV results in Figure 2A.

Figure 2. CVs (A) and EIS spectra (B) at different modification steps: (a) bare 3DOM MoS2-AuNPs electrode, (b) TDNs/3DOM MoS2-AuNPs, (c) passivated by MCH, (d) hybridization with the APT and (e) treated with 1 ng/mL AFB1. The work solution was 0.1 M KCl containing 5 mM [Fe(CN)6]3-/4-. Signal amplification using HRP/H1/AuNPs-SiO2@Fe3O4 nanoprobes. The event of AFB1 determination was reported by the HRP-catalyzed reduction of H2O2 using Thi as electrochemical marker. Since HRP undertook the task of amplifying the catalytic signal, it was necessary to increase the amount of HRP involved in the current signal generation as much as possible.35 Nanomaterials with large surface area are usually applied as the support for biomarker immobilization. Herein, AuNPs-SiO2@Fe3O4 magnetic beads were used as the nanoprobe for both the conjugation of H1 and HRP. To clarify the amplification effect of AuNPs-SiO2@Fe3O4 nanospheres, the comparison between AuNPs-SiO2@Fe3O4 and only AuNPs was performed. Figure S3 shows that the CVs of the APT/TDNs/3DOM MoS2-AuNPs in the absence and



presence of 1 fg/mL AFB1, using HRP/H1/AuNPs-SiO2@Fe3O4 and HRP/H1/AuNPs as nanoprobes, respectively. At -0.183 and -0.215 V, a pair of redox peaks originated from the electrochemical oxidation and reduction of Thi was observed (black dash curves).36 In the presence of AFB1, two different nanoprobes combined on the electrode surface, and the reduction peak currents of Thi showing in Figure S3A and S3B both increased (red solid curves), owing to the HRP catalysis. However, the current increment of 2.05 µA in Figure S3B was apparently larger than that of 0.43 µA in Figure S3A, which was 4.77-fold signal amplification. Thus, the AuNPs-SiO2@Fe3O4 nanobeads were much more appropriate for use in this work. Experimental condition optimization. To improve the performance of the aptasensor, various parameters were optimized using the control variable method, including the dosage of TDNs and APT, the hybridization time of TDNs with APT, the capture time of APT and AFB1 and the ratio of HRP vs H1. The dosage of TDNs. The amount of TDNs immobilized on the 3DOM MoS2-AuNPs surface must influence the performance of AFB1 aptasensor, since the fabrication of the sensing interface was the most important base. The TDNs modified electrode TDNs/3DOM MoS2-AuNPs was characterized by CV in 0.1 M KCl containing 5 mM [Fe(CN)6]3-/4-. Fixed the TDNs concentration as 1 µM, the peak current decreased along with the increase of TDNs dosage. When the dosage of TDNs increased from 4 to 10 µL, the peak current kept decreasing. Then, a plateau reached when the dosage exceeded 10 µL, implying the saturation of TDNs immobilization (Figure 3A). Therefore, from the economic perspective, 10 µL of TDNs was enough for use in this work. The dosage of APT and the hybridization time between APT and TDNs. For the sake of maximum efficiency of AFB1 capture, sufficient amount of APT should be immobilized on the electrode surface. Fixed the APT concentration as 10 µM, it could be seen from the curve a in Figure 3B, with the APT dosage increasing from 4 to 8 µL, the peak current of [Fe(CN)6]3-/4- decreased and then became stable. In the same way, the hybridization time between APT and TDNs was also optimized. Shown as the curve b in Figure 3B, the peak current decreased as the hybridization time increased from 0.5 to 2.5 h, and then became stable. Therefore, 8 µL of APT dosage and 2.5 h of hybridization were enough for the subsequent experiments. The reaction time of APT with AFB1. For improving the biosensing performance, it is vital to promote the signal acquirement. Herein, the HRP-catalytic current was the basis of quantitative AFB1 detection. The more HRP/H1/AuNPs-SiO2@Fe3O4 biomarker linked onto the electrode surface, the more sensitive the current signal could be detected and the more sensitive the aptasensor became. So, the reaction time of APT with AFB1 should be optimized, since it would influence the amount of the immobilized HRP/H1/AuNPs-SiO2@Fe3O4 biomarkers on the electrode surface substituted for the released APT. In view of the steric hindrance factor, three different concentrations of AFB1 as 1 fg/mL, 1 pg/mL and 1 ng/mL, respectively, were employed in this experiment. As described in Figure 3C, the stable maximum currents for these three situations were obtained within 30, 40 and 50 min, respectively. Therefore, in order to ensure the reaction time, 50 min of incubation was chosen for all experiment. The volume ratio of HRP vs H1. The immobilization amounts of HRP and H1 on the surface of Au– SiO2@Fe3O4 nanocarrier greatly influenced the aptasensor performance, since HRP participated in the catalytic current amplification while H1 was necessary for the hybridization of HRP/Au–SiO2@Fe3O4 with TDNs/3DOM MoS2-AuNPs. Owing to the fact that HRP and H1 were competitively immobilized onto the


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Au–SiO2@Fe3O4 surface, the dosage of HRP vs. H1 should be balanced and optimized. Fixed the concentrations of HRP and H1 as 2 mg/mL and 10 µM, respectively, the DPV peak currents corresponding to different volume ratios of HRP vs. H1 for 1 pg/mL AFB1 were recorded. From Figure 3D , we can see that the maximum peak current was achieved at the volume ratio of 3:2, which meant the dosages of HRP and H1 were 75 and 50 µL, respectively. So, this specific ratio was chosen in our study.

Figure 3. Effects of (A) the dosage of TDNs, and (B) the dosage of APT (curve a) and the hybridization time of TDNs and APT (curve b) on the CV peak currents of TDNs/3 DOM MoS2-AuNPs and APT-TDNs/3 DOM MoS2-AuNPs in 0.1 M KCl containing 5 mM [Fe(CN)6]3-/4-. Effects of (C) the reaction time of APT-TDNs/3 DOM MoS2-AuNPs and different concentrations of AFB1 as (a) 1 fg/mL, (b) 1 pg/mL and (c) 1 ng/mL, respectively; and (D) the volume ratio of HRP vs. H1 on the DPV peak currents in 0.01 M PBS (pH 7.4) containing 25 µM Thi and 1 mM H2O2. Amperometric determination of AFB1 with aptasensor. Upon the optimal conditions, the DPV curves were obtained. Figure 4A showed that the DPV peak current of Thi increased with the increase of AFB1 concentration. The background current of i0 was only the Thi response in the absence of AFB1, which just come from the electrostatic absorption of Thi on the APT-TDNs-3DOM MoS2-AuNPs surface. As shown in Figure 4B, a linear relationship between the absolute value of peak current changes (∆ip=|ip- i0|) and the minus logarithm of CAFB1 (-lg CAFB1) ranging from 0.1 fg/mL to 0.1 µg/mL was presented, with a detection limit of 0.01 fg/mL at 3σ (signal-to-noise ratio). The regression equation was ∆ip ＝ (0.402±0.015)lgCAFB1＋(9.647±0.184), with the correlation coefficient of 0.9903 (n=5). In order to further highlight the advantages of the aptasensor described in this work, the performance of our aptasensor was compared with other AFB1 aptasensors in literature. Analyze the data listed in Table 1, our electrochemical



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aptasensor based on the TDNs/3DOM MoS2-AuNPs interface and HRP/AuNPs-SiO2@Fe3O4 signal probe exhibited a lower detection limit and a wider linear range, indicating its potential applicability in real AFB1 detection.

Figure 4. (A) DPV responses of the developed aptasensor in 0.01 M PBS (pH 7.4) containing 25 µM Thi and 1 mM H2O2 after dealing with different concentrations of AFB1: (a) 0, (b) 0.01 fg/mL, (c) 0.1 fg/mL, (d) 1 fg/mL, (e) 0.01 pg/mL, (f) 0.1 pg/mL, (g) 1 pg/mL, (h) 0.01 ng/mL, (i) 0.1 ng/mL, (j) 1 ng/mL, (k) 0.01 µg/mL, (l) 0.1 µg/mL and (m) 1 µg/mL. (B) Calibration curve of the developed aptasensor for AFB1 detection. Table 1．．Comparison of the proposed aptasensor with the other AFB1 aptasensors Technique

Signal amplification

Linear range (ng/mL)

LOD (pg/mL)

Ref.

DPV

π-shape structure

0.007-5

2

2

Fluorescence

-

5-100

1600

37

Fluorescence

Zn(II)-ion

0.001-0.05

0.3

38

-7

-5

SWV

Telomerase, Exo III

1×10 -0.1

6×10

3

Fluorescence

DNase I

1-100

350

39

SERS

AuNS core–AgNP satellite

0.001-1

0.48

40

4

Colorimetric

-

0.1-1×10

100

41

Fluorescence

-

0.998-9.98×104

312

42

EIS

-

0.0312-31.2

15.6

43

5×10 -5

0.025

44

1×10-7-100

1×10-5

This work

RT-qPCR DPV

3DOM MoS2-AuNPs, HRP/AuNPs-SiO2@Fe3O4

-5

Specificity, reproducibility, stability of the AFB1 aptasensor. The specificity, reproducibility and stability were pivotal factors for the development of an aptasensor. To evaluate the specificity of this aptasenor, the proposed assay was challenged with other mycotoxins including AFB2, AFM1, ZEN and OTA. As shown in Figure S4, much higher ∆ip was obtained from the target AFB1 than from the other interferents. The excellent specificity was attributed to the nature of specific recognition between APT with its target molecule. The reproducibility tests were studied by using three batches of five independent aptasensors to detect 1 fg/mL, 1 pg/mL and 1 ng/mL AFB1, respectively. The RSD of the DPV current responses were 6.5, 5.9 and 4.2 %, respectively. The proposed aptasensor possesses good reproducibility, owing to the


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experimental conditions were precisely controlled in each fabrication step. The stability of the proposed AFB1 aptasensor was necessary to be investigated, and the comparison with ssDNA/3DOM MoS2-AuNPs as sensing interface was also carried out. Two aptasensors were fabricated at one time for each type, one was used immediately, and the other was stored at 4oC for one month. It was found that the DPV peak current responses for these two aptasensors to 1 ng/mL AFB1 retained 91 and 62% of their initial responses, respectively, exhibiting the excellent sensitivity and stability of the proposed apatasensor. This was attributed to the fact that the mechanical rigidity and structural stability of TDNs provided a stable basis for APT immobilization,45 and the 3DOM MoS2-AuNPs provided a biocompatible microenviorment for stabilizing the identification activity of biomolecules. Real samples detection. To check the feasibility and applicable potential of the aptasensor, the real sample (rice and wheat powder) extraction solution spiked with different concentrations of AFB1 were tested by standard addition method. In Table 2, the relative error (RE) values obtained by the aptasensor for the two samples ranged between -3.6 and 4.1 %, which was compared with the reference values obtained from the commercial HPLC-MS method, indicating the suitability of the developed AFB1 aptasensor in real samples. Table 2．．Detection of AFB1 in real samples by HPLC-MS and the proposed aptasensor. Sample

rice

wheat powder a

HPLC-MS

Proposed aptasensor

(ng/mL)a

(ng/mL)a

0.3

0.286

0.293

+2.4

2

1.0

1.009

0.986

-2.3

3

2.5

2.574

2.482

-3.6

1

0.3

0.291

0.303

+4.1

2

1.0

0.996

1.024

+2.8

3

2.5

2.497

2.465

-1.3

No.

Added (ng/mL)

1

RE (%)

Average of three measurements.

■ CONCLUSIONS Herein, a novel, highly sensitive, selective and stable electrochemical aptasensor for AFB1 was successfully developed. The highlights of this work included the following four aspects: (1) Nanomaterials brought great enhancement of the aptasensor performance. 3DOM MoS2-AuNPs film and Au–SiO2@Fe3O4 composites possessed such advantages as good conductivity, desirable biocompatibility and large surface area, forming the ideal carriers for high loading of TDNs, HRP and H1 biomolecules and fast electron transfer between electrode surface and the electroactive probe in solution; (2) the bio-identification between APT and the target molecule established the foundation for the specificity of the aptasensor; (3) the mechanical rigidity and structural stability of TDNs and nanomaterials, with the precise control of experimental parameters ensured the stability and reproducibility of the aptasensor; (4) The good specificity and stability enabled the aptasensor to be applied in real sample detections, and acquired the similar results with the commercial HPLC-MS technique. In addition, the proposed assay demonstrated a general strategy for the on-site monitoring of other mycotoxins in food matrices by simply employing various APT capturers. ■ ASSOCIATED CONTENT

Supporting Information Fabrication of the magnetic composite of AuNPs–SiO2@Fe3O4, schematic illustration of the TDNs (Scheme



S1), CVs and (B) EIS spectra at different electrodes: (a) bare Au slice electrode, (b) 3DOM MoS2, (c) 3DOM AuNPs and (d) 3DOM MoS2-AuNPs (Figure S1), TEM images of (A) Fe3O4, (B) SiO2@Fe3O4 (C) AuNPs and (D) AuNPs-SiO2@Fe3O4, and EDS spectrum of AuNPs-SiO2@Fe3O4 (Figure S2), signal amplification using HRP/H1/AuNPs-SiO2@Fe3O4 nanoprobes (Figure S3), DPV current changes of the developed AFB1 aptasensor in the presence of various mycotoxins including AFB1，AFB2，AFM1, ZEN and OTA (Figure S4), The sequences information of all oligonucleotides (Table S1) (PDF) ■ AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected] (XJ Chen); [email protected] (H Huang)

ORCID Xiaojun Chen: 0000-0002-8017-6844

Notes The authors declare no competing financial interest. ■ AKNOWLEDGEMENTS This work was financially supported by the National Natural Science Foundation of China (No. 21575064, 21476111, 21776136), National key research and development program (2017YFC1600604), Natural Science Foundation of Jiangsu Province (BK20151535), the Six Talent Peaks Project in Jiangsu Province (2016-SWYY-022), the Qinlan Project of Jiangsu Education Department, and the Program for Innovative Research Team in Universities of Jiangsu Province (2015).

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Table of Contents Graphic


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Aflatoxin B1 electrochemical aptasensor based on tetrahedral DNA

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