Colorimetric aptasensing of ochratoxin A using Au@Fe3O4 nanoparticles as signal indicator and magnetic separator

Author’s Accepted Manuscript Colorimetric aptasensing of ochratoxin A using Au@Fe3O4 nanoparticles as signal indicator and magnetic separator Chengquan Wang, Jing Qian, Kun Wang, Xingwang Yang, Qian Liu, Nan Hao, Chengke Wang, Xiaoya Dong, Xingyi Huang www.elsevier.com/locate/bios

PII: DOI: Reference:

S0956-5663(15)30559-5 http://dx.doi.org/10.1016/j.bios.2015.11.004 BIOS8134

To appear in: Biosensors and Bioelectronic Received date: 2 September 2015 Revised date: 23 October 2015 Accepted date: 2 November 2015 Cite this article as: Chengquan Wang, Jing Qian, Kun Wang, Xingwang Yang, Qian Liu, Nan Hao, Chengke Wang, Xiaoya Dong and Xingyi Huang, Colorimetric aptasensing of ochratoxin A using Au@Fe 3O4 nanoparticles as signal indicator and magnetic separator, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.11.004 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 galley proof before it is published in its final citable 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.

Colorimetric aptasensing of ochratoxin A using Au@Fe3O4 nanoparticles as signal indicator and magnetic separator Chengquan Wang,a,1 Jing Qian,b,1 Kun Wang,b,* Xingwang Yang,b Qian Liu,b Nan Hao,b Chengke Wang,a Xiaoya Dong,b Xingyi Huang a,* a

School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, P.

R. China b

Key Laboratory of Modern Agriculture Equipment and Technology, School of

Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013, P. R. China * Corresponding author: Tel.: +86 51188791800; Fax: +86 51188791708; E-mail addresses: [email protected] (K. Wang);

[email protected] (X. Y. Huang). 1

These authors contributed equally to this work.

1

Abstract Gold nanoparticles (Au NPs) doped Fe3O4 (Au@Fe3O4) NPs have been synthesized by a facile one-step solvothermal method. The peroxidase-like activity of Au@Fe3O4 NPs was effectively enhanced due to the synergistic effect between the Fe3O4 NPs and Au NPs. On this basis, an efficient colorimetric aptasensor has been developed using the intrinsic dual functionality of the Au@Fe3O4 NPs as signal indicator and magnetic separator. Initially, the amino-modified aptamer specific for a typical

mycotoxin,

ochratoxin

A

(OTA),

was

surface

confined

on

the

amino-terminated glass beads surafce using glutaraldehyde as a linker. Subsequently, the amino-modified capture DNA (cDNA) was labeled with the amino-functionalized Au@Fe3O4 NPs and the aptasensor was thus fabricated through the hybridization reaction between cDNA and the aptamers. While upon OTA addition, aptamers preferred to form the OTA-aptamer complex and the Au@Fe3O4 NPs linked on the cDNA were released into the bulk solution. Through a simple magnetic separation, the collected Au@Fe3O4 NPs can produce a blue colored solution in the presence of 3,3’,5,5’-tetramethylbenzidine and H2O2. When the reaction was terminated by addition of H+ ions, the blue product could be changed into a yellow one with higher absorption intensity. This colorimetric aptasensor can detect as low as 30 pg mL-1 OTA with high specificity. To the best of our knowledge, the present colorimetric aptasensor is the first attempt to use the peroxidase-like activity of nanomaterial for OTA detection, which may provide an acttractive path toward routine quality control of food safety.

2

Keywords:

Au@Fe3O4

nanoparticles;

Peroxidase-like

activity;

Colorimetric

aptasensor; Magnetic separation; Ochratoxin A 1. Introduction Colorimetric assays based on the idea that the color changes can be easily monitored with the naked eyes and do not require costly and sophisticated instrumentations, are of great significance in the accurate detection of targets in point-of-care diagnosis or field analysis (Song et al., 2011; Qian et al., 2015). The main challenge associated with colorimetric assay is transforming the detection events into color changes (Song et al., 2011). During the past few decades, a series of colorimetric assays have been fabricated based on the target induced aggregation of gold nanoparticles (Au NPs) (Alsager et al., 2015; Dwivedi et al., 2015; Ramezani et al., 2015). However, most of such sensing platforms are not applicable in complex samples, as the dispersion of Au NPs can be significantly affect by the presence of additional salt, proteins, DNA, and small molecules (Soh et al., 2015). In recent years, the tremendous development in nanotechnology has produced many unique nanomaterials with excellent peroxidase-like activity, such as CeO2 NPs (Asati et al., 2009), Co3O4 NPs (Mu et al., 2012), V2O5 nanowires (Andre et al., 2011), CdS NPs (Maji et al., 2012), and Au@Pt nanostructures (He et al., 2011). These enzyme mimetics have the ability to catalyze the oxidation of peroxidase substrates to produce distinguishable color change when enzymatically oxidized by H2O2, similar to that found in natural peroxidases, thus providing another strategy for colorimetric assays. Since the magnetic Fe3O4 NPs were found to possess the intrinsic peroxidase-like

3

activity (Gao et al., 2007), the Fe3O4 NPs-based colorimetric assays have attracted increasing attention with advances in magneto-controlled separation (Wei et al., 2008; Zhang et al., 2011; Yang et al., 2014; Yang et al., 2013; Liu et al., 2011). When replacing horseradish peroxidase (HRP), the Fe3O4 NPs displayed excellent performances in traditional enzyme linked immunosorbent assay (ELISA) as labels (Gao et al., 2007; 17, Yang et al., 2014; Yang et al., 2013). Further researches have demonstrated that the construction of Fe3O4 NPs-based hybrid materials can exhibit enhanced peroxidase-like activity, such as Fe3O4@Pt NPs (Ma et al., 2013), hollow Pt/Fe3O4 composites (Yang et al., 2014), graphene oxide (GO)-Fe3O4 nanocomposites (Dong et al., 2012), graphene quantum dots-Fe3O4 nanocomposites (Wu et al., 2014), Au-Fe3O4 dumbbell structure (Liu et al., 2013), and Au/Fe3O4/GO composites (Zhang et al., 2015). Some of these composites have been applied in the colorimetric detection of glucose (Yang et al., 2014; Dong et al., 2012) and Hg2+ (Zhang et al., 2015) or phenolic compound removal (Wu et al., 2014). Contaminated food can lead to more than 200 diseases and thus introduce 2 million deaths annually (Sharma et al., 2015). Among all these issues, mycotoxin pollution is one important branch. Ochratoxin A (OTA), the most common naturally occurring mycotoxin, is known to be nephrotoxic, hepatotoxic, neurotoxic, immunotoxic and teratogenic to human beings (Girolamo et al., 2011; Ma et al., 2013; Mishra et al., 2016). What makes it worse is that OTA molecule is stable enough and able to resist to most food processing steps. For the reasons, the highest OTA concentration in a variety of foodstuffs has been set by the European Commission, e.g.

4

7.4 nM for cereal products, 12.4 nM for roasted coffee, 5 nM for wine, and 7.5 nM for grape juice (Yang et al., 2013; Wu et al., 2012). Together with preventive measures, methods for OTA determination in contaminated raw materials are essential to avoid the risk of OTA consumption. Conventional methods for OTA quantification are based on instruments such as high performance liquid chromatography (HPLC) connected to fluorescence (Zhao et al., 2014) or tandem mass (Bazin et al., 2013) spectrometry. But high cost, long processing times, and the need of sophisticated instrumentation and trained personnel, have hindered their wide application (Yang et al., 2015; Wu et al., 2012). In comparison with antibodies, aptamers hold significant advantages such as high affinity and specificity, a better stability, ease-of labeling, target versatility, simple production, great reproducibility, commercially available, and easy storage (Barthelmebs et al., 2011; Zhang et al., 2012). Ever since the first report of the selected aptamer for OTA (Cruz-Aguado and Penner, 2008), different versions of aptasensors have been described combined with fluorescent (Wang et al., 2015; Wei et al., 2015), electrochemical (Bonel et al., 2011; Tong et al., 2012), chemiluminescence (Mun et al., 2014), conductometric (Dridi et al., 2015), and colorimetric (Soh et al., 2015; Yang et al., 2011; Liu et al., 2015; Lee et al., 2014) transducers. However, most of the reported colorimetric aptasensors were constructed by using Au NPs as signal indicator (Soh et al., 2015; Yang et al., 2011; Liu et al., 2015); the one using the peroxidase-like activity of nanomaterials has not been reported in literatures. In this work, Au NPs doped Fe3O4 (Au@Fe3O4) NPs have been synthesized by a

5

one-step solvothermal method. The peroxidase-like activity of the resultant Au@Fe3O4 NPs was determined and compared with the pure Fe3O4 NPs and Au NPs obtained by etching Fe3O4 away from the Au@Fe3O4 NPs. Due to the high peroxidase-like activity of Au@Fe3O4 NPs, they were successfully applied in fabrication of the colorimetric aptasensor for OTA to replace of HRP, in which the Au@Fe3O4 NPs were used as not only separation carriers, but also detection indicator. 2. Experimental 2.1. Reagents Diethylene glycol (DEG), ethylene glycol (EG), polyethylene glycol (PEG), HAuCl4·4H2O, (3-aminopropyl) triethoxysilane (APTS), and glass beads (GBs, 5 mm in diameter) were purchased from Sinopharm Chemical Reagent Co. Ltd (China). OTA, ochratoxin B (OTB), fumonisin B1 (FB1), aflatoxins B1 (AFB1), and 3,3’,5,5’-tetramethylbenzidine

(TMB)

were

obtained

from

Sigma-Aldrich.

Amino-modified capture DNA (cDNA): 5'-TTA CGC CAC TTA CAC CCG ATC-NH2-3' and amino-modified aptamer: 5'-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA-NH2-3' were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). DNA oligonucleotide stock solutions were prepared with 50 mM Tris-HCl buffer. Double-distilled water was used throughout the study. 2.2. Apparatus The morphologies of the samples were analyzed with transmission electron microscope technique (TEM, Hitach S-2400N, Japan). X-ray diffraction (XRD) spectra were performed on a Bruker D8 ADVANCE diffractometer (Germany) with

6

Cu Kα (λ = 1.5406 Å) radiation. Fourier transform infrared (FTIR) spectra were obtained from a Nicolet Nexus 470 FTIR spectrophotometer (Thermo Nicolet, USA). X-ray photoelectron spectroscopy (XPS) was recorded on ESCALAB 250 multitechnique surface analysis system (Thermo Electron Co., USA). Magnetic measurements were carried out using a BHV-55 Vibrating sample magnetometer. The UV-vis spectra and the time-dependent absorbance changes were collected on a UV-2450 spectrophotometer (Shimadzu, Japan). 2.3. Preparation of Au@Fe3O4 NPs Au@Fe3O4 NPs was prepared by a one-step solvothermal method. Typically, 2 mM of FeCl3 was dissolved in 20 mL of EG/DEG (1:1, v/v). In the next step, 1.5 g of NaAc and 1 g of PEG were dispersed in above suspension by sonication. Subsequently, 0.5 mL of HAuCl4 (2%) was added to above suspension and then the stirring was allowed to proceed for another 30 min to form a homogeneous suspension. The mixture was transferred into a Teflon-lined stainless-steel autoclave and heated at 200 °C for 6 h. The products were separated from the reaction medium under an external magnetic field, which was washed with ethanol repeatedly and dispersed in 5 mL of ethanol for later use. For comparison, pure Fe3O4 NPs were also prepared under the same condition without the addition of HAuCl4. Au NPs were obtained by etching Fe3O4 away from the Au@Fe3O4 NPs in 0.5 M H2SO4 (Lee et al., 2010). 2.4. Peroxidase-like catalytic activity study The catalytic activity study of the resultant Au@Fe3O4 NPs, pure Fe3O4 NPs, and

7

Au NPs was performed as follows: 50 μg of any of the nanoparticles was first mixed with 0.1 mmol of H2O2 and 0.5 mg of TMB in 1 mL acetate buffer solution (ABS, 0.2 M, pH 4.0). The absorption spectrum and photographs were taken after the mixture was incubated for 15 min at 40 oC in a water bath. 2.5. Aptasensor fabrication 400 μL of APTS was added to 5 mL of the Au@Fe3O4 NPs. After being vigorously stirred for 1 h, the mixture was centrifuged and washed with ethanol and water, to remove excess APTS. Subsequently, the aminated Au@Fe3O4 NPs were immersed in a 5% glutaraldehyde solution for 2 h at 37 °C, followed by washing, and re-dispersed in 5 mL of Tris-HCl buffer. In the next step, 200 μL of 100 μM cDNA was mixed with the glutaraldehyde-functionalized Au@Fe3O4 NPs. The mixture was sonicated for 5 min, plus incubating for 2 h at 37 °C on a shaker. Magnetite was utilized to separate the cDNA modified Au@Fe3O4 (cDNA-Au@Fe3O4) bioconjugates, followed by washing three times with Tris-HCl. For initial cleaning, fifty of GBs were pretreated according to the reported method of Wei et al. (2011) and the preparation of the aptamer modified GBs (GB-aptamer) was provided in the Supporting Information. Then the hybridization reaction between GBs-aptamer and cDNA-Au@Fe3O4 was allowed to proceed for 2 h at

37

°C.

After

rinsing

with

Tris-HCl

buffer

for

several

times,

the

GB-aptamer/cDNA-Au@Fe3O4 bioconjugations were redispersed in 40 mL Tris–HCl buffer and used as the aptasensor in further study.

8

2.6. Colorimetric detection of OTA One of the as-prepared aptasensor (GB-aptamer/cDNA-Au@Fe3O4) was immersed into 60 μL of the OTA-containing solutions prepared in 50 mM Tris-HCl buffer (pH 7.4, containing 120 mM NaCl, 20 mM CaCl2, and 5 mM KCl) for 60 min, followed by rinsing with buffer solution. After removal of the GB, the dissociated Au@Fe3O4 NPs in the solution were accumulated with external magnetic forces. When the supernatant was removed, the collected Au@Fe3O4 NPs were first mixed with 0.1 mmol of H2O2 and 0.5 mg of TMB in 1 mL of ABS (0.2 M, pH 4.0). When the mixture was incubated for 15 min at 40 oC in a water bath, the absorption spectrum and photographs were recorded when the enzymatic reaction was stopped by adding 50 μL of 2 M H2SO4. 3. Results and discussion 3.1. Characterization of the Au@Fe3O4 NPs Both the Au@Fe3O4 NPs and the nanoparticle doped in were sphere-like with a mean diameter of 150 nm and 20 nm, respectively (Fig.1A and B). Taking advantage of the stability differences between Au NPs and Fe3O4 NPs in acid solution that Fe3O4 NPs can be dissolved while Au NPs stay intact, some single-component Au NPs, verified by the XPS measurements (Fig. S1), were obtained by etching Fe3O4 away from the Au@Fe3O4 NPs in 0.5 M H2SO4. The nanoparticle doped in Au@Fe3O4 NPs was confirmed to be Au NPs snice its morphology and diamater are equal to that of the one wrapped inner the Au@Fe3O4 NPs (Fig. S2). The Au@Fe3O4 NPs have ever

9

dispersed into ethanol with vigorously ultrasound before TEM measurements, but there was no single Fe3O4 NPs could be observed, we therefore conclude that Au@Fe3O4 NPs are very stable. Following a similar procedure, the pure Fe3O4 NPs densely packed with Fe3O4 NPs were obtained without the introdution of HAuCl4, was determined to be approximately 400 nm (Fig. S3). None noanoparticles was observed from the TEM image after using the same etching steps. The chemical composition of Au@Fe3O4 NPs has been ascertained by XPS measurements. The peaks of C, O, and Fe elements were successfully observed in Fig. 2A. However, no Au peaks was detected even by measuring the high-resolution XPS spectrum of Au 4f regions. As the existence of Au element has been clearly indicated in Fig. S1, the constituent elements of the resulting Au@Fe3O4 NPs can still conclude to be C, O, Fe, and Au elements. All the C elements and part of O elements may come from the used solvent PEG. In comparision with EG and DEG, PEG with a long chain is eaiser to twine round the tiny Fe3O4 particles in Au@Fe3O4 NPs. Both the Au@Fe3O4 NPs (curve a in Fig. 2B) and pure Fe3O4 NPs (curve b in Fig. 2B) exhibited typical XRD diffraction peaks at 2θ values of 30.1°, 35.5°, 43.1°, 53.5°, 57.0°, and 62.6°, which were indexed to (220), (311), (400), (422), (511), and (440) planes of cubic structure of Fe3O4 (JCPDS no. 19-0629), respectively (Wan et al., 2012). As expected, four additional peaks centered at 38.1°, 44.3°, 64.5°, and 77.6° are appeared for Au@Fe3O4 NPs. These values are in good agreement with that of the Au NPs obtained from the etching Fe3O4 away from Au@Fe3O4 NPs (curve c in Fig. 2B), which can be ascribed to the (111), (200), (220), and (311) planes for the

10

face-centered-cubic gold (JCPDS No. 04-0784), respectively (Zhang et al., 2008). The diffraction peaks from the pure Fe3O4 NPs and Au NPs match well with those in Au@Fe3O4 NPs, suggesting the successful formation of the heterostructure (Lee et al., 2010). It was found that the as-obtained Au@Fe3O4 NPs could be completely accumulated within 2 s upon placement of a magnet and could be easily redispersed in solution by hand-shaking (inset of Fig. 2C), possessing the performance very similar to superparamagnetic particles (Xie et al., 2010). However, both the pure Fe3O4 NPs (curve a) and Au@Fe3O4 NPs (curve b) should be typical ferromagnetic in a way, considering the small magnetic remanence and coercivity (Fig. 2C). The saturation magnetization (Ms) value of the resulting pure Fe3O4 NPs was calculated to be 90.6 emu g-1 and that for Au@Fe3O4 NPs was 51.2 emu g-1. Compared with the pure Fe3O4 NPs, the obvious decrease in Ms of Au@Fe3O4 NPs can be explained by the diamagnetic contribution from the wrapped Au NPs, yet the value of both structured magnetization was still large enough for magnetic separation applications. In FTIR spectrum of the resulting Au@Fe3O4 NPs (Fig. 2D), a strong and broad absorption band in the range of 3000-3200 cm-1 can be assigned to O–H stretching vibrations coming from -OH groups in PEG and water adsorbed on the Au@Fe3O4 NPs. The absorption band that occurs at 1642 cm-1 is due to the O–H bending. The –CH– bending vibration peaks of CH2 in PEG molecule were observed at 1405 and 1325 cm-1 (Yang et al., 2012). The C–O–C symmetric stretching in PEG chain shows a weak peak at 1055 cm-1 (Yang et al., 2012). The peak located at 581 cm-1 could be

11

associated with the Fe–O stretch vibrations (Hou et al., 2011). All these results confirm that the PEG molecules have been effectively bounded with the Au@Fe3O4 NPs and thus their surfaces possess rich hydroxyl groups. 3.2. Peroxidase-like catalytic activity study The natural peroxidases can catalyze the oxidation of peroxidase substrate TMB in the presence of H2O2, and subsequently a blue product, oxidized TMB (oxTMB), was quickly formed with a maximum absorbance at 652 nm. The peroxidase-like activity of the synthesized Au@Fe3O4 NPs (curve b), pure Fe3O4 NPs (curve c), and Au NPs was examined by using TMB + H2O2 system. As shown in Fig. 3A, the solution was nearly colorless with a very low absorbance in the measured range when the nanoparticles were absence. With any of the nanoparticles, In contrast, the solutions displayed a blue color with maximum absorbance at 652 nm, but with obvious variations in absorbance intensity. The peroxidase-like activity of Au@Fe3O4 NPs was nearly 1.3-fold and 5.2-fold enhancement than that of the pure Fe3O4 NPs and the resultant Au NPs. Such catalytic enhancement can be assigned to the synergetic effect that occurs at the interface of Au NPs and Fe3O4 support in the heterostructure (Liu et al., 2013; Li et al., 2015; Lee et al., 2010). From the absorption spectra of the Au@Fe3O4 NPs, we observed a large red shift of the surface plasmon resonance band compared to that of the Au NPs by etching Fe3O4 away (Fig. S4), demonstrating a strong interfacial interaction between Au and Fe3O4 (Lee et al., 2010). This result confirmed that the enhanced catalytic activity of Au@Fe3O4 NPs was caused by the synergetic interaction between the two components rather than a simple

12

addition of the activities of them (Li et al., 2015). The time-dependent catalytic activity of these enzyme mimetics was displayed in Fig. 3B. Clearly, they exhibited different levels of activity over a reaction period of 15 min, in the order of Au@Fe3O4 NPs > pure Fe3O4 NPs > Au NPs > without the catalyst, suggesting Au@Fe3O4 NPs could be excellent peroxidase mimetics for the colorimetric assay with a strong response. To investigate whether the peroxidase activity of Au@Fe3O4 NPs is related to the solution pH, we compared the catalytic activities of Au@Fe3O4 NPs in a series of ABS solutions with varied pH values. As can be seen from Fig. 3C, higher catalytic activity was received in acidic solutions (pH 3.0–4.5) than that in near neutral solutions. The maximum catalytic activity was obtained in a solution with a pH value of 4.0. Similar to the natural peroxidase, the catalytic activity of Au@Fe3O4 NPs is also dependent on the reaction temperature. As indicated in Fig. 3D, the maximum catalytic activity of the Au@Fe3O4 NPs was obtained at a solution temperature of 40 °C. In a suitable concentration range of the substrate H2O2, typical Michaelis-Menten curve was obtained for Au@Fe3O4 NPs (Fig. S5). By using the Lineweaver-Burk plots of the double reciprocal of the Michaelis-Menten equation (Dong et al., 2012), the Michaelis-Menten constant (Km) and the maximal reaction velocity (Vmax) was calculated out and listed in Table S1. As can be seen, the apparent Km value of the Au@Fe3O4 NPs with H2O2 as the substrate was similar to that of the natural HRP (Gao et al., 2007) and much lower than that reported for Fe3O4 NPs (Gao et al., 2007)

13

and Au NPs (Luo et al., 2010), indicating a lower H2O2 concentration was required for the Au@Fe3O4 NPs than for Fe3O4 NPs and Au NPs to obtain the maximum activity (Dong et al., 2012). Besides, the decreasing Km value of Au@Fe3O4 NPs proves that the doping of Au NPs into Fe3O4 NPs effectively improved the catalytic affinity toward H2O2, indicating that the resulting Au@Fe3O4 NPs possess a higher affinity for H2O2 than Fe3O4 NPs and Au NPs (Dong et al., 2012). The affinity of the Au@Fe3O4 NPs is very close to that of natural HRP which makes the hybrids have high activities and extensive applications. 3.3. Aptasensor fabrication and its working principle To explore the practical application, we developed an aptasensor using the intrinsic dual functionality of the Au@Fe3O4 NPs as magnetic separator and signal indicator. The Fe3O4 NPs have been reported to be modified with SiO2, PEG or dextran to improve the biocompatiblity (Gao et al., 2007). As indicated by XPS and FTIR measurements, the as-obtained Au@Fe3O4 NPs possessed abundant PEG molecules and therefore no further modification steps was required to improve their biocompatiblity. These exposed hydroxyl groups from PEG molecules offers an ideal anchorage substrate for the chemical modification. In this work, APTS was selected to couple with the exposed hydroxyl groups on the Au@Fe3O4 surface to yield an amino-terminated self-assembled monolayer. The distal amino groups on Au@Fe3O4 surfaces were reacted with glutaraldehyde to bind with one of its aldehyde groups. The treated Au@Fe3O4 NPs were incubated with amino-modified cDNA for 2 h to immobilize the cDNA on the surface by the covalent bonds forming between the free

14

aldehyde groups and amino groups of the aptamer. The characterized P 2p peak from the phosphate backbone of cDNA was clearly observed at 133.5 eV from cDNA-Au@Fe3O4 (curve b, Fig. 4A); yet no clear peak was observed in the same region for Au@Fe3O4 NPs without cDNA immobilization (curve a, Fig. 4A), confirming cDNA was conjugated successfully onto the surface of Au@Fe3O4 NPs (Wang et al., 2006). Silanization of glass surfaces is a standard method to build up biocompatible layers for biomolecules attachment to solid surfaces. In this work, the GBs were first activated with 1:1 MeOH/HCl and concentrated H2SO4 to clean the surface and generate a high density of hydroxyl groups on the surface. The first step in fabrication of GB-aptamer was the reaction between APTS to modify the GBs with the amino groups, and the hydroxyl groups present on the GBs surface. Then the amino-modified aptamer was conjugated onto the amino-functionalized GBs, using glutaraldehyde as linkage. After the treatment with glycine to block the residual aldehyde groups and nonspecific binding sites, Au@Fe3O4 NPs were then brought onto the GBs surface through the hybridization reaction between cDNA and the surface

confined

aptamers,

to

form

the

sandwich

sensing

interface

of

GB-aptamer/cDNA-Au@Fe3O4. A typical sandwich aprasensor was designed for OTA and its working principle is illustrated in Fig. 4B. When OTA (10 ng mL-1) was introduced to the sensing system, the Au@Fe3O4 NPs linked on the cDNA were released into the bulk solution, due to the fact that the OTA prefer to form the OTA-aptamer complex. After removal of the

15

supernatant using a simple magnetic separation, the collected Au@Fe3O4 NPs can quantitatively convert TMB into oxTMB to produce a blue colored solution in the presence of H2O2 just as HRP does. The maximum absorbance of the reaction product was at 652 nm with an intensity of 0.16 (Fig. 4C). When the reaction was terminated by addition of H+ ions, the blue oxTMB could be changed into a yellow product with the maximum absorbance at 450 nm possessing a higher intensity of 0.36 (Fig. 4D). Control experiments showed that no Au@Fe3O4 NPs was collected when OTA concentration was 0 ng mL-1 because no obvious characteristic absorption peaks and color could be monitored (Fig. S6). 3.4. Colorimetric detection of OTA The effect of the binding time greatly influenced the final sensing of the target molecules. As shown in Fig. 5A, it was apparent that the absorption intensity of the released Au@Fe3O4 NPs obviously increased with the increasing binding time from 10 to 60 min and then reached a plateau in 60 min. This suggested that 60 min was enough and thus chosen as the incubation time in the following detection. As indicated in Fig. 4B, the more OTA molecules in the detection system, the more Au@Fe3O4 NPs were dissociated from the sensing interface and thus increased the absorption intensity of the solution. Based on this aptamer-switched signal-on aptasensing strategy, the intensity of the absorption at 450 nm was increased successively upon the incubation with an increasing amount of OTA (Fig. 5B). More importantly, the color variation of the reaction solution for increasing contents of OTA exhibited continuous color changes from light to deep yellow, which could be clearly

16

monitored with the naked eyes (top of Fig. 5B). By analyzing the absorption intensity (I) with OTA concentrations (Fig. 5C), a good linear relationship was obtained between I and the logarithm of the OTA concentrations (inset in Fig. 5C). The linear equation is I =0.1297+0.1905 log (c/ng mL-1) (R2=0.9810) over a wide concentration range of 0.5–100 ng mL-1. The detetion limit was calculated to be 30 pg mL-1 based on S/N=3. Its sensitivity is lower than that of the recently reported electrochemical (Xie et al., 2014; Huang et al., 2013) and fluorescent (Wang et al. 2015) aptasensor using certain signal amplification strategies but with the obvious advantage that semiquantitative detection can be performed with the naked eyes. However, its sensitivity is much higher than that observed in the existing colorimetric aptasensors with Au NPs (Soh et al., 2015; Yang et al., 2011; Liu et al., 2015) or hemin (Lee et al., 2014) as signal indicator. We thus provide a simple way for highly sensitive colorimetric detection of OTA using the peroxidase-like activity of nanomaterial. The selectivity of this aptasensor was evaluated by the comparison of the sensing results corresponding to 10 ng mL-1 of OTA and 50 ng mL-1 of OTB, FB1, and AFB1. As shown in Fig. 5D, the response signal to OTB was a bit high because OTB is the structural analog of the OTA and possesses the combination ability with OTA aptamer (Kuang et al., 2010). However, the structures of FB1 and AFB1 are distinct different from that of OTA, so the response signals are almost neglectable. All these results demonstrated that the selectivity of the developed colorimetric aptasensor is sufficient for the practical application. The reproducibility of the aptasensor was also investigated at the OTA concentration of 10 ng mL-1. The relative standard deviation

17

(RSD) for five measurements was 4.9%, indicating that the reproducibility of designed aptasensor was acceptable. 3.5. Analytical application in real samples The performance of the aptasensor showed that it could be used for OTA detection in cereal samples since the maximum limit set by the European Commission is 7.4 nM (equal to 3 ng mL-1). To verify applicability and accuracy, this colorimetric aptasensor was applied to peanut (without shell) or corn samples lacking of OTA from the supermarket and confirmed with ELISA Kit, but spiked with OTA. After the real samples were treated with our reported procedure (Wang et al., 2015), the extract was passed through a 0.45 mm syringe filter and then adjusted to pH 7.0, followed by dilution with Tris-HCl buffer to a final OTA concentration of 1, 10, 50 ng mL-1, respectively. As can be seen from Table 1, the recoveries of the spiked samples ranged from 92.0 to 108.1%, with RSD lower than 8.4%, demonstrating that this colorimetric aptasensor can be used for OTA detection in real samples with the satisfied results. 4. Conclusions Au@Fe3O4 NPs have been prepared by a simple method and their peroxidase-like activity was nearly 1.3-fold and 5.2-fold enhancement than that of the pure Fe3O4 NPs and the resultant Au NPs, due to the synergistic effect between Fe3O4 NPs and Au NPs. With the Au@Fe3O4 NPs as signal indicator and magnetic separator, an efficient colorimetric aptasensor has been developed for OTA detection in a wide linear range from 0.5 to 100 ng mL-1 with a detection limit of 30 pg mL-1. The aptasensor displayed an excellent selectivity against potentially interfering molecules and robust

18

operation in peanut samples. This aptasensing strategy provides an acttractive path toward routine quality control of food safety. The simplicity and performance of the aptasensing platform renders it applicable to other targets once the aptamer sequences are available. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at Acknowledgements The present work was supported by the National Natural Science Foundation of China (Nos. 21405063, 21175061, 21375050, and 31071549), the Natural Science Foundation of Jiangsu province (No. BK20130481), China Postdoctoral Science Foundation funded project (2015T80517), Project Sponsored by 2015 fourth "333 high level talent project" of Jiangsu province (No. BRA2015320), Natural Science Foundation of the Jiangsu Higher Education Institutions of China (No. 14KJA550001), and the Program Sponsored for Scientific Innovation Research of College Graduate in Jiangsu Province (KYLX_1072). References Alsager, O.A., Kumar, S.L., Zhu, B.C., Travas-Sejdic, J., McNatty, K.P., Hodgkiss, J.M., 2015, Anal. Chem. 87, 4201–4209. Andre, R., Natalio, F., Humanes, M., Leppin, J., Heinze, K., Wever, R., Schroeder, H.C., Mueller, W.E.G., Tremel, W., 2011, Adv. Funct. Mater. 21, 501–509. Asati, A., Santra, S., Kaittanis, C., Nath, S., Perez, J.M., 2009, Angew. Chem. Int. Ed.

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Table 1 Results of OTA detection in real cereal samples (n=3). Added (ng mL-1)

Found (ng mL-1)

Recovery (%)

RSD (%)

1

1

0.92

92.0

6.9

2

10

9.72

97.2

8.4

3

50

47.74

95.5

7.3

1

1

0.96

96.0

5.7

2

10

10.81

108.1

6.3

3

50

49.17

98.34

7.9

Sample Peanuts

Corn

24

Highlights •

Au@Fe3O4 NPs have been synthesized by a facile one-step solvothermal method.

• Au@Fe3O4 •

NPs possess high peroxidase-like activity due to the synergistic effect.

The rich -OH groups on the Au@Fe3O4 NPs facilitate their modification with

aptamer. •

An efficient colorimetric aptasensor for OTA was developed based on the

Au@Fe3O4 NPs. •

Au@Fe3O4 NPs were used as both signal indicator and magnetic separator.

25

Figure captions Fig. 1. TEM images of Au@Fe3O4 MNPs at low (A) and high (B) magnification. Fig. 2. (A) The wide scan XPS spectrum of Au@Fe3O4 NPs. Inset: the narrow-scan XPS of the Fe 2p regions. (B) XRD patterns of Au@Fe3O4 NPs (a), pure Fe3O4 NPs (b), and Au NPs (c). (C) Magnetic hysteresis loops of pure Fe3O4 (a) and Au@Fe3O4 (b) NPs. Inset: the separation-redispersion process of Au@Fe3O4 NPs. (D) FTIR spectrum of the as-obtained Au@Fe3O4 NPs. Fig. 3. Typical photographs and absorption spectra (A) and the time-dependent absorbance at 652 nm (B) of TMB + H2O2 solution in the absence (a) and presence of Au@Fe3O4 NPs (b), Fe3O4 NPs (c), and Au NPs (d). Dependence of the peroxidase-like activity of Au@Fe3O4 NPs on pH (C) and temperature (D). Fig. 4. (A) The narrow-scan P 2p XPS of Au@Fe3O4 (a) and cDNA-Au@Fe3O4 (b). (B) Schematic diagram of the basic principle for OTA aptasensing. The absorption spectra and the corresponding photographs of the reaction solution (catalyzed by the collected Au@Fe3O4 NPs when the aptasensor incubated with 10 ng mL-1 of OTA) before (C) and after (D) the addition of 2 M H2SO4. Fig. 5. (A) The absorption responses of the aptasensor under different binding time with OTA at the concentration of 10 ng mL-1. (B) The absorption intensity corresponding to various OTA concentrations (from a to j: 0.05, 0.1, 0.3, 0.5, 1, 5, 10, 50, 100, and 200 ng mL-1). (C) The absorption inensity in (B) was plotted as a function of concentration of OTA. Inset: the calibration curve for OTA detection. (D) The selectivity of the aptasensor.

26

Fig. 1

A

B

27

-50

-100 -10000 -5000 0 5000 10000 Applied Magnetic Field / Oe

440

200

311

72

D

4000

581

0

48 60 2 / Degree

1055

b

36

1405 1325

a

24

1642

50

1200

3150

100 C

300 600 900 Binding Energy / eV

220

c

Transmittance/ a.u.

Magnetization / emu g

-1

0

422 511

400

311

220

a

111

700 710 720 730 740 Binding Energy / eV

b

Intensity / a.u.

Fe 2p1/2

B Fe 2p3/2

O 1s Counts/ a.u.

Counts/ a.u.

A

C 1s

Fig. 2

3000 2000 1000 Wavenumber/ cm-1

28

Fig. 3

0.45 B

A

c

0.30

0.4 0.2

b c

0.0

d a 450

600 750 Wavelength/ nm

0.15

0

Relative activity/ %

100

80 60 40 20 3

4

pH

5

6

d a

0.00

900

100 C

Relative activity/ %

b

Absorbance

Absorbance

0.6

300 600 Time/ s

900

D

80 60 40 20

30 40 50 60 Temperature/ oC

29

Fig. 4

Counts/ a.u.

A

P 2p

a

b 125

0.4

C

D

0.3

Absorbance

0.6

130 135 140 145 Binding Energy/ eV

Absorbance

0.4 0.2 0.0

0.2

0.1

0.0

450

600 750 Wavelength/ nm

900

450

600 750 Wavelength/ nm

900

30

Fig. 5

B

Relative Absorbance

100 A

0.6 Absorbance

j

90 80 70

0.4 0.2 0.0

0

20 40 60 80 Incubation time/ min

a

300

0.6 C

Absorbance

Absorbance

0.4 0.6

Absorbance

0.4 0.2

0.4 0.2

600

D

0.3 0.2 0.1

0.0 -2

0.0

400 500 Wavelength/ nm

-1

0

1

2

log(cOTA/ ng mL-1)

0

50 100 150 cOTA/ ng mL-1

200

OTA

OTB

FB1

AFB1

31