Cauliflower-Inspired 3D SERS Substrate for Multiple Mycotoxins

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Cauliflower-Inspired 3D SERS Substrate for Multiple Mycotoxins Detection Jinjie Li, Heng Yan, Xuecai Tan, Zhicheng Lu, and Heyou Han Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04622 • Publication Date (Web): 22 Feb 2019 Downloaded from http://pubs.acs.org on February 22, 2019

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Analytical Chemistry

Cauliflower-Inspired 3D SERS Substrate for Multiple Mycotoxins Detection Jinjie Li†, §, Heng Yan‡, §, Xuecai Tan#, Zhicheng Lu†, Heyou Han*, † †State

Key Laboratory of Agricultural Microbiology, College of Food Science and

Technology, College of Science, Huazhong Agricultural University, Wuhan, Hubei 430070, P.R. China ‡ Hubei

Provincial Engineering and Technology Research Center for Food Quality and

Safety Test, Hubei Provincial Institute for Food Supervision and Test, Wuhan, Hubei 430075, P.R. China #School

of Chemistry and Chemical Engineering, Guangxi University for Nationalities,

Nanning 530008, P.R. China §Equal

contribution.

*Corresponding Author E-mail: [email protected].

1

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT: Surface-enhanced Raman spectroscopy (SERS) is a promising analytical tool, but simultaneous detection of multiple targets using SERS remains a challenge. Herein, a cauliflower-inspired 3D SERS substrate with intense hot spots was prepared through sputtering Au nanoparticles (Au NPs) on the surface of polydimethylsiloxane coated anodic aluminum oxide (PDMS@AAO) complex substrate. As a result, the cauliflower-inspired 3D SERS substrate achieved the highest SERS activities at a sputtering time of 8 min. Under the optimal conditions, this SERS substrate possessed a low detection limit of 10−12 M, excellent enhancement uniformity (relative standard deviation, RSD = 4.57%) and high enhancement factor (2.2×106) for 4mercaptobenzoic acid (4-MBA). Furthermore, the results of Raman showed that the 3D-Nanocauliflower SERS substrates could realize the simultaneous label-free detection for three mycotoxins (aflatoxin B1, deoxynivalenol, and zearalenone) in maize for the first time. It behaved good linear relationship between the concentrations and Raman intensities of aflatoxin B1, zearalenone, and deoxynivalenol. For the three mycotoxins, this method exhibited the limit of detection (LOD) of 1.8 ng/mL, 47.7 ng/mL, and 24.8 ng/mL (S/N = 3), respectively. The 3D-Nanocauliflower SERS substrates with dense hot spots presented remarkable SERS effect and activity, which could be act as a potential candidate for SERS substrate applied in the rapid and labelfree detection. KEYWORDS:

Surface-enhanced

Raman

spectroscopy,

3D-Nanocauliflower

substrate, hot spots, mycotoxins, label-free detection

2


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INTRODUCTION Maize is the number one cereal crop in the world and is mainly consumed as human food.1,2 However, maize is susceptible to pathological changes (for example, Gibberella ear rot), which are closely associated with mycotoxin contamination during postharvest handling, processing, and storage.3,4 About 25% of maize and maize based products in the world are contaminated with mycotoxins at different degrees5 and thus caused a large loss of food products. In addition, mycotoxins are carcinogenic, nephrotoxic, immunosuppressive, hepatotoxic, and mutagenic properties. When mycotoxins enter the food chain through contaminated maize, they can pose a huge threat to humans and livestock.6,7 Some mycotoxins could exist simultaneously in the food products and the most common mycotoxins in maize are aflatoxin B1 (AFB1), deoxynivalenol (DON), and zearalenone (ZON).2,8 Wherein, AFB1, recognized as a group I carcinogen, has strong carcinogenicity and toxicity to humans and animals.9,10 Also, DON could result in emesis, anorexia, hemorrhage, diarrhea, and digestive disorders11 and ZON might cause carcinogenicity, teratogenesis, abortion as well as estrogenic effect.12 Owing to the risk such as varied toxicity and stable existence in many food processing, mycotoxins have been a worldwide problem that causes the loss of the maize consumption and threaten the human health.13 Hence, it is essential to develop facile and sensitive method to monitor the mycotoxins in food matrices. Till now, conventional methods for detecting mycotoxins include high-performance liquid chromatography (HPLC),14 immunoaffinity based methods,15,16 liquid chromatography

combined

with

mass

spectrometry,17

and

enzyme-linked 3



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immunosorbent assays (ELISA).18 Though these methods can achieve good detection results, such as high sensitivity or selectivity. However, they are time-consuming, expensive and complicated, which became their bottlenecks in the actual applications.8,19 At present, some new methods have been developed for detecting mycotoxins, for example, surface plasmon resonance,20 aptamer bound electrochemical methods,21 SERS,22 DNAzyme labeled immunoassays,23 and microbead-based assays.24 Among these new methods, SERS is considered to be a robust technique for the rapid and accurate detection of mycotoxins owing to its excellent sensitivity, noninvasive detection capability, and unique fingerprint effect.25-29 Due to the electromagnetic field enhancement effects, Raman signals can be largely amplified when the target analytes are placed around the plasmonic nanoparticles.30-34 Further, it has been demonstrated that strong electromagnetic field enhancements are related to hot spots generated at interstitial junctions or nanogaps among metallic nanostructures through localized surface plasmon resonances (LSPR).34-40 Recently, SERS substrates with desired sensitivity, density, and reproducibility of hot spots are widely used to enhance the SERS signal.41-46 However, it is challenging to engineer hot spots and further improve the density and intensity of hot spots on SERS substrates. Thus, it is in great demand to develop SERS substrates with easy to prepare, sensitive, and dense hot spots for rapid and label-free detection. Cauliflower has a charming natural form with has large contact areas and many bulges, which are conductive to the formation of hot spots. Inspired by this, the combination of SERS and cauliflower structural substrate can be an effective 4


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strategy in SERS applications. According to the above facts, a 3D-Nanocauliflower SERS substrate with desired hot spots and good reproducibility was designed, which was used for the rapid and sensitive detection of three kinds of mycotoxins in maize. The preparation process was depicted in Scheme 1. First, anodic aluminum oxide (AAO) films were fabricated via two-step anodization. Then, PDMS (polydimethylsiloxane) was poured onto the surfacemodified AAO template and solidified to form PDMS@AAO. Finally, after removing the aluminum (Al) base, Au nanoparticles were sputtered on the surface of PDMS@AAO complex substrate. The obtained 3D-Nanocauliflower SERS substrate had tremendous contact area and noticeable SERS hot spots, which contributed to the strong enhancement of Raman signals. As a result, the simultaneous detection of multimycotoxins (including AFB1, DON, and ZON) in maize are successfully realized using the developed 3D-Nanocauliflower SERS substrate, which made it a promising candidate in rapid and label-free detection. EXPERIMENTAL SECTION Chemical Reagents. Trichlorooctadecylsilane (OTS, >85%) and 4-mercaptobenzoic acid (4-MBA, 90%) were obtained from Aladdin Co., Ltd. (Shanghai, China). Aflatoxin B1 (AFB1), zearalenone (ZON), and deoxynivalenol (DON) were purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Ultrapure aluminum (Al, 99.999%) was kindly provided by General Research Institute for Nonferrous Metals (Beijing, China). Other chemicals with analytical grade, like perchloric acid (HClO4), phosphoric acid (H3PO4), chromium oxide (Cr2O3), and oxalic acid were supplied by Sinopharm 5



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Chemical Reagent Co., Ltd. (Shanghai, China). Water purified by a Milli-Q system (resistivity of 18.2 MΩ cm) was used for sample preparation. Characterizations. An inVia Raman spectrometer (Renishaw, U.K.) coupled with a confocal microscope was used to determine the Raman data. The SERS spectra were measured using diode laser of 785 nm at low laser power (5 mW). The exposure time for each measurement was 10 s exposure with 1 accumulation. SEM images were observed using scanning electron microscopy (SEM, SU8000, Japan). Reflection spectra were recorded by ultraviolet-visible diffuse reflectance spectroscopy (TU-1901, Beijing Purkinje General Instrument Co.,Ltd.), with a smooth gold surface defined as a 100% reflection mirror for reference. Fabrication of AAO Array. AAO array was prepared according to the method described by Lee and our previous work47,48 with slight modification. Briefly, to exclude the effect of locally enhanced anodization due to the surface roughness, the ultra-pure Al foils were firstly electropolished in a mixture solution of ethanol and HClO4 (4:1, v/v). Then, the AAO templates were prepared using a two-step anodization of electropolished Al foils. In detail, the first anodization was conducted in 0.3 M oxalic acid at about 4 °C. The anodization time was 4 h at a constant voltage of 40 V. After that, the oxide layer of aluminum was detached from the Al foils through washing by mixture liquid (6 wt % H3PO4 and 1.8 wt % H2CrO4) at 60 °C for 1 h. Subsequently, another 30 min of anodization (the second time) was carried out under the same conditions. Finally, Al foils were treated in a H3PO4 solution (5 wt %) at 30 °C for 30 min to enlarge the pore diameter. 6


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Preparation of PDMS@AAO Complex. A self-assembled OTS monolayer was functionalized on the surface of AAO template for diminishing the surface energy between PDMS and AAO template. After that, a PDMS elastomer-curing agent mixture solution (mass radio of 10:1) was poured onto the prepared AAO template and solidified at 85 °C for 1 h. Then, the aluminum bases were removed by immersing the samples in a 0.1 M CuCl2 and 3 M HCl solution at 0 °C. To further remove impurities, the complex was washed by deionized water several times. Finally, the complex was segmented into 5×5 mm2 sections for further use. Preparation and Characterization of 3D-Nanocauliflower Substrate. The 3DNanocauliflower substrate was fabricated using the method described in previous work.49 Briefly, the surface of PDMS@AAO complex substrate was sputtered with Au at a ratio of 3.6 nm/min in a protective atmosphere of Ar2 with an ETD-3000 Ion sputtering (Beijing Elaborate Technology Development Ltd., Beijing, China). The sputtering operation was conducted at different periods of duration (every sputtering period consisted of a 1 min working duration coupled with a 1 min break) with 4 mA ion current under ~8 Pa pressure of Ar2 (99.999%). Next, the 3D-Nanoflower substrate was immersed in various concentration of 4-MBA ethanol solution (1 mL) for 4 h and then washed by ethanol to remove unbound molecular and dried at nitrogen atmosphere. Finally, the substrate was used to conduct the sensitivity, reproducibility, and stability analysis. Preparation of Food Samples. Different concentrations of AFB1, ZON, and DON were prepared by dissolving in 20% methanol solution. Measurements were taken at 5 7



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random points using a 5 μL drop of as-prepared mycotoxins solution, which were dropped onto the 3D-Nanocauliflower substrate and dried at room temperature. Real Food Sample Detection. After drying, maize samples (~1 g) were fully ground by mortar. Then, 2 mL of methanol water solution (20%, v/v) was added in maize samples. After sonication for 30 min, maize samples were centrifuged for 20 min at 12000 r/min. Subsequently, 1 mL supernatant was collected and transferred into other centrifugal tubes. Finally, AFB1, ZON, and DON standard samples at different concentrations were added in supernatant. The obtained samples were used as real samples for detection with the above-mentioned method. For multiple components detection, AFB1, ZON, and DON were added in supernatant with a final concentration of 0.05, 5 and 5 μg/mL, respectively. Data Analysis. All the Raman spectra were analyzed using a WiRE 2.0 software suite (Renishaw, U.K.). Spectral preprocessing includes two steps: smooth and baseline correction. Besides, the Raman enhanced effect of the 3D-Nanoflower substrate was theoretically estimated using the COMSOL Multiphysics software. The electric field distribution model was simulated based on the 3D-Nanoflower structures presented in SEM images.50 RESULTS AND DISCUSSION Preparation and Characterization of 3D-Nanocauliflower Substrate. The synthesis of 3D-Nanocauliflower substrate included two processes: prepare AAO arrays and sputter Au NPs. First, the preparation of AAO array was carried out based on the two-step anodization approach. Figure 1 presents the morphology images of the 8


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AAO template observed by SEM. The top-view image demonstrated that AAO template had highly ordered arrangement, and the size of the ordered domains was ~90 nm (Figure 1a). Besides, the side-view image indicated that AAO template had unique nanoarchitecture morphology and the depth of template was ~ 600 nm (Figure 1b). Furthermore, Figure 1c shows the template has ordered bottom morphology. All the morphology characterizations revealed that the AAO template was in agreement with other previous reports.48,51 Cracks often occur in the mold when transferring AAO to other substrate during the preparation process of the substrate. According to literature reported by Kim52, nanostructures without crack formation on the AAO mold surface could be obtained after adding PDMS with high ﬂexibility in the system. After formation of PDMS@AAO complex, Au nanoparticles were sputtered on the surface of PDMS@AAO array to prepare 3D-Nanocauliflower substrate (Scheme 1). The evolution of 3D-Nanocauliflower surface morphologies in relation to the sputtering time (4 to 14 min at an interval of 2 min) is showed in Figure 2. At an initial time of 4 min, few Au NPs and nanogaps were observed in the 3D-Nanocauliflower substrate (Figure 2a). With the increasing of sputtering time, the number of Au NPs and nanogaps gradually increased, giving rise to the increase of hot spots (Figure 2b-c). At 8 min, the substrate had plentiful Au NPs and nanogaps. With the further extension of sputtering time from 10 min to 14 min, the increasing number of nanocauliflowers were covered by big aggregated Au NPs, which may reduce the effective nanogaps (Figure 2d-f). Furthermore, the activities of SERS substrates with different sputtering time were 9



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measured by incubating with the same concentration of 4-MBA. As sputtering time increased from 2 to 8 min, a substantial increase in SERS intensity of 4-MBA at 1580 cm−1 was observed. However, the SERS intensity obviously dropped down as the deposition time further prolonged from 8 min to 14 min (Figure 3a, b). Moreover, from the reflectance spectra of 3D-Nanocauliflower substrate (Figure S4), an obvious reflectivity dip was evidently observed when the deposition time was 8 min. Therefore, the optimal deposition time was selected as 8 min, which was in accordance with the SEM results (Figure 2). Sensitivity, Reproducibility, and Stability of 3D-Nanocauliflower Substrate. 4-MBA was chosen to act as a signal molecule to determine the sensitivity, reproducibility, and stability of 3D-Nanocauliflower substrate. As shown in Figure 4a, two strongest characteristic SERS bands of 4-MBA at 1080 cm−1 and 1580 cm−1 are displayed. It was noted that the 4-MBA with a low concentration (10−12 M) could still be identified by the SERS substrate, which indicated that the 3D-Nanocauliflower substrate showed a lower limit of detection than other substrates reported in the literatures.53,54 Under such a low concentration, the molecule on the substrate is close to single molecule level. And a bi-analyte SERS method as described by Le Ru and Etchegion was used to demonstrate this phenomenon.55 This method is a contrast based spectroscopy technique using two molecules with different fingerprints at the same time. In this experiment, an aqueous solution of 4-MBA and R6G with same concentration was dispensed on the 3D-Nanocauliflower substrate for single molecules SERS detection with laser excitation of 785 nm. Raman intensity mappings of these two 10


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molecular vibration modes (4-MBA band at 1080 cm−1 and R6G band at 1360 cm−1) at concentrations of 10−10 M and 10−12 M are shown in Figure S1 a-d. When the aqueous solution at a low concentration of 10−12 M, the percentage of overlapping 4-MBA-R6G signal decreased and single molecule event had occurred (Figure S1 c-e). This result agreed with the literature reported by Le Ru and Etchegoins55. The bi-analyte nature of target molecules served as a strong evidence that the spectrum of one certain analyte from an individual pixel was attributed to single molecule. In addition, in order to investigate the background signal, the substrate is measured under the same conditions and reported in Figure S2. It was found that the characteristic band of 4-MBA at 1580 cm−1 was scarcely observed on 3D-Nanocauliflower substrate, suggesting that the substrate had a weak background interference during SERS detection. The SERS mapping image of 3D-Nanocauliflower substrate is performed to estimate the spot-to-spot reproducibility and is presented in Figure 4c. Each pixel at the spatial position on SERS substrate symbolized the Raman intensity of 4-MBA at 1580 cm−1. The uniformity pixel was clearly captured (Figure 4c), reflecting that 3DNanocauliflower substrate exhibited high spot-to-spot reproducibility. Furthermore, the reproducibility of substrate to substrate was further evaluated by recording the SERS signals of 40 random points from 5 substrates (Figure 4d). It was showed that relatively consistent Raman intensities were found in all SERS active points. Through statistical calculation, the relative standard deviation (RSD) of the Raman intensity at 1580 cm−1 was ~4.57% (Figure 4e), indicating that 3D-Nanocauliflower substrate possessed good reproducibility. Judging from above results, it could be deduced that the 3D11



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Nanocauliflower substrate exhibited great potential in practical applications with good sensitivity and excellent reproducibility. Besides, as shown in Figure 4b, the stability of 3D-Nanocauliflower substrate is recorded. The prepared SERS substrates were divided into two groups: one was stored in the air and the other was kept in N2. Three substrates were taken from each of the the two groups for Raman testing. The Raman intensity of substrate after 6 days of storage in N2 atmosphere still maintained at a high value (∼20 000). After exposing to N2 or air environment, the 3D-Nanocauliflower substrate showed nearly the same performance, which revealed that the SERS substrate owned good stability. With further extension of storage time to 21 days, the Raman intensity storing in air and N2 had slight drop, suggesting the negligible effect on the sample detection. To further estimate the SERS effect of 3D-Nanocauliflower substrate, the electric field distribution is simulated and shown in Figure S3. The electric field intensity from weak to strong was represented by the color from blue to red. The interparticle gaps of small Au NPs were estimated to be about 1-2 nm, which had the highest electric field intensity and corresponded to the locations of hot spots. The simulation results illustrated that the nanosized gaps of Au NPs contributed to the strong electric field intensity, which also demonstrated that the 3D-Nanocauliflower substrate possessed strong SERS enhanced effect. Moreover, according to methods and Eq 1 (see supporting information), the enhancement factor (EF) of the 3D-Nanocauliflower substrate was calculated to be 2.2×106 (the details for the EF calculation is provided in the Supporting Information), which further revealed that the 3D-Nanocauliflower 12


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substrate possessed remarkable SERS performance. Moreover, the comparison of the enhancement factor and reproducibility between 3D-Nanocauliflower substrate and with other SERS substrates is shown in Table S1, indicating the 3D-Nanocauliflower substrate have excellent performance. Detection of Mycotoxins. Till now, it was turned out that the 3D-Nanocauliflower substrate possessed excellent sensitivity, good reproducibility, and superior stability. The 3D-Nanocauliflower substrate was then employed to detect three mycotoxins (AFB1, ZON, and DON). Firstly, 5 μL of three mycotoxin solutions with different concentrations was added on the surface of the 3D-Nanocauliflower substrate separately and completely dried at room temperature. Then, the samples were measured and recorded by Raman spectrometer. Figure 5 presents the SERS spectra and linear calibration plot of different concentrations of AFB1, ZON, and DON. The Raman intensity of AFB1, ZON, and DON all increased with the increasing of mycotoxin concentrations (Figure 5a, 5c, and 5e). The characteristic peaks of AFB1, ZON, and DON are marked with band in Figure 5a, 5c, and 5e. Table 1 displays the major Raman peaks of AFB1, ZON, and DON, respectively. The characteristic bands at 1272 cm−1 (Figure 5a), 880 cm−1 (Figure 5c), and 1364 cm−1 (Figure 5e) showed remarkable variations and were selected as the standard peak to quantitatively analyze the AFB1, ZON, and DON. Figure 5b, 5d, and 5f presents the linear calibration plot between Raman intensity and the level of AFB1, ZON, and DON, respectively. The intensities of Raman peak at standard peaks were linear with the mycotoxin concentrations. And the linear regression equations are presented in Figure 5b, 5d, and 5f, respectively. The 13



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y value presented the intensity of Raman peak at standard peaks and x value presented the mycotoxin concentrations. Another important parameter in quantitative analysis is the limit of detection (LOD). Calculation details were described in the Supporting Information. Based on the signal-to-noise method which defined by The International Union of Pure and Applied Chemistry (IUPAC)56, the limit of detection (LOD) of SERS substrate for AFB1, ZON, and DON was 1.8 ng/mL, 47.7 ng/mL, and 24.8 ng/mL, respectively. In addition, Table S3 shows the maximum residue limits (MRLs) and LOD of AFB1, ZON, and DON in China, European Union, and our method. By comparison, we found that the values of our method are much lower than that of China and European Union, reflecting that the proposed SERS substrate showed excellent performance in mycotoxin detection. Moreover, Table S4 shows the recovery of AFB1, ZON, and DON in maize samples. Through spiking different concentrations of the target AFB1, ZON, and DON into the maize samples separately, the recovery rate is calculated and listed in Table S3. The recovery rate of AFB1, ZON, and DON was 94-110%, 97.8-104%, and 93-120%, respectively. The results verified that the 3D-Nanocauliflower substrate is acceptable in practical application. The principal components analysis (PCA) is conducted for further confirming the SERS performance of 3D-Nanocauliflower substrate on detecting the mycotoxins in maize and presented in Figure 6. AFB1, ZON, and DON separated into individual clusters clearly and the clinical sensitivity was over 98%, which demonstrated that the 3D-Nanocauliflower substrate could effectively identify and differentiate these 14


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mycotoxins. Detection of Multiple Components of Mycotoxins in Real Maize Samples. SERS is widely used to detect several substances due to exhibiting molecularly narrow band spectra. In this work, the 3D-Nanocauliflower substrate was used to detect three kinds of mycotoxins (AFB1, ZON, and DON) simultaneously. The characteristic bands of AFB1 (1271 cm−1, 1344 cm−1, and 1499cm−1), ZON (456 cm−1 and 1036 cm−1), and DON (659 cm−1 and 1365 cm−1) could be markedly distinguished through the spectrum (Figure 7), revealing that the 3D-Nanocauliflower substrate could rapid and efficient discriminate the three mycotoxins in real samples. CONCLUSIONS. In summary, the 3D-Nanocauliflower SERS substrates was successfully prepared through sputtering Au NPs on the surface of PDMS@AAO complex substrate. To achieve high SERS activities, the sputtering time was optimized and results showed that the 3D-Nanocauliflower SERS substrates had the highest denseness of hot spots and SERS intensity at sputtering time of 8 min. Compared to other substrates, this 3DNanocauliflower SERS substrates could simultaneously provide high enhancement factor (2.2×106), low detection limit (10−12 M, 4-MBA), and excellent enhancement uniformity (RSD = 4.57%). Moreover, the 3D-Nanocauliflower SERS substrates could increase the sensitivity, density, and reproducibility of hot spots illustrated by electricfield-distribution. Furthermore, the proposed SERS substrate could behave good linear relationship for the detection of AFB1, ZON, and DON, and the LODs were calculated to be 1.8 ng/mL, 47.7 ng/mL, and 24.8 ng/mL (S/N = 3), respectively. All the results 15



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demonstrated that the new 3D-Nanocauliflower SERS substrate can be successfully applied in the detection of mycotoxins, which could be act as a rapid and label-free detection method in food quality and safety.

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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. ORCID Heyou Han: 0000-0001-9406-0722 Author Contributions §J.L.

and H.Y. had equal contribution.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We gratefully acknowledge the financial support from The National Natural Science Foundation of China (21778020), Sci-tech Innovation Foundation of Huazhong Agriculture University (2662017PY042, 2662018PY024), Hubei Provincial Food and Drug Administration (201601012), Key Laboratory of Guangxi Colleges and Universities for Food Safety and Pharmaceutical Analytical (FPAC2017-ZD-01). The authors thank Fengrui Wu (Huazhong Agricultural University) for her help with the SEM characterization. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: SERS spectra of the 3D-Nanocauliflower substrate; bi-analyte SERS experiment by 17



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using 4-MBA and R6G molecules; reflectance spectra of 3D-Nanocauliflower substrate; SEM image of side-view of PDMS@AAO template; calculation of enhancement factor; the limit of detections of the 3D-Nanocauliflower substrate for the detection of different mycotoxins; table of the calculation of LODs for the different mycotoxins; comparison of maximum residue levels (MRLs) and SERS limit of detection (LOD) of three mycotoxins in maize; recovery of AFB1, ZON, and DON spiked in maize samples; comparison of the enhancement factor and reproducibility among different SERS substrates (PDF). Reference (1) Limay-Rios, V.; Schaafsma, A. W. Effect of prothioconazole application timing on Fusarium mycotoxin content in maize grain, J. Agric. Food Chem. 2018, 66, 4809-4819. (2) James, A.; Zikankuba, V. L. Mycotoxins contamination in maize alarms food safety in sub-Sahara Africa, Food Control 2018, 90, 372-381. (3) Geary, P. A.; Chen, G.; Kimanya, M. E.; Shirima, C. P.; Oplatowska-Stachowiak, M.; Elliott, C. T.; Routledge, M. N.; Gong, Y. Y. Determination of multi-mycotoxin occurrence in maize based porridges from selected regions of Tanzania by liquid chromatography tandem mass spectrometry (LC-MS/MS), a longitudinal study, Food Control 2016, 68, 337-343. (4) Hooker, D.; Schaafsma, A. Agronomic and environmental impacts on concentrations of deoxynivalenol and fumonisin B1 in corn across Ontario, Can. J. Plant Pathol. 2005, 27, 347-356. (5) Chilaka, C. A.; De Boevre, M.; Atanda, O. O.; De Saeger, S. The status of Fusarium 18


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Scheme 1. Schematic demonstration of preparation of the 3D-Nanocauliflower substrate and SERS measurement

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Table 1. Assignments and Raman Shift (cm−1) for SERS Spectra of AFB1, ZON, and DON AFB1 Band assignments C=C stretch; rings and special ring stretching C-C stretch and ring deformation C=C stretch, ring deformation CH3 bending β(C-H) ring deformation CH3 stretch + C-H stretch Ring deformation C-H stretch ZON Band assignments β(C-H)(CH3), β(C-H)(ring) C-H in-plane bending CH2 rocking C-H out-of-plane bending

Observed 1615 1550 1498 1345 1272 1203 1059 845

Reported11,9,57 1620 1550 1491 1355 1284 1202 1055 844

Observed 1517 1448 880 762

Reported9,58-60 1519 1447 876 760

Observed 1596 1553 1364 1002 881 663

Reported11,9,58,60 1596 1550 1358 1008 876 665

DON Band assignments β(C-H)(CH3), β(C-H)(ring) CH3 stretch CH3 bending CH3 stretch + C-H stretch CH2 rocking C-H in-plane bending

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Figure 1. The top (a), side (b), and bottom (c) view SEM images of AAO template.

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Figure 2. (a-f) Top-view SEM images of 3D-Nanocauliflower substrate with different sputtering deposition time (4 to 14 min) under the same deposition conditions.

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Figure 3. (a) SERS spectra of 4-MBA (10−5 M) molecules and (b) average Raman intensities at 1580 cm−1 peak of 3D-Nanocauliflower substrates with different sputtering deposition time (4 to 14 min).

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Figure 4. (a) SERS spectra of 4-MBA with different concentrations (10−6 to 10−12 M) using the 3D-Nanocauliflower substrates. (b) SERS intensities of 4-MBA (10−5 M) enhanced by the 3D-Nanocauliflower substrates stored in N2 atmosphere at times ranging from 0 to 21 days. (c) SERS intensity mapping of 4-MBA (10−5 M) measured at the 1580 cm−1 peak across a 60×60 μm2 piece of the 3D-Nanocauliflower substrate. (d) SERS spectra of 4-MBA (10−5 M) collected at 40 sites randomly from 5 3DNanocauliflower substrates and (e) intensity distribution at 1580 cm−1 corresponding to part D (the average intensity is indicated by blue line, and the light blue zones represent the ± 4.57% intensity variation).

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Figure 5. (a) SERS spectra of AFB1 with concentrations of 1, 0.5, 0.1, 0.05, 0.01, 0.005 μg/mL. (c) SERS spectra of ZON with concentrations of 50, 10, 5, 1, 0.5, 0.1 μg/mL. (e) SERS spectra of DON with concentrations of 10, 5, 1, 0.5, 0.1, 0.05 μg/mL. Linear calibration plot between the SERS intensity and AFB1 (b), ZON (d), and DON (f) concentration; inset, molecular structure of AFB1, ZON, and DON.

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Figure 6. Principle components analysis (PCA) plot of PC1 vs PC2 computed from the SERS spectra of AFB1, ZON, and DON.

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Figure 7. SERS spectra of multiple components of three mycotoxins (AFB1, ZON, and DON) in maize using the 3D-Nanocauliflower substrate.

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TABLE OF CONTENTS (TOC) GRAPHIC:

Fabrication of 3D-Nanocauliflower SERS Substrate and its application in multiple mycotoxins detection.

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Cauliflower-Inspired 3D SERS Substrate for Multiple Mycotoxins

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