3D plasmonic SERS aptasensor for rapid detection of aflatoxin B1 combined with Au@Ag bimetallic nanostars and Fe3O4@MoS2 magnetic nanoflowers

3D plasmonic SERS aptasensor for rapid detection of aflatoxin B1 combined with Au@Ag bimetallic nanostars and Fe3O4@MoS2 magnetic nanoflowers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article 3D plasmonic SERS aptasensor for rapid detection of aflatoxin B1 combined with Au@Ag bimetallic nanostars and Fe3O4@MoS2 magnetic nanoflowers Peifang Chen, Caiyun Jiang, Zhouping Wang, Hong-zhen Lian, Xiaoyuan Ma This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2437251/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract As a virulent metabolite, aflatoxin B 1 (AFB 1 ) presented in various cereal grain is tightly implicated in severe human diseases. In this study, 3D plasmonic nanohybirds of Raman molecule 4-mercaptobenzoic acid (4-MBA)-embedded and AFB 1 aptamer-modified bimetallic nanostars as probes bound to magnetic nanoflowers were fabricated and demonstrated as a high-performance SERS-active aptasensor to quantitatively analyze AFB 1 . Bimetallic Au@Ag SERS plasmonic nanoprobes with enhanced properties were capable of enhancing discriminative Raman peaks of 4-MBA. Then, the integration of iron tetroxide nanoparticles (Fe 3 O 4 NPs) and molybdenum disulfide nanosheets (MoS 2 NSs) with huge specific surface area constituted stable 3D Fe 3 O 4 @MoS 2 plasmonic nanoflowers, facilitating the bind of numerous aptamer-based SERS probes via the non-covalent interaction between MoS 2 NSs and aptamer, which were ideal candidates for SERS-active substrates. Additionally, Fe 3 O 4 NPs as magenetic core endowed 3D nanocomposites with specific magnetic separation characteristic that caused the collected SERS hotspots to exhibit superior signal response, and further strengthening the sensitivity in a complex food matrix. Aptamer-target AFB 1 specific recognition triggered linearly diminished 4-MBA signal intensity (I 4-MBA ) on the substrate to achieve a low detection limit of 58.9 pg/mL. Furthermore, the sensor has the potential to be a promising monitoring tool for trace contaminants. surface-enhanced raman spectroscopy aptasensor aflatoxin B1 magnetic nanoflowers Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Aflatoxin B 1 (AFB 1 ) derived from fungi species is difuran-coumarin compound with the most poisonous activities (Marchese et al. 2018). Over the last few decades, AFB 1 contamination in a wide variety of common foodstuffs has become an unavoidable global concern, containing grains (corn, wheat and peanuts) and snack foods (jerky, pistachios and almonds) (Mishra et al. 2022; Pleadin et al. 2015; Wu et al. 2020). In particular, for peanuts, the AFB 1 detection rate reached 33.8% from four major production regions in China (Yang et al. 2020). In Mexico, AFB 1 residues were found in 80% of investigated samples, 26% of the samples exceeded the EU maximum AFB 1 limit value (5 μg/kg) (Zuki-Orozco et al. 2018). AFB 1 contents measurement more than 182.28 μg/kg in Burkina Faso accounted for 41.50% of peanut samples (Bandé et al, 2022), suggesting an elevated contamination rate. Meanwhile, conventional heating treatments are hard to sufficiently degrade AFB 1 toxins with stable biochemical properties, even pasteurization. Prolonged exposure to low doses of the insidious AFB 1 poses a critical and potential threat to humans, and has been closely linked via epidemiological studies to chronic diseases consisting of malnourishment, growth disorders, and immunosuppression (Rushing and Selim 2019). Analytical approaches depending on large-scale instruments have been explored to accurately measure AFB 1 , such as thin layer chromatography (TLC) (Var et al. 2007), high-performance liquid chromatography (HPLC) (Mochamad and Hermanto 2017) and liquid chromatography-tandem mass spectrometry (LC-MS) (Janik et al. 2021). However, certain downsides of poor response, low efficiency, and excellent professionalism limit the application of methods in rapid food safety testing. Thus, designing simple, quick, and ultrasensitive detection methods based on signal amplification strategies of nanomaterials is conducive to real-time monitoring of trace amounts of toxicants. Surface-enhanced Raman scattering (SERS) is an advanced spectroscopy analysis technology, which has received great attention in food safety control, environmental monitoring, and disease diagnosis (Deng et al. 2022; Lu et al. 2020; Song et al. 2017). With the additional excellent properties of easy manipulation, resistance to discoloration and fluorescence, and fast response, sensitive detection of various mycotoxins and biomarkers is achieved (Pettine et al. 2020; Turan et al. 2022; Zhao et al. 2020). Since the SERS signal amplification on precious metal surfaces was elucidated by Jeanmaire in 1977 (Jeanmaire and Van Duyne 1977), the enhancement mechanism has developed into dominant electromagnetic enhancement (EM) excited by local surface plasmon resonance (LSPR) at present (Ding et al. 2017). Thus, SERS-based biosensing strategies mainly consist of target-guided direct detection, and probe-mediated indirect detection by virtue of the ability to generate molecular fingerprint information via EM. Although direct AFB 1 detection possesses a simple preparation process, it requires a combination of complex stoichiometry to distinguish the characteristic peaks to achieve quantification, showing low sensitivity (Shao et al. 2021). These issues are addressed via a designed AFB 1 labeled sensor based on Raman signal molecules with low background noise and significant characteristic peaks, realizing efficient indirect AFB 1 determination. In this sensor, the combination of Raman molecules, metal NPs (various morphology containing triangular, cubic, and star) (Wang and Guo 2020; Yang et al. 2020), and tailored aptamer (single-stranded oligonucleotides binding the target with strong specificity) (Pan et al. 2022) constitutes highly sensitive probes, such as Au@4-MBA@Ag NRs (Lin et al. 2020) and Au@4-MBA@Si NPs The components of the Au-Ag alloy and the tip effect of the anisotropic NPs enable significant enhancement of the SERS signal, improving the sensing sensitivity of the SERS-based approach. In recently reported magnetic substrates, Fe 3 O 4 NPs are widely applied in various biosensors, due to the advantages of low toxicity, low cost, excellent biocompatibility, and especially superparamagnetic properties (Guo et al. 2021; Tajik et al. 2021). But bare Fe 3 O 4 NPs exposed to air are susceptible to oxidation and agglomeration. The introduction of specific carriers is considered by researchers as an effective measure. Notably, MoS 2 NSs, layered two-dimensional (2D) materials, possess distinct benefits of large specific surface area, and multiple active sites, which are favorable for binding with SERS probes (Karaman et al. 2021; Rani et al. 2020). Thus, the complexes of MoS 2 NSs wrapped around Fe 3 O 4 NPs as magnetic substrates in combination of SERS aptasensor have the capability to quickly collect and concentrate SERS signals from complex systems, which greatly simplifies the test procedures, and improves sensitivity for trace analyses. In this work, a single-response SERS aptasensor was rationally prepared to detect AFB 1 for the first time with Fe 3 O 4 @MoS 2 NFs/Au@Ag NSs-AFB 1 apt nanohybirds. Firstly, Au-4MBA@Ag NSs-AFB 1 apt act not only as SERS enhancer of 4-MBA signal by sufficient SERS hot spots from sharp edges and tips but also as specific capture probes of AFB 1 in the presence of other interfering substances. Meanwhile, Fe 3 O 4 @MoS 2 NFs serve as the enrichment and amplification substrates of SERS signal when AFB 1 apt-functionalized SERS probes were immobilized on MoS 2 NSs covered on Fe 3 O 4 NPs via π-π interaction, exhibiting the strongest SERS signals. With the assistance of external magnets, 3D magnetic compound has the ability to purify the detection signal in a short time. The target AFB 1 was efficiently recognized by AFB 1 apt-modified SERS probes. Owing to the dissociation of the probes, the effective abatement of 4-MBA signal intensity in the substrate was showed. Therefore, the SERS sensor achieved ultrasensitive and good selective measure toward AFB 1 . 2. Materials And Methods Materials and reagent Chloroauric acid (HAuCl 4 ·3H 2 O), ascorbic acid (AA), sodium citrate tribasic dihydrate (Na 3 C 6 H 5 O 7 ·2H 2 O), silver nitrate (AgNO 3 ), ammonia (NH 3 ·H 2 O), ferric chloride (FeCl 3 ·6H 2 O), polyethylene glycol, ethylene glycol (EG), sodium acetate (CH 3 COONa), thiourea (CH 4 N 2 S), anhydrous ethanol (C 2 H 6 O), Tween 20 (Tween-20), ammonium heptamolybdate, sodium chloride (NaCl), hydrochloric acid (HCl), tris(hydroxymethyl)aminomethane (Tris), 2-mercaptoethanol solution (2-MCH), ethylenediaminetetraacetic acid (EDTA), magnesium chloride (MgCl 2 ) and Tris(2-carboxyethyl)phosphine (TCEP) were provided by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 4-mercaptobenzoic acid (4-MBA), aflatoxin B 1 (AFB 1 ), aflatoxin M1 (AFM1), ochratoxin (OTA) and fumonisin B1 (FB1) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Peanuts were bought from supermarket. All solutions used in the experiment were prepared using ultrapure water (18.2 MΩ) from a water purification system. Thiol-terminal AFB 1 aptamer (He et al. 2020) (SH-AFB 1 apt: 5'-SH-GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CC-3') was synthesized by Sangon Biological Engineering Technology & Co., Ltd (Shanghai, China), and purified by HPLC. 2.1. Instruments Transmission electron microscope (TEM) images of all nanomaterials were acquired by the JEM-21OOF microscope, operating at 200 kV (Tokyo, Japan). The UV-Vis absorption spectra were recorded by the UV-1800 spectroscopy (China). The Raman spectra of 4-MBA in the assemblies were measured by DXR2xi microscope (U.S.A.) equipped with 50× microscope lens and 632.8 nm laser excitation. And the origin software was used to smooth and correct the acquired Raman spectra for better data analysis. X-Ray Diffractomer (XRD) patterns of Fe 3 O 4 @MoS 2 NFs were obtained by D2 PHASER analyzer using Cu–Kα radiation (Switzerland). Zeta potential was determined using Zetasizer Nano ZS analyzer (Melvin, UK). Preparation of Au-4MBA@Ag NSs-AFB 1 apt Gold seeds were synthesized using classic sodium citrate reduction with some minor modifications (Frens 1972). HCl (1 M, 20 μL) was injected to 20 mL of HAuCl 4 ·3H 2 O (0.25 mM), which mixed thoroughly with 200 μL of Au NPs under shaking. AgNO 3 (2 mM, 200 μL) and freshly prepared AA (10 mM, 100 μL) were simultaneously added under vigorous shake. Once the solution turned blue-green, the Au NSs were purified by centrifugation (10 min) to stop the growth, and stored at -4℃ before use. 0.8 μL of 4-MBA (1 mM) in ethanol was incubated with 100 μL of Au NSs at room temperature for 5 hours. The unbound 4-MBA was removed by centrifuging (3000 rpm) for 10 min. Then, 0.1 M AgNO 3 with different volumes (0.5 μL, 1 μL, 2 μL, 3 μL, and 4 μL), 1 μL of AA (0.1 M), and 2 μL of NH 3 ·H 2 O were dripped under intense vortices, respectively. After incubation for 10 min, the Au-4MBA@Ag NSs were redissolved in ultrapure water by centrifugating for 10 min, Finally, the optimal volume of AgNO 3 was chosen by Raman intensity of 4-MBA to further optimize the analysis performance. To prepare functionalized nanoprobes, 250 μL of Au-4MBA@Ag NSs was resuspended in 0.05% Tween-20 solution. 5 μL of AFB 1 apt (50 μM, activated with TCEP solution in equal volume for 1 h) was added, and incubated for 1 h. NaCl solution was dropped every 30 min to complete aging (with a final concentration of 0.25 M). Afterward, the mixture maintained overnight at 37°C. The free nucleic acid was discarded under centrifugation to produce Au-4MBA@Ag NSs-AFB 1 apt. Preparation of Fe 3 O 4 @MoS 2 NFs Take 1.35 g of FeCl 3 ·6H 2 O powder into 18 mL of ethylene glycol solution. Then, 1.62 g of CH3COONa and 0.45 g of polyethylene glycol were successively added into the above mixture. The solution was thoroughly mixed well under sonicating for 30 min, and reacted at 200°C for 8 h in a high temperature reactor. The reaction products were washed three times with ethanol and ultrapure water by magnetic separation, and dried under vacuum at 60°C for 6 h to collect Fe 3 O 4 NPs. 20 mg of the obtained Fe 3 O 4 NPs powder was dispersed well in 10 mL of ultrapure water by ultrasonication. Then, 0.112 g of ammonium heptamolybdate and 0.365 mg of thiourea were sequentially added by vigorous sonicating for 30 min. Under 200°C, the mixture was placed in a high temperature reactor for 8 h. The resulting products were washed repeatedly with ethanol and ultrapure water. Finally, Fe 3 O 4 @MoS 2 NFs powder was obtained under vacuum drying at 60°C for 10 h. Construction of labeled-aptasensor for AFB 1 detection To construct Fe 3 O 4 @MoS 2 NFs-AFB 1 apt-Au@Ag NSs sensors, SERS probes were mixed with magnetic substrates at different volume ratios (1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, and 1:3) at room temperature for 20 min. The unattached nanoparticles were cleared by magnetic separation. Moreover, 2-MCH solution as blocker closed the unreacted sites to prevent nonspecific binding. The optimal volume ratio was determined by Raman intensity of 4-MBA at 1581 cm -1 . Next, AFB 1 standard solutions with different amounts (final concentrations of 0.1 ng/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL, and 1000 ng/mL, respectively) were reacted with 200 μL of the above solution at room temperature for 1 h. The complex was magnetically separated and washed twice to ensure signal drop. After that, 8 μL of Au-4MBA@Ag NSs-based 3D nanohybirds solutions were dropped onto aluminum foil and dried at 25℃. The SERS measure was performed at an excitation wavelength of 633 nm. The logarithmic value of AFB 1 concentration was used as the horizontal coordinate, and the Raman intensity of 4-MBA at 1581 cm -1 (I 1581 ) was employed as the vertical coordinate to determine the standard curve of the method. Selectivity evaluation To assess the selectivity of the SERS aptasensor, the control experiments were performed with interfering toxins including AFM 1 , OTA and FB 1 and AFB 1 under the same experimental conditions. The concentrations of the above toxins were set as 100 ng/mL. The comparison of SERS intensity directly reflected the specificity of aptamer-based sensor. Real samples detection Fresh peanuts were selected to analyze the utility of SERS aptamer sensors to detect AFB 1 in real samples. For pretreatment, the peanut samples were fully ground to powder firstly. 2 g of powder was dissolved in a mixture of methanol/water (5.6 mL/2.4 mL) under sonicating for 30 min to aid the extraction performance (Jing et al. 2009). The supernatant was collected by centrifuging (5000 rpm, 15 min). Lastly, AFB 1 standards were added at different final concentrations of 0.5 ng/mL, 5.0 ng/mL, and 50.0 ng/mL, respectively. After the above procedure analysis, the recovery results were calculated. 3. Results And Discussion 3.1. Detection strategy of the aptasensor for AFB 1 The working principle of designed 3D plasmonic SERS aptasensor for AFB 1 detection was illustrated in scheme 1. Bimetallic Au@Ag plasmonic nanostars were acquired by modifying 4-MBA on anisotropic Au NSs via Au-SH bond, further reducing AgNO 3 to form Ag outer shell. The enhanced EM excited by affluent hot spots of sharp tips significantly amplified the SERS response of 4-MBA embedded on bilayer Au-Ag. The specific core-shell nanostructure gave effectively protective effect on signal, improving the detection stability. Then, the coupled AFB 1 apt on Au@Ag stars employed as SERS probes could be capable of capturing target AFB 1 with high sensitivity and high specificity. Meanwhile, Fe 3 O 4 @MoS 2 plasmonic nanoflowers provided large number of active binding sites for SERS probes, generating the 3D magnetic SERS plasmonic substrates-aptamer-SERS plasmonic probes (Fe 3 O 4 @MoS 2 -AFB 1 apt-Au-4MBA@Ag NSs), which owned both SERS activities and magnetic features. Combined with the chemical enhancement effect of MoS 2 NSs on the multilayer plasma nanostructures, SERS signal was further magnified to the maximum. When AFB 1 appeared, the probes were separated from the nanoflowers owing to the specifical capture of aptamers, causing an obvious decrease of I 4-MBA . Thus, I 4-MBA was inversely related to the AFB 1 concentration, enabling sensitive detection in the peanuts. 3.2. Characterization and optimization of Au@Ag NSs-AFB 1 apt The TEM images for the morphology of the nanoparticles were shown in Fig. 1. Au NSs had great dispersion and showed sharply star-shaped morphology, which consisted of spherical core sized 34.53 nm ± 5.29 nm and rich sharp tips with a size of around 31.13 nm ± 6.71 nm (Fig. 1A). As shown in Fig. 1B, after the reduction of Ag ions was induced under ammonia-adjusted alkaline conditions, the Ag atoms were uniformly deposited to form Ag shells on the surface of monolayer Au NSs, which was due to the extremely similar lattice edge widths of both Au and Ag (Rodriguez-Gonzalez et al. 2005). The results showed that moderate amount of silver nitrate had little effect on the tip sharpness of the nanostars. In the UV-Vis spectra of Fig. 1C, as the formation of Au NSs prepared by Au seed growth method, the resonance plasmonic absorption peak of Au NPs located at 521 nm redshifted to around 691 nm. Thereby the clear characteristic absorption peak of Ag NPs appeared at 407 nm, indicating the successful modification of Ag shell outside Au NSs. The results showed the bimetallic Au@Ag plasmonic nanostars exhibited two absorption peaks at 407 nm and 549 nm in optical properties. Furthermore, the SERS properties of bimetallic nanomaterials were characterized by Raman spectrometer in Fig. 1D. There were no obvious Raman peaks on Au NSs. In contrast, Au-4MBA NSs occurred effectively enhanced the SERS signals of 4-MBA, which was owing to the multiple SERS "hot spots" at the tips. It was worth noting that Au@Ag NSs plasmonic structure-amplified 4-MBA signal was 4 times stronger than that of monolayer precious metal Au, which was in agreement with previous study (Jing et al. 2020). The above results clearly verified the successful preparation of Au-4MBA@Ag NSs for the further SERS detection of AFB1. In addition, the effect of the addition amount of 4-MBA and AgNO 3 on the plasmonic SERS probes was investigated. As shown in Fig. S1, the SERS intensity of 4-MBA was the strongest with the addition of 4-MBA (1 mM) up to 0.8 μL, suggesting the 4-MBA adsorption on the surface of Au NSs reached saturation. Based on the optimized addition value of Raman molecular, the optical features of materials modified with different amounts of AgNO 3 (1 M) were characterized by UV-Vis spectra shown in Fig. S2C. Along with the increase of the AgNO 3 volume to 4 μL, the resonance plasmonic absorption peak of Au@Ag NSs underwent a gradual blue shift, which was closely related to the aspect ratio of the star-shaped tip. Meanwhile, a new absorption peak at around 407 nm appeared and enhanced, proving the successful deposition of Ag shells and a gradual increase in thickness. All these results were tightly dependent on the dielectric properties around the material (Han et al. 2017). In Fig. S2 (A-B), the SERS intensity of 4-MBA at 1078 cm -1 showed an obvious increase in the range of 0.5 μL to 4 μL. Although the SERS signal was still enhanced when the modification amount exceeded 2 μL, the stability of signal measured by repeated tests significantly dropped, which strongly influenced the detection sensitivity. Moreover, the TEM image of Fig. S2D characterized the morphology of bimetallic Au@Ag NSs at the AgNO3 addition amount of 3 μL. It was found that the nanocomposites had completely tended to be spherical owing to the modification of extremely thick silver shells, which was also the direct cause of above signal instability. The results showed that the thickness of the Ag shell in Au NSs tips manifested positive correlation with the enhancement effects, while the change of morphology was significantly related to signal stability, showing that appropriate Ag shell thickness was a vital factor for stable detection (Mott et al. 2012). Thus, 0.8 μL of 4-MBA (1 mM) and 2 μL of AgNO 3 (0.1 M) were chosen as the optimal addition amounts for the preparation of SERS probes. The coupled AFB 1 apt on the surfaces of Au@Ag NSs was illustrated by UV-Vis spectroscopy and zeta potential measurements in Fig. S3. For Au@Ag NSs-AFB 1 apt, the absorbance of the supernatant decreased greatly, and the average zeta potential decreased to -33.9 mV, which was due to the characteristic absorption peak at 260 nm (Wu et al. 2012), and the presence of a negatively charged phosphate group of the introduced ssDNA (Zhu et al. 2021). Thus, the functionalized bimetallic nanoprobes were prepared successfully. 3. 3. Characterization and optimization of the fabricated Fe 3 O 4 @MoS 2 NFs In order to obtain magnetic substrates, Fe 3 O 4 @MoS 2 NFs was synthesized by secondary hydrothermal methods. As shown in Fig. 2A, Fe 3 O 4 NPs were mostly homogeneous spherical morphology with a particle size of around 231.77 nm ± 17.97 nm. The magnetic response feature of Fe 3 O 4 NPs was further verified by the separation test of the external magnetic field. The illustration of Fig. 2A showed that effective separation and enrichment by magnets were achieved within 15 s, indicating good paramagnetic properties of Fe 3 O 4 NPs. Fig. S4A revealed that 2D MoS 2 NSs (carbon-based nanomaterials) were highly transparent nanosheets with numerous folds. After another high temperature reaction, MoS 2 NSs were successfully combined around spherical Fe 3 O 4 NPs as revealed in Fig. 2B, forming multi-functional 3D core-shell Fe 3 O 4 @MoS 2 magnetic nanoflowers. Besides, the time of good magnetic separation was 30s for Fe 3 O 4 @MoS 2 NFs in the illustration of Fig. 2B, indicating that the load of moderate MoS 2 NSs had a weak influence on the magnetic effect, which was suitable to be SERS magnetic substrate in subsequent analysis. The combination of plasmonic Fe 3 O 4 NPs with good magnetic effect and MoS 2 NSs with large surface area was conducive to rapid enrichment of SERS signal in the complex detection system. Moreover, the crystalline morphology of materials was confirmed by XRD in Fig. 2C. The typical diffraction peaks of Fe 3 O 4 NPs at 30.6°, 35.9°, 43.5°, 53.9°, 57.4° and 62.9° were ascribed to (220), (331), (400), (422), (511) and (440) planes, which was consistent with their standard cards (JCPDS No. 19-0629). Then, the diffraction peaks at 18.1° (002), 36.1° (100), 43.9° (103) and 58.1° (110) proved the fabrication of MoS 2 NSs nanostructure. The characteristic peaks of Fe 3 O 4 NPs were measured for XRD spectra of Fe 3 O 4 @MoS 2 NFs, indicating that the crystal morphology and phases of Fe 3 O 4 NPs were preserved. The results were similar with those of Lu et al (Lu et al. 2021). As shown in Fig. 2D, the Zeta potential of the final composite was -19.5 mV as a result of the MoS 2 NSs with negative charge (-32.3 mV) as shells wrapping around the positive charged Fe 3 O 4 NPs cores (18.9 mV). Therefore, all the above results characterized the successful preparation of 3D magnetic Fe 3 O 4 @MoS 2 nanoflowers, which provided more active sites for the connection of the aptamers to facilitate the construction of next detection system. 3. 4. Feasibility and optimization of 3D SERS aptasensor for AFB 1 detection To obtain the designed assemblies to analyze AFB 1 , the TEM images were employed to characterize the surface morphology of SERS sensor. From Fig. 3A, the anisotropic bimetallic nanostars were successfully bound around 3D Fe 3 O 4 @MoS 2 NFs core to build assemblies, due to the aptamers were combined to the outer shell MoS 2 NSs through non-covalent bonds. In SERS spectra of Fig. 3B, Fe 3 O 4 @MoS 2 magnetic substrates showed no Raman peaks in the range from 900 cm -1 to 1800 cm -1 . However, compared with Au-4MBA@Ag NSs, the stronger 4-MBA SERS signal was observed on the plasmonic Fe 3 O 4 @MoS 2 NFs-AFB 1 apt-Au-4MBA@Ag NSs assemblies, owing to the fact that chemical enhancement of MoS 2 NSs synergistically enhanced SERS performance of the assemblies. After adding target AFB 1 , 4-MBA response significantly reduced, due to the decrease of SERS probes on the assemblies. Moreover, TEM images of the assemblies without and with AFB 1 were clearly shown in Fig. S5(A-B), indicating that the presence of AFB1 induced the dissociation of nanostars from the substrates due to the forming of Au@Ag NSs-AFB1apt/AFB 1 composites. Thus, all the above results demonstrated the feasibility of SERS sensor for quantitatively sensing AFB 1 based on significant changes in SERS signal peak intensity caused by changes in the composition of assembler. To further obtain highly sensitive SERS aptasensor, Fe 3 O 4 @MoS 2 NFs as magnetic substrates and Au-4MBA@Ag NSs-AFB 1 apt as SERS probes were mixed in different volume ratios at room temperature. As obtained from Fig. 3C, the SERS intensity of the assemblies at 1581 cm -1 reached the strongest at the volume ratio of 1:2, which was chosen as the optimal addition ratio. Besides, in order to evaluate the reproducibility and stability of the constructed AFB 1 SERS sensor, the original Raman spectra of random 15 points shown in Fig. 3D (waterfall plot) were collected on the assemblies. And the RSD value of Raman peak intensity (1581 cm -1 ) was calculated to be 7.6% (Fig. S6A), manifesting the good uniformity of signals on the SERS assemblies. Different storage times (1, 3, 5, 7, 9, 11, and 13 days) of 3D aptasensor were also studied (Fig. S6B). SERS signal intensity remained 86.5% on the around thirteenth day, revealing the good time-dependent stability of sensor. 3.5. Sensitivity of AFB 1 detection by SERS aptasensor To verify the detection capability of the aptasensor under optimized experimental conditions, a series of AFB 1 addition amounts were reacted with the SERS system at the final concentration from 0.1 ng/mL to 1000 ng/mL. As shown in Fig. 4A, the SERS signal intensity of 4-MBA molecular gradually decreased with the increase of AFB 1 concentration, which was owing to the release of a large amount of 4MBA-based SERS probes induced by AFB 1 from the magnetic substrates. The SERS peak intensity of 4-MBA at 1581 cm -1 (I 1581 ) as quantitative value showed negatively linearly correlation with the logarithmic value of AFB 1 concentration (log 10 C AFB1 ). After the linear fit in Fig. 4B, the regression equation was Y = 8541.55 - 2348.69X (R 2 = 0.986). The associated LOD was as low as 58.9 pg/mL calculated by the formula (LOD = 10 (Y+3SD-A)/B . Where Y and SD are the mean and standard deviation of the blank sample signal, respectively. A and B represent the intercept and slope of the curve, respectively. And signal-to-noise ratio was set as 3:1). The detection sensitivity of 3D plasma SERS nanoassemblies satisfied the EU requirements for the measure of the maximum limit of AFB 1 in cereal. Additionally, Table S1 gave a comparison between our work for AFB 1 detection and other previously reported analytical methods. The results showed that this study had obtained lower detection limits and wider detection range relative to the work of some researchers, but the sensitivity still needed to be further improved. 3. 6. Selectivity, reproducibility, and practicality evaluation To validate the selectivity of the aptasensor, AFM 1 , OTA and FB 1 toxins were selected to detect under the same experiment. In Fig. 5(A-B), I 1581 of the three interfering toxins changed weakly compared to that of the blank sample, while that of AFB 1 (100 ng/mL) decreased significantly, showing that the specific aptamer conferred good selectivity to the SERS sensor. To evaluate the reproducibility of the proposed sensor, AFB 1 (10 ng/mL) was analyzed repeatedly on the same batch and different six batches. It can be seen from the SERS intensity bar chart in Fig. S7 that this strategy presented relatively low and acceptable RSD values of I 1581 , which were 5.1% and 6.4%, respectively. To verify the practicality of the method in real samples, the peanuts at AFB 1 spiked concentrations of 0.5 ng/mL-50.0 ng/mL were selected for the recovery experiments. The satisfactory recoveries ranging from 97.9% to 99.4% with RSDs of 4.3%-9.3% were obtained as shown in Table 1, indicating that the assay was suitable for the detection of peanut, and had bright application prospect in food safety analysis. 4. Conclusions In all, a rational SERS aptasensor for quantitative detection of AFB 1 has been contrasted based on 3D plasmonic magnetic SERS assemblies (Au-4MBA@Ag NSs-AFB 1 apt-Fe 3 O 4 @MoS 2 NFs). The designed SERS-active platform has the following excellent performance: (1) rapid magnetic separation features, (2) good stability of substrates for long-term storage, (3) signal amplification function provided from abundant hot spots, (4) synergistic enhancement effect of bimetal Au-Ag and MoS 2 NSs on the SERS signal of 4-MBA. AFB 1 toxin was sensitively detected from 0.1 ng/mL to 100 ng/mL, achieving the LOD as low as 58.9 pg/mL. The high selectivity under interfering toxins, and good reproducibility within different batches measurement of this sensor were further determined. Moreover, the strategy can be capable of detecting AFB 1 in peanut samples with good recoveries of 97.9%-98.7%. Therefore, the proposed SERS sensor as effective detection technique has the potential to be applied to the monitoring of various hazards in the field of food safety. Declarations Acknowledgments The authors gratefully acknowledge instrument platform of school of Food Science and Technology, Jiangnan University, for technical supports. Funding This work was supported by the Social Development Fund Project of Wuxi (N20201001), the Excellent Scientific and Technological Innovation Team of Jiangsu Universities, the 333 High Level Talents Training Project of Jiangsu Province, and the National Natural Science Foundation of China (22176085 and 21874065). Competing Interests The authors declare no competing interests. References Bandé M, Traoré I, Nikiema F et al (2022) Aflatoxins contents determination in some foodstuffs in Burkina Faso and human health risk assessment. 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Mishra G, Panda BK, Ramirez WA, Jung H, Singh CB, Lee SH, Lee I (2022) Application of SWIR hyperspectral imaging coupled with chemometrics for rapid and non-destructive prediction of aflatoxin B1 in single kernel almonds. LWT 155: 112954. https://doi.org/10.1016/j.lwt.2021.112954 Mochamad L, Hermanto B (2017) High-performance liquid chromatography ultraviolet-photodiode array detection method for aflatoxin B1 in cattle feed supplements. Veterinary World 10: 932-938. https://doi.org/10.14202/vetworld.2017.932-938 . Mott DM, Dao Thi Ngoc A, Singh P, Shankar C, Maenosono S (2012) Electronic transfer as a route to increase the chemical stability in gold and silver core-shell nanoparticles. Advances in Colloid and Interface Science 185: 14-33. https://doi.org/10.1016/j.cis.2012.08.007 . Pan R, Liu J, Wang P, Wu D, Chen J, Wu Y, Li G (2022) Ultrasensitive CRISPR/Cas12a-driven SERS biosensor for on-site nucleic acid detection and its application to milk authenticity testing. Journal of Agricultural and Food Chemistry 70: 4484-4491. https://doi.org/10.1021/acs.jafc.1c08262 Pettine J, Choo P, Medeghini F, Odom TW, Nesbitt DJ (2020) Plasmonic nanostar photocathodes for optically-controlled directional currents. Nature communications 11: 1-10. https://doi.org/10.1038/s41467-020-15115-0 Pleadin J, Vulić A, Perši N, Škrivanko M, Capek B, Cvetnić Ž (2015) Annual and regional variations of aflatoxin B1 levels seen in grains and feed coming from Croatian dairy farms over a 5-year period. Food Control 47: 221-225. https://doi.org/10.1016/j.foodcont.2014.07.017 Rani R, Yoshimura A, Das S et al (2020) Sculpting artificial edges in monolayer MoS 2 for controlled formation of surface-enhanced Raman hotspots. ACS nano 14: 6258-6268. https://doi.org/10.1021/acsnano.0c02418 Rodriguez-Gonzalez B, Burrows A, Watanabe M, Kiely CJ, Liz-Marzan LM (2005) Multishell bimetallic AuAg nanoparticles: synthesis, structure and optical properties. Journal of Materials Chemistry 15: 1755-1759. https://doi.org/10.1039/b500556f . Rushing BR, Selim MI (2019) Aflatoxin B1: A review on metabolism, toxicity, occurrence in food, occupational exposure, and detoxification methods. Food and Chemical Toxicology 124: 81-100. https://doi.org/10.1016/j.fct.2018.11.047 . Shao Q, Zhang D, Wang C-e, et al (2021) Ag@ MIL-101 (Cr) film substrate with high SERS enhancement effect and uniformity. The Journal of Physical Chemistry C 125: 7297-7304. https://doi.org/10.1021/acs.jpcc.1c01167 Song C, Yang B, Zhu Y, Yang Y, Wang L (2017) Ultrasensitive sliver nanorods array SERS sensor for mercury ions. Biosensors & Bioelectronics 87: 59-65. https://doi.org/10.1016/j.bios.2016.07.097 . Tajik S, Beitollahi H, Jang HW, Shokouhimehr M (2021) A screen printed electrode modified with Fe 3 O 4 @polypyrrole-Pt core-shell nanoparticles for electrochemical detection of 6-mercaptopurine and 6-thioguanine. Talanta 232. https://doi.org/10.1016/j.talanta.2021.122379 . Turan E, Zengin A, Suludere Z, Kalkan NÖ, Tamer U (2022) Construction of a sensitive and selective plasmonic biosensor for prostate specific antigen by combining magnetic molecularly-imprinted polymer and surface-enhanced Raman spectroscopy. Talanta 237: 122926. https://doi.org/10.1016/j.talanta.2021.122926 Var I, Kabak B, Gok F (2007) Survey of aflatoxin B1 in helva, a traditional Turkish food, by TLC. Food Control 18: 59-62. https://doi.org/10.1016/j.foodcont.2005.08.008 . Wang X, Guo L (2020) SERS activity of semiconductors: crystalline and amorphous nanomaterials. Angewandte Chemie International Edition 59: 4231-4239. https://doi.org/10.1002/anie.201913375 Wu H, Wu J, Liu Y, Wang H, Zou P (2020) Target-triggered and T7 exonuclease-assisted cascade recycling amplification strategy for label-free and ultrasensitive fluorescence detection of aflatoxin B1. Sensors and Actuators B: Chemical 321: 128599. https://doi.org/10.1016/j.snb.2020.128599 Wu P, Gao Y, Zhang H, Cai CX (2012) Aptamer-guided silver-gold bimetallic nanostructures with highly active surface-enhanced Raman scattering for specific detection and near-infrared photothermal therapy of human breast cancer cells. Analytical chemistry 84: 7692-7699. https://doi.org/10.1021/ac3015164 . Yang B, Zhang C, Zhang X et al (2020) Survey of aflatoxin B1 and heavy metal contamination in peanut and peanut soil in China during 2017-2018. Food Control 118. https://doi.org/10.1016/j.foodcont.2020.107372 . Yang C, Qing C, Wang Q, Zhang X, Lou J, Liu Y (2020) Synthesis of the hybrid CdS/Au flower-like nanomaterials and their SERS application. Sensors and Actuators B: Chemical 304: 127218. https://doi.org/10.1016/j.snb.2019.127218 Zhao X, Campbell S, Wallace GQ, Claing A, Bazuin CG, Masson J-F (2020) Branched Au nanoparticles on nanofibers for surface-enhanced Raman scattering sensing of intracellular pH and extracellular pH gradients. ACS sensors 5: 2155-2167. https://doi.org/10.1021/acssensors.0c00784 Zhu A, Jiao T, Ali S, Xu Y, Ouyang Q, Chen Q (2021) SERS sensors based on aptamer-gated mesoporous silica nanoparticles for quantitative detection of Staphylococcus aureus with signal molecular release. Analytical chemistry 93: 9788-9796. https://doi.org/10.1021/acs.analchem.1c01280 . Zuki-Orozco BA, Batres-Esquivel LE, Ortiz-Perez MD, Juarez-Flores BI, Diaz-Barriga F (2018) Aflatoxins contamination in maize products from rural communities in San Luis Potosi, Mexico. Annals of Global Health 84: 300-305. https://doi.org/10.29024/aogh.918 . Schemes Scheme 1 is available in the Supplementary Files section Tables Table 1. Detection of AFB 1 spiked in real peanut samples with the proposed method. Samples Added concentration (ng/mL) Measurement (ng/mL) (±SD) Recovery (%) RSD (%) 1 0.5 0.489 ± 0.021 97.9 4.3 2 5.0 4.934 ± 0.304 98.7 6.2 3 50.0 49.237 ± 4.598 98.5 9.3 Additional Declarations No competing interests reported. Supplementary Files SupportingInformation.docx Scheme1.png Scheme 1. Schematic illustration of the developed 3D SERS aptasensor based on Au-4MBA@Ag NSs-AFB 1 apt- Fe 3 O 4 @MoS 2 NFs assemblies for AFB 1 detection. Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2437251","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":200783802,"identity":"c6fc5f95-7d62-4bb7-89de-a786af479c16","order_by":0,"name":"Peifang Chen","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Peifang","middleName":"","lastName":"Chen","suffix":""},{"id":200785257,"identity":"0c5dbbf5-e3dc-450a-9e0e-5a04a8933f26","order_by":1,"name":"Caiyun Jiang","email":"","orcid":"","institution":"Jiangsu Vocational Institute of Commerce","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Caiyun","middleName":"","lastName":"Jiang","suffix":""},{"id":200785258,"identity":"a1f31b53-85a3-43d4-8836-814ffb5ebfe5","order_by":2,"name":"Zhouping Wang","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhouping","middleName":"","lastName":"Wang","suffix":""},{"id":200785259,"identity":"e1107393-8bd2-4fca-bc64-aa753d93c168","order_by":3,"name":"Hong-zhen Lian","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYBACAwbmhgMMDDYMjA1AHg9xWhhBWtJI1AKkDkN4RGkx5z/YeODnjvN5zDMSGB+8bWOQNyekxbLhYMPB3jO3ixlnJDAbzm1jMNzZQMhhBxsbDvC23U5snJHAJs3bxpBgcICQlsOMDQf/tp0DaWH/TZyWY4wNh3nbDoBtYSZOyxmgFtm25MTGnofNknPOSRhuIKjl/OHDH9+22SVubE8++OFNmY08QVvgwLABHEESxKoHAnkS1I6CUTAKRsEIAwC+2kbsmrIH/gAAAABJRU5ErkJggg==","orcid":"","institution":"Nanjing University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hong-zhen","middleName":"","lastName":"Lian","suffix":""},{"id":200785260,"identity":"53eed054-c440-46b8-803e-609a311b8aa5","order_by":4,"name":"Xiaoyuan Ma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYLCCDzwScDZjAzE6GGeQrIWZB1k/QS267WfMpG1kLOTN+dce/FzAYCO74QDzswf4tJidSUuTzuGRMNw5412y9AyGNOMNB9jMDfBqOZB8DKSFccONMwbSPAyHEzcc4GGTwKvl/MM2aQseCXugFuPfPAz/idByA2gLA49E4obzPWZAWw4Qo+VZsmUPj0Tyhhs8ZtY8BsnGMw+zmRFwWI7hjZ89dbYbzp8xvs1TYSfbd7z5GV4tQMAiwdgDpCQSgAQoqJgJqAcp+cDwA0jxHyCsdBSMglEwCkYmAACgNkbaoUTCzAAAAABJRU5ErkJggg==","orcid":"","institution":"Jiangnan University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xiaoyuan","middleName":"","lastName":"Ma","suffix":""}],"badges":[],"createdAt":"2023-01-03 03:14:13","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-2437251/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-2437251/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":37106578,"identity":"9a0810b3-2667-443e-941b-28461053cfc8","added_by":"auto","created_at":"2023-05-16 18:59:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":342814,"visible":true,"origin":"","legend":"\u003cp\u003eTEM images of (A) Au NSs, and (B) Au-4MBA@Ag NSs with 2 μL AgNO\u003csub\u003e3\u003c/sub\u003e (0.1 M) added in Au-4MBA (100 μL); (C) UV-Vis spectra, and (D) SERS spectra of Au NSs, Au-4MBA NSs, and Au-4MBA@Ag NSs.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2437251/v2/cf1c251d582e21cab44a2119.png"},{"id":37106577,"identity":"12a1c71a-7534-422a-b9d0-5c22d5d6ffb9","added_by":"auto","created_at":"2023-05-16 18:59:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":275137,"visible":true,"origin":"","legend":"\u003cp\u003eTEM images of (A) Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs (inset: magnetic test and statistical analysis of diameters), and (B) Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e NFs (inset: magnetic test); (C) XRD pattern, and (D) Zeta potential measurement of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs, MoS\u003csub\u003e2\u003c/sub\u003e NSs and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e NFs.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2437251/v2/cc332394ef14e05f51f1710a.png"},{"id":37106580,"identity":"2658c007-4e4f-4865-ada1-8c5f893a84ec","added_by":"auto","created_at":"2023-05-16 18:59:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":330221,"visible":true,"origin":"","legend":"\u003cp\u003e(A) TEM images of SERS aptamer-based assemblies (Au-4MBA@Ag NSs-AFB\u003csub\u003e1\u003c/sub\u003eapt-Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e NFs); (B) SERS spectra variation diagram of SERS sensor for AFB\u003csub\u003e1\u003c/sub\u003e detection on the preparation process (containing Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e NFs, Au-4MBA@Ag NSs, assemblies without AFB\u003csub\u003e1\u003c/sub\u003e, and assemblies with AFB\u003csub\u003e1\u003c/sub\u003e); (C) SERS intensity at 1581 cm\u003csup\u003e-1\u003c/sup\u003e of different volume ratios (1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, and 1:3) of Au-4MBA@Ag NSs to Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e NFs; (D) Waterfall diagram of SERS spectra acquired from 15 random sites on the assemblies.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2437251/v2/b55d928774f6b1243fdb1f70.png"},{"id":37107360,"identity":"d9dfd4e3-76cf-4e45-9c9d-2062b097fcac","added_by":"auto","created_at":"2023-05-16 19:15:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":81735,"visible":true,"origin":"","legend":"\u003cp\u003e(A) SERS spectra in the detection of AFB\u003csub\u003e1\u003c/sub\u003e with different concentrations (from top to bottom: 0 ng/mL, 0.1 ng/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL and 1000 ng/mL); (B) Corresponding linear fitting of SERS intensity (I\u003csub\u003e1581\u003c/sub\u003e) with logarithmic AFB\u003csub\u003e1\u003c/sub\u003e concentrations (Log\u003csub\u003e10\u003c/sub\u003eAFB\u003csub\u003e1\u003c/sub\u003e concentration)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2437251/v2/979b556350ef2f0e4c96b36a.png"},{"id":37106581,"identity":"c8610593-dccc-49c0-88fb-d86be7fd18d7","added_by":"auto","created_at":"2023-05-16 18:59:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":64172,"visible":true,"origin":"","legend":"\u003cp\u003e(A) SERS spectra, and (B) SERS intensities at 1581 cm\u003csup\u003e-1\u003c/sup\u003e in the presence of blank and four toxin samples including AFM1, FB1, OTA, and AFB\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2437251/v2/4bd255f70f28bc77679007cd.png"},{"id":37107370,"identity":"da2175f1-576a-4109-8fa0-f588dc2fbc92","added_by":"auto","created_at":"2023-05-16 19:15:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1381404,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2437251/v2/0f51755e-8162-4cf4-9dde-f5d9a82551c0.pdf"},{"id":37106679,"identity":"8afa4e0c-b77e-4857-b244-321c01aecc0b","added_by":"auto","created_at":"2023-05-16 19:07:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2140229,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-2437251/v2/2f93ab7660be990185507310.docx"},{"id":37106681,"identity":"0c312fb5-4867-4d26-a909-aa8be090813a","added_by":"auto","created_at":"2023-05-16 19:07:03","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":185888,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1.\u003c/strong\u003e Schematic illustration of the developed 3D SERS aptasensor based on Au-4MBA@Ag NSs-AFB\u003csub\u003e1\u003c/sub\u003eapt- Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e NFs assemblies for AFB\u003csub\u003e1\u003c/sub\u003e detection.\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-2437251/v2/a1e65931966ace2a5d5912e9.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e3D plasmonic SERS aptasensor for rapid detection of aflatoxin B1 combined with Au@Ag bimetallic nanostars and Fe3O4@MoS2 magnetic nanoflowers\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAflatoxin B\u003csub\u003e1\u003c/sub\u003e (AFB\u003csub\u003e1\u003c/sub\u003e) derived from fungi species is difuran-coumarin compound with the most poisonous activities\u0026nbsp;(Marchese et al. 2018). Over the last few decades, AFB\u003csub\u003e1\u003c/sub\u003e contamination in a wide variety of common foodstuffs has become an unavoidable global concern, containing grains (corn, wheat and peanuts) and snack foods (jerky, pistachios and almonds)\u0026nbsp;(Mishra et al. 2022; Pleadin et al. 2015; Wu et al. 2020). In particular, for peanuts, the AFB\u003csub\u003e1\u003c/sub\u003e detection rate reached 33.8% from four major production regions in China\u0026nbsp;(Yang et al. 2020). In Mexico, AFB\u003csub\u003e1\u003c/sub\u003e residues were found in 80% of investigated samples, 26% of the samples exceeded the EU maximum AFB\u003csub\u003e1\u003c/sub\u003e limit value (5 \u0026mu;g/kg)\u0026nbsp;(Zuki-Orozco et al. 2018). AFB\u003csub\u003e1\u003c/sub\u003e contents measurement more than 182.28 \u0026mu;g/kg in Burkina Faso accounted for 41.50% of peanut samples\u0026nbsp;(Band\u0026eacute; et al, 2022), suggesting an elevated contamination rate. Meanwhile, conventional heating treatments are hard to sufficiently degrade AFB\u003csub\u003e1\u003c/sub\u003e toxins with stable biochemical properties, even pasteurization. Prolonged exposure to low doses of the insidious AFB\u003csub\u003e1\u003c/sub\u003e poses a critical and potential threat to humans, and has been closely linked via epidemiological studies to chronic diseases consisting of malnourishment, growth disorders, and immunosuppression\u0026nbsp;(Rushing and Selim 2019).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnalytical approaches depending on large-scale instruments have been explored to accurately measure AFB\u003csub\u003e1\u003c/sub\u003e, such as thin layer chromatography (TLC)\u0026nbsp;(Var et al. 2007), high-performance liquid chromatography (HPLC)\u0026nbsp;(Mochamad and Hermanto 2017)\u0026nbsp;and liquid chromatography-tandem mass spectrometry (LC-MS)\u0026nbsp;(Janik et al. 2021). However, certain downsides of poor response, low efficiency, and excellent professionalism limit the application of methods in rapid food safety testing. Thus, designing simple, quick, and ultrasensitive detection methods based on signal amplification strategies of nanomaterials is conducive to real-time monitoring of trace amounts of toxicants.\u003c/p\u003e\n\u003cp\u003eSurface-enhanced Raman scattering (SERS) is an advanced spectroscopy analysis technology, which has received great attention in food safety control, environmental monitoring, and disease diagnosis\u0026nbsp;(Deng et al. 2022; Lu et al. 2020; Song et al. 2017). With the additional excellent properties of easy manipulation, resistance to discoloration and fluorescence, and fast response, sensitive detection of various mycotoxins and biomarkers is achieved\u0026nbsp;(Pettine et al. 2020; Turan et al. 2022; Zhao et al. 2020). Since the SERS signal amplification on precious metal surfaces was elucidated by Jeanmaire in 1977\u0026nbsp;(Jeanmaire and Van Duyne 1977), the enhancement mechanism has developed into dominant electromagnetic enhancement (EM) excited by local surface plasmon resonance (LSPR) at present\u0026nbsp;(Ding et al. 2017). Thus, SERS-based biosensing strategies mainly consist of target-guided direct detection, and probe-mediated indirect detection by virtue of the ability to generate molecular fingerprint information via EM. Although direct AFB\u003csub\u003e1\u003c/sub\u003e detection possesses a simple preparation process, it requires a combination of complex stoichiometry to distinguish the characteristic peaks to achieve quantification, showing low sensitivity\u0026nbsp;(Shao et al. 2021). These issues are addressed via a designed AFB\u003csub\u003e1\u003c/sub\u003e labeled sensor based on Raman signal molecules with low background noise and significant characteristic peaks, realizing efficient indirect AFB\u003csub\u003e1\u003c/sub\u003e determination. In this sensor, the combination of Raman molecules, metal NPs (various morphology containing triangular, cubic, and star)\u0026nbsp;(Wang and Guo 2020; Yang et al. 2020), and tailored aptamer (single-stranded oligonucleotides binding the target with strong specificity)\u0026nbsp;(Pan et al. 2022)\u0026nbsp;constitutes highly sensitive probes, such as Au@4-MBA@Ag NRs\u0026nbsp;(Lin et al. 2020)\u0026nbsp;and Au@4-MBA@Si NPs The components of the Au-Ag alloy and the tip effect of the anisotropic NPs enable significant enhancement of the SERS signal, improving the sensing sensitivity of the SERS-based approach.\u003c/p\u003e\n\u003cp\u003eIn recently reported magnetic substrates, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs are widely applied in various biosensors, due to the advantages of low toxicity, low cost, excellent biocompatibility, and especially superparamagnetic properties\u0026nbsp;(Guo et al. 2021; Tajik et al. 2021). But bare Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs exposed to air are susceptible to oxidation and agglomeration. The introduction of specific carriers is considered by researchers as an effective measure. Notably, MoS\u003csub\u003e2\u003c/sub\u003e NSs, layered two-dimensional (2D) materials, possess distinct benefits of large specific surface area, and multiple active sites, which are favorable for binding with SERS probes\u0026nbsp;(Karaman et al. 2021; Rani et al. 2020). Thus, the complexes of MoS\u003csub\u003e2\u003c/sub\u003e NSs wrapped around Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs as magnetic substrates in combination of SERS aptasensor have the capability to quickly collect and concentrate SERS signals from complex systems, which greatly simplifies the test procedures, and improves sensitivity for trace analyses.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this work, a single-response SERS aptasensor was rationally prepared to detect AFB\u003csub\u003e1\u003c/sub\u003e for the first time with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e NFs/Au@Ag NSs-AFB\u003csub\u003e1\u003c/sub\u003eapt nanohybirds. Firstly, Au-4MBA@Ag NSs-AFB\u003csub\u003e1\u003c/sub\u003eapt act not only as SERS enhancer of 4-MBA signal by sufficient SERS hot spots from sharp edges and tips but also as specific capture probes of AFB\u003csub\u003e1\u003c/sub\u003e in the presence of other interfering substances. Meanwhile, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e NFs serve as the enrichment and amplification substrates of SERS signal when AFB\u003csub\u003e1\u003c/sub\u003eapt-functionalized SERS probes were immobilized on MoS\u003csub\u003e2\u003c/sub\u003e NSs covered on Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs via \u0026pi;-\u0026pi; interaction, exhibiting the strongest SERS signals. With the assistance of external magnets, 3D magnetic compound has the ability to purify the detection signal in a short time. The target AFB\u003csub\u003e1\u003c/sub\u003e was efficiently recognized by AFB\u003csub\u003e1\u003c/sub\u003eapt-modified SERS probes. Owing to the dissociation of the probes, the effective abatement of 4-MBA signal intensity in the substrate was showed. Therefore, the SERS sensor achieved ultrasensitive and good selective measure toward AFB\u003csub\u003e1\u003c/sub\u003e.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials and reagent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChloroauric acid (HAuCl\u003csub\u003e4\u003c/sub\u003e\u0026middot;3H\u003csub\u003e2\u003c/sub\u003eO), ascorbic acid (AA), sodium citrate tribasic dihydrate (Na\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO), silver nitrate (AgNO\u003csub\u003e3\u003c/sub\u003e), ammonia (NH\u003csub\u003e3\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO), ferric chloride (FeCl\u003csub\u003e3\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO), polyethylene glycol, ethylene glycol (EG), sodium acetate (CH\u003csub\u003e3\u003c/sub\u003eCOONa), thiourea (CH\u003csub\u003e4\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eS), anhydrous ethanol (C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO), Tween 20 (Tween-20), ammonium heptamolybdate, sodium chloride (NaCl), hydrochloric acid (HCl), tris(hydroxymethyl)aminomethane (Tris), 2-mercaptoethanol solution (2-MCH), ethylenediaminetetraacetic acid (EDTA), magnesium chloride (MgCl\u003csub\u003e2\u003c/sub\u003e) and Tris(2-carboxyethyl)phosphine (TCEP) were provided by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 4-mercaptobenzoic acid (4-MBA), aflatoxin B\u003csub\u003e1\u003c/sub\u003e (AFB\u003csub\u003e1\u003c/sub\u003e), aflatoxin M1 (AFM1), ochratoxin (OTA) and fumonisin B1 (FB1) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Peanuts were bought from supermarket. All solutions used in the experiment were prepared using ultrapure water (18.2 M\u0026Omega;) from a water purification system.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThiol-terminal AFB\u003csub\u003e1\u003c/sub\u003e aptamer\u0026nbsp;(He et al. 2020)\u0026nbsp;(SH-AFB\u003csub\u003e1\u003c/sub\u003eapt: 5\u0026apos;-SH-GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CC-3\u0026apos;) was synthesized by Sangon Biological Engineering Technology \u0026amp; Co., Ltd (Shanghai, China), and purified by HPLC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1. Instruments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransmission electron microscope (TEM) images of all nanomaterials were acquired by the JEM-21OOF\u0026nbsp;microscope, operating at 200 kV (Tokyo, Japan). The UV-Vis absorption spectra were recorded by the UV-1800 spectroscopy\u0026nbsp;(China). The Raman spectra of 4-MBA in the assemblies were measured by DXR2xi microscope\u0026nbsp;(U.S.A.)\u0026nbsp;equipped with 50\u0026times; microscope lens and\u0026nbsp;632.8 nm laser excitation. And the\u0026nbsp;origin software was used to smooth and correct the acquired Raman spectra for better data analysis. X-Ray Diffractomer (XRD) patterns of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e NFs were obtained by D2 PHASER analyzer using Cu\u0026ndash;K\u0026alpha; radiation\u0026nbsp;(Switzerland).\u0026nbsp;Zeta potential was determined using Zetasizer Nano ZS analyzer (Melvin, UK).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAu-4MBA@Ag NSs-AFB\u003csub\u003e1\u003c/sub\u003eapt\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGold seeds were synthesized using classic sodium citrate reduction with some minor modifications\u0026nbsp;(Frens 1972). HCl (1 M, 20 \u0026mu;L) was injected to 20 mL of HAuCl\u003csub\u003e4\u003c/sub\u003e\u0026middot;3H\u003csub\u003e2\u003c/sub\u003eO (0.25 mM), which mixed thoroughly with 200 \u0026mu;L of Au NPs under shaking. AgNO\u003csub\u003e3\u003c/sub\u003e (2 mM, 200 \u0026mu;L) and freshly prepared AA (10 mM, 100 \u0026mu;L) were simultaneously added under vigorous shake. Once the solution turned blue-green, the Au NSs were purified by centrifugation (10 min) to stop the growth, and stored at -4℃ before use.\u003c/p\u003e\n\u003cp\u003e0.8 \u0026mu;L of 4-MBA (1 mM) in ethanol was incubated with 100 \u0026mu;L of Au NSs at room temperature for 5 hours. The unbound 4-MBA was removed by centrifuging (3000 rpm) for 10 min. Then, 0.1 M AgNO\u003csub\u003e3\u003c/sub\u003e with different volumes (0.5 \u0026mu;L, 1 \u0026mu;L, 2 \u0026mu;L, 3 \u0026mu;L, and 4 \u0026mu;L), 1 \u0026mu;L of AA (0.1 M), and 2 \u0026mu;L of NH\u003csub\u003e3\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO were dripped under intense vortices, respectively. After incubation for 10 min, the Au-4MBA@Ag NSs were redissolved in ultrapure water by centrifugating for 10 min, Finally, the optimal volume of AgNO\u003csub\u003e3\u003c/sub\u003e was chosen by Raman intensity of 4-MBA\u0026nbsp;to further optimize the analysis performance.\u003c/p\u003e\n\u003cp\u003eTo prepare functionalized nanoprobes, 250 \u0026mu;L of Au-4MBA@Ag NSs was resuspended in 0.05% Tween-20 solution. 5 \u0026mu;L of AFB\u003csub\u003e1\u003c/sub\u003eapt (50 \u0026mu;M, activated with TCEP solution in equal volume for 1 h) was added, and incubated for 1 h. NaCl solution was dropped every 30 min to complete aging (with a final concentration of 0.25 M). Afterward, the mixture maintained overnight at 37\u0026deg;C. The free nucleic acid was discarded under centrifugation to produce Au-4MBA@Ag NSs-AFB\u003csub\u003e1\u003c/sub\u003eapt.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e NFs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTake 1.35 g of FeCl\u003csub\u003e3\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO powder into 18 mL of ethylene glycol solution. Then, 1.62 g of CH3COONa and 0.45 g of polyethylene glycol were successively added into the above mixture. The solution was thoroughly mixed well under sonicating for 30 min, and reacted at 200\u0026deg;C for 8 h in a high temperature reactor. The reaction products were washed three times with ethanol and ultrapure water by magnetic separation, and dried under vacuum at 60\u0026deg;C for 6 h to collect Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs.\u003c/p\u003e\n\u003cp\u003e20 mg of the obtained Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs powder was dispersed well in 10 mL of ultrapure water by ultrasonication. Then, 0.112 g of ammonium heptamolybdate and 0.365 mg of thiourea were sequentially added by vigorous sonicating for 30 min. Under 200\u0026deg;C, the mixture was placed in a high temperature reactor for 8 h.\u0026nbsp;The resulting products were washed repeatedly with ethanol and ultrapure water. Finally, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e NFs powder was obtained under vacuum drying at 60\u0026deg;C for 10 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of labeled-aptasensor for AFB\u003csub\u003e1\u003c/sub\u003e detection\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo construct Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e NFs-AFB\u003csub\u003e1\u003c/sub\u003eapt-Au@Ag NSs sensors, SERS probes were mixed with magnetic substrates at different volume ratios (1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, and 1:3) at room temperature for 20 min. The unattached nanoparticles were cleared by magnetic separation. Moreover, 2-MCH solution as blocker closed the unreacted sites to prevent nonspecific binding. The optimal volume ratio was determined by Raman intensity of 4-MBA at 1581 cm\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eNext, AFB\u003csub\u003e1\u003c/sub\u003e standard solutions with different amounts (final concentrations of 0.1 ng/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL, and 1000 ng/mL, respectively) were reacted with 200 \u0026mu;L of the above solution at room temperature for 1 h. The complex was magnetically separated and washed twice to ensure signal drop. After that, 8 \u0026mu;L of Au-4MBA@Ag NSs-based 3D nanohybirds solutions were dropped onto aluminum foil and dried at 25℃. The SERS measure was performed at an excitation wavelength of 633 nm. The logarithmic value of AFB\u003csub\u003e1\u003c/sub\u003e concentration was used as the horizontal coordinate, and the Raman intensity of 4-MBA at 1581 cm\u003csup\u003e-1\u003c/sup\u003e (I\u003csub\u003e1581\u003c/sub\u003e) was employed as the vertical coordinate to determine the standard curve of the method.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSelectivity evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the selectivity of the SERS aptasensor, the control experiments were performed with interfering toxins including AFM\u003csub\u003e1\u003c/sub\u003e, OTA and FB\u003csub\u003e1\u003c/sub\u003e and AFB\u003csub\u003e1\u003c/sub\u003e under the same experimental conditions. The concentrations of the above toxins were set as 100 ng/mL. The comparison of SERS intensity directly reflected the specificity of aptamer-based sensor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReal samples detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFresh peanuts were selected to analyze the utility of SERS aptamer sensors to detect AFB\u003csub\u003e1\u003c/sub\u003e in real samples. For pretreatment, the peanut samples were fully ground to powder firstly. 2 g of powder was dissolved in a mixture of methanol/water (5.6 mL/2.4 mL) under sonicating for 30 min to aid the extraction performance\u0026nbsp;(Jing et al. 2009). The supernatant was collected by centrifuging (5000 rpm, 15 min). Lastly, AFB\u003csub\u003e1\u003c/sub\u003e standards were added at different final concentrations of 0.5 ng/mL, 5.0 ng/mL, and 50.0 ng/mL, respectively. After the above procedure analysis, the recovery results were calculated.\u003c/p\u003e"},{"header":"3. Results And Discussion","content":"\u003cp\u003e\u003cstrong\u003e3.1. Detection strategy of the aptasensor for AFB\u003csub\u003e1\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe working principle of designed 3D plasmonic SERS aptasensor for AFB\u003csub\u003e1\u003c/sub\u003e detection was illustrated in scheme 1. Bimetallic Au@Ag plasmonic nanostars were acquired by modifying 4-MBA on anisotropic Au NSs via Au-SH bond, further reducing AgNO\u003csub\u003e3\u003c/sub\u003e to form Ag outer shell. The enhanced EM excited by affluent hot spots of sharp tips significantly amplified the SERS response of 4-MBA embedded on bilayer Au-Ag. The specific core-shell nanostructure gave effectively protective effect on signal, improving the detection stability. Then, the coupled AFB\u003csub\u003e1\u003c/sub\u003eapt on Au@Ag stars employed as SERS probes could be capable of capturing target AFB\u003csub\u003e1\u003c/sub\u003e with high sensitivity and high specificity. Meanwhile, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e plasmonic nanoflowers provided large number of active binding sites for SERS probes, generating the 3D magnetic SERS plasmonic substrates-aptamer-SERS plasmonic probes (Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e-AFB\u003csub\u003e1\u003c/sub\u003eapt-Au-4MBA@Ag NSs), which owned both SERS activities and magnetic features. Combined with the chemical enhancement effect of MoS\u003csub\u003e2\u003c/sub\u003e NSs on the multilayer plasma nanostructures, SERS signal was further magnified to the maximum. When AFB\u003csub\u003e1\u003c/sub\u003e appeared, the probes were separated from the nanoflowers owing to the specifical capture of aptamers, causing an obvious decrease of I\u003csub\u003e4-MBA\u003c/sub\u003e. Thus, I\u003csub\u003e4-MBA\u003c/sub\u003e was inversely related to the AFB\u003csub\u003e1\u003c/sub\u003e concentration, enabling sensitive detection in the peanuts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Characterization and optimization of Au@Ag\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;NSs-AFB\u003csub\u003e1\u003c/sub\u003eapt\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe TEM images for the morphology of the nanoparticles were shown in Fig. 1. Au NSs had great dispersion and showed\u0026nbsp;sharply\u0026nbsp;star-shaped morphology, which consisted of spherical core sized 34.53 nm \u0026plusmn; 5.29 nm and rich\u0026nbsp;sharp tips with a size of around 31.13 nm \u0026plusmn; 6.71 nm (Fig. 1A).\u0026nbsp;As shown in Fig. 1B, after the reduction of Ag ions was induced under ammonia-adjusted alkaline conditions, the Ag atoms were uniformly deposited to form Ag shells on the surface of monolayer Au NSs, which was due to the extremely similar lattice edge widths of both Au and Ag\u0026nbsp;(Rodriguez-Gonzalez et al. 2005). The results showed that moderate amount of silver nitrate had little effect on the tip sharpness of the nanostars. In the UV-Vis spectra of Fig. 1C, as the formation of Au NSs prepared by Au seed growth method, the resonance plasmonic absorption peak of Au NPs located at 521 nm redshifted to around 691 nm. Thereby the clear characteristic absorption peak of Ag NPs appeared at 407 nm, indicating the successful modification of Ag shell outside Au NSs. The results showed the\u0026nbsp;bimetallic Au@Ag plasmonic nanostars\u0026nbsp;exhibited two absorption peaks at 407 nm and 549 nm in optical properties. Furthermore, the SERS properties of bimetallic nanomaterials were characterized by Raman spectrometer in Fig. 1D. There were no obvious Raman peaks on Au NSs. In contrast, Au-4MBA NSs occurred effectively enhanced the SERS signals of 4-MBA, which was owing to the multiple SERS \u0026quot;hot spots\u0026quot; at the tips. It was worth noting that Au@Ag NSs plasmonic structure-amplified 4-MBA signal was 4 times stronger than that of monolayer precious metal Au, which was in agreement with previous study\u0026nbsp;(Jing et al. 2020). The above results clearly verified the successful preparation of Au-4MBA@Ag NSs for the further SERS detection of AFB1.\u003c/p\u003e\n\u003cp\u003eIn addition, the effect of the addition amount of 4-MBA and AgNO\u003csub\u003e3\u003c/sub\u003e on the plasmonic SERS probes was investigated. As shown in Fig. S1, the SERS intensity of 4-MBA was the strongest with the addition of 4-MBA (1 mM) up to 0.8 \u0026mu;L, suggesting the 4-MBA adsorption on the surface of Au NSs reached saturation. Based on the optimized addition value of Raman molecular, the optical features of materials modified with different amounts of AgNO\u003csub\u003e3\u003c/sub\u003e (1 M) were characterized by UV-Vis spectra shown in Fig. S2C. Along with the increase of the AgNO\u003csub\u003e3\u003c/sub\u003e volume to 4 \u0026mu;L, the resonance plasmonic absorption peak of Au@Ag NSs underwent a gradual blue shift, which was closely related to the aspect ratio of the star-shaped tip. Meanwhile, a new absorption peak at around 407 nm appeared and enhanced, proving the successful deposition of Ag shells and a gradual increase in thickness. All these results were tightly dependent on the dielectric properties around the material\u0026nbsp;(Han et al. 2017). In Fig. S2 (A-B), the SERS intensity of 4-MBA at 1078 cm\u003csup\u003e-1\u003c/sup\u003e showed an obvious increase in the range of 0.5 \u0026mu;L to 4 \u0026mu;L. Although the SERS signal was still enhanced when the modification amount exceeded 2 \u0026mu;L, the stability of signal measured by repeated tests significantly dropped, which strongly influenced the detection sensitivity. Moreover, the TEM image of Fig. S2D characterized the morphology of bimetallic Au@Ag NSs at the AgNO3 addition amount of 3 \u0026mu;L. It was found that the nanocomposites had completely tended to be spherical owing to the modification of extremely thick silver shells, which was also the direct cause of above signal instability. The results showed that the thickness of the Ag shell in Au NSs tips manifested positive correlation with the enhancement effects, while the change of morphology was significantly related to signal stability, showing that appropriate Ag shell thickness was a vital factor for stable detection\u0026nbsp;(Mott et al. 2012). Thus, 0.8 \u0026mu;L of 4-MBA (1 mM) and 2 \u0026mu;L of AgNO\u003csub\u003e3\u0026nbsp;\u003c/sub\u003e(0.1 M) were chosen as the optimal addition amounts for the preparation of SERS probes.\u003c/p\u003e\n\u003cp\u003eThe coupled AFB\u003csub\u003e1\u003c/sub\u003eapt on the surfaces of Au@Ag NSs was illustrated by UV-Vis spectroscopy and zeta potential measurements in Fig. S3. For Au@Ag NSs-AFB\u003csub\u003e1\u003c/sub\u003eapt, the absorbance of the supernatant decreased greatly, and the average zeta potential decreased to -33.9 mV, which was due to the characteristic absorption peak at 260 nm\u0026nbsp;(Wu et al. 2012), and the presence of a negatively charged phosphate group of the introduced ssDNA\u0026nbsp;(Zhu et al. 2021). Thus, the functionalized bimetallic nanoprobes were prepared successfully.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.\u003c/strong\u003e\u003cstrong\u003e3. Characterization and optimization of the fabricated\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e NFs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to obtain magnetic substrates, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e NFs was synthesized by secondary hydrothermal methods. As shown in Fig. 2A, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs were mostly homogeneous spherical morphology with a particle size of around 231.77 nm \u0026plusmn; 17.97 nm. The magnetic response feature of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs was further verified by the separation test of the external magnetic field. The illustration of Fig. 2A showed that effective separation and enrichment by magnets were achieved within 15 s, indicating good paramagnetic properties of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs. Fig. S4A revealed that 2D MoS\u003csub\u003e2\u003c/sub\u003e NSs (carbon-based nanomaterials) were highly transparent nanosheets with numerous folds. After another high temperature reaction, MoS\u003csub\u003e2\u003c/sub\u003e NSs were successfully combined around spherical Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs as revealed in Fig. 2B, forming\u0026nbsp;multi-functional 3D core-shell Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e magnetic nanoflowers. Besides, the time of good magnetic separation was 30s for Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e NFs in the illustration of Fig. 2B, indicating that the load of moderate MoS\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eNSs had a weak influence on the magnetic effect, which was suitable to be SERS magnetic substrate in subsequent analysis. The combination of plasmonic Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs with good magnetic effect and MoS\u003csub\u003e2\u003c/sub\u003e NSs with large surface area was conducive to rapid enrichment of SERS signal in the complex detection system. Moreover, the crystalline morphology of materials was confirmed by XRD in Fig. 2C. The typical diffraction peaks of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs at 30.6\u0026deg;, 35.9\u0026deg;, 43.5\u0026deg;, 53.9\u0026deg;, 57.4\u0026deg; and 62.9\u0026deg; were ascribed to (220), (331), (400), (422), (511) and (440) planes, which was consistent with their standard cards (JCPDS No. 19-0629). Then, the diffraction peaks at 18.1\u0026deg; (002), 36.1\u0026deg; (100), 43.9\u0026deg; (103) and 58.1\u0026deg; (110) proved the fabrication of MoS\u003csub\u003e2\u003c/sub\u003e NSs nanostructure. The characteristic peaks of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs were measured for XRD spectra of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e NFs, indicating that the crystal morphology and phases of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs were preserved. The results were similar with those of Lu et al\u0026nbsp;(Lu et al. 2021). As shown in Fig. 2D, the Zeta potential of the final composite was -19.5 mV as a result of the MoS\u003csub\u003e2\u003c/sub\u003e NSs with negative charge (-32.3 mV) as shells wrapping around the positive charged Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs cores (18.9 mV). Therefore, all the above results characterized the successful preparation of 3D magnetic Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e nanoflowers, which provided more active sites for the connection of the aptamers to facilitate the construction of next detection system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.\u003c/strong\u003e\u003cstrong\u003e4. Feasibility and optimization of 3D SERS aptasensor for AFB\u003csub\u003e1\u003c/sub\u003e detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo obtain the designed assemblies to analyze AFB\u003csub\u003e1\u003c/sub\u003e, the TEM images were employed to characterize the surface morphology of SERS sensor. From Fig. 3A, the anisotropic\u0026nbsp;bimetallic\u0026nbsp;nanostars were successfully bound around 3D Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e NFs core to build assemblies, due to the aptamers were combined to the outer shell MoS\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eNSs through non-covalent bonds. In SERS spectra of Fig. 3B, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e magnetic substrates showed no Raman peaks in the range from 900 cm\u003csup\u003e-1\u003c/sup\u003e to 1800 cm\u003csup\u003e-1\u003c/sup\u003e. However, compared with Au-4MBA@Ag NSs, the stronger 4-MBA SERS signal was observed on the plasmonic Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e NFs-AFB\u003csub\u003e1\u003c/sub\u003eapt-Au-4MBA@Ag NSs assemblies, owing to the fact that chemical enhancement of MoS\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eNSs synergistically enhanced SERS performance of the assemblies. After adding target AFB\u003csub\u003e1\u003c/sub\u003e, 4-MBA response significantly reduced, due to the decrease of SERS probes on the assemblies. Moreover, TEM images of the assemblies without and with AFB\u003csub\u003e1\u003c/sub\u003e were clearly shown in Fig. S5(A-B), indicating that the presence of AFB1 induced the dissociation of nanostars from the substrates due to the forming of Au@Ag NSs-AFB1apt/AFB\u003csub\u003e1\u003c/sub\u003e composites. Thus, all the above results demonstrated the feasibility of SERS sensor for quantitatively sensing AFB\u003csub\u003e1\u003c/sub\u003e based on significant changes in SERS signal peak intensity caused by changes in the composition of assembler.\u003c/p\u003e\n\u003cp\u003eTo further obtain highly sensitive SERS aptasensor, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e NFs as magnetic substrates and Au-4MBA@Ag NSs-AFB\u003csub\u003e1\u003c/sub\u003eapt as SERS probes were mixed in different volume ratios at room temperature. As obtained from Fig. 3C, the SERS intensity of the assemblies at 1581 cm\u003csup\u003e-1\u003c/sup\u003e reached the strongest at the volume ratio of 1:2, which was chosen as the optimal addition ratio. Besides, in order to evaluate the reproducibility and stability of the constructed AFB\u003csub\u003e1\u003c/sub\u003e SERS sensor, the original Raman spectra of random 15 points shown in Fig. 3D (waterfall plot) were collected on the assemblies. And the RSD value of Raman peak intensity (1581 cm\u003csup\u003e-1\u003c/sup\u003e) was calculated to be 7.6% (Fig. S6A), manifesting the good uniformity of signals on the SERS assemblies. Different storage times (1, 3, 5, 7, 9, 11, and 13 days) of 3D aptasensor were also studied (Fig. S6B). SERS signal intensity remained 86.5% on the around thirteenth day, revealing the good time-dependent stability of sensor.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. Sensitivity of AFB\u003csub\u003e1\u003c/sub\u003e detection by SERS aptasensor\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo verify the detection capability of the aptasensor under optimized experimental conditions, a series of AFB\u003csub\u003e1\u003c/sub\u003e addition amounts were reacted with the SERS system at the final concentration from 0.1 ng/mL to 1000 ng/mL. As shown in Fig. 4A, the SERS signal intensity of 4-MBA molecular gradually decreased with the increase of AFB\u003csub\u003e1\u003c/sub\u003e concentration, which was owing to the release of a large amount of 4MBA-based SERS probes induced by AFB\u003csub\u003e1\u003c/sub\u003e from the magnetic substrates. The SERS peak intensity of 4-MBA at 1581 cm\u003csup\u003e-1\u003c/sup\u003e (I\u003csub\u003e1581\u003c/sub\u003e) as quantitative value showed negatively linearly correlation with the logarithmic value of AFB\u003csub\u003e1\u003c/sub\u003e concentration (log\u003csub\u003e10\u003c/sub\u003eC\u003csub\u003eAFB1\u003c/sub\u003e). After the linear fit in Fig. 4B, the regression equation was Y = 8541.55 - 2348.69X (R\u003csup\u003e2\u003c/sup\u003e = 0.986). The associated LOD was as low as 58.9 pg/mL calculated by the formula (LOD = 10\u003csup\u003e(Y+3SD-A)/B\u003c/sup\u003e. Where Y and SD are the mean and standard deviation of the blank sample signal, respectively. A and B represent the intercept and slope of the curve, respectively. And signal-to-noise ratio was set as 3:1). The detection sensitivity of 3D plasma SERS nanoassemblies satisfied the EU requirements for the measure of the maximum limit of AFB\u003csub\u003e1\u003c/sub\u003e in cereal. Additionally, Table S1 gave a comparison between our work for AFB\u003csub\u003e1\u003c/sub\u003e detection and other previously reported analytical methods. The results showed that this study had obtained lower detection limits and wider detection range relative to the work of some researchers, but the sensitivity still needed to be further improved.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.\u003c/strong\u003e\u003cstrong\u003e6. Selectivity, reproducibility, and practicality evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate the selectivity of the aptasensor, AFM\u003csub\u003e1\u003c/sub\u003e, OTA and FB\u003csub\u003e1\u003c/sub\u003e toxins were selected to detect under the same experiment. In Fig. 5(A-B), I\u003csub\u003e1581\u003c/sub\u003e of the three interfering toxins changed weakly compared to that of the blank sample, while that of AFB\u003csub\u003e1\u003c/sub\u003e (100 ng/mL) decreased significantly, showing that the specific aptamer conferred good selectivity to the SERS sensor. To evaluate the reproducibility of the proposed sensor, AFB\u003csub\u003e1\u003c/sub\u003e (10 ng/mL) was analyzed repeatedly on the same batch and different six batches. It can be seen from the SERS intensity bar chart in Fig. S7 that this strategy presented relatively low and acceptable RSD values of I\u003csub\u003e1581\u003c/sub\u003e, which were 5.1% and 6.4%, respectively. \u003csub\u003e\u0026nbsp;\u003c/sub\u003eTo verify the practicality of the method in real samples, the peanuts at AFB\u003csub\u003e1\u003c/sub\u003e spiked concentrations of 0.5 ng/mL-50.0 ng/mL were selected for the recovery experiments. The satisfactory recoveries ranging from 97.9% to 99.4% with RSDs of 4.3%-9.3% were obtained as shown in Table 1, indicating that the assay was suitable for the detection of peanut, and had bright application prospect in food safety analysis.\u0026nbsp;\u003c/p\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn all, a rational SERS aptasensor for quantitative detection of AFB\u003csub\u003e1\u003c/sub\u003e has been contrasted based on 3D plasmonic magnetic SERS assemblies (Au-4MBA@Ag NSs-AFB\u003csub\u003e1\u003c/sub\u003eapt-Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e NFs). The designed SERS-active platform has the following excellent performance: (1) rapid magnetic separation features, (2)\u0026nbsp;good stability of substrates for long-term storage,\u0026nbsp;(3) signal amplification function provided from abundant hot spots, (4) synergistic enhancement effect of bimetal Au-Ag and MoS\u003csub\u003e2\u003c/sub\u003e NSs on the SERS signal of 4-MBA. AFB\u003csub\u003e1\u003c/sub\u003e toxin was sensitively detected from 0.1 ng/mL to 100 ng/mL,\u0026nbsp;achieving the LOD as low as 58.9 pg/mL. The high selectivity under interfering toxins, and good reproducibility within different batches measurement of this sensor were further determined. Moreover, the\u0026nbsp;strategy\u0026nbsp;can be capable of detecting AFB\u003csub\u003e1\u003c/sub\u003e in peanut samples with good recoveries of 97.9%-98.7%. Therefore, the proposed SERS sensor as effective detection technique has the potential to be applied to the monitoring of various hazards in the field of food safety.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge instrument platform of school of Food Science and Technology, Jiangnan University, for technical supports.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Social Development Fund Project of Wuxi (N20201001), the Excellent Scientific and Technological Innovation Team of Jiangsu Universities, the 333 High Level Talents Training Project of Jiangsu Province, and the National Natural Science Foundation of China (22176085 and 21874065).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eBand\u0026eacute; M, Traor\u0026eacute; I, Nikiema F et al (2022) Aflatoxins contents determination in some foodstuffs in Burkina Faso and human health risk assessment. 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ACS nano 14: 6258-6268.\u0026nbsp;https://doi.org/10.1021/acsnano.0c02418\u003c/p\u003e\n\u003cp\u003eRodriguez-Gonzalez B, Burrows A, Watanabe M, Kiely CJ, Liz-Marzan LM (2005) Multishell bimetallic AuAg nanoparticles: synthesis, structure and optical properties. Journal of Materials Chemistry 15: 1755-1759.\u0026nbsp;\u003ca href=\"https://doi.org/10.1039/b500556f\"\u003ehttps://doi.org/10.1039/b500556f\u003c/a\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRushing BR, Selim MI (2019) Aflatoxin B1: A review on metabolism, toxicity, occurrence in food, occupational exposure, and detoxification methods. Food and Chemical Toxicology 124: 81-100.\u0026nbsp;\u003ca href=\"https://doi.org/10.1016/j.fct.2018.11.047\"\u003ehttps://doi.org/10.1016/j.fct.2018.11.047\u003c/a\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eShao Q, Zhang D, Wang C-e, et al (2021) Ag@ MIL-101 (Cr) film substrate with high SERS enhancement effect and uniformity. 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Analytical chemistry 84: 7692-7699.\u0026nbsp;\u003ca href=\"https://doi.org/10.1021/ac3015164\"\u003ehttps://doi.org/10.1021/ac3015164\u003c/a\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYang B, Zhang C, Zhang X et al (2020) Survey of aflatoxin B1 and heavy metal contamination in peanut and peanut soil in China during 2017-2018. Food Control 118.\u0026nbsp;\u003ca href=\"https://doi.org/10.1016/j.foodcont.2020.107372\"\u003ehttps://doi.org/10.1016/j.foodcont.2020.107372\u003c/a\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYang C, Qing C, Wang Q, Zhang X, Lou J, Liu Y (2020) Synthesis of the hybrid CdS/Au flower-like nanomaterials and their SERS application. Sensors and Actuators B: Chemical 304: 127218.\u0026nbsp;https://doi.org/10.1016/j.snb.2019.127218\u003c/p\u003e\n\u003cp\u003eZhao X, Campbell S, Wallace GQ, Claing A, Bazuin CG, Masson J-F (2020) Branched Au nanoparticles on nanofibers for surface-enhanced Raman scattering sensing of intracellular pH and extracellular pH gradients. ACS sensors 5: 2155-2167.\u0026nbsp;https://doi.org/10.1021/acssensors.0c00784\u003c/p\u003e\n\u003cp\u003eZhu A, Jiao T, Ali S, Xu Y, Ouyang Q, Chen Q (2021) SERS sensors based on aptamer-gated mesoporous silica nanoparticles for quantitative detection of Staphylococcus aureus with signal molecular release. Analytical chemistry 93: 9788-9796.\u0026nbsp;\u003ca href=\"https://doi.org/10.1021/acs.analchem.1c01280\"\u003ehttps://doi.org/10.1021/acs.analchem.1c01280\u003c/a\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eZuki-Orozco BA, Batres-Esquivel LE, Ortiz-Perez MD, Juarez-Flores BI, Diaz-Barriga F (2018) Aflatoxins contamination in maize products from rural communities in San Luis Potosi, Mexico. Annals of Global Health 84: 300-305.\u0026nbsp;\u003ca href=\"https://doi.org/10.29024/aogh.918\"\u003ehttps://doi.org/10.29024/aogh.918\u003c/a\u003e.\u0026nbsp;\u003c/p\u003e"},{"header":"Schemes","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section\u003c/p\u003e"},{"header":"Tables","content":"\u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eTable 1.\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eDetection of AFB\u003csub\u003e1\u003c/sub\u003e spiked in real peanut samples with the proposed method.\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"width: 4.7e+2pt;margin-left:-.45in;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82.25pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 2.25pt medium 1.5pt;padding: 0in 5.4pt;height: 31.15pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eSamples\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144.55pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 2.25pt medium 1.5pt;padding: 0in 5.4pt;height: 31.15pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eAdded concentration (ng/mL)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111.8pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 2.25pt medium 1.5pt;padding: 0in 5.4pt;height: 31.15pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eMeasurement (ng/mL) (\u0026plusmn;SD)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.45pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 2.25pt medium 1.5pt;padding: 0in 5.4pt;height: 31.15pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eRecovery (%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58.2pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 2.25pt medium 1.5pt;padding: 0in 5.4pt;height: 31.15pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eRSD (%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82.25pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144.55pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111.8pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;vertical-align:middle;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e0.489 \u0026plusmn; 0.021\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.45pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;vertical-align:middle;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e97.9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58.2pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;vertical-align:middle;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e4.3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82.25pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144.55pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e5.0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111.8pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e4.934 \u0026plusmn; 0.304\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.45pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e98.7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58.2pt;border: medium none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e6.2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82.25pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 2.25pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144.55pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 2.25pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e50.0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111.8pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 2.25pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e49.237 \u0026plusmn; 4.598\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.45pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 2.25pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e98.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58.2pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 2.25pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e9.3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Calibri\",sans-serif;margin:0in;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"surface-enhanced raman spectroscopy, aptasensor, aflatoxin B1, magnetic nanoflowers","lastPublishedDoi":"10.21203/rs.3.rs-2437251/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2437251/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAs a virulent metabolite, aflatoxin B\u003csub\u003e1\u003c/sub\u003e (AFB\u003csub\u003e1\u003c/sub\u003e) presented in various cereal grain is tightly implicated in severe human diseases. In this study, 3D plasmonic nanohybirds of Raman molecule 4-mercaptobenzoic acid (4-MBA)-embedded and AFB\u003csub\u003e1\u003c/sub\u003e aptamer-modified bimetallic nanostars as probes bound to magnetic nanoflowers were fabricated and demonstrated as a high-performance SERS-active aptasensor to quantitatively analyze AFB\u003csub\u003e1\u003c/sub\u003e. Bimetallic Au@Ag SERS plasmonic nanoprobes with enhanced properties were capable of enhancing discriminative Raman peaks of 4-MBA. Then, the integration of iron tetroxide nanoparticles (Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs) and molybdenum disulfide nanosheets (MoS\u003csub\u003e2\u003c/sub\u003e NSs) with huge specific surface area constituted stable 3D Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e plasmonic nanoflowers, facilitating the bind of numerous aptamer-based SERS probes via the non-covalent interaction between MoS\u003csub\u003e2\u003c/sub\u003e NSs and aptamer, which were ideal candidates for SERS-active substrates. Additionally, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs as magenetic core endowed 3D nanocomposites with specific magnetic separation characteristic that caused the collected SERS hotspots to exhibit superior signal response, and further strengthening the sensitivity in a complex food matrix. Aptamer-target AFB\u003csub\u003e1\u003c/sub\u003e specific recognition triggered linearly diminished 4-MBA signal intensity (I\u003csub\u003e4-MBA\u003c/sub\u003e) on the substrate to achieve a low detection limit of 58.9 pg/mL. Furthermore, the sensor has the potential to be a promising monitoring tool for trace contaminants.\u003c/p\u003e","manuscriptTitle":"3D plasmonic SERS aptasensor for rapid detection of aflatoxin B1 combined with Au@Ag bimetallic nanostars and Fe3O4@MoS2 magnetic nanoflowers","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2023-05-16 18:58:58","doi":"10.21203/rs.3.rs-2437251/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2023-01-10 16:36:37","doi":"10.21203/rs.3.rs-2437251/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ddc9f6f1-8c80-4078-92cf-6470e51916cf","owner":[],"postedDate":"May 16th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-03-02T17:14:34+00:00","versionOfRecord":[],"versionCreatedAt":"2023-05-16 18:58:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-2437251","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2437251","identity":"rs-2437251","version":["v2"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}