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

doi:10.21203/rs.3.rs-2437251/v1

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

https://doi.org/10.21203/rs.3.rs-2437251/v1

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As a virulent metabolite, aflatoxin B₁ (AFB₁) 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 AFB₁ aptamer-modified bimetallic nanostars as probes bound to magnetic nanoflowers were fabricated and demonstrated as a high-performance SERS-active aptasensor to quantitatively analyze AFB₁. 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 (Fe₃O₄ NPs) and molybdenum disulfide nanosheets (MoS₂ NSs) with huge specific surface area constituted stable 3D Fe₃O₄@MoS₂ plasmonic nanoflowers, facilitating the bind of numerous aptamer-based SERS probes via the non-covalent interaction between MoS₂ NSs and aptamer, which were ideal candidates for SERS-active substrates. Additionally, Fe₃O₄ 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 AFB₁ 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

Aflatoxin B₁ (AFB₁) derived from fungi species is difuran-coumarin compound with the most poisonous activities (Marchese et al. 2018). Over the last few decades, AFB₁ 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 AFB₁ detection rate reached 33.8% from four major production regions in China (Yang et al. 2020). In Mexico, AFB₁ residues were found in 80% of investigated samples, 26% of the samples exceeded the EU maximum AFB₁ limit value (5 µg/kg) (Zuki-Orozco et al. 2018). AFB₁ 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 AFB₁ toxins with stable biochemical properties, even pasteurization. Prolonged exposure to low doses of the insidious AFB₁ 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 AFB₁, 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 AFB₁ 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 AFB₁ labeled sensor based on Raman signal molecules with low background noise and significant characteristic peaks, realizing efficient indirect AFB₁ 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, Fe₃O₄ 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 Fe₃O₄ NPs exposed to air are susceptible to oxidation and agglomeration. The introduction of specific carriers is considered by researchers as an effective measure. Notably, MoS₂ 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 MoS₂ NSs wrapped around Fe₃O₄ 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 AFB₁ for the first time with Fe₃O₄@MoS₂ NFs/Au@Ag NSs-AFB₁apt nanohybirds. Firstly, Au-4MBA@Ag NSs-AFB₁apt 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 AFB₁ in the presence of other interfering substances. Meanwhile, Fe₃O₄@MoS₂ NFs serve as the enrichment and amplification substrates of SERS signal when AFB₁apt-functionalized SERS probes were immobilized on MoS₂ NSs covered on Fe₃O₄ 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 AFB₁ was efficiently recognized by AFB₁apt-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 AFB₁.

2.1. Materials and reagent

Chloroauric acid (HAuCl₄·3H₂O), ascorbic acid (AA), sodium citrate tribasic dihydrate (Na₃C₆H₅O₇·2H₂O), silver nitrate (AgNO₃), ammonia (NH₃·H₂O), ferric chloride (FeCl₃·6H₂O), polyethylene glycol, ethylene glycol (EG), sodium acetate (CH₃COONa), thiourea (CH₄N₂S), anhydrous ethanol (C₂H₆O), 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 (MgCl₂) and Tris(2-carboxyethyl)phosphine (TCEP) were provided by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 4-mercaptobenzoic acid (4-MBA), aflatoxin B₁ (AFB₁), 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 AFB₁ aptamer (He et al. 2020) (SH-AFB₁apt: 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 Fe₃O₄@MoS₂ NFs were obtained by D2 PHASER analyzer using Cu–Kα radiation (Switzerland). Zeta potential was determined using Zetasizer Nano ZS analyzer (Melvin, UK).

2.2. Preparation of Au-4MBA@Ag NSs-AFB₁apt

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 HAuCl₄·3H₂O (0.25 mM), which mixed thoroughly with 200 µL of Au NPs under shaking. AgNO₃ (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 AgNO₃ 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 NH₃·H₂O 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 AgNO₃ 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 AFB₁apt (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-AFB₁apt.

2.3. Preparation of Fe₃O₄@MoS₂ NFs

Take 1.35 g of FeCl₃·6H₂O 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 Fe₃O₄ NPs.

20 mg of the obtained Fe₃O₄ 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, Fe₃O₄@MoS₂ NFs powder was obtained under vacuum drying at 60°C for 10 h.

2.4. Construction of labeled-aptasensor for AFB₁ detection

To construct Fe₃O₄@MoS₂ NFs-AFB₁apt-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, AFB₁ 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 AFB₁ concentration was used as the horizontal coordinate, and the Raman intensity of 4-MBA at 1581 cm^-1 (I₁₅₈₁) was employed as the vertical coordinate to determine the standard curve of the method.

2.5. Selectivity evaluation

To assess the selectivity of the SERS aptasensor, the control experiments were performed with interfering toxins including AFM₁, OTA and FB₁ and AFB₁ 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.

2.6. Real samples detection

Fresh peanuts were selected to analyze the utility of SERS aptamer sensors to detect AFB₁ 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, AFB₁ 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.

3.1. Detection strategy of the aptasensor for AFB₁

Scheme 1.

The working principle of designed 3D plasmonic SERS aptasensor for AFB₁ 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 AgNO₃ 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 AFB₁apt on Au@Ag stars employed as SERS probes could be capable of capturing target AFB₁ with high sensitivity and high specificity. Meanwhile, Fe₃O₄@MoS₂ plasmonic nanoflowers provided large number of active binding sites for SERS probes, generating the 3D magnetic SERS plasmonic substrates-aptamer-SERS plasmonic probes (Fe₃O₄@MoS₂-AFB₁apt-Au-4MBA@Ag NSs), which owned both SERS activities and magnetic features. Combined with the chemical enhancement effect of MoS₂ NSs on the multilayer plasma nanostructures, SERS signal was further magnified to the maximum. When AFB₁ 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 AFB₁ concentration, enabling sensitive detection in the peanuts.

3.2. Characterization and optimization of Au@Ag NSs-AFB₁apt

Figure 1.

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 AgNO₃ 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 AgNO₃ (1 M) were characterized by UV-Vis spectra shown in Fig. S2C. Along with the increase of the AgNO₃ 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 AgNO₃ (0.1 M) were chosen as the optimal addition amounts for the preparation of SERS probes.

The coupled AFB₁apt on the surfaces of Au@Ag NSs was illustrated by UV-Vis spectroscopy and zeta potential measurements in Fig. S3. For Au@Ag NSs-AFB₁apt, 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 Fe₃O₄@MoS₂ NFs

Figure 2.

In order to obtain magnetic substrates, Fe₃O₄@MoS₂ NFs was synthesized by secondary hydrothermal methods. As shown in Fig. 2A, Fe₃O₄ NPs were mostly homogeneous spherical morphology with a particle size of around 231.77 nm ± 17.97 nm. The magnetic response feature of Fe₃O₄ 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 Fe₃O₄ NPs. Fig. S4A revealed that 2D MoS₂ NSs (carbon-based nanomaterials) were highly transparent nanosheets with numerous folds. After another high temperature reaction, MoS₂ NSs were successfully combined around spherical Fe₃O₄ NPs as revealed in Fig. 2B, forming multi-functional 3D core-shell Fe₃O₄@MoS₂ magnetic nanoflowers. Besides, the time of good magnetic separation was 30s for Fe₃O₄@MoS₂ NFs in the illustration of Fig. 2B, indicating that the load of moderate MoS₂ NSs had a weak influence on the magnetic effect, which was suitable to be SERS magnetic substrate in subsequent analysis. The combination of plasmonic Fe₃O₄ NPs with good magnetic effect and MoS₂ 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 Fe₃O₄ 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 MoS₂ NSs nanostructure. However, only the characteristic peaks of Fe₃O₄ NPs were measured for XRD spectra of Fe₃O₄@MoS₂ NFs, indicating that the crystal morphology and phases of Fe₃O₄ NPs were preserved, and the content and crystallinity of MoS₂ NSs were low. 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 MoS₂ NSs with negative charge (-32.3 mV) as shells wrapping around the positive charged Fe₃O₄ NPs cores (18.9 mV). Therefore, all the above results characterized the successful preparation of 3D magnetic Fe₃O₄@MoS₂ 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 AFB₁ detection

Figure 3

To obtain the designed assemblies to analyze AFB₁, 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 Fe₃O₄@MoS₂ NFs core to build assemblies, due to the aptamers were combined to the outer shell MoS₂ NSs through non-covalent bonds. In SERS spectra of Fig. 3B, Fe₃O₄@MoS₂ 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 Fe₃O₄@MoS₂ NFs-AFB₁apt-Au-4MBA@Ag NSs assemblies, owing to the fact that chemical enhancement of MoS₂ NSs synergistically enhanced SERS performance of the assemblies. After adding target AFB₁, 4-MBA response significantly reduced, due to the decrease of SERS probes on the assemblies. Moreover, TEM images of the assemblies without and with AFB₁ 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/AFB₁ composites. Thus, all the above results demonstrated the feasibility of SERS sensor for quantitatively sensing AFB₁ based on significant changes in SERS signal peak intensity caused by changes in the composition of assembler.

To further obtain highly sensitive SERS aptasensor, Fe₃O₄@MoS₂ NFs as magnetic substrates and Au-4MBA@Ag NSs-AFB₁apt 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 AFB₁ 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 AFB₁ detection by SERS aptasensor

Figure 4.

To verify the detection capability of the aptasensor under optimized experimental conditions, a series of AFB₁ 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 AFB₁ concentration, which was owing to the release of a large amount of 4MBA-based SERS probes induced by AFB₁ from the magnetic substrates. The SERS peak intensity of 4-MBA at 1581 cm^-1 (I₁₅₈₁) as quantitative value showed negatively linearly correlation with the logarithmic value of AFB₁ concentration (log₁₀C_AFB1). After the linear fit in Fig. 4B, the regression equation was Y = 8541.55–2348.69X (R² = 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 AFB₁ in cereal. Additionally, Table S1 gave a comparison between our work for AFB₁ 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

Figure 5.

To validate the selectivity of the aptasensor, AFM₁, OTA and FB₁ toxins were selected to detect under the same experiment. In Fig. 5(A-B), I₁₅₈₁ of the three interfering toxins changed weakly compared to that of the blank sample, while that of AFB₁ (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, AFB₁ (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 I₁₅₈₁, which were 5.1% and 6.4%, respectively. To verify the practicality of the method in real samples, the peanuts at AFB₁ spiked concentrations of 0.5 ng/mL-50.0 ng/mL were selected for the recovery experiments. The satisfactory recoveries ranging from 97.9–99.4% 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.

Table 1

Detection of AFB₁ 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
Notes: “AFB₁” means “aflatoxin B₁”; “SD” means “standard deviation”; “RSD” means “relative standard deviation”.

In all, a rational SERS aptasensor for quantitative detection of AFB₁ has been contrasted based on 3D plasmonic magnetic SERS assemblies (Au-4MBA@Ag NSs-AFB₁apt-Fe₃O₄@MoS₂ 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 MoS₂ NSs on the SERS signal of 4-MBA. AFB₁ 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 AFB₁ in peanut samples with good recoveries of 97.9%-99.4%. 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.

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 Jiangsu Agriculture Science and Technology Innovation Fund (JASTIF; CX (19)2005), the Social Development Fund Project of Wuxi (N20201001), and the Excellent Scientific and Technological Innovation Team of Jiangsu Universities.

Author Contribution

Peifang Chen: conceptualization, investigation, methodology, data curation, writing-original draft. Chenbiao Li: formal analysis, preparation, software. Xiaoyuan Ma: funding acquisition, methodology, supervision, writing - review and editing. Zhouping Wang: resources, supervision. Caiyun Jiang: funding acquisition.

Data Availability

The datasets generated during the current study are available from the corresponding author upon reasonable request.

Competing Interests

The authors declare no competing interests.

Conflict of Interest

Peifang Chen declares that she has no conflict of interest. Chenbiao Li declares that he has no conflict of interest. Xiaoyuan Ma declares that she has no conflict of interest. Zhouping Wang declares that he has no conflict of interest. Caiyun Jiang declares that she has no conflict of interest.

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Scheme 1 is available in the Supplementary Files section

No competing interests reported.

Scheme1..png
Scheme 1. Schematic illustration of the developed 3D SERS aptasensor based on Au-4MBA@Ag NSs-AFB₁apt- Fe₃O₄@MoS₂ NFs assemblies for AFB₁ detection.
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3D plasmonic SERS aptasensor for rapid detection of aflatoxin B1 combined with Au@Ag bimetallic nanostars and Fe3O4 @MoS2 magnetic nanoflowers

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Materials and reagent

2.1. Instruments

2.2. Preparation of Au-4MBA@Ag NSs-AFB₁apt

2.3. Preparation of Fe₃O₄@MoS₂ NFs

2.4. Construction of labeled-aptasensor for AFB₁ detection

2.5. Selectivity evaluation

2.6. Real samples detection

3. Results And Discussion

3.1. Detection strategy of the aptasensor for AFB₁

3.2. Characterization and optimization of Au@Ag NSs-AFB₁apt

3.3. Characterization and optimization of the fabricated Fe₃O₄@MoS₂ NFs

3.4. Feasibility and optimization of 3D SERS aptasensor for AFB₁ detection

3.5. Sensitivity of AFB₁ detection by SERS aptasensor

3.6. Selectivity, reproducibility, and practicality evaluation

4. Conclusions

Declarations

References

Schemes

Additional Declarations

Supplementary Files

Status:

Version 1

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

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Materials and reagent

2.1. Instruments

2.2. Preparation of Au-4MBA@Ag NSs-AFB1apt

2.3. Preparation of Fe3O4@MoS2 NFs

2.4. Construction of labeled-aptasensor for AFB1 detection

2.5. Selectivity evaluation

2.6. Real samples detection

3. Results And Discussion

3.1. Detection strategy of the aptasensor for AFB1

3.2. Characterization and optimization of Au@Ag NSs-AFB1apt

3.3. Characterization and optimization of the fabricated Fe3O4@MoS2 NFs

3.4. Feasibility and optimization of 3D SERS aptasensor for AFB1 detection

3.5. Sensitivity of AFB1 detection by SERS aptasensor

3.6. Selectivity, reproducibility, and practicality evaluation

4. Conclusions

Declarations

References

Schemes

Additional Declarations

Supplementary Files

Status:

Version 1

2.2. Preparation of Au-4MBA@Ag NSs-AFB₁apt

2.3. Preparation of Fe₃O₄@MoS₂ NFs

2.4. Construction of labeled-aptasensor for AFB₁ detection

3.1. Detection strategy of the aptasensor for AFB₁

3.2. Characterization and optimization of Au@Ag NSs-AFB₁apt

3.3. Characterization and optimization of the fabricated Fe₃O₄@MoS₂ NFs

3.4. Feasibility and optimization of 3D SERS aptasensor for AFB₁ detection

3.5. Sensitivity of AFB₁ detection by SERS aptasensor