Determination of aflatoxin B1 in Pixian Douban based on aptamer magnetic solid-phase extraction

Abstract

Aflatoxin B1 (AFB₁) is considered as the most prevalent and toxic mycotoxin in food, and is the indispensable index in the monitoring of Pixian Douban, a traditional chinese fermented bean paste from Sichuan. However, the effeciency of AFB₁ detection in Pixian Douban is influenced by the traditional extraction, which is usually complex and time consuming. Therefore, an aptamer-based magnetic solid-phase extraction method was designed for the pretreatment of AFB₁ in this sample, for which Fe₃O₄ was synthesized via the solvothermal method and then a Fe₃O₄@SiO₂–NH₂ with a core–shell structure was prepared, followed by an AFB₁-aptamer attachment. The validation was performed via an enzyme-linked immunosorbent assay and compared with HPLC-MS/MS. The linearity range of this method was 0.5–2.0 ng mL ⁻¹ with R² of 0.981, and recoveries of AFB₁ ranged from 80.19% to 113.92% with RSDs below 7.28% with no significant differences compared to HPLC-MS/MS. The three-time reusability efficiencies of aptamer-MNPs were averaged at 78.24%. The results proved that aptamer-MNPs were high-performance adsorbents for extracting and enriching AFB₁, facilitating quick and effective detection of AFB₁ in Pixian DouBan samples.

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1. Introduction

Pixian Douban is a fermented condiment with chinese regional characteristics. Its fermentation was recorded to be accompanied with a process called “sun and night dew” treatment. The complex changes of temperature to a certain extent can affect the various Aspergillus and enzymes that play a biochemical role, and form unique flavour characteristics widely used in Sichuan Cuisine. However, due to the relatively extensive process of traditional production, Douban's fermentation is liable to be contaminated by other Aspergillus and subsequently cause aflatoxin pollution.^1,2 The evidence shows that AFB₁ has the highest pollution frequency in this food.³ It has been reported that AFB₁ can lead to liver cancer and other diseases in mammals,^2,4 which is classified as type I carcinogen by international cancer research institutions.^1,5 In order to reduce food safety risk of Pixian Douban, AFB₁ has been regarded as a necessary item in Pixian Douban's Geographical Indication Product Standard of GB/T 20560-2006. Traditional detection methods mainly include thin layer chromatography, high performance liquid chromatography, gas chromatography, capillary electrophoresis and immunoassay.^3,6,7 Generally speaking, these detection methods are relatively wide-applied in the practical detection right now. However, the broad bean is rich in nutrients such as protein, amino acids, enzymes, pigments, lipids and other components,⁸ which is too complex to be easily detected with high efficiency so far. The shortcomings of traditional detection methods, such as long detection time, high detection cost and poor repeatability as well as not environment friendly, pose great challenges to the effective monitoring of AFB₁ in Douban. Therefore, it is necessary to establish an efficient, simple, and robust method for detection of AFB₁ in Pixian Douban.

Methanol solvent extraction is adopted in the commercial ELISA kit method, which is a solid–liquid extraction for AFB₁. Then a quantitative analysis can be performed according to the colour depth after coloration. However, the blending of chilli in Pixian Douban brings bright red colouring matter into the extract, which shows the background interference in ELISA determination. Aptamers have several advantages, such as strong specificity, high affinity, wide target range, in vitro chemical synthesis, low molecular weight, and good stability. As a result, aptamers have been widely used in the fields of analytical chemistry, medicine, environmental monitoring, and food safety control.^9–12 In addition, magnetic solid-phase extraction (MSPE) is a new solid-phase extraction (SPE) method based on the use of magnetic adsorbents, in which suitable adsorbents are combined with magnetic materials.^13,14 In the MSPE method, magnetic adsorbents are critical for the efficient enrichment of analytes. Ferrosoferric oxide (Fe₃O₄) is widely used in sample pretreatment because of its characteristics of magnetic adsorption, low price, easy synthesis, and surface modification.^15–17 Nevertheless, Fe₃O₄ is unstable and easily oxidized by air. Surface modification of Fe₃O₄ with appropriate functional groups can prevent oxidation and improve its durability and adsorption.¹³ The modification and functionalization of Fe₃O₄ are conducted mainly by using graphene,¹⁸ graphene oxide,¹⁹ carbon quantum dots,²⁰ the polymer,²¹ and so on. Silica is one of the best coating materials for Fe₃O₄ magnetic nanoparticles (MNPs), because it owns characteristics of easy surface modification, high chemical stability, and environmental compatibility.^17,22 Currently, aptamer-Fe₃O₄-based composite adsorption materials are gradually applied to various samples pretreatments, such as metal elements,²³ bacteria,^21,24 pesticides,²⁵ veterinary drug,²⁶ toxins,²⁷ nucleotides,²⁸ and other samples. Recently, the extraction method of AFB₁ from dairy products and edible vegetable oils,^29–31 such as SPE and MSPE, have been extensively studied in a fast, simple, inexpensive and safe method.

The deep eutectic solvent–based matrix solid-phase dispersion (DES-MSPD) method has currently been established for extracting and enriching aflatoxins (AFB₁, AFB₂, AFG₁, and AFG₂) in various crops.³² A simple modified SPE combined with high-performance liquid chromatography (HPLC)-fluorescence detection was established for detecting AFB₁ and AFB₂, and amine-functionalized MNPs were successfully employed in SPE for the first time for adsorbing AFB₁ and AFB₂ in drugs³³ In addition, aptasensors are considered as an emerging strategy for quantifying AFB₁ with high selectivity and sensitivity.³⁴

In this work, the functionalization of Fe₃O₄@SiO₂–NH₂ using ssDNA aptamers (referred to as aptamer-MNPs) was designed to prepare AFB1-specific nanoparticles with excellent magnetic responses. Moreover, various adsorption conditions were tested to determine the optimal conditions for the adsorption of AFB₁ in Pixian Douban to the prepared nanomaterials. The variables tested included the amount of aptamer-MNPs, extraction time, elution time, elution types, and elution volumes. An efficient method for extracting AFB₁ from Pixian Douban was established, which lays the foundation for the subsequent analysis of AFB₁ in food.

2. Materials and methods

2.1. Materials and reagents

The main text of the article should appear here with headings as appropriate. Ferric chloride hexahydrate (FeCl₃·6H₂O) and AFB₁ standard were purchased from Shanghai Yuanye Biological Co., Ltd. (Shanghai, China). Poly (4-styrenesulfonic acid-co-maleic acid) sodium salt (PSSMA) was purchased from Huaxia Reagent Co., Ltd. (Harbin, China). Tetraethyl silicate (TEOS), 3-aminopropyltriethoxysilane (APTES), and glutaraldehyde were purchased from Shanghai McLean Company (Shanghai, China). The AFB₁ kit was purchased from Jiangsu Suwei Co., Ltd. (Jiangsu, China). Hydroxymethylaminomethane (Tris) and streptavidin were purchased from Shanghai Shenggong Co., Ltd. Pixian Douban was purchased from the local supermarket. Biotinylated aptamer (5′-GT TGG GCA CGT GTT GTC TCT CTG TGT CTC GTG CCC TTC GCT AGG CCC ACA-Biotin-3′) was synthesized by Shanghai Shenggong Bioengineering Co., Ltd. (Shanghai, China). Further, NaAc, NaOH, ethanol, NH₃·H₂O, KCl, CaCl₂ were purchased from Cologne Chemicals Co., Ltd. (Chengdu, China).

2.2. Methods

The preparation of aptamer-MNPs comprises two steps: the preparation of Fe₃O₄@SiO₂–NH₂ nanoparticles and the functionalization of AFB₁-aptamer, as shown in Scheme 1a.

Scheme 1 Schematic illustration of the synthesis process of aptamer-MNPs nanocomposite (a); a schematic diagram for the extraction and detection of AFB1 in Pixian Douban samples (b).

2.2.1. Preparation of Fe₃O₄@SiO₂–NH₂ nanoparticles.
2.2.1.1. Preparation of Fe₃O₄ nanoparticles. The preparation method for Fe₃O₄ nanoparticles reported by previous researchers³⁵ was slightly modified. 1.08 g of FeCl₃·6H₂O, 3.0 g of NaAc, and 1.2 g of PSSMA were added in 40 mL of ethylene glycol. The mixture was uniformly stirred in an oil bath at 50 °C. Then, 0.6 g of NaOH was added to the mixture and continuously stirred until the solution became dark. Subsequently, the mixture was transferred to a stainless-steel autoclave, and Fe₃O₄ nanoparticles were obtained after being heated at 190 °C for 9 h. Fe₃O₄ nanoparticles were uniformly dispersed in 30 mL of distilled water (Fe₃O₄ concentration was approximately 0.8 wt%) and stored at 4 °C.
2.2.1.2. Preparation of Fe₃O₄ @SiO₂. 12 mL of dispersion were dispersed in 70 mL of ethanol and transferred to a round-bottomed flask. After stirring for 15 min, 0.4 mL of TEOS and 4 mL of NH₃·H₂O were added twice at 20 min intervals into the reaction mixture. Subsequently, Fe₃O₄@SiO₂ nanoparticles were obtained after stirring for 80 min.
2.2.1.3. Preparation of Fe₃O₄ @SiO₂–NH₂. 50 mg of Fe₃O₄@SiO₂, 6 mL of distilled water, and 40 mL of anhydrous ethanol were added into the flask for ultrasonic dispersion. Then, 3 mL of ammonia was added to the mixture and stirred for 15 min. Furthermore, 10 mL of anhydrous ethanol and 2 mL of the APTES mixture were added and stirred for 10 h to obtain Fe₃O₄@SiO₂–NH₂. The upper liquid was discarded by magnetic separation, washed using PBS (2.7 mmol L⁻¹ of KCl, 137 mmol L⁻¹ of NaCl, 2 mmol L⁻¹ of KH₂PO₄ and 10 mmol L⁻¹ of Na₂HPO₄, and pH = 7.4) thrice, dispersed in 10 mL of PBS, and stored at 4 °C.

2.2.2. AFB₁-aptamer functionalization. 20 mL of PBS, 40 mg of Fe₃O₄@SiO₂–NH₂, and 5 mL of 25% glutaraldehyde-water solution were subjected to oscillation reaction at 110 r per min for 2 h. The reaction solution was removed via magnetic separation. After washing with PBS, it was dispersed in 16 mL of PBS (containing 960 μg streptavidin) and was subjected to oscillation for 12 h. After conducting magnetic separation and discarding the reaction solution, washing three times with PBS and dispersed in 20 mL of PBS to obtain avidin-modified nanomagnetic beads. 40 mg of avidin magnetic beads were dispersed in 40 mL of PBS (containing an AFB₁-aptamer concentration of 400 pmol ml⁻¹) and oscillated at 28 °C for 2 h. Aptamer-MNPs were obtained via magnetic separation and stored in 40 mL of Tris–HCl (10 mM of Tris, 120 mM of NaCl, 5 mM of KCl, 20 mM of CaCl₂, and pH = 8.5).

2.2.3. Characterization of synthesized nanomaterials. The synthesized nanomaterials were characterized via particle size analyzer, transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT–IR), X-ray diffraction (XRD), and UV-visible (UV-Vis) spectrophotometer.

2.2.4. Extraction and detection of AFB₁. A new method for detecting AFB₁ in Pixian Douban was developed using aptamer-MNPs adsorbents along with ELISA (Scheme 1b).
2.2.4.1. Aptamer-MNPs extraction of AFB₁. 10.0 g of Pixian Douban, 50 ml of 50% aqueous methanol solution, and 20 mL of n-hexane were subjected to oscillation for 15 min and transferred to the separator funnel to stratify the solution. 10 mL of the solution at the lower layer was collected and concentrated via rotary evaporation. 10% of methanol-Tris-HCl was added to redissolve and obtain a crude sample extract. After 1 mL of crude extract and 5 mg of aptamer-MNPs were incubated with shaking for 30 min, the aptamer-MNPs were separated using a magnet. The separated aptamer-MNPs were washed with Tris–HCl, and 1 mL was eluted with methanol for 10 min. The eluate was collected and concentrated to near dryness, and then redissolved in 1 mL of PBS solution. 50 μL of the solution was used for ELISA.
2.2.4.2. Detection of AFB₁ by ELISA. 200 μL of detergent was added to each hole of the ELISA plate. After 1 min, the detergent was shake off and patted dry. Repeat the washing plate once. In each well, 50 μL of the standard solution or sample solution, and 50 μL of enzyme-labeled antigen solution were added, shaken and mixed well, incubated for 30 min at 37 °C, the detergent was shaken off the reaction solution, and patted dry. 200 μL of detergent was added and placed for 2 min, patted dry and repeat the washing plate for four times. 50 μL of the chromogenic substrate a and 50 μL of chromogenic substrate b were added, shaken and mixed, incubated at 37 °C for 15 min, and 50 μL of terminating solution was added. After shaking well, the absorbance at a wavelength of 450 nm was determined via ELISA.
2.2.4.3. Comparison detection of AFB₁ by HPLC-MS/MS. HPLC-MS/MS analysis was compared to the present work as the way of replacing ELISA test in the spike recovery of Pixian Douban samples. After Aptamer-MNPs treatment, the extractive was redissolved with acetonitrile-water (60 + 40 v/v). HPLC-MS/MS system (Agilent 6460) detection conditions:³⁸ AFB₁ were chromatographed on ACQUITY BEH-18 column (1.7 μm particle size, 110 × 2.1 mm i. d., waters) and separated using gradient elution with 2 mmol L⁻¹ ammonium acetate aqueous solution and acetonitrile as mobile phase A and B, respectively (both acidified with 0.2% formic acid). The gradient program was as follows: at time zero 10% solvent B; at 2 min 35% solvent B; linear gradient to 60% solvent B within 7 min, at 9 min 90% solvent B then 10% B isocratic for 3 min. The flow rate was 0.3 mL min⁻¹. The ionization was carried out with an ESI interface in positive mode as follows: spray capillary voltage was 4.0 kV; sheath gas flow rate 11 L min⁻¹, respectively; temperature of sheath gas 350 °C. The mass spectrometric analysis was performed in MRM. For fragmentation of AFB₁ [M + H]⁺ ions is 313 m/z., the detected and quantified fragment ions were: 241 and 269 m/z for AFB₁. Quantitative determination was performed by MassHunter software (Agilent). All analyses were performed with the SPSS 23.0 software (SPSS Incorporated, USA). Statistical significance was analyzed using ANOVA. In all statistical comparisons, values of P ≤ 0.05 were considered as of significance, and values of P ≤ 0.01 were considered to be markedly significant.

3. Results and discussion

3.1. Characterization

3.1.1. Characterization of nanometer magnetic beads by Fe₃O₄, Fe₃O₄@SiO_2, and Fe₃O₄@SiO₂–NH₂. TEM images (Fig. S1†) and Fig. S2† show that the prepared Fe₃O₄ were regular spheres, with a particle size of 20–50 nm, and mainly concentrated around 35 nm. This shows that the prepared Fe₃O₄@SiO₂ particles were regular spheres. It had a clear core–shell structure, and the thickness of the SiO₂ shell was approximately 15 nm. The presence of Fe₃O₄@SiO₂ afforded a quite different morphology (Fig. S1a and S1b†). It can be seen that Fe₃O₄@SiO₂ had a homogeneous cover layer on the surface, which can be attributed to the successful coating of SiO₂ on Fe₃O₄. The addition of PSSMA during the preparation of Fe₃O₄ can negatively charge the surface of the magnetic beads, increase the stability of the Ostwald ripening process, and allow Fe₃O₄ to uniformly grow and stably disperse in the solution.³⁵ In addition, SiO₂-modified Fe₃O₄ has better dispersibility.

The XRD pattern of Fe₃O₄ samples is shown in Fig. S3.† The XRD spectra of Fe₃O₄ show spinel ferrites, which fully matches with the diffraction card JCPDS (PDF-#76-1849). The diffraction peaks at 2θ = 18.06°, 30.13°, 35.47°, 43.03°, 53.19°, 56.89°, and 62.58°, corresponding to 111, 220, 311, 400, 422, 511, and 440 plates, respectively, indicate a magnetic phase and the crystalline cubic spinel structure of Fe₃O₄.²³

The magnetic separation of Fe₃O₄@SiO₂ from deionized water was performed to test the practical magnetic response-ability (Fig. S4†). Fe₃O₄@SiO₂ particles were uniformly dispersed in deionized water. Under the presence of magnetic field, Fe₃O₄@SiO₂ was rapidly separated from the solution and assembled in the corner near the magnet.

The FT-IR spectra of Fe₃O₄ and Fe₃O₄@SiO₂–NH₂ are depicted in Fig. S5.† The absorption peak at 3381.39 cm⁻¹ was attributed to the stretching vibration of the hydroxyl group, indicating that a certain number of hydroxyl groups exist on the surface of Fe₃O₄. In the FT-IR spectra of Fe₃O₄@SiO₂–NH₂, the absorption intensity of the Fe–O group decreased with the addition of silica. The Si–O–Si group exhibited a strong absorption intensity at 1100.21 cm⁻¹ owing to the silica coating, indicating that Fe₃O₄ had been successfully coated with SiO₂.³⁶ In addition, the Absorption peaks at 1627.94 and 3328.89 cm⁻¹ correspond to bending and stretching vibrations of the–NH₂ group,¹⁶ respectively. The peaks at 2856.35 and 2923.24 cm⁻¹ correspond to symmetric and asymmetric stretching vibrations of the –CH₂ group, respectively, which are attributed to the hydrolysis of the amination reagent APTES. These absorption peaks confirmed the successful modification of Fe₃O₄. The absorption peaks at 588.63 and 583.22 cm⁻¹ are attributed to the characteristic absorption peak of the Fe–O group. After the surface modification of Fe₃O₄, the characteristic absorption peak slightly shifted, but the properties of Fe₃O₄ did not change.

3.1.2. Characterization of avidinized Fe₃O₄ @ SiO₂–NH₂ and aptamer-MNPs. In this experiment, Fe₃O₄@SiO₂–NH₂ of avidin magnetic beads and aptamer-MNPs were characterized via the ultraviolet absorption of avidin at 280 nm and aptamer at 260 nm. Fig. S6a† shows that the absorbance at 280 nm considerably decreased, which indicated that the content of avidin in the solution decreased. The avidin covalently bound to glutaraldehyde and was connected to the surface of magnetic beads. In addition, the experiment continued to explore the relationship between dosage of magnetic beads and concentration of avidin. The experimental results show that when 1 mg of magnetic beads (Fe₃O₄@SiO₂–NH₂) were incubated with 24 μg of avidin, and almost all the avidin is coupled to the surface of the magnetic beads to attain saturation. The absorbance value at 260 nm considerably decreased (Fig. S6b†), indicating a decrease with the concentration of aptamer decreasing. Aptamer bound to the surface of avidin magnetic beads. In addition, the experiment was continued to investigate the aptamer-MNPs binding ability, and the results showed that the aptamer concentrated on the surface of 1 mg avidin magnetic beads (Fe₃O₄@SiO₂–NH₂) attained saturation after being incubated with 400 pmol of aptamer.

3.2. The establishment of detection standard curve

In this experiment, a commercial AFB₁ ELISA kit was used for performing the quantitative analysis of AFB₁ extracted using aptamer-MNPs. The standard curve of ELISA was obtained after step 2.2.4.2. As shown in Fig. 1, the linearity of the developed method was observed over a range of 0.5–2.0 ng mL⁻¹, with a correlation coefficient of 0.981.

Fig. 1 Standard curve of AFB₁.

3.3. Optimization of sample preparation conditions

In the MSPE procedure, the extraction and elution processes are crucial in realizing satisfactory recovery of analytes. To obtain higher extraction recoveries, AFB₁ in Pixian Douban was enriched with aptamer-MNPs. The main experimental parameters that affect the recovery of the extraction were optimized, such as the amount of aptamer-MNPs, extraction time, and elution time of the sample.

3.3.1. The influence of the amount of aptamer-MNPs on AFB₁ recoveries. The amount of magnetic adsorbent is crucial in realizing satisfactory recoveries of the target analytes.¹³ Fig. 2. Shows that the AFB₁ recoveries varied with the amount of aptamer-MNPs. The dosage of adsorbents is proportional to the adsorption rate within a certain range. The larger the dosage of magnetic adsorbents, the more active sites on the surfaces of the adsorbents.^13,37 With increasing the amount of aptamer-MNPs from 3 mg to 5 mg, the recovery of AFB₁ gradually increased. The recovery of AFB₁ did not considerably increase as the number of magnetic beads increased but decreased slightly. The low AFB₁ recovery rate at 6 mg may be attributed to the experimental operation error. Finally, 5 mg is determined to be the optimum amount of aptamer-MNPs to ensure the good recovery of each compound and is used in subsequent experiments.

Fig. 2 The effect of aptamer-MNPs amount on the recovery of AFB₁ (n = 3).

3.3.2. The influence of extraction time on AFB₁ recoveries. Adsorption is an equilibrium process, and sufficient contact time between the sample solutions and sorbents should be provided to establish the extraction equilibrium.³⁷ Therefore, the binding of aptamer-MNPs to AFB₁ under different extraction times was investigated in this experiment. As shown in Fig. 3, with increasing the extraction time, the adsorption capacity of AFB₁-MNPs to AFB₁ increased gradually. When the extraction time increased to 30 min, the extraction efficiency of AFB₁ by aptamer-MNPs was the highest. When the extraction time was prolonged, the recovery rate of AFB₁ decreased gradually, which indicated that when the extraction time was 30 min, the aptamer's recognition and capture of AFB₁ had attained a saturated state.

Fig. 3 The effect of extraction time on the recovery of AFB₁ (n = 3).

3.3.3. The influence of the desorption time on AFB₁ recoveries. In this study, methanol was used to elute AFB₁ from aptamer-MNPs and the effect of different desorption times on the recovery rate was investigated Fig. 4 shows that the recovery rate decreases with increasing the desorption time and the best recovery rate is obtained when the elution time is 5 min.

Fig. 4 The effect of desorption time on the recovery of AFB₁ (n = 3).

3.3.4. The influence of other factors on AFB₁ recoveries. In this experiment, the influence of the types of solvents was investigated in addition to optimizing the three key factors affecting extraction efficiency. The experiment compared two types of elution solvents: methanol and acetonitrile. The two elution solvents had almost similar influence on the recoveries of AFB₁, indicating that both methanol and acetonitrile could be used as elution solvents. Considering the cost and toxicity of the reagent, methanol was chosen as the elution solvent. In addition, this study also explored the effects of different volumes of elution (1 and 3 mL) on the recoveries of AFB₁ (Fig. 5). Compared with 1 mL of methanol solution, 3 mL of methanol solution may cause a greater loss in the process of volatilization and redissolution after elution reducing the extraction efficiency. Therefore, the experiment decided to use 1 mL of methanol solution.

Fig. 5 The effect of the elution volume on the recovery of AFB₁ (n = 3).

3.4. Method performance validation

3.4.1. Spike recovery experiment. The AFB₁ content in the samples determined by using ELISA was 3.4 μg kg⁻¹, less than the national standard limit of 5.0 μg kg⁻¹. AFB₁ was spiked into Pixian Douban samples at concentrations of 0.5 μg L⁻¹, 1.0 μg L⁻¹, 2.0 μg L⁻¹, and 10.0 μg L⁻¹, and the experiment was performed according to step 2.2.4.1. A comparative evaluation of the spiked samples was performed by using the present method and HPLC-MS/MS, respective. Qualitative identification was conducted by using MRM scan in HPLC-MS/MS method shown in Fig. S7b and c;† then the linear regression analysis was carried out in Fig. S7a.† The recoveries and the corresponding RSDs were presented in Table 1. The recoveries of proposed measurement of AFB₁ ranged from 80.19% to 113.92% with RSDs lower than 7.28% (n = 3), while those of the compared HPLC-MS/MS were 88.63% to 101.10% with RSDs below 3.44% (n = 3). The P values of the test concentrations of the two methods were 0.259, 0.365, 0.432 and 0.452 respectively, which were all greater than 0.05(P > 0.05), indicating that there was no significant statistical difference between the methods. Thus, the extraction method is efficient and feasible.

Table 1 Recoveries of AFB₁ in spiked sample by compared methods (n = 3)

Tagged value/(ng mL^−1)	ELISA Detected value/(ng mL^−1)	Recovery/(%)	RSD/(%)	HPLC-MS/MS Detected value/(ng mL^−1)	Recovery/(%)	RSD/(%)
0.5	0.42	84.29	3.27	0.44	88.63	1.37
1.0	1.14	113.92	7.28	0.89	89.88	2.91
2.0	1.60	80.19	2.34	1.87	93.50	1.32
10.0	9.37	93.70	2.30	10.11	101.10	3.44

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3.4.2. The reusability of aptamer-MNPs. The reusability of the material was investigated based on the recovery as an index, and the result is depicted in Fig. 6. The reusability of the selected membrane for aptamer-MNPs indicated efficiencies of 87.34%, 81.20%, 66.17%, and 57.02% for the 1^st, 2^nd, 3^rd, and 4^th cycles, respectively. The results show that the aptamer-MNPs prepared using this method exhibit reusability when used in the MSPE for detecting AFB₁.

Fig. 6 Reusability in four cycles of the aptamer-MNPs SPME adsorbents for extraction of AFB₁ in Pixian Douban samples.

Compared with traditional SPE, MSPE affords enhanced extraction efficiencies and avoids time-consuming and labor-intensive extraction steps, which are particularly important when considering large sample sets.¹³ Table 2 shows a comparison of sample preparation procedures with other methods in the literature. The LOD and LOQ obtained from the present method are comparable to or lower than those obtained from other methods. The MSPE method simplifies the sample preparation procedure because the adsorption processes are fast and the magnetic adsorbents can be easily separated from the sample solution under the applied external magnetic field.³⁰ The MSPE method established in this study can identify target molecules with high specificity and realize rapid separation and enrichment under an external magnetic field, which saves the operation of centrifugation and filtration in other extraction methods, greatly simplifying the experimental steps, and reducing the experiment time. In addition, aptamer has strong chemical stability and storage stability; therefore, it can be used in various reaction systems and can be preserved for a long time without any reduction in its functional activity. Aptamer and nanomagnetic beads have the advantages of simple preparation, easy modification, and good stability.³⁹ Therefore, the specific recognition of aptamer and the rapid separation as well as enrichment of nanomagnetic beads are integrated to prepare a new type of extraction material, and the pretreatment method for the detection of AFB₁ is demonstrated to have great significance and value.

Table 2 Comparison of sample preparation procedures and LOQs with different methods^a

Method/(ng mL^−1)	Sample	Adsorbent	LOD (ng mL^−1)	LOQ (ng mL^−1)	Linear range (ng mL^−1)	Ref.
a Note: “-” means the sample preparation procedures without aptamer@Fe3O4@SiO2–NH2.
MSPE-aptamer-ELISA	Pixian Douban	Aptamer@Fe3O4@SiO2–NH2	0.17	0.48	0.5–2	Present work
ELISA	Pixian Douban	—	0.41	1.26	0.5–2	Comparison 1
MSPE-aptamer-HPLC-MS/MS	Pixian Douban	Aptamer@Fe3O4@SiO2–NH2	0.07	0.22	0.5–10	Comparison 2
MSPE-UHPLC	Milk	PEG-MWCNTs-MNP	0.01	0.03	1.25–40	30
MSPE-HPLC	Vegetable oils	PDA@Fe3O4-MWCNTs	0.20	0.60	1–50	31
MSPD-HPLC	Crops	TBAC-hexyl alcohol DES	0.10	0.33	0.1–100	32

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Conclusions

In this study, aptamer-MNPs were prepared and adopted as the adsorbents for AFB₁ in Pixian Douban samples. Aptamer-MNPs effective in extracting and enriching the targeted compound AFB₁ and exhibited a good magnetic response. A new detection method for AFB₁ in Pixian Douban samples was developed by combining aptamer-MNPs with ELISA. The linearity of the method was 0.5–2 ng mL⁻¹ with a correlation coefficient of 0.981, and the content of detections for AFB₁ was 3.4 μg kg⁻¹. The recoveries of AFB₁ ranged from 80.19% to 113.92% with RSDs lower than 7.28%, and the reusability of the selected membrane for aptamer-MNPs indicated efficiency of 87.34%, 81.20%, and 66.17% for the 1^st, 2^nd, and 3^rd cycles, respectively. The results proved that aptamer-MNPs were high-performance adsorbents for extracting and enriching AFB₁, which can quickly and effectively detect AFB₁ in Pixian Douban samples.

Author contributions

All authors contributed to the study conception and design. Material preparation, data collection, analysis, and revision were performed by Chi Xu, Kun Shao, Yaning Song and Hongyun Tian. The first draft of the manuscript was written by Chaoyi Zeng and the previous versions of the manuscript were commented by Xiao Yang. The resources and supervision were provided by Zhenming Che. The conceptualization and writing – review and editing were done by Yukun Huang. All authors read and approved the final manuscript.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was partly supported by the National Natural Science Foundation of China (31801647), Sichuan Science and Technology Program (2020YFN0153, 2020YFN0151, 2022JDTD0028), Research project of Sichuan Cuisine Development research Center (CC22Z27).

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Footnotes

† Electronic supplementary information (ESI) available. See https://doi.org/10.1039/d2ra02763a

‡ The authors contributed equally in this work.

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