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

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 R 2 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. This journal is © The Royal Society of Chemistry.

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 drugs[33] 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.

Materials and methods

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).

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).

Preparation of Fe3O4@SiO2–NH2 nanoparticles

Preparation of Fe3O4 nanoparticles

The preparation method for Fe3O4 nanoparticles reported by previous researchers[35] 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.

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.

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.

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).

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.

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).

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.

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.

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.

Results and discussion

Characterization

Characterization of nanometer magnetic beads by Fe3O4, Fe3O4@SiO2, 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.

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.

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.

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.

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).

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).

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).

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).

Method performance validation

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.

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

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 1st, 2nd, 3rd, and 4th 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.

Comparison of sample preparation procedures and LOQs with different methodsa

Method/(ng mL−1)	Sample	Adsorbent	LOD (ng mL−1)	LOQ (ng mL−1)	Linear range (ng mL−1)	Ref.
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

Note: “-” means the sample preparation procedures without aptamer@Fe3O4@SiO2–NH2.

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 1st, 2nd, and 3rd 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.