Magnetic beads carrying poly(acrylic acid) brushes as “nanobody containers” for immunoaffinity purification of aflatoxin B₁ from corn samples

Abstract

The magnetic-bead-based immunoaffinity extraction (M-IAE) method has been widely used for the purification of aflatoxin B₁ (AFB₁) from complex food and feed matrices. However, this method suffers from several inherent disadvantages that limit its practical application, including low reusability and limited saturated adsorption capacity. Herein, we report an improved M-IAE for the highly efficient purification of AFB₁ from corn samples. This method involves the expression of anti-AFB₁ nanobodies (Nbs), which possess high degeneration resistance, to replace conventional antibodies; and magnetic beads carrying poly(acrylic acid) brushes (MB@PAA) were fabricated as an “Nb container” for improving AFB₁ adsorption capacity. The MB@PAA shows a high loading capacity for anti-AFB₁ Nbs at 623 μg g⁻¹, which is 19-fold the magnitude of that of conventional MB@Nbs. Meanwhile, the resultant MB@PAA@Nbs exhibit good AFB₁ adsorption, with a maximum adsorption capacity of 0.23 mg g⁻¹, which is 35-fold superior to that of the conventional MB@Nbs. MB@PAA@Nbs can be reused at least 10 times, without obvious loss of the capture efficiency for AFB₁. The reliability and practicability of the proposed MB@PAA@Nbs for AFB₁ extraction were further evaluated using AFB₁-spiked corn samples. In brief, the proposed MB@PAA@Nbs-based immunoaffinity extraction method is a highly promising, novel sample pre-treatment platform for AFB₁ as well as other mycotoxins.

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1 Introduction

Mycotoxin contamination in food and feed materials has gained increasing attention as mycotoxin overexposure poses a potential threat to human and animal health.^1–3 Various analytical techniques, including high-performance liquid chromatography,⁴ gas chromatography,⁵ liquid chromatography coupled with mass spectrometry,⁶ and enzyme-linked immunosorbent assay (ELISA),⁷ have been developed for the sensitive determination of mycotoxins. In these methods, a proper pretreatment for the extraction and clean-up of mycotoxins is required to ensure the accuracy and sensitivity of the assay especially for the detection of trace amounts of analyte in a complex biological matrix.⁸

Several existing methods have been proposed for mycotoxin extraction and clean-up, such as liquid–liquid extraction (LLE), solid-phase extraction (SPE), molecular imprinted polymers (MIPs), and immunoaffinity extraction (IAE). LLE is a traditional method for mycotoxin extraction, which is based on the different solubilities of the toxin in two immiscible phases employed for the removal of background interferences. However, the process is time consuming and requires a large amount of organic solvent.⁹ On the other hand, SPE technology has been widely used for mycotoxin sample pretreatment because of its high binding capacities and low cost. Nevertheless, this method lacks specificity and is easily affected by changes in the environment because using SPE cartridges for analyte extraction is mainly based on the differences in the chemical and physical properties of the analyte and the materials used.¹⁰ Meanwhile, MIPs are tailor-made polymers with high selectivity for a given analyte. Nevertheless, the MIP method suffers from several shortcomings, such as polymer swelling in unfavorable solvents, slow binding kinetics of analytes, and potential sample contamination by template bleeding.^11,12 Antibody-based IAE is considered as the most effective method for mycotoxin extraction and clean-up. But, this method is expensive because each column can only be used once. Moreover, immunoaffinity materials still require the filling of the column, which makes the operation process complicated and time consuming.^13–15

By contrast, magnetic-bead-based IAE (M-IAE) exhibits many advantages over the traditional IAE. The magnetic adsorbent can interact homogeneously with the sample solution to achieve higher extraction efficiency. Furthermore, magnetic adsorbent can be performed in suspensions containing solid or oily components because of the application of an external magnetic field, which allows the rapid and convenient separation of analytes from these sample matrices.^16,17 Many efforts have been devoted to the application of M-IAE for the pre-concentration and clean-up of various mycotoxins, including ochratoxins,¹⁸ zearalenone,¹⁹ deoxynivalenol,²⁰ fumonisin B₁,²¹ and aflatoxin B₁ (AFB₁).²² However, the M-IAE method still possesses several inherent drawbacks that limit its popularization. These drawbacks include the method's low reusability and limited saturated adsorption capacity. Nanobodies (Nbs), which exhibit a unique antibody format naturally existing in the Camelidae species, are the smallest functional antigen-binding fragments.²³ Compared with conventional intact antibodies, Nbs possess many advantages including small size, high expression yield, and high stability even in extreme conditions, such as extremely low or high pH, high temperature, and organic solvent content.²⁴ These properties allow the wide application of Nbs in clinical diagnosis, medical therapy, food safety, and environmental monitoring.^25,26

To our best knowledge, only a few investigations proposed the usage of Nbs instead of conventional antibodies in IAE for the pre-concentration and clean-up of mycotoxin. Herein, we report an enhanced M-IAE method to facilitate its practical application. Aflatoxin B₁ was selected as the model analyte; it is a highly toxic mycotoxin and has been classified as a group I carcinogen by the International Agency for Research on Cancers.^27,28 To maximize the capture amounts of AFB₁, magnetic beads carrying poly(acrylic acid) brushes (MB@PAA) were synthesized and designed as “Nb containers” (MB@PAA@Nbs) for improving AFB₁ adsorption capacity. The Nb loading capacity and AFB₁ capturing efficiency of the resultant MB@PAA@Nbs were compared with carboxyl-modified magnetic beads. Moreover, the performance of MB@PAA@Nbs, including its reusability, reliability, and practicability, was further evaluated.

2 Experimental section

2.1 Materials and reagents

N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (EDC), bovine serum albumin (BSA), and AFB₁ were purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Ethanethiol was obtained from J&K Chemicals. Acrylic acid (AA), 2,20-azobisisobutyronitrile (AIBN), K₃PO₄, and K₃PO₄·H₂O were obtained from Aladdin Industrial Corporation (Shanghai, China). (4-(Chloro)phenyl)trimethoxysilane was purchased from Gelest, Inc. (Pennsylvania, America). Hydroxylated and carboxylated Fe₄O₃ nanoparticle doped magnetic silicon beads (HMBs and CMBs, respectively) and KBsphere™ Ni-IDA were purchased from Knowledge & Benefit Sphere Tech. Co., Ltd (Suzhou, China). BCA Protein Quantitation Kit was purchased from Com Win Biotech Co., Ltd (Beijing China). The commercial AFB₁ ELISA kit was provided by Wuxi Zodoboer Biotech. Co., Ltd (Wuxi, China). The other reagents were of analytical grade or higher and were purchased from Sinopharm Chemical Corp. (Shanghai, China).

2.2 Synthesis of MB@PAA

As shown in Scheme 1A, HMBs carrying the poly(acrylic acid) brushes (MB@PAA) were synthesized via the surface-initiated reversible addition–fragmentation chain transfer (RAFT) polymerization method, including the synthesis of RAFT and free RAFT chain transfer agent (RAFT CTA and free RAFT CTA, respectively), the preparation of MB@RAFT, and the synthesis of MB@PAA. RAFT CTA and free RAFT CTA were synthesized according to a previously reported method.²⁹ MB@RAFT and MB@PAA were prepared through the following protocol. First, 59 μL of RAFT was added to a 13 mL ethanol solution containing 428 mg of HMBs. The mixture was refluxed under N₂ protection for 24 h. The resultant product was separated by magnetic shelf and washed with ethanol thrice. Yellow MB@RAFT (150 mg) was re-suspended with 10 mL of N,N-dimethylformamide (DMF), and 3.24 g AA, 0.01 g AIBN, and 0.11 g free RAFT CTA were added to the DMF solution. Afterward, the mixture was transferred into a Schlenk tube. After five cycles of freeze–pump–thaw, the system was closed and incubated at 70 °C in an oil bath thermostat. After 3 h polymerization reaction, the MB@PAA was separated from the DMF solution and washed with ethanol for five times to fully remove impurities.

Scheme 1 Schematic for MB@PAA@Nbs preparation and magnetic separation of AFB₁ with MB@PAA@Nbs.

The Fourier-transform infrared (FTIR) spectra of the HMBs, MB@RAFT, and MB@PAA were obtained over the range of 400–4000 cm⁻¹ using a Nicolet 5700 FTIR spectrometer (Thermo Fisher Scientific, Inc., USA). The carboxyl group density on the surface of the MB@PAA was determined as previously described,³⁰ with some modifications. Briefly, the electrode of an FE30 conductivity meter (Mettler Toledo, Shanghai, China) was immersed in 10 mL of ultrapure water containing 10 mg of MB@PAA. NaOH (0.1 M) was added dropwise under gentle stirring. The titration curve was plotted using the conductivity of the solution against the titrant volume of the NaOH solution. The carboxyl group density was calculated as D_s = [(V₂ − V₁) × C]/m, where D_s (mmol g⁻¹) is the density of the carboxyl group on the MB@PAAs; V₁ and V₂ are the NaOH titrant volumes at the first and second inflection points of the titration curve, respectively; C is the concentration of the NaOH solution; and m is the mass of the MB@PAA. The thermogravimetric analysis (TGA) curves of MB@PAA@BSAs and CMB@BSAs were obtained from a simultaneous thermal analyzer (Perkin Elmer, USA). The morphologies of MB@PAA@BSAs and MB@PAA were determined using JEM-2100 scanning electron microscopy (SEM) (JEOL, Japan).

2.3 Expression and purification of anti-AFB₁ Nb

Phage particles carrying the anti-AFB₁ Nbs were obtained from an immune alpaca phage-display VHH library constructed by our laboratory. The DNA fragment (G8) that coded for the anti-AFB₁ Nbs was cloned into the pET-25b (+) vector (Novagen, Billerica, MA, USA) and transformed into Escherichia coli Rosetta (DE3) cells to express soluble anti-AFB₁ Nbs. The single positive colony was inoculated into 5 mL LB medium containing 5% (v/v) glucose, 0.01% (v/v) ammonia benzyl, and chloramphenicol, and incubated at 37 °C overnight with shaking at 250 rpm. Then, 0.5 mL of the overnight culture was transferred to 50 mL of the LB medium for additional culture. When the culture reached OD₆₀₀ = 0.6–0.8, 50 μL (0.1 mM) of isopropyl β-D-1-thiogalactopyranoside was added to induce the protein expression by culture for another 6 h. The cells were harvested by centrifugation at 5000 rpm for 10 min and then re-suspended with 10 mL PBS buffer for the ultrasonic cell-break. The protein supernatant was separated by centrifugation at 8000 rpm for 20 min. Anti-AFB₁ Nbs containing 6× His tag were purified with KBsphere™ Ni-IDA metal affinity column according to the manufacturer's instruction. The size of Nbs was assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to a standard protocol, followed by staining with Coomassie Brilliant Blue dye. The concentration of anti-AFB₁ Nbs was determined by the BCA Protein Quantitation Kit.

2.4 Preparation of MB@PAA@Nbs

The MB@PAA@Nbs conjugates were prepared according to the “chemical conjugation after electrostatic entrapment” (CCEE) method as previously described,³¹ with some modification. In brief, 0.1 mg MB@PAA was suspended in 0.5 mL of 10 mM 2-(N-morpholino) ethanesulfonic acid buffer (MES) (pH = 5.0, containing 0.05 wt% Tween-20), and then 60 μg of anti-AFB1 Nbs was added at room temperature. After MB@PAA electrostatical adsorption of anti-AFB₁ Nbs for 15 min, 0.72 mg EDC was added for covalent coupling of the carboxyl group of MB@PAA with the amino group of Nbs at ambient temperature for 2 h. The obtained MB@PAA@Nbs complex was separated by the magnetic shelf, washed with PBST buffer (PBS, pH = 7.4, containing 0.05 wt% Tween-20) for three times, and then stored in 0.2 mL PBS (containing 0.02% sodium azide) at 4 °C for further use. The binding efficiency of anti-AFB₁ Nbs onto the MB@PAA was evaluated by determining the amount of unbound anti-AFB₁ Nbs in the supernatant using the BCA Protein Quantitation Kit.

2.5 Adsorption behavior of MB@PAA@Nbs

The capture efficiency (CE), elution efficiency (EE), and adsorption isotherm behavior of MB@PAA@Nbs were evaluated through the following experiments. First, 0.1 mg of MB@PAA@Nbs was added into 0.5 mL PBS buffer containing 20% methanol with AFB₁ final concentrations from 2 ng mL⁻¹ to 200 ng mL⁻¹ and then incubated at room temperature with gentle shaking. After adsorption for 1 h, the MB@PAA@Nbs + AFB₁ complex was separated by magnetic shelf and washed with 20% methanol–PBS once. The bound AFB₁ onto the MB@PAA@Nbs was eluted by 200 μL pure methanol twice (100 μL each time). The AFB₁ concentrations in the supernatant and eluent were determined by a commercial ELISA kit. The CE of MB@PAA@Nbs was calculated according to the percentage fraction of those captured by MB@PAA@Nbs and the total AFB₁ spiked concentration as described by the equation CE (%) = [(N − N_u)/N] × 100%. On the other hand, EE was calculated according to the percentage fraction of those in the eluent and the total AFB₁ content by the equation EE (%) = (N_e/N) × 100%, where N is the total spiked AFB₁ content (ng), N_u is the unbound AFB₁ in the supernatant (ng), and N_e is the amount of AFB₁ in the eluent (ng mL⁻¹). The equilibrium adsorption capacity was calculated as q_e = [(C₀ − C_e)V]/M, where q_e (mg g⁻¹) is the equilibrium adsorption capacity; C₀ and C_e are the initial and equilibrium concentrations (mg L⁻¹), respectively, of AFB₁ in the adsorption solution supernatant; V (L) is the volume of supernatant; and M (g) is the weight of the MB@PAA@Nbs.

2.6 Evaluation of the MB@PAA@Nbs

To evaluate the performance of MB@PAA@Nbs, the adsorption capacity of anti-AFB₁ Nbs onto MB@PAA and the CE of MB@PAA@Nbs for AFB₁ were compared with those of the CMBs and MB@Nbs, respectively. Briefly, 60 μg of anti-AFB₁ Nbs were coupled with 0.1 mg of MB@PAA and 1.0 mg of CMBs according to the CCEE method, respectively. The resultant MB@PAA@Nbs (0.1 mg) and MB@Nbs (1.0 mg) were used to capture 5 ng AFB₁ in 0.5 mL of PBS buffer containing 20% methanol. The recycling ability of MB@PAA@Nbs was performed by the evaluation of the CE of the MB@PAA@Nbs. Briefly, 0.1 mg of fresh MB@PAA@Nbs was used to capture 10 ng AFB₁ in the 0.5 mL absorption solution. After elution with pure methanol, the used MB@PAA@Nbs was regenerated in PBS buffer for over 12 h and then used for AFB₁ adsorption in the successive 10 independent experiments. In addition, the recovery and variable coefficient of MB@PAA@Nbs for AFB₁-spiked actual corn samples were used to evaluate the practicality of the proposed immuno-adsorption materials. The AFB₁ extraction and clean-up of corn samples using the MB@PAA@Nbs are illustrated in Scheme 1B. Briefly, 1.0 g of homogenized corn sample was mixed with 5.0 mL of 70% methanol using an ultrasonicator and then centrifuged at 8000 rpm for 10 min. The supernatant was centrifuged at 10 000 rpm for another 5.0 min to discard the precipitate and then diluted threefold with ultrapure water. The MB@PAA@Nbs (0.1 mg) were added to 0.5 mL of the diluted extract. The mixture was then incubated at room temperature for 1 h. Subsequently, the MB@PAA@Nbs + AFB₁ complex was separated using an external magnetic field and washed with 0.1 mL of PBS. The AFB₁ was eluted by 100 μL pure methanol twice (50 μL per each time), and the elution was kept at 4 °C for ELISA detection.

3 Results and discussion

3.1 Production of anti-AFB₁ Nbs

The DNA fragment that encoded anti-AFB₁ Nbs was amplified and cloned into a pET-25b (+) expression vector. Rosetta (DE3) was used as host strain for Nb expression. The expression of anti-AFB₁ Nbs was determined by a 15% SDS-PAGE. The results were shown in Fig. S1,† indicating that the anti-AFB₁ Nbs expressed were soluble proteins in the supernatant portion of the cell disruption. The molecular weight of the anti-AFB₁ Nbs is about 21 kDa. The recombinant anti-AFB₁ Nbs was purified using a KBsphere™ Ni-IDA metal-affinity column. A nonspecific binding protein was washed with 50 mM imidazole solution, whereas the purified Nbs were eluted using 100 mM imidazole buffer. Protein concentration was measured using a BCA Protein Quantitation Kit, and the yield of the purified protein reached up to about 20.16 ± 0.26 mg L⁻¹ of bacterial culture (purification yields are expressed as means ± standard deviation of three independent experiments).

3.2 Characterization of MB@PAA

MB@PAA was synthesized via a RAFT polymerization method. The FTIR spectra of HMB (Fig. 1A) showed three typical absorption peaks of 585.24, 1098.72, and 3405.33 cm⁻¹, corresponding to the Fe–O, Si–O–Si, and Si–OH of Fe₃O₄ and SiO₂. After coupling with RAFT CTA, a characteristic signal at 632.521 cm⁻¹ corresponding to the C–S stretching vibration was observed in the FTIR spectra of MB@RAFT. Two peaks at 2926.92 and 1722.46 cm⁻¹ in the spectrum of MB@PAA represented the stretching vibration of the CH₂– and C=O bands that confirmed the modification of the PAA brushes. Moreover, the carboxyl group density on the resulting MB@PAA was determined by conductometric titration using a conductivity meter to measure signal changes. As shown in Fig. 1B, the curve shows a good agreement with the mode of alkali titrate–weak acid, and the carboxyl density is calculated to be 0.55 mmol g⁻¹ MB@PAA. TGA analysis of MB@PAA@BSAs and CMB@BSAs was conducted, and results indicated that the protein loading capacity of MB@PAA largely improved compared with that of the CMBs (Fig. 1C). The morphologies of the MB@PAA@BSAs and MB@PAA were determined using SEM. Fig. 1D displays that the surface of MB@PAA is porous and relatively rough, whereas the surface of MB@PAA@BSAs became smoother with many collapses, indicating successful BSA conjugation.

Fig. 1 Characterization of materials: (A) FTIR spectrum of HMBs (a), MBs@RAFT (b), and MB@PAA (c); (B) conductometric titration graph of MB@PAA; (C) TGA graph of CMB–BSAs and MB@PAA@BSAs; (D) SEM micrograph of MB@PAAs and MB@PAA@BSAs.

3.3 Production of MB@PAA@Nbs

The anti-AFB₁ Nbs and MB@PAA conjugates were prepared using the “CCEE” method. For electrostatic entrapment, the pH value of the coupling buffer was considered as the most important factor that affects the adsorption efficiency of Nbs on the surface of MB@PAAs. The effect of buffer pH on Nb coupling efficiency was determined using 1.0 mg MB@PAA adsorption with 600 μg Nbs at the pH range of 4.0 to 6.0. Fig. 2A indicates that the maximum Nb adsorption values were achieved at 400 ± 9 μg per mg MB@PAA in MES buffer at pH 5.0. Fig. 2B shows the effect of EDC concentration on the coupling efficiency of the Nbs. The maximum Nb coupling amount was 424 ± 11 μg per mg MB@PAA at EDC concentration of 0.2 mM. Under the optimized coupling condition, the saturated labeling content of Nbs onto the MB@PAA was determined using 1.0 mg of MB@PAA conjugated with a series of amounts of anti-AFB₁ Nbs from 100 μg to 1.2 mg. As shown in Fig. 2C, the maximum binding capacity of the Nbs for MB@PAA (1 mg) was achieved at 623 ± 23 μg, when 900 μg of anti-AFB₁ Nbs was coupled with 1 mg of MB@PAA. Moreover, the MB@PAA@Nbs containing different concentrations of anti-AFB₁ Nbs were used to capture the AFB₁, and the CE for AFB₁ was used to evaluate the reserved bioactivity of anti-AFB₁ Nbs on the surface of the MB@PAA. Fig. 2C indicates that the CE increased with increasing Nb content by 600 μg per mg MB@PAA and reached a maximum at 86.7% ± 4%. Subsequently, the CE of MB@PAA@Nbs for AFB₁ sharply decreased with the continual increase of Nb concentration. Thus, 600 μg anti-AFB₁ Nbs per mg MB@PAA was considered as the optimal amount of Nbs for the preparation of MB@PAA@Nbs. The Nb loading capacity and AFB₁ adsorption efficiency of MB@PAA@Nbs were further compared with those of carboxyl-modified magnetic beads. The results are shown in Fig. 2D, which indicates that the adsorption capacity of MB@PAA for Nbs is about 19-fold higher than that of conventional CMBs, and the CE of MB@PAA@Nbs for AFB₁ is 35-fold higher than that of MB@Nbs.

Fig. 2 Production of MB@PAA@Nbs and the outperform of MB@PAAs and MB@PAA@Nbs: (A) optimization of pH, (B) optimization of EDC concentration, (C) saturation binding of anti-AFB1 Nbs on MB@PAA and relationship between the adsorption amounts of anti-ANbs on MB@PAA and it's capture efficiency. Here, the additive amount of AFB₁ is 10 ng with 0.1 mg MB@PAA@Nbs in 0.5 mL 20% methanol–PBS solution. (D) Outperform of MB@PAAs and MB@PAA@Nbs compared with conventional CMBs, the additive amount of AFB₁ is 5 ng with 0.1 mg MB@PAAs and MB@PAA@Nbs, respectively, in 0.5 mL 20% methanol–PBS solution.

3.4 Adsorption isotherm of AFB₁ on MB@PAA@Nbs

The equilibrium isotherm was obtained by capturing different concentrations of AFB₁ from 2.0 ng mL⁻¹ to 200 ng mL⁻¹ using 0.1 mg of MB@PAA@Nbs as absorbent materials at room temperature. Fig. 3 indicates that the Langmuir equation is more appropriate to describe the adsorption behavior. The Langmuir equation is given by C_e/q_e = 1/(K_L × q_m) + C_e/q_m, where q_m is the maximum adsorption capacity of MB@PAA@Nbs (mg g⁻¹), C_e is the equilibrium concentration of AFB₁ in solution (mg L⁻¹), q_e is the amount of AFB₁ adsorbed on the MB@PAA@Nb unit mass at equilibrium concentration (mg g⁻¹), and K_L is the Langmuir constant related to the affinity of the binding sites (L mg⁻¹). The linear curve of equilibrium isotherm is C_e/q_e = 4.3103C_e + 0.0255, which was obtained by plotting C_e against C_e/q_e (R² = 0.9941). The q_m and K_L were 0.23 mg g⁻¹ and 196 L mg⁻¹, respectively. The higher correlation coefficient (R² > 0.99) indicates that the Langmuir isotherm is more suitable for the kinetics of AFB₁ adsorption because of the homogeneous and monolayer adsorption of the anti-AFB₁ Nbs.

Fig. 3 Adsorption isotherm and Langmuir model simulation (inset) for the adsorption of AFB₁ on MB@PAA@Nbs. Aqueous solution (0.5 mL) containing AFB₁ concentration from 2.0 ng mL⁻¹ to 200 ng mL⁻¹ and 0.1 mg MB@PAA.

3.5 Performance of MB@PAA@Nbs

To maximize the eluting AFB₁ from the MB@PAA@Nbs + AFB₁ complex, the percentage fraction of methanol in the elution buffer was optimized. Fig. S2† shows that the EE of AFB₁ increased with increasing methanol concentration and reached a value of 89.85% ± 3.4% when 100% pure methanol was used as eluent. Therefore, 100% methanol is considered as the optimal methanol concentration for AFB₁ elution and used in the subsequent experiments. To estimate the reusability of the designed MB@PAA@Nbs, the CE of AFB₁ was measured through the repeated use of MB@PAA@Nbs for 10 times with 1 day intervals. Fig. 4 shows that the CE of MB@PAA@Nbs for AFB₁ remains relatively high at 80.7% after the repeated use for 10 times, which had no significant difference with that of the first use (p > 0.05). This finding can be attributed to the superior properties of Nbs, especially the resistance to extreme environment.

Fig. 4 Recycle times of MB@PAA@Nbs, the additive amounts of AFB₁ is 10 ng mixed with 0.1 mg MB@PAA@Nbs in 0.5 mL 20% methanol–PBS solution.

The practicability of the MB@PAA@Nbs was evaluated by performing M-IAE using AFB₁-spiked corn samples. The recoveries of the intra- and inter-day assays were used to estimate the accuracy of the separation of the actual sample. Three AFB₁-spiked corn samples at concentrations of 5, 10, and 20 μg kg⁻¹ were prepared for the intra- and inter-day assay precision analysis. Table 1 indicates that the average recoveries of the intra-day assay ranged from 96.4% to 104.4% with relative standard deviations (RSDs) of 2.3% to 11.0%, and the inter-day assay recoveries ranged from 95.7% to 103.6% with RSDs ranging from 5.8% to 9.0%. These above-mentioned results suggest that the MB@PAA@Nbs satisfy the requirements for the practical application of a simple and rapid AFB₁ clean-up.

Table 1 Recovery efficiency of AFB₁ with MB@PAA@Nbs by ELISA in AFB₁ spiked corn samples

AFB1 spiked corn sample (μg kg^−1)	Intra-day assay		Inter-day assay
	Capture efficiency (%, n = 3)	RSD (%)	Capture efficiency (%, n = 3)	RSD (%)
5	104.4	2.3	98.4	9.0
10	96.4	8.5	103.6	7.5
20	99.2	11.0	95.7	5.8

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4 Conclusions

We reported a new MB@PAA and anti-AFB₁ Nb nanocomposite (MB@PAA@Nbs) that can replace the conventional conjugates of MB and anti-AFB₁ antibody for AFB₁ separation. In this proposed nanocomposite, MB@PAA was used to increase the anti-AFB₁ Nb loading capacity through the PAA brushes, and the anti-AFB₁ Nbs were employed to replace the conventional intact antibody for rendering the reusability of the MB@PAA@Nbs complex due to its excellent properties, especially the resistance to extreme environment. The resulting MB@PAA (1 mg) shows a high loading capacity of 623 μg anti-AFB₁ Nbs at equilibrium state, which is 19-fold higher than that of the conventional MB without PAA brushes. By using the resultant MB@PAA@Nbs for the separation of AFB₁, the maximum adsorption capacity reaches to as high as 0.23 mg g⁻¹, which is 35-fold higher than that of conventional MB@Nbs. Moreover, the MB@PAA@Nbs can be reused for at least 10 times without obvious loss of CE. The reliability and practicability of the proposed MB@PAA@Nbs for AFB₁ separation were tested using AFB₁-spiked corn samples. Overall, the developed MB@PAA@Nbs-based IAE technology demonstrated great potential as a novel platform for the pre-treatment of other mycotoxins in food samples.

Author contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Conflict of interest

The authors declare no competing financial interest.

Acknowledgements

This work was supported by a grant from the National Basic Research Program of China (2013CB127804), “Twelfth Five-Year Plan” for National Science and Technology Support Program (2013BAD19B02 and 2012BAK17B02), the Research Program of the State Key Laboratory of Food Science and Technology, Nanchang University (SKLF-ZZB-201306).

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Footnote

† Electronic supplementary information (ESI) available: The SDS-PAGE graph of anti-AFB₁ Nbs; optimization of methanol concentration for AFB₁ elution. See DOI: 10.1039/c5ra15843e

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