Polystyrene Microsphere-Based Immunochromatographic Assay for Detection of Aflatoxin B1 in Maize

Aflatoxin B1 (AFB1), a mycotoxin, is hepatotoxic, carcinogenic, and nephrotoxic in humans and animals, and contaminate a wide range of maize. In this study, an immunochromatographic assay (ICA) based on polystyrene microspheres (PMs) was developed for sensitive and quantitative detection of AFB1 in maize. The amounts of PMs, the condition for activating carboxyl groups of PMs, the amount of monoclonal antibody (mAb), and the volume of the immune probe were optimized to enhance the performance PMs-ICA for point-of-care testing of AFB1 in maize. The PMs-ICA showed the cut-off value of 1 ng/mL in phosphate buffer (PB) and 6 µg/kg in maize samples, respectively. The quantitative limit of detection (qLOD) was 0.27 and 1.43 µg/kg in PB and maize samples, respectively. The accuracy and precision of the PMs-ICA were evaluated by analysis of spiked maize samples with recoveries of 96.0% to 107.6% with coefficients of variation below 10%. In addition, the reliability of PMs-ICA was confirmed by the liquid chromatography-tandem mass spectrometry method. The results indicated that the PMs-ICA could be used as a sensitive, simple, rapid point-of-care testing of AFB1 in maize.


Introduction
Aflatoxins (AFTs), a class of mycotoxins, are toxic and carcinogenic secondary metabolites produced by A. flavus, A. parasiticus, and the rare A. nomius [1]. Aflatoxin B 1 (AFB 1 ), aflatoxin B 2 (AFB 2 ), aflatoxin G 1 (AFG 1 ), and aflatoxin G 2 (AFG 2 ) are the common AFTs [2], and AFB 1 presents hepatotoxic, carcinogenic, and nephrotoxic in humans and animals [3][4][5]. The International Agency for Research on Cancer (IAMC) listed AFB 1 as a Group I carcinogen [6]. AFB 1 demonstrated high chemical stability against elevated temperature through food processing, making the prevention of their entrance into the food supply chain difficult [7]. To ensure food safety and protect human health, the maximum allowable limits of AFB 1 in maize have been set by many countries and regions. For examples, 20 µg/kg is the maximum allowable limits in maize and its products in China (GB 2761-2017) and 2 µg/kg is the maximum limit for peanuts and cereals in the European Commission (EC) [8]. However, a recent survey showed that AFB 1 had a high prevalence in cereals, which, in most of the cases, it exceeded the EC allowed limit [9]. In order to ensure the concentration of AFB 1 in maize is less than the maximum allowable limits, it is necessary to develop an accurate, effective detection method to monitor the concentration of AFB 1 . Currently, the common methods for AFB 1 detection include instrumental analysis methods and immunoassay. Instrumental analysis methods, such as Fourier transform infrared spectroscopy (FTIR) [10], high-performance liquid chromatography (HPLC) [11][12][13], liquid chromatography-tandem mass spectrometry (LC-MS/MS) [14,15], etc., provide accurate and reliable quantitative detection results. However, they need expensive equipment, complex sample preparation, professional skilled personnel, and much time. Moreover, these techniques are not suitable for point-of-caring detection. For the immunoassay, enzyme-linked immunosorbent assay (ELISA) and lateral flow immunochromatographic assay (LFICA) are common methods. LFICA, as a typical point-of-care technology [16], has been widely applied to food analysis [17,18], environmental monitoring [19], and in vitro diagnosis [20,21]. Comparing the ELISA, LFICA is used for the detection of AFB 1 , which is rapid, low cost, simple detection steps, and more suitable for a large number of samples screening [22][23][24]. Colloidal gold and fluorescent microspheres (FMs) are the two most common label materials for LFICA [25,26]. However, CG-LFICA is often limited by sensitivity [27], and FMs-LFICA has the advantages in sensitivity and signal intensity, but it requires a fluorescence reader to obtain results [28]. In order to obtain high sensitivity and brighter colors than CG-ICA, the detection results can be obtained by the naked eye instead of a reader similar to FMs-ICA. In the study, based on PMs, an ICA was developed for sensitive and quantitative detection of AFB 1 in maize. The principle of PMs-ICA is based on the completive reaction among the coating antigen (AFB 1 -BSA) coated on the T line, the analyte in the samples and the polystyrene microspheres-monoclonal antibody (PMs-mAb) conjugate. The amounts of PMs, the condition for activating carboxyl groups of PMs, the amount of mAb, and the volume of the immune probe were optimized to enhance the performance PMs-ICA for point-of-care testing of AFB 1 in maize. The developed detection method shows high sensitivity, good accuracy, and precision for detection of AFB 1 in maize. . Anti-AFB 1 mAb and coating antigen (AFB 1 -BSA) were prepared in our laboratory (Preparation of coating antigen and Anti-AFB1 mAb were described in Supplementary File).

Labeling of mAbs with PMs
The EDC activated ester method was used to couple carboxy-modified PMs with the mAbs [29]. The principle of conjugation is presented in Figure 1B. Initially, PMs were suspended in 1 mL of MES or PBS solution, then 15 µL of EDC (0.05 mg/mL) and 18 µL of NHS (0.05 mg/mL) solution were added to the above solution, and stirred with a shaker at 200 rpm for 15 min to active carboxyl groups of PMs. After activation, the mixture was centrifuged at 14,000 rpm for 15 min at 4 • C to discard the excess activation solution and the precipitate was dissolved in borate buffer [30] solution (0.05 mol/L, pH 8.0) solution. The mAb was added and coupled with PMs and shaken at 200 rpm for 30 min; 40 µL of BSA (10%, w/v) was then added to block the sites on the surface of PMs under shaking. After conjugation, the mixture was centrifuged at 14,000 rpm for 15 min at 4 • C, and repeated twice to discard the supernatant. The PMs-mAb conjugate was dissolved in PBS BSA (10%, w/v) was then added to block the sites on the surface of PMs under shaking. After conjugation, the mixture was centrifuged at 14,000 rpm for 15 min at 4 °C, and repeated twice to discard the supernatant. The PMs-mAb conjugate was dissolved in PBS (0.01 mol/L, pH 7.4) containing BSA (0.5%, w/v) and Tween-20 (0.05%, v/v), and was stored at 4 °C for future use.

Fabrication of PMs-ICA Strips
The PMs-ICA strip consists of a sample pad, a NC membrane, an absorbent pad, and a PVC plate ( Figure 1A). The appropriate concentration of coating antigen (0.25 mg/mL, AFB1-BSA) and goat anti-mouse IgG antibody (0.4 mg/mL), diluted by the coating buffer (PBS, 0.01 mol/L, pH 7.4), were coated onto the NC membrane to form the test line (T line) and control line (C line), with the dispense platform (XYZ 3060, BioDot, Irvine, CA, USA) at a jetting rate of 0.8 µL/cm. Then, the NC membrane was dried at 37 °C overnight. The sample pad was pretreated with 0.5% sucrose, 0.3% polyvinylpyrrolidone, 0.5% BSA, and 0.5% Tween-20 in PB solution (0.05 mol/L, pH 7.4), and dried at 37 °C overnight. The sample pad and absorbent pad were cut into 15 and 25 cm, respectively. Subsequently, the NC membrane, the sample pad, and the absorbent pad were sequentially pasted on the PVC plate with an overlap of 1-2 mm. Finally, the assembled plate was cut into 3.05 mm wide strips and kept in a desiccator at room temperature.

Fabrication of PMs-ICA Strips
The PMs-ICA strip consists of a sample pad, a NC membrane, an absorbent pad, and a PVC plate ( Figure 1A). The appropriate concentration of coating antigen (0.25 mg/mL, AFB 1 -BSA) and goat anti-mouse IgG antibody (0.4 mg/mL), diluted by the coating buffer (PBS, 0.01 mol/L, pH 7.4), were coated onto the NC membrane to form the test line (T line) and control line (C line), with the dispense platform (XYZ 3060, BioDot, Irvine, CA, USA) at a jetting rate of 0.8 µL/cm. Then, the NC membrane was dried at 37 • C overnight. The sample pad was pretreated with 0.5% sucrose, 0.3% polyvinylpyrrolidone, 0.5% BSA, and 0.5% Tween-20 in PB solution (0.05 mol/L, pH 7.4), and dried at 37 • C overnight. The sample pad and absorbent pad were cut into 15 and 25 cm, respectively. Subsequently, the NC membrane, the sample pad, and the absorbent pad were sequentially pasted on the PVC plate with an overlap of 1-2 mm. Finally, the assembled plate was cut into 3.05 mm wide strips and kept in a desiccator at room temperature.

Optimization of Key Parameters
To evaluate the influence of the amounts of PMs on the performance of PMs-ICA and obtain appreciate amounts of PMs to couple with anti-AFB 1 mAb, different amounts of PMs were compared, including 5, 10, 15, 20, 25, 30 µL. The negative control groups (PBS, Biosensors 2021, 11, 200 4 of 12 0.01 mol/L, pH 7.4) and positive test groups (1 ng/mL AFB 1 ) were tested at the same condition.
To activate carboxyl groups on the surface of PMs more efficiently, six buffer solution were selected to be tested, including 0.05 mol/L MES (pH 5, 5.5, 6, 6.5) and 0.01 mol/L PBS (pH 7.0, 7.4). The negative control groups (PBS, 0.01 mol/L, pH 7.4) and positive test groups (1 ng/mL AFB 1 ) were tested at the same condition to evaluate the influence of the condition for activating carboxyl groups on the surface of PMs.
The amount of mAb was optimized because it directly affected the color intensity of the T line, and the sensitivity of PMs-ICA. 2, 2.5, 3, 3.5, 4 µL of mAb (1 mg/mL) were selected to couple with PMs. The results of the color intensity of the T Line on the negative control groups (PBS, 0.01 mol/L, pH 7.4) and the inhibition effect on the positive test groups (1 ng/mL AFB 1 ) were tested and considered simultaneously to determine the amount of optimal mAb.
Applying PMs-ICA to real samples, the volume of PMs-Ab conjugate (immune probe) was a key factor affecting the color intensity of the T line and the sensitivity of PMs-ICA. Moreover, 3, 4, 5, 6 µL of immune probes were added to tested and compared in spiked maize samples. The negative control groups (negative maize samples) and positive test groups (negative maize samples spiked 6 µg/kg AFB 1 ) were tested to decide the volume of the immune probe.

Sample Preparation
A total of 5 g of maize samples to a 50 mL centrifuge tube, then 10 mL of methanol/water (7:3, v/v) was added, and the mixture was shaken for 2 min on a vortex shaker. After the mixture was centrifuged at 5000 rpm for 5 min, and the supernatant was collected and filtered through 0.22 µm filter, 100 µL of supernatant was added to 200 µL of PB (0.02 mol/L, pH 7.4) for detection.

Test Procedure
A total of 150 µL of standard or sample solution and a certain amount of PMs-mAbs conjugates were mixed into microplate wells and incubated for 3 min at room temperature. Then, the prepared test strips were vertically inserted into the micropores for another 5 min chromatographic reaction, and the sample pad was rejected immediately. For qualitative detection, the results of ICA strips can be observed by the naked eye. For quantitative detected, the ICA strips were scanned by an HP ScanJet (HP ScanJet g3110, Shanghai, China), and the digital images of strips were analyzed by the TotalLab TL120 software (Nonlinear Dynamics, Newsletter, UK) [31].

Sensitivity and Specificity of PMs-ICA
A series of concentrations of AFB 1 , including 0, 0.125, 0.25, 0.5. 0.75, 1 ng/mL were prepared, the sensitivity of PMs-ICA was evaluated under optimal condition, and each concentration of AFB 1 was detected by three parallel experiments. For qualitative detection, the cut-off value was defined as the concentration of AFB 1 , resulting in no red bands on the T line judged by the naked eye [32]. For quantitative detection, the digital images scanned with a scanner were analyzed by TotalLab TL120 software. B/B0 was calculated and the calibration curve fitted by OriginPro 9.1 software (OriginLab Corp., Northampton, MA, USA) was obtained by plotting B/B0 against the concentration of AFB 1 . B and B0 denoted the color intensity of the T line in the positive control group and the test negative group, respectively. The qLOD was defined as the concentration giving 80% B/B 0 from the calibration curve [33,34]. For the sensitivity of PMs-ICA in maize samples, the negative maize samples were brought from the local market and spiked. The sensitivity was evaluated under optimal conditions, with AFB 1 at concentrations of 0, 0.75, 1.5, 3, 4.5, 6 µg/kg.

Accuracy and Precision
The accuracy (expressed as recovery) and precision (expressed as coefficient of variation (CV)) of the PMs-ICA were evaluated, and were determined after spiking with AFB 1 at concentrations of 1, 3, 5, 12 µg/kg in maize samples; the results were detected after sample preparation.

Detection of Real Maize Samples
To evaluate the reliability of PMs-ICA, 20 real maize samples brought from the local market and provided by Shanxi Institute of Feed and Veterinary Drug control were detected by PMs-ICA and LC-MS/MS. The LC-MS/MS method was performed according to our lab's previous study, with slight modifications [35]. Briefly, the dry grated samples (10.0 g) were extracted with 40.0 mL of acetonitrile/water/acetic acid (79:20:1, v/v) by vigorous stirring for 10 min. Then, the mixture was centrifuged at 5000 g for 10 min. Purification of the extract was conducted using 0.5 mL of the final extract diluted with same amount of acetonitrile/water/acetic acid (20:79:1, v/v), and then a second purification was conducted using a syringe filter (0.22 µm). Chromatographic separation was conducted with an Agilent C 18 column (InfinityLab Poroshell 120 EC-C18, 4.6 × 150 mm 2.7-Micron. The mobile phase consisted of 0.1% acetic acid (mobile phase A) and 100% methanol (mobile phase B), and the mobile phase flow rate was 700 µL/min. The gradient elution procedure was carried as follows: 0-5 min, 5-90% B; 5-7 min, 90% B; 7-7.1 min, 90-5% B, 7.1-10 min, 5% B. The column was kept at 30 and the sample injection volume was 10 µL. Mass analyses were carried out by electrospray ionization (ESI) sources in positive-ion mode. The spray voltage was 5.5 kV. The capillary temperature was set at 550 • C. Curtain gas, spray gas, and auxiliary gas maintained at a pressure of 30, 50 and 50 psi, respectively. Figure 1 illustrates the schematic process of detection AFB 1 by PMs-ICA. Figure 1A presents the structure of the test strips, including a sample pad, a NC membrane, an absorbent pad, and a PVC plate; the AFB 1 -BSA and goat anti-mouse antibody IgG were coated on the NC membrane form the T line and C line, respectively. Figure 1A shows the process of labeling of mAbs with PM, the carboxyl groups of PMs were firstly activated by EDC solution, then the mAb was added to couple with PMs. Based on the competitive inhibition interaction between the free AFB 1 in the samples and the coating antigen (AFB 1 -BSA) spaced on the NC membrane, the PMs-ICA was developed. If there was no AFB 1 in the samples, the PMs-Ab conjugates bound to the antigen coated on the T Line to form a red band ( Figure 1C,D). If there was AFB 1 in the samples, AFB 1 bound to the PMs-Ab conjugates to form a PMs-Ab-AFB 1 complex in the well, and the binding sites of mAb would be firstly occupied by AFB 1 . The excessive PMs-Ab conjugates would be captured by the antigen, and the color intensity of T line would gradually decrease to zero with the concentration of AFB 1 increase ( Figure 1C,D). Regardless of whether there is AFB 1 in the sample, PMs-mAb conjugate would pass through the T line and react with the goat anti-mouse antibody IgG coated on the C line, which was used to ensure the validity of the PMs-ICA.

The Amount of PMs
PMs, the label of the PMs-ICA, were directly related the color intensity of the T line. Hence, the amounts of PMs were optimized to get enough color intensity at the lowest cost. As shown in Figure 2A, for the negative control groups, the color intensity of the T line gradually enhanced until the amounts of PMs reached 15 µL, the color intensity of the T line was no longer enhanced. For positive test groups, the T line of each group was colorless. Considering the price of PMs, 15 µL of PMs were selected for the next optimization experiments.

The Amount of PMs
PMs, the label of the PMs-ICA, were directly related the color intensity of the T line. Hence, the amounts of PMs were optimized to get enough color intensity at the lowest cost. As shown in Figure 2A, for the negative control groups, the color intensity of the T line gradually enhanced until the amounts of PMs reached 15 µL, the color intensity of the T line was no longer enhanced. For positive test groups, the T line of each group was colorless. Considering the price of PMs, 15 µL of PMs were selected for the next optimization experiments.

The Condition for Activating Carboxyl Groups of PMs
In order to enable PMs to couple with Ab, the carboxyl groups of PMs must be activated by the EDC method. The condition for activating carboxyl groups of PMs is an important factor [36], 0.05 mol/L MES (pH 5.0, 5.5, 6.0, 6.5) and 0.01 mol/L PBS (pH 7.0, 7.4) were prepared for the solution to active carboxyl groups. As presented in Figure 2B, no matter what the negative control groups or positive test groups were, the PMs-ICA had the best color intensity, and the same inhibition effect, activating by 0.5 mol/L MES (pH 5.5). Therefore, the MES (0.05 mol/L, pH 5.5) solution was chosen to activate carboxyl groups for next optimization experiments.

The Amounts of mAb
The amounts of mAb directly affect the sensitivity of the immunoassay [32]; 2, 2.5, 3, 3.5, 4 µL of mAb were selected to couple with PMs, respectively, and the sensitivity was tested. For the negative control groups, the color intensity of the T line gradually increased as the amount of mAb increased, but the color intensity was (basically) stable when the

The Condition for Activating Carboxyl Groups of PMs
In order to enable PMs to couple with Ab, the carboxyl groups of PMs must be activated by the EDC method. The condition for activating carboxyl groups of PMs is an important factor [36], 0.05 mol/L MES (pH 5.0, 5.5, 6.0, 6.5) and 0.01 mol/L PBS (pH 7.0, 7.4) were prepared for the solution to active carboxyl groups. As presented in Figure 2B, no matter what the negative control groups or positive test groups were, the PMs-ICA had the best color intensity, and the same inhibition effect, activating by 0.5 mol/L MES (pH 5.5). Therefore, the MES (0.05 mol/L, pH 5.5) solution was chosen to activate carboxyl groups for next optimization experiments.

The Amounts of mAb
The amounts of mAb directly affect the sensitivity of the immunoassay [32]; 2, 2.5, 3, 3.5, 4 µL of mAb were selected to couple with PMs, respectively, and the sensitivity was tested. For the negative control groups, the color intensity of the T line gradually increased as the amount of mAb increased, but the color intensity was (basically) stable when the amount of antibody was added, 3 µL, which should be due to the limited carboxyl groups on the surface of the PMs ( Figure 2C). For the positive test groups, all T lines were colorless. To obtain the best result with the least amount of mAb, 3 µL of mAb was decide for the next optimization experiments.

The Immune Probe Amount
Applying PMs-ICA to actual samples-the PMs-Ab conjugate was used as the immune probe and its volume would directly affect the performance of PMs-ICA. Hence, under the other optimal conditions, 3, 4, 5, 6 µL of the immune probe was added to the well for testing and comparing in spiked maize samples. The results of optimization are shown in Figure 2D, for the negative control groups, the color intensity of the T line gradually increased with the volume of the immune probe increasing. For the positive test groups, the T line appeared as a faint red band when the volume of the immune probe reached 4 µL. Considering the color intensity of the T line and the sensitivity of PMs-ICA, when the volume of the immune probe was 3 µL, the color intensity of the T line was enough and the sensitivity was better than the more immune probe. Therefore, 3 µL of the immune probe was added to the detected AFB 1 in maize.
Furthermore, to reduce the time of labeling of mAb with PMs, the time of PMs couple with mAb, and the time of blocking PMs to bind the site to the mAb, were investigated ( Figure S1). The length of time, the color intensity of the T line, the sensitivity, and the non-specific adsorption were comprehensively considered. The final optimal conditions of labeling of mAbs with PMs are summarized in Table S1.

Sensitivity and Specificity of PMs-ICA
The sensitivity and specificity are important factors to evaluate the performance of ICA. Hence, under the optimal condition, the sensitivity and dynamic interval of PMs-ICA in the buffer were determined by testing a series of concentrations, AFB 1 standards, from 0 to 1 ng/mL. The color intensity of the T line of PMs-ICA decreased until it disappeared with the concentration of the AFB 1 increase. Generally, the cut-off value was defined as the concentration of AFB 1 resulting in no red bands on the T line, judged by the naked eye. The results (Figure 3) show that the cut-off value was 1 ng/mL. Furthermore, the color intensity of the T line was calculated with the software TotalLab TL120, and the B/B 0 value was calculated. The calibration curve fitted by OriginPro 9.1 software (OriginLab Corp., Northampton, MA, USA) was obtained by plotting B/B 0 against the concentration of AFB 1 . The qLOD was defined as the concentration giving 80% B/B 0 from the calibration curve. As shown in Figure 3 the calibration curve presented an inverse linear relationship with a high correlation coefficient (R 2 = 0.9957), and the qLOD was 0.27 ng/mL, derived from the calibration curve. In addition, as shown in Table 1, the PMs-ICA for detection AFB 1 has better sensitivity and simpler operation than other LFICAs, based on nanoparticles such as colloidal gold nanoparticles, fluorescent microspheres, quantum dots, etc. [37][38][39][40], and the results could be obtained by the naked eye compared to fluorescence sensors. For the sensitivity of PMs-ICA in real samples, the maize samples ( Figure 4) were tested. The blank maize samples confirmed by LC-MS/MS were prepared according to the sample preparation listed in Section 2.5. Briefly, 5 g maize samples were extracted with 10 mL of methanol/water (7:3, v/v) and centrifuged; the supernatant was diluted three times by PB to eliminate matrix effects. The sensitivity was also determined after spiking with AFB 1 at concentrations of 0, 0.75, 1.5, 3, 4.5, 6 µg/kg in maize samples using the same method as in PB. As shown in Figure 4 and Figure S2, the cut-off value was 6 µg/kg, and the qLOD was 1.43 µg/kg.
To evaluate the specificity of the PMs-ICA, AFB 2 , AFG 1 , AFM 1 , ZEN, OTA, DON, FB 1 and T-2 toxins, which were often found in maize, were chosen as interfering compounds. The results were shown in Table 2, and these results indicated the PMs-ICA for AFB 1 detection would not be affected by other mycotoxins.   To evaluate the specificity of the PMs-ICA, AFB2, AFG1, AFM1, ZEN, OTA, DON, FB1 and T-2 toxins, which were often found in maize, were chosen as interfering compounds. The results were shown in Table 2, and these results indicated the PMs-ICA for AFB1 detection would not be affected by other mycotoxins.

Accuracy and Precision
For the accuracy and precision, blank maize samples were spiked with 1, 3, 5, 12 µg/kg in maize samples ( Table 3). Recovery of PMs-ICA for detection of AFB 1 in maize samples was from 96.0% to 107.6% with corresponding CV of 2.5% to 8.4%. These results indicate that PMs-ICA has good accuracy and reproducibility for the detection of AFB 1 in maize samples.

Detection of Real Maize Samples
To further evaluate the suitability of PMs-ICA for screening in maize samples, 20 real maize samples were detected by PMs-ICA and LC-MS/MS. Among these samples, four samples (nos. 6,9,14, and 18) detected by PMS were 1.1, 1.2, 0.7, 1.9, respectively, whereas those detected by LC-MS/MS were 1.2, 1.1, 0.9, 1.8, respectively. Other samples were not detected by either method ( Table 4). The detection demonstrated that the developed PMs-ICA would be reliable for detection of AFB 1 in maize samples.

Conclusions
In summary, an ICA, based on PMs, was developed for sensitive and quantitative detection of AFB 1 in maize. The PMs-ICA was more sensitive than CG-ICA and could obtain results by the naked eye instead of utilizing a reader, such as the FMs-ICA. The developed PMs-ICA showed the cut-off value of 1 ng/mL in PB and 6 µg/kg in maize samples, respectively. The qLOD was 0.27 and 1.43 µg/kg in PB and maize samples, respectively. The accuracy and precision of the PMs-ICA were evaluated by analysis of spiked maize samples with recoveries of 96.0% to 107.6% and coefficients of variation below 10%. In addition, the reliability of PMs-ICA was confirmed by the LC-MS/MS method. The results indicated that the PMs-ICA could be used as a sensitive, simple, rapid point-of-care testing of AFB 1 in maize.