Sensitive Aflatoxin B1 Detection Using Nanoparticle-Based Competitive Magnetic Immunodetection

Abstract

Food and crop contaminations with mycotoxins are a severe health risk for consumers and cause high economic losses worldwide. Currently, different chromatographic- and immuno-based methods are used to detect mycotoxins within different sample matrices. There is a need for novel, highly sensitive detection technologies that avoid time-consuming procedures and expensive laboratory equipment but still provide sufficient sensitivity to achieve the mandated detection limit for mycotoxin content. Here we describe a novel, highly sensitive, and portable aflatoxin B1 detection approach using competitive magnetic immunodetection (cMID). As a reference method, a competitive ELISA optimized by checkerboard titration was established. For the novel cMID procedure, immunofiltration columns, coated with aflatoxin B1-BSA conjugate were used for competitive enrichment of biotinylated aflatoxin B1-specific antibodies. Subsequently, magnetic particles functionalized with streptavidin can be applied to magnetically label retained antibodies. By means of frequency mixing technology, particles were detected and quantified corresponding to the aflatoxin content in the sample. After the optimization of assay conditions, we successfully demonstrated the new competitive magnetic detection approach with a comparable detection limit of 1.1 ng aflatoxin B1 per ml sample to the cELISA reference method. Our results indicate that the cMID is a promising method reducing the risks of processing contaminated commodities.

Keywords: frequency mixing technology; immunofiltration; magnetic beads; mycotoxin.

Figure A1

ELISA checkerboard titration determining suitable aflatoxin B1-BSA (AFB1-BSA) coating concentrations ranging from 0.1 µg·mL⁻¹ to 5 µg·mL⁻¹ and monoclonal antibody (mAb) AFB1_002 concentrations ranging from 19.5 ng·mL⁻¹ to 1250 ng·mL⁻¹. Absorbance was measured at 405 nm by indirect readout after using a mouse-specific secondary antibody conjugated to horseradish peroxidase and application of respective substrate (n = 1).

Figure A2

Magnetic immunodetection checkerboard titration determining suitable aflatoxin B1-BSA (AFB1-BSA) coating concentrations and aflatoxin B1-specific biotinylated monoclonal antibody AFB1_002 concentrations. The readout was done by detecting 700 nm streptavidin-functionalized magnetic particles coupled to biotinylated AFB1_002 using FMMD (n = 1).

Figure 1

(A) Handheld, portable frequency mixing-based readout device and (B) schematic cross-section of detection head composed of driving coil providing the low driving frequency (f₂), the excitation coil providing the high excitation frequency (f₁) and detection unit based on a detection coil detecting the resulting mixing frequency signal of magnetic particles (MPs; f₁ + 2f₂) together with the directly induced signal and the reference coil detecting only the directly induced signal. The finally resulting measuring signal does not contain the directly induced excitation due to the opposite winding direction of detection and reference coil.

Figure 2

Schematic overview of the competitive magnetic immunodetection principle. (A) Immunofiltration column coated with aflatoxin B1-BSA mycotoxin conjugate with bound biotinylated monoclonal antibodies targeting aflatoxin B1. Magnetic particles functionalized with streptavidin bind to antibodies and can be detected by FMMD. (B) After pre-incubation of biotinylated, monoclonal antibodies with serially diluted free aflatoxin B1, the sample is flushed through an aflatoxin B1-BSA coated immunofiltration column. Non-saturated antibodies bind to the coated antigen and are retained within the matrix. The higher the mycotoxin content within the sample, the more antibodies are saturated and are flushed through the column. Afterward, streptavidin-functionalized magnetic particles are applied onto the column, bind to retained antibodies and can be detected using FMMD.

Figure 3

Competitive ELISA-based calibration curves of different pairs of aflatoxin B1-BSA coating and aflatoxin B1-specific monoclonal antibody AFB1_002 concentrations pairs. As a competitor, free aflatoxin B1 in dilutions ranging from 0.006 ng·mL⁻¹ up to 50,000 ng·mL⁻¹ in sample buffer was used. The indirect readout was done at 405 nm after the application of mouse-specific secondary antibody conjugated with horseradish peroxidase and respective substrate. Each data point represents the mean ± SD (n = 3).

Figure 4

Averaged calibration curve with 0.2 µg·mL⁻¹ AFB1-BSA conjugate coating and 150 ng·mL⁻¹ mAb with aflatoxin B1 competitor concentrations ranging from 0.006 ng·mL⁻¹ to 500,000 ng·mL⁻¹. Measuring signal was achieved by indirect readout at 405 nm with mouse-specific secondary antibody conjugated to horseradish peroxidase and respective substrate LOD: limit of detection; IC₅₀: half maximal inhibitory concentration. Each data point represents the mean ± SD (n = 12).

Figure 5

Dose-dependent measuring signal of 700 nm streptavidin-functionalized magnetic particles after applying 2.5 µg·mL⁻¹ biotinylated AFB1_002 monoclonal antibody onto 2 µg·mL⁻¹ AFB1-BSA coated immunofiltration columns. Each data point represents mean ± SD (n = 2).

Figure 6

cMID calibration curves with 2 µg·mL⁻¹ AFB1-BSA conjugate coating and 2.5 µg·mL⁻¹ biotinylated mAb in combination with (A) 700 nm streptavidin-functionalized magnetic particles, and (B) 70 nm streptavidin-functionalized magnetic particles with aflatoxin B1 competitor concentrations ranging from 0.006 ng·mL⁻¹ to 500,000 ng·mL⁻¹. LOD, limit of detection; IC₅₀, half maximal inhibitory concentration. Each data point represents the mean ± SD (n = 2).