Chemicals and reagents
Mycotoxin standards—aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), aflatoxin M1 (AFM1), ochratoxin A (OTA), zearalenone (ZEA), T-2 toxin (T2), HT-2 toxin (HT-2), deoxynivalenol (DON), and fumonisin B1 (FB1) were purchased from Romer Labs (Biopure, Singapore). Goat-anti mouse (GAM) antibody (Code: KPL5210-0187) and rabbit anti-goat (RAG) antibody (Code: ab6697) were purchased from SeraCare (MA, USA) and Abcam (Cambridge, UK), respectively. All specific-mycotoxin antibodies and mycotoxin-conjugated bovine serum albumin (BSA) were obtained from Queen’s University Belfast (QUB), UK. Monoclonal antibodies against AFB1, T2, and DON were produced as described in detail by Oplatowska-Stachowiak et al.55 and Meneely et al.56,57 Mycotoxin-conjugated BSA was synthesized with slight modification56,58,59,60,61. Water and methanol (LC/MS grade) were supplied by Merck (Darmstadt, Germany). Other chemical reagents were purchased from Sigma-Aldrich (MO, USA).
Preparation of organic dye M424-labeled monoclonal antibody (GAM-M424 conjugation)
A luminescent organic dye named M424 was synthesized and linked to GAM antibody via a Schiff base-formation-reductive reaction as previously described by Charlermroj et al.21, with slight modifications. Briefly, 50 μL of 10 mg/mL M424 in dimethylsulfoxide (DMSO) was mixed with 500 μL of 1 mg/mL GAM antibody in phosphate buffer saline (PBS, 50 mM pH 7.4) before the introduction of 5.5 μL of 5 M cyanoborohydride in 1 N sodium hydroxide. After shaking at room temperature for 2 h, the mixture was added with 12 µL of ethanolamine (pH 6.6) to block any unreacted aldehyde sites of M424 and incubated at room temperature for 15 min with shaking. The supernatant containing a GAM-M424 conjugate was collected after centrifugation at 10,000 × g for 5 min and stored at 4 °C.
Fabrication of microarray lateral flow immunoassay strip (µLFIA)
The µLFIA strips were fabricated using a non-contact microarray dispenser (1520 CE) equipped with Biojet Elite™ dispenser (BioDot, Irvine, CA, USA) as previously described by Charlermroj et al.21. Mycotoxin-conjugated BSAs, including AFB1-BSA, T2-BSA, ZEA-BSA, DON-BSA, and FB1-BSA, were spotted on the microarray signal pad in quadruplicate with 500 μm in diameter with the following spotting parameters: 15 nL/spot, 200 μs open-time, 700-μm center-to-center spacing for both rows and columns and 60% relative humidity. The fluorescence signals from these four identical spots were averaged for further analysis. BSA and RAG antibodies were spotted in duplicate as negative and positive controls, respectively, while 0.15 mg/mL of M424 was used to mark the four corners of the µLFIA strip to facilitate spot localization for signal analysis. AFB1-BSA and BSA were spotted at 1 µg/mL, while the other mycotoxin-conjugated BSAs were spotted at 0.5 µg/mL. After spotting, each signal pad was dried at 37 °C for 2 h and immersed in 1 mL of optimized 10 mM borate buffer (pH 8.0) containing 0.5% polyvinyl pyrrolidone, 0.25% Triton X-100, and 1% BSA. Then, the signal pad was re-dried overnight at 37 °C. On a conjugate pad, a mixture of five monoclonal antibodies specific for target mycotoxins and M424-GAM antibody conjugate were spotted. To assemble a lateral flow strip, all the membrane pads (sample pad, conjugate pad, signal pad, and absorbent pads) were pasted onto a backing board (Fig. 5a) and cut into strips with widths of 4.0 mm using a guillotine cutter. Each strip was then inserted into a two-piece plastic cassette and stored at 4 °C.
a µLFIA components and the spot layout of mycotoxin-bovine serum albumin (BSA), negative control, positive control, and dye M424 reference panel on a signal pad. b Expected results from the μLFIA strip using a sample with and without the target/relevant mycotoxins.
Microarray-strip reader
A cost-effective, rapid, and portable microarray reader was employed for on-site mycotoxin detection. The optical setup and signal correction of this reader, as reported in a previous study62, effectively compensate for fluorescence variation caused by intensity fluctuations and spatial non-uniformity in the excitation light. A light-emitting diode (LED: LST1-01G01-UV01-00, New Energy), in conjunction with an iris diaphragm (SM1D12, Thorlabs) and a bandpass filter (FF01-365/2-25, Semrock), generated a 365 nm wavelength light beam directed towards a μLFIA strip test using a dichroic beamsplitter (MD453, Thorlabs). The fluorescence emissions originating from the strip test were filtered by a 450 nm long-pass filter (FEL0450, Thorlabs) and then conveyed to an 8-bit camera (12.3 MP HQ, Raspberry Pi Foundation). Optomechanical components corresponding to these materials were created using a DLP 3D printer (Prusa SL1, Prusa Research). Using the Raspberry Pi platform (Raspberry Pi 4 model B, Raspberry Pi Foundation) programmed with Python 3, a microarray fluorescence image of the strip test with a resolution of 2592 × 1952 pixels was captured. The fluorescence signals from each spot, represented by the green channel, were extracted, while the corresponding background signals were obtained from the dark areas adjacent to those spots. An in-house software package for the reader was designed and developed to provide user-friendliness through a built-in 4.3-inch touchscreen (Cytron Technologies, Thailand). This software package comprised the backend for microarray-spot image acquisition and processing, mainly implemented using the OpenCV module, and the frontend, which offered a straightforward graphical user interface, using the PyQt module and Qt Designer.
Optimization of microarray lateral flow immunoassay multiplex detection conditions
In order to improve the robustness and sensitivity of the μLFIA, the following key parameters were optimized: concentrations of monoclonal antibodies, GAM-M424 conjugate, and running buffer components. More than 40 combinations of mixed anti-mycotoxin antibodies, ranging from 3 to 100 µg/mL, were investigated in this study. Briefly, 10 µL of anti-mycotoxin antibody mixtures at different concentrations were spotted on the conjugate pads. Then, 100 µL PBS containing 0.1% tween 20 and 0.5% BSA was introduced into the sample pad. After strip incubation, microarray spot signal intensities were captured and processed using the microarray reader to select the optimal concentrations of monoclonal antibodies.
In terms of GAM-M424 conjugation, different concentrations of the GAM-M424 conjugate (25, 75, 100, 110, 120, 130, 140, and 150 µg/mL) were tested to determine the optimal concentration that yielded the lowest background and highest signal intensity. Each GAM-M424 conjugate concentration and the optimized concentrations of anti-mycotoxin antibodies were fixed onto a conjugate pad. PBS containing 0.1% tween 20 and 0.5% BSA without target mycotoxins was used as a running buffer. After the experiments were completed, the test strips were incubated at 70 °C for 1 h. Microarray spot intensities on the strip tests were captured, processed, and analyzed.
Moreover, the running buffer of the developed method was optimized. Two detergents (tween 20 and Triton X-100) over a range of concentrations (0.1%, 0.2%, 0.5%, and 1%) were evaluated as a running buffer. In addition, six different molecular weights (MW) of PEG: MW100 (5%, 10%, and 20%), MW600 (5%, 10%, and 20%), MW3350 (5%, 7.5%, and 10%), MW6000 (0.5%, 1%, 2.5%, and 5%), MW8000 (1%, 2.5%, 3%, 4%, 5%, and 6%), and MW20,000 (1%, 1.5%, 2%, 2.5%, and 5%) were assessed. Optimized concentrations of mixed anti-mycotoxin antibodies and GAM-M424 conjugate were utilized for this experiment. Microarray spot images were captured and processed following strip incubation at room temperature.
Development of a green mycotoxin extraction solution for the lateral flow strip
A simple, rapid, and green sample preparation procedure was developed for the simultaneous extraction of five mycotoxins (AFB1, T2, ZEA, DON, and FB1). A wide range of surfactants and solutions were tested. PEG with a molecular weight of 20,000 (PEG 20K) was found to be compatible with the µLFIA strip and yield the highest recoveries of the target mycotoxins. Subsequently, six different concentrations (0.01%, 0.1%, 0.2%, 0.5%, 1%, and 2%) of PEG 20K supplemented with 20% ethanol (to improve mycotoxins recovery) were examined using LC–MS/MS to determine for the extraction capacity for target mycotoxins in rice. The recovery value was calculated as follows: %Recovery=(measured concentration of spiked sample/spiked concentration)×100. PEG 20K at 0.1% with 20% ethanol was found to give the highest mycotoxin recovery and was later used for further experiments, including optimization of sample extraction time and µLFIA assay validation.
Performance evaluation of microarray lateral flow immunoassay strip in rice sample
The specificity of µLFIA strip test for single and multiple mycotoxin detection was tested through a competitive assay format using both target and non-target mycotoxins, including AFB1, AFB2, AFG1, AFG2, AFM1, T2, HT-2, ZEA, DON, FB1, OTA, and different mycotoxin combinations (2-Plex, 3-Plex, 4-Plex, and 5-Plex).
In terms of sensitivity, a matrix-matched calibration curve was constructed for the quantitation of each target mycotoxin. The sensitivity and linearity of the assay were evaluated using the following mycotoxin concentration ranges: 0–2000 ng/mL for AFB1, ZEA, and T2 and 0–4000 ng/mL for DON and FB1. These concentration ranges were selected to cover the established MPL for mycotoxins in cereals and toxin levels commonly found in cereal samples in Asia19,21,24. The standard curves were plotted with the y-axis as log (%B/B0) and the x-axis as log (concentrations of mycotoxins). B is the signal intensity value of the mycotoxin-contaminated sample, and B0 is the signal intensity value of the blank sample. In addition, 20 blank (mycotoxin-free) samples were analyzed to estimate the limit of detection (LOD) and limit of quantification (LOQ) of the µLFIA. The LOD was calculated from a 3-fold of standard deviation obtained from the blank sample without any mycotoxin (n = 12), divided by the slope of a matrix-matched calibration curve, while LOQ was calculated from a 10-fold of standard deviation obtained from the blank sample without any mycotoxin (n = 12), divided by the slope of the matrix-matched calibration curve.
Moreover, the precision and accuracy of the assay were determined. Mycotoxin-free rice samples, confirmed by using a validated LC–MS/MS, were spiked at three different concentrations of multi-mycotoxin standards: low (L1), medium (L2), and high (L3) levels. The samples were mixed thoroughly and kept in a fume hood overnight to allow solvent evaporation. The final spiked concentration of each mycotoxin was 100, 200, and 500 µg/kg. These concentrations were also confirmed using a validated LC–MS/MS method described in the “Methods” section. The precision of the µLFIA was determined through intra-day (repeatability) and inter-day precisions (reproducibility). The intra-day precision was carried out by analysis of three replicates on the same day at three different concentration levels, while inter-day precision was evaluated by repeating the same procedure over three consecutive days. The data were used to calculate within-laboratory accuracy and precision and expressed as percentage relative standard deviation (%RSD). The recovery of each target compound was also calculated as the spot intensity ratio of blank samples spiked before and after sample extraction and expressed as a percentage. The µLFIA extraction recovery results were compared with the recovery results obtained using the validated LC–MS/MS method.
Test procedure for the microarray lateral flow immunoassay strip
The optimized assay conditions were used for the simultaneous analysis of five target mycotoxins in rice. Briefly, 1 g of contaminated rice samples was weighed into 15 mL propylene tubes. Subsequently, 5 mL of extraction solution containing 0.1% PEG 20K with 20% ethanol in 10 mM acetate buffer pH 5.0 was added to each tube. The tube was manually vortexed for 1 min and the mixture was filtered into a new Eppendorf tube using a 0.45 µm filter. The filtrate was diluted with an equal volume of running buffer solution containing 3.9% PEG 20K, 2% tween 20, and 1% BSA in 10 mM acetate buffer (pH 5.0) and briefly vortexed. An aliquot of 100 µL of the mixture was introduced into the sample pad of μLFIA strip. After incubation at room temperature for 15 min, semi-qualitative results from the strip were observed via the naked eye under ultraviolet (UV) light at 312 nm (ENB-260C/FE, New York, USA), while the quantitative results were obtained using the portable microarray reader. The total analysis time was ~17 min, and the sample-to-result workflow is illustrated in Fig. 6.
The developed and validated microarray lateral flow immunoassay strip with the green sample extraction enables the detection of multiple mycotoxins within 17 min using a portable microarray reader device.
To detect the mycotoxins, a direct competitive immunoassay approach was utilized due to their low molecular weights. Without any mycotoxin contamination, the specific monoclonal antibody fixed on the conjugate pad would bind to GAM-424 fluorescent organic dye before moving down the strip, and the antibody complex would be able to bind to the mycotoxin standard spots on the strip. With mycotoxin contamination, the antibody complex would bind to the mycotoxin present in the test sample, impeding it from binding to the standard mycotoxin-BSA spots on the strip (Fig. 5). As the specific antibodies are unable to react with mycotoxins on the signal pad, the signal intensity would be lower than that from the mycotoxin-free sample. The higher the level of mycotoxins in the test sample, the lower the spot signal intensity and vice versa (Fig. 5).
Sample preparation for LC–MS/MS analysis
One gram of blank (mycotoxin-free) and spiked rice samples were weighed into a 15 mL Eppendorf tube. Then, 5 mL of 70% methanol at pH 3 was added. The tube was vortexed for 30 min and centrifuged at 100 rpm for 10 min. An aliquot of the supernatant was transferred into a new Eppendorf tube and diluted with an equal volume of acetonitrile with 2% acetic acid. The mixture was vortexed and filtered through a 0.22 µm PTFE filter into a glass vial for LC–MS/MS analysis.
The qualitative and quantitative determination of mycotoxins were carried out on an Agilent 1260 Infinity II HPLC system coupled to an Ultivo triple quadrupole mass spectrometry, equipped with a jet stream electrospray ionization source (LC–MS/MS). The analytes were separated through gradient elution on Gemini 5 μm C18 110 Å 50 × 4.6 mm i.d. column maintained at 30 °C. The mobile phases consisted of water (A) and methanol (B), both containing 0.1% formic acid and 5 mM ammonium hydroxide. The following gradient elution protocol was followed for mycotoxin analysis: 0–2 min (60% A), 2–4 min (50% A), 4–6 min (20% A), and 6–9 min (60% A). The total run time was 9 min with a flow rate of 0.9 mL/min and sample injection volume of 2 μL.
The mass spectrometer (MS) was operated in dynamic multiple reaction monitoring mode (dMRM), filtering one precursor and two product ions of each mycotoxin. The MS parameters, such as collision energy and fragmentor voltage, and ion transitions, were optimized for each compound. The ion source was operated in both positive and negative modes, with a gas temperature of 250 °C, a gas flow of 10 L/min, a nebulizer pressure of 10 psi, and a capillary voltage of 4 kV or −4 kV. Data acquisition and analyses were performed using the Agilent Mass Hunter software package, which includes acquisition for Ultivo (Ver 1.1.2222), Optimizer (Ver 1.1.2222), quantitative analysis (Ver 10.0.707.0), and qualitative analysis (Ver 10.0.10305.0).
Statistical analysis
Statistical analysis was carried out using SPSS version 26.0 for Windows, and significant differences (p < 0.05) between means were determined by Duncan’s multiple range test.
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- Source: https://www.nature.com/articles/s41538-024-00342-2