Microfluidics-Based Time-Resolved Fluorescence Immunoassay for the On-Site Detection of Aflatoxins B1 Zearalenone and Deoxynivalenol in Cereals
Abstract
:1. Introduction
2. Experimental Section
2.1. Reagents and Instruments
2.2. Preparation of Antigens
2.3. Production of MAb
2.4. Indirect Competitive ELISA
2.5. Characterization of MAb
2.6. Antibody Specificity Determination
2.7. Prepare TRFMs-Probes
2.8. Assembly of the Microfluidic Chip
2.9. Sample Pretreatment
2.10. Microfluidics Assay Procedure
3. Results and Discussion
3.1. The Mab’s Characterization
3.2. Antibody Specificity Determination
3.3. Indirect Competitive ELISA
3.4. Optimization of the Microfluidics
3.4.1. Optimization of the Microspheres
3.4.2. Coating Buffer Selection
3.4.3. Coating Antigen
3.5. Sensitivity of the Microfluidics
3.6. Spiked Sample Analysis
3.7. Natural Sample Analysis
3.8. Comparison of the Microfluidics
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Conflicts of Interest
References
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Mycotoxin Analyte | Structure | IC50 (ng/mL) | CR (%) |
---|---|---|---|
AFB1 | - | 0.08 | 100% |
AFB2 | 0.67 | 12% | |
AFM1 | 1.11 | 7% | |
AFM2 | 10.7 | 1% | |
AFG1 | 11.56 | 1% | |
AFG2 | 15.53 | 1% | |
ZEN, DON, FB1, OTA, T-2 | >10,000 | <0.1 | |
ZEN | - | 6.41 | 100% |
ZAN | 28.82 | 22% | |
α-ZOL | 56.24 | 11% | |
β-ZOL | 53.31 | 12% | |
α-ZAL | 44.28 | 14% | |
β-ZAL | 48.22 | 13% | |
AFB1, DON, FB1, OTA, T-2 | >10,000 | <0.1 | |
DON | - | 16.31 | 100% |
3-AcDON | 66.28 | 25% | |
15-AcDON | >10000 | <0.1 | |
NIV | >10,000 | <0.1 | |
ZEN, AFB1, FB1, OTA, T-2 | >10,000 | <0.1 |
Target Analyte | AFB1 | ZEN | DON |
---|---|---|---|
Coating antigen | AFB1-OVA | ZEN-OVA | DON-OVA |
Coating concentration (μg/mL) | 0.42 | 0.34 | 0.46 |
Coating buffer | 0.05 M carbonate buffer (pH 9.6) | ||
Coating condition | 14 h, 4 °C | ||
MAb | Anti-AFB1 MAb | Anti-ZEN MAb | Anti-DON MAb |
Standard analyte | AFB1 | ZEN | DON |
Competition condition | 30 min, 37 °C | ||
HRP- IgG dilution | 1:5000 | ||
HRP-IgG incubation condition | 30 min, 37 °C |
Analyte | Spiked Level (µg/kg) | Microfluidics Detection Level (µg/kg) | Visual | Ic-ELISA Detection Level (µg/kg) | ||||
---|---|---|---|---|---|---|---|---|
Mean ± SD | Recovery Rate (%) | CV (%) | Mean ± SD | Recovery Rate (%) | CV (%) | |||
AFB1 | 0 | ND | NC | NC | − − − | ND | NC | NC |
5 | 4.75 ± 0.01 | 95 | 1 | ± ± ± | 5.33 ± 0.35 | 107 | 7 | |
25 | 22.9 ± 1.18 | 92 | 5 | + + + | 25.08 ± 0.21 | 100 | 1 | |
50 | 50.65 ± 1.13 | 101 | 2 | + + + | 51.1 ± 0.39 | 102 | 1 | |
ZEN | 0 | ND | NC | NC | − − − | ND | NC | NC |
50 | 52.28 ± 2.92 | 105 | 6 | ± ± ± | 50.66 ± 1.20 | 101 | 2 | |
250 | 258.05 ± 8.19 | 103 | 3 | + + + | 257.83 ± 11.11 | 103 | 4 | |
500 | 510.00 ± 6.68 | 102 | 1 | + + + | 513.32 ± 8.17 | 103 | 2 | |
DON | 0 | ND | NC | NC | − − − | ND | NC | NC |
50 | 54.20 ± 2.31 | 108 | 4 | ± ± ± | 55.35 ± 2.56 | 111 | 5 | |
250 | 258.55 ± 3.45 | 103 | 1 | + + + | 261.65 ± 5.34 | 105 | 2 | |
500 | 512.68 ± 4.99 | 103 | 1 | + + + | 501.65 ± 5.13 | 100 | 1 |
Sample | LC-MS (μg/kg) | Microfluidics (μg/kg) | |||||||
---|---|---|---|---|---|---|---|---|---|
AFB1 | ZEN | DON | AFB1 | AFB1-Visual | ZEN | ZEN-Visual | DON | DON-Visual | |
1 * corn | 1.31 | 388.33 | 715.54 | 1.48 | ± a | - c | + | - | + |
2 * corn | 2.44 | 133.11 | 189.15 | 256 | ± | - | + | - | + |
3 * corn | 1.53 | 298.54 | 85.07 | 1.66 | ± | - | + | - | + |
4 * wheat | 52.81 | 634.36 | 2258.37 | - | + b | - | + | - | + |
5 * wheat | 3.62 | 1105.28 | 3125.8 | 4.02 | ± | - | + | - | + |
6 * wheat | 85.31 | 18.36 | 276.81 | - | + | 18.66 | ± | - | + |
7 * brown rice | 2.11 | 33.84 | 71.26 | 2.46 | ± | - | + | 70.55 | ± |
8 * brown rice | 2.32 | 18.42 | 65.54 | 2.64 | ± | 19.16 | + | 66.39 | ± |
Method | Target Analytes | Test Sample | Mechanical System | vLOD | cLOD | Quantitative Device | Reference |
---|---|---|---|---|---|---|---|
Microfluidics | T2/OTA/AFB1/ ZEN | Corn, wheat | Syringe pump | - | 0.12/0.03/1.24/0.58 μg/kg (sample) | Fluorescent strip reader | [21] |
Microfluidics | AFB1/OTA/ DON | Maize, wheat | Magnetic valves | - | 3/100/100 μg/kg (sample) | [22] | |
Microfluidics | AFB1/OTA/ DON | feed | Syringe valves | - | <40/0.1~0.2/<10 ng/mL (buffer) | Smart phone | [23] |
QDs-LFIA | FB1/AFB1/ OTA | Maize, rice, wheat | capillarity | - | 1.58/0.002/0.059 ng/mL (buffer) | ESE-Quant LFR Fluorescence reader | [24] |
TRFMs-LFIA | ZEN/AFB1 | Maize | capillarity | - | 0.07/0.05 ng/mL (buffer) | Homemade portable Fluorescence | [25] |
GNPs-LFIA | FB1/ZEN/ DON | Maize, wheat | capillarity | 60/6/12.5 ng/mL (buffer) | - | - | [26] |
Multicolor-LFIA | ZEN/T-2/AFB1 | Maize, feed | capillarity | 2/30/0.5 ng/mL (buffer) | - | - | [27] |
SPR chip | T-2/HT-2, DON | Wheat, maize | - | - | 12.1/29/31 ng/mL (sample) | SPR reader- | [28] |
Microchip coupled with ELISA and electrochemical | ZEA | Corn | - | - | <1 μg/L | MK3 microplate reader- | [29] |
Microfluidics | AFB1/ZEN/ DON | Corn, wheat, rice | capillarity | 1.5/5.0/10 ng/mL (buffer) | 0.003/0.05/1.09 ng/mL (buffer) | Fluorescent strip reader | present work |
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Wang, X.; Lu, D.; Huang, Q.; Yang, J. Microfluidics-Based Time-Resolved Fluorescence Immunoassay for the On-Site Detection of Aflatoxins B1 Zearalenone and Deoxynivalenol in Cereals. Foods 2022, 11, 1319. https://doi.org/10.3390/foods11091319
Wang X, Lu D, Huang Q, Yang J. Microfluidics-Based Time-Resolved Fluorescence Immunoassay for the On-Site Detection of Aflatoxins B1 Zearalenone and Deoxynivalenol in Cereals. Foods. 2022; 11(9):1319. https://doi.org/10.3390/foods11091319
Chicago/Turabian StyleWang, Xu, Disha Lu, Qingfeng Huang, and Jinyi Yang. 2022. "Microfluidics-Based Time-Resolved Fluorescence Immunoassay for the On-Site Detection of Aflatoxins B1 Zearalenone and Deoxynivalenol in Cereals" Foods 11, no. 9: 1319. https://doi.org/10.3390/foods11091319