1. Biosensor for Detecting Mycotoxins in Grains Sundaram Gunasekaran University of Wisconsin-Madison In collaboration with Senay Simsek North Dakota State University Funding from The Andersons

2. Mycotoxins Toxic, secondary fungal metabolites that occur naturally and unavoidably Enter our food chain directly from the use of mycotoxins-contaminated foods or indirectly from the growth of toxigenic fungi on food MycotoxinFDA Action Level (ppm) Aflatoxin0.0005-0.3 Fumonisin2-100 Zearalenone1-3 Vomitoxin (DON)1-30 Cause acute or chronic diseases to human and animals; reduce feed quality. Co-contaminants are common (e.g., aflatoxin and fumonisin in corn, DON and ochratoxin in wheat) (FDA, 2011)

3. Current Methods Thin layer chromatography (TCL) Liquid chromatography (HPLC) Gas chromatography-mass spectrometry (GC-MS) Immunoassay (ELISA) AflatoxinFoodMethodDetection limit (ppb) B1, B2, G1B1, B2, G1 Maize (Corn)ELISA20 B1, B2, G1,G2B1, B2, G1,G2 MaizeTLC5 B1, B2, G1,G2B1, B2, G1,G2 Maize, peanut butter HPLC5 B1B1 Animal feedTLC/fluorescence4 Expensive equipment, skilled operator, extensive sample pre- treatment, time- consuming

4. Biosensors Devices that use biological components to react or bind with a target molecule and transduce this event into a detectable signal -- antibody (Ab)-antigen (Ag) binding Highly specific molecular recognition property of antigens by antibodies (i.e., immunosensors) leads to greatly selective and sensitive assays Can incorporate of nanotechnologies to greatly enhance analytical performance

5. Electrochemical Techniques Cyclic Voltammetry (CV) Monitor redox reaction of chemical species on working electrode Differential Pulsed Voltammetry (DPV) Effect of charging current is minimized, so high sensitivity is achieved. Capable of detecting trace amount of chemicals Counter electrode(CE) Reference electrode ( RE ) Working electrode(WE) Screen-printed electrode (SPE) Modify

6. Hand-held Portability

7. Incorporation of Nanomaterials Carbon nanotubes Large surface area Superior electrical and thermal conductivity Chemical inertness Strong mechanical strength CNTs could be functionalized with various functional groups, giving the potential for antibody, protein immubolization

8. Electrochemical Immunosensing Abbreviations: IC: indirect competitive assay, AMP: amperometry, DC: direct competitive assay, AuNPs: gold nanoparticles, NC: no competitive, PB: Prussian blue, DPV: differential pulse voltammetry, EIS: electrochemical impedance spectroscopy, ELIME-assay: enzyme-linked immune-magnetic electrochemical assay, GCE: glassy carbon electrode, AChE: achetylcholinesterase enzyme, PANi-PSSA: polyaniline- polystyrenesulfonicacid

9. Motivation Due to widespread co-occurrence of multiple toxins in food matrices and their possible additive or synergistic adverse effects, we need a system to sensitively and simultaneously detect multiple toxins. Currently, such multi-toxin detection methods are not available on a rapid, easy-to-use and portable biosensor platform.

10. Research Design Insulating effect of antibody-toxin complex will reduce DPV signals, which is used for quantifying toxin amount Start with a single-channel sensor for detecting aflatoxin B 1 and fumonisin, and then proceed to the multi-channel sensor fabrication Increases electrical conductivity and prepare for antibody immobilization Specifically recognize toxins Modify SPE with CNT-COOH Modify SPE with CNT-COOH Immobilize antibody Measure DPV Determine toxin level

11. Immuno-electrode Preparation

12. Effect of PDDA/CNT-COOH Layers CV (left) and DPV (right) after 1 to 5 (i to v) layers deposited on electrode in PBS (10 mM, pH 7.4) solution containing 1mM [Fe(CN) 6 ] 3-/4- Peak current significantly increases after 3 layers, which suggests a great improvement of electrode conductivity. Adding 4 th and 5 th layer did not help much. (iii)

13. SEM of (PDDA/CNT-COOH) 3 Modified Electrode Fairly uniform and high surface coverage of (PDDA/CNT-COOH) CNT-COOH

14. Characterizing Functionalized Electrode CV (left) and DPV (right) of (i) bare SPE, (ii) 3 layers of (PDDA/CNT-COOH) deposited on electrode, (iii) (PDDA/CNT-COOH)3-aAFB1 immunoelectrode in PBS (10 mM, pH 7.4) solution containing 1 mM [Fe(CN)6] 3-/4-

15. Detection Scheme

16. Calibration Curves DPV signals (left) and calibration curves (right) for different AFB 1 concentration ranges with optimal antibody loading for each range.

17. Detection of Fumonisin Detection of Fumonisin Ab 5 ug/mL Ab 10 ug/mL 17

18. Multi-channel Sensing Antibodies modified working electrodes Four different toxins Four detection channels (working electrodes) Simultaneous detection of different mycotoxins such as aflatoxin B 1, ochratoxin A, deoxynivalenol (DON), and fumonisin

19. Thank you!