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

 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

• Cause acute or chronic diseases to Mycotoxin FDA Action Level

(ppm)
human and animals; reduce feed
Aflatoxin 0.0005-0.3
quality.
Fumonisin 2-100
• Co-contaminants are common (e.g.,
Zearalenone 1-3
aflatoxin and fumonisin in corn, DON
Vomitoxin (DON) 1-30
and ochratoxin in wheat)

(FDA, 2011)
 Current Methods
•Thin layer chromatography (TCL)
•Liquid chromatography (HPLC)
•Gas chromatography-mass spectrometry (GC-MS)
•Immunoassay (ELISA)

Aflatoxin Food Method Detection

limit (ppb)
B 1, B 2, G 1 Maize (Corn) ELISA 20
Expensive
B1, B2, G1 ,G2 Maize TLC 5 equipment, skilled
operator, extensive
sample pre-
B1, B2, G1 ,G2 Maize, peanut HPLC 5
butter treatment, time-
consuming
B1 Animal feed TLC/fluorescence 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
 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)
Modify
Reference
electrode(RE)

Working
electrode(WE)

Screen-printed electrode (SPE)

Hand-held Portability
 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
 Electrochemical Immunosensing
Method Effective range (ng/mL) LOD (ng/mL)
IC/IPA on 96-well SPEs
0.05–2 0.03
IC/DPV on an 8x ELIME-array 0.8-9 0.6
IC/AMP on SPEs 0.1–10 0.09
DC/AMP on SPEs 0.15–0.25 0.15
IC/DPV in GCE doped with AuNPs 0.6–2.4 0.07
NC/AMP 0.1–12 0.05
AChE inhibition/AMP on SPEs modified with PB 10 to 60 2
Enzyme Biosensor (AFOx)/AMP using CNTs 1-225 0.5
DC/EIS on silica-gel+ionic liquid films 0.1–10 0.001
DC/EIS on electropolymerized PANi-PSSA films 0.1-6 0.1

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
 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.
 Research Design
Start with a single-channel sensor for detecting aflatoxin B1 and
fumonisin, and then proceed to the multi-channel sensor fabrication

Modify SPE Immobilize Determine

Measure DPV
with CNT-COOH antibody toxin level

Increases electrical
Specifically Insulating effect of antibody-toxin
conductivity and
recognize complex will reduce DPV signals, which
prepare for antibody
toxins is used for quantifying toxin amount
immobilization
Immuno-electrode Preparation
 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-

(iii)

• Peak current significantly increases after 3 layers, which suggests a great

improvement of electrode conductivity. Adding 4th and 5th layer did not help much.
SEM of (PDDA/CNT-COOH)3 Modified Electrode

CNT-COOH

• Fairly uniform and high surface coverage of (PDDA/CNT-COOH)

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-
Detection Scheme
 Calibration
• Curves
DPV signals (left) and calibration
curves (right) for different AFB1
concentration ranges with optimal
antibody loading for each range.
 Detection of Fumonisin
Ab 5 ug/mL FUM 2-10 PPM FUM 10-50 PPM Ab 10 ug/mL
16 9
14 8 f(x) = − 0.1749 x + 10.476
7 R² = 0.993531491755055
12 f(x) = − 0.555 x + 14.64
CURRENT /uA

CURRENT /uA
10 R² = 0.991502100333156 6
8 5
6 4
4 3
2 2
1
0
1 2 3 4 5 6 7 8 9 10 0
5 10 15 20 25 30 35 40 45 50
FUM CONCENTRATION(ug/ml)
FUM CONCENTRATION(ug/ml)
FUM 0-0.8 PPM
16 FUM 0-100 PPM
7
6 14
12 f(x) = − 0.119785714285714 x + 13.587619047619
CURRENT/uA

5 f(x) = − 6.355 x + 5.83533333333333

CURRENT /uA
R² = 0.990319061644188
4 R² = 0.903565733164457 10
3 8
2 6
1 4
0 2
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
0
FUM CONCENTRATION (ug/ml) 0 10 20 30 40 50 60 70 80 90 100
FUM CONCENTRATION(ug/ml)
17
 Multi-channel Sensing

• Simultaneous detection
of different mycotoxins
such as aflatoxin B1,
ochratoxin A,
deoxynivalenol (DON),
and fumonisin

Four detection channels

(working electrodes)

Antibodies modified
working electrodes

Four different toxins

Thank you!