Transcript Document

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
human and animals; reduce feed
quality.
FDA Action Level
(ppm)
Aflatoxin
0.0005-0.3
Fumonisin
2-100
Zearalenone
1-3
Vomitoxin (DON)
1-30
• Co-contaminants are common (e.g.,
aflatoxin and fumonisin in corn, DON
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, G1
Maize (Corn)
ELISA
20
B1, B2, G1 ,G2
Maize
TLC
5
B1, B2, G1 ,G2
Maize, peanut
butter
HPLC
5
B1
Animal feed
TLC/fluorescence
4
Expensive
equipment, skilled
operator, extensive
sample pretreatment, timeconsuming
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)
Reference
electrode(RE)
Modify
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
IC/IPA on 96-well SPEs
IC/DPV on an 8x ELIME-array
IC/AMP on SPEs
DC/AMP on SPEs
IC/DPV in GCE doped with AuNPs
NC/AMP
AChE inhibition/AMP on SPEs modified with PB
Enzyme Biosensor (AFOx)/AMP using CNTs
DC/EIS on silica-gel+ionic liquid films
DC/EIS on electropolymerized PANi-PSSA films
Effective range (ng/mL)
LOD (ng/mL)
0.05–2
0.8-9
0.1–10
0.15–0.25
0.6–2.4
0.1–12
10 to 60
1-225
0.1–10
0.1-6
0.03
0.6
0.09
0.15
0.07
0.05
2
0.5
0.001
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: polyanilinepolystyrenesulfonicacid
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
with CNT-COOH
Increases electrical
conductivity and
prepare for antibody
immobilization
Immobilize
antibody
Specifically
recognize
toxins
Measure DPV
Determine
toxin level
Insulating effect of antibody-toxin
complex will reduce DPV signals, which
is used for quantifying toxin amount
Immuno-electrode Preparation
Bare electrode
NH
NH
4-ABA
-
-
-
C O
-
O C
PDDA
O
LBL
CNT-COOH
C O
O C
O
EDC/NHS
coupling
N
O
O
OH
HO
N
O
C O
O C
O
An body
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-/48.E-06
(iv)
(v)
Current/A
4.E-06
0.E+00
(i)
-4.E-06
(ii)
-8.E-06
(v)
(iii)
-1.E-05
-2.E-05
-0.4
-0.2
0
0.2
Poten al/V
0.4
0.6
0.8
• 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
An body
modifica on
No toxin
incuba on
DPV detec on S0
a
c
Aflatoxin B1
An body
PBS buffer
Func onalized CNT
a
Carbon counter electrode
b
Carbon working electrode
c
Silver reference electrode
incuba on
b
S0
Toxins
ec o n S t
DPV det
St
0%
6.E'06%
4
4.E'06%
2
0.09%
ppb%
2.E'06%
0
0.E+00%
'0.3% '0.2% '0.1%
-2
0%
0.1%
Poten, al/V)
0.2%
0.3%
Current/A)
0.02
0.04
10
0
0.06
0.08
0.1
y = -5.03x + 6.245
R² = 0.95741
8
6.E'06%
6
4.E'06%
4
0.9 ppb
2.E'06%
0.E+00%
'0.4%
2
0
'0.2%
0%
Poten, al/V)
0.2%
0
0.4%
0.2
0.4
10
8.E'06%
Current/A)
0
0.4%
8.E'06%
6.E'06%
0
0.6
0.8
1
y = -0.5533x + 6.167
R² = 0.9512
8
6
4.E'06%
4
9 ppb
2.E'06%
0.E+00%
'0.3% '0.2% '0.1%
2
0
0%
0.1%
0.2%
0.3%
0
0.4%
2
4
6
8
10
Poten, al/V)
8.E'06%
6
y = -0.0354x + 3.6168
R² = 0.85114
6.E'06%
Current/A)
• DPV signals (left) and calibration
curves (right) for different AFB1
concentration ranges with optimal
antibody loading for each range.
y = -58.184x + 4.9427
R² = 0.80682
6
Current/A)
Calibration Curves
8
8.E'06%
0
4
90 ppb
2
4.E'06%
2.E'06%
0.E+00%
'0.3% '0.2% '0.1%
0
0%
0.1%
Poten, al/V)
0.2%
0.3%
0.4%
0
20
40
60
80
100
Detection of Fumonisin
16
14
12
10
8
6
4
2
0
FUM 10-50 PPM
FUM 2-10 PPM
CURRENT /uA
CURRENT /uA
Ab 5 ug/mL
y = -0.555x + 14.64
R² = 0.9915
0
2
4
6
8
FUM CONCENTRATION(ug/ml)
10
10
9
8
7
6
5
4
3
2
1
0
Ab 10 ug/mL
y = -0.1749x + 10.476
R² = 0.9935
0
10
20
30
40
FUM CONCENTRATION(ug/ml)
50
9
8
7
6
5
4
3
2
1
0
CURRENT /uA
CURRENT/uA
FUM 0-0.8 PPM
y = -6.355x + 5.8353
R² = 0.9036
0
0.2
0.4
0.6
0.8
FUM CONCENTRATION (ug/ml)
1
18
16
14
12
10
8
6
4
2
0
FUM 0-100 PPM
y = -0.1198x + 13.588
R² = 0.9903
0
20
40
60
80
FUM CONCENTRATION(ug/ml)
100
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
DPV Signals
S02
St2
S01
St1
S03
St3
S04
St4
Four different toxins
Channel 1
Channel 2
Channel 3
Channel 4
Thank you!