Extraction, Concentration and Detection of Toxins
Extraction, Concentration and Detection of Toxins
Extraction, Concentration and Detection of Toxins
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<strong>Extraction</strong>, <strong>Concentration</strong> <strong>and</strong><br />
<strong>Detection</strong> <strong>of</strong> <strong>Toxins</strong><br />
Mansel W. Griffiths, Keith Warriner, Luba Brovko<br />
(Food Science, University <strong>of</strong> Guelph)<br />
Subrrayl Reddy (School <strong>of</strong> Biomedical <strong>and</strong> Molecular Sciences,<br />
University <strong>of</strong> Surrey)
Objectives<br />
• Fabricate hydrogel imprinted polymers for concentration<br />
<strong>of</strong> Staphylococcus aureus enterotoxin B<br />
• Construct impedimetric immuno-sensors to detect<br />
enterotoxin B<br />
• Develop a pathogenicity biosensor
Staphylococcal Enterotoxin B<br />
• Bacterial toxin<br />
– Staphylococcus aureus<br />
• Easily soluble in water<br />
• Very resistant to temperature fluctuations<br />
– Withst<strong>and</strong>s boiling for several minutes<br />
– Freeze dried remains active for one year
Enterotoxin B<br />
• Mw 28 kDa<br />
• 240 Amino Acids<br />
• LD 50 30ng/Kg<br />
• <strong>Detection</strong> limits 1ng
Development <strong>of</strong> Hydrogel-based Molecularly<br />
Imprinted Polymers (HydroMIPs) for Protein<br />
Recognition<br />
Dr Sub Reddy <strong>and</strong> Quan Phan<br />
School <strong>of</strong> Biomedical <strong>and</strong> Molecular Sciences<br />
University <strong>of</strong> Surrey, UK
Protein Imprinted Polymers<br />
Polymerisation<br />
Electrostatic <strong>and</strong> hydrogen<br />
bonding interactions between<br />
monomer <strong>and</strong> protein<br />
Loose network<br />
Protein imprinted<br />
polymer<br />
Template<br />
elution<br />
Rebinding
Molecularly Imprinted Polymer Hydrogels:<br />
‘Nano-play-dough!’<br />
TEMED<br />
Polyacrylamide
Retention <strong>of</strong> enterotoxin by polymer<br />
10% AcOH SDS washes after 1mg/40µl enterotoxin B reloaded into<br />
enterotoxin B MIP (10% cross linking)
Points to consider<br />
• Effect <strong>of</strong> crosslinking density on SEB imprinting<br />
• Effect <strong>of</strong> pH <strong>of</strong> imprinting<br />
• Effect <strong>of</strong> co-polymerisation using other functional<br />
monomers (hydroxy-acrylamide;<br />
hydroxyethylmethacrylate (HEMA))<br />
• Optimised method <strong>of</strong> eluting SEB<br />
• Method <strong>of</strong> quantifying eluted SEB from HydroMIP<br />
matrix.
Impedimetric Immuno-assay based on<br />
Modified Conducting Polymer Electrodes<br />
Keith Warriner<br />
University <strong>of</strong> Guelph
Conducting Polymer Electrodes<br />
• Free st<strong>and</strong>ing conducting polymer films<br />
• Low cost (
What are conducting polymers?<br />
Polyconjugated polymers with<br />
high electrical conductivities, low<br />
ionization potentials, high<br />
electronic affinities<br />
Applications<br />
Stealth (Radar Absorbing)<br />
LED display<br />
Fuel cell<br />
Organic computers<br />
Sensors
Formation <strong>of</strong> conducting polymer<br />
electrode<br />
Preconditioning:<br />
Pyrrole(0.5M) & SDS(1mM)0<br />
Microporous polycarbonate membrane<br />
(45mm ,10μm pore size)<br />
Polymerization:<br />
FeCl 3<br />
(0.4M) & lactic acid (0.1M)<br />
Polymer electrode
Electrochemical characterization <strong>of</strong><br />
polypyrrole electrodes<br />
Cyclic voltammetry<br />
Impedance<br />
spectroscopy<br />
H<strong>and</strong>held FRA<br />
Solartron Impedance Analyser
Reusable Electrode assembly<br />
Reaction well<br />
Polymer film<br />
Top plate with<br />
reaction well<br />
(inside view)<br />
Copper connections
Principle<br />
Antibody<br />
Antigen<br />
BSA<br />
Silane<br />
Polypyrrole<br />
Paramagnetic bead
Sensor Response to Salmonella<br />
0<br />
-500<br />
Z"<br />
-1000<br />
-1500<br />
-2000<br />
-2500<br />
Background<br />
Sal Extract<br />
-3000<br />
1 10 100 1000 10000 100000<br />
Frequency
<strong>Detection</strong> Strategy<br />
Silane<br />
Polypyrrole<br />
Antigen<br />
BSA blocking agent<br />
Antibody<br />
Paramagnetic bead
Myoglobin <strong>Detection</strong><br />
• Myoglobin model for SEB<br />
• 100ng Myoglobin<br />
• BSA blocking agent<br />
• Anti-myoglobin dynabeads<br />
-1 00 00<br />
-7500<br />
-5000<br />
Z''<br />
Myoglobin<br />
BSA blocking agent<br />
Dynabeads 7 anti -myogl obin<br />
Dynabeads<br />
BSA<br />
Myoglobin<br />
-2500<br />
0<br />
0 2500 5000 7500 10000<br />
Z'
Future Work<br />
• Further optimize myoglobin detection protocol.<br />
• Repeat experiments with SEB<br />
• Integrate hydrogel MIP with detection platform
Cell-based Biosensors<br />
Mansel W. Griffiths, Luba Brovko, Nidham Jamaldeen<br />
University <strong>of</strong> Guelph
Principle <strong>of</strong> toxicity biosensor<br />
[ATP] [Ca 2+ ]
Expression <strong>of</strong> luciferase <strong>and</strong> aequorin genes in<br />
HeLa cells in the presence <strong>of</strong> human complement<br />
component C9<br />
a) luciferase<br />
b) aequorin
Other luminescent toxicity biosensors
The future looks bright
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