Biosensors for Pesticide Biosensors for Pesticide Detection in Food Detection in Food Yu-Ho Won, Ravi Sinha, Silvana Yu Ho Won , Ravi Sinha, Silvana Andreescu, Lia Stanciu School of Materials Engineering Purdue University USDA Purdue Meeting USDA –Purdue Meeting October 27-28, 2009 Contents 1. Introduction 2. Amperometric Pesticide Biosensors 2 1 AChE ZnO sol 2-1. AChE-ZnO-sol 2-2. Biomagnetic glasses 3. Summary Hybrid Inorganic-Biological Materials for Enzymatic Sensors Objective Investigate novel material platforms for Objective the design of stable pesticide biosensors. Inorganic Biological materials Biological Materials Hybrid composite materials for biosensor application Hybrid composite materials for biosensor application Hybrid Inorganic-Biological Materials for Enzymatic Sensors Acetylcholinesterase Pesticides very toxic, used in agriculture (AChE) agriculture Contaminate several food products inhibited by organophosphorus pesticides • low solubility in water • extraction and concentration in organic media might be needed for detection in potential to the detection of pesticides with media might be needed for detection in food samples • higher toxicity of their oxidized forms application in food industry Portable Enzyme Sensor for on Portable Enzyme Sensor for on-site Detection of site Detection of Portable Enzyme Sensor for on Portable Enzyme Sensor for on site Detection of site Detection of Pesticides in Food Products Pesticides in Food Products Amperometric Biosensor Enzyme immobilized nanocomposites •low cost / mass production i l / di bl •single use / disposable •suitable for portable device • simple handling • no skilled technicians id d i Pesticides Application in food samples •rapid detection Amperometric Biosensor Design Inhibited by pesticides pesticides + R-COO - +H + Cl - (CH 3 ) 3 N + -CH 2 -CH 2 -SH (CH 3 ) 3 N + -CH 2 -CH 2 -S-CO-CH 3 Cl - H O AChE AChE thiocholine acetylthiocholine H 2 O Detection method : amperometric determination of thiocholine, produced by the enzymatic hydrolysis of acetylthiocholine substrate: + 2H + + 2e - + 2Cl - vs. Ag/AgCl (CH 2 ) 2 N + (CH 3 ) 3 (CH 2 ) 2 N + (CH 3 ) 3 S S Cl - (CH 3 ) 3 N + -CH 2 -CH 2 -SH thiocholine 2 Ag/AgCl 2 2 3 3 dithio-bis-choline
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Biosensors for Pesticide Biosensors for Pesticide Detection in FoodDetection in Food
Yu-Ho Won, Ravi Sinha, SilvanaYu Ho Won, Ravi Sinha, SilvanaAndreescu, Lia Stanciu
• low solubility in water• extraction and concentration in organic media might be needed for detection in
potential to the detection of pesticides with
li i i f d i dmedia might be needed for detection in
food samples• higher toxicity of their oxidized forms
application in food industry
Portable Enzyme Sensor for onPortable Enzyme Sensor for on--site Detection of site Detection of Portable Enzyme Sensor for onPortable Enzyme Sensor for on site Detection of site Detection of Pesticides in Food ProductsPesticides in Food Products
Amperometric Biosensor
Enzyme immobilized
nanocomposites
•low cost / mass productioni l / di bl•single use / disposable
•suitable for portable device• simple handling• no skilled technicians
id d iPesticides Application in
food samples
•rapid detection
Amperometric Biosensor Design
Inhibited by pesticidespesticides
+ R-COO- + H+Cl-
(CH3)3N+-CH2-CH2-SH(CH3)3N+-CH2-CH2-S-CO-CH3
Cl-
H OAChEAChE
thiocholineacetylthiocholineH2O
Detection method: amperometric determination of thiocholine, produced by theenzymatic hydrolysis of acetylthiocholine substrate:
+ 2H+ + 2e- + 2Cl-vs.
Ag/AgCl (CH 2)2N+(CH 3)3
(CH 2)2N+(CH 3)3S
SCl-
(CH3)3N+-CH2-CH2-SHthiocholine
2
Ag/AgCl 2 2 3 3dithio-bis-choline
Research directions
1st STEP 2nd STEPDevelopment of inorganic
materials for enzyme immobilization in which E i bili i i immobilization in which
enzymes remains fully stable during
manufacturing, storage
Enzyme immobilization into the inorganic support
manufacturing, storage and use
3rd STEP
Fabrication of biosensor&
Detection of pesticides Detection of pesticides in the food sample
Part 1. AChE-ZnO
Immobilization of AChE in ZnO
• Preparation of ZnO sol for AChE immobilization
• ZnO is Biocompatible.
• Screen printed electrodes are prepared with AChE-ZnO.
• Electrostatic interaction between ZnO and AChE- ZnO Isoelectric point: 9.5
AChE i l t i i t - AChE isoelectric point: 5.5
at pH 7.4, ZnO is positively charged and AChE is negatively g g ycharged
AChE-ZnO Biosensor
Calibration curves (A) and sensor stability (B) of ZnO-AChE biosensors prepared with various ratios of ZnO:DI water.
AChE-ZnO Biosensor
Inhibition curves of ZnO-AChE biosensor by paraoxon after 10, 20 and 30 min.incubation (logarithmic scale) Measurement conditions: 0 01 M PBS appliedincubation (logarithmic scale). Measurement conditions: 0.01 M PBS, appliedpotential: 235 mV vs Ag/AgCl reference electrode, 1 mM ATChCl substrate. Thecurrent response is measured before (I0) and after (I) pesticide incubation.