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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
School of Materials Engineering
Purdue University
USDA Purdue MeetingUSDA –Purdue MeetingOctober 27-28, 2009
Contents
1. Introduction
2. Amperometric Pesticide Biosensors
2 1 AChE ZnO sol2-1. AChE-ZnO-sol
2-2. Biomagnetic glassesg g
3. Summary
Hybrid Inorganic-Biological Materialsfor Enzymatic Sensorso y at c Se so s
Objective
Investigate novel material platforms for
Objective
g pthe design of stable pesticide biosensors.
InorganicBiological gmaterials
BiologicalMaterials
Hybrid composite materials for biosensor applicationHybrid composite materials for biosensor application
Hybrid Inorganic-Biological Materialsfor Enzymatic Sensors
AcetylcholinesterasePesticides
o y at c Se so s
very toxic, used in agriculture
y(AChE)
agricultureContaminate several food
products
inhibited by organophosphoruspesticides
• 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.
I I% inhibition = X 100I0 - II0
Detection limit ~10-7 M of Paraoxon
Part 2. Biomagnetic glasses
BiomagneticBiomagnetic glasses glasses -- BiosensorBiosensor
Inorganic materials Biomaterials
InorganicGlasses
EnzymeGlasses
y
Magnetic nano
G6PDH
nano-particles
Silica shell
AChEHRP
PesticideBiosensorsLL i ii i Biosensors-- LongLong--operation timeoperation time
-- RenewableRenewable
Hi h f Hi h f -- High surface areaHigh surface area
PURDUE MATERIALS PURDUE MATERIALS
Biomagnetic glasses: preparation, characterization and biosensor applicationscharacterization and biosensor applications
IGEPAL CO 520IGEPAL CO 520
TEOSTEOS
Fe O NPs in Hexane Fe3O4 NPs in DI water Fe3O4/silica shell
SDSSDS
Fe3O4 NPs in Hexane Fe3O4 NPs in DI water Fe3O4/silica shell
EnzymeEnzyme
CellCell
Enzyme immobilized
Biomagnetic glasses
Fe3O4/silica shellSPE
Experimental (Fe3O4 nanoparticle)
• Synthesis method of iron oxide nanoparticle
Iron-oleatecomplex
Ironchloride
CondenserIron-oleatecomplex
Thermo-
complex
Tree neckFlask
couple
Flask
Heating mantle
[Ref] J. N. Park, and T. H. Hyeon, Nat. Mat. 3, 891 (2004)
Experimental (Silica shell)
Make Hydrophilic Fe3O4 from Hydrophobic
Add NH4OH (Catalyst)
Add TEOS (Precursor)
Stir the solution for <48 hrs
[Ref.] D. K. Yi et al., JACS, 127, 4990 (2005)
IGEPAL CO-520Hexane
Precipitate using Methanol
HexaneFe3O4 nanoparticle
Ammonia hydroxideTEOS(RT)
Wash by Ethanol
Mechanical stirrer
Disperse Silica coated Fe3O4 in DI water
Results (Fe3O4 nanoparticle)
• TEM images of Fe3O4 NPs
( ) (b)(a) (b)
20 nm 20 nm20 nm 20 nm
13 nm Fe3O4 NPsIn hexane
13 nm Fe3O4 NPsIn DI waterIn hexane In DI water
Surface modification by SDS
* SDS: Sodium dodecyl sulfate
Results (Fe3O4 nanoparticle)
• XRD pattern of Fe3O4 nanoparticle
(311)Magnetic
property of Fe3O4 NPs !
(220)(400)
(511)(440)
( )(422)(222)
XRD pattern is consist with Fe3O4 phase!
Results (Silica shell)
TEOS M
• TEM of Fe3O4/m-silica nanocomposites
TEOS M
Fe3O4
Sili
TEOS = 10 mM TEOS = 20 mM
Silica shell
Fe3O4 core: 13 nmFe3O4 core: 13 nmSilica shell: 20 nm
Silica shell: 5 nm23 nm
53 nm
Results (Silica shell)• SEM of Fe3O4/m-silica nanocomposites
50 nm
100 nm
The surface of silica shells is porous!
Biomagnetic glasses - Biosensor
• SEM image of SPE • Calibration curve of SPEgwith biomagnetic glasses
Calibration curve of SPE
500 nm500 nm
Electrostatic interaction !!
• Measurement condition0.01 M PBS (pH 7.4)Applied potential 235 mV vs. Ag/AgClElectrostatic interaction !! Applied potential 235 mV vs. Ag/AgClATChCl: 1.0 mMAChE: 3.53 mU
Biomagnetic glasses - Biosensor
• Immobilized AChE vs. free AChE
• The SPE with biomagnetic glasses shows higher current response.
• Biomagnetic glasses shows higher stability.
Fe O /m silica nanocomposite is great to immobilize AChE enzymeFe3O4/m-silica nanocomposite is great to immobilize AChE enzyme.
The biosensor with biomagenteic glasses is sensitive and stable.
Biomagnetic glasses - Biosensor
• Inhibition curve• Calibration curve of SPEs
100
% inhibition = X 100I0 - I
I
60
70
80
90
bi
tion
%
% inhibition X 100I0
30
40
50
60
Inhi
b
1E-9 1E-8 1E-7 1E-6 1E-5
30
Concentration of Paraoxon (M)
• 7 SPEs with biomagnetic glasses were prepared to measure the inhibition curve.
• SPEs showed variation less than 10 % at the calibration curve.
• The detection limit is 4.6 X 10 -9 M of paraoxon.
Summary1. Synthesis of Biomagnetic glasses
2. Fabrication and Testing of biosensor
20 nm 100 nm
• The detection limit is80
90
100
% The detection limit is4.6 X 10 -9 M of paraoxon.
40
50
60
70
In
hibi
tion
1E-9 1E-8 1E-7 1E-6 1E-5
30
Concentration of Paraoxon (M)