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)