NANOPARTICLES IN FOOD BIOSENSING

J.M. Pingarrón* L. Agüí and P. Yáñez-SedeñoDepartment of Analytical Chemistry. Faculty of Chemistry

University Complutense of Madrid28040-Madrid SPAIN

pingarro@quim.ucm.es

NANOJASP’2010

Barcelona, December 2010

Preparation of nanostructured electrode surfacesPreparation of nanostructured electrode surfacesPreparation of nanostructured electrode surfacesPreparation of nanostructured electrode surfaces

Advances in sensors technology

Development of several (bio)assay-transductor strategies

Applications of nanotechnology

Research line combining:Research line combining:

- Wide range of approaches- Use or not of biological systems

Products, processes and systems operating in nanometric magnitude

Improved charge transfer reactions

Electrocatalytic ability: lower detection potential

Antifouling capability

SensitivitySensitivity

SelectivitySelectivity

RepeatabilityRepeatability

AdvantagesAdvantages

Nanostructured electrode surfacesNanostructured electrode surfacesNanostructured electrode surfacesNanostructured electrode surfaces

Electrochim. Acta., 53 (2008) 5848

ELECTROCHEMICAL BIOSENSORS BASED ON GOLD NANOPARTICLE-MODIFIED ELECTRODES

Gold nanoparticlesGold nanoparticles

Ability to provide a stable surface for biomolecules immobilization retaining their biological activity

Ability to provide a stable surface for biomolecules immobilization retaining their biological activity Permit direct electron-transfer between redox proteins and bulk electrode materials no need for electron-transfer mediators

High surface-to-volume ratio

High surface energy

Ability to decrease the distance between proteins and metal particles

Functioning as electron- conducting pathways between the prosthetic groups and the electrode surface

Permit direct electron-transfer between redox proteins and bulk electrode materials no need for electron-transfer mediators

High surface-to-volume ratio

High surface energy

Ability to decrease the distance between proteins and metal particles

Functioning as electron- conducting pathways between the prosthetic groups and the electrode surface

Au

Au

AuUseful interfaces for electrocatalysis of redox processes of H2O2 or NADHUseful interfaces for electrocatalysis of redox processes of H2O2 or NADH

ELECTROCHEMICAL BIOSENSORS FOR FOOD ANALYSIS

DEVELOPMENT AND INNOVATIONDEVELOPMENT AND INNOVATION

FOOD SAFETYFOOD SAFETY FOOD QUALITYFOOD QUALITY

EFFICIENT TRACEABILITY SYSTEMSEFFICIENT TRACEABILITY SYSTEMS

Development of detection, analysis and diagnosis Development of detection, analysis and diagnosis

methodsmethods

RapidRapid

SensitiveSensitive

Automated screening Automated screening

AMPEROMETRIC BIOSENSOR FOR HYPOXANTHINE BASED ON IMMOBILIZED XOD ON NANOCRYSTAL GOLD-CARBON PASTE

ELECTRODES

• Hypoxanthine (Hx) is formed as a product of nucleotide Hypoxanthine (Hx) is formed as a product of nucleotide catabolism during the degradation processes in foodstuffs of catabolism during the degradation processes in foodstuffs of animal origin.animal origin.• Hx is accumulated mostly in the animal muscle and its levels Hx is accumulated mostly in the animal muscle and its levels are used as an index of fish and meat freshness in the food are used as an index of fish and meat freshness in the food industryindustry

XODHx + O2 → X + H2O2

XODX + O2 → Uric acid + H2O2

Gold nanoparticle preparation: Electrodeposition from a HAuCl4 solution on the bulk electrode material

Determination of hypoxanthine based on the enzyme reaction catalyzed by XOD

LOD at 0.00 V: 2.2x10LOD at 0.00 V: 2.2x10-7-7 mol L mol L-1-1

KKmmapp app = 18x10= 18x10-6-6 mol L mol L-1-1

Useful lifetime = at least 15 daysUseful lifetime = at least 15 days

SEM of a GA-BSA-XOD-nAu-CPE biosensor

Determination of hypoxanthine in sardines and chicken meat using the GA-BSA-XOD-nAu-Determination of hypoxanthine in sardines and chicken meat using the GA-BSA-XOD-nAu-CPE biosensorCPE biosensor

Sample

Non-spiked 1

2

3

Spiked 1

3

2

Sardines

Added (mg/100g) Found (mg/100g) Recovery (%)

Chicken

Added (mg/100g) Found (mg/100g) Recovery (%)

- 145 -

- 138 -- 152 -

- 225 105

69.6 213 103

- 210 95

- 91.7 -

- 85.0 -- 94.7 -

- 157.2 103

60.7 145.1 99

- 160.2 103

Mean recoveries (Mean recoveries ( = 0.05): = 0.05): 101 101 ± 8 % sardines± 8 % sardines 102±3% chicken meat102±3% chicken meat

Sens. Actuators B. 113 (2006) 272

AMPEROMETRIC BIOSENSOR FOR HYPOXANTHINE BASED ON IMMOBILIZED XOD ON NANOCRYSTAL GOLD-CARBON PASTE

ELECTRODES

Bienzyme amperometric biosensor using gold nanoparticles-modified electrodes

for the determination of inulin in foods

Anal. Biochem., 375 (2008) 345-353

(C6 H10 O5 )n (n=35)

INULIN

Prebiotic ingredient added to functional foodsPrebiotic ingredient added to functional foods

Vegetal origin: chicory root, artichoke

O

H

HO

H

HO

H

O

OHHH

OH

CH2

OH

OH

OHO

O

CH2OH

CH2OH

OH

OH

O

n

DETERMINATION METHODS

HPLC UV, RI, ED

1st enzymebiosensor

1st enzymebiosensor

This work

INULIN

Determination of interest in:

- MONITORING OF PROCESSES- inuline extraction- fructose production

- QUALITY CONTROL - diethetic and children’s foods- component of dietary fiber

FOOD INDUSTRY

- ECONOMIC AND LEGISLATIVE- added value for functional foods - ingredients establish prices

inherent specificity

simplicity

rapidity

real time analysis

Biosensor advantagesBiosensor advantages

AuEAuE CystCyst AuAucolcol TTFTTF FDHFDH InulinaseInulinase

BIENZYME BIOSENSOR FOR INULIN

2e2e

PQQPQQ

PQQHPQQH22

FRUCTOSEFRUCTOSE

5-CETO-D-FRUCTOSE5-CETO-D-FRUCTOSE

2TTF2TTF

2TTF2TTF++

INULININULIN

Redox mediator

E = +0.2 VPBS 0.05 M, pH 4.5

Gold nanoparticle preparation: By adding sodium citrate to a boiling HAuCl4 By adding sodium citrate to a boiling HAuCl4 aqueous solution HAuCl4/sodium citrate aqueous solution HAuCl4/sodium citrate Particle size Particle size

Stability

More than 5 monthsStorage conditions:

0.05 M phosphate buffer, pH 4.5, a 4ºC

0 20 40 60 80 100 120 140 1600.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

-3s

+3s

i, A

time, days

BIENZYME BIOSENSOR FOR INULIN

20 nA

100 s

dextrose

inulin additions

glucose lactose maltose saccharose

[INTERFERENT] / [INULIN] = 1[INTERFERENT] / [INULIN] = 1

Interferences

10% Erel

BIENZYME BIOSENSOR FOR INULIN

Sample

Sample preparation by size exclusion SPE

2 μA

t, min

Bio-Gel P-6 (Bio-Rad)

fructosefructose

inulininulin

BIENZYME BIOSENSOR FOR INULIN

Chicory powder (18.5% inulin)19.5 ± 0.4%, RSD = 2%, n = 6

Prebiotic food “Mas Vital”(2.0% inulin)1.8 ± 0.1%, RSD = 5%, n = 6

0

3

6

9

12

15

0 2 4 6 8 10 12 14 16 18 20

t, min

i, nA

COLLOIDAL GOLD-CARBON NANOTUBES COMPOSITE ELECTROCHEMICAL BIOSENSORS

Hybrid nanoparticles/nanotubes materialsBiocompatible materials with important electroanalytical features

Aucoll-CNT-Teflon electrodeAucoll-CNT-Teflon electrode

0 200 400 600 800 1000-20

4

8

12

16

i, A

E, mV

Aucoll-CNTs-Teflon

CNTs-Teflon (30:70)

graphite-Teflon (30:70)

Slope values of the calibration plot over (1.0-5.0)x10-3 M H2O2, at Eapp=+0.5 V

0.0083μA mM-1 ; 2.1 μA mM-1; 4.3 μA mM-1

Other advantages: Much lower noise level Rapidity

J. Electroanal. Chem., 603 (2007) 1

COLLOIDAL GOLD-CARBON NANOTUBES COMPOSITE ELECTROCHEMICAL BIOSENSORS

Analytical characteristics and kinetic parameters for glucose biosensors based on GOx–CNT electrodes

BIOSENSOR Edet, V Linear range, Slope, LOD, Useful KMapp

mM mA/M μM lifetime

30 1day 1.2 33 0.1-8 +0.5 vs Ag/AgClGOx-CNT-Teflon

14..9 3 months 2.6 17 0.05 – 1 +0.5 vs Ag/AgClGOx-Aucoll-CNT-Teflon

2 40

25

50

75

100

Current, %

Days

6 8

COLLOIDAL GOLD-CARBON NANOTUBES COMPOSITE ELECTROCHEMICAL BIOSENSORS

GOx –Aucoll – CNT-Teflon biosensor

GOx – CNT-Teflon biosensor

1.0 x 10-3 M glucose; Eapp=+0.5 V

Suitable electrode material for NADH detection

Suitable for the preparation of dehydrogenase biosensors

Aucoll-CNT-Teflon

electrocatalytic activityEnhanced electrode kinetics

Aucol CNT

NADH amperometric detection

0 0.2 0.4 0.6 0.8 E, V

50

40

30

20

10

i, µA

Aucoll-CNT-Teflon

CNT-Teflon

Aucoll-graphite-Teflon

graphite-Teflon

enhanced currents at less positive potentials

Aucoll-CNT-Teflon

NADH amperometric detection

Aucoll-CNT-Teflon

CNT-Teflon

Aucoll-graphite -Teflon

graphite -Teflon

50 s

10A

50 s

0.5 A

50 s

0.5 A

Eapp.=+0.3 V; NADH, 1.0 x 10-4M

Aucoll-CNT-Teflon

repeatability

t90%= 10-15 s

rapidity

RSD = 3.7 %(n=10)

NADH, 2.0 x 10-4 M

Analytical characteristics

Electrode Edet, V Linear range, mM

Slope, µA/mM

LOD, µM

Reference

TB-CNT +0.021 - 24 15 Lawrence, 2006

DHB-CNT-GCE -0.05 0.005-0.4 1.66 0.1 Retna, 2006

CNT-epoxy +0.55 hasta 1.0 29.7 - Pumera, 2006

CNT-Chit-GCE +0.6 0.005-0.8 8.7 0.5 Tsai, 2007

PVA-CNT-GCE +0.6 hasta 2.0 5.88 20 Tsai, 2007a

MB-CNT-GCE -0.1 hasta 0.5 0.52 4.8 Zhu, 2007

MB-Chit-CNT-GCE -0.14 hasta 0.08 5.9 0.5 Chakraborti, 2007

PDAB-CNT-GCE +0.07 0.002- 4.0 - 0.5 Zeng, 2007

CNT-sol-gel +0.3 hasta 0.65 2.31 12.4 Zhu, 2007a

Aucolll-CNT-Teflon +0.3 0.010-1.0 37.7 3.0 This work

CNT-Teflon +0.3 0.010-1.0 17.3 - This work

NADH DETECTION

Aucoll-CNT-Teflon

Redox mediator

- the highest calibration plot slope value- low detection potential with no mediator

2e

NAD+

NADH

CH3CH2OH

CH3CHO

ADH

ADH-Aucoll-CNT-Teflon

Alcohol dehydrogenase biosensor based on a colloidal gold-carbon nanotubes composite electrode

Alcohol dehydrogenase biosensor based on a colloidal gold-carbon nanotubes composite electrode

ETHANOL

Electrochim. Acta, 53 (2008) 4007-4012

Analytical characteristics ADH-Aucoll-CNT-Teflon

ETHANOL DETERMINATION

ElectrodE Eap, VLinearrange,

mM

Slope, µA/mM

LOD, µM

Reference

ADH-MB-CNT-CPE 0.0 0.05 - 10.0 0.597 5Santos,

2006

ADH-PVA-CNT-GCE +0.7 up to 1.5 0.196 13 Tsai, 2007

ADH-PDDA-CNT-GCE +0.1 0.5-5.0 - 90 Liu, 2007

ADH- Aucoll -CNT-Teflon +0.3 0.02-1.0 2.27 4.7 This work

ADH-CNT-Teflon +0.3 0.10-10.0 1.8 32 This work

Redox mediator

higher slope value even with no mediator

APPLICATION ADH-AucolL-CNT-Teflon

SAMPLEReference material AO6191

4.8 + 0.4 1.0 + 0.1 5.5 +0.3

4.3 + 0.6 <1 5.5

Ethanol concentration, g / 100 ml* Ethanol concentration, g / 100 ml*

Found

Declared

Sample FREE Sample WITH

* mean value + ts / √n (n = 3)

RESULTSsample

a)us stirringb)dilution

CO2

analyticalsolution

glucosinolates

DETERMINATION OF GLUCOSINOLATE DETERMINATION OF GLUCOSINOLATE IN VEGETABLESIN VEGETABLES

Β-thioglucoside-N-hydroxysulfatesFound in cabbage and broccoliIngredient in functional foodsAnticarcinogenic properties

MYR/GOx-Aucoll-CNT-Teflon

2

H2O

2

O2

2eH O

2

O2

H2O

glucose

GOxFAD

FADH

H O

GOxFAD

MYR

Electroanalysis, 21 (2009) 1527

CONCLUSIONS

Gold nanoparticles allows the construction of electrochemical biosensors exhibiting enhanced performances with respect to other designs

Gold nanoparticles allows the construction of electrochemical biosensors exhibiting enhanced performances with respect to other designs

The unique properties of gold nanoparticles concerning immobilization of biomolecules retaining their biological activity, and as efficient conducting interfaces with electrocatalytic ability makes them a powerful tool to modify electrode materials and to construct robust and sensitive biosensors.

The unique properties of gold nanoparticles concerning immobilization of biomolecules retaining their biological activity, and as efficient conducting interfaces with electrocatalytic ability makes them a powerful tool to modify electrode materials and to construct robust and sensitive biosensors.

They can be powerful analytical tools to be applied to the food industry. Applications in this field comprise the whole food chain, from the primary production to the final distribution to the consumer, which implies an enormous potential of application to food traceability.

They can be powerful analytical tools to be applied to the food industry. Applications in this field comprise the whole food chain, from the primary production to the final distribution to the consumer, which implies an enormous potential of application to food traceability.

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