1. NANOPARTICLES IN FOOD BIOSENSING
J.M. Pingarrón* L. Agüí and P. Yáñez-Sedeño
Department of Analytical Chemistry. Faculty of Chemistry
University Complutense of Madrid
28040-Madrid SPAIN
NANOJASP’2010
Barcelona, December 2010

2. Preparation of nanostructured electrode surfaces
Research line combining:
Advances in sensors technology
Development of several (bio)assay-transductor strategies
Applications of nanotechnology
- Wide range of approaches
- Use or not of biological systems
Products, processes and systems operating in nanometric magnitude

3. Nanostructured electrode surfaces
Advantages
Improved charge transfer reactions
Sensitivity
Selectivity
Repeatability
Electrocatalytic ability: lower detection potential
Antifouling capability

4. ELECTROCHEMICAL BIOSENSORS BASED ON
GOLD NANOPARTICLE-MODIFIED ELECTRODES
Gold nanoparticles Au
Ability to provide a stable surface for biomolecules immobilization retaining their biological activity Au
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
Useful interfaces for electrocatalysis of redox processes of H2O2 or NADH
Electrochim. Acta., 53 (2008) 5848

5. ELECTROCHEMICAL BIOSENSORS FOR FOOD ANALYSIS
DEVELOPMENT AND INNOVATION
FOOD SAFETY
FOOD QUALITY
EFFICIENT TRACEABILITY SYSTEMS
Development of detection, analysis and diagnosis methods
 Rapid
 Sensitive
 Automated screening

6. AMPEROMETRIC BIOSENSOR FOR HYPOXANTHINE BASED ON
IMMOBILIZED XOD ON NANOCRYSTAL GOLD-CARBON PASTE ELECTRODES
Gold nanoparticle preparation: Electrodeposition from a HAuCl4 solution on the bulk electrode material
Hypoxanthine (Hx) is formed as a product of nucleotide catabolism during the degradation processes in foodstuffs of animal origin.
Hx is accumulated mostly in the animal muscle and its levels are used as an index of fish and meat freshness in the food industry
Determination of hypoxanthine based on the enzyme reaction catalyzed by XOD
XOD
Hx + O2 → X + H2O2
X + O2 → Uric acid + H2O2

7. AMPEROMETRIC BIOSENSOR FOR HYPOXANTHINE BASED ON
IMMOBILIZED XOD ON NANOCRYSTAL GOLD-CARBON PASTE ELECTRODES
LOD at 0.00 V: 2.2x10-7 mol L-1
Kmapp = 18x10-6 mol L-1
Useful lifetime = at least 15 days
Sens. Actuators B. 113 (2006) 272
SEM of a GA-BSA-XOD-nAu-CPE biosensor
Determination of hypoxanthine in sardines and chicken meat using the GA-BSA-XOD-nAu-CPE biosensor
Sample
Sardines
Chicken
Added (mg/100g)
Found (mg/100g)
Recovery (%)
Added (mg/100g)
Found (mg/100g)
Recovery (%)
Non-spiked 1 2 3
Spiked 1 2 3
Mean recoveries ( = 0.05): 101 ± 8 % sardines 102±3% chicken meat

8. for the determination of inulin in foods
Bienzyme amperometric biosensor using gold nanoparticles-modified electrodes
for the determination of inulin in foods
Anal. Biochem., 375 (2008)

9. INULIN 1st enzyme biosensor DETERMINATION METHODS HPLC
Prebiotic ingredient added to functional foods
Vegetal origin: chicory root, artichoke
DETERMINATION METHODS
HPLC
UV, RI, ED
1st enzyme
biosensor
This work
(C6 H10 O5 )n (n=35)

10. Determination of interest in:
INULIN
Biosensor advantages
FOOD
INDUSTRY
Determination of interest in:
inherent specificity
simplicity
MONITORING OF PROCESSES
inuline extraction
fructose production
rapidity
real time analysis
- QUALITY CONTROL
- diethetic and children’s foods
- component of dietary fiber
ECONOMIC AND LEGISLATIVE
added value for functional foods
ingredients establish prices

11. BIENZYME BIOSENSOR FOR INULIN
Gold nanoparticle preparation: By adding sodium citrate to a boiling HAuCl4 aqueous solution HAuCl4/sodium citrate  Particle size  2e
PQQ
PQQH2
FRUCTOSE
5-CETO-D-FRUCTOSE
2TTF
2TTF+
INULIN
Redox mediator
E = +0.2 V
PBS 0.05 M, pH 4.5
AuE
Cyst
Aucol
TTF
FDH
Inulinase

12. 0.05 M phosphate buffer, pH 4.5, a 4ºC
BIENZYME BIOSENSOR FOR INULIN
Stability 20 40 60 80
100
120
140
160
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
-3s
+3s i, m A
time, days
More than 5 months
Storage conditions:
0.05 M phosphate buffer, pH 4.5, a 4ºC

13. BIENZYME BIOSENSOR FOR INULIN
Interferences
20 nA
100 s
dextrose
glucose
lactose
maltose
saccharose
inulin additions
10% Erel
[INTERFERENT] / [INULIN] = 1

14. Sample preparation by size exclusion SPE
BIENZYME BIOSENSOR FOR INULIN
Sample preparation by size exclusion SPE 3 6 9 12 15 2 4 8 10 14 16 18 20
t, min
i, nA
Sample
inulin
Bio-Gel P-6
(Bio-Rad)
fructose
2 μA
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
t, min

15. COLLOIDAL GOLD-CARBON NANOTUBES COMPOSITE ELECTROCHEMICAL BIOSENSORS
Hybrid nanoparticles/nanotubes materials
Biocompatible materials with important electroanalytical features
Aucoll-CNT-Teflon electrode
200
400
600
800
1000 -2 4 8 12 16 i, m A
E, mV
graphite-Teflon (30:70)
CNTs-Teflon (30:70)
Aucoll-CNTs-Teflon
Slope values of the calibration plot over ( )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

16. GOx-Aucoll-CNT-Teflon
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
GOx-Aucoll-CNT-Teflon
+0.5 vs Ag/AgCl
0.05 – 1
3 months
14..9
GOx-CNT-Teflon
+0.5 vs Ag/AgCl
0.1-8
1day 30

17. COLLOIDAL GOLD-CARBON NANOTUBES COMPOSITE ELECTROCHEMICAL BIOSENSORS
Current, %
100 75 50 25 2 4 6 8
Days
GOx –Aucoll – CNT-Teflon biosensor
GOx – CNT-Teflon biosensor
1.0 x 10-3 M glucose; Eapp=+0.5 V

18. Enhanced electrode kinetics electrocatalytic activity
Aucoll-CNT-Teflon
Aucol
Enhanced electrode kinetics
electrocatalytic activity
CNT
Suitable electrode material for NADH detection
Suitable for the preparation of dehydrogenase biosensors

19. enhanced currents at less positive potentials
NADH amperometric detection
Aucoll-CNT-Teflon
enhanced currents at less positive potentials 50 40 30 20 10
i, µA
0.2
0.4
0.6
0.8
E, V
Aucoll-CNT-Teflon
Aucoll-graphite-Teflon
CNT-Teflon
graphite-Teflon

20. NADH amperometric detection Aucoll-CNT-Teflon
repeatability
t90%= s
rapidity
RSD = 3.7 %
(n=10)
NADH, 2.0 x 10-4 M
10 mA
50 s
Aucoll-CNT-Teflon
CNT-Teflon
Aucoll-graphite -Teflon
0.5 mA
50 s
graphite -Teflon
0.5 mA
50 s
Eapp.=+0.3 V; NADH, 1.0 x 10-4M

21. Analytical characteristics Aucoll-CNT-Teflon
NADH DETECTION
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
1.66
0.1
Retna, 2006
CNT-epoxy
+0.55
hasta 1.0
29.7
Pumera, 2006
CNT-Chit-GCE
+0.6
8.7
0.5
Tsai, 2007
PVA-CNT-GCE
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
Chakraborti, 2007
PDAB-CNT-GCE
+0.07
Zeng, 2007
CNT-sol-gel
+0.3
hasta 0.65
2.31
12.4
Zhu, 2007a
Aucolll-CNT-Teflon
37.7
3.0
This work
CNT-Teflon
17.3
Redox mediator
the highest calibration plot slope value
low detection potential with no mediator

22. ADH-Aucoll-CNT-Teflon
Alcohol dehydrogenase biosensor based on a colloidal gold-carbon nanotubes composite electrode
ADH-Aucoll-CNT-Teflon 2e
NADH
CH3CHO
NAD+
CH3CH2OH
ETHANOL
ADH
Electrochim. Acta, 53 (2008)

23. Analytical characteristics ADH-Aucoll-CNT-Teflon
ETHANOL DETERMINATION
ElectrodE
Eap, V
Linear
range, mM
Slope,
µA/mM
LOD, µM
Reference
ADH-MB-CNT-CPE
0.0
0.597 5
Santos, 2006
ADH-PVA-CNT-GCE
+0.7
up to 1.5
0.196 13
Tsai, 2007
ADH-PDDA-CNT-GCE
+0.1 - 90
Liu, 2007
ADH- Aucoll -CNT-Teflon
+0.3
2.27
4.7
This work
ADH-CNT-Teflon
1.8 32
Redox mediator
higher slope value even with no mediator

24. * mean value + ts / √n (n = 3)
ADH-AucolL-CNT-Teflon
APPLICATION
sample
RESULTS
CO2
a)us
stirring
b)dilution
Ethanol concentration, g / 100 ml*
analytical
solution
SAMPLE
Reference material AO6191
<1
5.5
Sample FREE
Sample WITH
Found
Declared
* mean value + ts / √n (n = 3)

25. DETERMINATION OF GLUCOSINOLATE
glucosinolates
DETERMINATION OF GLUCOSINOLATE
IN VEGETABLES
Β-thioglucoside-N-hydroxysulfates
Found in cabbage and broccoli
Ingredient in functional foods
Anticarcinogenic properties
MYR/GOx-Aucoll-CNT-Teflon 2 H O 2e
glucose
GOx
FAD
FADH
MYR
Electroanalysis, 21 (2009) 1527

26. CONCLUSIONS
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.
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.

27. Thank you