Main ObjectiveTo provide a set of sensors able to be used as screening analytical devices to assess the secondary metabolites content and their efficacy as antioxidants

ARRAY OF BIO-SENSORS & SENSORS

overall responsesMAIN OBJECTIVE

OBJECTIVE I –Metabolites content TP (based on PPox)

Ob. I.a. Tyrosinase immobilisation

Ob. I.b. Lacasse immobilisation

OBJECTIVE II –Metabolites efficacy Aox Capacity Evaluation

Ob. II. a. Low density lipoprotein peroxidation

Ob. II. b. Enzymes’ use (SOD/XOD & cyt) and (SOD/XOD)

OBJECTIVE III – Response validation

TP results Folin-Ciocalteu; 280 nm maximum

absorbence; HPLC/GC content

AoxC results free radicals scavenging estimation via

TEAC; ORAC; DPPH modelsValidation – inter-laboratory

comparison (statistics)

Tyr

O2

H2O

: tyrosinaseTyrTyrosinase from mushroom (SIGMA)

Ia. Tyrosinase

(EC 1.14.18.1)

2 e-

Electrode

Reduction -200 mV/AgAgCl

OBJECTIVE I –Metabolites content - TP

Calibration :Calibration :Substrate: 1,2-benzenediol = catechol

Counter electrode

Reference electrode

Ag/AgClWorking electrode

Automation :Liquid microdispense system

SILIFLOW

Immobilisation of the biological element Entrapment PVA polymer

Deposition of the biological element

Biosensor design

Tyrosinase-based biosensor performance assessment

Response time (s)

Sensitivity ( A mM-1)

Linear range (M) R2 Detection limit

( M) 20 74 1x10-6- 1 x10-4 0.999 0.05

y = 74,311x + 14,452 R 2 = 0,9991

0

2000

4000

6000

8000

10000

12000

14000

0 50 100 150 200 250 300 [catechol]

(µM)

I (nA)

Conditions * SPE* Tyr: 0.2U immobilised in PVA 50:50* PBS 0.1 M, pH 7.4, 0.1M KCl* E=-200mV vs. Ag/AgCl

(For each standard, the average value of intensity is determined after 5 measurements)

Tyrosinase-based biosensor performance assessment

OH

OH

Laccase

OH

OH

O

O

2e; 2H+

electrode

amperometric response; reduction ELECTRODE

Ib. Laccase

Laccase (EC 1.10.3.2) –benzenediol oxidoreductase

Lacasse from Trametes versicolores (SIGMA)

Calibration:Calibration:Substrate : 1,2-benzenediol = catechol ABTS

-enzyme on chitosan/chitosan-CNT matrix (2mgCNT/mLCHIT sol)

Immobilisation of the biological element

Biosensor design - solid supports: Au (cleaned and annealed)

ITO

Deposition processes: • on Au during CHIT electrodeposition –1.5V vs Ag/AgCl

• on etched ITO (H2O2:NH3:H2O=1:1:5), solution casting (from 1% in acetic acid 0.5 %)

Laccase-based biosensor performance assessment Response characteristics, citrate buffer, pH = 4.50

1.8 x10-44.08x10-65x10-6- 5x10-5 I(µA)=0.23xC(µM)- 0.02

+0.35 VABTSlacc/MWNT-Chi/Au

KappM

value(molL-1)

LoD(molL-1)

Linearity range

(molL-1)

Ecuation of calibration curve

Applied pot.

Substr.Electrode

6.3x10-42.5x10-61x10-6-5x10-5 I(nA)=21.975xC(µM) + 2.7

-0.2 VPyrocat

2.7x10-51.11x10-81x10-7-3x10-6I(nA)=37.59xC(µM) +0.14

+0.30 VABTSlacc/MWNT-Chi/ITO

100 s

120 s

Resp. time

5 measurementsCatechol/ lacc/MWNT-Chi/ITO

10 measurementsABTS/ lacc/MWNT-Chi/Au

Operational stabilitySubstrate/ElectrodeStability

Laccase-based biosensor performance

55.2-0,150 V, pH=4.50; citrate bufferRosmarinic acid

1200

150

280

263

-0,150 V, pH=4.50; citrate bufferResveratrol

-0,150 V, pH=4.50; citrate bufferGallic acid

-0,150 V, pH=4.50; citrate bufferCaffeic acid

-0,150 V, pH=4.50; citrate bufferCatechol

Reaction conditions Response, nA/mMReaction time 4 min

MetaboliteResponse towards interests metabolites

Inhibition %

137.33.3

9.3560.3C2H5OH28203.3

92.159033

118.20.3

30.6326.473.3

82.4375.733

CH3OH 2.500.3

10010033

DMSO10 min0 min

% inhibitor (vol)Solvent Solvent effect on Lacasse activity

OBJECTIVE I –Metabolites content - TP

Laccase and Tyrosinase - based biosensors- suitable to estimate TP content

OBJECTIVE II –Metabolites efficacy- Aox capacity

IIa. Low density lipoprotein (LDL) based sensor

Mechanisms involved in Aox Capacity determinationMechanisms involved in Aox Capacity determination HAT:HAT: ArOH ArOH + ROO+ ROO ROOH + ArO ROOH + ArO (ORAC assay) (ORAC assay) ET:ET: ROO ROO + ArOH + ArOH ROO - + ArOH+ …… ROO - + ArOH+ …… HAT + ETHAT + ET ( (DPPH assay, ABTS –TEAC assayDPPH assay, ABTS –TEAC assay))

HO.

565 nm

B – phycoerythrin fluorescence B – phycoerythrin fluorescence

quenching quenching AAPH

ORAC assay

Aox

AAPH + H2Ot=37°C

2HO. + AAP.

LH + HO.+O2 LOO.+H2O LOOH+HO.Inactive el

Active el

LDL approach

AAPH + H2Ot=37°C

2HO. + AAP.

Low density lipoprotein (LDL) based sensorLDL deposition: sol-casting from 36 ppm solution, (overnight ) on Au (cleaned and sonicated to oxide traces removal). Calibration against Trolox

FTIR spectra of LDL layer after reaction with .OH free radical (inset: AFM 3D image of deposed LDL layer on Au support)

0

10

20

30

40

50

60

1 3 5 7 9 11 13timp (min)

% lipoperoxides formation Electrode less Aoxcontent

Electrode higher Aoxcontent (36 ppm)

El 1

Low density lipoprotein (LDL) based sensor

II.b.Biosensors for the determination of antioxidant capacity (O2• - )

SODH2O2

2H+

SOD : superoxide dismutase (EC 1.15.1.1)

1st strategy

XOD : xanthine oxidase (EC 1.1.3.22)

Xanthine

Uric Acid

XODO2

O2• - Electrode

2 e-

response

II.b.Biosensors for the determination of antioxidant capacity (O2• - )

Cyt. cHeme (Fe3+)

Cyt. cHeme (Fe2+)

COOHS

COOH

S

COO-

S

S

COO-S

COO-

S

COOH

O2

O2 -

e-

H2O2catalase

O2 + H2O

(hypo)xanthine

O2

XOD

uric acid

XOD: Xanthine oxidase

Gol

d el

ectr

ode

2nd strategy

XOD : xanthine oxidase (EC 1.1.3.22)

II.b.Biosensors for the determination of antioxidant capacity (O2• - )

SPE-Au cleaned; MercaptoUndecanol:MercaptoUndecanoicAcid (3.75:1.25 mM) SAM formation via sol-casting; SAM activation using 200 mM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and 50 mM NHS (N-hydroxysuccinimide); cyt C bounding by adsorption from 50 mM Cyt c solution (in 5 mM K-PBS pH=7)

Cyclic voltammogram of covalently immobilized Cyt c

Cyt c-modified electrode response to superoxide generation

II.b.Biosensors for the determination of antioxidant capacity (O2• - )

buffer+catalase (10U/ml) + HX (50M)

8.7nA

buffer+catalase (10U/ml) + HX (50M)

9.2nA

buffer+catalase (10U/ml) + HX (50M)

9nA

Time

Current

Conditions:• 1 h incubation in 250mU/ml XOD; XOD adsorption on the electrode surface• 0.1 M PBS + 0.1 mM EDTA pH=7.5• E: 150 mV

OBJECTIVES I & II –Metabolites content & efficacy

To develop a flow system to assess the metabolites content and efficacy

Actual status of prototype

OBJECTIVE III – Results validation, in terms of Aox capacityOBJECTIVE III – Results validation, in terms of Aox capacity

)/( gmolm

xSSSS

xfxnTEACblankTrolox

blanksampleTroloxsample 1

−−

=

gallic acidcaffeic acid

cholorgenic acidcucurmincathecolresorcinol

rosmarinic acidresveratrol

3-hidroxy flavone DPPHABTS

0

5000

10000

15000

20000

25000TEAC µmol/g

DPPH

ABTS

TEAC DPPH (µmol/g dry base) TEAC ABTS (µmol/g dry base)

SAMPLE Initial RT

storage Refrigerator

storage Initial RT

storage Refrigerator

storage

Basil phenolics 1671.371 749.255 1009.101 878.873 541.581 690.169

Dandelion phenolics 1101.097 1035.742 1137 .014 1717.513 1082.764 953.989

Soybean isoflavone 90.291 61.340 86.812 415.747 265.143 92.740 Soybean purified isoflavone 302.466 32.788 113.412 241.447 1067.557 830.804

Mint flavonoid 670.558 1188.808 674.513 1191.015 1726.139 1715.686 Mint purifi ed flavonoids 1470.126 1182.808 1384.944 1705.336 2403.178 1935.765

TEAC stability test during 1 month from initial determination, samples provided by Partner 4-ISS Poland

Sample TEACABTS

µmol/gTEACDPPH

µmol/gSOF1-CO 393.103 456.961

SOF2-CO 549.264 633.184

SOF3-CO 459.018 442.334

SOF4-CO 560.078 648.943

S30-CO 463.960 475.417

SCINn 199.405 120.161

SOF2-COEO 918.648 514.704

SOF3-COEO 23.065 ND

S30-COEO 382.880 ND

S33UPISEO 1668.687 72.887

TEAC assays, samples provided