main objective
DESCRIPTION
Main Objective To 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 responses. MAIN OBJECTIVE. Ob. I.a. Tyrosinase immobilisation. - PowerPoint PPT PresentationTRANSCRIPT
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