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Main Objective

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.

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Main Objective

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  1. 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

  2. ARRAY OF BIO-SENSORS & SENSORS overall responses MAIN OBJECTIVE Ob. I.a. Tyrosinase immobilisation OBJECTIVE I –Metabolites content TP (based on PPox) Ob. I.b. Lacasse immobilisation Ob. II. a. Low density lipoprotein peroxidation OBJECTIVE II –Metabolites efficacy Aox Capacity Evaluation Ob. II. b. Enzymes’ use (SOD/XOD & cyt) and (SOD/XOD) TP results Folin-Ciocalteu; 280 nm maximum absorbence; HPLC/GC content OBJECTIVE III – Response validation AoxC results free radicals scavenging estimation via TEAC; ORAC; DPPH models Validation – inter-laboratory comparison (statistics)

  3. Ia. Tyrosinase O2 Electrode Tyr 2 e- H2O Reduction -200 mV/AgAgCl Tyr (EC 1.14.18.1) : tyrosinase Tyrosinase from mushroom (SIGMA) Calibration : Substrate: 1,2-benzenediol = catechol OBJECTIVE I –Metabolites content - TP

  4. Reference electrode Ag/AgCl Working electrode Immobilisation of the biological element Entrapment PVA polymer Deposition of the biological element Automation : Liquid microdispense system SILIFLOW Counter electrode Biosensor design

  5. Tyrosinase-based biosensor performance assessment Conditions * SPE * Tyr: 0.2U immobilised in PVA 50:50 * PBS 0.1 M, pH 7.4, 0.1M KCl * E=-200mVvs. Ag/AgCl

  6. (For each standard, the average value of intensity is determined after 5 measurements) Tyrosinase-based biosensor performance assessment

  7. Calibration: Substrate : 1,2-benzenediol = catechol ABTS Ib. Laccase Laccase (EC 1.10.3.2) –benzenediol oxidoreductase Lacasse from Trametes versicolores (SIGMA)

  8. Immobilisation of the biological element -enzyme on chitosan/chitosan-CNT matrix (2mgCNT/mLCHIT sol) 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 %)

  9. Substrate/Electrode Resp. time Operational stability Response characteristics, citrate buffer, pH = 4.50 Catechol/ lacc/MWNT-Chi/ITO 120 s 5 measurements ABTS/ lacc/MWNT-Chi/Au 100 s 10 measurements Electrode Substr. Applied pot. Ecuation of calibration curve Linearity range (molL-1) LoD (molL-1) KappM value (molL-1) lacc/ MWNT-Chi/Au ABTS +0.35 V I(µA)=0.23xC(µM)- 0.02 5x10-6- 5x10-5 4.08x10-6 1.8 x10-4 Stability lacc/ MWNT-Chi/ITO ABTS +0.30 V I(nA)=37.59xC(µM) +0.14 1x10-7-3x10-6 1.11x10-8 2.7x10-5 Pyrocat -0.2 V I(nA)=21.975xC(µM) + 2.7 1x10-6-5x10-5 2.5x10-6 6.3x10-4 Laccase-based biosensor performance assessment

  10. Response towards interests metabolites Metabolite Reaction conditions Response, nA/mM Reaction time 4 min Solvent effect on Lacasse activity Solvent % inhibitor (vol) Inhibition % 0 min 10 min Catechol -0,150 V, pH=4.50; citrate buffer 263 DMSO 0.3 8.2 11 3.3 26.47 30.63 Caffeic acid -0,150 V, pH=4.50; citrate buffer 280 33 100 100 C2H5OH 0.3 6 9.35 Gallic acid -0,150 V, pH=4.50; citrate buffer 150 3.3 20 28 33 90 92.15 Rosmarinic acid -0,150 V, pH=4.50; citrate buffer 55.2 CH3OH 0.3 0 2.5 3.3 7.3 13 Resveratrol -0,150 V, pH=4.50; citrate buffer 1200 33 75.7 82.43 Laccase-based biosensor performance

  11. OBJECTIVE I –Metabolites content - TP Laccase and Tyrosinase - based biosensors-suitable to estimate TP content

  12. Aox ORAC assay HO. B – phycoerythrin fluorescence quenching AAPH LDL approach 565 nm Inactive el Active el OBJECTIVE II –Metabolites efficacy- Aox capacity IIa. Low density lipoprotein (LDL) based sensor Mechanisms involved in Aox Capacity determination HAT: ArOH+ ROO ROOH + ArO (ORAC assay) ET: ROO + ArOH ROO - + ArOH+ …… HAT + ET (DPPH assay, ABTS –TEAC assay)

  13. FTIR spectra of LDL layer after reaction with .OH free radical (inset: AFM 3D image of deposed LDL layer on Au support) Low density lipoprotein (LDL) based sensor LDL deposition: sol-casting from 36 ppm solution, (overnight ) on Au (cleaned and sonicated to oxide traces removal). Calibration against Trolox

  14. Low density lipoprotein (LDL) based sensor

  15. 1st strategy Electrode H2O2 SOD 2 e- 2H+ response XOD : xanthine oxidase (EC 1.1.3.22) SOD ANTIOX : superoxide dismutase (EC 1.15.1.1) II.b.Biosensors for the determination of antioxidant capacity (O2• - ) Xanthine O2• - XOD O2 Uric Acid

  16. S S COO- S S S S COOH COOH COOH COO- COO- XOD : xanthine oxidase (EC 1.1.3.22) (hypo) xanthine XOD O2 + H2O H2O2 catalase O2 uric acid II.b.Biosensors for the determination of antioxidant capacity (O2• - ) 2nd strategy Gold electrode Cyt. c Heme (Fe3+) O2 e- Cyt. c Heme (Fe2+) O2 - XOD: Xanthine oxidase

  17. Cyclic voltammogram of covalently immobilized Cyt c 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)

  18. Current 9nA 8.7nA 9.2nA buffer+catalase (10U/ml) buffer+catalase (10U/ml) buffer+catalase (10U/ml) + HX (50mM) + HX (50mM) + HX (50mM) Time II.b.Biosensors for the determination of antioxidant capacity (O2• - ) Cyt c-modified electrode response to superoxide generation • 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

  19. OBJECTIVES I & II –Metabolites content & efficacy To develop a flow system to assess the metabolites content and efficacy Actual status of prototype

  20. OBJECTIVE III – Results validation, in terms of Aox capacity

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

  22. Sample TEACABTS µmol/g TEACDPPH µmol/g SOF1-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

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