hau seminar 1 powerpoint copy

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Human-in-a-chipPresented byNguyen Van Hau

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a microfluidic device

of the metabolism-dependent antioxidant activityfor evaluating the dynamics

of nutrientspresented by:Nguyen Van Hau

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IntroductionExperimental

Results & discussionConclusion

outline

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1Introduction

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Play an important role in human health

introduction

Anti-aging

Maintain good health

Protect the liver Support the immune

system Avoid dangerous diseases

Benefit of antioxidants with human health

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Fig 1. Free radical formation process in human body

antioxidantsFree radicals

Linked moleculesFree radicals

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Definition:An antioxidant is a

molecule that inhibits the oxidation of other molecules

Fig 2. How an antioxidant reduce a free radical

antioxidants

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antioxidants analysisAntioxidants activity

The rate constant of the reaction between

a unique antioxidant and a given free radical

Antioxidants sources Glutathione Vitamins: C, E... Enzymes: catalase... Flavonoids

Fig 3. Quercetin (an antioxidants compounds)

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introductionThe effect of metabolism processto antioxidant activity? metabolism

process Antioxidant compounds

Antioxidant activity

Antioxidant activity of some fruits

http://acaiology.com/orac-oxygen-radical-absorbance-capacity/

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PHASE I PHASE II

Xenobitic

OxidationReductionHydrolysisHydration

DethioacetylationIsomerization

GlucosidationSulfation

MethylationAcetylation

Amino acid conjugationGlutathione conjugation

HydrophilicHydrophobic

liver metabolism process

G.Gordon Gibson, Paul Skett, 2001

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microfluidic systemScope of this researchEffect of

metabolism processto antioxidant activity

introduction

Mimic the liver metabolism Determine antioxidants activity

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objective

objectiveEvaluating the effect of the liver metabolism on the antioxidant activity of nutrients by a microfluidic system

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2Experimental

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Quercetin

Quercetin radical

+ +

DPPH free radical

Antioxidant compounds

+ DPPH stable molecule

antioxidants analysisDPPH assay

Spectrophotometric assay based on the scavenging of DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals (DPPH•) (m=517 nm)

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DPPH assay

DPPH+

DPPH

517 nm

antioxidants analysis

Abs

Concentration of Trolox

Fig 5. Absorbance of DPPH + 0.12 mM with different Trolox

concentration

Fig 4. Spectrum of DPPH+ and DPPH

Aurelia Magdalena Pisoschi, 2009.

16 Fig 7. Lab on a chip technique

Standard/ sample

Concentrationof analyte

reagent UV-Vis spectrophotometerCalibration curve

reagentsample

3x3 cm chipMeasurement zone

Reaction zo

ne

Fig 6. Bath colorimetry technique

Light source

Detector

2 mm

100 m

17 Fig 8. Liver metabolism-antioxidant analysis-chip

antioxidant analysis

DPPH• + AH DPPHH + A•

microfluidic system

liver metabolism reaction

Quercetin Metabolic products

enzymes

PDMS: Polymethyl dimethylsiloxane

PDMS microfluidic system

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Photomask

UV light

Focus lensWafer

Photolithography technique principleAn example of a commercial photomask

Photolithography techniqueTranferring geometry shapes on the photomask to the surface of the

wafer which cover with a photoresists

chip fabrication

http://www.science.gc.ca/

http://www.bit-tech.net/

Silicon Wafer1. Wafer preparation

pdms chip fabrication

Cleaning the wafer

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SU-8 photoresists2. Coating photoresists

pdms chip fabricationProperties is changed when exposured to UV light Spin-coating at 1700 rpm

for 30s

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Photomask3. Exposure

Photomask

Photo-polymerization SU-8

pdms chip fabrication

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pdms chip fabricationUV light

3. Exposure Photo-polymerization SU-8

Photomask

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Cross-linking SU-8

Uncross-linking SU-8

4. Stripping

pdms chip fabrication

Chip master

Photomask

Washing un-treated SU-8

Unsoluble in eluent (-butylaractone)

Soluble in eluent (-butylaractone)

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Uncured PDMS5. Fabricating PDMS stampHigh viscosity liquid

PDMS: Polymethyl dimethylsiloxane

pdms chip fabrication

PDMS curing conditionsTemperature : 80oCTime : 3h

High viscosity liquid SolidUncured PDMS Cured PDMScuring

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Cured PDMSPDMS chipSolid

pdms chip fabrication

Cross-linking

Peeling the PDMS out of the master

Treating with FOTS

High viscosity liquid SolidUncured PDMS Cured PDMScuring

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Cured PDMS

Glass substrate

pdms chip fabrication6. Bonding Bonding PDMS chip +

glass substrateby O2 plasma treatment

for 30s

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How to mimic the liver metabolism

in microfluidic system

Enzymes

Liver enzyme

s

Liver metabolis

m

100 M

2 mm

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PDMS

Glass substrate

encapsulation enzymes in the micro-channel

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1. Introducing the solution into the micro channel

Enzymes+PEGDA+AAPH High

viscosity liquidPEGDA: Poly(EthyleneGlycol) DiAcrylate

AAPH: 2,2’-azobis(2-methylpropionamidine) dihydrochloride

PEGDA PEGDAHigh viscosity liquid Solid

Cross-linkingUV light

encapsulation enzymes in the micro-channel

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Photomask

Photomask

2. Exposure Exposure for 17s

UV light

encapsulation enzymes in the micro-channel

Enzymes+PEGDA+AAPH High

viscosity liquidPEGDA: Poly(EthyleneGlycol) DiAcrylate

AAPH: 2,2’-azobis(2-methylpropionamidine) dihydrochloride

PEGDA PEGDAHigh viscosity liquid Solid

Cross-linkingUV light

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Photomask

Stripping un-treated PEGDA with PBS buffer

Enzymes

PEGDA pillar

3. Stripping

Enzymes is encapsulated in PEGDA pillars inside the chip channel

encapsulation enzymes in the micro-channel

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liver enzymes

HomogenizationCentrifugation @100,000

xg

S9-fraction(supernatant)

Phase I and II enzymes

Easy to use, cheap

Needs co-factor

microsome-fraction CYP450, UGT

enzymes Easy to use, cheap Needs co-factor

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Optics fiber

led spectrometer set-up

Fig 9 . Fiber-coupled miniature spectrometer (USB4000) set-up

Microfluidic system set-up

Bath method set-upoceanoptics.com

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mathematical modeling Plug flow reactor-PFR

PFR parameterVolume of channel 2.96x10-8 m3

Volume of flow rate 5.41x10-11 m3/sQuercetin concentration

0.1, 0.05, 0.02

mol/m3

DPPH concentration

0.25 mol/m3

V : the reactor volumeF0 : molar flow rate of DPPH moleculesr1 : reaction ratex : conversion of DPPH+ to DPPH

V=F0∫0

x 1−r 1

dx

Reaction constant: 2.807x10-2 m3mol-1s-1

real chip system

computer simulationvs

Examing the effect of volumetric flow rate by computer model

Compare the results by computer model – real chip experiments

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mathematical modeling Finite element analysis

Computer simulation by COMSOL Multiphysics

COMSOL parameterQuercetin concentration

0.4, 0.2, 0.08

mol/m3

DPPH concentration

0.5 mol/m3

Velocity of ethanol 8.3x10-4 m/sVelocity of quercetin

8.3x10-4 m/s

Velocity of DPPH 16.6x10-4 m/sDiffusivity 1.26x10-8 m2/sDensity 1000 kg/

m3

Viscosity 0.01 kg/m.s

Reaction constant: 2.807x10-2 m3mol-1s-1

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mathematical modeling Plug flow reactor-PFR Finite element analysis

PFR parameterVolume of channel 2.96x10-8 m3

Volume of flow rate 5.41x10-11 m3/sQuercetin concentration

0.1, 0.05, 0.02

mol/m3

DPPH concentration

0.25 mol/m3

Computer simulation by COMSOL Multiphysics

COMSOL parameterQuercetin concentration

0.4, 0.2, 0.08

mol/m3

DPPH concentration

0.5 mol/m3

Velocity of ethanol 8.3x10-4 m/sVelocity of quercetin

8.3x10-4 m/s

Velocity of DPPH 16.6x10-4 m/sDiffusivity 1.26x10-8 m2/sDensity 1000 kg/

m3

Viscosity 0.01 kg/m.s

V : the reactor volumeF0 : molar flow rate of DPPH moleculesr1 : reaction ratex : conversion of DPPH+ to DPPH

V=F0∫0

x 1−r 1

dx

Reaction constant: 2.807x10-2 m3mol-1s-1 Reaction constant: 2.807x10-2 m3mol-1s-1

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3Results - Discussion

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Blank channelno metabolism reaction

studying the performance of microfluidic system

no encapsulate enzyme

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optimization microfluidic system The precipitation of DPPH inside the channel

At interface between two compartment

Extra ethanol stream

Quercetin in PBS buffer

DPPH in ethanol

Ethanol

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a) Precipitation of DPPH in the channel b) Finite element simulation of the mixing phenomena at the interface

and the actual picture of the interface after adding ethanol in the buffering

channelFig 12. Minimization the precipitation of DPPH inside the channel

DPPH

Quercetin

optimization microfluidic system

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a) Predicting final amounts of scavenged radicals by PFR

b) Concentration of DPPH predicted by finite element modeling

Fig 13. Determining optimal flow rate by analytical mathematical model

optimization microfluidic system Determing optimal flow rate by computer model

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a) Predicting final amounts of scavenged radicals by PFR

Fig 13 Determining optimal flow rate by analytical mathematical model

optimization microfluidic system

PFR modelThe realtionship between conversion-flow rate

V=F0∫0

x 1−r 1

dx

The using flow rate is suitable Flow rate: 5.41x10-11 m3s-1

Determing optimal flow rate by PFR computer model

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b) Concentration of DPPH predicted by finite element modeling

Fig 13. Determining optimal flow rate by analytical mathematical model

optimization microfluidic system

Homogenous environment inside the

channel

Verifying optimal flow rate value from PFR model by finite element modeling

Supporting the PFR model

Flow rate: 5.41x10-11 m3s-1

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radical scavenging reaction kinetics on a chip Examing the reaction kinetics on the chip

real chip system computer simulationvs

Reaction constant (k)

Predicting the radical scavengingby computer model

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radical scavenging reaction kinetics on a chip Determining reaction constant (k)

First-order reaction

DPPH• + AH DPPHH + A•

A-H: quercetin

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radical scavenging reaction kinetics

a) Time-dependent of the DPPH concentration by bath method

(cuvette)

b) Initial reaction rate (at 1min)

Fig 14. Time dependent antioxidant activity of quercetin by usual colorimetry method

20M

50M 100M

Determining reaction constant (k)

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radical scavenging reaction kinetics

b) Initial reaction rate (at 1min)Fig 14. Time dependent antioxidant activity of quercetin by usual colorimetry method

20M

50M 100M

k = 2.807 x 10-2 m3mol-1s-1

−d CDPPHdt

= k  CDPPHCquercetin

Slope of the slotk

Determining reaction constant (k)

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radical scavenging reaction kinetics

Fig 15. Time dependent antioxidant activity of quercetin on the chip system

Examing the reaction kinetics on the chipDPPH• + AH DPPHH + A•

A-H: quercetin

Radical scavenged amount

radical scavenging reaction kinetics

Fig 16. Measured and predicted amount of radical scavenging

Quercetin in PBS buffer

DPPH in ethanol

Ethanol

Precipitation of quercetin

real chip system computer simulationvs49

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Adding more parameters to

computer model Solubility of quercetin in

solution Solubility of DPPH in solution

radical scavenging reaction kinetics

Fig 16. Measured and predicted amount of radical scavenging

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Channelwith encaplsulated enzymes

studying effect of the metabolism processto antioxidant activity

Quercetin is metabolized before enter 2nd part

52Fig 17. Antioxidant activity of quercetin after various metabolic conditions

radical scavenging reaction kinetics

Co-factor: co-factor for glucuronidation

Quercetin

Metabolized

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PHASE I PHASE II

Quercetin

OxidationReductionHydrolysisHydration

DethioacetylationIsomerization

GlucosidationSulfation

MethylationAcetylation

Amino acid conjugationGlutathione conjugation

Hydrophobic Hydrophilic

Fig 17. Antioxidant activity of quercetin after various metabolic conditions

No metabolism

Phase I onlyPhase I + 1 reaction phase IIPhase I + Phase II

radical scavenging reaction kinetics

Co-factor: co-factor for glucuronidation

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4Conclusion

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Evaluating the antioxidant activity of nutrients after liver metabolism process

Developing an optical detection system for real-time tracking of the reaction occurring on the chip

Indicating the correction well between computer simulation and experiment results at the low concentration of quercetin

Comparing the antioxidant activity of quercetin after various metabolic reaction

conclution

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acknowledgementsAssoc. Prof. Dr. Napaporn Youngvises

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Your questions is welcome...

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performance of led spectrometer

Fig 10. Transmission intensity of the spectrometer system at various

wavelengths

Fig 11. Measured absorbance at various concentrations of DPPH on the chip

517 nm Using cuvette

Using chip

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3’ O-methylquercetin

Quercetin-3’-O-sulphate

Quercetin-3’-O-glucurinide

3’ O-methylquercetin-7-glucuronide (10,11,18)

quercetin

Eula Maria de M. B. Costa, Fabiana Cristina Pimenta, et al, 2008.

Metabolic profile of quercetinQuercetinPHASE IDeglycosidation

PHASE IIGlucuronidationSulfationO-methylation

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How an antioxidant reduce a free radical

Ascorbate free radical formation

Antioxidants structuralConjugated systemResonance

structure

61 Fig 18. Initial reaction rate with various ethanol volume fraction in the solvent

Effect of ethanol fraction on radical scavenging activity

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Microsomal reaction in static system

Fig 18. Amount of radical scavenged of quercetin under various condition

Quercetin trapped inside a PEGDA hydrogel pillar

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PEGDA property Rapid linking under illumination of UV

light Porousity structure

encapsulation enzyme in pedga hydrogel

Advantage of encapsulation enzyme into hydrogel

Increasing stability Biocompatibility of the matrix Non-toxic Fast linking time Ease of patterning

SEM image of PEGDA 3400 PEGDA pillars

Z.Amelia, K.Arpita, M.Mohsen, C.Michael, AMER March 2013