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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