Seminar: A microfluidic device for evaluating the dynamics of the metabolism-dependt antioxidant activity of nutrients (J Lee et al., ‎2014)

1. Human-in-a-chip
Presented by
Nguyen Van Hau
1

2. a microfluidic device
he metabolism-dependent antioxidant activity
for evaluating the dynamics
of nutrients
presented by:Nguyen Van Hau
2

3. Introduction
Experimental
Results & discussion
Conclusion
outline
3

4. 1Introduction
4

5. 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

5

6. Fig 1. Free radical formation process in human body
antioxidants
Free radicals
Linked molecules
Free radicals
6

7. Definition:
An antioxidant is a molecule
that inhibits the oxidation of other
molecules
Fig 2. How an antioxidant reduce a free
radical
antioxidants
7

8. antioxidants analysis
Antioxidants 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)
8

9. introduction
The effect of metabolism
process
to antioxidant activity
? metabolism
process Antioxidant
compounds
Antioxidant activity
Antioxidant activity of some
fruits
http://acaiology.com/orac-oxygen-radical-absorbance-capacity/9

10. PHASE I PHASE II
Xenobitic
Oxidation
Reduction
Hydrolysis
Hydration
Dethioacetylation
Isomerization
Glucosidation
Sulfation
Methylation
Acetylation
Amino acid conjugation
Glutathione conjugation
Hydrophilic
Hydrophobic
liver metabolism process
G.Gordon Gibson, Paul Skett,
10

11. microfluidic system
Scope of this
research
Effect of metabolism
process
to antioxidant activity
introduction
 Mimic the liver metabolism
 Determine antioxidants activity
11

12. objective
objective
Evaluating the effect of the liver metabolism on the antioxidant activity of nutrients by a
microfluidic system
12

13. 2Experimental
13

14. Quercetin Quercetin radical+ +
DPPH free radical Antioxidant
compounds
+ DPPH stable molecule
antioxidants analysis
DPPH assay
Spectrophotometric assay based on the scavenging of DPPH (2,2-diphenyl-1-
picrylhydrazyl) radicals (DPPH•) (m=517 nm)
14

15. 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.
15

16. Fig 7. Lab on a chip technique
andard/ sample
Concentration
of analyte
reagent UV-Vis spectrophotometerCalibration curve
reagentsample
3x3 cm chip
Measurement zone
Fig 6. Bath colorimetry technique
Light source
Detector
100 m
16

17. Fig 8. Liver metabolism-antioxidant analysis-chip
antioxidant analysis
DPPH• + AH  DPPHH + A•
microfluidic system
liver metabolism reaction
Quercetin Metabolic
products
enzymes
MS: Polymethyl dimethylsiloxane
PDMS microfluidic
system
17

18. Photomask
UV light
Focus lens
Wafer
Photolithography technique principle An example of a commercial photomask
Photolithography technique
Tranferring geometry shapes on the photomask to the surface of the wafer which
cover with a photoresists
chip fabrication
http://www.science.gc.http://www.bit-18

19. Silicon Wafer
1. Wafer preparation
pdms chip fabrication
 Cleaning the wafer
19

20. SU-8 photoresists
2. Coating photoresists
pdms chip fabrication
Properties is changed when exposured to
UV light Spin-coating at 1700 rpm for 30s
20

21. Photomask
3. Exposure
Photomask
 Photo-polymerization SU-8
pdms chip fabrication
21

22. pdms chip fabrication
UV light
3. Exposure
 Photo-polymerization SU-8
Photomask
22

23. 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)
23

24. Uncured PDMS5. Fabricating PDMS stamp
High viscosity liquid
PDMS: Polymethyl
dimethylsiloxane
pdms chip fabrication
 PDMS curing conditions
Temperature : 80oC
Time : 3h
High viscosity liquid Solid
Uncured PDMS Cured PDMScuring
24

25. 25
Cured PDMSPDMS chip
Solid
pdms chip fabrication
Cross-linking
 Peeling the PDMS out of the
master
 Treating with FOTS
High viscosity liquid Solid
Uncured PDMS Cured PDMScuring
25

26. Cured PDMS
Glass substrate
pdms chip fabrication
6. Bonding
 Bonding PDMS chip + glass
substrate
by O2 plasma treatment for 30s
26

27. How to mimic the liver
metabolism
in microfluidic system
Enzymes
Liver
enzymes
Liver
metabolism
100 M
2 mm
27

28. PDMS
Glass substrate
encapsulation enzymes in the micro-channel
28

29. 1. Introducing the solution into the micro channel
Enzymes+PEGDA+AAPH
 High viscosity
liquid
PEGDA: Poly(EthyleneGlycol) DiAcrylate
AAPH: 2,2’-azobis(2-methylpropionamidine) dihydrochloride
PEGDA PEGDA
High viscosity liquid Solid
Cross-linking
UV light
encapsulation enzymes in the micro-channel
29

30. Photomask
Photomask
2. Exposure
 Exposure for 17s
UV light
encapsulation enzymes in the micro-channel
Enzymes+PEGDA+AAPH
 High viscosity
liquid
PEGDA: Poly(EthyleneGlycol) DiAcrylate
AAPH: 2,2’-azobis(2-methylpropionamidine) dihydrochloride
PEGDA PEGDA
High viscosity liquid Solid
Cross-linking
UV light
30

31. 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
31

32. liver enzymes
Homogenization
Centrifugation @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
32

33. Optics fiber
led spectrometer set-up
Fig 9 . Fiber-coupled miniature spectrometer (USB4000) set-up
 Microfluidic system set-up  Bath method set-up
oceanoptics.com
33

34. mathematical modeling
 Plug flow reactor-PFR
PFR parameter
Volume of channel 2.96x10-8 m3
Volume of flow rate 5.41x10-11 m3/s
Quercetin
concentration
0.1, 0.05,
0.02
mol/
m3
DPPH concentration 0.25 mol/
m3
V : the reactor volume
F0 : molar flow rate of DPPH
molecules
r1 : reaction rate
x : conversion of DPPH+ to DPPH
V = F0
0
x
1
−r1
dx
Reaction constant: 2.807x10-2 m3mol-1s-1
real chip system
computer simulation
vs
 Examing the effect of volumetric
flow rate by computer model
 Compare the results by computer
model – real chip experiments
34

35. mathematical modeling
 Finite element analysis
Computer simulation by
COMSOL Multiphysics
COMSOL parameter
Quercetin
concentration
0.4, 0.2,
0.08
mol/
m3
DPPH concentration 0.5 mol/
m3
Velocity of ethanol 8.3x10-4 m/s
Velocity of quercetin 8.3x10-4 m/s
Velocity of DPPH 16.6x10-4 m/s
Diffusivity 1.26x10-8 m2/s
Reaction constant: 2.807x10-2 m3mol-1s-1
35

36. mathematical modeling
 Plug flow reactor-PFR  Finite element analysis
PFR parameter
Volume of channel 2.96x10-8 m3
Volume of flow rate 5.41x10-11 m3/s
Quercetin
concentration
0.1, 0.05,
0.02
mol/
m3
DPPH concentration 0.25 mol/
m3
Computer simulation by
COMSOL Multiphysics
COMSOL parameter
Quercetin
concentration
0.4, 0.2,
0.08
mol/
m3
DPPH concentration 0.5 mol/
m3
Velocity of ethanol 8.3x10-4 m/s
Velocity of quercetin 8.3x10-4 m/s
Velocity of DPPH 16.6x10-4 m/s
Diffusivity 1.26x10-8 m2/s
V : the reactor volume
F0 : molar flow rate of DPPH
molecules
r1 : reaction rate
x : conversion of DPPH+ to DPPH
V = F0
0
x
1
−r1
dx
Reaction constant: 2.807x10-2 m3mol-1s-1 Reaction constant: 2.807x10-2 m3mol-1s-1
36

37. 3Results - Discussion
37

38. Blank channel
no metabolism reaction
studying the performance of microfluidic system
no encapsulate enzyme
38

39. 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
39

40. 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 channel
Fig 12. Minimization the precipitation of DPPH inside the channel
DPPH
Quercetin
optimization microfluidic system
40

41. 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
41

42. a) Predicting final amounts of scavenged
radicals by PFR
Fig 13 Determining optimal flow rate by analytical mathematical model
optimization microfluidic system
PFR model
The realtionship between conversion-flo
rate
V = F0
0
x
1
−r1
dx
The using flow rate is suitable
 Flow rate: 5.41x10-11 m3s-1
Determing optimal flow rate by PFR computer model
42

43. 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
43

44. 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 scavenging
by computer model
44

45. radical scavenging reaction kinetics on a chip
 Determining reaction constant (k)
First-order reaction
−
dCDPPH
dt
=k CDPPHCquercetin
DPPH• + AH  DPPHH + A•
A-H: quercetin
45

46. 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
20M
50M 100M
 Determining reaction constant (k)
46

47. radical scavenging reaction kinetics
b) Initial reaction rate (at 1min)
Fig 14. Time dependent antioxidant activity of quercetin by usual colorimetry method
20M
50M 100M
k = 2.807 x 10-2 m3mol-1s-1
−
dCDPPH
dt
=k CDPPHCquercetin
Slope of the slot
k
 Determining reaction constant (k)
47

48. radical scavenging reaction kinetics
Fig 15. Time dependent antioxidant activity of quercetin on the chip
system
 Examing the reaction kinetics on the chipDPPH• + AH  DPPHH + A•
A-H: quercetin
Radical scavenged amount
48

49. 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 simulationvs
49

50. 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
50

51. Channel
with encaplsulated enzymes
studying effect of the metabolism process
to antioxidant activity
Quercetin is metabolized before enter 2nd part
51

52. Fig 17. Antioxidant activity of quercetin after various metabolic conditions
radical scavenging reaction kinetics
Co-factor: co-factor for glucuronidation
Quercetin
Metabolized
52

53. PHASE I PHASE II
Quercetin
Oxidation
Reduction
Hydrolysis
Hydration
Dethioacetylation
Isomerization
Glucosidation
Sulfation
Methylation
Acetylation
Amino acid conjugation
Glutathione conjugation
Hydrophobic Hydrophilic
Fig 17. Antioxidant activity of quercetin after various metabolic conditions
No metabolism
Phase I only
Phase I + 1 reaction phase
Phase I + Phase II
radical scavenging reaction kinetics
Co-factor: co-factor for glucuronidation53

54. 4Conclusion
54

55.  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
55

56. acknowledgements
soc. Prof. Dr. Napaporn Youngvises
56

57.  Your questions is welcome...
57

58. 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
58

59. 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.
bolic profile of quercetinQuercetin
PHASE I
Deglycosidation
PHASE II
Glucuronidation
Sulfation
O-methylation
59

60. antioxidant reduce a free radical
Ascorbate free radical formation
Antioxidants structural
Conjugated  system
Resonance
structure60

61. Fig 18. Initial reaction rate with various ethanol volume fraction in the solvent
hanol fraction on radical scavenging activity
61

62. omal reaction in static system
Fig 18. Amount of radical scavenged of quercetin under
various condition
Quercetin trapped
inside a PEGDA
hydrogel pillar62

63. 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 Marc63