The core of the system is an integrated chip, the NutriChip, which, as a demonstrator of an artificial and miniaturized gastrointestinal tract, will be able to probe the health potential of dairy food samples, using a minimal biomarker set identified through in vivo and in vitro studies. The project will develop innovative CMOS circuits at the nano-scale for high signal-to-noise ratio optical detection and propose a special microfluidic system closely integrating cell-based materials within the chip. The NutriChip will be tested for screening and selection of dairy products with specific health-promoting properties, in particular immunomodulatory properties. The CMOS detection chip will be used to image down to single immune cells. For the biochemical validation of the NutriChip platform, the response of the immune cells upon the application of food will be examined by monitoring the Toll-like receptors 2 and 4, key molecules bridging metabolism and immuno-regulation in nutrition.

1. NutriChip
A technological platform for nutrition
analysis to promote healthy food

2. Contributing partners & competences
Group Hurrell
Human nutrition
 strategies to combat micronutrient
deficiencies and chronic diseases
 theoretical aspects of nutrition Group Vergères:
Group Gijs:
Biochemistry & Physiology
 microfluidics of dairy products
 bioMEMS  Human nutrition
 cell-based & particle  Nutrigenomics
handling systems  Biochemistry
NutriChip ALP
Project
Group Ramsden: Group Carrara:
 interfacial interactions in Integrated Nano-Bio-systems
aqueous systems  use of CMOS design and
 cellomics (cell-on-chip) technology for bio-sensing
 complex systems purposes
modeling and design
NutriChip

3. General objective
To develop a microfluidic analytical platform for
screening the health-promoting properties of milk and
dairy products.
NutriChip

4. The NutriChip platform
The NutriChip project provides a platform for testing the impact
of dairy food digestion on human health by monitoring
inflammation biomarkers.
• Immuno-competent
artificial micro-gastro-
intestinal tract ( GIT)
• Interfaced with an
integrated control system
• High-resolution, high-S/N
CMOS imager
• Validated by human
nutrition trial
NutriChip

5. NutriChip: development phases
1 2 3 4 5 6 7
Active phases
NutriChip

6. In vitro digestion of milk
Digestion of pasteurized milk
pancreatic juice
gastric pH 6.5-7
saliva
juice +
pH 6.8 bile Application
pH 2-3 Analysis of
pH 6.5-7 on the cell
past.milk macro-nutrient
5 min 2h 2h digestion culture
system
(modified from Versantvoort et al., 2005)
Model Versantvoort: used for the detection of bioavailability of
mycotoxins
Aim in our study: detect effect on inflammation
NutriChip

7. Protein analysis
Digestion of pasteurized milk: analysis of macronutrient digestion
in the case of proteins
M1 M2 M3 M4
M1 = 5 min Saliva
M2 = 120 min Gastric juice
M3 = 120 min Pancreatic juice + bile
M4 = 120 min Pancreatic juice
Proteins of digestion
enzymes
milk
proteins
beta-lactoglobulin
Major milk proteins (caseins) are degraded with saliva and gastric juice.
The whey protein beta-lactoglobulin gets only digested in the presence of both
pancreatic juice and bile.
NutriChip

8. Artificial GIT (Transwell)
• ‘Macroscopic’ in vitro cellular
system that allows to set cell
culture parameters and
responds to
lipopolysaccharide (LPS) and
milk stimuli, and allows
monitoring of biomarkers IL-6
and IL-1 , TLR-2/4.
• This setting mimics the passage
of nutrients through the human
GIT (digestion, transport trough
the epithelial cell layer, and the
activation of the underlying
immune system).
NutriChip

9. Caco-2 differentiation and integrity
2 hours 0.2% serum
21 days 10% in FBS
culture medium Detection of biomarkers
Treatment
Caco-2 seeding in with dairy
Transwell products
• Alkaline phosphatase (AP) activity 900
800 Alk.P. and Lct expression in Caco-2
Relative amounts [%]
(signature of tight epithelial cell 700
600
junctions) 500
400
• Lactase expression (signature of 300
200
Caco-2 differentiation) 100
• Trans-epithelial electrical resistance
0
0 5 10 15 20 25
(TEER) time [days]
• Permeability to Lucifer Yellow ap lct
NutriChip

10. Application of digested milk
Apply directly on the epithelial cell layer
(without filtration)
In vitro digestion
With filtration <30 kDa (Centricon column
to remove Caco-2 cytotoxic enzymes
originating from bile)
Trans-epithelial electrical resistance
UT : untreated
NF M: non-filtered digested
milk
NF W: non-filtered digestion
buffer
M<30 kDa : digested milk
passed through >30kDa
exclusion column
NutriChip

11. IL-1 / IL-6 expression by THP-1 cells
IL-1
IL-6
T. T. McDonald, Nature Medicine, 16(11), 1194, 2010
• Differentiation of THP-1 cells by phorbol 12-myristate 13-acetate (PMA)
macrophage-activating factor
• IL-6 basal level is very low and IL-1 basal level is relatively high upon
stimulation with lipopolysaccharide(LPS)
NutriChip

12. micro-GIT (NutriChip)
• Co-culturing of epithelial cells
(Caco-2) and immune cells (THP-1)
on both sides of a miniaturized
porous membrane.
• The device integrates the processes
used in typical cell culture
experiments in a single self-
contained microfluidic system.
• Major functions include repeated
cell growth/passage cycles, reagent
introduction and real time monitoring
of culture environment.
NutriChip

13. micro-GIT (Nutrichip)
NutriChip

14. micro-GIT (NutriChip)
PDMS bonded chip with polycarbonate membrane
NutriChip

15. Fluorescence imaging: CMOS sensor
Optical/CMOS detection unit
• High resolution photodiode-based pixels.
• High signal-to-noise detection CMOS
circuits
• Pixel spatial super-resolution
• Automatic image processing
NutriChip

16. Fluorescence imaging: CMOS sensor
• Small area active pixel sensors
• 4T Active Pixel Sensor ✓
- 0.18 m standard CMOS process
• High signal-to-noise ratio Courtesy of Rene Beuchat
• Noise Reduction Circuitry: ✓ LAP, EPFL
- Switched Capacitor Fully Differential Offset Compensated Correlated Double Sampling
• High resolution
• 12-14 bit ADC:
- Successive Approximation or Cyclic Analog to Digital Converter per Column
• Compact interface with -aGIT
NutriChip

17. Fluorescence imaging: CMOS sensor
Fully Differential
Switched Capacitor
Correlated Double
Sampling
Buffers
Active Pixel Sensor Blocks
Multi-
phase
FD CLK
SC GEN
CDS Fully Differential
Switched Capacitor
Correlated Double
FD
Sampling with Offset
SC CDS
Compensation
With
Offset
Comp
Signal to Noise Ratio of the
Two CDS Architectures SNR
comparison
Imager_v1 – Tape-out March, 2011
UMC 0.18 Standard CMOS Process
G. Koklu, Y. Leblebici, S. Carrara, A Switched Capacitor Fully Diffferential Correlated Double Sampling Circuit
for CMOS Image Sensors, ISMICT 2011, Montreux, Switzerland
NutriChip

18. Synthetic image generation
Simulated data are used as a tool to test and validate
image processing methods.
(i) Generation of random fluorophore cluster
using a Monte-Carlo approach
• Cell population
• For each cell: fluorophore clusters
generation
(ii) Imaging simulation from the location of the
fluorophores
• Simulation of the optical system
(convolution with the point spread function)
• Simulation of the CCD/CMOS imager
(shadowing, noise, exposure time,
sampling…)
NutriChip

19. Nutritional trials
Validation of the NutriChip results in a
human nutrition trial
 Testing the ability of dairy products to
decrease IL-6 and TLR-2/4 on the
surface of immune cells following daily
ingestion of these products for 4 weeks.
 Postprandial inflammation.
 The results from the whole set of
analytical parameters will be discussed
globally to draw mechanistic
conclusions regarding the physiological
and nutritional properties of the dairy
products tested in the human trial.
NutriChip

20. 1st postprandial trial
High-fat (HF) meals
Bread Macronutrients:
500 kcal 1‘000 kcal 1‘500 kcal
Salami Fat:
Eggs Carbohydrates: 21%
Protein: 18%
Palm oil
Subjects
20 healthy volunteers
20 obese volunteers
Blood sampling
Day: d1, d8, d15
Time: 0h (fasting)
1h, 2h, 4h, 6h (postprandial)
Analytics:
Metabolism: glucose, TG, insulin
Inflammation: hs CRP, IL-6, TNF-α, IL-1β, IL-8, IL-10, TLR-2, TLR-4
Nutrigenomics: serum metabolomics, blood cell transcriptomics
NutriChip

21. Summary
• Caco-2 cells confluency and biomarker detection in
Transwell device demonstrated
• 1st generation micro-gastro intestinal tract device
realized; cell co-culture started
• First tape-out of CMOS detection chip, work on
super-resolution algorithms
• Major released amino acids due to milk in vitro
digestion identified and protein digestion studied in
vitro
• 1st human nutrition study started
• NutriChip scope will be extended to study the Ca
bio-availability
NutriChip

22. Thanks for your attention!
The NutriChip team
NutriChip

23. Backup slides
Freitag, 3. Februar 2012 Departement/Institut/Gruppe
NutriChip 1

24. Motivation and relevance
• Consumption of suitable food & food supplements can
contribute to the prevention of diseases (e.g. diabetic disorders,
cardiovascular diseases, cancer).
• Boosting the nutritional profile helps dairy products effectively
to compete with new established functional foods.
Per capita consumption of livestock products
Region Meat (kg per year) Milk (kg per year)
1964-1966 1997-1999 2030 1964- 1997- 2030
1966 1999
World 24.2 36.4 45.3 73.9 68.1 89.5
Developing countries 10.2 25.5 36.7 28.0 44.6 65.8
Industrialized countries 61.5 88.2 100.1 185.5 212.2 221.0
Transition countries 42.5 46.2 60.6 156.6 159.1 178.7
FAO/WHO consultation on food consumption and exposure assessment to chemicals in food. Geneva, Switzerland, Feb 1997
NutriChip

25. Characterization of milk products
Collection of data of 2-D gel electrophoresis and LC-MS identification in
an interactive database
Information about identified proteins (Uniprot)
Result tables and comparison between different
dairy products possible
From 15 selected dairy products ~ 2000 proteins were
identified (450 were different proteins)
 Each product has a unique proteome and might
produce a different inflammatory response
NutriChip

26. Application of digested milk
Conclusion: digested products (milk or buffer) have to be passed through
a Centricon <30 kDa to loose their cytotoxic properties. The data suggest
that elimination of digestion enzymes is a crucial step to perform these
experiments
NutriChip

27. Characterization of milk products
• To test the ability of undigested and
digested milk to inhibit the elevated
expression of TLR-2/4 and IL-6 on
THP-1 human immune cells.
• To establish a standard operating
procedure to prepare in vitro digested
milk.
• Selection of the pro-inflammatory
environment used to activate TLR-2/4
and IL-6 .
NutriChip

28. TLR-2/4 expression on THP-1 cells
Tlr-2 mRNA expression
TLR-2 mRNA expression
250
200
150
[%]
100
50
0
ctrl 6h 24h
350
W. Strober, Nature Medicine, 16(11), 1195, 2010 Tlr-4 mRNA expression
TLR-4 mRNA expression
300
• LPS stimulation induces TLR-2/4 250
200
expression on surface of THP-1
[%]
150
macrophages
100
• TLR-2 up-regulation is more rapid 50
than TLR-4 one. 0
• Both TLR-2 and TLR-4 inductions ctrl 6h 24h
are relatively strong.
NutriChip

29. TLR-2/4 expression
immunofluorescence)
Readout of 2 TLR-2 and -4 immunofluorescence
NutriChip

30. Chronic inflammation
Inflammation is a major contributor to many chronic diseases, including
obesity.
An unhealthy diet may induce an elevated postprandial inflammation and
contribute, via a positive feedback loop, to the development of low grade
systemic chronic inflammation.
Margioris, Current Opinion in Clinical Nutrition and Metabolic Care 2009, 12:129–137
NutriChip

31. Microfluidics-based aGIT
 Continuous perfusion flow
of media which ensures fresh
medium & waste removal &
dynamic cell culture
NutriChip

32. Distribution of fluorophore clusters
Counting the amount of clusters with 1,2,3,… fluorophores
gives an estimation of the amount of stained biomarkers within
the image field of view.
(i) The light intensity distribution received by
the imager from a cluster with c fluorophores is
modeled by:
æ J ö
I(x, y) = ç Õ Fj (x, y)÷ cI
ç ÷
Random variable due to measurement processes
è j=1 ø Number of fluorophores in the cluster
Intensity of a single-fluorophore (constant)
(ii) A fitting algorithm can estimate the amount of clusters with a given
amount of fluorophores using:
• This model
• Calibration data (intensity distribution for c=1)
• Measured data (intensity distribution of the n
image signal) yi = å ac fc (i)
S.A Mutch et al., Biophysical journal, Vol 92, April 2007, 2926-2943
c=1
NutriChip

33. Postprandial inflammation
Postprandial inflammation is a normal reaction of the immune system
induced by food consumption.
Postprandial inflammation may be sustained in patients with a disturbed
metabolism, e.g. in the case of obesity or diabetes.
Margioris, Current Opinion in Clinical Nutrition and Metabolic Care 2009, 12:129–137
NutriChip

34. Nutritional trials
Study Content Aim
1st Postprandial inflammation study with -Dose-and time-response relation of
high fat meals different kcal meals on inflammatory
markers in blood
-Difference in postprandial
response between healthy
and obese subjects
2nd Postprandial inflammation study -Effect of milk on postprandial
comparing high fat meal with inflammation (time-response)
effect of milk product and soy (isocaloric)
3rd Long-term intervention study with milk -Long-term effect of milk on chronic
and isocaloric soy product inflammation
(fat content 3.5%)
Analysis: classical clinical parameters, inflammation markers (IL-6, TNF-α), proteins of interest
(TLR-4, TLR-2), metabolomics, transcriptomics
NutriChip

35. Outlook: Ca-NutriChip platform
Variety of dairy
product-based Ca
concentrations Stimuli: various dairy
Epithelial Cells products
(Caco-2)
Porous membrane
Ca2+ Ca2+ Ca2+ Ca2+
 Transported Ca2+
through Caco-2 cell
Ca2+ Ca2+ Ca2+ Ca2+
layer
Target cells (Ca2+ sink)  Ca2+ uptake by target
cells
 THP-1 cells
Ca-NutriChip  Osteoblast-like cells
FRET
NutriChip

36. General Objectives
 To extend the functionality of the original Nutrichip
platform with a nutrikinetic capability, investigating
the bio-availability of calcium from dairy food as a
model.
 To quantitatively monitor the adsorption and
transport of calcium through the epithelial cell layer
as well as it’s uptake by target cells.
 To develop the Ca-Nutrichip platform, capable of
investigating other types of nutrition constituents in
future.
NutriChip

37. Motivation
 There is an increased interest in the role that nutrients may
play in preventing or alleviating the effect of major diseases
(e.g., some types of cancer and cardiovascular diseases).
The bioavailability (adsorption and transport) of an ingested
nutrient is even more relevant than the total amount in the
original food.
The NutriChip structure, with Caco-2 cells, can be used as a
basis to assess human Calcium bioavailability from dairy
food.
Extending the scope of NutriChip with a new functionality (Ca
bioavailability) would widen its applications in two fields with
growing interest, namely nutrikinetics & pharmacokinetics.
NutriChip

38. Ca-NutriChip workflow
 Establishment of an in vitro Transwell culture system that
responds to Ca stimuli in dairy food.
 Design & Fabrication of Ca-NutriChip (Ca-assay, different
cell types, magnetic beads).
 Testing milk with Ca-NutriChip.
 Screening a number of dairy product with Ca-NutriChip.
 Matrix formulation: Protein, fat, and vitamin D will be
varied in the dairy products to investigate their role in Ca
uptake.
NutriChip