nu tri chip
TRANSCRIPT
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NutriChip
Annual Plenary Meeting, Zrich, April 27th 2012
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NutriChipProject
NutriChipGroup Gijs: microfluidics bioMEMS
cell-based & particlehandling in systems
Group Ramsden: interfacial interactions inaqueous systems
cellomics (cell-on-chip) complex systems
modeling and design
Group Carrara:Integrated Nano-Bio-systems use of CMOS design andtechnology for bio-sensingpurposes
Group HurrellHuman nutrition strategies to combatmicronutrient deficiencies andchronic diseases theoretical aspects ofnutrition
Group Vergres:Biochemistry & Physiologyof dairy products
Human nutrition Nutrigenomics Biochemistry
http://www.microsens.ch/http://www.ayanda-biosys.com/index.html -
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General objective
To develop a microfluidic analytical platform forscreening the health-promoting properties of
milk and dairy products.
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60000 kg
(40000 pills)
20 kg
Food more than a confounding factor
For each gram ingested, the number of medical publications is 30000
times higher for drugs than for food (G. Vergres).
. . at the same time, 30-40% of cancers are associated with nutrition.
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NutriChip: biological principle
MilkLPSHigh-fat dietHigh-calory diet
Epithelium
Immune cell
Cytokines
lipopolysaccharide (LPS)
interleukins(IL),Tumor Necrosis Factor-a(TNF-a), ..
Can milk act againstinflammation?
Gastro-intestinal
Tract (GIT)
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NutriChip: biological principle
MilkLPSHigh-fat dietHigh-calory diet
Epithelium
Immune cell
Cytokines ?
lipopolysaccharide (LPS)
Interleukins (ILs),Tumor Necrosis Factor-a (TNF-a), ..
T. T. McDonald, Nature Medicine 16, 1194 (2010).
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NutriChip: a gut-on-a-chip
GastrointestinalTract (GIT)
Epithelial Cells(Caco-2)
Cytokinesdetection
Differentiatedmonocytes (U937)
Functionalizedmagnetic beads
Epithelial cellsculturing &
differentiation
Monocytesculturing &
differentiation
Epithelial cells (Caco-2)
Immune cells
Immunomagnetic
assay
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NutriChip project
Transwell-based GIT
NutriChip(icro-GIT)
CMOS imager
Human study
Calcium bio-availabality+ Ca-NutriChip
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Research in Progress
In vitrodigestion
Artificial Gastro-Intestinal Tract
High-resolutionImager Human Study
Transwell system Micro-GIT
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In vitrodigestion of dairy food
30 kDafiltration
- Gel electrophoresis- size-excl. High Performance Liquid Chromatography- OPA labeling of di-and tripeptides, free amino acids
37 oC
Analysis ofdigested product :
OPA: ortho-phtalaldehyde
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In vitrodigestion of dairy food
Pasteurized milk:undigested and
digestedat different stages
Undigested Digested
5 min Saliva
120 mingastric juice
30 minpancreaticjuice & bile
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Analysis by liquid chromatography-mass spectrometry (LC-MS) or MS
Peptide length distribution after in vitrodigestion of milk
In vitrodigestion of dairy food
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In vitrodigestion of dairy food
Quantification of individual free amino acids (AA) by HPLC
after Stage 3 digestion (Saliva, Gastric, Pancreatic juices and Bile)
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CMOS camera
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United Microelectronics Corp.0.18 m process
Mini ASIC :1525 m x 1525 m
Different photodiodes Different pixel architectures Different noise reduction circuits Multiphase clock generator
CMOS chip
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Synthetic image generation
Generation of random fluorophore clusterusing a Monte-Carlo approach Cell population
For each cell: fluorophore clustersgeneration
Imaging simulation from the location of thefluorophores Simulation of the optical system
(convolution with the point spread function) Simulation of the CCD/CMOS imager
(shadowing, noise, exposure time,sampling)
Simulated data used to test and validate fluorescence image
processing methods
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Image processing
Thresholding Adaptive image thresholdingFluorescent pixels
Average fluorescent
pixel intensity per cell
Number of fluorescentpixels per cell
Quantifying and evaluating the amount offluorescent targets from epi-fluorescencemicroscopy images = estimating the numberof fluorescent targets
Objective
~ measured number offluorescent targets
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Image processing
0 0.5 1 1.5 24
0
2000
4000
6000
8000
# surface receptors per cell
#
fluorescentpixels
percell
0 0.5 1 1.5 2
x 104
0
0.2
0.4
0.6
0.8
1
# surface receptors per cell
Fluore
scentpixels
average
intensity
Number of Fluorescent Pixels
Method (NFPM)
Average Fluorescent Intensity
Method (AFIM)
AFIM is linking number of fluorescent targets per
cell
average fluorescent pixel intensity
NFPM is linking number of fluorescent targets per
cell fluorescent pixel intensity per cell
Linear behavior, when pixels are notsaturated, can be used for low Nr:
~ Use of arctan fit function:
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Transwell: Caco-2 differentiation and integrity
0
100200
300
400
500
600
700
800
900
0 5 10 15 20 25
Relativeamounts[%]
time [days]
Alk.P. and Lct expression in Caco-2
ap lct
Alkaline phosphatase (AP) activity(signature of tight epithelial celljunctions)
Lactase expression (signature of
Caco-2 differentiation) Trans-epithelial electrical resistance
(TEER) Permeability to Lucifer Yellow
Caco-2 seeding inTranswell
21 days 10% in FBSculture medium
2 hours 0.2% serum
Treatmentwith dairy
products
Detection ofbiomarkers
T ll C 2 l t
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Transwell: Caco-2 layer response tostimulation
0
200
400
600
800
1000
1200
0 20 40 60 80 100 1 20
IL-6
[pg
/m
l]
TNF-a [ng/ml]
0
200
400
600
800
1000
1200
0 4000 8000 12000
IL-6
[pg
/m
l]
LPS [ng/ml]
Caco-2 layer is sensitive toTNF-, but not to LPS, in
terms of basolateral IL-6induction
IL-6 is measured by ELISA
TNF-a apical
TNF-a basolateral
0
20
40
60
80
100
120
140
160180
UT apical basolateral both
IL-6
[pg/ml]
T ll i t it f C 2 l
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LPS, TNF-a apical
Transwell: integrity of Caco-2 layerwithTNF-a and LPS stimulation
020406080
100120140
160180
Re
lativeTEER
LPS (1) 1 g/mlLPS (10) 10 g/ml
TNF-a applied at10 ng/mlconcentration
050100
150200250300350400450
Relative4
kDaFITC
p
ermea
bility
T ll lt t TNF d
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TNF-a, LPS apical
Transwell: co-culture response to TNF-a andLPS stimulation
0
1020
304050
60
70
80
Baso
latera
lIL-6[
pg
/m
l]
LPS (1) 1 g/mlLPS (10) 10 g/ml
TNF-a applied at10 ng/mlconcentration
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NutriChip: schematic and assay principle
Stimuli
TLRs
Cytokines
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Media flow velocity should be lessthan 0.1 m/s for safe shear stress oncells
Contours of wall shearstress (Pascal)
Contours of velocitymagnitude (m/s)
Flow
NutriChip: simulation of shear stress
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A procedure for irreversiblebonding of the porous membranein between two PDMS layers wasdeveloped
Cell culture -chamberwith perfusion channels
NutriChip: microfabrication aspects
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Epithelial cells (Caco-2) and monocytes (U937) cells are being separatelycultured in the microfluidic chips
Caco-2 U937
Cells on the NutriChip
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On-Chip immunomagnetic assay
Differential response of monocytic cells to LPS / milk stimulation
Ca NutriChip: Nutrichip for study of
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Ca-NutriChip: Nutrichip for study of
nutrikinetics
Free Ca2+ detection principle using FURA-2 dye
Free Ca2+
Exciter
Ca2+ binding to FURA-2 modulates itsemission intensity
Ratio
Ca NutriChip Nutrichip for study of
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Digestion of different cheeses & determination of the amount of Ca
in the digests by a colorimetric assay
Gruyre
Emmentaler
Tilsiter
Sbrinz
Vacherin fribourgeois
Appenzeller
3 mg ofproteindigestedwithout CaCl2in digestionjuices
Semi-hard and hard cheeses are rich sources of calcium
Ca-NutriChip: Nutrichip for study ofnutrikinetics
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Meal A
Meal B
Meal C
38 persons 3 times @ Inselhospital, Bern to consume
meals A, B, and C 5 blood samplings per Person/ Day/Testmeal 570 blood sampling time points
Every blood sampling time point: Serum (cytokines): measuring IL-6 in blood samples
obtained before and after ingestion of the meal Serum (insulin) Plasma (blood lipids, cholesterol, glucose, hs CRP) Serum (metabolites,)
500kcal
1000 kcal
1500 kcal
Human nutrition study
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Human nutrition study
In the media:
Salami essen fr die Forschung, St. Galler Tagblatt24.02.2012
Das Inselspital mstet Mnner, Blick am Abend22.02.2012
Essen fr die Forschung, NZZ am Sonntag19.02.2012
Mnner gesucht fr Salami-Zmorge, Berner Zeitung18.02.2012
Salami-Esser fr Studie gesucht, 20 Minuten
17.02.2012 Inselspital sucht Mnner fr Salami-Zmorge
Medienecho Infonlinemed16.02.2012 Inselspital sucht Mnner fr Salami-Zmorge
Medienmitteilung Medienmitteilung(Originaltext)16.02.2012
1 Test Meal A Test Meal B Test Meal C
2 Test Meal C Test Meal A Test Meal B
3 Test Meal B Test Meal C Test Meal A
4 Test Meal A Test Meal C Test Meal B
5 Test Meal C Test Meal B Test Meal A6 Test Meal C Test Meal A Test Meal B
7 Test Meal A Test Meal C Test Meal B
8 Test Meal B Test Meal C Test Meal A
9 Test Meal C Test Meal A Test Meal B
10 Test Meal C Test Meal B Test Meal A
11 Test Meal A Test Meal B Test Meal C
12 Test Meal C Test Meal A Test Meal B
13 Test Meal B Test Meal C Test Meal A
14 Test Meal C Test Meal A Test Meal B
15 Test Meal A Test Meal C Test Meal B
16 Test Meal B Test Meal A Test Meal C
17 Test Meal B Test Meal C Test Meal A18 Test Meal C Test Meal A Test Meal B
19 Test Meal C Test Meal B Test Meal A
20 Test Meal B Test Meal C Test Meal A
21 Test Meal B Test Meal A Test Meal C
22 Test Meal C Test Meal A Test Meal B
23 Test Meal B Test Meal C Test Meal A
24 Test Meal B Test Meal A Test Meal C
25 Test Meal B Test Meal C Test Meal A
26 Test Meal C Test Meal B Test Meal A
27 Test Meal A Test Meal B Test Meal C
28 Test Meal C Test Meal A Test Meal B
29 Test Meal A Test Meal B Test Meal C
30 Test Meal B Test Meal A Test Meal C31 Test Meal B Test Meal A Test Meal C
32 Test Meal A Test Meal C Test Meal B
33 Test Meal C Test Meal A Test Meal B
34 Test Meal B Test Meal A Test Meal C
35 Test Meal B Test Meal C Test Meal A
36 Test Meal C Test Meal A Test Meal B
37 Test Meal A Test Meal B Test Meal C
38 Test Meal A Test Meal C Test Meal B
39 Test Meal B Test Meal A Test Meal C
40 Test Meal B Test Meal A Test Meal C
Leansubjects
Obeses
ubjects
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Human study: first results
Cumulative Distribution Function of the area under the curve (AUC)
for the 0 h to 6 h blood sampling period for:- hs-CRP, C reactive protein, a marker of inflammation- glucose
Values for glucose and hs-CRP are systematically higher in the obesesubjects.
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March 15th 2012
St t f th j t
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Status of the project
A viable cell co-culture system in Transwell is established
Second version of the NutriChip device being fabricated
Demonstration of fluorescence imaging using custom-
assembled CMOS camera Establishment of protocol to prepare in vitrodigested food
Ca-availability in dairy products investigated
First results obtained from human nutrition study
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Thanks for your attention!
The NutriChip team
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Backup slides
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Postprandial Inflammation
A. N. Margioris, Current Opinion in Clinical Nutrition and Metabolic Care 2009, 12:129137
1 2 3 4 5 6 7 8
Postprandial time in hours
Inflammationand
oxid
ationmarkers
Meal
Maladaptationdysmetabolism
NormalAdaptation +2SD
Meals high in calories, carbohydrate and Saturatedfatty acidsproduce a strong postprandialimmune/inflammatory response;
Postprandial stress as an indicator of
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1 2 3 4 5 6 7 8
Postprandial time in hours
Inflammationand
oxidationmarkers
Meal
Maladaptationdysmetabolism
NormalAdaptation
+2SD
A. N. Margioris, Current Opinion in Clinical Nutrition and Metabolic Care 2009, 12:129137
Postprandial stress as an indicator ofmetabolic health
Meals high in calories, carbohydrate and Saturated fatty acids producea strong postprandial immune/inflammatory response;
Characterization of milk products
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Characterization of milk products
Collection of data of 2-D gel electrophoresis and LC-MS identification inan interactive database
Information about identified proteins (Uniprot)
Result tables and comparison between differentdairy products possible
From 15 selected dairy products ~ 2000 proteins wereidentified (450 were different proteins) Each product has a unique proteome and might
produce a different inflammatory response
In vitro digestion of milk
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In vitrodigestion of milk
Digestion of pasteurized milk
past.milk
salivapH 6.8
gastric
juicepH 2-3
pancreatic juicepH 6.5-7
bilepH 6.5-7
5 min 2 h 2 h
Applicationon the cell
culturesystem
(modified from Versantvoort et al., 2005)
Model Versantvoort: used for the detection of bioavailability ofmycotoxins
Aim in our study: detect effect on inflammation
+ Analysis ofmacro-nutrient
digestion
Protein analysis
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M1 M2 M4M3
M1= 5 min SalivaM2= 120 min Gastric juiceM3= 120 min Pancreatic juice + bileM4= 120 min Pancreatic juice
milkproteins
Proteins of digestionenzymes
Protein analysis
beta-lactoglobulin
Digestion of pasteurized milk: analysis of macronutrient digestionin the case of proteins
Major milk proteins (caseins) are degraded with saliva and gastric juice.The whey protein beta-lactoglobulin gets only digested in the presence of bothpancreatic juice and bile.
In Vitro digestion of dairy food
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Proteins aredegraded in the invitrodigestion
experiments
In Vitro digestion of dairy food
Undigested
Digested
In Vitro digestion of dairy food
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Determination of free amino acids, di-and tripeptides1 HF g stand
2 HF kcal stand
5 Gruyere g stand
7 Gruyere kcal stand
9 yoghurt nature g stand
11 yoghurt nature kcal stand
13 milk g stand
15 milk kcal stand
In Vitro digestion of dairy food
Undigested Digested Not filtered Digested, filtered
The CMOS Imaging System
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Quantitative Comparison between a Custom-Designed
CMOS and a Commercial CCD Camera
The CMOS Imaging System
CMOS Sensor - BeforeNoise Removal
CMOS Sensor - Master DarkFrame (MDF)
CMOS Sensor - After FPNRemoval and Median Filt. (3, 3)
CCD Sensor - After MedianFiltering (3, 3)
CMOS Sensor - After Filteringand Thresholding
CCD Sensor - After Filteringand Thresholding
Fluorescence imaging: CMOS sensor
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Small area active pixel sensors
4T Active Pixel Sensor- 0.18 m standard CMOS process
High signal-to-noise ratio Noise Reduction Circuitry:
- 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
Fluorescence imaging: CMOS sensor
CMOS sensor and camera
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CMOS sensor and camera
A commercial CMOS imager was compared with
a CCD imager
A custom-built camera system was implemented
and tested
High quality images were obtained using the
CMOS camera
CMOS imaging chip is in test
CMOS sensor
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CMOS sensor
Fully Differential
Switched CapacitorCorrelated DoubleSampling with Offset
Compensation
Signal to Noise Ratio of theTwo CDS Architectures
FDSC
CDS
FDSC CDS
WithOffsetComp
ActivePixelSensorBlocks
Buffers
Imager_v1 Tape-out March, 2011currently under test
UMC 0.18 Standard CMOS Process
Multi-phaseCLKGEN
Fully DifferentialSwitched CapacitorCorrelated Double
Sampling
SNRcomparison
Transwell based GIT
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Transwell-based GIT
Biologically active in vitrocellular systemthat responds to the stimuli LPS/Milk,by modifying concentrations of IL-1/6,TNF-
Trans-epithelialelectrical resistance(TEER) of Caco-2layer
Alkaline Phosphatase
& Lactase aredifferentiationindicators
Epithelial
(Caco-2)
Immune cells(U937)
Ca-NutriChip
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Ca-NutriChipNutrichip with a nutrikinetic capability
Ca Ratio imaging usingFURA-2 & FRET
Ca-NutriChip
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Ca-NutriChipDigestion of Emmental cheese with/without CaCl2 in the digestion juices
Emmental Cheese(1,2,3 and 4g)
digested with/without CaCl2
Amount of free amino acids Amount of free fatty acids
Ca-NutriChip
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Ca-NutriChipDigestion of different dairy products to determine Ca2+ amount in the digests
5min 2h 2h
200 L salivapH 6.8
400 L gastric juicepH 2-3
400 L pancreaticjuice pH 6.5-7
200 L bilepH 6.5-7
&
+ +
Saliva contains 2 mmol/L of Ca2+
Gastric juice contains 0.6 mmol/L of Ca2+
Pancreatic juice contians 0.6 mmol/L of Ca2+
Bile contains 3.7 mmol/L of Ca2+
CaCl2 OMITTED from the digestion!
Filtration of thedigestthrough 30
kDa
Ca-NutriChip
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Ca-NutriChipDigestion with/without CaCl2 in the digestion juices
Milk
YogurtGruyreCheese
digestedwith &
withoutCaCl2
Amount of free amino acids
Peptide lengthAmount of free fatty acids
Ca-NutriChip
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Amount of free amino acids
mMo
ffreeaminoacids
Ca NutriChipDigestion with/without CaCl2 in the digestion juices
The efficency of digestion is not influenced by omittingcalcium chloride from the digestion juices
Fat & proteins in milk, yogurt and cheese are well digested.
Ca-NutriChip
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Ca NutriChip
Stimuli: various dairyproducts
Ca-NutriChip
Epithelial Cells
(Caco-2)
Ca2+Ca2+ Ca2+ Ca2+
Transported Ca2+through Caco-2 cells
Ca2+ uptake by targetcells
THP-1 cells
Osteoblast-like cells
Magnetic beads
Ca2+Ca2+ Ca2+ Ca2+
Target cells (Ca2+ sink)
Variety of dairyproduct-based Caconcentrations.
Extending the functionality of Nutrichip with a nutrikinetic capability
Ca-NutriChip: Nutrichip for study
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No Ca2+
No FRET
+ Ca2+
CFP & YFP are close
FRET
440 nm
480 nm 530 nm
Ratio
Intracellular Ca2+ imaging with Frster resonance energy transfer (FRET)
p p yof nutrikinetics
Human nutrition study
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Human nutrition study
High-fat (HF) meals
BreadSalamiEggsPalm oil
Blood samplingDay: d1, d8, d15Time: 0h (fasting)
1h, 2h, 4h, 6h (postprandial)
Analytics:Metabolism: glucose, TG, insulinInflammation: hs CRP, IL-6, TNF-, IL-1, IL-8, IL-10, TLR-2, TLR-4Nutrigenomics: serum metabolomics, blood cell transcriptomics
Subjects20 healthy volunteers20 obese volunteers
500 kcal 1000 kcal 1500 kcalMacronutrientsFatCarbohydrates: 21%Proteins: 18%
The NutriChip Platform
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NutriChip(micro- Gastro-Intestinal Tract)
CMOSImager
Optics
ImagerProcessor
Interfacing andcontrol unit
The NutriChip Platform
Dairy food analysisresults
Publications and media coverage
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Publications and media coverage
Journals4 submissions
Conferences25 oral and poster presentations
Media coverage
http://www.sf.tv/http://www.cdrf.org/