Nu Tri Chip

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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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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



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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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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




10 Test Meal C Test Meal B Test Meal A






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












30 Test Meal B Test Meal A Test Meal C31 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
http://www.sf.tv/


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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


Undigested

Digested



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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


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



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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/

Nu Tri Chip

Documents

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