NutriChip A technological platform for nutrition

analysis to promote healthy food

NutriChip

NutriChip Project

Contributing partners & competences

ALP

Group Gijs: microfluidics bioMEMS cell-based & particle

handling systems

Group Vergères:Biochemistry & Physiology of dairy products Human nutrition Nutrigenomics Biochemistry

Group Ramsden: interfacial interactions in

aqueous systems cellomics (cell-on-chip) complex systems

modeling and design

Group Carrara: Integrated Nano-Bio-systems use of CMOS design and

technology for bio-sensing purposes

Group HurrellHuman nutrition strategies to combat micronutrient

deficiencies and chronic diseases theoretical aspects of nutrition

NutriChip

General objective

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

NutriChip

The NutriChip platform

• 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

The NutriChip project provides a platform for testing the impact of dairy food digestion on human health by monitoring inflammation biomarkers.

NutriChip

-aGIT

aGIT

stud

y

CMOS d

etecti

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syste

mTe

sting

milk

on

NutriC

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Human

nut

rition

trial

1 2 3 4 5 6 7

Integ

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Nut

riChip

platfo

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NutriC

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NutriChip: development phases

Active phases

NutriChip

In vitro digestion of milk

Digestion of pasteurized milk

past.milk

salivapH 6.8

gastric juice

pH 2-3

pancreatic juicepH 6.5-7

bile pH 6.5-7

5 min 2 h 2 h

Application on the cell

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

+Analysis of

macro-nutrient digestion

NutriChip

M1 M2 M4M3

M1 = 5 min SalivaM2 = 120 min Gastric juiceM3 = 120 min Pancreatic juice + bileM4 = 120 min Pancreatic juice

milkproteins

Proteins of digestion enzymes

Protein analysis

beta-lactoglobulin

Digestion of pasteurized milk: analysis of macronutrient digestion in 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 both pancreatic juice and bile.

NutriChip

• ‘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-1b, 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).

Artificial GIT (Transwell)

aGIT

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NutriChip

Caco-2 differentiation and integrity

0100200300400500600700800900

0 5 10 15 20 25

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time [days]

Alk.P. and Lct expression in Caco-2

ap lct

Caco-2 seeding in Transwell

21 days 10% in FBS culture medium

2 hours 0.2% serum

Treatmentwith dairy products

• Alkaline phosphatase (AP) activity (signature of tight epithelial cell junctions)

• Lactase expression (signature of

Caco-2 differentiation)• Trans-epithelial electrical

resistance (TEER)• Permeability to Lucifer

Yellow

Detection of biomarkers

NutriChip

Application of digested milk

In vitro digestion

Apply directly on the epithelial cell layer (without filtration)

With filtration <30 kDa (Centricon column to remove Caco-2 cytotoxic enzymes originating from bile)

UT : untreatedNF M: non-filtered digested milk NF W: non-filtered digestion buffer

M<30 kDa : digested milk passed through >30kDa exclusion column

Trans-epithelial electrical resistance

NutriChip

IL-1 b / IL-6 expression by THP-1 cells

• Differentiation of THP-1 cells by phorbol 12-myristate 13-acetate (PMA) macrophage-activating factor

• IL-6 basal level is very low and IL-1b basal level is relatively high upon stimulation with lipopolysaccharide(LPS)

T. T. McDonald, Nature Medicine, 16(11), 1194, 2010

IL-6

IL-1b

NutriChip

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.

µ-GIT

NutriChip

micro-GIT (Nutrichip)

NutriChip

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

NutriChip

Optical/CMOS detection unit

Fluorescence imaging: CMOS sensor

• High resolution photodiode-based pixels.

• High signal-to-noise detection CMOS circuits

• Pixel spatial super-resolution

• Automatic image processing

CMOS sys

tem

NutriChip

• Small area active pixel sensors

• 4T Active Pixel Sensor ✓- 0.18 mm 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 m-aGIT

Fluorescence imaging: CMOS sensor

Courtesy of Rene Beuchat LAP, EPFL

NutriChip

Fluorescence imaging: CMOS sensor

Fully Differential Switched Capacitor Correlated Double

Sampling with Offset Compensation

Signal to Noise Ratio of the Two CDS Architectures

FDSC

CDS

FDSC CDS

WithOffset Comp

Act

ive

Pix

el S

en

sor

Blo

cks

Bu

ffe

rs

Imager_v1 – Tape-out March, 2011UMC 0.18 Standard CMOS Process

Multi-phase CLKGEN

Fully Differential Switched Capacitor Correlated Double

Sampling

SNR comparison

G. Koklu, Y. Leblebici, S. Carrara, A Switched Capacitor Fully Diffferential Correlated Double Sampling Circuit for CMOS Image Sensors, ISMICT 2011, Montreux, Switzerland

NutriChip

Synthetic image generation

(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…)

Simulated data are used as a tool to test and validate image processing methods.

NutriChip

Validation of the NutriChip results in a human nutrition trial

Nutritional trials

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.

Nutritio

n trai

l

NutriChip

1st postprandial trialHigh-fat (HF) mealsBread SalamiEggsPalm oil

Blood samplingDay: d1, d8, d15Time: 0h (fasting)

1h, 2h, 4h, 6h (postprandial)

Analytics: Metabolism: glucose, TG, insulinInflammation: hs CRP, IL-6, TNF-α, IL-1β, IL-8, IL-10, TLR-2, TLR-4Nutrigenomics: serum metabolomics, blood cell transcriptomics

Subjects20 healthy volunteers20 obese volunteers

500 kcal 1‘000 kcal 1‘500 kcalMacronutrients:Fat:

Carbohydrates: 21%Protein: 18%

NutriChip

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

Thanks for your attention!

The NutriChip team

NutriChip

Backup slides

Dienstag, 18. April 2023

1

Departement/Institut/Gruppe

NutriChip

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 1964-1966

1997-1999

2030

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

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

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

• 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 .

Characterization of milk products

Milk

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NutriChip

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ctrl 6h 24h

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Tlr-2 mRNA expression

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Tlr-4 mRNA expression

• LPS stimulation induces TLR-2/4 expression on surface of THP-1 macrophages

• TLR-2 up-regulation is more rapid than TLR-4 one.

• Both TLR-2 and TLR-4 inductions are relatively strong.

TLR-2/4 expression on THP-1 cells

TLR-2 mRNA expression

TLR-4 mRNA expressionW. Strober, Nature Medicine, 16(11), 1195, 2010

NutriChip

Readout of 2 TLR-2 and -4 immunofluorescence

TLR-2/4 expression immunofluorescence)

NutriChip

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

Microfluidics-based aGIT

Continuous perfusion flow of media which ensures fresh medium & waste removal & dynamic cell culture

NutriChip

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:

Random variable due to measurement processes

Number of fluorophores in the cluster

Intensity of a single-fluorophore (constant)

• This model• Calibration data (intensity distribution for c=1)• Measured data (intensity distribution of the

image signal)S.A Mutch et al., Biophysical journal, Vol 92, April 2007, 2926-2943

(ii) A fitting algorithm can estimate the amount of clusters with a given amount of fluorophores using:

NutriChip

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

Nutritional trialsStudy Content Aim

1st Postprandial inflammation study with high fat meals

-Dose-and time-response relation of different kcal meals on inflammatory markers in blood-Difference in postprandial response between healthy and obese subjects

2nd Postprandial inflammation study comparing high fat meal with effect of milk product and soy (isocaloric)

-Effect of milk on postprandial inflammation (time-response)

3rd Long-term intervention study with milk and isocaloric soy product

-Long-term effect of milk on chronic 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

Outlook: Ca-NutriChip platform

Porous membrane

Stimuli: various dairy products

Ca-NutriChip

Epithelial Cells (Caco-2)

Ca2+Ca2+ Ca2+ Ca2+ Transported Ca2+

through Caco-2 cell layer

Ca2+ uptake by target cells

THP-1 cells

Osteoblast-like cells

Ca2+Ca2+ Ca2+ Ca2+

Target cells (Ca2+ sink)

Variety of dairy product-based Ca concentrations

FRET

NutriChip

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

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

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.