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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.

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  1. 1. NutriChipA technological platform for nutrition analysis to promote healthy food
  2. 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 ProjectGroup Ramsden: Group Carrara: interfacial interactions in Integrated Nano-Bio-systemsaqueous systems  use of CMOS design and cellomics (cell-on-chip) technology for bio-sensing complex systems purposesmodeling and design NutriChip
  3. 3. General objectiveTo develop a microfluidic analytical platform forscreening the health-promoting properties of milk anddairy products. NutriChip
  4. 4. The NutriChip platformThe NutriChip project provides a platform for testing the impactof dairy food digestion on human health by monitoringinflammation 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. 5. NutriChip: development phases1 2 3 4 5 6 7 Active phases NutriChip
  6. 6. In vitro digestion of milkDigestion 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 cellpast.milk macro-nutrient 5 min 2h 2h digestion culture system (modified from Versantvoort et al., 2005)Model Versantvoort: used for the detection of bioavailability ofmycotoxinsAim in our study: detect effect on inflammation NutriChip
  7. 7. Protein analysisDigestion of pasteurized milk: analysis of macronutrient digestionin 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-lactoglobulinMajor 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. NutriChip
  8. 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. 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. 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. 11. IL-1 / IL-6 expression by THP-1 cells IL-1 IL-6T. 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. 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. 13. micro-GIT (Nutrichip) NutriChip
  14. 14. micro-GIT (NutriChip)PDMS bonded chip with polycarbonate membrane NutriChip
  15. 15. Fluorescence imaging: CMOS sensorOptical/CMOS detection unit• High resolution photodiode-based pixels.• High signal-to-noise detection CMOS circuits• Pixel spatial super-resolution• Automatic image processing NutriChip
  16. 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. 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 comparisonImager_v1 – Tape-out March, 2011UMC 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. 18. Synthetic image generationSimulated data are used as a tool to test and validateimage processing methods.(i) Generation of random fluorophore clusterusing a Monte-Carlo approach• Cell population• For each cell: fluorophore clusters generation(ii) 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…) NutriChip
  19. 19. Nutritional trialsValidation of the NutriChip results in ahuman 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. 20. 1st postprandial trialHigh-fat (HF) mealsBread Macronutrients: 500 kcal 1‘000 kcal 1‘500 kcalSalami Fat:Eggs Carbohydrates: 21% Protein: 18%Palm oilSubjects20 healthy volunteers20 obese volunteersBlood 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 NutriChip
  21. 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. 22. Thanks for your attention! The NutriChip team NutriChip
  23. 23. Backup slidesFreitag, 3. Februar 2012 Departement/Institut/Gruppe NutriChip 1
  24. 24. Motivation and relevance• Consumption of suitable food & food supplements cancontribute to the prevention of diseases (e.g. diabetic disorders,cardiovascular diseases, cancer).• Boosting the nutritional profile helps dairy products effectivelyto 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 60.6 156.6 159.1 178.7FAO/WHO consultation on food consumption and exposure assessment to chemicals in food. Geneva, Switzerland, Feb 1997 NutriChip
  25. 25. Characterization of milk productsCollection of data of 2-D gel electrophoresis and LC-MS identification inan 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. 26. Application of digested milkConclusion: digested products (milk or buffer) have to be passed througha Centricon <30 kDa to loose their cytotoxic properties. The data suggestthat elimination of digestion enzymes is a crucial step to perform theseexperiments NutriChip
  27. 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. 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. 29. TLR-2/4 expression immunofluorescence)Readout of 2 TLR-2 and -4 immunofluorescence NutriChip
  30. 30. Chronic inflammationInflammation is a major contributor to many chronic diseases, includingobesity.An unhealthy diet may induce an elevated postprandial inflammation andcontribute, via a positive feedback loop, to the development of low gradesystemic chronic inflammation. Margioris, Current Opinion in Clinical Nutrition and Metabolic Care 2009, 12:129–137 NutriChip
  31. 31. Microfluidics-based aGIT  Continuous perfusion flow of media which ensures fresh medium & waste removal & dynamic cell culture NutriChip
  32. 32. Distribution of fluorophore clustersCounting the amount of clusters with 1,2,3,… fluorophoresgives an estimation of the amount of stained biomarkers withinthe image field of view.(i) The light intensity distribution received bythe imager from a cluster with c fluorophores ismodeled 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 givenamount 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. 33. Postprandial inflammationPostprandial inflammation is a normal reaction of the immune systeminduced by food consumption.Postprandial inflammation may be sustained in patients with a disturbedmetabolism, e.g. in the case of obesity or diabetes. Margioris, Current Opinion in Clinical Nutrition and Metabolic Care 2009, 12:129–137 NutriChip
  34. 34. Nutritional trialsStudy Content Aim1st 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 subjects2nd 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. 35. Outlook: Ca-NutriChip platformVariety of dairyproduct-based Caconcentrations 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 cellsCa-NutriChip  Osteoblast-like cells FRET NutriChip
  36. 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. 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. 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