Date post: | 03-Apr-2018 |
Category: |
Documents |
Upload: | nanoterach |
View: | 216 times |
Download: | 0 times |
of 54
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
1/54
NutriChip
Annual Plenary Meeting, Bern, May 30th 2013
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
2/54
NutriChip
Project
NutriChipGroup Gijs:
microfluidics
bioMEMS
cell-based & particlehandling in systems
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
Group Vergres:Biochemistry & Physiology
of dairy products
Human nutrition
Nutrigenomics
Biochemistry
http://www.microsens.ch/http://www.ayanda-biosys.com/index.html7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
3/54
General objective
To develop a microfluidic analytical platformmimicking the gastro-intestinal tract for
screening the health-promoting properties ofmilk and dairy products.
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
4/54
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.
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
5/54
NutriChip: biological principle
MilkLPS
High-fat diet
High-calory diet
Epithelium
Immune cell
Cytokines
lipopolysaccharide (LPS)
Interleukins (IL),
Tumor Necrosis Factor-a (TNF-a), ..
Can milk act against
inflammation ?
Gastro-
intestinal
Tract (GIT)
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
6/54
NutriChip: biological principle
MilkLPS
High-fat diet
High-calory diet
Epithelium
Immune cell
Cytokines ?
lipopolysaccharide (LPS)
Interleukins (ILs),
Tumor Necrosis Factor-a (TNF-a), ..
T. T. McDonald, Nature Medicine 16, 1194 (2010).
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
7/54
NutriChip: a gut-on-a-chip
Gastrointestinal
Tract (GIT)
Epithelial Cells
(Caco-2)
Cytokines
detection
Differentiated
monocytes (U937)
Functionalized
magnetic beads
Epithelial cellsculturing &
differentiation
Monocytesculturing &
differentiation
Epithelial cells (Caco-2)
Immune cells
Immunomagnetic
assay
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
8/54
NutriChip project
CMOS imagersystem
Transwell-
based GIT
Human study
Calcium bio-
availabality+ Ca-NutriChip
Nutrichip
(micro-GIT)
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
9/54
CMOS camera
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
10/54
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 first chip-prototype: test photodiodes
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
11/54
Noise Reduction: Correlated Double Sampling perColumn
Column Scanning: Shift Registers
Row Scanning: 6 bit Counter
United
Microelectronics Corp.
0.18 m process
Mini ASIC:
3240 m x 1525 mResolution:
64 x 40 pixel array
Testing from July 2013
CMOS pixel array chip
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
12/54
Synthetic image generation
Generation of random fluorophore cluster
using a Monte-Carlo approach
Cell population
For each cell: fluorophore clustersgeneration
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 used to test and validate fluorescence image
processing methods
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
13/54
Synthetic image processing
Thresholding Adaptive image thresholding
Fluorescent pixels
Average fluorescent
pixel intensity per cell
Number of fluorescent
pixels per cell
Quantifying and evaluating the amount of
fluorescent targets from epi-fluorescence
microscopy images = estimating the number
of fluorescent targets
Objective
~ measured number offluorescent targets
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
14/54
Original experimental
image
Fluorescent image processing
Codes developed in Matlab and translated for CMOS-embedded
application.
Discretization of
fluorescent spots
Removal of low-varying
background by Top-HatFiltering
O ll l i f th
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
15/54
Overall conclusions for the
CMOS imager system
n-well/p-sub type photodiodes are recommended for higher sensitivity
n+/p-sub type photodiodes have the smallest area but cannot be used
for low-intensity light imaging applications
Optimum fluorophores need to emit light at wavelengths from 630 nm
to 780 nm to reach the highest responsivityand sensitivityof the
CMOS detector.
Local thresholding method was proven to be the best for
classifying/localizing fluorescent biomarkers on CaCo-2 cells
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
16/54
In vitrodigestion of dairy food
30 kDa
filtration
- Gel electrophoresis
- size-excl. High Performance Liquid Chromatography
- OPA labeling of di-and tripeptides, free amino acids
37 oC
Analysis of
digested product :
OPA: ortho-phtalaldehyde
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
17/54
In vitrodigestion of dairy food
Pasteurized milk:
undigested and
digestedat different stages
Undigested Digested
5 min Saliva
120 min
gastric juice
30 minpancreatic
juice & bile
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
18/54
Analysis by liquid chromatography-mass spectrometry (LC-MS) or MS
Peptide length distribution afterin vitro digestion of milk
In vitrodigestion of dairy food
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
19/54
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 cell
junctions)
Lactase expression (signature of
Caco-2 differentiation) Trans-Epithelial Electrical
Resistance (TEER)
Permeability to FITC-dextran or
Lucifer Yellow
Caco-2 seeding in
Transwell
21 days 10% in FBS
culture medium
2 hours 0.2% serum
Treatment
with digested
products
Detection of
biomarkers
T ll C 2 l t
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
20/54
Transwell: Caco-2 layer response to
stimulation
0
200
400
600
800
1000
1200
0 20 40 60 80 100 1 20
IL-6
[pg/ml]
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 to
TNF-, 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 lt t TNF d
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
21/54
TNF-a, LPS apical
Transwell: co-culture response to TNF-a andLPS stimulation
0
1020
30
4050
6070
80
Baso
latera
lIL-6
[pg
/m
l]
LPS (1) 1 g/mL
LPS (10) 10 g/mLTNF-a applied at 10
ng/mL concentration
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
22/54
Overall conclusions for the Transwell system
TNF-a induces a decrease of TEER in Caco-2
U937 immune cells (human leukemic monocyte lymphoma cell line)
treated with phorbol ester PMA express TNF-a
TNF-a induces IL-8 basolateral secretion in co-culture experiments
LPS induces IL-6 expression and secretion in U937 cells
The in vitro digested products do not markedly influence Caco-2 or
U937 cells and do not interfere with LPS or TNF-a application
Ca availability in dairy products investigated
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
23/54
NutriChip design
Design of a miniaturized device for culturing Caco-2 cells with concurrent4-point measurement of the Trans-Epithelial Electrical Resistance (TEER)
4-p
ointTEER
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
24/54
NutriChip realization
Fluidic inlet top layer
Fluidic inlet bottom layer Top electrode (current)
Bottom electrode (voltage)
Channel height: 250 m 20 m
Fluidic outlet bottom layerBottom electrode (current)
Fluidic outlet top layer
Top electrode (voltage)
1 mm
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
25/54
Caco-2 cell layer characterization
Apparent permeability coefficient for FITC-labeled dextran:
Papp (cm/s) = P (mol/s) / (A (cm2) C0(mol/mL))
with
Pthe permeability rate, C0 the initial concentration in the upper chamber,
andA the surface area of the monolayer
Papp 710-4 cm/s
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
26/54
Caco-2 cell layer characterization
4-point measurement of the Trans-Epithelial Electrical Resistance (TEER)
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
27/54
Caco-2 cell layer characterization
Transwell
NutriChip
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
28/54
On-chip immunomagnetic assay
Differential response of monocytic cells to LPS stimulation
Overall conclusions for the
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
29/54
Overall conclusions for the
miniaturized GIT (NutriChip)
Microfluidic model of the gastrointestinal tract realized
Caco-2 culture performed
Layer integrity tested by FITC-dextran transport study
TEER results confirmed confluency of the cell monolayerobtained after 6 days of culture
Magnetic-bead based immunoassay developed
Ca-transport study through the cell monolayer in progress
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
30/54
Meal A
Meal B
Meal C
37 persons
3 times @ Inselhospital, Bern to consume
meals A, B, and C
5 blood samplings per Person/Day/Test meal 570 blood sampling time points
Objective:
Evaluate the effect of a high-fat meal on postprandialmetabolism and inflammation in normal weight and
obese subjects
500kcal
1000 kcal
1500 kcal
Human nutrition study
18 19
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
31/54
Meal A
Meal B
Meal C
Analysis: Classical clinical parameters: glucose, insulin,
triglycerides, total cholesterol, high-density
lipoprotein(HDL)-cholesterol
Inflammation mediators: C-reactive protein (high-sensitivity CRP), IL-6, endotoxin (lipopolysaccharide
LPS))
Antihyperglycemic hormone : glucagon-like peptide-1
(GLP-1)
Other technologies: transcriptomics, metabolomics
500kcal
1000 kcal
1500 kcal
Human nutrition study
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
32/54
Postprandial glucose and insulin response
0
1
2
3
4
5
6
7
8
0 2 4 6
mmol/L
500kcal1000kcal
0
1
2
3
4
5
6
7
8
0 2 4 6
mmol/L 500 kcal
1000 kcal
1500 kcal
0
20
40
60
80
100
120
140
160
0 2 4 6
mU/L
500 kcal
1000 kcal
1500 kcal
time (h)
time (h)
time (h)
Glucose
Insulin
Glucose
time (h)
Insulin
+iAUC meal A
0
20
40
60
80
100
120
140
160
0 2 4 6
m
U/L 500 kcal
1000 kcal1500 kcal
+iAUC: positive
incremental area under the
curve
D f l d i li
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
33/54
Dose-response for glucose and insulin
0
0.5
1
1.5
2
2.5
3
3.5
4
4.55
500 kcal 1000 kcal 1500 kcal
Glucose 0h - 6h
0
50
100
150
200
250
300
350
400
500 kcal 1000 kcal 1500 kcal
Insulin 0h 6h
Significant increase in +iAUC from meal A to meal B to meal C
(glucose only in obese, insulin for both groups) Significant difference between obese and normal weight in the dose-
response
+iAUC for meal C in normal weight still below +iAUC for meal A in
obese
meal Bmeal A meal C
+iAUC: positive incremental area under the curve
meal Bmeal A meal C
x
b
a
c z
y
a
ab
b
x
y
z
+iAUC(mU/L*h)
+iAUC(mmol/L*h)
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
34/54
Overall conclusions for the human study
Methodological basis for nutrition intervention study onhuman subjects with a different metabolic status
Dose-dependent effect of a high-fat meal on metabolic
parameters demonstrated
Difference between normal weight and obese in the doseresponse (metabolic parameters, IL-6)
Triglycerides may play a role in endotoxin absorption
Obese had higher baseline levels of GLP-1, but responded
similar or less strong (meal C) to the different meals
compared to the normal weight
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
35/54
March 15th 2012
Thanks for your attention!
The NutriChip team
http://www.sf.tv/7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
36/54
Backup slides
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
37/54
In vitrodigestion of dairy food
Quantification of individual free amino acids (AA) by HPLC
after Stage 3 digestion (Saliva, Gastric, Pancreatic juices and Bile)
Transwell: integrity of Caco-2 layer
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
38/54
LPS, TNF-a apical
Transwell: integrity of Caco-2 layer
with TNF-a and LPS stimulation
020406080
100120140
160180
Re
lativeTEER
LPS (1) 1 g/mL
LPS (10) 10 g/mL
TNF-a applied at10 ng/mL
concentration
050100
150200250300350400450
Relative4
kDaFITC
permea
bility
P di l I fl i
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
39/54
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
Maladaptation
dysmetabolism
Normal
Adaptation +2SD
Meals high in calories, carbohydrate and Saturated
fatty acidsproduce a strong postprandial
immune/inflammatory response;
Postprandial stress as an indicator of
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
40/54
1 2 3 4 5 6 7 8
Postprandial time in hours
Inflammationand
oxidationmarkers
Meal
Maladaptation
dysmetabolism
Normal
Adaptation
+2SD
A. N. Margioris, Current Opinion in Clinical Nutrition and Metabolic Care 2009, 12:129137
Postprandial stress as an indicator of
metabolic health
Meals high in calories, carbohydrate and Saturated fatty acids produce
a strong postprandial immune/inflammatory response;
Characterization of milk products
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
41/54
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 wereidentified (450 were different proteins)
Each product has a unique proteome and might
produce a different inflammatory response
In vi tro digestion of milk
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
42/54
In vitrodigestion of milk
Digestion of pasteurized milk
past.milk
saliva
pH 6.8
gastric
juicepH 2-3
pancreatic juice
pH 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
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
43/54
N t iChi i l ti f h t
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
44/54
Media flow velocity should be less
than 0.1 m/s for safe shear stress on
cells
Contours of wall shear
stress (Pascal)
Contours of velocity
magnitude (m/s)
Flow
NutriChip: simulation of shear stress
N triChip microfabrication aspects
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
45/54
A procedure for irreversiblebonding of the porous membrane
in between two PDMS layers was
developed
Cell culture -chamber
with perfusion channels
NutriChip: microfabrication aspects
Cells on the NutriChip
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
46/54
Epithelial cells (Caco-2) and monocytes (U937) cells are being separately
cultured in the microfluidic chips
Caco-2 U937
Cells on the NutriChip
Ca-NutriChip
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
47/54
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 acidsAmount of free fatty acids
Ca-NutriChip
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
48/54
Ca-NutriChipDigestion of different dairy products to determine Ca2+ amount in the digests
5min 2h 2h
200 L saliva
pH 6.8
400 L gastric juice
pH 2-3
400 L pancreatic
juice pH 6.5-7
200 L bile
pH 6.5-7
&
+ +
Saliva contains 2 mmol/L ofCa2+
Gastric juice contains 0.6 mmol/L ofCa2+
Pancreatic juice contians 0.6 mmol/L ofCa2+
Bile contains 3.7 mmol/L ofCa2+
CaCl2 OMITTED from the digestion!
Filtration of the
digest
through 30
kDa
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
49/54
Ca-NutriChip: NutriChip for study of
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
50/54
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 of
protein
digested
without CaCl2
in digestion
juices
Semi-hard and hard cheeses are rich sources of calcium
Ca NutriChip: NutriChip for study of
nutrikinetics
Ca-NutriChip
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
51/54
Amount of free amino acids
mMo
ffreeaminoacids
Ca-NutriChipDigestion with/without CaCl2 in the digestion juices
The efficency of digestion is not influenced by omitting
calcium chloride from the digestion juices
Fat & proteins in milk, yogurt and cheese are well digested.
Ca-NutriChip
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
52/54
Ca NutriChip
Stimuli: various dairy
products
Ca-NutriChip
Epithelial Cells
(Caco-2)
Ca2+Ca2+ Ca2+ Ca2+
Transported Ca2+
through Caco-2 cells
Ca2+ uptake by target
cells
THP-1 cells
Osteoblast-like cells
Magnetic beads
Ca2+Ca2+ Ca2+ Ca2+
Target cells (Ca2+ sink)
Variety of dairy
product-based Ca
concentrations.
Extending the functionality of Nutrichip with a nutrikinetic capability
Ca-NutriChip: Nutrichip for study
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
53/54
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)
Ca NutriChip: Nutrichip for study
of nutrikinetics
Publications and media coverage
7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013
54/54
Publications and media coverage
Journals4 submissions
Conferences25 oral and poster presentations
Media coverage
http://www.sf.tv/http://www.cdrf.org/