Sensors For Food validation project Optical Fibre Biosensors · confidentieel IWT VIS-Traject...

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IWT VIS-Traject Sensors For Food

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Sensors For Food validation project

Optical Fibre Biosensors

8th User group meeting

April 15th 2015

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User group members

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Welcome

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Agenda

RECEPTION WITH COFFEE & LUNCH 12:30-13:00

1 WELCOME & REVIEW AGENDA 13:00-13:10

2 PRESENTATION OF RESULTS (DD-KUL) + DISCUSSION (ALL) 13:10-14:20

3PRESENTATION OF FUTURE WORKPLAN (DD-KUL) + DISCUSSION

(ALL)14:20-14:50

COFFEE BREAK 14:50-15:05

4 PRODUCT AND BUSINESS UPDATE FILIP DELPORT 15:05-15:30

5REVIEW AND FORECAST - EXPECTATIONS (ALL)

PROJECT BAROMETER IWT (VDG)15:30-15:50

6 SENSORS FOR FOOD NEWS (VDG) 15:50-16:10

7 CONCLUSIONS & ACTIONS - DATE NEXT MEETING (ALL) (V) 16:10-16:15

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Conclusions and actions previous GG

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Workplan

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Validation project: Optical fiber biosensors

FO Users Group Meeting 8th user group meeting, Wieze, 15-04-2015

Devin Daems ● Filip DelportDragana Spasic ● Prof. Jeroen LammertynMeBioS – Biosensor group

Presentation outline

• Project overview

• Discussion of different cases

o Part I: Celery research case

o Part II: MPA research case

o Part III: Salmonella research case

o Part IV: Ochratoxin A research case

• Progress overview

• Discussion and action points

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Project overview

IWT SFF project

Case 1:

Celery and celeriac

Case 2:

Milk protein allergens

Case 3:

Salmonella

Case 4:

Ochratoxin A

Other projects for FO-SPR technology development

e.g. IWT – LA, Nanodiag.

No IP on system development will be generated in this project

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Overview of chosen research lines

• Research cases started:

o Milk protein allergens

o Salmonella

o Celery

o Ochratoxin A

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Nathalie left the group

+

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Part I:

Celery allergen research case

Celery case - overview

• HRM protocol

• qPCR protocol

• Bioassay development

• Standard curve (LOD)

• Sample testing• Testing synthetic DNA

• Testing real samples

(In collaboration with UHasselt)

Literature study

• Allergen(s) analysis

• Primer selection

FO-SPR technology

Reference technology

Sample preparation

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Literature study

• Celery

o Apium graveolens

• Root (celeriac)

• Sticks

o Class II food allergen

o Homology to other vegetable proteins

• No successful ELISA (so far)

• DNA based detection

• PCR/HRM analysis

France

Germany

Switzerland

Prevalence to peanut/celery allergy

Api g1

Api g2Api g5

Api g3Celery

allergens

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Celery research case

Aim: Development of a FO-SPR biosensor for the detection

of celery

• Aspects to be considered:

o Different food matrices

o low LOD needed (< 10-18 M)

o Easiness/simplicity of application

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qPCR + HRM

Select optimal sample

preparation method

Overview celery detection

Reference technology New technology

FO-SPR

DNA extract

from samples

Benchmark

(synthetic DNA)

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HRM

• HRM analysis

o Applications

• Mutations

• Polymorphisms

• Epigenetic differences

o Advantages

• Cost effective

• Fast

• Simple

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qPCR on synthetic target

• Selecting celery primers

o API, Mtd, Mtd2 and Mtd3

Mtd 3 selected, with lowest Non Template Control (NTC)

• Calibration curves

o 30 cycles, 1 nM – 10 fM

• Melting analysis on end products

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qPCR on real samples

Name Sample type Name Sample type

DNA1 Stem (Retsch mixer) 10-10 Synthetic DNA target 10-10 M

DNA2 Stem (mortar + pestle) 10-12 Synthetic DNA target 10-12 M

DNA3 Leaves (mortar + pestle) NTC Non-template control

Cycle

5 10 15 20 25 30

Flu

ore

scence

15

10

5

0

10-10 DNA3 10-12

DNA2

DNA1

NTC

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qPCR: conclusions

• qPCR:

o DONE:

• Good primer/probe set selected Mtd3

• Calibration curve: 1 nM – 10 fM

• NTC controlled in this region

o TO DO:

• Extend calibration curve

• Lower concentrations 10-18 M

• Spiked samples

• Selection of the best DNA extraction method

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FO-SPR celery detection: melting assay

• FO-SPR DNA based assay

o Comparable to HRM

o DNA melting analysis

• Start with synthetic target

• Two probe regions

- Carrot

- Celery

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FO-SPR celery detection: PCR

• Comparable to qPCR and HRM

• Combine solution phase PCR and DNA melting assay on

fiber

• Start with synthetic target

• 1 probe region Mtd3

• PCR amplified target

• From celery samples

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FO-SPR PCR: celery detection

• Selected primer (Mtd3)

o Possible to perform PCR on synthetic DNA

10-11 M 10-12 M

NTC

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FO-SPR celery detection: PCR

• Calibration curves

o 32 cycles, 0.1 nM – 1 fM

• Melting analysis during PCR

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Problem qPCR and FO-SPR: primer-dimer

• Possible solution: PCRmix without SybrGreen

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FO-SPR celery detection

• PCR mix without SybrGreen

• Possible to perform PCR (Here: 10-16 M)

• Some technical problems raw data

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• Preliminary results different concentrations (raw data)

• 40 cycles, 0.1 pM – 1 aM

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• Preliminary results different concentrations (raw data)

• 40 cycles, 0.1 pM – 1 aM

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DNA based celery detection

• Conclusions

o qPCR and FO-SPR with SybrGreen

• Good primer/probe set selected

• Calibration curve: 1 nM – 1 fM

• Problem with NTC

o FO-SPR without SybrGreen

• Calibration curve: 0.1 pM – 1 aM

• Future work

o Data analysis and reproducibility synthetic target

o Spiked sample detection

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DNA extraction methods celery

Homogenisation method Extraction method

0 No homogenisation

0.1 Homogenisation in cleaning water

1.0

Homogenisation in CTAB buffer

No further extraction

1.1 DNA precipitation with NaAcetate and EtOH

1.2 First chloroform extraction followed by DNA

precipitation as in 1.1

8.1 Homogenisation in QIAGEN

Dneasy plant kit buffer QIAGEN extraction

8.2 No homogenisation

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Part II

Milk protein allergen research case

MPA research case: overview

• ELISA protocol

• HRM protocol

• qPCR protocol


• Standard curve (LOD, LOQ)

• Sample testing• Testing food samples

• Testing reference samples

Literature study

• Allergen(s) analysis

• Bioreceptor search

• Primer selection

FO-SPR technology


Sample preparation

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Milk allergens: literature study

• Casein

o αS1, αS2, β and κ

o 80% of cow milk proteins

o Solid part of milk

• Whey

o α-lactalbumin and β-lactoglobulin (BLG)

o BLG: 10% of cow milk proteins

o Liquid part of milk

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MPA research case


of casein



o Comparable LOD with ELISA


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FO-SPR technology

FO-SPR bioassay

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MPA bioassay

• Requirements

o Detecting “total milk”

• Casein detection

• BLG detection

o Available Abs (casein and BLG)

o Protocols for Ab immobilization

o Control over unspecific binding

• Food matrix: milk, chocolate, eggs, additives, …

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Available antibodies

• Tested:

o Commercial AB does not bind casein from:

• Standard ELISA solutions

• Casein solution in 1M NaOH

• Milk powder solution

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Available antibodies

• Tested:

o French National Institute for Agricultural Research

(INRA)

• Purification of best performing antibody (BBC1 56)

• Low yield: 85 µg/ml

• BLItz

- Positive result for detection in diluted milk

- Negative result for detection in diluted milk powder

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Possible casein antibodies

• Polyclonal antibodies

o Different companies (Abcam, Biorbyt, Abbiotect,

GeneTex and Antibodies-online)

o From rabbit

o ± € 250

• Contact University of Nebraska – Lincoln

o Food Allergy Research and Resource program

o Work together with Neogen (f.e. casein, ochratoxin, E.

coli, etc.)

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Case 1: Immuno-assay on FO-SPR

• Objective

o Develop FO-SPR immuno-assay for target detection in

biological matrix (here: serum)

• Protocol

o Surface chemistry: EDC/NHS

o Aim to detect: 5 ng/ml in 1/100 serum dilution

o Sandwich assay: antibody conjugated gold

nanoparticles

target

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Case 1: Calibration curve

0 10 20 30 40 50 60 70 80

0

2

4

6

8

10

12

14

16

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SP

R s

hif

t (n

m)

IFX concentration (ng/ml)

y = Bmax*x/(Kd + x)

Bmax = 27.912, Kd = 105.248

Adj. R2= 0.999

• 100-fold serum dilution

o 6 repetitions

o LOD: 2.1 ng/ml (0.21

mg/ml in whole serum)

• 200-fold serum dilution

o 4 repetitions

o LOD: 0.67 ng/ml (0.13

mg/ml in whole serum)

0 10 20 30 40 50 60 70 80

0

2

4

6

8

10

12

14

16

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SP

R s

hif

t (n

m)

IFX concentration (ng/ml)

y = Bmax*x/(Kd+x)

Bmax = 72.863, Kd = 304.657

R2

= 0.996

Target Target

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Case 1: Real samples

• Error bars: standard error

Sample1 Sample2 Sample3 Sample4 Sample5

ELISA (3 repetitions) 3.43 ± 0.13 <0.3 19.15 ± 1.51 6.35 ± 0.58 4.04 ± 0.5

FO-SPR (4 repetitions) 3.83 ± 0.12 0.22 ± 0.06 22.33 ± 1.63 6.1 ± 0.25 4.03 ± 0.21

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Case 2: Competitive immuno-assay

• Objective

o Develop FO-SPR immuno-assay for target detection

(here: progesteron) in milk

• Protocol

o Surface chemistry: EDC/NHS

o Aim to detect: 1 ng/ml in 1/2 milk dilution

o Competitive inhibition assay

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Case 2: Competitive inhibition assay

• Immobilisation

o Immobilisation buffer: 10 mM sodium acetate pH 5.5

o Immobilisation concentration: 50 mg/ml

0

1

2

3

4

5

6

7

8

SP

R s

hif

t (n

m)

Immobilisation

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Case 2: Competitive inhibition assay

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Case 2: Calibration curves on 1 fiber

• LOD PBS: 1.05 ng/mL (sensitivity 0.35 ng/mL)

• LOD milk standards: 0.23 ng/mL (sensitivity 0.08 ng/mL)

• LOD spiked milk: 4.10 ng/mL (sensitivity 0.81 ng/mL)

PBS Milk standards Spiked Milk

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MPA research case: conclusions

• Antibodies:

o Screening of 1 commercial and 15 INRA Abs did not

provide a selective bioreceptor

o Preferable option: contacting University of Nebraska

• Cases on fiber

o Case 1: FO-SPR immuno-assay in serum

o Case 2: Competitive inhibition FO-SPR assay in milk

• Future work

o Calibration curves with good antibody

o FO-SPR detection of casein in real samples

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Part III

Salmonella research case

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Salmonella research case: overview

• ELISA SP protocol

• Alternative SP protocol(s)

• Extraction efficiency


• Standard curve (LOD)

• Sample testing• Testing synthetic DNA

• Testing real samples

(In collaboration with UHasselt)

Literature study

• Bacteria analysis

• Bioreceptor search

FO-SPR technology


Sample preparation

Overview literature

• Methods for Salmonella detection

o Classical culture methods

• Advantages: cheap, open access, standardized

• Disadvantages: laborious and time consuming (2-5 days)

o Alternative (rapid) methods

• Novel culture techniques

• Immunomagnetic separation

• ELISA based assays, lateral flow assays

• Molecular techniques (PCR and DNA hybridization based

assays)

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FO-SPR Salmonella detection

• FO-SPR as an alternative/rapid method for Salmonella

detection

• Two strategies

o Protein/Ab based bioassay

• Advantages: no sample preparation needed

• Disadvantages: assay sensitivity/specificity depends on Ab

o DNA based bioassay (see part I: celery)

• Advantages: high sensitivity/specificity

• Disadvantages: requires sample preparation

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Salmonella research case


of Salmonella



o Low LOD needed (102-103 CFU/mL)


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qPCR + HRM

Select optimal sample

preparation method

Overview Salmonella detection

Reference technology New technology

FO-SPR

DNA extract

from samples

Benchmark

(synthetic DNA)

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qPCR on synthetic target

• Testing E. coli primers

o NTC ok for 32 cycles

• Different concentrations

o 50 cycles, 1 pM – 0.1 fM

• Melting analysis on end products

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DNA based Salmonella detection

• Combination of solution phase PCR and DNA melting

assay on fiber (cf. celery detection)

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FO-SPR E. Coli detection: melting assay

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FO-SPR E. Coli detection: PCR

• Possible to perform PCR on FO-SPR:

o Target synthetic DNA concentration in PCR mix: 10-10 M

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FO-SPR E. Coli detection: PCR

• Possible to perform PCR on FO-SPR:

o Overview of different cycles during PCR

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DNA based Salmonella detection

• Conclusions

o qPCR and FO-SPR with SybrGreen

• Good primer/probe set selected

• LOD with SybrGreen in PCR mix on qPCR: 10-16 M

• Melting curves on FO-SPR with synthetic DNA

• Preliminary PCR experiments on FO-SPR with synthetic DNA

o Future work

• Calibration curve with synthetic on FO-SPR

• Analysis of extracted DNA samples

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Progress overview

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Discussion and action points

• Feedback and suggestions for future work

o Celery case

• Reproducibility of PCR on fiber

• Analysis of DNA sample extracts

o Milk protein case

• Selection and testing of good antibodies

o Salmonella case

• Optimization of PCR on fiber

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Agenda





(ALL)14:20-14:50







Sensors for Food

User group

Filip Delport

MeBioS

15-4-2015

Outline

• Sensor tip

• New setup

• Partnerships

• Business

FO-SPR gold coated tip: SEDI

• Pricing

o Remains too high

o Quality unsatisfactory• Initial wavelength: 639,47 +/- 10,42 nm

• Dip depth: 0,15 +/- 0,053 IU

• Sensitivity: 1735,30 +/- 129,78 nm/RIU

• 2 fibers out of 16 should be discarded

o Slow production time

o Not willing to invest

FO-SPR gold coated tip: In house production

• Upside of own production

o Allow control over quality

o Allow control over modifying process

o Bio-functionalization on freshly sputtered probes

• Upgrading production equipment

o Lowering hands on time

o Increasing reproducibility

Fiber cutter and stripper 5.000

Fiber cladding removal 4.000

Automatic fiber core cutter 8.000

Ultrasonic cleaner 2.000

Mould for connector 20.000

Sputter coater Titanium 50.000

Sputter coater gold 50.000

Connector mounting station 500

Lyophilisation station 15.000

CNC robot x2 8.000

Dedicated holders and tools 2.000

Packaging stage 10.000

Fiber pigtail polisher 7.000

Waste bins 300

Chemicals cabinet 3.000

Fridge 700

Gold targets 1.000

Argon Gas 75

Chemicals 1.000

2 tower computers 3.000

2 laptops 3.500

FO-SPR probe alternatives

• Sputtering

o Triple? holder dual target? sputter clock

o Need for Ti? Need for integrated equipment?

• 3SAE

o Ring of fire

• Nyfors (currently investigating feasability)

o Auto cut and strip• 3 min per prep to 20 sec per prep

o Automated high precision diamond cleave blade• 2 min per cleave to 15 sec per cleave

New setup_A: DNA

• Operational

• Debugging

o Standby errors

o Start settings

o Temperature

• Hardware

o Orientation of probes

o Power on/off light sources

o Light source lifetime

New functionalization robot

• Robot adapted for holder incorporation (950 €)

• Holder and fixation (500 €)

• Electronics (100 €)

• Gripper (500 €)

• Current design

o 10 simultaneous probes

o Fixed manually

• Next gripper design

o Automated probe handling

o Parallel continuous functionalization

• 2 measurements per 2h40 to 40 min

Partnering

• Unitron + Comate (NDA) (144.000 €)

o Design and engineering

o External design and rendering

o Prototyping (3 setups)

o CE marking

o UI

o …

• Vision4Care (NDA)

o Several direct and web meetings

o Introducing technology to allergen diagnostic companies

o Momentarily on hold

Business

• Business plan: Overhaul

o Netscientific to Farad/Gaston

o Start with research device

o No ready application

o Aim to provide a quick development of a POC?

• Present technology to diagnostic SME’s?

o Need contacts!

Future work

• Debug DNA setup

• Fix hardware issues

• Cost efficiency of optical fiber tips production!

• Talking to customers/partner!

Thank you

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Agenda





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IWT Gebruikerspoll

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IWT Gebruikerspoll

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IWT Gebruikerspoll

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Terugblik : wat neemt u tot hiertoe mee

• Bedrijven

Wat was voor u het interessantste uit deze vergadering

Wat heeft u bijgeleerd

Vooruitblik : verwachtingen

• Allen

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Terugblik & Vooruitblik

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Agenda





(ALL)14:20-14:50







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Goedgekeurd!

Startdatum 01/02/2016

Nieuw VIS-traject i-FAST

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Vierde jaar Sensors For Food

• Einddatum project: 30 november 2015

Sensors for Food platform

• GG9: laatste GG met voorstelling nieuwe resultaten

Wanneer? (september?), waar?

• GG10: eind GG (locatie?)

Voorstelling eindrapport

SWOT analyse (allen)

November (doodle)

• Slotevent met demo’s: februari –maart 2016

• Impactmeeting

Korte online vragenlijst

Peilen naar concrete impact bij de deelnemers

• Succesverhalen -> bedrijfsbezoeken

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Sensors For Food nieuws

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Impactmeting

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Impactmeting

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Interactieve training Experimenteel ontwerp

• Succesvolle eerste editie op 26 maart (Food Pilot Melle)

• Wegens grote interesse: tweede editie op 25 juni (KU Leuven)

PAT en datamanagement voor de voedingsindustrie

• 27 augustus

• Montil, Affligem

Dataverwerking en –analyse/diagnose

• 23 september

• KU Leuven

Statistische procesmonitoring en –sturing/controle

• 15 oktober

• UGent (Farmacie)

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Activiteiten Sensors for Food

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• 6-7-8 mei, Kortrijk Expo

• 7 mei: Studiedag “Hoe helpen robotica en sensortechnologie de voedingsindustrie vooruit?”

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Activiteiten SFF

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Agenda





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IWT VIS-Traject Sensors For Food

86

Sensors For Food validation project

Optical Fibre Biosensors

8th User group meeting

April 15th 2015

Sensors For Food validation project Optical Fibre Biosensors · confidentieel IWT VIS-Traject...

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