confidentieel
IWT VIS-Traject Sensors For Food
1
Sensors For Food validation project
Optical Fibre Biosensors
8th User group meeting
April 15th 2015
confidentieel 3
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
confidentieel 6
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
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
8
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
9
Overview of chosen research lines
• Research cases started:
o Milk protein allergens
o Salmonella
o Celery
o Ochratoxin A
10
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
13
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
14
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
15
qPCR + HRM
Select optimal sample
preparation method
Overview celery detection
Reference technology New technology
FO-SPR
DNA extract
from samples
Benchmark
(synthetic DNA)
16
HRM
• HRM analysis
o Applications
• Mutations
• Polymorphisms
• Epigenetic differences
o Advantages
• Cost effective
• Fast
• Simple
17
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
18
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
19
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
20
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
21
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
22
FO-SPR PCR: celery detection
• Selected primer (Mtd3)
o Possible to perform PCR on synthetic DNA
10-11 M 10-12 M
NTC
23
FO-SPR celery detection: PCR
• Calibration curves
o 32 cycles, 0.1 nM – 1 fM
• Melting analysis during PCR
24
FO-SPR celery detection
• PCR mix without SybrGreen
• Possible to perform PCR (Here: 10-16 M)
• Some technical problems raw data
26
FO-SPR celery detection
• Preliminary results different concentrations (raw data)
• 40 cycles, 0.1 pM – 1 aM
27
FO-SPR celery detection
• Preliminary results different concentrations (raw data)
• 40 cycles, 0.1 pM – 1 aM
28
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
29
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
30
MPA research case: overview
• ELISA protocol
• HRM protocol
• qPCR protocol
• Bioassay development
• Standard curve (LOD, LOQ)
• Sample testing• Testing food samples
• Testing reference samples
Literature study
• Allergen(s) analysis
• Bioreceptor search
• Primer selection
FO-SPR technology
Reference technology
Sample preparation
32
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
33
MPA research case
Aim: Development of a FO-SPR biosensor for the detection
of casein
• Aspects to be considered:
o Different food matrices
o Comparable LOD with ELISA
o Easiness/simplicity of application
34
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, …
36
Available antibodies
• Tested:
o Commercial AB does not bind casein from:
• Standard ELISA solutions
• Casein solution in 1M NaOH
• Milk powder solution
37
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
38
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.)
39
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
40
Case 1: Calibration curve
0 10 20 30 40 50 60 70 80
0
2
4
6
8
10
12
14
16
18
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
18
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
41
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
42
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
43
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
44
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
46
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
47
49
Salmonella research case: overview
• ELISA SP protocol
• Alternative SP protocol(s)
• Extraction efficiency
• Bioassay development
• Standard curve (LOD)
• Sample testing• Testing synthetic DNA
• Testing real samples
(In collaboration with UHasselt)
Literature study
• Bacteria analysis
• Bioreceptor search
FO-SPR technology
Reference 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)
50
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
51
Salmonella research case
Aim: Development of a FO-SPR biosensor for the detection
of Salmonella
• Aspects to be considered:
o Different food matrices
o Low LOD needed (102-103 CFU/mL)
o Easiness/simplicity of application
52
qPCR + HRM
Select optimal sample
preparation method
Overview Salmonella detection
Reference technology New technology
FO-SPR
DNA extract
from samples
Benchmark
(synthetic DNA)
53
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
54
DNA based Salmonella detection
• Combination of solution phase PCR and DNA melting
assay on fiber (cf. celery detection)
55
FO-SPR E. Coli detection: PCR
• Possible to perform PCR on FO-SPR:
o Target synthetic DNA concentration in PCR mix: 10-10 M
57
FO-SPR E. Coli detection: PCR
• Possible to perform PCR on FO-SPR:
o Overview of different cycles during PCR
58
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
59
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
61
confidentieel 62
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
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
confidentieel 73
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
confidentieel
Terugblik : wat neemt u tot hiertoe mee
• Bedrijven
Wat was voor u het interessantste uit deze vergadering
Wat heeft u bijgeleerd
Vooruitblik : verwachtingen
• Allen
77
Terugblik & Vooruitblik
confidentieel 78
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
confidentieel
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
80
Sensors For Food nieuws
confidentieel
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)
83
Activiteiten Sensors for Food
confidentieel
• 6-7-8 mei, Kortrijk Expo
• 7 mei: Studiedag “Hoe helpen robotica en sensortechnologie de voedingsindustrie vooruit?”
84
Activiteiten SFF
confidentieel 85
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