sample-in-result-out: lab-on-a-chip technology on its way...
TRANSCRIPT
Sample-in-result-out: Lab-on-a-Chip
technology on its way to an integrated
analytical system for B-Agents
Claudia Gärtner, microfluidic ChipShop GmbH
JIP CBRN workshop, Brussels, 15th of September 2011
1. Miniaturization – What does this mean
2. The overall goal
3. Technical & biological elements
4. Summary & conclusion
OUTLINE
Requires a multidisciplinary team
SAMPLE-IN-RESULT-OUT
PROJECT, TEAM & RESSOURCES
1. microfluidic ChipShop GmbH
(Germany), SME
Microfluidics
2. Clemens GmbH (Germany), SME
3. Institut für Mikrobiologie der
Bundeswehr (Germany), Institute
4. Friedrich-Löffler-Institut (Germany),
Institute
5. Joanneum Research (Austria),
Institute
Array
6. Bertin Technologies (France),
Industry
1. microfluidic ChipShop GmbH
(Germany), SME
2. Friedrich-Löffler-Institut (Germany),
Institute
3. IMM, Institut für Mikrotechnik Mainz
(Germany), Insitute
4. University of Roviria Spain
5. iMICROQ (Spain), SME
6. Institute for Physical Biology,
(Slovenia) SME
7. Bertin Technologies (France),
Industry
8. Cedralis (France) SME
Microfluidics – Present and Future
Inkjet printer heads Lab-on-a-Chip Blood vessels in
the lung
MICROFLUIDICS
LAB-ON-A-CHIP – WHAT DO WE GAIN
Minimization of analysis times – from hours to minutes or seconds
Reduction of sample volumes by orders of magnitude
Novel ways to obtain information
Increased sample and data throughput
Increased performance
Simpler handling of complex analytical tasks
New ways to conduct a reaction
Realization of portable systems for on-site-analysis
Little space necessary
On-line measurement is possible
TARGET PATHOGENS
Bacteria:
• Yersinia pestis
• Francisella tularensis
• Burkholderia mallei
• Burkholderia pseudomallei
• Brucella melitensis
• Brucella abortis
• Coxiella burnetti
Later:
• Bacillus anthracis
• Ortho pock virus
SAMPLE-IN-RESULT-OUT
WHAT DOES THIS MEAN
Sample enrichment
Filtration
DNA extraction
DNA amplification
Detection – different options
The development of a system for the mobile and
doubtless identification of biological pathogens.
Concept Design and fluidic
tests
Integration into
system
cone interface
septum
needle
filter
Design cone-chip
interface
Prototype
Time: 15 min
Fixed sampling
protocol
on-chip membrane:
mesh width of 5 µm
Module 1:Chip with
Integrated filter
membranes
STEP 1: SAMPLE ENRICHMENT
Modul 1
chamber volume
25µl
Fluidical tests:
Design Fluidical and
functional tests
Integration into
chip
Module 2
chamber volume
120 µl
Time: 3 min
Fixed lysis
protocol
Evaluation of process
time, temperature and
efficiency
STEP 2: DNA EXTRACTION
Modul 1
44 -Zyklen 2-step PCR-
Chip Modul 1
41-Zyklen 3-step PCR-Chip
qPCR sucessful
tested in 41 Cycle
chip
PCR in biplex
developed and
pathogen combination
successful tested
try reagent storage
established
together with WP8
partners
Design Fluidical and
functional tests
Integration into
chip
20 min
PCR
protocol
STEP 3: PCR
WP2 Detection modul
2
Modul 2
Chemiluminescent
Reagenz: PS-ATTO
Single lane readout
device succsessful tested
PCR-ELISA
• Fixed hybridisation
conditions
• Fixed hybridisation
protocol
18-well chip
for establishment
of PCR-ELISA
Design Fluidical and
functional tests
Integration into
chip
Fixed PCR-ELISA
protocol
SAMPLE-IN-RESULT-OUT
INTEGRATION ON CHIP
Yersinia Pestis PCR
template conc.: 10 pg/ µl ~ 1900 copies/ µl
Targets: IS200 178 bp
Caf1 240 bp
pla 232 bp
Chip: PCR performed on 41-cycle 3-step Chip
with 2-step thermal setup:
95°C, 60°C, 60°C 41-cycle-chip _
IS200 caf1 pla IS + IS +
caf1 pla
PROOF-OF-CONCEPT: YERSINIA PESTIS
SINGLEPLEX AND BIPLEX PCR ON CHIP
41-cycle PCR-Chip
300 bp
200 bp
100 bp
Results: singleplex IS200 positive
Caf1 positive
pla positive
Results: biplex IS200 + Caf1 positive / positive
IS200 + pla positive / positive
COMPLETE PROCESS WITH SINGLE
MODULES – THE SET-UP
Thermocycler
External valve block
Liquid reservoirs
Waste
Sample take-up
Chip with lyophilized
reagents
2-way valve
AND FINALLY – THE INTEGRATED
SYSTEM
But: Just a first version in a series of lab-on-a-chip
instrument set-ups!
CONCLUSION
All single steps (lysis, nucleic acid extraction, dried reagent
storage, PCR) work on chip.
Combination of single modules work.
Novel PCR concept massively decreases the overall raction
time (less then 20 minutes for 41 cycles compared to more
then 1 hour with the Roche Light Cycler).
Target timeline 30 min with further optimization realistic.
….still some time to go to the ultimate universal
analytical tool!
CONCLUSION PART 2
NEXT STEPS
Process set-up for the integrated system.
Time optimization needs to be done – goal of 20 – 30 minutes
for the overall process.
Evaluation of detection methods in the overall set-up.
Complete process set-up in the integrated chip.
Parallelization of several PCR reactions and multiplexing
needs to be done.
CONTACT &
ACKNOWLEDGEMENT
Dr. Claudia Gärtner
microfluidic ChipShop GmbH
Stockholmer Str. 20
D – 07747 Jena
Germany
Phone: + 49 (0) 36 41– 64 31 21
Phone: + 49 (0) 36 41– 64 31 21
Acknowledgement for partial support of the work via:
EDA-project PathoID Chip
European Community´s Seventh Framework Programme
(FP7/2007-2013) under grant agreement n° [261810]