Briefing by ChipShop

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

OUTLINE
1. Miniaturization – What does this mean
2. The overall goal
3. Technical & biological elements
4. Summary & conclusion

SAMPLE-IN-RESULT-OUT
Requires a multidisciplinary team

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
Microfluidics – Present and Future
Blood vessels in
the lung
Inkjet printer heads
Lab-on-a-Chip

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
The development of a system for the mobile and
doubtless identification of biological pathogens.
Sample enrichment
Filtration
DNA extraction
DNA amplification
Detection – different options

STEP 1: SAMPLE ENRICHMENT
cone interface
septum
needle
Time:
15 min
filter
on-chip membrane:
mesh width of 5 µm
Module 1:Chip with
Integrated filter
membranes
Prototype
Design cone-chip
interface
Concept
Design and fluidic
tests
Integration into
system
Fixed sampling
protocol

STEP 2: DNA EXTRACTION
Modul 1
chamber volume
25µl
Time:
3 min
Module 2
chamber volume
120 µl
Evaluation of process
time, temperature and
efficiency
Fluidical tests:
Design
Fluidical and
functional tests
Integration into
chip
Fixed lysis
protocol

STEP 3: PCR
qPCR sucessful
tested in 41 Cycle
chip
Modul 1
41-Zyklen 3-step PCR-Chip
PCR in biplex
developed and
pathogen combination
successful tested
20 min
44 -Zyklen 2-step PCR-
Chip Modul 1
try reagent storage
established
together with WP8
partners
Design
Fluidical and
functional tests
Integration into
chip
PCR
protocol

WP2 Detection modul
Chemiluminescent 2
Reagenz: PS-ATTO
18-well chip
for establishment
of PCR-ELISA
PCR-ELISA
• Fixed hybridisation
conditions
• Fixed hybridisation
protocol
Single lane readout
device succsessful tested
Modul 2
Design
Fluidical and
functional tests
Integration into
chip
Fixed PCR-ELISA
protocol

SAMPLE-IN-RESULT-OUT
INTEGRATION ON CHIP

PROOF-OF-CONCEPT: YERSINIA PESTIS
SINGLEPLEX AND BIPLEX PCR 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
300 bp
200 bp
100 bp
Results: singleplex
IS200
Caf1
pla
positive
positive
positive
41-cycle PCR-Chip
Results: biplex
IS200 + Caf1 positive / positive
IS200 + pla positive / positive

COMPLETE PROCESS WITH SINGLE
MODULES – THE SET-UP
Waste
2-way valve
Chip with lyophilized
reagents
Sample take-up
Liquid reservoirs
Thermocycler
External valve block

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.

CONCLUSION PART 2
….still some time to go to the ultimate universal
analytical tool!

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
Claudia.Gaertner@microfluidic-ChipShop.com
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]