UNCLASSIFIED
PathoID
Publishable Executive Summary
PathoID-Chip – Robust and autonomous airborne threat identification system as lab-on-a-chip
device with integrated optoelectronic sensors and combined pathogen enrichment
Document reference: JIP-FP Executive Summary _111202 V5.doc
Issue: Date: 2011-12-02
Pages: 4
Contract n° A- 102-RT-GC
Under the Joint Investment Programme
on Force Protection
A-0223-RT-GC
Institut für Mikrobiologie der
Bundeswehr
Project PathoID-Chip
Publishable Executive Summary
UNCLASSIFIED
1 Introduction
This document summaries the activities and results of the project PathoID Chip carried out by
a consortium consisting of the companies microfluidic ChipShop, Clemens GmbH, Bertin
Technologies, and the institutes Johanneum Research, Institut für Microbiologie der
Bundeswehr, and Friedrich Löffler Institut.
This project was managed and funded in the frame of the EDA R&T Joint Investment
Programme on Force Protection A-0120-RT-GC by the Contributing Members: Austria,
Belgium, Cyprus, Czech Republic, Estonia, Finland, France, Germany, Greece, Hungary,
Ireland, Italy, the Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden
2 Objectives
The terroristic attacks with non conventional weapons as the B-agents attacks in the US of
October 2001, or the avian flu of recent years in Asia and Europe demonstrate the need for a
rapid, safe and portable detection and identification methods to securely determine an
infection or contamination of people, animals, food or sensitive infrastructure.
Therefore, this project targeted at the development of an autonomous lab-on-a-chip based
standoff CBRNE detection system that combines sample enrichment of airborne pathogenes,
lab-on-a-chip lysis, and chemiluminescence-based quantification and identification of PCR
products, using printed photosensors.
The single objectives are summarized below:
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Development of an integrated microfluidic chip performing a process from biological
”sample in” to “answer out” and, as a consequence thereof, containing modules for
debris filtration, pathogen lysis, PCR based amplification, and, finally, analyte
detection by hybridization on a microarray.
Portable Pathoanalyzer instrument to drive the lab-on-a-chip platform and to doubtless
identify airborne pathogen bacteria. This system includes an integrated airborne
pathogen detection system, fluid handling (pumps and valves), temperature control,
fully integrated optoelectronic sensor for direct chemiluminescent detection of PCR,
and data analysis.
Instrumentation & control software for the technical processes, managing the
interfaces between the individual modules, defining the transfer points for the sample,
the protocols to be done via recognizing the inserted chip types, managing the
handling of the microfluidic chips and the periphery to run them.
Analysis software for the qualitative and quantitative analysis of PCR via a
hybridisation and the interpretation of these data.
Established protocols and running chip systems with integrated reagents as ready-touse kit for the target pathogens tested under simulated aerosol application.
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Project PathoID-Chip
Publishable Executive Summary
UNCLASSIFIED
3 Project organization
Entity
Company (leading entity)
microfluidic ChipShop GmbH
Country
Point of contact
Germany
Dr. Claudia Gärtner
claudia.gaertner@microfluidic-chipshop.com
Company
Clemens GmbH
Germany
Thomas Clemens
t.clemens@clemens-gmbh.de
Bertin Technologies
France
Nicolas Rouch
rouch@bertin.fr
Research centre
Institut für Mikrobiologie der
Bundeswehr
Germany
Dr. Wolf Splettstößer
WolfSplettstoesser@Bundeswehr.org
Friedrich Loeffler Institut
Germany
Dr. Heinrich Neubauer
Heinrich.Neubauer@fli.bund.de
Joanneum Research
Austria
Dr. Stefan Köstler
stefan.koestler@joanneum.at
5 Project Results
The unexpected manifestation of uncertainties and angst which have been caused by the very
recent EHEC outbreak, demonstrates that the threat of infections by harmful bacteria is not
obsolete. Needless to say that the “EHEC episode” again illustrated the enormous destructive
power of bacteria misused as biological weapons. Fortunately, this particular bacterial hazard
lasted relatively short but illustrated at least two important facts: Bacterial pathogens are far
away from being beaten and a very rapid identification of the attacking germ is mandatory.
The aim of the project was to develop a portable device which allows identification of
dangerous bacterial strains within 60 minutes. The instrument meets two very important
requirements. At first, the user is not a technically skilled expert but a soldier or nurse for
instance and, secondly, it is expected that the germ in question threatens the citizens via an air
borne infection.
Accordingly, the whole process of pathogen
identification starts with air sampling. Our air
sampling device is capable to identify less than
10,000 bacteria out of one litre of surrounding air.
These bacteria are resuspended in approximately
one ml of water, which is used to transport the
germs into a microfluidic chip (see figure 1) that
has been clicked into the device. Inside the chip the
microbes are destroyed by boiling and the released
DNA is transported to the next station, the PCR
area. PCR is a very important molecular biological
Figure 1: Integrated chip
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Project PathoID-Chip
Publishable Executive Summary
UNCLASSIFIED
technique which facilitates an enormous multiplication (or amplification) of DNA pieces, and
the resulting DNA amplificate tells us whether the investigated pathogen is present or not.
The PCR runs through multiple cycles encompassing two or – in most cases - three different
temperatures. To do so, the PCR solution meanders in a channel system over these different
temperature zones in our chip. Thereby, the solution is rapidly heated up or cooled down
when leaving one zone and entering the other.
The heaters are implemented in an instrumental device (see figure 2). In addition to that, the
device has to elicit and to control the movement of the liquid into and inside the chip by
actuation of special syringe pumps (here: socalled tank drives). Membrane valves help to
organize the flow direction of liquids. Therefore,
they are closed by pneumatic or mechanic
pushing of a flexible foil into an adequate
channel structure sealing respective passage.
For the detection, we have adapted the so-called
PCR-ELISA to our requirements. Here, the
amplified and denatured, pathogen-indicating
sequence will specifically bind to an immobilized
reporter sequence, if present. The trapped
pathogen DNA will send out a signal (after some
Figure 2: PathoAnalyser Instrument
additional steps) which can be monitored by an
appropriate detection unit within the device. We
have tested colorimetric, fluorimetric, and chemiluminescent techniques.
Obviously there was a learning outcome for the further developments: Besides identifying and
optimizing the modules and combining them to an integrated device, also the module not
performing in a satisfying manner due to physical restraints and requiring a strategy change
was identified and further optimization purpose was identified: The module to be exchanged
is the air collecting system. A special miniaturized device, Coriolis Nano, was developed but
the outcome was that a bigger version simply due to physical laws is required, namely the
Coriolis Micro. The module that needs in particular optimization effort is the PCR module in
order to highly increase sensitivity of the unit.
On the prototypic level, the integrated instrumental device works well by fluidic means. All
biological assays have been successfully tested on single module level and optimization needs
have been identified. The protocol transfer in the integrated version of chip and instrument is
still ongoing and gives further reason for optimism.
With future development work to be carried out, this platform is the base for the first
integrated lab-on-a-chip device carrying out the sample-in result-out vision from airborne
pathogens.
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