Development of an Inline Rapid Detection System for Waterborne Pathogens Otto J1, Behrmann O2, Blättel V3, Dame G2, Dietze S4, Dilcher M5, Elsässer D6, Hakenberg S2, Heese C7, Hufert F5, Hügle M2, Karthe D8, Kunze A6, Scharaw B4, Sedehizade F9, Seidel M6, Vosseler S3, Westerhoff T4, Tiehm A1 1 DVGW - Technologiezentrum Wasser, Karlsruhe (TZW), Germany 2 Institute of Microsystems Technology, Freiburg University, Germany 3 R-Biopharm AG, Darmstadt, Germany 4 Advanced System Technology (AST) Branch of Fraunhofer IOSB, Ilmenau, Germany 5 Center for Biological Systems Analysis, Freiburg University, Germany
6 Institute of Hydrochemistry and Chemical Balneology, Technical University of Munich, Germany 7 GWK Precision Technology GmbH, Munich, Germany 8 Helmholtz Centre for Environmental Research, Magdeburg, Germany 9 Berliner Wasserbetriebe (BWB), Berlin, Germany
Tel.: +49 721 9678 136. E-Mail:
[email protected] Tel.: +49 721 9678 137. E-Mail:
[email protected] 18th International Symposium on Health-Related Water Microbiology, September 13th-19th 2015 - Lisbon, Portugal
M OTIVATION Providing clean water is one of the essentials of modern societies. The currently used cultivation techniques do not allow prompt monitoring and control. However, the ability to continuously detect waterborne contaminants is desirable and would allow immediate reactions on emergencies.
C ONCEPT The main goal of the project EDIT is the development of a concentration and detection system for the inline monitoring of waterborne organisms in water. The concept integrates innovative techniques to detect health related microorganisms by a sequence of concentration, extraction and molecular biological detection with live / dead differentiation.
H ygiene Omodular n L ine M onitoring design M ACRO -C ONCENTRATION
L AB ON CHIP
Continuous concentration of 1m³ to 1mL
Micro-concentration to 5µL Extraction
R AW
Bacteria and viruses are concentrated with a
L i v e / d e a d d i ff e r e n t i a t i o n
continuous cross-flow-ultrafiltration (CUF). 1000 liters of water are reduced to 20 liters in one hour. The
The sample is further concentrated using a free
resulting concentrate is reduced to 20 mL via
flow electrophoresis on a microchip. During
monolithic affinity chromatography (MAF) for turbid
sample flow through, the organisms are collected
samples or a combined CUF-MAF for clear samples.
at a gel front in an electric field.
1000L
Cross-flow-ultrafiltration (CUF)
PMA is used to distinguish between living and dead organisms. Using an electro-thermal lysate
Current state: 70% recovery for concentrating 1000 L in one hour (MS2).
step, the nucleic acids are extracted and purified Free flow electrophoresis microchip
Kunze A., Pei L., Elsässer D., Niessner R., Seidel, M. High performance concentration method for viruses in drinking water. J. Virol. Methods 222, 132–137 (2015).
via electrophoresis.
Current state: Extraction and purification procedures tested
successfully.
Live /dead differentiation shows promising results for defined conditions Pei L., Rieger M., Lengger S., Ott S., Zawadsky C., Hartmann N.M., Selinka H.-C., Tiehm A., Niessner R., Seidel M. Combination of Crossflow Ultrafiltration, Monolithic Affinity Filtration, and Quantitative Reverse Transcriptase PCR for Rapid Concentration and Quantification of Model Viruses in Water. Environ. Sci. Technol. 46, 10073-10080. (2012)
(Chlorine, ozone, thermal disinfection). Hakenberg S., Hügle M., Meyer P., Behrmann O., Dame G., Urban G. A. Fenton fragmentation for faster electrophoretic on chip purification of amplifiable genomic DNA. Biosensors and Bioelectronics, 67, 49–52. (2014)
P ROCESSING
D ETECTION /M ICROARRAY
Result analysis and alarm signal
Detection of pathogens via isothermal amplification
All data from each module are recorded and
Nucleic acids of target organisms are multiplied via isothermal
evaluated in an online accessible database.
amplification in parallel. Species-specific primers are labelled on
The records can be accessed via smartphone
the microarray surface. The label is detected via protein binding and
or tablet app (QR-Code). An alarm signal is
chemiluminescence reaction. The resulting light signal is captured
created in case of detections exceeding a set
by a CCD camera.
value. Mobile processing app
Current state: Isothermal amplification of E. faecalis and phiX174 working (multiplex). Target bacteria: E. coli, E. faecalis, P. aeruginosa, C. jejuni, K. pneumophila Target viruses: Norovirus GI+GII, Adenovirus, Enterovirus, MS2, phiX174
Project consortium
Lengger S., Otto J., Elsässer D., Schneider O., Tiehm A., Fleischer J., Niessner R., Seidel M. Oligonucleotide microarray chip for the quantification of MS2, ΦX174, and adenoviruses on the multiplex analysis platform MCR 3. Analytical and Bioanalytical Chemistry 406, 3323–3334. (2014)
A CKNOWLEDGEMENT Financial support by the German Ministry of Education and Research is gratefully acknowledged (EDIT project, INIS research priority, BMBF grant no. 033W010).
MCR3 microfluidic chip system.
