A NOVEL MOBILE MONITORING SYSTEM FOR FAST AND
AUTOMATED BACTERIA DETECTION IN WATER
Christoph Heller, Ulrich Reidt, Andreas Helwig, Florian Klettner, Gerhard Müller, Alois Friedberger
EADS Innovation Works, IW-SI – Sensors, Electronics & Systems Integration, 81663 Munich, Germany
Leonhard Meixner, Karl Neumeier
FhG-IZM Fraunhofer-Institut für Zuverlässigkeit und Mikrointegration, Hansastr. 27d, 80686 Munich, Germany
Petra Lindner, Ramona Molz and Hans Wolf
University of Regensburg, 93053 Regensburg, Germany
Keywords: Micromechanical filters, Micro fluidic system, Fluorescence detection, Bacteria sampling, Potable water.
Abstract: Standard detection methods for viable bacteria in potable water are time consuming due to a required
cultivation step. Fast and automated detection of water borne microorganisms with high sensitivity and
selectivity is still a challenge. We report on a novel biosensor using micromechanical filters with nano sized
pores to capture and enrich bacteria on the filter surface and subsequent detection using fluorescent probes.
The whole process is fully automated by integrating the sieves into a fluidic system together with a high
performance fluorescence detector. The results show the effective retention of bacteria on the filter surface,
which are then accessible for different staining procedures. As an example, we use special fluorescent dyes
that bind to or intercalate in the DNA molecules of the bacteria. After detection, the microfilters undergo
cleaning and conditioning steps to be ready for the next measurement.
1 INTRODUCTION
Under normal circumstances, our drinking water is
of very high quality. According to the current
directive of the European Community, no viable E.
coli may be present in a 250 mL sample of potable
water. To ensure this high quality, water works and
water suppliers are obliged to test the water quality
at regular intervals.
For these tests, classical methods with cultivation
on special growth media are still being used for
routine applications. The water samples are plated
on culturing plates and incubated at various
temperatures. Depending on the type of bacteria, this
cultivation step can take up to several days. After
incubation, the number of viable bacteria is
determined by visually counting the bacterial
colonies (Brenner, 1996; Gracias, 2004). Obviously,
this procedure is very tedious and time consuming
and requires skilled and experienced laboratory
personnel. The fact that a contamination with some
bacterial species (such as Legionella) will only be
detected after several days may lead to the outbreak
of epidemics. This can be a potential danger to
people with a weak immune system.
In the rare event of bacterial growth in parts of a
water supply network, all pipes, valves, pumps, etc.,
have to be thoroughly cleaned and re-examined
before they can be approved for further use. The
time consuming procedure of classical bacterial
testing will therefore lead to rather long down times
of a water supply system. The same is true for the
supply of clean production water in pharmaceutical
industry and for mobile water supply systems (e.g.
in ships).
To avoid such problems, a system is needed that
can continuously and automatically sample water
and test it quickly for the presence or absence of
bacteria or other pathogens.
Here, we describe such a system that has been
developed in our group. At the core of this device,
there is a micromechanical filter (sieve) having a
large number of pores (about 10
7
) with a diameter of
0,45 µm (Figure 1). When passing water through
such filters, any bacteria present in the sample will
384
Heller C., Reidt U., Helwig A., Klettner F., Müller G., Friedberger A., Meixner L., Neumeier K., Lindner P., Molz R. and Wolf H. (2009).
A NOVEL MOBILE MONITORING SYSTEM FOR FAST AND AUTOMATED BACTERIA DETECTION IN WATER.
In Proceedings of the International Conference on Biomedical Electronics and Devices, pages 384-389
DOI: 10.5220/0001545203840389
Copyright
c
SciTePress
be effectively captured and retained on the filter
surface. Once on the filter, they are easily accessible
to any kind of specific or non-specific staining
method (e.g. with DNA intercalating dyes, enzyme
substrates, membrane stains, labelled antibodies or
hybridization probes). Fluorescent labels are used in
our monitoring equipment.
Micromechanical filters are highly discriminative
and show a much better filtration performance
(higher filtration rate) than conventional filters of the
same size. Their absolutely flat surface does not
allow any particle to penetrate deeper into the filter’s
structure. Any bacteria retained by the filter will be
present at the surface, allowing easy detection and
washing off afterwards. Some work has been
reported on using microfilters which successfully
separate particles by means of filtration (Hsiai,
2002; Xing, 1999). These filters have pore diameters
of 6-12 µm. Ogura et al. reported the separation of
deformed red blood cells using microfilters with
larger pore size (Ogura, 1991a; b). Since the average
size of bacteria, for example E. coli, is
approximately 1µm, theses filters are not suitable for
the discriminative enrichment of bacteria.
In this paper we describe the successful capture,
labelling and detection of E. coli on the surface of
micromechanical sieves. We have integrated the
MEMS filters into a microfluidic system, allowing
fully automated water analysis with this biosensor
system.
2 MATERIALS AND METHODS
2.1 Capture and Enrichment of
Bacteria
The micromechanical filters are composed of a
Si
3
N
4
membrane (5 x 5 mm, approx. 1 µm thick)
supported by a silicon frame. The membrane is
perforated, each pore with a diameter of 450 nm
(Figure 1). In contrast to tissue or membrane based
filters, particles are not trapped inside a three
dimensional filter structure but on the surface.
Therefore, the particles are directly accessible.
Previous experiments show that bacteria trapped on
the surface can be easily removed after filtration
(Reidt, in press). A further advantage of the
micromechanical filters is based on the fact that all
pores are simultaneously etched after a
photolithographic patterning step. This ensures that
all pores have exactly the same diameter and their
spacing is completely homogenous (Figure 1).
Figure 1: REM picture of a micromechanical filter surface
with 0,45 µm diameter pore size (top view) after filtration
of drinking water.
2.2 A Fully Automated System for
Water Analysis
Using the micromechanical filters (sieves), we have
developed a fully automated system for capturing,
labelling and detecting bacteria in water. The sieves
are housed in a microfluidic chamber with a glass
window in very close distance above the filter
surface. There is a water inlet close to the filter and
one outlet in opposite position. Filter and glass
window form a microfluidic channel, where liquid
can be pumped from the inlet to the outlet passing in
parallel to the filter surface (cross flow). A second
outlet is positioned on the back side of the filter,
allowing for flow through (dead end) filtration.
Using a double selector valve, inlet and outlets can
be activated to perform both types of flow. There is
also the possibility to swap inlet and outlet for back
flushing the filter (Figure 2). The maximum
differential pressure over the microsieve is limited to
2 bar to avoid breaking the filter.
For detection, we use a standard setup with
coaxial excitation and emission light paths. Light
from a high power LED is collected and passed
through an emission filter (485 +/-12 nm band pass).
It is then reflected at 45° angle by a dichroic mirror
(505 nm) and focussed onto the filter surface. The
fluorescent light passes the mirror, is filtered
through a 520 +/-15 nm band pass filter and
focussed onto a photomultiplier tube (Figure 3).
Device control and data acquisition is performed by
LabView software on a laptop computer.
The precision pump (0 - 10 bar, 0.01 - 70
mL/min) was purchased from HNP (Parchim,
Germany), whereas the selector valves were
obtained from Valco-VICI (Schenkon, Switzerland).
As detector we use a Perkin-Elmer MP962 photo
A NOVEL MOBILE MONITORING SYSTEM FOR FAST AND AUTOMATED BACTERIA DETECTION IN WATER
385
Figure 2: Scheme of the fluidic path in the automated detection system. L1 – L8: different liquids; V1: selector valve, V2:
double selector valve, P: pump, W: waste, G: glass window, Ch: fluidic chamber, i: inlet, o1, o2: outlets, Det: detection
system, S: micromechanical sieve.
Figure 3: Scheme of the optical path in the automated
detection system. LED: high power LED with lens, L1 –
L3: lenses, A1, A2: apertures, Ex: excitation filter, Di:
dichroic mirror, Em: emission filter, PM: photo multiplier,
Ch: fluidic chamber.
multiplier; excitation is performed by a blue high
power LED (Luxeon). Optical filters were from
AHF Analysentechnik (Tübingen, Germany) and
LabView software as well as a DAQ board were
from National Instruments. Figure 4 shows a picture
of the complete biosensor system which is small
enough to be used as a mobile system.
Figure 4: Picture of the automated monitoring system for
bacteria in potable water.
2.3 Bacteria Labelling
For non specific labelling, 30 µL of a 100 fold
dilution of SYBRGreen I (Invitrogen, Karlsruhe,
Germany) was added to 1 mL bacterial suspension
(about 10
8
cells/mL of heat inactivated E. coli
O157:H7 from KPL, Gaithersburg, USA) and
incubated for 15 min in the dark. From this stock
solution serial dilutions were made in water and the
exact numbers of bacteria were determined by
P
L1
V1
V2
Ch
G
De
t
o2
o1
i
S
W
L2 L3
L4
L5 L6
L7 L8
L2
E
Di
E
L3
PM
L1
A
1
A
2
LED
Ch
BIODEVICES 2009 - International Conference on Biomedical Electronics and Devices
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counting in an improved Neubauer counting
chamber.
For immunodetection (specific detection) on the
filter surface captured bacteria were overlaid with
antibody solution specific for E. coli O157:H7
(polyclonal anti-E. coli O157:H7-Alexa 488 (ABL
Advanced Biomedical, UK) 2.4 µg/ml) and
incubated for 15 min. Non-bound antibodies were
removed by an extensive washing step (100 ml,
PBS) and bacteria recognized by the antibodies were
identified by fluorescence microscopy (Reidt, in
press).
3 RESULTS
3.1 Suitability of Micromechanical
Filters for Capturing and
Enrichment of Bacteria
In a first step, the suitability of micromechanical
sieves for capturing bacteria was tested. 20 mL of
various dilutions (10
2
, 10
3
and 10
5
cells/mL) of a
freshly prepared overnight culture of E. coli XL1-
Blue were passed through the filter. After filtration,
bacteria were removed from the filter surface by
rinsing with 5 mL PBS (0.14 M NaCl, 0.01 M KCl,
0.008 M Na
2
HPO
4
, 0.0015 M KH
2
PO
4
, pH 7.4). In
each case 100 µL of rinsing solution and filtrate
(flow through) were plated on LB agar (10 g
tryptone, 5 g yeast extract, 5 g NaCl to 1 liter H
2
O)
and incubated overnight at 37°C. Cell counts
obtained by plating initially applied bacterial
solutions (original solution) and from rinsing
solutions correlate very well, indicating that bacteria
can easily and efficiently be removed from the filter.
A major finding of all filter experiments was that
in no case any bacteria could be detected in the 100
µL samples from the flow-through. The
impermeability of the filters for bacteria was further
confirmed by examination of the complete filtrate
solution for the presence of bacteria. This
demonstrates a highly efficient and reliable filtering
process using the inorganic microsieves (Reidt, in
press).
3.2 Bacteria Detection
For unspecific detection we use fluorescent dyes that
bind to or intercalate in the DNA of the bacteria.
Upon binding, the fluorescent signal of the dyes
increases by a factor of 1.000-10.000.
Figure 5: Upper: Microscopic image of part of the
microsieve after filtering a water sample containing
SYBRGreen I stained bacteria. The width of an active
filter stripe is 180 µm. Lower: Same area, with
fluorescence microscopy. The bright spots represent
bacteria.
In our case inactivated E. coli O157:H7 are
stained with SYBRGreen I. Water samples are
spiked with different amounts of fluorescently
labelled bacteria and analyzed in the monitoring
system. Figure 5 shows a microscopic image of the
filter after analysis and the corresponding
fluorescent image visualizing the bacteria. So far, a
limit of detection of 40.000 cells on a single
microfilter could be achieved (Figure 6). The
detection limit can be significantly improved as it is
currently limited by the optical detection system not
yet being optimized.
With the system, it is also possible to first pass a
water sample (with non labelled bacteria) through
the microsieve and then stain the cells on the filter
surface.
Obviously, it should be possible to perform other
specific or non specific labelling procedures. For
example, we have also tested the immunodetection
180 µ
m
A NOVEL MOBILE MONITORING SYSTEM FOR FAST AND AUTOMATED BACTERIA DETECTION IN WATER
387
of non-pathogenic E. coli O157:H7 on
micromechanical filters. It could be demonstrated
that E. coli serotype O157:H7 could be detected
specifically by the antibodies while neither the
micromechanical filter surface nor the negative
control (E. coli DH5α) interacted with the antibodies
(Reidt, in press).
0
1.000
2.000
3.000
4.000
5.000
6.000
7.000
0 200 400 600 800 1.000 1.200
Number of bacteria (x 1000)
Signal (kHz)
Figure 6: Detection of fluorescently labelled bacteria with
a fully automated detection system.
Depending on the source, there may be potable
water available in some regions with a quite high
density of particles. This could cause problems in an
automated water analysis system. One concern could
be a background fluorescence signal originating
from particles. Although the optical system is not
optimized yet, we could not observe a significant
signal from particles.
Another issue might be the blocking of the filters
by the dirt. In fact, water flow rate of tap water
through the sieves is reduced in comparison to pure
water. However, a reasonable flow rate is still
sustained (Figure 7). If the system should be used
for water with a high density of particles, the
microfilter area can simply be increased (currently it
is 5 x 5 mm).
0
5
10
15
0 5 10 15 20
Volume (m L)
Flowrate (mL/min)
Figure 7: Flow rate in mL/min of tap water through a
microsieve (microfilter) with size 5 x 5 mm.
4 DISCUSSION
We have shown that micromechanical filters can
retain bacteria from water with high efficiency and
reliability. Also, bacteria can directly be detected on
the surface of the micromechanical filters using
fluorescently labelled antibodies or DNA binding
dyes. The microfilter platform is very flexible, i.e.
other specific or non specific labelling procedures
can be applied such as membrane probes, enzyme
substrates, nucleic acid hybridization probes or PCR.
Furthermore, the micromechanical filters have
been integrated into a fully automated fluid delivery
and detection system. Micromechanical filters with
appropriate pore sizes could also be an effective tool
for recovering other pathogens such as viruses and
protozoa.
During the last years, a few bacteria detection
methods have been developed as alternatives to
cultivation techniques, mainly based on flow
cytometry (e.g., Sakamato, 2005) or solid phase
cytometry (e.g., Aurell, 2004; Lisle, 2004). Flow
cytometry is fast, but is only capable of analyzing a
volume of a few microlitres per minute and thus
requires preconcentration of the drinking water
sample. In solid phase cytometry, the water is
filtered through a porous membrane which has then
to be transferred to a laser scanning device. Total
analysis time is about 4 hours.
In both cases, a number of manual steps is
required, making automation difficult, whereas our
method offers the possibility of fully automated and
rapid analysis of potable water samples.
ACKNOWLEDGEMENTS
We thank Erwin Yacoub-George and Waltraud Hell
(FhG-IZM) as well as Eberhard Rose and Thomas
Ziemann (EADS) for their valuable assistance. Part
of this work has been supported through the project
OptoZell funded by the German ministery for
education and research, BMBF (funding agency:
VDI).
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