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

) 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

 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

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

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

L2 L3

L5 L6

L7 L8

LED

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

, 10

and 10

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

HPO

, 0.0015 M KH

, 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

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

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).

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 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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