development of an evanescent-field fibre optic sensor for

7/31/2019 Development of an Evanescent-field Fibre Optic Sensor For

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Biosensors & Bioelectronics 16 (2001) 399408

Development of an evanescent-field fibre optic sensor forEscherichia coli O157:H7

A.P. Ferreira a,*, M.M. Werneck b, R.M. Ribeiro b

a Department of Sanitation and En6ironmental Health, National School for Public Health, Oswaldo Cruz Foundation, Rio de Janeiro, Brazilb Biomedical Engineering Program, Federal Uni6ersity of Rio de Janeiro, PO Box 68564, 21945-970, Rio de Janeiro, Brazil

Received 6 March 2000; received in revised form 14 March 2001; accepted 12 April 2001

Abstract

An intensity-modulated fibre optic sensor was developed for Escherichia coli O157:H7. The interaction between the whole

natural bacteria and the guided lightwave was carried out by means of evanescent-field coupling. A correlation between optical

response and the current number of bacteria was achieved. The device sensitivity had been calibrated for initial number of bacteria

(N0) from 10800. The sensor sensitivity was 0.016 (90.001) dB/h/N0. The sensing mechanism starts together with the log phase

leading the present sensor response to be five to ten times faster than conventional bacteriological techniques. 2001 Elsevier

Science B.V. All rights reserved.

Keywords: Fibre optic sensor; Evanescent field; Escherichia coli O157:H7

www.elsevier.com/locate/bios

1. Introduction

Escherichia coli has been recognized as a common-

place microorganism found in the intestinal tract of

human and other warm-blooded animals. E. coli typi-

cally colonizes the infant is gastrointestinal tract within

hours of life. Usually it remains harmlessly confined to

the intestinal lumen, only becoming pathogenic in the

debilitated or immunosuppressed host, or when gas-

trointestinal barriers are violated. Therefore, E. coli has

served as an indicator for faecal contamination in water

and food.

Enterohemorrhagic Escherichia coli O157:H7 is anemerging pathogen; which is the cause of foodborne

illness, the major cause of serious outbreaks and spo-

radic cases of hemorrhagic colitis and hemolytic-uremic

syndrome (Pickering et al., 1994; Siegler, 1995; Nataro

and Kaper, 1998). The major impact of bacterial in-

duced diarrhoea disease is on children under the age of

10 and on elderly people living in less-developed coun-

tries, in which bacterial diarrhoea diseases remain a

significant public-health problem. It represents also the

greatest challenge for both physician and scientific re-searches. E. coli O157:H7 is the cause of acute and

persistent diarrhoea disease and ongoing morbidity that

has malnutrition as one of its major contributors

(Huilan et al., 1991). The level of biohazard is high due

to the extremely low dose (10 microorganisms) required

for infection (Nataro and Kaper, 1998).

The greatest drawback for pathogen-detection tech-

niques is the lack of reliable and sensitive means for

measuring their presence in real time. Evanescent-wave-

coupling sensor technology is a suitable technique for

microorganism measurement in its natural form that

may be applied to the biosensor development takingadvantage of the current understanding of the whole

cell architecture of the microbial (Watts et al., 1994;

Hutchinson, 1995; Bousse, 1996; Muller et al., 1997;

Nath et al., 1997).

The biological and medical areas such as living cells

and bacteria detection remain a challenging technique.

The advantages of fibre optic sensing are well known

and have been reported in the literature (Smoczynski et

al., 1993; Ligler et al., 1993; Karube and Nakanishi,

1994). In the last few years fibre optic sensors have been

increasingly employed in biological sensing because of

their electrical isolation, electromagnetic-interference

immunity, compactness, lightweight, sensitivity, in-line

* Corresponding author. Tel.: +55-21-598-2746; fax: +55-21-270-7352.

E-mail address: [email protected] (A.P. Ferreira).

0956-5663/01/$ - see front matter 2001 Elsevier Science B.V. All rights reserved.

PII: S 0 9 5 6 - 5 6 6 3 ( 0 1 ) 0 0 1 4 9 - X


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A.P. Ferreira et al. /Biosensors & Bioelectronics 16 (2001) 399408400

implementation by means of evanescent-field coupling,

potential low cost and biological compatibility. The

optical sensor has revealed to present a faster response

than conventional techniques of clinical laboratory

analysis. One of the major advantages of using the

coupling with the guided lightwave evanescent field is

the development of in-line fibre optic devices. The fibre

is not interrupted and the light is not removed from the

fibre. The signal processing takes place inside the opti-cal fibre thus characterizing an intrinsic sensor.

A fibre optic biological sensor has been designed in

our laboratory and was described elsewhere (Ferreira et

al., 1999a,b). In this work, using the same measurement

principle, the sensor has been employed to quantify E.

coli O157:H7.

This paper describes for the first time, to the best of

our knowledge, experimental results on sensitivity cali-

bration of an intensity-modulated fibre optic evanescent

sensor for the E. coli O157:H7 as a whole bacterium.

The complete bacteria growth phases were optically

sensed and time resolved with reproducibility. Electronand optical microscopy were also employed for a de-

tailed understanding of the interaction dynamics be-

tween the bacteria and the optical fibre probe.

2. Principles

2.1. The bacteria growth e6olution

As reported from the literature (Pelczar et al., 1993),

the bacterium growth evolution follows a well-knownpattern that comprises four phases, as is briefly de-

scribed below. The LAG phase begins once the bacteria

are isolated in a suitable biological culture medium.

Growth is very slow at first (little or no division), while

the organism is adjusting its metabolism to the food

and nutrients in its new environment.

The log (or logarithmic) phase starts once the

metabolic machinery is running and the bacteria start

multiplying exponentially, doubling in number (binary

fission) at every few minutes. The time required for the

bacteria population to double after the lag phase is

called generation time g:

g=t

n. (1)

Where n is the number of generations that occur in

the period of time t (in min). A more practical formula

may be derived from the definition stated above and

gives:

g=t

3.3(log10N log10N0). (2)

Where, N is the current number of bacteria at time t

and N0 is the initial number of bacteria. The current

population N(t) at the log phase can be derived from

Eq. (2):

N(t)=No2t/g (3)

As more and more bacteria are competing for food

and nutrients the booming growth stops and the num-

ber of bacteria stabilizes in the stationary phase. At the

same time dying and dividing the organisms are at

equilibrium. Death is due to reduced nutrients, pHchanges, toxic waste and reduced oxygen. In some cases

cells do not die but they are not multiplying. Eventu-

ally, when the death rate exceeds the growth rate, the

cells enter the death phase. The population is dying in

a geometric fashion so there is more death than new

cells, and the population may be entirely destroyed.

2.2. The sensing mechanism of the sensor

The sensing mechanism of the sensor described here

relies upon the attenuation of a guided lightwave by

means of evanescent-field coupling that takes place atthe sensitive fibre. As the bacteria grow in the neigh-

bourhood of the sensitive fibre, the guided lightwave is

changed in intensity.

The culture medium is adherent to the cladding of

the almost unclad sensitive fibre. Normally, light travel-

ling inside the fibre experiences total internal reflection

when light beams strike the interface between the fibre

core and the cladding. This occurs when the angle of

incidence is greater than the critical angle. Thus, a

propagating optical wave through the optical fibre is

bounded in its core, but allowed to interact with theoutside medium by evanescent-field coupling when this

field is exposed (Rahnavardy et al., 1997). The evanes-

cent field of the guided lightwave penetrates beyond the

core surface and exponentially decreases in its magni-

tude an order of a wavelength away from the core/clad-

reflecting interface. The evanescent-wave penetration

depth (dp) is calculated by:

dp=u

2y(n

2 sin2qn cl2 )1/2

, (4)

Where dp is the distance by which the magnitude of

the electric field decays of l/e to its intensity at thecore/clad interface, q is the internal incident-ray angle

with the normal to the core/cladding interface, u is the

vacuum wavelength of the light beam and nco and nclare the core and cladding index of refraction,

respectively.

The evanescent-field coupling interaction mechanism

will cause optical attenuation of the guide lightwave

when any material (gas, liquid or solid) gets into con-

tact with the surface of the etched fibre clad. The loss of

power is proportional to the intrinsic bulk absorption

and scattering (Rayleigh and Mie). Thus, as a light ray

progresses down the multimode fibre by bouncing off


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A.P. Ferreira et al. /Biosensors & Bioelectronics 16 (2001) 399408 401

the core-cladding interface, it loses power at each

bounce. While the loss per bounce is small, the accumu-

lated loss after a large number of bounces can be

significant even fbr a modest length of the probe fibre.

When a light ray travelling in a medium of a particu-

lar refractive index impinges on the boundary of an-

other slightly attenuation medium of lower refractive

index, it suffers the following approximate fractional

loss of power on refraction:

T=4(q/q c

2)

qc2q2 uh

4yncl

. (5)

Where T is the fractional loss of power, qc is the

critical angle of two media, q is incidence angle at the

boundary between the media (measured with respect to

the plane of the interface, rather than its normal), u is

the optical wavelength, h is the bulk-attenuation coeffi-

cient of the culture medium at the region of contact,

and ncl is the (real part of the) refractive index of theclad at the boundary (Snyder and Love, 1983).

Thus, the fractional power reflected back into the

original medium is (1T) and the fraction of the

original power remaining after N reflections is (1T)N.

In this formula qBqc and should not be too close to it

for the approximation to be valid. This means that it

applies to rays that would have been well guided in the

absence of loss. Furthermore the tighter such guidance

(i.e. the smaller q/qc), the lower the loss on reflection.

The optical attenuation can be generated by two

interrelated physical mechanisms: variation of the cladrefractive index ncl as well as of its absorption coeffi-

cient h. When the bacteria are allowed to grow around

the optical fibre, both effects can occur:

1. In the lag phase enzymes are released because of the

bacteria metabolism, causing the change of the in-

dex of refraction. Although this issue is not the aim

of this paper, the interdependence between absorp-

tion coefficient and index of refraction has been

experimentally shown and is theoretically described

by the Kramers-Kronig Eqs. (Nussenzveig, 1972).

2. Due to the existence of an increasing number of

bacteria in contact with the fibre during the logphase, the medium becomes more opaque as time

goes by. Consequently the intrinsic absorption also

changes.

Both effects may change the mechanism of interac-

tion between the evanescent field and the outside mate-

rial medium, coupling out different amounts of the

fibre-guided lightwave. Therefore, the measurement of

the light power at the exit of the fibre shows indirectly

the bacteria growth. As explained in more detail in

Section 4, the decreased optical power is mainly related

to the number of bacteria present in the volume occu-

pied by the evanescent field around the fibre.

3. Experimental

The sensor described here may be envisaged as com-

prising by three parts: The optical circuitry, the evanes-

cent probe fibre (sensitive fibre) and the biological

culture medium.

3.1. The optical circuitry set -up

Fig. 1 shows a schematic drawing of the biosensor

set-up. The optical source is a 3-mW CW GaAlAs laser

(AsGa Microeletronica, Brasil) with graded-index mul-

timode fibre pigtail, emitting at 840 nm. The output

fibre was spliced with a bi-directional optical fibre

coupler (CPqD Telebras, Brasil). Half of the emitted

optical power propagates over the probe fibre (sensing

element). The light modulated by the growing bacteria

exits the fibre and is detected by the photodiode PD2providing the electrical signal Samplein. The other half

of the optical power is detected by the photodiode PD1from which an electrical reference signal Refin is gener-

ated. Both electrical signals are amplified and measured

by a twochannel calibrated optical-power meter

(Graseby Optronics, USA). An AID board controlled

by the LabVIEW software (National Instruments Cor-

poration) digitises both output signals from the optical-

power meter (Sampleout. and Refout) by means of its

IEEE-488 interface. The AID board collects 800 points

per min of both Sampleout and Refout recording and

storing the respective averages for a period of 24 h per

measurement cycle.

3.2. The e6anescent optical probe fibre

In order to expose the evanescent lightwave field, 20

cm of a graded-index multimode optical fibre (62.5/125

mm) was clad-stripped by chemical etching. The etching

was performed by means of hydrofluoric acid solution

(38%) during 11 min in order to leave between 0.5 to 1

mm of clad over the core of the fibre. After this time,

the chemical reaction was stopped by immersion in

demonised water and then in phosphate-buffered saline

(PBS) with pH 7.4 for 15 min, in order to remove allremains of water from the probe. The exact etching

Fig. 1. Optical circuitry of the sensor.


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time was determined by monitoring diameters in a

previous measurement using a calibrated optical micro-

scope. This method has been detailed described by

Muller and co-workers (Muller et al., 1998).

After the chemical etching the fibre was wound into

a single loop, with a total sensitive surface area of

approximately 40.0 mm2, in which the evanescent

field can be accessed. The probe fibre was put over

the culture medium in which the bacteria grew, selec-tively.

3.3. Selecti6e culture medium for E. coli O157:H7

All of the described experiments in this paper were

performed using Escherichia coli O157:H7 CDC EDL-

933 (ATCC 43894), which was supplied by the Centre

for Disease Control and Prevention (CDC/USA).

In order to promote the E. coli O157:H7 growth, the

selective culture medium employed was MacConkey

Sorbitol Agar (SMAC) Difco 0079177 (Difco Labo-

ratories, Brasil) at an incubation temperature of 35 C.After optical measurements with the sensor, the bacte-

ria underwent microbiological identity tests in order to

ensure that only that specific microorganism had grown

on each Petri plate. The identity tests were made in

accordance with Farmer and Davis (1985).

Although biochemical characteristics associated

with the great majority of E. coli O157:H7 serotypes

are well known, there are not much data available on

their identification. It must be stressed that about 75 to

94% of E. coli strains quickly ferment D-sorbitol,

while the O157:H7 strain does not (Thompson et al.,1990).

It is also referred to this pathogen its inability to

produce b-glucuronidase which hydrolyses-4-methyl-

umbelliferyl-D-glucuronide. This does not happen with

other serbtypes of E. coli. Another parameter is that

more than 90% of E. coli O157:H7 strains produce one

or two unique biochemical profile numbers on a Mi-

croScan conventional gram-negative identification

panel (Baxter Diagnostics, Inc., California) and other

D-sorbitol negatives were not detected by this technique

(Abbott et al., 1994).

Finally, E. coli is a facultative anaerobe, whichmeans that it can survive in the presence or absence of

oxygen, it is a gram-negative bacterium and is not

fastidious in its nutritional requirements.

3.4. Bacteriological preparations for the sensor

calibration

In order to calibrate the sensor for its sensitivity,

several tests were performed using different N0.

E. coli O157:H7, available lyophilised in ampoules,

was restored with 1.0 ml of PBS, pH 7.4, inoculated in

several Petri plates with SMAC and incubated at 35 C

for 24 h. At the end of this period, the purity of the

material was checked and the Petri plates were stored at

4 C for further dilution.

For obtaining a dilution with N0=10, 20, 30, 40, 50,

60, 70, 80 and 90 microorganisms, were used 100 ml of

PBS, pH 7.4, whereas for N0=100, 200, 400 and 800

the volume used was 500 ml of PBS, pH 7.4. For each

sample the cells were counted using the Coulter Coun-

ter (Beckman, USA), which allows an accuracy of91%. Finally, the probe fibre was rested upon the Petri

plates with the culture as described above, the dilution

with a known number of microorganisms was poured

onto the sensitive area of the probe and the hardware

and software were started.

In all cases the SMAC was supplemented with glyc-

erol at 0.2%, which allows the maintenance of the

residual humidity and provides a better interaction

between the microorganisms and the sensitive area.

This was done in order to avoid the culture going dry

during the tests and thus inserting another variable into

the process.

3.5. Taking pictures of the process

For a direct observation of the microorganisms in-

teraction with the probe fibre, photographs were taken

employing the scanning electron microscope and the

optical microscope.

3.5.1. Scanning electron microscopy

Micrographs were taken at 316 min (N0=400) and

410 min (N0=20), corresponding to the elapsed timeafter which the sensor reaches half of the optical-signal

drop. Cells were fixed with 2% (v/v) glutaraldehyde in

PBS, pH 7.4, for 3 h at 4 C. After washing 3 times

with PBS, pH 7.4, 4% (v/v) of osmium tetroxide was

added to each sample during 1 h at 4 C. After dehy-

dration in a graded series of ethanol dilutions, cells

were subjected to a critical point drying with CO2(Samdri-780A, Tousimis Research Corporation). Sam-

ples were covered with ag 0 Id thin film by an ion

sputtering (JFC-1 100 Ion Sputter, JEOL) and exam-

ined under a digital scanning microscope (Carl Zeiss

DSM94OA). Photographs were taken with Agfa APX100 black and white film.

3.5.2. Optical microscopy

The optical microscopy pictures were taken after 398

min (N0=100), corresponding to the stationary optical

signal. Cells were fixed with 2% (v/v) glutaraldehyde in

PBS, pH 7.4, for 3 h at 4 C and then washed 3 times

with PBS, pH 7.4. The images in differential interfer-

ence contrast were obtained with the Zeiss Axioplan 2

microscope (Carl Zeiss) and photographed with a Po-

laroid MC2OO (Carl Zeiss) using Kodak Tmax 100

film.


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Fig. 2. Scanning electron micrograph of E. coli O157:H7 (log phase)

that are in physical contact with the probe fibre. Two levels of

magnification are shown.

Fig. 3. Optical phase-contrast micrograph of the E. coli 0 1 57:H7

after Iout(t ) reached the stationary state. Two levels of magnification

are shown on 3(ab).

physical contact with the probe fibre. The optical mi-

crograph shows an almost physical isolation of the E.

coli O157:H7 cluster (on top of the probe fibre) from

the others colonies. Because of the short range of the

evanescent field, the former were optically probed whilethe later were not.

In order to characterize the sensitivity of the sensor,

several measurements were carried out, each one during

a 24 h interval (1440 min), employing thirteen different

values of N0. These values were varied from 10800 E.

coli O157:H7 bacteria samples. Fig. 4 shows the plot of

4. Results and discussion

4.1. Presentation of the experimental results

Fig. 2 and Fig. 3 display micrographs of the probe

fibre showing the physical contact with the E. coli

O157:H7 growing around it.

Fig. 2 shows the micrograph taken by means of the

scanning electron microscopy on two levels of magnifi-

cation. It displays the fibre-bacteria physical contact

during the Iout(t) drop (log phase) as shown in Fig. 4and Fig. 5. Fig. 2(a) shows an island-like cluster of E.

coli O157:H7 on top of the probe fibre. Notice that the

optical fibre, as evaluated by the scale shown at the left

of the picture, is only about 60 mm in diameter, which

is approximately the same as the one of the core. Fig.

2(b) shows with a greater spatial resolution an as-like

amorphous structure of the E. coli O157:H7 cluster in

which the bacteria are stacked as foam.

Fig. 3 shows a micrograph taken from a phase-con-

trast optical microscopy with two levels of magnifica-

tion. It displays the fibre-bacteria physical contact, after

Iout(t) has reached the stationary phase, which are inFig. 4. Optical response Iout(t) of the sensor parameterised by theinitial number N0=0, 10, 80 and 800 of E. coli O157:H7.


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Fig. 5. Line 1 and line 2: The optical response Iout(t) of the sensor for two similar tests with N0=200, referred to the left vertical axis (the

superimposed straight lines are averaging of the data used to calibrate the sensor). Line 3: The estimated number of bacteria, as predicted by Eq.

(3), referred to the right axis.

the temporal optical response Iout(t) (in arbitrary units)of three of such measurements (N0=10, 80 and 800)

plus the blank test (N0=0).

Fig. 5 shows two separated tests for N0=400 (lines 1

and 2) and the theoretical current number of bacteria

N(t) representing the population of the colony along its

life evolution, as previewed by Eq. (3) (line 3). The

superimposed straight lines are eye-guide lines repre-

senting an averaging of the experimental plot used to

calibrate the sensor (see Section 4.6). The theoretical

plot also shown at Fig. 5 is the representation of Eq.

(3).

As explained on the text (see Sections 4.2 and 4.6) the

superimposed straight lines represent a mathematical

idealization that helps on the sensor calibration. The

arrows guide the reader for the correct vertical axis

corresponding the two plots.

As seen from the two tests performed with N0=200

shown in Fig. 5 it is possible to infer that the results of

the optical measurements were quite reproducible. The

good reproducibility of the optical response of the

sensor is guaranteed by the systematic procedure that

was employed for the biological culture as well the

sensing probe fibre insertion.

4.2. Analysis of the sensor response Iout

(t) in the lag

phase of the culture

The plots displayed in Fig. 4 show different phases of

Iout(t) for any used N0. In the first phase, it may be

observed a DC level Iout(t)=ILAG with an almost simi-

lar time range. The ILAG level is assigned to the sensor

response in which the E. coli O157:H7 remains in its lag

phase during ZtLAG time delay.

Fig. 6 shows a plot of DtLAG

against N0

for all

measurements. The linear relationship with an almost

null angular coefficient was fitted with an average of27094 min or approximately 4.5 h, meaning a re-

peatability of $1.5%. DtLAG may be attributed to the

time range that E coli 0 1 57:H7 spent in its lag phase,

despite their initial number N0.

Since enzymes are usually released by E. coli

O157:H7 along the lag phase, the average refractive

index of the culture medium ncm is likely to vary with

time. As described in Section 2, the variation of ncmmay cause changes in Iout(t), as stated by Eq. (5).

However, the measurements shown in Fig. 6 suggest

that the sensor, while probing the lag phase, is insensi-

tive to this effect. A possible explanation for this is that

the refractive index changed very little and the variation

of the optical response was buried into the noise. It is

possible to observe in Fig. 2 that the optical signals

present a slight slope during this phase. Therefore it

possible to infer that the technique used in this work is

Fig. 6. Time delay in the lag phase DtLAG (min) against the initialnumber N0 of E. coli O157:H7.


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more sensitive to the bacteria division (growth) than to

the variation of the index of refraction of the culture

medium itself.

4.3. Analyses of the sensor response Iout

(t) in the log

phase

Following the lag phase, E. coli O157:H7 starts the

log phase in which the size growth and replication takeplace. The curves displayed in Fig. 4 show that just

after approximately 270 min the sensor output signal

Iout(t) begins to drop gradually. The linear decreasing

of Iout(t) may be attributed to the sensor tracking of the

log phase evolution when the optical attenuation, as

sensed by the evanescent field, monotonically increases.

It should be carelully examined from the electron scan-

ning micrograph shown in Fig. 2 that the colony does

not cover the lull surface of the probe fibre.

If the attenuation coefficient h and the refractive

index ncm of the culture medium change in opposite

directions, as the bacteria grow, the critical angle qcincreases. As a consequence, shown by Eq. (5), the

effect of increasing the attenuation coefficient is par-

tially offset by improved optical guidance, for rays of a

given incidence angle, and the final effect on the optical

power output is unnoticeable. However if the attenua-

tion coefficient h and the refractive index ncm of the

culture medium change in the same direction, as bacte-

ria grow, the critical angle qc decreases. Both effects are

now coherently summing up and the fractional loss T is

reinforced, for rays of a given incidence angle, and the

final effect is a decrease of Iout(t).By extrapolation from the lag phase, it is unlikely

that the enzymes produced by the E. coli O157:H7,

even in the log phase, may affect the sensor Iout(t) for

all N0 employed. In this way, along the log phase, only

the attenuation coefficient a suffered appreciable

changes, thus affecting Iout(t). In other words, the sen-

sor described here seems to be insensitive to the average

refractive index changes, if it changes at all. Only

intrinsic absorption as well Rayleigh and Mie scattering

affect the optical signal Iout(t).

4.4. Analyses of the sensor response Iout(t) in thestationary phase

All plots displayed in Fig. 4 show that after the end

of the Iout(t) drop, the sensor response reaches another

DC level (ISTAT). Following the log phase, the E. coli

0157:117 stops their size growth and replications, thus

reaching the stationary phase in which Iout(t)=ISTAT.

A careful examination of Fig. 4 and Fig. 5 shows

that, in the so-assigned log phase Iout(t) turn to a lower

derivative, a little before reaching the stationary phase,

it is likely that between the purely log and stationary

phase, a transition region takes place. From some

instant of time, a fraction of E. coli O157:H7 stops its

growth and replication, thus reaching its own station-

ary phase. In this way, the smaller derivative of Iout(t)

is assigned to the coexistence of E. coli O157:H7 clus-

ters over the probe fibre, at both the log and the

stationary phase thus affecting the optical response of

the sensor.

It is also noticeable in Fig. 4, for all measurements

performed with different N0 (until N0=800), that ISTATis always smaller than the previous one. The sensor

response Iout(t) has shown an optical-dynamic reserve

in the log phase that makes it clear that E. coli

O157:H7 really reached the stationary phase. Thus, the

ISTAT level (lower than ILAG) may be assigned to the

stationary phase of the E. coli O1 57:H7 evolution,

when the attenuation, as sensed by the evanescent field,

remains unchanged.

After the clad was stripped from the probe fibre, a

surface area of approximately 40.0 mm2 has been

achieved, and the evanescent field was exposed. On the

other hand, it should be carelully examined from theoptical micrograph shown by Fig. 3(a) and (b), that E.

coli O157:H7 at the stationary phase were able to cover

almost all the surface area of the probe fibre.

4.5. Analyses of the sensor response Iout

(t) in the death

phase

For a 24 h monitoring, the optical sensor response

Iout(t) did not show any remarkably change after the

stationary phase was reached.

Although the E. coli O157:H7 starts its death phasesome time after the end of the log phase, the sensor

described here does not seem to optically discriminate

the stationary phase from the death phase.

4.6. Calibration of the sensor

In Fig. 5 three eye guidelines were drawn for each

Iout(t) regime of the sensor and E. coli O157:H7 phase.

Along with the lag phase, an averaged unitary normal-

ized straight line was outlined. Just at the beginning of

the log phase, Iout(t) suddenly dropped with a sharp

derivative. It was smoothed by a non-null angular-co-efficient straight line. A similar procedure was per-

formed at the stationary phase extrapolated from

t=. But the steady state straight line outlined had an

ISTAT DC level lower than the ILAG DC level.

Two crossing points among the three straight lines

may be viewed in Fig. 5. The first one fix the boundary

between the lag and the log phase and the second one,

analogously, fix the boundary between the log and the

stationary phase.

The ordinate difference between the two cross points

provides the optical attenuation DIout

(in dB) for each

N0. Similarly, the abscissa difference between the two


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Fig. 7. Sensor calibration curve. It is shown the time derivative iLOGof Iout(t) in the log phase against N0. A linear dependence was fitted.

ference on the detection of E. coli 0 1 57:H7 was

observed.

Although not shown here, it was also carried out a

measurement in which none E. coli Ol 57:H7 were

present in the culture mediumnegative control or

blank measurement. In this case, the output signal

Iout

(t) did not change even after 24 h of monitoring,

and the noise pattern observed was similar to the one

presented in Fig. 4 (with the presence of E. coliO157:H7). In other words, none microorganism (except

E. coli O157:H7) could grow.

It is difficult to infer the causes of the noise superim-

posed in the output signal seen in Fig. 4 particularly

because it is an ultra-low-frequency noise. Nevertheless

it is possible to point out possible mechanisms that

could cause it:

1. Thermal and acoustic instabilities, affecting the op-

tical circuitry in every point, perturb the lightwave

before it reaches the multimode fibre optic 3dB

coupler. This may cause deviations of the coupler

ratio from the expected value of 3dB. The deviations

may be specifically caused by the mode coupling

and polarization-dependent loss (PDL) effects (Ren-

ner, 1998).

2. Electronic noise, due to instabilities of the detection

and amplification circuitry.

The sensor response relies on the interaction between

the lightwave and the bacteria causing the optical atten-

uation at the probe fibre. The optical interaction arises

from the evanescent-field coupling since there is a phys-

ical contact between the fibre and the bacteria, as

shown in Fig. 2 and Fig. 3. The physical contactbetween the SiO2 (probe fibre) and the whole E. coli

O157:H7 occurs because the bacteria are allowed to

grow around the probe fibre. This mechanism greatly

simplifies the construction of the sensor, when com-

pared with 6ther processes, for instance the silanization

technique employed by Weetall (1993) for a true

biosensor.

A bacteria colony may grow indefinitely if an infinite

amount of nutrients would be available, as well as if

other conditions, such as temperature, pH etc., would

be optimised. However, using culture medium in a Petriplate, it is obvious that there is a strong limitation and

the biological medium constrains the bacteria growth in

an available time range. In this study, preliminary

experiments have shown that E. coli O157:H7 is able to

grow for up to about 72 h, using similar Petri plates

and culture medium as those employed for the optical

measurements shown in Fig. 4. Therefore, the question

that arises is: why, since all conditions remains the

same, E. coli O157:H7 reached the stationary phase in

an average of 5 h (see Fig. 4), when this should happen

only in about 72 h? This behaviour may be explained in

the following way:

cross points provides the time width DtLOG (in h) of the

log phase for each N0. The indirectly measured (from

Fig. 5) time derivative iLOG of Iout(t) in the log phase

varies with N0 and may be calculated from:

iLOG(N0)DIout

DtLOG. (6)

Fig. 7 shows a plot of the sensor sensitivity bLOG(dB/h) at the log phase when the initial number of

bacteria is changed from N0=10N0=400. Although

the available data is not enough to draw statistical

conclusions it is possible to fit the data with a straight

line with a regression coefficient of 0.985 and standard

deviation of 0.351. This gives us a provisional calibra-

tion curve as shown in Fig. 7.

By employing the slope of optical response at the log

phase, i.e. the rate of increase in bacterial numbers, the

angular coefficient was calculated to be DiLOG/DN0=

(0.016 i 0.001) dB/h/N0. It means that for each E. coli

O157:H7 bacterium inoculated upon the Petri plate the

speed of the sensor response at the log phase increases

by 0.016 dB/h. For N0=800 the angular coefficient is

given as iLOG(800)$6 dB/h.

Therefore, it is possible to extract from the output

signal Ii(t), the N0 by measuring the angular coefficient

iLOG of the log phase. The N0 is directly related to the

degree of contamination of the sample, when applyingthe system in vivo.

4.7. General analyses

It has been reported that fibre optic evanescent-field

coupling presents some drawbacks when used as a

biological sensor (Ramsden, 1997). In other biological

processes as well, some bio-components may interfere

in the detection of the true target. However, as was

already explained at Section 3.3, the biological proce-

dures herein employed were not only intrinsically highly

selective, but also absolute. This explains why no inter-


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It is known that E. coli O157:H7, as well as some

other bacteria, do not grow inside the bulk of the

culture medium, but only at the surface. However, a

careful examination of Fig. 2(b) suggests that E. coli

O157:H7 grows and stacks one over each other, up to 3

layers, generating voids like foam. The as-like amor-

phous structure of E. coli O157:H7 cluster means that

the lightwave evanescent field would not be attenuated

(absorbed and scattered) in the voids or in places wherethe stacked bacteria are \\1 mm far away from the

surface of the probe fibre.

The cells of E. coli O157:H7 that happen to be all

over the Petri plate, without physical contact with the

probe fibre, go on growing for 72 h because there are

enough nutrients. However, these optically isolated

bacteria do not affect the sensor response Iout(t), since

they are out of reach of the sensing mechanism, the

evanescent field, that extends only about one or slightly

over a, wavelength from the core-clad interface. On the

other hand, the bacteria that touched the fibre during

the inoculation, together with those that happen togrow over and around the fibre, are within the evanes-

cent field, and are sensed and monitored.

However, since the probe fibre was rested upon the

surface of the biological medium prior to the optical

measurements, the lightweight probe fibre was not com-

pletely buried inside the gel-like culture medium, be-

cause of the superficial tension and the nutrients in

excess slowly slide down from the top of the probe

fibre. Also due to the surface tension, only a thin film

of culture medium will remain touching the fibre.

Therefore, the bacteria that happen to be isolated in theprobe fibre will grow and reproduce until the exhaus-

tion of this limited amount of nutrients. The mi-

crograph shown in Fig. 2(a), taken while the sensor

reached half of the optical-signal drop, show the bacte-

ria clusters isolated along the centre of the fibre width.

This is the initial level of the culture, but due to

evaporation the level drops leaving the clusters behind.

The calibration of the sensor sensitivity with N0, as

shown by Fig. 7, features a linear dependence of

iLOG(=DIout/DtLOG) from N0=10 until N0=400. For

N0=800 a deviation was observed from the linear

dependence, which suggests a possible saturation of thesensor response.

4.8. Possible impro6ements of the sensor

In order to increase the sensitivity and the time

derivative (speed at the log phase) of the sensor in its

presently basic configuration, some simple improve-

ments are suggested:

1. Optimisation of the wavelength for a better sensitiv-

ity. For instance, by using a longer wavelength we

would have a larger sensitivity volume because the

evanescent field would also be larger;

2. The use of a longer probe fibre (the actual measures

20 cm) will attenuate more light and therefore will

present a higher sensitivity for the same bacteria

concentration;

3. At present, it is possible to detect only one microor-

ganism per Petri plate at a time. One approach to

overcome this limitation would be the miniaturiza-

tion of the probe fibre such as that an array of them

might be employed. Several different microorgan-isms may be selectively sensed, for instance, by

multiplexing different wavelengths into a single opti-

cal fibre bus (wavelength-division multiplexing

WDM).

5. Conclusions

An evanescent-field and intensity-modulated fibre op-

tic sensor for detection and monitoring of E. coli

O157:H7 has been described and calibrated. These bac-

teria have been successlully detected and quantified inits natural form, 510 times faster than conventional

bacteriological techniques. With this system it is possi-

ble to conclude which bacteria have been inoculated

and its concentration. The development of this highly

sensitive and selective probe for real-time pathogen

detection has improved biological sensing. With minor

modifications the method can be used to test for food

contamination as well as for clinical essays and envi-

ronmental monitoring. Further investigations are under

way in our laboratories and will be object of luture

publications.

Acknowledgements

We would like to thank the Escola Nacional de

Saude Publica of the Fundacao Oswaldo Cruz (ENSP/

FIOCRUZ) for the bacteriological support on this

work; the Foundation for the Research Support of the

State of Rio de Janeiro (FAPERJ) and the Brazilian

National Research Centre (CNPq/PADCT) for finan-

cial support. We are also in dept with Mr. Jose Roberto

da Rocha Bernardo, MSc, and Dr. Ulisses GarciaCasado Lins, from the Federal University of Rio de

Janeiro, for their assistance in electron scanning and

optical microscopy, respectively.

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