Biosensors & Bioelectronics 16 (2001) 399–408

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 En�ironmental Health, National School for Public Health, Oswaldo Cruz Foundation, Rio de Janeiro, Brazilb Biomedical Engineering Program, Federal Uni�ersity 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 wholenatural bacteria and the guided lightwave was carried out by means of evanescent-field coupling. A correlation between opticalresponse and the current number of bacteria was achieved. The device sensitivity had been calibrated for initial number of bacteria(N0) from 10–800. The sensor sensitivity was 0.016 (�0.001) dB/h/N0. The sensing mechanism starts together with the log phaseleading the present sensor response to be five to ten times faster than conventional bacteriological techniques. © 2001 ElsevierScience 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 ofhuman and other warm-blooded animals. E. coli typi-cally colonizes the infant is gastrointestinal tract withinhours of life. Usually it remains harmlessly confined tothe intestinal lumen, only becoming pathogenic in thedebilitated or immunosuppressed host, or when gas-trointestinal barriers are violated. Therefore, E. coli hasserved as an indicator for faecal contamination in waterand food.

Enterohemorrhagic Escherichia coli O157:H7 is anemerging pathogen; which is the cause of foodborneillness, the major cause of serious outbreaks and spo-radic cases of hemorrhagic colitis and hemolytic-uremicsyndrome (Pickering et al., 1994; Siegler, 1995; Nataroand Kaper, 1998). The major impact of bacterial in-duced diarrhoea disease is on children under the age of10 and on elderly people living in less-developed coun-tries, in which bacterial diarrhoea diseases remain asignificant 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 andpersistent diarrhoea disease and ongoing morbidity thathas malnutrition as one of its major contributors(Huilan et al., 1991). The level of biohazard is high dueto the extremely low dose (10 microorganisms) requiredfor infection (Nataro and Kaper, 1998).

The greatest drawback for pathogen-detection tech-niques is the lack of reliable and sensitive means formeasuring their presence in real time. Evanescent-wave-coupling sensor technology is a suitable technique formicroorganism measurement in its natural form thatmay be applied to the biosensor development takingadvantage of the current understanding of the wholecell 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 cellsand bacteria detection remain a challenging technique.The advantages of fibre optic sensing are well knownand have been reported in the literature (Smoczynski etal., 1993; Ligler et al., 1993; Karube and Nakanishi,1994). In the last few years fibre optic sensors have beenincreasingly employed in biological sensing because oftheir electrical isolation, electromagnetic-interferenceimmunity, compactness, lightweight, sensitivity, in-line

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

E-mail address: aldo@ensp.fiocruz.br (A.P. Ferreira).

0956-5663/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S 0 9 5 6 -5663 (01 )00149 -X

A.P. Ferreira et al. / Biosensors & Bioelectronics 16 (2001) 399–408400

implementation by means of evanescent-field coupling,potential low cost and biological compatibility. Theoptical sensor has revealed to present a faster responsethan conventional techniques of clinical laboratoryanalysis. One of the major advantages of using thecoupling with the guided lightwave evanescent field isthe development of in-line fibre optic devices. The fibreis not interrupted and the light is not removed from thefibre. The signal processing takes place inside the opti-cal fibre thus characterizing an intrinsic sensor.

A fibre optic biological sensor has been designed inour laboratory and was described elsewhere (Ferreira etal., 1999a,b). In this work, using the same measurementprinciple, the sensor has been employed to quantify E.coli O157:H7.

This paper describes for the first time, to the best ofour knowledge, experimental results on sensitivity cali-bration of an intensity-modulated fibre optic evanescentsensor for the E. coli O157:H7 as a whole bacterium.The complete bacteria growth phases were opticallysensed 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 e�olution

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 bacteriaare isolated in a suitable biological culture medium.Growth is very slow at first (little or no division), whilethe organism is adjusting its metabolism to the foodand nutrients in its new environment.

The log (or logarithmic) phase starts once themetabolic machinery is running and the bacteria startmultiplying exponentially, doubling in number (binaryfission) at every few minutes. The time required for thebacteria population to double after the lag phase iscalled generation time g :

g=tn

. (1)

Where n is the number of generations that occur inthe period of time t (in min). A more practical formulamay be derived from the definition stated above andgives:

g=t

3.3(log10N− log10N0). (2)

Where, N is the current number of bacteria at time tand N0 is the initial number of bacteria. The current

population N(t) at the log phase can be derived fromEq. (2):

N(t)=No×2t/g (3)

As more and more bacteria are competing for foodand nutrients the booming growth stops and the num-ber of bacteria stabilizes in the stationary phase. At thesame time dying and dividing the organisms are atequilibrium. Death is due to reduced nutrients, pHchanges, toxic waste and reduced oxygen. In some casescells do not die but they are not multiplying. Eventu-ally, when the death rate exceeds the growth rate, thecells enter the death phase. The population is dying ina geometric fashion so there is more death than newcells, and the population may be entirely destroyed.

2.2. The sensing mechanism of the sensor

The sensing mechanism of the sensor described hererelies upon the attenuation of a guided lightwave bymeans 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 ischanged in intensity.

The culture medium is adherent to the cladding ofthe almost unclad sensitive fibre. Normally, light travel-ling inside the fibre experiences total internal reflectionwhen light beams strike the interface between the fibrecore and the cladding. This occurs when the angle ofincidence is greater than the critical angle. Thus, apropagating optical wave through the optical fibre isbounded in its core, but allowed to interact with theoutside medium by evanescent-field coupling when thisfield is exposed (Rahnavardy et al., 1997). The evanes-cent field of the guided lightwave penetrates beyond thecore surface and exponentially decreases in its magni-tude an order of a wavelength away from the core/clad-reflecting interface. The evanescent-wave penetrationdepth (dp) is calculated by:

dp=�

2�(n�2 sin2�−n cl

2 )1/2 , (4)

Where dp is the distance by which the magnitude ofthe electric field decays of l/e to its intensity at thecore/clad interface, � is the internal incident-ray anglewith the normal to the core/cladding interface, � is thevacuum wavelength of the light beam and nco and ncl

are the core and cladding index of refraction,respectively.

The evanescent-field coupling interaction mechanismwill cause optical attenuation of the guide lightwavewhen any material (gas, liquid or solid) gets into con-tact with the surface of the etched fibre clad. The loss ofpower is proportional to the intrinsic bulk absorptionand scattering (Rayleigh and Mie). Thus, as a light rayprogresses down the multimode fibre by bouncing off

A.P. Ferreira et al. / Biosensors & Bioelectronics 16 (2001) 399–408 401

the core-cladding interface, it loses power at eachbounce. While the loss per bounce is small, the accumu-lated loss after a large number of bounces can besignificant 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 refractiveindex, it suffers the following approximate fractionalloss of power on refraction:

T=4(�/� c

2)

�� c2−�2

� ��

4�ncl

�. (5)

Where T is the fractional loss of power, �c is thecritical angle of two media, � is incidence angle at theboundary between the media (measured with respect tothe plane of the interface, rather than its normal), � isthe optical wavelength, � 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 theoriginal medium is (1−T) and the fraction of theoriginal power remaining after N reflections is (1−T)N.In this formula ���c and should not be too close to itfor the approximation to be valid. This means that itapplies to rays that would have been well guided in theabsence of loss. Furthermore the ‘tighter’ such guidance(i.e. the smaller �/�c), the lower the loss on reflection.

The optical attenuation can be generated by twointerrelated physical mechanisms: variation of the cladrefractive index ncl as well as of its absorption coeffi-cient �. When the bacteria are allowed to grow aroundthe 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 aimof this paper, the interdependence between absorp-tion coefficient and index of refraction has beenexperimentally shown and is theoretically describedby the Kramers-Kronig Eqs. (Nussenzveig, 1972).

2. Due to the existence of an increasing number ofbacteria in contact with the fibre during the logphase, the medium becomes more opaque as timegoes by. Consequently the intrinsic absorption alsochanges.

Both effects may change the mechanism of interac-tion between the evanescent field and the outside mate-rial medium, coupling out different amounts of thefibre-guided lightwave. Therefore, the measurement ofthe light power at the exit of the fibre shows indirectlythe bacteria growth. As explained in more detail inSection 4, the decreased optical power is mainly relatedto 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 biologicalculture medium.

3.1. The optical circuitry set-up

Fig. 1 shows a schematic drawing of the biosensorset-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 outputfibre was spliced with a bi-directional optical fibrecoupler (CPqD — Telebras, Brasil). Half of the emittedoptical power propagates over the probe fibre (sensingelement). The light modulated by the growing bacteriaexits the fibre and is detected by the photodiode PD2

providing the electrical signal Samplein. The other halfof the optical power is detected by the photodiode PD1

from which an electrical reference signal Refin is gener-ated. Both electrical signals are amplified and measuredby a twochannel calibrated optical-power meter(Graseby Optronics, USA). An AID board controlledby the LabVIEW software (National Instruments Cor-poration) digitises both output signals from the optical-power meter (Sampleout. and Refout) by means of itsIEEE-488 interface. The AID board collects 800 pointsper min of both Sampleout and Refout recording andstoring the respective averages for a period of 24 h permeasurement cycle.

3.2. The e�anescent optical probe fibre

In order to expose the evanescent lightwave field, 20cm of a graded-index multimode optical fibre (62.5/125�m) was clad-stripped by chemical etching. The etchingwas performed by means of hydrofluoric acid solution(38%) during 11 min in order to leave between 0.5 to 1�m of clad over the core of the fibre. After this time,the chemical reaction was stopped by immersion indemonised 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.

A.P. Ferreira et al. / Biosensors & Bioelectronics 16 (2001) 399–408402

time was determined by monitoring diameters in aprevious measurement using a calibrated optical micro-scope. This method has been detailed described byMuller and co-workers (Muller et al., 1998).

After the chemical etching the fibre was wound intoa single loop, with a total sensitive surface area ofapproximately 40.0 mm2, in which the evanescentfield can be accessed. The probe fibre was put overthe culture medium in which the bacteria grew, selec-tively.

3.3. Selecti�e culture medium for E. coli O157:H7

All of the described experiments in this paper wereperformed using Escherichia coli O157:H7 CDC EDL-933 (ATCC 43894), which was supplied by the Centrefor Disease Control and Prevention (CDC/USA).

In order to promote the E. coli O157:H7 growth, theselective culture medium employed was MacConkeySorbitol Agar (SMAC) Difco 0079–17–7 (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 toensure that only that specific microorganism had grownon each Petri plate. The identity tests were made inaccordance with Farmer and Davis (1985).

Although biochemical characteristics associatedwith the great majority of E. coli O157:H7 serotypesare well known, there are not much data available ontheir identification. It must be stressed that about 75 to94% 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 toproduce �-glucuronidase which hydrolyses-4-methyl-umbelliferyl-D-glucuronide. This does not happen withother serbtypes of E. coli. Another parameter is thatmore than 90% of E. coli O157:H7 strains produce oneor two unique biochemical profile numbers on a Mi-croScan conventional gram-negative identificationpanel (Baxter Diagnostics, Inc., California) and otherD-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 ofoxygen, it is a gram-negative bacterium and is notfastidious in its nutritional requirements.

3.4. Bacteriological preparations for the sensorcalibration

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 inseveral Petri plates with SMAC and incubated at 35 °C

for 24 h. At the end of this period, the purity of thematerial was checked and the Petri plates were stored at4 °C for further dilution.

For obtaining a dilution with N0=10, 20, 30, 40, 50,60, 70, 80 and 90 microorganisms, were used 100 �l ofPBS, pH 7.4, whereas for N0=100, 200, 400 and 800the volume used was 500 �l of PBS, pH 7.4. For eachsample the cells were counted using the Coulter Coun-ter (Beckman, USA), which allows an accuracy of�1%. Finally, the probe fibre was rested upon the Petriplates with the culture as described above, the dilutionwith a known number of microorganisms was pouredonto the sensitive area of the probe and the hardwareand software were started.

In all cases the SMAC was supplemented with glyc-erol at 0.2%, which allows the maintenance of theresidual humidity and provides a better interactionbetween the microorganisms and the sensitive area.This was done in order to avoid the culture going dryduring the tests and thus inserting another variable intothe process.

3.5. Taking pictures of the process

For a direct observation of the microorganism’s in-teraction with the probe fibre, photographs were takenemploying the scanning electron microscope and theoptical microscope.

3.5.1. Scanning electron microscopyMicrographs 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-signaldrop. Cells were fixed with 2% (v/v) glutaraldehyde inPBS, pH 7.4, for 3 h at 4 °C. After washing 3 timeswith PBS, pH 7.4, 4% (v/v) of osmium tetroxide wasadded to each sample during 1 h at 4 °C. After dehy-dration in a graded series of ethanol dilutions, cellswere 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 ionsputtering (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 microscopyThe optical microscopy pictures were taken after 398

min (N0=100), corresponding to the stationary opticalsignal. Cells were fixed with 2% (v/v) glutaraldehyde inPBS, pH 7.4, for 3 h at 4 °C and then washed 3 timeswith PBS, pH 7.4. The images in differential interfer-ence contrast were obtained with the Zeiss Axioplan 2microscope (Carl Zeiss) and photographed with a Po-laroid MC2OO (Carl Zeiss) using Kodak Tmax 100film.

A.P. Ferreira et al. / Biosensors & Bioelectronics 16 (2001) 399–408 403

Fig. 2. Scanning electron micrograph of E. coli O157:H7 (log phase)that are in physical contact with the probe fibre. Two levels ofmagnification are shown.

Fig. 3. Optical phase-contrast micrograph of the E. coli 0 1 57:H7after Iout(t) reached the stationary state. Two levels of magnificationare shown on 3(a–b).

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) fromthe others colonies. Because of the short range of theevanescent 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 duringa 24 h interval (1440 min), employing thirteen differentvalues of N0. These values were varied from 10–800 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 probefibre showing the physical contact with the E. coliO157:H7 growing around it.

Fig. 2 shows the micrograph taken by means of thescanning electron microscopy on two levels of magnifi-cation. It displays the fibre-bacteria physical contactduring 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 theoptical fibre, as evaluated by the scale shown at the leftof the picture, is only about 60 �m in diameter, whichis approximately the same as the one of the core. Fig.2(b) shows with a greater spatial resolution an as-likeamorphous structure of the E. coli O157:H7 cluster inwhich 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, afterIout(t) has reached the stationary phase, which are in

Fig. 4. Optical response Iout(t) of the sensor parameterised by theinitial number N0=0, 10, 80 and 800 of E. coli O157:H7.

A.P. Ferreira et al. / Biosensors & Bioelectronics 16 (2001) 399–408404

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 (thesuperimposed 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 1and 2) and the theoretical current number of bacteriaN(t) representing the population of the colony along itslife evolution, as previewed by Eq. (3) (line 3). Thesuperimposed straight lines are eye-guide lines repre-senting an averaging of the experimental plot used tocalibrate the sensor (see Section 4.6). The theoreticalplot also shown at Fig. 5 is the representation of Eq.(3).

As explained on the text (see Sections 4.2 and 4.6) thesuperimposed straight lines represent a mathematicalidealization that helps on the sensor calibration. Thearrows guide the reader for the correct vertical axiscorresponding the two plots.

As seen from the two tests performed with N0=200shown in Fig. 5 it is possible to infer that the results ofthe optical measurements were quite reproducible. Thegood reproducibility of the optical response of thesensor is guaranteed by the systematic procedure thatwas employed for the biological culture as well thesensing probe fibre insertion.

4.2. Analysis of the sensor response Iout(t) in the lagphase of the culture

The plots displayed in Fig. 4 show different phases ofIout(t) for any used N0. In the first phase, it may beobserved a DC level Iout(t)=ILAG with an almost simi-lar time range. The ILAG level is assigned to the sensorresponse in which the E. coli O157:H7 remains in its lagphase during �tLAG time delay.

Fig. 6 shows a plot of �tLAG against N0 for allmeasurements. The linear relationship with an almost

null angular coefficient was fitted with an average of270�4 min or approximately 4.5 h, meaning a re-peatability of �1.5%. �tLAG may be attributed to thetime 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. coliO157:H7 along the lag phase, the average refractiveindex of the culture medium ncm is likely to vary withtime. As described in Section 2, the variation of ncm

may cause changes in Iout(t), as stated by Eq. (5).However, the measurements shown in Fig. 6 suggestthat the sensor, while probing the lag phase, is insensi-tive to this effect. A possible explanation for this is thatthe refractive index changed very little and the variationof the optical response was buried into the noise. It ispossible to observe in Fig. 2 that the optical signalspresent a slight slope during this phase. Therefore itpossible to infer that the technique used in this work is

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

A.P. Ferreira et al. / Biosensors & Bioelectronics 16 (2001) 399–408 405

more sensitive to the bacteria division (growth) than tothe variation of the index of refraction of the culturemedium itself.

4.3. Analyses of the sensor response Iout(t) in the logphase

Following the lag phase, E. coli O157:H7 starts thelog phase in which the size growth and replication takeplace. The curves displayed in Fig. 4 show that justafter approximately 270 min the sensor output signalIout(t) begins to drop gradually. The linear decreasingof Iout(t) may be attributed to the sensor tracking of thelog phase evolution when the optical attenuation, assensed by the evanescent field, monotonically increases.It should be carelully examined from the electron scan-ning micrograph shown in Fig. 2 that the colony doesnot cover the lull surface of the probe fibre.

If the attenuation coefficient � and the refractiveindex ncm of the culture medium change in oppositedirections, as the bacteria grow, the critical angle �c

increases. As a consequence, shown by Eq. (5), theeffect of increasing the attenuation coefficient is par-tially offset by improved optical guidance, for rays of agiven incidence angle, and the final effect on the opticalpower output is unnoticeable. However if the attenua-tion coefficient � and the refractive index ncm of theculture medium change in the same direction, as bacte-ria grow, the critical angle �c decreases. Both effects arenow coherently summing up and the fractional loss T isreinforced, for rays of a given incidence angle, and thefinal effect is a decrease of Iout(t).

By extrapolation from the lag phase, it is unlikelythat the enzymes produced by the E. coli O157:H7,even in the log phase, may affect the sensor Iout(t) forall N0 employed. In this way, along the log phase, onlythe attenuation coefficient a suffered appreciablechanges, thus affecting Iout(t). In other words, the sen-sor described here seems to be insensitive to the averagerefractive index changes, if it changes at all. Onlyintrinsic absorption as well Rayleigh and Mie scatteringaffect 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 endof the Iout(t) drop, the sensor response reaches anotherDC level (ISTAT). Following the log phase, the E. coli0157:117 stops their size growth and replications, thusreaching the stationary phase in which Iout(t)=ISTAT.

A careful examination of Fig. 4 and Fig. 5 showsthat, in the so-assigned log phase Iout(t) turn to a lowerderivative, a little before reaching the stationary phase,it is likely that between the purely log and stationaryphase, a transition region takes place. From some

instant of time, a fraction of E. coli O157:H7 stops itsgrowth 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 thestationary phase thus affecting the optical response ofthe sensor.

It is also noticeable in Fig. 4, for all measurementsperformed with different N0 (until N0=800), that ISTAT

is always smaller than the previous one. The sensorresponse Iout(t) has shown an optical-dynamic reservein the log phase that makes it clear that E. coliO157:H7 really reached the stationary phase. Thus, theISTAT level (lower than ILAG) may be assigned to thestationary 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, asurface area of approximately 40.0 mm2 has beenachieved, and the evanescent field was exposed. On theother 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 coveralmost all the surface area of the probe fibre.

4.5. Analyses of the sensor response Iout(t) in the deathphase

For a 24 h monitoring, the optical sensor responseIout(t) did not show any remarkably change after thestationary phase was reached.

Although the E. coli O157:H7 starts its death phasesome time after the end of the log phase, the sensordescribed here does not seem to optically discriminatethe stationary phase from the death phase.

4.6. Calibration of the sensor

In Fig. 5 three eye guidelines were drawn for eachIout(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 ofthe log phase, Iout(t) suddenly dropped with a sharpderivative. It was ‘smoothed’ by a non-null angular-co-efficient straight line. A similar procedure was per-formed at the stationary phase extrapolated fromt=�. But the steady state straight line outlined had anISTAT DC level lower than the ILAG DC level.

Two crossing points among the three straight linesmay be viewed in Fig. 5. The first one fix the boundarybetween the lag and the log phase and the second one,analogously, fix the boundary between the log and thestationary phase.

The ordinate difference between the two cross pointsprovides the optical attenuation �Iout (in dB) for eachN0. Similarly, the abscissa difference between the two

A.P. Ferreira et al. / Biosensors & Bioelectronics 16 (2001) 399–408406

Fig. 7. Sensor calibration curve. It is shown the time derivative �LOG

of Iout(t) in the log phase against N0. A linear dependence was fitted.

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

Although not shown here, it was also carried out ameasurement in which none E. coli Ol 57:H7 werepresent in the culture medium—negative control or‘blank measurement’. In this case, the output signalIout(t) did not change even after 24 h of monitoring,and the noise pattern observed was similar to the onepresented in Fig. 4 (with the presence of E. coliO157:H7). In other words, none microorganism (exceptE. 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 particularlybecause it is an ultra-low-frequency noise. Neverthelessit is possible to point out possible mechanisms thatcould cause it:1. Thermal and acoustic instabilities, affecting the op-

tical circuitry in every point, perturb the lightwavebefore it reaches the multimode fibre optic 3dBcoupler. This may cause deviations of the couplerratio from the expected value of 3dB. The deviationsmay be specifically caused by the mode couplingand polarization-dependent loss (PDL) effects (Ren-ner, 1998).

2. Electronic noise, due to instabilities of the detectionand amplification circuitry.

The sensor response relies on the interaction betweenthe lightwave and the bacteria causing the optical atten-uation at the probe fibre. The optical interaction arisesfrom the evanescent-field coupling since there is a phys-ical contact between the fibre and the bacteria, asshown in Fig. 2 and Fig. 3. The physical contactbetween the SiO2 (probe fibre) and the whole E. coliO157:H7 occurs because the bacteria are allowed togrow around the probe fibre. This mechanism greatlysimplifies the construction of the sensor, when com-pared with 6ther processes, for instance the silanizationtechnique employed by Weetall (1993) for a truebiosensor.

A bacteria colony may grow indefinitely if an infiniteamount of nutrients would be available, as well as ifother conditions, such as temperature, pH etc., wouldbe optimised. However, using culture medium in a Petriplate, it is obvious that there is a strong limitation andthe biological medium constrains the bacteria growth inan available time range. In this study, preliminaryexperiments have shown that E. coli O157:H7 is able togrow for up to about 72 h, using similar Petri platesand culture medium as those employed for the opticalmeasurements shown in Fig. 4. Therefore, the questionthat arises is: why, since all conditions remains thesame, E. coli O157:H7 reached the stationary phase inan average of 5 h (see Fig. 4), when this should happenonly in about 72 h? This behaviour may be explained inthe following way:

cross points provides the time width �tLOG (in h) of thelog phase for each N0. The indirectly measured (fromFig. 5) time derivative �LOG of Iout(t) in the log phasevaries with N0 and may be calculated from:

�LOG(N0)��Iout

�tLOG

. (6)

Fig. 7 shows a plot of the sensor sensitivity �LOG

(dB/h) at the log phase when the initial number ofbacteria is changed from N0=10–N0=400. Althoughthe available data is not enough to draw statisticalconclusions it is possible to fit the data with a straightline with a regression coefficient of 0.985 and standarddeviation 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 logphase, i.e. the rate of increase in bacterial numbers, theangular coefficient was calculated to be ��LOG/�N0=(0.016 i 0.001) dB/h/N0. It means that for each E. coliO157:H7 bacterium inoculated upon the Petri plate thespeed of the sensor response at the log phase increasesby 0.016 dB/h. For N0=800 the angular coefficient isgiven as �LOG(800)�6 dB/h.

Therefore, it is possible to extract from the outputsignal Ii(t), the N0 by measuring the angular coefficient�LOG of the log phase. The N0 is directly related to thedegree of contamination of the sample, when applyingthe system in vivo.

4.7. General analyses

It has been reported that fibre optic evanescent-fieldcoupling presents some drawbacks when used as abiological sensor (Ramsden, 1997). In other biologicalprocesses as well, some bio-components may interferein the detection of the true target. However, as wasalready explained at Section 3.3, the biological proce-dures herein employed were not only intrinsically highlyselective, but also absolute. This explains why no inter-

A.P. Ferreira et al. / Biosensors & Bioelectronics 16 (2001) 399–408 407

It is known that E. coli O157:H7, as well as someother bacteria, do not grow inside the bulk of theculture medium, but only at the surface. However, acareful examination of Fig. 2(b) suggests that E. coliO157:H7 grows and stacks one over each other, up to 3layers, generating voids like ‘foam’. The as-like amor-phous structure of E. coli O157:H7 cluster means thatthe lightwave evanescent field would not be attenuated(absorbed and scattered) in the voids or in places wherethe stacked bacteria are � �1 �m far away from thesurface of the probe fibre.

The cells of E. coli O157:H7 that happen to be allover the Petri plate, without physical contact with theprobe fibre, go on growing for 72 h because there areenough nutrients. However, these ‘optically isolated’bacteria do not affect the sensor response Iout(t), sincethey are out of reach of the sensing mechanism, theevanescent field, that extends only about one or slightlyover a, wavelength from the core-clad interface. On theother hand, the bacteria that touched the fibre duringthe 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 thesurface of the biological medium prior to the opticalmeasurements, the lightweight probe fibre was not com-pletely buried inside the gel-like culture medium, be-cause of the superficial tension and the nutrients inexcess slowly slide down from the top of the probefibre. Also due to the surface tension, only a thin filmof 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 sensorreached 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 toevaporation the level drops leaving the clusters behind.

The calibration of the sensor sensitivity with N0, asshown by Fig. 7, features a linear dependence of�LOG(=�Iout/�tLOG) from N0=10 until N0=400. ForN0=800 a deviation was observed from the lineardependence, which suggests a possible saturation of thesensor response.

4.8. Possible impro�ements of the sensor

In order to increase the sensitivity and the timederivative (speed at the log phase) of the sensor in itspresently 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 wewould have a larger sensitivity volume because theevanescent field would also be larger;

2. The use of a longer probe fibre (the actual measures20 cm) will attenuate more light and therefore willpresent a higher sensitivity for the same bacteriaconcentration;

3. At present, it is possible to detect only one microor-ganism per Petri plate at a time. One approach toovercome this limitation would be the miniaturiza-tion of the probe fibre such as that an array of themmight be employed. Several different microorgan-isms may be selectively sensed, for instance, bymultiplexing 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. coliO157:H7 has been described and calibrated. These bac-teria have been successlully detected and quantified inits natural form, 5–10 times faster than conventionalbacteriological techniques. With this system it is possi-ble to conclude which bacteria have been inoculatedand its concentration. The development of this highlysensitive and selective probe for real-time pathogendetection has improved biological sensing. With minormodifications the method can be used to test for foodcontamination as well as for clinical essays and envi-ronmental monitoring. Further investigations are underway in our laboratories and will be object of luturepublications.

Acknowledgements

We would like to thank the Escola Nacional deSaude Publica of the Fundacao Oswaldo Cruz (ENSP/FIOCRUZ) for the bacteriological support on thiswork; the Foundation for the Research Support of theState of Rio de Janeiro (FAPERJ) and the BrazilianNational Research Centre (CNPq/PADCT) for finan-cial support. We are also in dept with Mr. Jose Robertoda Rocha Bernardo, MSc, and Dr. Ulisses GarciaCasado Lins, from the Federal University of Rio deJaneiro, for their assistance in electron scanning andoptical microscopy, respectively.

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