July 15, 2003 / Vol. 28, No. 14 / OPTICS LETTERS 1233

Optical waveguide sensor for on-line monitoring of bacteria

Róbert Horváth, Henrik C. Pedersen, and Nina Skivesen

Optics and Fluid Dynamics Department, Risø National Laboratory, DK-4000 Roskilde, Denmark

Dávid Selmeczi and Niels B. Larsen

Danish Polymer Center, Risø National Laboratory, DK-4000 Roskilde, Denmark

Received February 14, 2003

A grating-coupled planar optical waveguide sensor is presented for sensing of bacteria by evanescent waves.The waveguide design results in increased depth of penetration into the sample volume, which makes itsuitable for detecting micrometer-sized biological objects. We tested the sensor’s performance by monitoringthe adhesion of Escherichia coli K12 cells to the sensor surface. © 2003 Optical Society of America

OCIS codes: 170.0170, 130.2790.

In recent years sensor systems for the detection ofpathogenic bacteria have received renewed interest, es-pecially within the fields of food safety, medical diag-nostics, and biological warfare. This is so because ofthe increasing numbers of illnesses caused by bacteria,which account for as many as 40% of the 50 million an-nual deaths worldwide.1 The main goal for the newsensor systems has been to encompass a rapid alterna-tive to the time-consuming culturing methods, whichusually require several days to give a result.

Among others, optical techniques have been usedto meet this challenge and have produced quite afew biosensor detection instruments, such as surfaceplasmon resonance sensors,2 resonant mirror sensors,3

waveguide sensors,4,5 and interferometric sensors.6

Most optical sensors rely on evanescent wave sensing,in which small changes in the refractive index closeto the sensor surface are probed by an evanescentfield. For bacterial sensing, the sensor’s surface isthen usually coated with a layer of specific antibod-ies, which are capable of binding specific types ofbacteria to the sensor surface. Hence the refractive-index change occurs as a result of the surface attach-ment of bacterial cells, which have different refractiveindices from the surrounding medium, which is usuallywater.

Because the optical evanescent f ield decays expo-nentially from the sensor surface into the samplevolume, only changes within the penetration depthof the evanescent field are probed. Under normalconditions this penetration depth has an upper limitof 100–200 nm. Thus, for a bacterial cell typically1 3 mm in size, only a small fraction of the cell is ac-tually probed by the evanescent field, which results ina severe reduction in sensitivity.2,3 As a consequence,optical sensors play only a marginal role in bacterialsensing today. In contrast, the so-called ELISA(Enzyme-Linked Immunosorbent Assay),7 introducedmore than 20 years ago, still dominates the marketof rapid sensors.1 However, as the ELISA requiresa relatively high number of bacterial cells for a de-tectable signal, most of the commercial ELISA testsstill require at least 18–24 h of incubation. Hencethere is still a large demand for bacterial sensorsthat are sufficiently rapid to be used as true on-linesensors.

0146-9592/03/141233-03$15.00/0

In an effort to reach this goal we recently suggesteda new planar optical waveguide sensor design withso-called reverse symmetry.8,9 In this design the useof a substrate layer that supports the waveguidingfilm and that has a refractive index smaller than thatof water implies that the penetration depth of theevanescent field has no upper limit. Thus, simplyby choosing the correct thickness of the waveguidingfilm, one can adjust the penetration depth to covervirtually any size of biological object, ranging fromproteins (3–20 nm), through viruses (approximately50–200 nm) and bacteria �0.5 5 mm�, and poten-tially all the way to parasites and mammalian cells�5 50 mm�. In this Letter we investigate the perfor-mance of the reverse symmetry waveguide sensor asa bacteria sensor.

The central part of the waveguide sensor systemis shown in Fig. 1. A waveguiding polystyrene film,with a thickness of 160 nm and a refractive index of1.57, is supported by a 1-mm-thick substrate layerof nanoporous silica, which, in turn, is supportedby a 1.6-mm glass plate. The refractive index ofthe nanoporous layer is 1.22, considerably lowerthan water’s refractive index of 1.33. A sinusoidalsurface-relief grating, with a periodicity of 479 nmand a profile depth of 10 nm, is imprinted into thepolystyrene f ilm to couple light into the waveguide.

Fig. 1. Reverse symmetry waveguide sensor: PLL,poly-L-lysine.

© 2003 Optical Society of America

1234 OPTICS LETTERS / Vol. 28, No. 14 / July 15, 2003

To bind the bacteria onto the sensor surface we coatthe waveguide with a 4-nm-thick layer of poly-L-lysine,which is a protein layer that provides a positive chargeon the surface, resulting in strong electrostatic bindingto bacterial cells.10

A cuvette with inlet and outlet tubing is placedon top of the waveguide, with the tubing connectedto a peristaltic pump. The cuvette leaves an areaof 8 mm2 exposed to the sample. The whole sensorsystem is mounted upon a high-precision, computercontrolled goniometer. An unexpanded, fixed He–Nelaser beam with a wavelength of 632.8 nm and withmixed TE and TM polarization illuminates the cou-pling grating, which is positioned at the center ofthe goniometer. During rotation, the incoupled lightintensity is measured by a photodetector positionedat the end of the waveguide. The detector signalis monitored and analyzed by a computer with ananalog-to-digital converter card.

We prepared a bacterial solution by suspendingand washing a single Escherichia coli K12 colonyfrom an agar plate in 10-mL phosphate-bufferedsaline (PBS), resulting in a concentration of3 3 107 cells�mL. These nonpathogenic cells arerod shaped, with an approximate size of 1 mm 3 3 mm.

In the experiment the cuvette was initially rinsedthrough with PBS buffer at a f low rate of 0.29 mL�min.The resultant incoupled light intensity versus angleof incidence a is shown in Fig. 2. Two sharp reso-nant peaks can be seen that correspond to the TE0 andTM0 modes of the waveguide with incoupling anglesaTE � 3.128 deg and aTM � 1.592 deg. From thesevalues one can calculate penetration depths of 278 and592 nm for the TE and the TM modes, respectively,by using the three-layer waveguide model presentedin Refs. 11 and 12. Hence, if a 1-mm-thick bacterialcell attaches to the sensor surface, more than half of itwill be covered by the TM evanescent f ield.

The TE and TM peak positions were continuouslymonitored in real time at a sampling rate of 40 s;see Fig. 3. After 40 min of PBS f low, the bacterialsolution was applied while the f low speed was heldconstant. This caused a continuous change in peakpositions caused by the accumulating E. coli cellattachments to the sensor surface. After a further45 min the bacterial solution was replaced withPBS buffer again, resulting in stabilization of theincoupling angles as shown in Fig. 3. The stabi-lization indicates that the bacterial attachment isirreversible; i.e., the cells are not removed by the liq-uid f low.

The slightly positive slopes that appear in Fig. 3in the raw data points during the two PBS washingperiods indicate that some drift in peak positionsoccurs during washing. The slopes were found to be4.2 3 1027 and 7.7 3 1027 deg�s for the TE and TMcurves, respectively. The appearance of these slopes,we believe, is due to small amounts of water beingabsorbed by the polystyrene film, which graduallyreduces the refractive index of the film and, in turn,increases its thickness.13 However, as the drift isquite constant in the present experiment, it can easilybe compensated for in data processing.

Concerning noise, it may be noted from Fig. 3 thatthere seems to be a repetitive jump in the peak po-sitions every �7 min. We believe that this noise isdue to an inhomogeneous liquid pressure caused bythe peristaltic pump. The noise can, however, be sup-pressed by a factor of 4 by use of a 15-point averagingprocedure. The results of the drift compensation andthe noise reduction are represented by the processeddata points in Fig. 3. The noise levels were found tobe 1024 deg for both the TE and the TM data.

The total changes in peak positions that weredue to the attached bacterial cells were measuredto be DaTE � 0.0160 deg and DaTM � 0.0205 deg.To quantify the corresponding cell density at thesensor surface we investigated the waveguide undera microscope; see Fig. 4. Different areas of thesensor surface were photographed, and the numberof bacteria were counted by the computer program

Fig. 2. Intensity of incoupled light versus angle ofincidence.

Fig. 3. Peak position versus time. Upper traces, rawdata points taken directly from the detector; lower traces,processed data to which drift compensation and 15-pointaveraging have been applied.

July 15, 2003 / Vol. 28, No. 14 / OPTICS LETTERS 1235

Fig. 4. Microscope image of the waveguide surface afterE. coli attachment.

ImageTool (Health Science Center, University ofTexas, San Antonio). From these investigations wefound that the bacteria covered the sensor surfacewith an average density of 12, 400 cells�mm2. Byusing the total shifts in peak positions given above, weobtained average single-cell sensitivities of 1.3 3 1026

and 1.65 3 1026 �deg�cell��mm2 for the TE and theTM modes, respectively. In terms of coverage ratio,assuming an average coverage of 2 mm2�cell, the totalsurface coverage ratio amounts to r � 2.5 3 1022,which leads to angular coverage sensitivities ofDaTE � 0.65r deg and DaTM � 0.83r deg. The corre-sponding sensitivities for the resonant mirror sensorwere found to be 806.9r arc sec or 0.22r deg,3 nearlyfour times less than the TM sensitivity of the presentsensor.

If we consider the previously determined noiselevel of 1024 deg, we arrive at detection limits of78 cells�mm2 (TE mode) and 60 cells�mm2 (TMmode), which correspond to surface coverages of1.6 3 1024 and 1.2 3 1024, respectively. The limitof detection for the resonant mirror was estimatedat 6 8 3 106 cells�mL, corresponding to 75 arc sec(according to Fig. 3 of Ref. 3) or to a surface coverageof 9.3 3 1022, which is 775 times higher than the TMdetection limit of the present sensor.

In conclusion, we have presented a reverse sym-metry waveguide sensor with an integrated grating

coupler that is capable of detecting and monitoringthe adhesion of bacterial cells. Using E. coli K12cells, we have demonstrated a peak angle sensitivityof 1.65 3 1026 �deg�cell��mm2 and a detection limit of60 cells�mm2 for the TM mode, which has the highestsensitivity. The TM sensitivity and detection limitthat we found are, respectively, approximately 4 timeshigher and nearly 800 times lower than for the reso-nant mirror sensor.

The authors thank S. Wallace (Nanopore, Inc.) forpreparing the nanoporous samples, P. Klemm (Biocen-trum, DTU, Denmark) for providing bacteria, L. Odd-ershede (Niels Bohr Institute, Copenhagen, Denmark)for helping with the biochemistry, and L. Knudsen,J. Stubager, and B. Sass (Risø National Laboratory,Denmark) for technical assistance. This researchwas supported by Danish Technical Research Councilgrant 26-01-0211. H. C. Pedersen’s e-mail address ishenrik.pedersen@risoe.dk.

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