Monitoring of living cell attachment and spreading

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have been reported, but since whole cells are typically. 1–10 μm in .... 14R. Horvath, J. Voros, R. Graf, G. Fricsovszky, M. Textor, L. R. Lindvold,. N. D. Spencer ...


Monitoring of living cell attachment and spreading using reverse symmetry waveguide sensing Robert Horvath,a兲 Henrik C. Pedersen, and Nina Skivesen Risø National Laboratory, Optics and Plasma Research Department, DK-4000 Roskilde, Denmark

David Selmeczi and Niels B. Larsen Risø National Laboratory, Danish Polymer Centre, DK-4000 Roskilde, Denmark

共Received 17 September 2004; accepted 7 December 2004; published online 7 February 2005兲 The effect of the attachment and spreading of living cells on the modes of a grating coupled reverse symmetry waveguide sensor is investigated in real time. The reverse symmetry design has an increased probing depth into the sample making it well suited for the monitoring of cell morphology. As a result, significant changes in the incoupling peak height and peak shape were observed during cell attachment and spreading. It is suggested that the area under the incoupling peaks reflects the initial cell attachment process, while the mean peak position is mostly governed by the spreading of the cells. © 2005 American Institute of Physics. 关DOI: 10.1063/1.1862756兴 Initiated by the pioneering work of Tiefenthaler and Lukosz,1 the field of optical waveguide sensing has experienced an ever-growing interest for more than 20 years. Most applications have been directed towards the monitoring of biomolecular binding events at the sensor surface,2–4 which is due to the fact that the probing depth of the evanescent field has an upper limit of 100– 150 nm. Recently the alternative reverse symmetry waveguide sensor was reported,5–7 in which a low-refractive-index layer introduced between the substrate and the waveguiding film was shown to increase the probing depth beyond the 100– 150 nm limit, resulting in a very sensitive detection of micron-sized bacterial cells. A fast-growing activity in biosensor research is the investigation of whole-cell behavior, such as the attachment, spreading8–10 and proliferation11 of animal cells on solid surfaces. A few attempts at using waveguide sensors in this area have been reported, but since whole cells are typically 1 – 10 ␮m in size, a probing depth of 150 nm results in a rather poor sensitivity to changes in cell morphology. With the reverse symmetry configuration, however, the probing depth is in principle unlimited, meaning that the whole cell or at least a considerable fraction of the cell can be covered by the evanescent field, see Figs. 1共a兲 and 1共b兲. In the present letter we investigate the performance of the reverse symmetry waveguide sensor while being exposed to normal human dermal fibroblast 共NHDF兲 cells. The presented waveguide chip consists of a glass substrate coated with two dielectrics: a substrate layer of nanoporous silica with a thickness of 1 ␮m and a refractive index nS = 1.2 and a waveguiding film of polystyrene with a thickness dF = 147 nm and a refractive index nF = 1.58. Together with an aqueous cover medium, this waveguide configuration assumes a reverse symmetry in the sense that the refractive index of the substrate layer is less that the refractive index of water 共1.33兲. Light is coupled into the waveguide via a coupling grating that is heat embossed into the surface of the waveguiding film. Here, the grating period is 478 nm and the grating depth is 8 nm.

The characteristic sensing parameters, i.e., the probing depths and the cover index sensitivities of the TE and TM modes, are listed in Table I along with the corresponding parameters of a conventional symmetry waveguide 共ASI2400␮V, Microvacuum Ltd., Hungary兲 used previously for the detection of living cells.4 It is seen that by using the reverse symmetry configuration a sevenfold improvement in TM probing depth and a fivefold improvement in TM cover index sensitivity are obtained. The TM penetration depth of 727 nm is sufficient to detect refractive index changes deep inside the cells as well as cell morphology changes far from the surface. The characteristic sensing parameters of the reverse symmetry sensor chip can be further increased by choosing a film thickness closer to the cutoff thickness.5 Before use, the reverse symmetry waveguide chip was plasma treated for 10 s in air plasma and was subsequently dipped into an aqueous solution of poly-l-lysine 共PLL兲. This procedure deposits a 5-nm-thick PLL layer on the polystyrene film, which serves as an adhesion layer for the cells. Immediately after the PLL treatment, the waveguide was inserted in the sensor setup with an open cuvette placed on top.


Author to whom correspondence should be addressed; electronic mail: [email protected]

FIG. 1. 共a兲 Conventional and 共b兲 reverse symmetry waveguide designs.

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Appl. Phys. Lett. 86, 071101 共2005兲

Horvath et al.

TABLE I. Characteristic sensing parameters, probing depth and cover index sensitivity, for the presented reverse symmetry waveguide 共nF = 1.58, dF = 147 nm, nS = 1.2兲 and a conventional waveguide 共ASI2400␮V, Microvacuum Ltd., Hungary, nF = 1.8, dF = 180 nm, nS = 1.53兲. Cover probing depth 共nm兲

Reverse Conventional

TE 291 111

TM 728 105

Cover index sensitivity TE 0.4 0.08

TM 0.684 0.13

The waveguide chip was then exposed to different aqueous solutions by pipetting the liquids in and out of the cuvette. The coupling grating on the chip was illuminated from below by a He–Ne laser beam while the chip was rotated via a computer-controlled rotation stage and the incoupled light was detected at the end facet of the waveguide by a fibercoupled photoreceiver.6 The experimental setup was thermostated at 36.5 ° C. Initially, the waveguide was exposed to serumsupplemented, CO2-independent cell culture medium for 78 min until stable incoupling peaks were obtained; see Fig. 2共a兲 共70 min peaks兲. Subsequently, 30 ␮l of cell suspension with a concentration of 100 cells/ ␮l was applied to the cuvette. This resulted in significant reductions of the peak heights shown in Fig. 2共a兲 at t = 82, 85, and 86 min, respectively. At t ⬇ 100 min peculiar changes in the peak structures started to appear, see Figs. 2共b兲 and 2共c兲 resulting in second-

FIG. 3. NHDF cells on the surface of the waveguide.

ary peaks taking over at angles about 0.3° 共TE兲 and 0.6° 共TM兲 higher than the original peaks. The positions of the secondary peaks measured at t = 189 min in Figs. 2共b兲 and 2共c兲 correspond to cover refractive indices of 1.346 共TM兲 and 1.343 共TE兲. These values are very close to 1.35, which is the reported average refractive index of living cells.12 One possible reason for the small discrepancy could be the presence of the so-called aqueous gap, some tens of nanometers thick, that typically exists between the cell and the surface.13 The peak splitting is significantly different from the results obtained from using a conventional waveguide geometry,14 in which only peak broadenings of 5% 共TE兲 and 20% 共TM兲 were observed. This difference, we believe, is due to the improved cover index sensitivity 共Table I兲 of the reverse symmetry waveguide, which causes the angular splitting between the water peak and the cell peak to increase by a factor of 5, thereby making it possible to resolve both peaks. The measured change in peak shape seemed to be saturating after 190 min. After this, the experiment was stopped and the waveguide was investigated under microscope, see Fig. 3. These investigations revealed that the cells covered the surface of the waveguide with a concentration of approximately 390 cells/ mm2 and a surface coverage of 85%. The standard procedure, which is to follow the positions of the incoupling peaks and from this calculate the effective refractive index of the modes, is obviously not feasible in the present situation. To get quantitative information, two different methods were tested. First, the area under the peaks was displayed versus time, which is shown in Fig. 4共a兲. As is seen, the appearance of cells leads to a rapid decrease in peak area for both TE and TM modes. The decrease in peak area reached a saturation level after 8 – 10 min. This time correlates quite well with the time of sedimentation that we observed from parallel microscopy tests. The main physical effect that governs the peak height change is therefore probably scattering of the evanescent optical field from the optically inhomogeneous cell layer at the surface. This is unlike the so-called cutoff peak reduction reported previously for a reverse symmetry waveguide, in which both the positions and the heights of the peaks changed upon changing the cover refractive index homogeneously.15 As a second method, the mean peak positions of the peak structures were calculated, as shown in Fig. 4共b兲. Unlike the peak area the mean peak position continued to increase even after the cell attachment phase. This, we believe,

FIG. 2. Incoupling peaks measured at different times during cell attachment and spreading. Downloaded 15 Feb 2005 to Redistribution subject to AIP license or copyright, see


Appl. Phys. Lett. 86, 071101 共2005兲

Horvath et al.

the cell sedimentation and attachment while mean peak positions of the complex peak structures can be used for monitoring cell spreading. Based on the results presented, we believe that the reverse symmetry waveguide design opens interesting possibilities in cell research, both in terms of basic cell-surface interactions and in terms of important applications such as cell based toxicity screening. This work was supported by the Danish Technical Research Council, Grant No. 26-01-0211. The authors express their special thanks to L. Hubert, J. Stubager, B. Sass, and L. Knudsen for their help with this work. K. Tiefenthaler and W. Lukosz, Proc. SPIE 514, 215 共1984兲. R. E. Kunz, Sens. Actuators B 38, 13 共1997兲. 3 W. Lukosz, Sens. Actuators B 29, 37 共1995兲. 4 J. Voros, J. J. Ramsden, G. Csucs, I. Szendro, S. M. de Paul, M. Textor, and N. D. Spencer, Biomaterials 23, 3699 共2002兲. 5 R. Horvath, L. R. Lindvold, and N. B. Larsen, Appl. Phys. B: Lasers Opt. 74, 383 共2002兲. 6 R. Horvath, H. C. Pedersen, and N. B. Larsen, Appl. Phys. Lett. 81, 2166 共2002兲. 7 R. Horvath, H. C. Pedersen, N. Skivesen, D. Selmeczi, and N. B. Larsen, Opt. Lett. 28, 2473 共2003兲. 8 J. J. Ramsden, S.-Y. Li, E. Heinzle, and J. E. Prenosil, Cytometry 19, 97 共1995兲. 9 J. Voros, R. Graf, G. L. Kenausis, A. Bruinink, J. Mayer, M. Textor, E. Wintermantel, and N. D. Spencer, Biosens. Bioelectron. 15, 423 共2000兲. 10 T. S. Hug, J. E. Prenosil, and M. Morbidelli, Biosens. Bioelectron. 16, 865 共2001兲. 11 T. S. Hug, J. E. Prenosil, P. Maier, and M. Morbidelli, Biotechnol. Bioeng. 80, 213 共2002兲. 12 13 K. F. Giebel, C. Bechinger, S. Herminghaus, M. Riedel, O. Leiderer, U. Weiland, and M. Bastmeyer, Biophys. J. 76, 509 共1999兲. 14 R. Horvath, J. Voros, R. Graf, G. Fricsovszky, M. Textor, L. R. Lindvold, N. D. Spencer, and E. Papp, Appl. Phys. B: Lasers Opt. 72, 441 共2001兲. 15 R. Horvath, N. Skivesen, and H. C. Pedersen, Appl. Phys. Lett. 84, 4044 共2004兲. 1 2

FIG. 4. The area 共a兲 and mean peak position 共b兲 of the incoupling peaks vs time.

is related to the cell spreading which causes an increasing amount of high-index material to be located within the evanescent field of the modes, which therefore continues to push the mean peak positions towards higher angles. In conclusion, a grating-coupled waveguide sensor with the so-called reverse symmetry design was tested as a quantitative sensor for living cell attachment and spreading. Unlike with conventional waveguide sensors, we have observed a significant change in the incoupling peak height and peak shape. The area under the peaks can be used for monitoring

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