Integrated optical input grating couplers as direct affinity sensors

Biosensors & Bioelectronics 8 (1993) 129-147

Integrated optical input grating couplers as direct affinity sensors* Ph. M. Nellen 81 W. Lukoszt Optics Laboratory,

Swiss Federal Institute (Received 4 November

of Technology

Zurich, CH-8093 Zurich, Switzerland

1992; accepted 1 December

1992)

Abstract: An input grating coupler instrument with planar monomode waveguides as sensors was used for real-time monitoring of protein adsorption and of affinity- and immunoreactions. The adsorbed proteins studied were avidin and immunoglobulins (h-IgG). In affinity- and immunosensing, adsorbed monolayers of these proteins formed the receptors on the waveguide surface for the corresponding ligand in the sample. The formation of complexes between avidin and biotinylated protein A (prot A) and biotinylated bovine serum albumin (BSA), between h-IgG and anti-hIgG, and between h-IgG and prot A were investigated. With an improved sensor instrument, changes of the effective refractive indices Nru and NrM of the TEo and TM0 guided modes at two laser wavelengths’,+, = 632-g nm and h2 = 514.5 nm were measured. From these data not only the thickness dFf but also the refractive index I+ and the surface coverage I’ of an adsorbed or bound single isotropic adlayer F’ were determined at both wavelengths. This model was extended to include (i) a possible optical anisotropy a F, of an (adsorbed single) adlayer and (ii) the formation of double adlayers (F’ + F”) in affinity reactions, adlayer F’ describing the immobilized receptors and adlayer F’ the bound ligands. The SiOrTiOz waveguides, fabricated by a sol-gel process, are microporous and, in aqueous buffer solutions, show a slow but persistent increase of the measured effective refractive indices with time. This drift effect is greatly diminished in compact SiOz-TiOz waveguides annealed at a temperature of T = 900°C (instead of T = 500°C). Keywords: integrated optical sensors, direct optical affinitysensors, optical waveguides, grating couplers.

1. INTRODUCTION

We have previously demonstrated that input grating couplers on planar optical waveguides can be used as integrated optical sensors for

* Poster presentation at Biosensors ‘92, Geneva, 20-22 May 1992. t To whom correspondence should be addressed. 0956-.5663/93/%06.00 0

and immuno-

protein adsorption studies and for direct immunosensing (Nellen & Lukosz, 1991). In this paper, we report on the real-time monitoring of (i) adsorption of the proteins avidin and immunoglobulin G (h-IgG), (ii) affinity reactions between immobilized (adsorbed) avidin and biotinylated proteins (bovine serum albumin (BSA) and protein A (prot A)) and between prot A and h-IgG, (iii) immunoreactions between immobilized (adsorbed) antigens and antibodies

1993 Elsevier Science Publishers Ltd.

129

Ph. M. Nellen & W. Lukosz

(h-IgG and anti-h-IgG), and (iv) a sequence of affinity- and immunoreactions. The affinity- and immunosensing experiments show the sensitivity of the method and compare continuous-flow and stopped-flow sample injection techniques. During these adsorption processes or reactions, described as formation of a single adlayer F’ on the waveguide, not only its thickness dr, but also its refractive index nr,, and thus the surface coverage I’, are determined as functions of time. That the thicknesses measured at two wavelengths, hi= 632.8 nm and A2 = 514.5 nm, agree well, corroborates the reliability of the method. For single adlayers F’, first results were obtained with an extended model taking into account the anisotropy caused by preferential orientation of the protein molecules. Affinity reactions were modelled as formation of a double adlayer (F’ + F’), adlayer F’ describing the immobilized receptors, and adlayer F” the bound ligands; the thicknesses (&’ and &) and refractive indices (Itr, and nF) of both adlayers were determined. The paper also reports on investigations of the drift effect of microporous and compact SiO*-TiOZ waveguides in aqueous buffer solutions, and their behaviour under changes in salt concentrations. We start by recalling the sensor principle and then briefly describe our multiwavelengths operated input grating coupler instrument and the data evaluation, which starts from the measured effective refractive indices NTEOand NTMOof the guided modes and determines the optical parameters of the adsorbed adlayers or bound affinityor immunocomplexes.

2. SENSOR PRINCIPLE The fundamental mechanism of integrated optical sensors makes use of the evanescent field of guided modes which decays exponentially into the sample medium covering the planar waveguide. The evanescent field probes changes in refractive index of the sample medium only near the interface to the waveguiding film F, more precisely within the penetration depth Azo. Integrated optical sensors respond to changes in the effective refractive indices N of the guided modes induced by the following mechanisms (see Fig. 1): (a) changes of the refractive index nc of the sample medium C; (b) unspecific adsorption 130

Biosensors & Bioelectronics

of molecules out of a gaseous or liquid sample C either on the surface of the waveguiding film F (which we model as formation of an adlayer F’ with refractive index nr’ and thickness dr’); or (c)-in the case of a microporous waveguiding film F-in its pores (changing its refractive index nr); and (d) specific binding of analyte molecules (in affinity- or immunoreactions) to receptor molecules immobilized on the waveguide surface (which we model as changes of the parameters &’ and & of a single adlayer F’ or as formation of a second adlayer F” with refractive index nF” and thickness &, on the first adlayer F’). The input grating coupler sensor is one possible method to excite the waveguide modes and to measure their refractive indices. In our laboratory, we also work with other IO sensors, namely an output grating coupler instrument (Lukosz, Clerc & Nellen, 1991; Clerc & Lukosz, 1992) and a difference interferometer (Lukosz & Stamm, 1991; Stamm & Lukosz, 1992).

3. MULTIWAVELENGTHS OPERATION OF INPUT GRATING COUPLER INSTRUMENT We built an input grating coupler instrument which measures the effective refractive indices NTEo and NTMo of the TEo and TM, modes of a planar monomode waveguide with a resolution of AN,, = 2 X 10m6 (see Fig. 2). We briefly recall the measuring principle (see also Nellen & Lukosz, 1991; Nellen, 1992): laser light is coupled into the waveguide by a surface relief grating on the waveguide. From the optimum angle of incidence (Y~ the effective refractive index N is calculated from the relation N = nair sin ai+ 1NA

(1)

where hair is the refractive index of air, 1 = ? 1, *2, . . . is the diffraction order, A is the wavelength in vacuum, and A is the grating period. The optimum incoupling angle cyIis determined from the peaks of the powers of the incoupled guided modes which are measured by photodetectors Di and D2 at the ends of the waveguide in an angular scan (Fig. 2a). Previously, we used the sensor instrument to the effective measure refractive indices NTEo and NrMo only at a single wavelength hi, usually at the He-Ne laser wavelength Ai = 632.8 nm. We operate the improved version of

Integrated grating couplers

Biosensors & Bioelectronics

+AnF 1nv d,, +Ad,

dF “F

I

F’

I

X

Y I

Fig. 1. Sensor effects. Integrated optical sensors respond to: a) refractive index changes of the sample C; b) aakorption of molecules on the waveguide surface (forming an adlayer F); c) sorption of molecules in micropores of the waveguiding film F; and d) binding of analyte molecules to a chemically selective capture layer F’, thus forming a second adlayer F” (affinity sensing). Schematically represented is the field of a guided mode. S, substrate; F, waveguiding film; C, sample; AZ,-, penetration depth of the evanescent field.

the instrument at two wavelengths; two pairs of effective refractive indices of the TE,, and TM, modes at two wavelengths (A, = 632.8 nm and the argon laser wavelength A2 = 514.5 nm) are measured quasi-simultaneously with an angular scan of only ha = 10” (Fig. 2b). Compared to single-wavelength operation where repetition rates of I O-5 s-l were possible, the larger angular scans Aa! lead to a lower repetition rate of twice per minute. A typical measurement with the double-wavelenths operated instrument is shown in Fig. 6; all other figures in this paper refer to measurements at Ai. The mechanical rotation of the waveguide is computer controlled; signal detection and the determination of the four effective refractive indices are automated.

4. DATA EVALUATION The data evaluation starts from the measured effective refractive indices A&,,(t) and NT%(t). From the values NT&t I 0) and NThlg(f 5 0) measured just before the starting time (t = 0) of

an experiment, the optical parameters, i.e., the thickness dF and the refractive index nr of the individual waveguide used, are determined from the mode equation of a one-layer waveguide F between the substrate S and the buffer solution C, whose refractive indices ns and nc, respectively, have to be known. During the actual adsorption or affinity sensing experiment (t > 0), the optical parameters of the single adlayer F’ or double adlayers F’ and F” are evaluated from the data N,,(f) and A&,,(t) as follows. (1) In the single-adlayer model F’, the thickness dv,(t) and refractive index nr(t) of adlayer F’ are calculated rigorously from the mode equation of a two-layer waveguide consisting of waveguide F and adlayer F’ between substrate S and sample C. The surface concentration of coverage I’(t), i.e., the adsorbed mass per unit area, is calculated from the relation I’ = (nF’ - no)(d?r/dc)-‘d,,

(2) where dnldc = O-188 ml g-’ is the increment of refractive index of aqueous solutions 131

Biosensors & Bioelectronics

Ph. M. Nellen & W. Lukosz

. .

.

.

.

** .

-. :. :. 10"

a+

0”

c>

.

1-

-

-

syringes

peristaltic pump

va!ve

~ cuvette

waste

Fig. 2. Input grating coupler sensor instrument. a) Multiwavelengths operation: the angles of incidence of the two laser beams are chosen in such a way that the TEe and TM, modes at both wavelengths hl and A2 are excited in an angular scan of only 10” by the di~r~ction order 1 = -Z. The sensor head, consisting of w~veguide W and cuvette Cu, is mounted on a rotation stage R. The powers of the incoupled modes are measured with two photodetectors Dt and D2. b) Measured incoupling curves during a lo” angular scan. Each dot corresponds to a measured signal value at angular intervals of 18 arcseconds. c) Fluid handling system.

132

Integrated grating couplers

Biosensors & Bioelectronics

with protein concentration c (Nellen & Lukosz, 1991). (2) In the case of the double-adlayer model, first the optical parameters & and nr’ of the (adsorbed) adlayer F’ are determined. Then the thickness d&f) and the refractive index n&t) of the second adlayer F’, formed, for example, by the binding of ligand molecules to the adlayer F’ of receptor molecules, are determined by rigorously solving the mode equation for the three-layer waveguide consisting of waveguide F and adlayers F’ and F between substrate S and sample C. The surface concentration I”(t) is determined from the relation r, = (IzF”- no)(dnldc)-‘&

(3)

In the measurements at two different wavelengths Ai and AZ, the refractive indices and thicknesses of the film F and adlayers F’ are determined independently at each wavelength. The thicknesses dr and dF should be independent of wavelength, while the refractive indices will differ somewhat due to dispersion. The agreement of the geometric thicknesses measured at Ai and AZrepresents a quality control of our method. We found that the characterization of the waveguiding films F at two wavelengths Ai = 6328 nm and = 514.5 nm leads to differences in the A2 normalized waveguide thickness of AdF/dF =Z1%. For protein adlayer thicknesses dFg the differences were larger, i.e., AdFs/dFs S 10%. We assumed the waveguiding film F itself and the adlayers F’ and F” to be homogeneous and isotropic media. But we also present below the first results of an extended model in which an optical anisotropy of a single adlayer F’ is taken into account.

5. MICROPOROUS AND COMPACT SI02-TI02 WAVEGUIDES In our experiments, we used planar optical monomode Si02-Ti02 waveguiding films on either glass substrates (Pyrex or Schott glass AF45) or fused silica substrates. We fabricated the films by a dipcoating method (sol-gel process). Prior to a firing process that transformed the gel-like film into a hard glassy oxide material, surface relief gratings with l/A = 2400 lines per

mm were embossed into the film with a diffraction grating as a die. After the normal firing process at 500°C for one hour the waveguide had refractive indices of nr = 1.75-1.79; the thickness (typically dF = 160-190 nm) is mainly determined by the withdrawal velocity from solution (Merck Liquicoat@) in the dipcoating process. Using longer firing times of up to 12 h and/or higher temperatures up to 9OO”C,which is only possible with silica substrates, waveguides with higher refractive indices up to nr = 186 were fabricated. Higher refractive indices indicate more compact, less microporous waveguiding films. A semi-quantitative test for the microporosity of the waveguides works as follows (Nellen, 1992): the waveguides are dried in flowing nitrogen for several hours. When they are exposed to ambient air of about 50% relative humidity, water adsorbs on the waveguide surface and forms an adlayer of thickness d,.; it also adsorbs in the micropores of the waveguiding film, thus changing its refractive index nr; in an example, for a microporous waveguide with nr = 1.735 and dF = 191 nm, we found dFs = 3.5 nm and Anr = O-02. When the waveguides are exposed to water or aqueous buffer solutions, a drift effect occurs, i.e., a slow but persistent increase of the effective refractive indices NTEo and NTMo with time. The drift effect can be disturbing when adsorption processes or affinity reactions are monitored. We have no consistent physical explanation or model of the drift effect. The magnitude of the drift effect depends on the parameters of the waveguide fabrication process and especially on the resulting porosity. With higher firing temperatures, denser and less microporous films were obtained. Figures 3 and 4 show the drift effect in the first hour of exposure and in 24 h, respectively, for three different waveguides I)-III): waveguide I) on a Pyrex substrate (T = 500°C for 2 h, n F = 1.757, dF = 146 nm), waveguide II) on AF45 glass (T = 600°C for 3.2 h plus 700°C for O-5 h, nr = 1.793, dF = 161 nm), and waveguide III) on a fused silica substrate (T = 800°C for l-3 h, nF = 1.829, dF = 159 nm). All three waveguides were exposed to deionized water for at least 1 h. Then the water was exchanged with a phosphate buffer solution (PBS). A non-drifting waveguide would respond only with an instantaneous change AN = (t3N/&zc)Anc in the effective refractive indices NTEOand NrMo induced by the cover index change of Ant = O-002 between water and PBS (refractometer effect). All wave133

Ph. M. Nellen & W. Lukosz

Biosensors & Bioelectronics N TEo

1.5410-

NTMo

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1.0

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time (hours)

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Fig. 3. Response of SiOrTi02 waveguides to buffer solutions in the first hour of exposure. Shown are the measured effective refractive indices NTEO(left) and N TM0 (right) for three different waveguides I), ZZ) and ZZZ)having different microporosity. Note that in all cases a range of AN = I x 10W3 is shown. The arrows (& ) indicate the time when the water in the cuvette was exchanged with phosphate buffer solution (PBS), pH 7.4.

guides I), II) and III) showed an instantaneous response. But the changes in the effective refractive indices of waveguide I) were about 10 times larger than calculated for the cover index change Ant. Afterwards, all three waveguides drifted. The shapes of the curves are different for the three waveguides. After one hour the temporal changes of the effective refractive indices were: 0 for waveguide I) AN,lAt = 2.3 x AN-JAt = 2.0 x l for waveguide II) AN,,tAt = 4.6 x AN%lAt = 3.3 X 0 for waveguide III) 134

lop4 h-l and 1O-4 h-l, 10T4 h-l and 1O-4 h-l, and

AN,,lAt AN-JAt

= 1.5 x 10m5 h-l and = 2.5 x 1O-5 h-l.

Twenty-four hours later the following values were measured: l

l

l

for waveguide I) ANrnjAt = 1.3 X lop5 h-l AN-,/At = 1.7 x 1O-5 h-‘, for waveguide II) AN&At = 4.2 X low5 h-l AN+r&At = 2.9 X 10m5 h-l, for waveguide III) AN&At = 1.3 X lop5 h-l AN-,,lAt = 2.3 x 1O-5 h-l.

and

and and and

For waveguides I) and II) the drift has diminished. For waveguide II) the drift effect is most pro-

1.6245 1.6235 1.5380 15370 15360 0

5

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time fhwrsl

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nounced. The drift of waveguide III) at the beginning of the experiment was smaller, by a factor of ten, than those of the others; it remained nearly constant over the whole 24 h period. Waveguides fired at higher tem~ratures (T = 9OO*C)showed a very small drift effect of the order of AN~A~= (l-2) X 10e6 h-l. With the resolution of ANGLE = 2 X 10m6of the grating coupler instrument, this drift effect is only noticeable in an e~riment lasting several hours. For sensor experiments of less than one hour the drift effect is therefore negligible. In another series of experiments, we exposed microporous and compact waveguides to aqueous saline solutions of varying salt concentrations (and to buyers with varying pH value). We found that responses (i.e., changes in the effective refractive i~~ces) occur, which are higher for microporous and much reduced for compact waveguides.

cl

5

10

15

20

time (h~u~s~ -+

Figure 5 pertains to such an experiment in which the concentrations of NaCl and KC1 were varied. The exposure time was 1 h; &o% of the response occurs after the first 5 min. The result of these ex~e~ments can be quantitatively explained by induced changes in the re~active index nF of the micro~orous waveguide. From the measured effective refractive index changes AN,,, and ANT&, we calculated the refractive index changes AnF and the cover index changes Ant by solving the mode equation. The calculated values of nc for the various salt concentrations, ranging from c = 5 X 10m5mol 1-l to 5 X 10-Z mol l-r, agreed well with the values of nc measured independently with an Abbe refractometer. The refractive index changes Ahn, (of up to 1 X 10N3) are not completely reversible; they can ob~o~sly not be explained simply by an exchange of the solution with refractive index nc in the micropores, because the values of AnF

135

Ph. M. Nellen & W. Lukosz

0

1ti4

Biosensors & Bioelectronics

1G3

KY2

16'

c (mol/l) -+ Fig. 5. Response of microporous waveguides to changes in electrolyte (NaCl or KCl) concentration c. The changes in the refractive index nF at A, = 632-8 nm of the waveguiding film F versus concentration c (logarithmic scale) are shown.

are greater than the refractive index change Ant. Probably, salt or ions are absorbed in the micropores. In an analogous experiment with a compact waveguide (not shown in Fig. 5), the refractive film index remained constant within AnF 5 5 x 10w5. For affinity- and immunosensing, the influence of changes in salt concentration and pH value on the waveguide should be minimized. Therefore, we now think it advisable to perform such experiments with compact waveguides (firing temperature T 2 SOO’C) on silica substrates. However, such waveguides were available only for the experiments shown in Fig. 6; for all other experiments microporous waveguides were used. Immediately after a careful cleaning of the waveguides (their surface is hydrophilic) they were mounted on the rotation stage and exposed to PBS. To reduce the disturbing influence of the drift effect, this exposure lasted at least 1 h for waveguides with a small drift effect and up to 24 h for waveguides with a higher drift effect, before the actual sensor experiment was started.

6. EXPERIMENTAL We used cuvettes made of polymethylmethacrylate (PMMA) with volumes of 1.7, 15.4 and 136

154 ~1 and depths of 50, 100 and 1000 pm, respectively. The outlet of the cuvette is connected to a peristaltic pump that sucks the fluid through the cuvette into a waste reservoir (Fig. 2~). We carried out the experiments in two different modes: (i) in the continuous-flow mode, the sample solution is pumped through the cuvette at flow rates of 0.3 ml min-l down to 10 ~1 min-’ during the whole measuring time; and (ii) in the stopped-flow mode, at first in continuous flow at a flow rate of O-3 ml min-l, the cuvette of volume 154 ~1 was refilled six times; then the pump stopped and the last fill remained in the cuvette. The sensor experiments were carried out at room temperature (23.5”C). The proteins were dissolved in a phosphate buffer solution of pH 7.4 (PBS: 6.5 mM phosphate buffer, 131 mM sodium chloride). With an Abbe refractometer the values of the refractive index of PBS were measured to be: nc = 1.333 at A1 = 632.8 nm and nc = 1.337 at AZ = 514.5 nm. For protein solutions the refractive index increases linearly with concentration c with a refractive index increment of dnldc = 0.188 mg 1-l (Sober, 1976). For low protein concentrations (c < 100 pg ml-‘) the values of nc were approximated by those of PBS. The suppliers of the proteins used for our adsorption and affinity reaction studies were: Fluka, Buchs, Switzerland, for human immunoglobulin G (h-IgG, M, = 150000 daltons) and bovine serum albumine (BSA, M, = 67000 daltons); Dakopatts, Denmark, for rabbit antihuman-immunoglobulin G (anti-h-IgG); Pierce, Rockford, Illinois, USA, for avidin (M, = 67000 daltons), protein A (prot A, M, = 42 000 daltons), biotinylated prot A (biotin-(prot A)) and biotinylated BSA (biotin-LC-BSA, M, = 68 000 daltons).

7. ADSORPTION

EXPERIMENTS

We studied the adsorption of avidin and h-IgG on the waveguide surface. We found that both proteins were found to adsorb nearly irreversibly (i.e., they desorb very slowly) and that they preserve their functionality, i.e., they still react with the corresponding ligand or antigen. Figure 6 shows an avidin adsorption experiment measured at two wavelengths A1 = 632.8 nm and AZ = 514.5 nm. The measurements were made in the continuous-flow mode with a flow rate of

Integrated grating couplers

Biosensors & Bioelectronics

17

h2= c

514.5 nm

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0 25

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-

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Fig. 6. Avidin adsorption, measured at two wavelengths (Al = 632.8 nm and A2 = 514-5 nm), shows good agreement in the calculated adlayer thicknesses dF’ and surface coverages r ‘.

15 ~1 min-’ and a cuvette of volume 1.7 ~1. The protein concentration was increased from c = 5 nM in steps by a factor of 5 every 35 min to finally c = 74 PM. At the highest concentration, the adsorption started to saturate. The determined adlayer parameters (at c = 74 PM) were: nF’ = 1.43, dFr = 4.5 nm and I&, = 2.29 ng mm-* at Ai; and nr, = 1.44, dFf = 4.1 nm and I&, = 2.24 ng mme2 at AZ. In this and other examples, we found that the adlayer thicknesses dF’ and surface coverages l? determined at the two wavelengths agreed quite well. (That in this experiment the surface coverages are lower than in the experiment reported in Fig. 7 may be connected with different surface properties of the waveguides fired at temperatures of 800°C and 500°C respectively.) Figure 7 shows the adsorption isotherm of avidin on a waveguide surface, i.e., the equilibrium surface coverage Id versus concentration c. The results can be fitted by a Langmuir adsorption isotherm

r:/rk,, = CI(C + c*)

(4)

where &,, corresponds to a monolayer coverage and c* is a constant; the usual intepretation of C * = k&kads (the ratio of the dissociation and adsorption constants kdiss and kads) cannot be used here because the adsorption is practically irreversible. The constants were found to be I+, = 3.7-3.9 ng mme2 and c* = 240-260 nM. The experiments were carried out in the stopped-flow mode; all values of IL were determined after 37 min. In all adsorption experiments with different proteins such as avidin (see Fig. 6) or h-IgG (Fig. 8), the following striking result was obtained. Evaluations with the model of a homogeneous and isotropic adlayer F’ gave r’(t) curves which fluctuated much less than the curves for both r+(t) and dFV(t). In the first phase of adsorption, i.e., at low surface coverages r’/I&, =S 0.3, the calculated VdUeS of dFPwere unrealistically small 137

Ph. M. Nellen & W. Lukosz

Biosensors & Bioelectronics

4

3 f “E E a 5

2

i

1

0 0.001 0.01

0.1

1

10

loo

c f@U + Fig. 7. Adsorption isotherm of avidin on SiO,TiOz waveguide surfaces. Shown are the equilibrium surface coverages r:(c) versus avidin concentration c (logarithmic scale) in buffer solution at constant pH 7.4 and constant temperature T = 23.5”C. Filled squares, experimental values; solid line, fitted Langmuir adrorption isotherm.

(compared with the sizes of the molecules) and those of nF’ were much higher than expected for protein adlayers. A possible explanation of this effect, according to Lukosz (1992), is an anisotropy of the adsorbed adlayer F’ . An anisotropy factor +’ = iS defined where &’ = (nFP)OiS the (~F*)J(~F*)w ordinary and (nF,)eO the extraordinary refractive index. The optical axis is assumed to be parallel to the z-axis. The anisotropy results from a preferential orientation of the non-sphericallysymmetrical molecules. In this model oblate or prolate ellipsoids of rotation, respectively, correspond to molecules ‘lying’ (+’ > 1) and ‘standing’ (aF’ < 1) on the surface. The unrealistically high values of &, obtained with the isotropic adlayer model are explained as a consequence of a small anisotropy +’ > I. The model of an anisotropic adlayer introduces a third unknown parameter, either (+)eo or +‘. Obviously, from just two measured effective refractive indices, NTEo and NT%, it is impossible to calculate the three adlayer parameters &,, & and uF’ independently. However, the following results can be given, which show the magnitude of the anisotropy and which indicate whether the molecules ‘stand’ or ‘lie’ on the surface. The 138

evaluation procedure was as follows. At the highest surface coverage I&,, the anisotropy factor was assumed to be &’ = 1; the measured effective refractive indices ZVTEoand ZVTMowere evaluated with the isotropic adlayer model, giving the values nr’ and dr, = (&‘)m_ Then the rest of the experimental data was evaluated for nr’ and +’ by rigorously solving the mode equation of the two-layer waveguide consisting of the isotropic film F and the anisotropic adlayer F under the assumption that the thickness of the adlayer reIIIainS COnStant, dF = (dF’)mm. At a concentration of c = 0, the adlayer F’ of thickness (&)max does not contain any molecules; and consequently &’ = nc and +’ = 1. With increasing concentration c, molecules begin to fill the adlayer, thereby increasing its refractive index nr’. Depending on the shape of the molecules, the anisotropy factor increases or decreases from the intial value of + = 1. Figure 8 compares evaluations made with the isotropic and the anisotropic model for the adsorption (in the stopped-flow mode) of hIgG, starting from the same measured effective refractive indices N,,(t) and N%(t). The following parameters were calculated: in the isotropic model, &(t) and &(f) with a&t) = 1; and, in the aniSOtrOpiC model, n&t) and +(f) with d&t) = (dF’)max = 58 nm. As expected for the anisotropic model, the refractive index nr’ increases with increasing concentration c from the cover index nc (empty adlayer) up to the value of &’ = 1.44 at the highest surface coverage. The anisotropy factor uF’ starts at a value of OF’ = 1 (empty adlayer) and increases to a maximum of + = lXlO5 at the concentration of c = 266 nM, where FYI&, = O-41. At this concentration, the ordinary refractive index nr’ = (nF,)O of the adlayer F’ is nF’ = l-380; the value of the extraordinary refractive index is therefore +/+ = (+)eo = 1.373. Because the thickness & is left constant in this evaluation, the aIIiSOtrOpy f&Or uF’ iS forced again t0 approach UF’ = 1 at r:/r&,= 1. In spite of the incomplete evaluation scheme, this interpretation of the data seems to be very prOIIIiSing. The nr(t) and +(f) curves show smaller fluctuations than +(f) and &(f) in the isotropic interpretation. From + > 1 it can be concluded that most of the Y-shaped h-IgG molecules lie horizontally on the surface. Similar interpretations of the data for human immunoglobulin A (A4, = 160000 daltons) and

Integrated grating couplers

Biosensors & Bioelectronics

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ilme (rnlnuter) -+

Fig. 8. Aakorption of h-ZgG: comparison of isotropic and anisotropic adlayer models. The isotropic model (left) shows unrealistically high nFS and small dFS values at small surface coverages rllr;, s 0.3. The anisotropic model (right) shows an optical anisotropy factor ap > 1, indicating that the h-ZgG molecules tend to lie on the waveguide surface. The fluctuations in np are smaller in the anisotropic model.

avidin showed that these molecules, too, predominantly ‘lie down’ on the surface. With (dFP)max = 5 nm, h-IgA had a maximum anisotropy factor of aF’ = l-003 with nF, = l-36 at a concentration of c = 125 nM, where l?i/r& = O-27. For avidin the values were (dF’)m_ = 4.5 nm, +’ = l-006, = 1.36 at a concentration of c = 588 nM, nF corresponding to I’i/rA, = 0.43. In order to obtain independent values for n&t), a&(t) and a&t), more than two effective refractive indices N have to be measured. This requires thicker multimode waveguides, which guide not only the TE,, and TM0 modes but also the TE1 and TM1 modes.

8. AFFINITY

SENSING

EXPERIMENTS

For integrated optical affinity sensors, receptor molecules have to be immobilized on the waveguide surface and, in the case of grating coupler

sensors, in the grating region. The corresponding ligand molecules, if present in the sample solution, bind specifically to the receptor molecules on the surface; the sensor directly responds to the formation of the receptor-ligand complex. For immunosensors, antibodies (Abs) or antigens (Ags) are immobilized on the waveguide surface; they bind their corresponding Ags or Abs, respectively. In our experiments, the receptor molecules were immobilized on the waveguide surface by adsorption, not by chemical binding. The strongest affinity reaction known is the binding of biotinylated proteins to immobilized avidin (Wilchek & Bayer, 1990). Figures 9a and 9b show that nanomolar concentrations of biotinLC-BSA can be easily detected with adsorbed avidin adlayers. The adsorbed surface coverage of avidin was I” = 3.2 ng mmp2 in both experiments a) and b). For experiment a) the optical parameters of the avidin adlayer were nF’ = l-46 and dF, = 4.7 nm; for experiment b) ItF, 139

Biosensors & Bioelectronics

Ph. M. Nellen & W. Lukost

biotin-LC-ESA

23.5nM H

$j

+;“*

Od

-4.7 nM

I

biotin-LC-BSA

588nM

i

t 118nM

time (minutes)+

time (minutes]+

Fig. 9. Avidin-biotin-LC-BSA reaction: comparison of surface coverage changes AT’(t) versus time observed in a) stopped-flow mode and b) continuous-flow mode at a biotin-LC-BSA concentration of c = 4.7 n,u.

of = 1.47 and dFg = 4.4 nm. A concentration c = 4.7 nM of biotin-LC-BSA was unambiguously detected in less than 5 min. That the observed increase in AT’ was not caused by unspecific adsorption of biotin-LC-BSA is proved by the observation that (previous) addition of nonbiotinylated BSA, even at a 125 times higher concentration, did not produce any increase in AY. For experiment a) the stopped-flow mode was used. For each concentration from c = 4.7 nM to c = 588 nM, the cuvette was flushed six times in 3 min (exchange time ta = 30 s); then the flow was stopped for the remaining 55 min. At a concentration of c = 4.7 nM biotin-LC-BSA the surface coverage increased by an amount of Ar’ = 50 pg mmP2 in the first 3 min, which is a factor of 8 higher than the minimum detectable surface coverage change AI&” = 6 pg mm-*. After 50 min the total change was AI” = 170 pg mm-*. That AT’ saturates with time is caused by depletion of the solution. Successively increasing the concentration and injecting the solutions as described above led then to a saturation of the binding reaction at the concentration of c = 588 nM. The total amount of bound biotin-LC-BSA was Al? = 1.27 ng mm-*; this means that one avidin molecule binds O-4 biotin-LC-BSA molecules. Experiment b) was carried out in the continuous-flow mode and with a cuvette of volume 15.4 ~1. During the whole experiment, with a concentration of c = 4.7 nM biotin-LC-BSA, the flow rate was 50 ~1 mine1 (exchange time tb = 18.5 s). In the first 3 min the surface coverage 140

increased by an amount of Ar’ = 70 pg mm-*, which is a factor of 11 higher than AI&, = 6 pg mm-*. After 50 min a surface coverage change of Ar’ = 0.79 ng mm-* was reached. Within the 80 min shown in Fig. 9b, 1-O ng mm-* biotin-LC-BSA bound to the avidin monolayer, which corresponds to 80% of the saturation value. Since the avidin-biotin binding reaction is known to be very fast, the observed reaction rates in the continuous-flow mode were limited by diffusion. Then, for small coverages, the surface coverage Ar’ increases linearly with time t, which can be explained semi-quantitatively as follows. In the exchange time t, in which the solution in the cuvette is exchanged, molecule within a distance AZ(&) = 2(Dt&)ln reach the surface, where D is the diffusion constant. In time t the cuvette is refilled 12 = t/la times; consequently, the coverage change is Ar’(t) = cAz(t,)t/t, = 2ct(D&J l’*. From the measured surface coverage changes during the first 3 min of flow in case a) where Ar’ = 50 pg mm-*, and in case b) where AT’ = 70 pg mm-*, estimates for the diffusion coefficient of biotinLC-BSA are obtained as D(BSA) = (1.8 and 2.0) x lop5 mm* s- l. This value is of the same order of magnitude as the value for a similar molecule, human serum albumin (HSA, M, = 69000 daltons), D(HSA) = 6.1 x 10e5 mm* s-l, given in the literature (Sober, 1976). Figure 10 shows the avidin-biotin-LC-BSA concentrations. reaction at sub-nanomolar Before, the adsorption of the avidin adlayer had been monitored; its parameters were determined

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time (minutes) + Fig. 10. Avidin-biotin-LC-BSA reaction at sub-nanomolar concentrations. The effective refractive indices NTE~and &M,, and the calculated adlayer parameters of the avidin-biotin-LC-BSA complex I+, dp and r’ are shown versus time. The first arrow ( J ) indicates

the start of the blocking step; the following arrows indicate the injection of the biotin-LC-BSA solutions at concentrations given.

to be nF? =

1.47, dF* = 4.5 nm and r’ = 3-2 ng mmw2. Washing with blocking buffer (BSA, O-6 PM) removed 3% of the adsorbed avidin. We used a cuvette of volume 15.4 ~1 and worked in the continuous-flow mode with a flow

rate of 100 ~1 min-‘. At concentrations c = 188 and 941 PM and c = 4.7 nM of biotin-LC-BSA, a linear increase of AT’ with time f was observed until saturation set in. In the continuous-flow mode, the slopes d(AI”)ldt were expected to increase practically linearly with c. The ratios of the concentrations c were chosen to be 1:5:25, the measured ratios of the slopes were d(AI”)ldt [(pg mme2) min-‘1: 0.95:2.28:11.17 = 2.1:5.1:25. That the slope at c = 188 nM is higher is probably due to the drift effect, which was AN/At = (5-9) x 10e6 h-l. In this experiment, we found that one avidin molecule bound about 0.36 biotinLC-BSA molecules. The experiments show that avidin, adsorbed on the waveguide surface, did not lose its capability to bind biotinylated molecules. Avidin has four biotin binding sites. In all our experiments, we found that only about 0.3-0.4 biotinLC-BSA molecules were bound to one avidin molecule. Each BSA molecule is biotinylated with about 8-12 biotin molecules (Pierce, ImmunoTechnology, Catalogue and Handbook, 1992). Therefore, one biotin-LC-BSA molecule could bind to several avidin molecules. Probably not all of the four biotin binding sites of the adsorbed avidin were accessible, because of steric hindrance. In another series of experiments, the binding of biotin-(prot A) to an adsorbed avidin adlayer was studied. The experiment shown in Fig. 11 was done in the continuous-flow mode with a flow rate of 150 ~1 min-’ and a cuvette of volume 15.4 ~1. The’ adsorbed avidin adlayer had the parameters nF’ = l-43, dF, = 5.2 nm and r’ = 2.54 ng mmp2. With non-biotinylated prot A at concentration c = 1 PM no response in Ar’(t) was observed, which means that neither a binding reaction nor an unspecific adsorption occurred. But biotin-(prot A) at a concentration of c = 7.5 nM could clearly be detected (Fig. lla). The surface coverage I” increased linearly with time until the flow was stopped after 55 min. Saturation was already observed at a concentration of c = 30 nM of biotin-(prot A). The total change in surface coverage was AT’ (biotin-(prot A)) = 0.52 ng mmp2; this means that one avidin molecule bound only 0.3 biotin-(prot A) molecules. Prot A has four binding sites which can bind immunoglobulins at their F,-part. In order to test the activity of these binding sites of the biotin-(prot A) bound to the avidin adlayer, a 141

Ph. h4. Nellen & W. Lukosr

Biosensors & Bioelectronics

3. I

3.0 2.9

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time (minutes) + Fig. 11. Affinity reactions between a) adsorbed avidin and biotinylated prot A, and b) binding of h-IgG to the avidin-biotin-(prot A) complex. Shown are the changes in surface coverage AT’(t) versus time.

concentration of c = 660 nM of h-IgG was injected. The surface coverage changed by an amount of Ar’(h-IgG)) = 3.15 ng mme2, as shown in Fig. llb; the thickness &, changed by A&(h-IgG) = 8.5 nm. About l-7 h-IgG molecules bound to one biotin-(prot A) molecule. This means that about two of the four binding sites of prot A were not accessible, probably because of steric hindrance due to the avidin-biotin binding part of the bound biotin(prot A) molecules. The ratios of avidin: biotin(prot A): h-IgG were 3:1:1.7. The experiment proves that prot A keeps its functionality (binding to the F,-part of h-IgG) when immobilized by binding to adsorbed avidin. We found the same result when prot A was immobilized by direct physical adsorption on the waveguide surface (see Fig. 15). We studied the immunoreaction between antiIgG and h-IgG adsorbed on the waveguide surface in several series of experiments (in the stopped-flow mode). Figure 12 summarizes the results for the saturation value of the surface 142

coverage AI’ of anti-h-IgG (normalized with respect to the surface coverage I’ of adsorbed h-IgG) versus anti-h-IgG, concentration c, in solution. The results can be fitted by a curve in the form of a Langmuir isotherm (cf. eqn. 4) with c* = 300 nM. We found that the maximum number of anti-h-IgG molecules bound per hIgG molecule to be 2-l. In all the above experiments, the formation of the receptor-ligand complex was described by changes in the optical parameters nr’ and dF’ Of a Single, homogeneous and isotropic adlayer F’. The refractive index &’ was a median value and the thickness dFr that of the complex. A different interpretation of a binding reaction is the double-adlayer model. The immobilized molecules are described as the adlayer F’ whose parameters (&?, dFs and I’) remain constant during the affinity reaction. The binding molecules are assumed to form a second adlayer F” with optical parameters nF”, dFg and r’. The values + and dFj have to be determined before the beginning of the binding

Biosensors & Bioelectronics

Integrated grating couplers

I

anti-h-IgG

i

T 2.0 g I

1.5 -

S z

1.0 -

I E 6 -

0.5 8 6 4

c (Ml + Fig. 12. Results of immunosensing experiments: binding of anti-h-ZgG to h-ZgG adsorbed on the waveguide. Plotted are the normalized surface coverage changes AT’(anti-h-ZgG)lZ’(h-ZgG) versus anti-h-ZgG concentration c in solution. The different symbols characterize different series of experiments.

experiment, then the values of nF, dF, and I” can be obtained. Figure 13 shows the calculated values of +(t), d&f) and r”‘(t) versus time t in the immunoreaction between adsorbed h-IgG and anti-h-IgG. For these experiments the stoppedflow mode was used. The values of the adsorbed h-IgG adlayer F’ were nFP = l-48, & = 5.5 nm and I’ = 4.2 ng mm-*. At an anti-h-IgG concentration of c = 3.3 PM, we found the values of nF’ = 1.43, dF- = 12 nm and I” = 6.5 ng mm-* for the adlayer F” in saturation. The comparison of these values with those of the single-adlayer model (nr, = l-45, AdF’ = 11.5 nm, AI’ = 6.8 ng mm-‘) shows that the refractive index of the anti-h-IgG-h-IgG complex, nF’ = 1.45, is indeed a median value between the refractive indices of the adsorbed h-IgG adlayer, nFS = l-48, and the bound anti-h-IgG adlayer, nF’ = l-43, respectively. It can be concluded that the bound anti-h-IgG adlayer is less compact than the immobilized antigens. The increases in the thicknesses are comparable, i.e., AdFS = d,.; both models give the same additional surface coverage AI’ = I”.

2 0

3

50

I loo

I 150

I 2m

time (minutes) +

I

250

Fig. 13. Zmmunoreaction between anti-h-ZgG and adsorbed h-ZgG interpreted with the double-adlayer model. In the calculation of the parameters nF, dF and Z” of the adlayer F” of bound anti-h-ZgG, we assumed the previously measured values dF = 5-5 nm and nF = 1.48 (Z’ = 4.2 ng mme2) for the adlayer F (monolayer of immobilized h-ZgG) to be constant during the immunoreaction.

For the anti-h-IgG concentration of c = 27 nrq very high values of nF and extremely small dF, values were obtained. For higher concentrations, nFP becomes smaller and dF- has values in the nanometre range. For concentrations smaller than c = 27 nM the double-adlayer model was not applicable at all; the numerical methods did not find any values for nF’ and dF,. This behaviour is similar to results obtained in adsorption experiments where, for small surface coverages rvr ,._, high nFSVdUeS and very Smdl dF< VdUeS were evaluated with the isotropic adlayer model. Either an anisotropy of adlayer F” has to be taken into account or, for small antibody concentrations, the formation of the immunocomplex antibody-antigen can probably not be explained by stacked molecules, i.e., the antibody molecules on top of the antigen molecules. Rather, it is possible that the antibody can nestle into the antigen adlayer F’, thereby increasing the density 143

Ph. M. Nellen & W. Lukosz

of the protein adlayer, which then increases the refractive index 1~~. Indeed, for small antibody concentrations, the increase in surface coverage I’ was not caused by an increase in thickness &, but by a slight increase in the refractive index nr,. However, for larger surface coverages I” the double-adlayer model is a good alternative description of an ongoing binding reaction on the waveguide surface. For proteins that do not adsorb well on the SiO*-TiOZ waveguide surface, more accurate values of the refractive index and the thickness of a monolayer could be obtained by first coating the waveguide with a binding adlayer (F’) for these proteins, then monitoring their binding to this adlayer F’, and finally applying the double-layer model to the bound proteins (F’). An avidin-biotin-LC-BSA affinity reaction, interpreted by both the single- and the doubleadlayer model, is shown in Fig. 14. Avidin was first immobilized by adsorption. An intensive washing with PBS removed less than 5% of the adsorbed avidin molecules. In a blocking test with BSA (c = l-5 PM, not biotinylated) no unspecific binding was found. After these pretreatments the adsorbed avidin monolayer had the following parameters: +’ = 1.45, dFn = 5-l nm and I’ = 3.2 ng mmp2. Then a concentration of c = l-5 PM biotin-LC-BSA was flushed through the cuvette in the stopped-flow mode. Saturation was reached after 50 s. Evaluation with the single-adlayer model showed a slight decrease in the refractive index nF’, a thickness increase of AdFs = 2.4 nm and an increase in surface coverage of AI’ (biotin-LC-BSA) = l-25 ng mm-*. More information was obtained with the double-adlayer model. The calculated refractive index of the second adlayer F”, IZ~, = 1.41, the thickness dFs = 3-l nm and the surface coverage of I”’ = l-25 ng mm-* agreed reasonably well with the values obtained in our experiments (not shown) for an adsorbed BSA adlayer (r+ = l-405, dFv = 4.0 nm and I’ = 1.6 ng mrnp2 at a concentration Of C = 75 /AM). We also monitored sequences of affinity- and immunoreactions on the same waveguide. An example is shown in Fig. 15. The experiment was done in the stopped-flow mode. The physical adsorption of prot A was monitored; with a concentration of c = 12 PM, a surface coverage of I’ = O-8 ng mm-* of prot A was obtained. Only 10% of the adsorbed amount of prot A 144

Biosensors & Bioelectronics

was removed by washing. The remaining free sites on the surface were blocked with BSA (a solution of concentration c = 75 PM). The surface coverage increase of Ar’(BSA) = O-7 ng mm-* showed that in this case the blocking step with BSA was necessary. The resulting adlayer F’, consisting of a mixture of prot A and BSA, had the parameters nr’ = l-595, dFp = 1-l nm and r’ = 1.5 ng mm-*. For the first affinity reaction an h-IgG solution of concentration c = 33 PM was used; saturation was obtained. In the single-adlayer model (Fig. 15, left), the adlayer thickness increased from dFv = 1 nm to dFT = 5 nm, i.e., Ad,.(h-IgG) = 4 nm, and its refractive index decreased from nr’ = 1’595 to nr’ = 1.483. In the double-adlayer model (Fig. 15, right) the h-IgG adlayer F” had a refractive index of nF’ = 1.443 and a thickness of dF = 4.5 nm, values which agree well with the results obtained for monomolecular h-IgG adlayers directly adsorbed on a waveguide. Both models gave the same additional surface coverage of AI’(h-IgG) = I” = 2.7 ng mm-*. Precisely one h-IgG molecule is bound per prot A molecule. A subsequent washing with PBS removed only 10% of the surface coverage. The second step is the immunoreaction between anti-h-IgG and h-IgG immobilized on the surface by prot A. For a concentration of c = 667 nM of anti-h-IgG the following changes in adlayer parameters were obtained: in the single-adlayer model the refractive index r+ decreased to a value of nr, = 1.433, indicating that the adlayer F’ became less dense. The thickness dFp increased by Ad,(anti-h-IgG) = 11.5 nm and the surface coverage I’ by AI’(anti-h-IgG) = 5.3 ng mm-*. In the double-adlayer model, the combined antih-IgG-h-IgG adlayer F” had the parameters nF’ = 1.409, dFg = 19.9 nm and I” = 8-O ng mm-*. This means that the anti-h-IgG adlayer has a thickness of Ad,(anti-h-IgG) = 15.4 nm. The surface coverage increase of 5.3 ng mm-* corresponds to two anti-h-IgG molecules bound to one h-IgG molecule. Washing with PBS removed practically no molecules. The third step is the affinity reaction between prot A, at concentration c = 2.4 PM, and the immunoglobulins on the surface. Prot A is known to bind the F, parts of the immunoglobulins. For this binding we observed a thickness increase of 2 nm and a surface coverage increase of 1 ng mm-*. Washing removed then about 15% of the prot A molecules.

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145

Biosensors & Bioelectronics

Ph. M. Nellen & W. Lukost L

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146

Integrated grating couplers

Biosensors & Bioelectronics

This means that 0.67 prot A molecules bind to one anti-h-IgG molecule. The anti-h-IgG molecules can be expected to be bound with their Fab part to the h-IgG molecules, thus exposing their F, parts to the approaching prot A molecules. Figure 15 shows that during binding reactions refractive indices )2F, or nF are not constant, thus indicating structural changes in the protein adlayers. This illustrates that the simultaneous determination of thickness and refractive index as functions of time is an advantageous feature of our measurement and evaluation method, whereas other optical methods have to assume a constant value for the refractive index in order to be able to determine the thickness and surface coverage of adsorbed or bound protein adlayers.

from the measured effective refractive indices either from the single-adlayer model (&‘, & and I”) or the double-adlayer model (+, &+ and I”). The drift effect, i.e. a rather disturbing persistent increase of the effective refractive indices of the guided modes when the SiOZ-TiOZ waveguides are exposed to aqueous buffer solution, can be greatly reduced by using compact, less microporous waveguides of higher refractive index nF = 1.86 as compared with nF = 1.75-1.79. Further, these compact waveguides, fired at temperatures of T = 800-900°C (instead of 5OO”C), are practically insensitive to changes in salt concentration.

REFERENCES 9. CONCLUSIONS We have demonstrated the potential of integrated optical input grating couplers for real-time monitoring of adsorption processes, and of affinityand immunoreactions. Not only the thicknesses but also the refractive indices (and the surface coverages) of the adlayers formed by adsorption or binding can be determined as functions of time. Results for protein adsorption, obtained with the multi-wavelengths operated input grating instrument by quasi-simultaneous coupler measurements at wavelengths A1 = 632.8 nm and A2 = 514.5 nm, agreed well in thicknesses and surface coverages. For low surface coverages Y/I&,, C 0.3, however, the assumption of an isotropic protein adlayer leads to unrealistically small thicknesses &? (compared with the sizes of the protein molecules) and too high values of the refractive indices nF’. First results indicate that evaluations which take into account an anisotropy of the adlayers are very promising. Since the anisotropy is caused by preferential orientation of the nonspherically symmetrical molecules, information about protein orientation on the surface can be obtained. Affinity reactions between preadsorbed receptor molecules (e.g. avidin) and ligands (e.g. biotinylated BSA or prot A) in solution could be detected at sub-nanomolar concentrations in a detection time of less than 15 min. For the adsorbed and bound molecules refractive index, thickness and surface coverage were calculated

Clerc, D. & Lukosz W. (1992). Integrated optical output-grating-coupler as refractometer and (bio-)chemical sensor. 1st Europ. Conf. Optical Chemical Sensors and Biosensors, Graz, April. Also: Sens. Actuat. B, 11, 461-5. Lukosz, W. (1992). Integrated optical and surface plasmon sensors for direct affinity sensing: influence of an anisotropy of adsorbed or bound protein adlayers. Biosensors ‘92 conf., Geneva, May (to be submitted to Biosens. Bioelectronics). Lukosz, W. & Stamm, Ch. (1991). Integrated optical difference interferometer as relative humidity sensor and refractometer. Sens. Actuat. A, 25, 185-8.

Lukosz, W., Clerc, D. & Nellen, Ph. M. (1991). Input and output grating couplers as integrated optical biosensors. Sens. Actuat. A. 25-7, 181-4. Nellen, Ph.M. (1992). PhD thesis No. 9871. ETH, Zurich. Nellen, Ph.M. & Lukosz, W. (1991). Model experiments with integrated optical input grating couplers as direct immunosensors. Biosens. Bioelectron., 6, 517-25.

Sober, H.A. (ed.) (1976). Handbook of Biochemistry and Molecular Biology, Vol. 2, Proteins. CRC Press, Cleveland, Ohio. 3rd edn., ed. G.D. Fasman . Stamm, Ch. & Lukosz, W. (1992). Integrated optical difference interferometer as refractometer and chemical sensor. 1st Europ. Conf. Optical Chemical Sensors and Biosensors, Graz, April. Also: Sem. Actuat. B, 11, 177-81. Wilchek, M. & Bayer, E.A. (1990). Avidin-biotin technology. In: Methods in Enzymology, Vol. 184, eds. J.N. Abelson and M.I. Simon. Academic Press Inc., New York. 147