Integrated optical output grating coupler as refractometer and (bio-)chemical sensor

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Sensors and ActuatorsB, 1I ( 1993) 461-455

Integrated optical output grating coupler as refractometer and (bio-) chemical sensor D. Clerc and W. Lukosz Optics Laboratory, Swiss Federal Institute of Technology, 8093 Ziirich (Switzerland)

Abstract We report on a newly built output grating coupler instrument. We have used it successfully as a refractometer for liquid samples, to monitor the adsorption of proteins (avidin) on the waveguide surface, to determine the refractive index and thickness of the adsorbing (mono)laycrs with high precision, and to investigate the afhnity reaction between adsorbed avidin molecules as receptors and biotinylated proteins (bovine serum albumin) as the analyte in the sample, as a model of a direct affinity biosensor with a low detection lhnit.

1. Introductioll

2. output grating coupler instmment

Previously, we demonstrated the successful use of output grating couplers on planar waveguides as integrated optical sensors [ 1,2]. In the present paper, we

The principle of the output grating coupler sensor is as follows [l-3]: the guided TE, and TM,, modes in a planar waveguide are excited by endfire coupling and are coupled out by a surface relief grating. From the measured outcoupling angles tl, the effective refractive indices N of the guided modes are determined with the relation

report on a newly built very precise output grating coupler instrument and its use as: (1) a refractometer for liquid samples; (2) a monitor of the adsorption of proteins (avidin), including a precise determination of the refractive index and thickness of the adsorbing (mono)layers as a function of time; and (3) an affinity sensor (avidin-biotin binding). The primary sensor response, as in all integrated optical sensors, is the effective refractive-index changes AN,,,(r) and ANTMO(t)of the guided modes, which are induced by: (1) changes in refractive index nc of the liquid sample covering the waveguide; (2) adsorption of molecules on the waveguiding surface; and (3) binding of analyte (ligand) molecules to corresponding receptor molecules immobilii on the waveguide surface. The new instrument permits the time-dependent changes ANT%(f) and ANTMO(t)of the effective refractive indices of the guided TE,, and TM, modes in a planar waveguide to be measured during the actual sensor experiment with high precision and resolution, and the absolute values NTEoand NTMOof the effective refractive indices to be measured before the actual start of the experiment. We stress that the latter information is needed to characterize the individual waveguide precisely and to calculate its sensitivities, which are required for the theoretical interpretation of the sensor experiments. An attractive feature of the fully optoelectronic instrument is its fast temporal resolution (adjustable from 80 us to 30 s).

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where L is the vacuum wavelength, A the grating period and I = 1 the diffraction order. The angles o! are measured with position-sensitive photodetectors (PSDs). The new instrument is schematically shown in Fig. 1. It works in two different measuring modes: (1) The absolute values of the effective refractive indices NTEoand NTMOare sequentially measured with two lasers A and B, which excite counter-propagating modes. The resolution is ANni, N 2 x 10V5. A set of such measurements can be repeated every 5 s. In more detail, the measurement works as follows: both lasers A and B are linearly polarized under 45” with respect to the normal on the waveguide. Not shown in Fig. l(a) are the computer-controlled electromechanical shutters in front of both lasers. Using these shutters, we f&t excite the TEO and TM, modes propagating in the sx direction and subsequently the modes propagating in the --x direction. Also not shown in Fig. l(a) is a moving slit just above the long PSD, which permits the positions uT~ and uTMoto be measured sequentially. Instead of the moving slit, a faster electro-optical control of the polarization states of lasers A and B could be used.

@ 1993 - Else&r Sequoia.AU rights reserved

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mean values of AN,,(t) and ANTMa(t)in a time interval of duration Mz are formed. For M = 10 or M = 100 this averaging, and the storage of the data on the hard disk of a PC, takes about 50 or lOOms, respectively. The effective index changes ANTEo(f)and ANTMO(t)can be recorded at arbitary sampling intervals At ( 2 50 ms). The opto-electronic instrument itself has a resolution ANti, N 5 x lo-‘. However, fluctuations in the measured effective refractive indices AN(f) of the microporous SiO,-TiO, waveguides in contact with liquid samples limit the resolution to about AN,,,i, Y (l-2) x 10-h.

3. Refractometer

PSD

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platform (b) Fig. 1. Schematic of the output grating coupler instrument. (a) Measurement of the (absolute) values of the effective refractive indices NTeoand NTMousing two He-Ne lasers A and B linearly polarized at 45”with respect to the normal on the planar waveguide. S, substrate; F, waveguidingfilm; C, sample in tlow cell Cu made of PMMA (of depth 90 pm and volume 4 PI) covering the waveguidein the grating region; l,, cylindricallenses (focal length f = I. 1mm);L, spherical lens of focal length f = 300mm; ADC, 16 bit analog-to-digital converter; PC, computer; PSD, positionsensitive photodetector measuring (sequentially) the positions u, and ua of the foci of the outcoupled beams of the two counterpropagating modes. The effective indices arc calculated with the -M/A, which derives from the relation N u n,(u,-u,)/(Y) outcouplmg eqn. (l), were i = 633nm is the wavelength, A the grating period (l/A = 2400mm-‘) and I = I the diffraction order. (b) Detailed view of the focal plane. Mounted on a common platform are one long PSD (UDT type 34/SL76, 2.5 x 76mn-13 for the absolutemeasurementsand two shorterPSDs(UDT type 34/SL5-2,1 x 5 mm*) for the simultaneousmeasurements of both time-dependent effective refractive-index changes ANW(r) II Au’b(t)lf and ANTMO(f) = AuTM”(t)lf:Translation of the platform in the direction perpendicular to the u direction permits switching between the two measuring modes.

(2) The changes AN%(t) and ANTMO(t)of the effective refractive indices are measured simulzaneously as functions of time t with submillisecond temporal resolution. For these measurements the TE, and TM, modes are excited by a single laser A, which is linearly polarized under 45” with respect to the normal on the planar waveguide. The signals from the PSDs are sampled every T = 80 ps. If a submillisicond response time is not required, we use an oversampling method. By averaging over M (where M = 10, 10’ or 103) position data, the

When the instrument is used as a refractometer, the liquid sample continuously flows through the cell at a constant flow rate. The working principle of the instrument is as follows: (1) Before the actual experiment, at time t Q 0. a reference liquid of known refractive index n, flows through the cell and the absolute effective refractive indices NTb(0) and NTMO(0)are measured. From the mode-guiding condition of a single-layer waveguide, the thickness dF and the refractive index nF of the individual waveguiding film F are determined. (2) At times t r 0, with the sample flowing through the cell, the effective refractive-index changes AN=,,+(t) and A&,(t) are measured. From the values Nm(t) = N&f) +ANN,,(t) and N&9 = NTMo(0)+ ANTMO(f),the refractive index q(t) of the sample and the refractive index n,(t) of the waveguiding ti F are determined as functions of time t by rigorous solution of the waveguiding condition (where the refractive index ns of the substrate S is known and the value of the thickness $ was obtained from the waveguide characterization procedure (1). That nF(r) is not strictly constant is a consequence of the microporosity of the SiO,-TiO, waveguides used. The liquid in the micropores in the iihn F is slowly exchanged by convection and/or diffusion. The refractive-index change dnF is approximately given by A+ = (1 - q) Ann,, where q is the packing density, (1 - q) the relative volume of pores or voids, and &p the change in refractive index of the liquid in the pores. From such measurements of Ann,, the microporosity of the waveguides was determined to be typically (1 -4) = 0.05-0.15. Due to the finite lengths of the PSDs, the maximum effective index changes that can be measured are AN,, N 0.015. This corresponds to a maximum refractive-index difference between sample and reference liquid of Ar1~~0.1. Figures 2 and 3 show results of refractometry with alcohols. Methanol is chosen as the reference liquid.

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The refractive indices of ethanol and an 1:l mixture of methanol and ethanol are determined. The flow rate was 6 d/s. Figure 4 shows results of refractometry with aqueous sucrose solutions with concentrations c = 0.04, 0.2 and 1%. The reference liquid was pure water. The flow rates were 6 M/s. The Ant values determined were perfectly linear with concentration c; we obtained dn/dc = O.l3Oml/g (in excellent agreement with measurements using a commercially available Abbe refractometer). The resolution presently obtained was [L\ncltin N 5 x 10e5, corresponding to a sucrose concentration of 0.04%; it is limited by temperature fluctuations AT N +0.2 “C. The maximum sucrose concentration that can be measured (with water as reference liquid) is c_ = 40%, corresponding to Ann,N 0.1, The results show that thanks to the simultaneous measurements of AN,(f) and AA&,,(I) and the subsequent evaluation of both nc(f) and n,(t), the instrument can be successfully used as a refractometer even with microporous waveguides. However, we expect improvements in resolution and accuracy by the use of less microporous compact waveguiding I&S, such as dipcoated SiO*-TiO* fihns on silica substrates which can be fired at higher temperatures. However, a remaining small microporosity is probably unavoidable. Therefore the method described abdve remains valuable and even indispensable for very precise and accurate refractometric measurements.

time (minutes) Fig. 2. Output grating coupler used as a refractometer for alcohols. The measured effective refractive-index changes AA&(f) and A&.&), and the determined refractive indices n&r) of the sample C and rip(t))of the microporous waveguiding film F for repeated exchange of methanol and ethanol are shown vs. time 1. SiO,-TiOz waveguide parameters: dF = 164nm and ns = 1.47.

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time (minutes) Fig. 3. Output grating coupler used as refractometer for alcohols. Determined refractive index n,(t) of sample vs. time t for methanol, ethanol and 1:I mixture of both alcohols at a temperature T = (24.5 f 0.2)“C.

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time (minutes) Fig. 4. Refractometer for aqueous sucrose solutions. Refractive indices n&r) of sample and n*(t) of waveguidingtilm vs. time t at a temperature T=(24.5 kO.2) “C. Sucrose concentrations were e, = 0.04%, c, = 0.2% and c, = I% (co= pure water). SiO,-TiO, waveguideparameters: dF = 194nm and ns = 1.47.

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A review of other types of integrated optical refractometers is given by Ulrich [4]. An advantageous feature of the SiO,-TiO, waveguides used by us is their high sensitivity aN/&. The reason is that the SiO*TiOt waveguides are much thinner than the waveguides normally employed in integrated optics, such as waveguides indiffused in glass. Because of the small thickness of the SiOz-TiOz waveguides, the total volume of micropores is small. Therefore the outdiffision of the previous sample and mixing with the new sample to be measured can be neglected if the refractometer is used in the constant-flow mode.

solution; i.e., the values of N%(O) and NTMO(0)are measured, and the refractive index nF and the thickness dF of the individual waveguiding film F are determined from the mode-guiding condition of the single-layer waveguide. (2) From the measured effective index changes AN(t), i.e., from NT&(t) = N%(O) +ANTEo(t) and NTMO(t)E NTMO(0)+AN,,,(t), the thickness dF,(t) and refractive index +(t) of the total adlayer F’ (comprising both the adsorbed and the bound molecules) are determined by rigorously solving the mode-guiding condition of the two-layer waveguide consisting of the films F and F’between the substrate S and the sample C. The surface coverage r(t) is calculated from the relation

4. Protein adsorption and atlhity sensing l?(t) = (dn/d[c])-‘[n&) The instrument was used to monitor (1) the adsorption of proteins on the waveguide surface and (2) affinity reactions as functions of time. In affinity sensing, the receptors are adsorbed or immobilized on the waveguide; they capture the ligand molecules contained in the sample. In such measurements we proceeded as follows: (1) Before the actual experiment at time t < 0, the waveguide characterization takes place in the buffer

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time (minutes) Fig. 5. Adsorption of avidin. The surface coverage r’, the refractive index nFsand the thickness & of the (monomolecular) avidin adlayer F’ on the waveguide surface are shown vs. time t. Avidin concentration c = 0.5 mg/ml in PBS solution. Sampling time At =O.l s. Flow rate was 36 @/min. SiO,-TiO, waveguide parameters: dF = 194 nm, nF = 1.75 and ns = 1.47.

Fig. 6. Affinity reaction between avidin and biot&LC-BSA. Shown vs. time t are the surface coverage AT’(r), which was determined from the measured effective refractive-index changes AN&t) and ANTMD(r).(I), BSA concentration c = 440 ng/ml; (II), biotin-LC-BSA c = 220 ng/ml plus BSA c = 220 ng/ml; (III), BSA c = 1.3 pg/ml; (IV), biotin-LC-BSA c = 660 q/ml plus BSA c =66Ong/ml; (V), BSA c =4 w/ml. Sampling time AI =O.S s. During the whole experiment the flow rate was 36 pl/min. The same SiO,-TiO, waveguide as in Fig. 5 was used.

where dn/d[c] = 0.188 ml/g is the refractive-index increment of proteins in aqueous solutions with concentration [c] (see [ 1,2]). In the following example, we investigated the affinity reaction between avidin and biotinylated protein molecules. We used avidin of molecular weight M, N 67 000 Da (Pierce, catalogue no. 21121) and biotinLC-BSA with M, = 68 000 Da (Pierce, catalogue no. 29130). Bovine serum albumin (BSA) was labelled via longchain (LC) spacers with about nine biotin molecules. As a test for unspecific adsorption, we used unlabelled BSA (Fluka, no. 05470) with A4, = 67 000 Da. The biochemicals were dissolved in an aqueous saline phosphate buffer solution (PBS) at pH 7.4 at temperature T = 23 “C; the refractive index of PBS is n, = 1.333. Figure 5 shows the results for the adsorption of avidin from a solution of concentration c = 0.5 mg/ml in PBS. The saturation of r’(t) shows that a monomolecular avidin adlayer is formed; its parameters are dF, = 3.9 mu, nF. = 1.48 and Y = 3.0 ng/mm*. The experimental resolution in surface coverage is Armin N 3-6 pg/mm2. Figure 6 shows the affinity reaction between an adsorbed avidin layer and biotin-LC-BSA. From the effective index changes AN%(I) and A&,,,(t), and the determined additional surface coverage AT’(t) due to the bound biotin-BSA molecules, we conclude: (1) a biotin-BSA concentration of 220 ng/ml G 3.2 nM produces a strong and clear response. Much lower biotin-BSA concentrations should be detectable. (2) Non-labelled BSA even at much higher concentrations shows practically no effect, i.e., it is neither bound nor unspecifically adsorbed. In the experiment a constant flow rate of 36 l.d/min was used; the depth of the cuvette

was only 90 pm. Therefore the increase in surface coverage, d(AT’)/d& is neither limited by diffusion nor by depletion of the sample, but is reaction controlled. With such experiments we plan to determine the forward-reaction rate constant of the affinity reaction between adsorbed avidin and biotinylated molecules.

5. Conclusions We have successfully used a newly built output grating coupler instrument as an integrated optical refractometer. A satisfactory resolution was achieved despite the microporosity of the SiO,-TiO, waveguides. As a model for affinity sensors, binding of biotinylated bovine serum albumin molecules in the sample to a monolayer of avidin adsorbed on the waveguide surface was investigated. With more compact waveguides further improvements in accuracy and resolution can be expected, not only in refractometry but also in affinityreaction-based biosensing.

References 1 W. Lukosz, D. Clerc, Ph. M. Nellen, Ch. Stamm and P. Weiss, Output grating couplers on planar optical waveguides as direct immunosensors, Biosensors Bioeiecfron., 6 (1991) 227-232. 2 W. Lukosz, D. Clerc and Ph. M. Nellen, Input and output grating couplers as integrated optical biosensors, Sensors and Actuarors A, 25-27 (1991) 181-184. 3 W. Lukosz, Th. Brenner, V. Briguet, Ph. Nellen and P. Zeller, Output grating couplers on planar waveguides as integrated optical sensors, Proc SPIE, Vol. 1141, 1989, pp. 192-200. 4 R. Ulrich, Fiber-optic refractometry, Is? Europ. Conf. Optical Chemical Sensors and Biosensors, Graz, Austria, April 12-15, 1992