Integrated optical difference interferometer as refractometer and chemical sensor

Integrated optical difference interferometer as refractometer and chemical sensor

117 Sensors and Actuators B, II (1993) 177-181 Integrated optical difference interferometer as refractometer and chemical sensor Ch. Stamm and W. Lu...

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Sensors and Actuators B, II (1993) 177-181

Integrated optical difference interferometer as refractometer and chemical sensor Ch. Stamm and W. Lukosz Optics Laboratory, Swiss Federal Institute of Technology, 8093 Ziirich (Switzerland)

Abstract We have successfullyused an integrated optical (IO) difference(or polarimetric)interferometer( 1)as a refractometer for liquid samples, (2) for monitoring protein adsorption, and (3) as an affinity sensor. The affinity reaction between avidin adsorbed on the waveguide surface and biotinylated bovine serum albumin is investigated. The simplicity of the IO difference interferometer is advantageous; only a planar waveguide is required. We use dipcoated SiO*-TiO* waveguides (prepared by a sol-gel process) on silicon wafers with SiOz buffer layers as substrates; depending on the firing temperature, the M-200 nm thick waveguiding films are microporous or compact, the latter showing superior stability in sensor applications.


1. Introduction Recently, we introduced a novel integrated optical (IO) difference interferometer; we demonstrated the

sensor principle by using it as a relative humidity sensor [l]. The object of the present paper is to demonstrate the successful use of the difference interferometer (1) as a refractometer, (2) in monitoring protein adsorption, and (3) as an affinity sensor. Attractive features of this IO difference interferometer are as follows: the actual sensor element is just a planar waveguide; no microstructuring is required for its fabrication, in contrast to other well-known IO interferometers such as MachZehnder interferometers. If very thin waveguides of high refractive index are used (such as the 140-200 nm thick SiOz-TiOz waveguides of refractive index about nF = 1.8 described below), the interferometer has a high sensitivity both as a refractometer and as an affinity sensor.

2. The IO difference interferometer

A schematic of the difference interferometer is shown in Fig. 1. Polarized light is endcoupled into a planar waveguide so that the TE,, and the TM, modes are coherently excited. The two modes propagate on a common path and interact over a length L with the sample, which interaction causes time-dependent changes A&%(t) and A&,+,(t) of their effective indices. Therefore, a time-dependent phase difference between the TE, and the TM, modes A&) = [email protected]&) - AC+&)


= [email protected]/n)



Afi(t) = ANT&) -AN&r)


occurs ct the end of the waveguide. This phase difference [email protected](t) is measured as a function of time t as described in more detail in ref. 1. The four photodetectors’D,-D, measure the intensities r,, Jt) =&{l +cos[A&t) +6,,]}


and I3,4(t) = &{l + sin[A&t) t 6,,]}


where &, agd To are proportional to the incoupled power and @ais a constant phase difference. The phase difference [email protected](t) is determined as follows: from the relation A&f) + A& = arctan{([Z,(0 t 12(t)l[Z3(t) - Utll)

/WI(4 - Mtm 0) + ml) >


and from the signs 0, [Z,(t) -Z2(t)] or [Z3(t)-&(t)], a phase value [email protected](t) + Q, lying in the interval --II to t n is calculated. To this calculated phase, the value 2nm is added, where the integer m is unambhuously chosen so that the time-dependent function [email protected](tj) is continuous at successive sampling points $ = j At. The sampling interval At has to be chosen accordingly short. Our system permits the choice of any sampling interval At > At,, = 80 ps. Laser power fluctuations and changes of the attenuation of the guided modes by interaction wit_hthe sample do not influence the measurement of [email protected](t). Therefore, [email protected](t) can-be determined with an experimental resolution of AOmi,62n/lOOO.

@ 1993 ~

Elsevier Sequoia. All rights reserved


Fig. 1. Schematic of the difference interferometer. (a) Cross-sectional view: laser, 10mW He-Ne laser (A= 632.8nm); L, interaction length; WG, waveguide;BS, beam splitter; I,, cylindricallens; I, cylindrical or sperical lens; 1/2, half-wave plate; 1/4, quarter-wave plate; W, and Wl, Wollaston prisms; Di, photodetectorsj = l-4; ADC, 16 bit analog-to-digitalconverter; PC, personal computer. (b) Detailed cross-sectional view of wavegnide and flowcell; S, substrate(Si); BL, buffer layer (3.6 pm SiO,); F, waveguiding film (140-200 nm SiO,-TiO,); PL, protection layer (2.0 pm SiO,), C, sample in flow cell or cuvette Cu made of PMMA (length Lx = 19mm, width

L, = 10mm, depth h = 0.5mm, and volume V = 95 pl). From eqn. (1) it follows that for an interaction length L = 12 mm, for example, effective index changes A??,,+ = 5 x lo-’ can be resolved at a wavelength 1 = 632.8 nm.

3. Waveguides For the difference interferometer we used SiOz-TiOz waveguiding films fabricated by dipcoating (sol-gel process). The substrates were silicon wafers (of 500 pm standard thickness) with a thermally grown SiO, buffer layer about 3.6 pm thick. The use of such Si/SiOz substrates has two main advantages (as compared with glass substrates): (1) The Si/SiO* substrates (covered with the SiOl buffer layer and the SiOz-Ti02 waveguiding fihn) can be easily scribed with a diamond and cleaved along a crystal plane. This procedure yields endfaces of good optical quality, which are required for in- and outcoupling. (2) The maximum processing temperatures with glass substrates are limited to about T = 550-650 “C; with Si/SiO* substrates firing temperatures up to T = 850 “C were used. Two types of SiOz-TiO* waveguides were fabricated: (A) films fired at T = 500 “C for 1 h; (B) fihns which after the first heat treatment were additionally fired at T = 850 “C for 1 h. As described in more detail below, the waveguides (A) are microporous; while waveguides (B) are compact, the remaining microporosity being estimated to be less than a few percent. The surfaces of the waveguides were covered (except for a window of length L, where the, sample interacts with the waveguide) with an evaporated SiO, protection

layer. This layer prevents any perturbation of the modes by the flow cell, which is pressed against the protection layer (see Fig. l(b)). The evaporated SiOz layers are not ideal protective layers, since they are microporous and pervious to both liquid water and vapour. The ends of the waveguide covered by the protection layer, but not by the flow cell, interact with ambient air. Therefore the fluctuations in relative humidity (to which the SiO,-TiO, waveguides are very sensitive) Jimit the stability of the interferometer’s response [email protected](t). Thus its resolution for effective index changes is presently only AI?,,,,, = 1 x 10e6, i.e., one order of magnitude worse than expected.

4. The difference interferometeras a refractometer The difference interferometer responds to changes Ann, of the refractive index n, of liquid samples C. The changes of the effective index difference N are approximately given by Afi =




Figure 2 shows the calculated sensitivity a%/&, of the difference interferometer as a refractometer for refractive index changes of aqueous solutions versus waveguide thickness dF. With calculated values of a$/&, r -0.1, a difference interferometer with an instrumental resolution of Afi,,,i, = 5 x lo-* or 1 x 10m6, respectively, used as a refractometer, has a theoretical resolution of An, = 5 x lo-’ or 1 x IO-‘. Figure 2 also shows that the (compact) waveguide (B) with the higher refractive index nF is slightly more sensitive than the (microporous) waveguide (A) with the lower index nF.








dP (nm)


Fig. 2. Calculated sensitivities afi/& (-) and aB/an, (- - -) vs. waveguide thickness dF, which are related to the difference interferometer’s sensitivity as a refractometer and to the effect of (ad)sorption in poresor voids of the microporefilm F. Parameters: ns(Si0,) = 1.46; n,(H,O) = 1.33; 1 = 632.8nm. (A) Microporous waveguide with nF = 1.76; (B) compact waveguide with RF= 1.86.

Figure 3 shows experimental results, obtained with a microporous waveguide (A), for two different alcohols, methanol with n, = 1.327 and ethanol with nc = 1.359. Figure 4 shows the response of the difference interferometer with waveguides (A) and (B) for aqueous sucrose solutions of different concentrations. An evaluation of this experiment with a waveguide (B), presented in Fig. 5, clearly shows the linear$y between the changes of measured phase difference [email protected](f)and the concentration changes AC. With an interaction_ length L = 12 mm and a calculated sensitivity ZV/&rc = -0.106, we find for the concentration dependence of the refractive index of the sucrose solution dn,/dc = 1.28 x 10e4 l/g, in agreement with our measurements with a commercially available Abbe refractometer. Figure 6 compares the responses of microporous (A) and compact (B) Si02-TiOz waveguides to an increase in sucrose concentration. In the microporous waveguide (A) the response to the A+ change is swamped by a

!z lj

-’ -2 -3 10 time




Fig. 4. DitTerence interferometer used as a refractometer for aqueous sucrose solutions. (Top) microporous waveguide (A) with dF= 196nm; (bottom) cornet waveguide (B) with dF= 144nm. The phase difference [email protected](r) vs. time t is shown. Sucrose concentrations are c, = 0 (pure water), c, = 0.4, c, = 2.0 and C~= 10.0 g/l; flow rate 1.1 ml/min.



z1 -




o-1 -2-\









AC s~cmse (g /liter) Fig. 5. Experimental sensitivity of diierence interferometer used as a refractometer for sucrose solutions. Measured phase difference [email protected] change ACin sucrose concentration c. Waveguide type (B), dF = I44 nm.

1.2 0.8 r (u 0.4 1% 0 IizTlL 1



time (minutes)



time (minutes)

Fig. 3. Difference interferometer used as a refractometer for alcohols. The phase diierence A&r) vs. time f is shown when (left) methanol in the flow cell is displaced by ethanol and (right) ethanol is replaced by methanol again. The arrows (7, 1) indicate the beginning of a pumping period of 135 s in which 1 ml of the new sample is pumped through the cuvette. Microporous waveguide type (A) with dF = 176 nm.


-0.4 -0.8 0


time (minutes)



time (minutes)

Fig. 6. Response of difference interferometer with (left) microporous waveguide (A) and (right) compact waveguide (B) to the displacement of pure water by sucr_ose solution of concentration of c = 2 g/l. The phase difference A(t) vs. time t is shown. Same waveguides as in Fig. 4.


stronger effect of opposite sign, which we interpret as the exchange of water in the pores by sucrose solution. The refractive index nF of the waveguiding film F increases by An, = (1 - q) An,, where q is the packing density, (1 -4) the relative volume of the pores, and An, the refractive index change of the medium in the pores. This variation hnF in turn induces a change Ai = (&‘?/&,) An, in the effective index difference. The experiments prove convincingly the superiority of compact SiO,-TiO, waveguides (B) in refractometry. Affinity sensors can also be expected to benefit from the increased stability of the compact waveguides (B). We also plan to use the difference interferometer for gas sensing. The waveguides have to be coated with adlayers which selectively absorb certain analyte molecules. Such coatings, for example for hydrocarbons, are known from the literature [3]. A coating thickness of a few microns is sticient; essential for the sensor effect is the part of the coating adjacent to the waveguiding film F into which the evanescent fields of the guided modes penetrate. The sensor response to the refractive index change is induced by the absorption of the analyte molecules. For a first test of this gas-sensing principle, we used an evaporated porous SiOz adlayer about 2 pm thick to monitor changes in the relative humidity of ambient air.

Fig. 7. The calculatedsensitivities&f/ad,. of two diierent waveand (B) vs. waveguidethicknessdp show the difference interferometer’sensitivities s for the adsorptionof proteinsand as an aflinitysensor.Assumed refractive index of adsorbed adlayer nF. = I .48 (avidin). Parameters: waveguide (A) with np = 1.76 and guides (A)

waveguide (B) with nF = 1.86; nc = 1.33 for water or PBS; n, = 1.46; I = 632.8 nm.

with M, = 67 000 Daltons. 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 8 shows results for the adsorption of avidin in an experiment where the concentration c avidin in PBS was increased in steps. The flow rate was constant at 62 @/min. From eqn_s. (1) and (7), we find that the phase difference [email protected] is proportional to the adlayer thickness

5. Protein adsorption and affinity sensing


We used the difference interferometer to monitor the adsorption of a protein (avidin) on the waveguide surface and the afEn,ity reaction between the adsorbed receptors (avidin) and the ligand molecules (biotinylated bovine serum albumin (BSA)) in the sample. The adsorption or binding of molecules on the waveguide surface induces-a change in the effective refractive index difference N of

[email protected] = (L/&Vi$d,.)

AI? = (&/ad,,)




Introducing the surface coverage r(t) = (dn/d[c]) -‘[+ - n,]d,,(t)



where the adsorbed adlayer F’is modelled as a homogeneous isotropic layer of refractive index nE, and thickness dF. The corresponding sensitivities aN/ad,. versus waveguide thickness dF for waveguides of types (A) and (B) are shown in Fig. 7; they were calculated assuming a value nF, = 1.48 for the avidin adlayer F’ [2]. In the experiments we used avidin of molecular weight M, E 67 000 Daltons (Pierce, catalogue no. 21121) and biotin-LC-BSA with M, = 68 000 Daltons (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)






time (minutes) Fig. 8. Adsorption of avidin. The measured phase difference [email protected](t)vs. time I is shown. At times indicated by arrows (1) the avidm concentration is i&eased from c =0 (pure PBS) to c = 320 ng/ml and then each time by a factor five to finally c = I mg/ml. Constant flow rate of 62 pl/min. Waveguide type(B) with dF = 144 nm.


where dn/d[c] = 0.18 ml/g is the concentration dependence of the refractive index of aqueous protein solutions, we obtain Ad(t)/2n = (L/n)(a~/ad,,)(dn/d[cl)[~~, -no] -‘F’(r) (10) For the waveguide (B) of thickness dF = 144 nm we obtain [email protected]/2n = -2.05dFt, where dFc is given in nm, and [email protected]= -2.46F’, where F’ is given in ng/mm2. For the adsorbed avidin monolayer we obtain dF8= 4.1 nm and F’= 3.45 ng/mm’. The optimum and presently achieved resolution limits of A~~i”/2n: = 1 x 10m3and 2 x 10m2,respectively, correspond to the resolution limits Fk, = O-4-8.1 pg/mm2 in surface coverage. Figure 9 shows results of an affinity reaction between avidin and biotin-LC-BSA. For this experiment, an avidin monolayer (with F’(avidin) = 3.3 ng/mm2) had been adsorbed on the waveguide from a solution of concentration c = 2.5 mg/ml. The minimum biotin-LC-BSA concentration c at which a strong and clear response was observed is c = 500 ng/rni= 7.5 nM. _



time (minutes)

6. Conclusions We have demonstrated that the difference interferometer can be successfully used as a refractometer, as a gas sensor (relative humidity changes), in the monitoring of protein adsorption (avidin), and in affinity sensing (avidin-biotin system). The use of Si/SiO, substrates (silicon wafers with SiO, buffer layers) instead of glass substrates has opened a route to fabricate more compact, less microporous waveguiding films by dipeoating (sol-gel process) and thus to improve the stability of the IO sensors for refractometry and affinity sensing.


sAIlo*rLc-uy =Q4m

Ii V

Effects limiting the resolution are small refractive index changes Aa, in the sample, caused by temperature fluctuations and by spatial and temporal variations in total protein concentration in the sample. These disturbing effects could be eliminated by choosing waveguides with a certain thickness dF = a,, at which [afi/&],, =dF = 0. However, waveguides of this thickness 2, do not have the maximum sensitivity dN/ad,. (see Fig. 7).



Fig. 9. Affinity reaction betwee_navidin and biotin-LCBSA. The measured phase difference [email protected]$)vs. time r is shown, (I), BSA concentration c = 1 pgglml;(II), biotin-LC-BSA c = 500ng/ml plus BSA c = 500ng/ml; (III), BSA c = 5 )rglml (IV), biotinLC-BSA c = 2.5 ug/ml plus BSA c =2.5 pg/ml; (V), BSA c = 25 &ml; (VI), biotin-LC-BSA c = 12.5ug/ml plus BSA c = 12.5ug/ml. Same waveguide and flow rate as in Fig. 8.

We thank Dr P. Miiller of Rnecht Optik, Stein am Rhein, Switzerland, for fabricating the Si02 protection layers on our waveguides, E. Hausammann for mechanical engineering work and G. Natterer for his support in electronics.

References 1 W. Lukosz and Ch. Stamm, Integrated optical interferometer as relative humidity sensor and differential refractometer, Sensors and Actuators A, 25-27 (1991) 185-188. 2 D. Clerc and W. Lukosz, Integrated optical grating coupler as refractometer and (bio-)chemict$ sensor, Sensors and Acfwtom B, 11 (1992) 46-465. 3 G. Gauglitz and J. Ingenhoff, Integrated optical sensors for halogenated and non-halogenated compounds, Sensors and Actuafors B, II (1992) 207-212,