TIO S

CHEMICAL

ELSEVIER

Sensors and Actuators B 29 (1995) 37 50

Integrated optical chemical and direct biochemical sensors

W. Lukosz
Optics Laborato~3'. Swiss Federal Institute o[' Technoh)gy. 8093 Zurich. Switzerland

Abstract
An overview is given on work by the author's group on integrated optical (IO) sensors. The sensors make use of guided waves
or modes in optical waveguides, in particular of the orthogonally polarized TE0 and TM 0 modes in very thin planar waveguides
of high refractive index. The principle of all direct (No)chemical waveguide or IO sensors is as follows. A chemically selective
coating on the waveguide surface binds the analyte molecules contained in the gaseous or liquid sample. Thus, the refractive index
of the medium near the waveguide surface (more precisely, within the penetration depth Az of the evanescent field of the guided
wave) is changed. This effect in turn induces changes ANsE,, and ANTM,, of the effective refractive indices NTE,, and Nl.,a,' of the
guided modes. For example, in biochemical affinity sensors the chemically selective coating contains receptor molecules that
specifically or selectively bind certain ligands as analyte molecules; in particular, in immunosensors or immunoassays the receptors
are antibodies (or antigens, respectively) and the analyte molecules are the corresponding antigens (or antibodies). These 'direct'
affinity or immunosensors eliminate the use of (e.g., fluorescently) labelled reagents. Effective refractive-index changes AN can also
be induced by two other effects; namely by unspecific adsorption of molecules on the (uncoated) waveguide surface (or in pores
of a waveguiding film F itself if it is microporous) and by refractive-index changes Ant of the liquid sample. In the latter case the
IO sensors work as refractometers. The effective refractive indices N give the phase velocity vph = c/N of the guided modes, where
c is the velocity of light in vacuum. This means that the effective refractive-index changes AN can be measured by various optical
means. Consequently, a number of different types of 10 sensors can be used, in particular, grating couplers and interferometers.
In the paper, I report on our own work on IO sensors including: the discovery of the basic sensor effect with grating couplers as
sensors for relative-humidity changes, the theory of the sensor sensitivities, and experimental results obtained with three different
types of IO sensors developed in our laboratory, namely input grating couplers, output grating couplers and the difference or
polarimetric interferometer. The experiments have been performed with dip-coated SiO2 TiO 2 waveguiding films of refractive
index nt ~ 1.75 1.88, on glass, silica and silicon wafers with oxidized buffer layers as substrates. The sensors working as
refractometers are tested, for example, with glucose solutions of different concentrations. The adsorption of proteins (h-IgG, BSA,
protein A, avidin) is monitored in real time. Not only the surface concentration F, with a resolution of a few pg mm 2 but also
the thickness d F. and the refractive index n v. of the adsorbing (mono)layers are determined as functions of time. Also
immunoreactions (e.g., between h-IgG and anti-h-IgG, and between IgGs and protein A) and affinity, reactions (between avidin
and biotinylated proteins, such as biotin BSA) are observed in real time. The feasibility of IO immunosensors or affinity sensors
or immunoassays with sub-nanomolar detection limits is demonstrated.
Keywor&': Biochemical sensors; Chemical sensors; Integrated optical sensors

1. Introduction
In 1983, a new integrated optical (IO) sensor effect
was discovered in the a u t h o r ' s group when the response
of high refractive index S i O 2 - T i O 2 p l a n a r waveguides
only 100 1 5 0 n m thick to relative-humidity changes
was observed in i n p u t grating coupler experiments [1,2].
This paper gives a n overview of o u r s u b s e q u e n t work.

'~ Plenary paper.

0925-4005/95/$09.50 >) 1995 ElsevierScience S.A. All rights reserved
SSDI 0925-4005(95)01661 -E

The basic IO sensor effect is explained in terms of the

effective refractive-index changes A N of the guided
modes in Section 2. The theory of the optical sensor
sensitivities is discussed in Section 3, where it also
becomes a p p a r e n t why very thin waveguides of high
refractive index are required for IO sensors; waveguides
for optical c o m m u n i c a t i o n purposes, which have mode
diameters of a few micrometres, c o m p a r a b l e with the
core diameters of m o n o m o d e fibres, are n o t suitable. In
Section 4, the principles of IO chemical a n d direct
biochemical sensors are delineated. (Bio)chemistry has

38

W. Lukos: ' Sensors and Actuators B 29 (1995) 37 50

to guarantee the selectivity or specificity for the analyte,

while integrated optics can 'only' provide the high
sensitivity. The effective refractive-index changes AN
are translated into readily measurable physical quantities in different types of IO sensors described in Section
5. In Section 6, a few typical results obtained with input
and output grating couplers and with the difference
interferometer, in relative-humidity sensing, refractometry, real-time protein-adsorption studies and in model
experiments of affinity sensing and immunosensing are
shown. To keep this paper reasonably shorL only resuits obtained in our own group are discussed. Also the
very interesting subject of a comparison between IO
and other optical direct (bio)chemical sensors, such as
surface plasmon resonance sensors or thin-film interference sensors, is not included.

2. Physics of the 1 0 sensor effect

The guided waves or modes in planar optical waveguides are TE,, (transverse electric or s-polarized)
modes and TM,,, (transverse magnetic or p-polarized)
modes, where m = 0 , 1. . . . is the mode number. In
sensor applications, the effective refractive index N is
the most important physical quantity of the guided
modes. The (monochromatic) modes propagate down
the waveguide with the phase velocity vp, = c/N, where
c is the velocity of light in vacuum and N is the effective
refractive index of the mode; N depends on polarization
(TE or TM), mode number m, (vacuum) wavelength ~,
the properties of the waveguiding film F, i.e., its thickness d~/2 in units of 2 and its refractive index n~, and
on the refractive indices n s and no, respectively, of the
substrate S and of the medium C covering the waveguide (see Fig. 1). The field of a guided wave penetrates
as an evanescent wave a small distance A-c into the

medium C, which in sensor applications is the sample

covering the waveguide. More precisely, the evanescent
field decays exponentially proportional to e x p ( - =/Azc)
with distance : from the waveguide surface, where
A_c, _ (2/,2rr)[N 2 _ nc 2] l 2

(1)

is the penetration depth.

The basic IO sensor effect is caused by interaction of
the evanescent wave of the guided mode with the
sample. The evanescent field 'senses' changes in the
refractive-index distribution near the waveguide surface. Thus changes AN in the effective refractive indices
of the guided modes are induced. This is the basic or
primary IO sensor effect on which ell[ IO sensors are
based.
The effective refractive-index changes AN can be
induced by two different effects as follows:
(1) the formation of an adlayer F' of adsorbed or
bound molecules, which are transported by convection
or diffusion from the bulk of the gaseous or liquid
sample C to the waveguide surface. This adlayer is
modelled as a homogeneous layer F' of thickness d~.
and refractive index nv,.
(2) changes Ant of the refractive index nc of the
homogeneous (liquid) sample C covering the waveguide
surface.
Only in the case of microporous waveguides, a third
sensor effect (3) can occur, namely the adsorption or
desorption of molecules in pores of the waveguiding
film F itself, which primarily changes its refractive
index nv by AnF and in turn leads to an effective
refractive-index change AN. The field distribution inside
the waveguide F itself, and not the evanescent field in
C, is responsible for effect (3).
If all the effects (1)-(3) are simultaneously present,
the resulting effective refractive-index changes are

AN=

dV.+k(nc/~ A n t +

Any

(2)

How the derivatives or sensitivity constants (~?N/2dv.),

(#N/(?nc) and (~N/(?n~=) depend on the optical parame-

dF,t F' ~
de F

/"

Fig. 1. Basic 10 sensor effect. Changes AN of the effective refractive

index N of a 'guided mode are induced by changes of the refractive-index distribution n(z) in the vicinity of the waveguide surface, i.e.,
within the penetration depth Az c of the evanescent field in the sample
C. Sensor effect (1): molecules transported by convection or diffusion
adsorb on the surface forming an adlayer F' of thickness d v and
refractive index n~.. Sensor effect (2): homogeneous change Anc of
refractive index of (liquid) sample C.

ters of the waveguide F, substrate S and sample C is

discussed in Section 3. Effect (1) makes it possible to
monitor in real time the adsorption of molecules on the
waveguide surface or their desorption from the surface,
respectively. Effect (2) is the basis for the application of
IO sensors as differential refractometers. Effects (1) and
(2) are also the basis of (No)chemical sensors (see
Section 4). Effect (3) can be exploited in relative-humidity sensing and in gas sensing. In other cases, such as in
refractometry and in affinity sensing, effect (3) can be
rather disturbing and compact waveguides with very
low microporosity are preferable.
As expressed in Eq. (2), the effects (1) (3) contribute
additively to the effective refractive-index changes AN.
In order to differentiate between the two or three

39

W. Lukosz ,' Sensors and Actuator,s B 29 (1995) 37 50

different effects, it is therefore necessary to measure the

effective refractive-index changes of the corresponding
number of guided modes at the same wavelength 2. In
our work, we used very thin so-called monomode
waveguides in which only modes with mode number
m = 0, i.e., the TEo and TM 0 modes, can propagate.
Consequently we only measured the effective refractiveindex changes ANvE,~ and ANTM. of the TE0 and TMo
modes. This permits, for example, (provided that the
microporosity effect is negligible) to differentiate adsorption (or desorption, respectively) or, in (bio)
chemical sensing, binding of an analyte, from refractive-index changes Anc of the sample, caused, for example, by temperature variations. If the sample's
refractive-index changes An c are either negligible or are
precisely known, from the two measured effective refractive-index changes ANTE,, and ANTM,, two parameters of the adsorbed (or bound) adlayer F' can be
determined, namely its thickness d~-, and refractive index nv, provided that the adlayer F' is isotropic. A
possible anisotropy of protein adlayers F' is discussed
in Section 6.3.
The basic IO sensor effect explained above leads to
generic types of IO chemical and biochemical sensors
for the detection or quantification of various analytes;
this we describe in Section 4. In the next section we
discuss which types of waveguides are required in order
to attain IO or waveguide sensors with high optical
sensltl'vltles.

3. Optical sensor sensitivities and 'history' of the I 0

sensor effect

The optical sensitivity constants (3.N/~dF,), (~,N/~nc)

and (~N/~nv) introduced in Eq. (2) are the differential
changes in effective refractive index of a guided mode
for a small change in: (1) the thickness dF, of an
adsorbed or bound adlayer F'; (2) the refractive index
nc of the (liquid) sample; and (3) the refractive index nv
of the microporous waveguiding film F. We calculated
the optical sensitivity constants using two independent
methods.
The first method [3,4] starts from the exact modeguiding condition for a planar two-layer waveguide
consisting of the waveguiding film F (of thickness dv
and refractive index nv) with an adsorbed or bound
adlayer F' (of arbitrary thickness dr, and refractive
index nv,) sandwiched between the substrate S and the
sample C of refractive indices ns and nc, respectively,
and then considers the limit of very thin adlayers F'
with dr, << 2. The second method [5] is a perturbation
theoretical approach applied to a planar waveguide
with any transverse refractive-index distribution. Both
methods gave the same results for single-layer step-index waveguides.

However, the perturbational approach gives more

physical insight, in that the basic formula shows the
relation between the effective refractive-index changes
AN of the guided mode and the changes in refractive
index in the vicinity of the waveguide surface (within a
layer of the order of the penetration depth Az c) that
are 'probed' by the evanescent field of the guided mode.
For the TE modes, we found for A(N 2) ~ 2N AN,
+-J

+~

A(N2) = f Ag(z)[u(z)]2 dz ; f [u(z)]2

(3)

where u(z) is the transverse electric-field distribution of

the unperturbed guided mode as a function of the
coordinate z perpendicular to the planar waveguide,
Ae(z) = 2n(z) An(z) is the change in dielectric permittivity ~:(z) and An(z) the change in refractive-index distribution; for the corresponding formula for TM modes
see Ref. [5].
The main results are:
(i) The optical sensitivity constants depend on the
polarization (TE or TM) of the mode, the mode number m (m = 0, 1. . . . ), the waveguide thickness dv/Tt in
units of ,i, and on the refractive indices ns, nv, nv, and
H(-.

(ii) From Eq. (3) we conclude that a high field

strength of the 'probing' evanescent field (in the numerator of Eq. (3)) for a given power of the guided wave
(which is proportional to the denominator in Eq. (3)),
or, in other words, a strong confinement of the transverse field distribution of the guided wave yields high
sensitivity constants (~N/'cdv~,) and (~N/~?nc)
. . . . . . Both constants are inversely proportional to the so-called effective waveguide thickness ct~fr, which for TE waves is the
sum of the waveguide thickness dv and the penetration
depths Azc and Az s of the evanescent fields into sample
C and substrate S, i.e., d~jr- dv + Azc + Azs.
Therefore, to obtain high sensitivities ~N/(?dv, and
~N/'&lc, very thin waveguiding films F have to be used.
But for a mode to be guided in a planar waveguide, the
waveguiding film F must have a thickness dv somewhat
larger than the minimum or 'cut-off thickness
( dmF ~)

[,~ = I j . ( n F 2 - - nS2)

12

arctan( \ n("c J

2p

2 __

L(nF~-ns")J

1/2

;1
33

which depends on the mode number m = 0, 1. . . . and

on the polarization, with p = 0 and 1 for TE and TM
modes, respectively. From Eq. (4) we learn that waveguides with cut-off thicknesses (a?F)o~' of the TEo and
TMo modes much smaller than the wavelength 2 can
only be realized if the difference n v - n s between the
refractive indices of the waveguiding film F and the
substrate S is large, e.g., nv - ns >~0.3. The consequence
is that with very thin high-refractive-index films F on

40

W. Lukosz / Sensors and Actuators B 29 (1995) 3 7 - 5 0

glass or silica substrates high optical sensitivity constants (#N/~dv,) can be obtained. Such very thin waveguides were originally of no importance in the past,
because IO components were only of interest for optical
communication purposes and for fibre sensors, and
therefore, they had to be compatible with monomode
fibres. In order to achieve low coupling losses between
IO components and fibres, the waveguide thicknesses or
mode diameters have to be matched to the core diameter (typically about 5-10~tm) of monomode fibres.
(Planar) waveguides satisfying these requirements, such
as waveguides fabricated by diffusion processes just
below the surfaces of either glasses or electro-optic
crystals such as LiNbO3, have very low optical sensor
sensitivities; the sensitivity constant (?~N/~dv,) is one to
two orders lower than for very thin high-refractive-index films.
This dependence of the optical sensitivity constants
on waveguide parameters has some bearing on the
history of the basic IO sensor effect. This effect was
discovered, by serendipity, in our group because we
worked with only about 120-150rim thick planar
SiO2-TiO2 waveguides of refractive index nv ~ 1.75.
The original object of the work [6] was actually to find
out whether or not grating couplers could be fabricated
on dip-coated SiO2 TiO2 waveguides (produced by a
sol-gel process) on glass substrates by embossing at
room temperature a surface relief grating with submicron grating constant A into the gel films, which were
subsequently 'fired' at temperatures of about 500 C to
become hard amorphous films. Since sol-gel films
shrink considerably in depth (by a factor of typically
three to four) [7], the eventual success of this new
method to fabricate surface relief gratings with 1/A
= 1200-3600 lines per mm was not guaranteed from
the outset. Fortunately, the lateral submicrometre grating structure is preserved in the fabrication process; the
shrinkage in depth reduces the surface modulation embossed into the gel film, which is determined by the
surface relief modulation (typically about 100 150 nm)
of the commercially available diffraction grating used
as a die. But very shallow surface relief gratings (with a
modulation depth of several to a few tens of nanometres) are fortunately well suited as grating couplers,
having coupling lengths of the order of millimetres.
When these SiO2 TiO2 waveguides provided with
surface relief gratings were used for incoupling experiments with red He Ne laser light,, the unexpected and
surprising finding was that the intensity of the light
coupled into the waveguide at a constant angle of
incidence ~ was not constant in time as is to be expected from the incoupling condition (see Section 5.1)
but varied erratically; expressed differently, the optimum incoupling angle ~t(t) varied, which corresponds
to a time-dependent effective refractive-index change
AN(t). Soon we found that the effect was caused by

~"
7

'~
1D

Z
t'o

TM - T E o

-s

400

d F (nm)
9

(o

TM o - TE o

to
,,

-9

400

d F (nm)
Fig. 2. C a l c u l a t e d sensitivities #NwE,,/~dv., ~NTM,,/~d v for T E o and
T M 0 modes, and sensitivity - #,~/{~dv, of difference interferometer vs.
waveguide thickness d v for a d s o r p t i o n of H 2 0 molecules from a
gaseous sample, where ~'q------NTwo - N T M ., n c = l ;
n v-l.33;
2 = 633 rim. Top, waveguide of type (a) with p a r a m e t e r s n v = 1.80
and n s = 1.47; b o t t o m , w a v e g u i d e of type (b) with p a r a m e t e r s
n v = 2.01 and n s = 1.46.

variations of the relative humidity in the environment

of the coupler grating on the waveguide. The effect can
be induced by the experimenter exhaling towards the
waveguide or holding a finger near the waveguide (not
touching it, of course). This first IO sensor experiment
was reported at the European Conference on Integrated
Optics held in Florence in 1983 [1]. Nowadays, we
routinely perform this experiment to demonstrate the
basic IO sensor effect. (The sensitivity of the SiO2-TiO2
waveguides with respect to relative-humidity changes is
greatly enhanced by their microporosity; in highly
porous waveguides, the sensor effect (3) can even be
predominant [8 10].)
In Figs. 2 5 we present optical sensitivity constants
versus waveguide thickness dv calculated for two different high-index waveguiding films F, i.e., waveguides of
type (a) with nv = 1.8 and n s = 1.47 and waveguides of
types (b) with nv = 2.01 and ns = 1.46. Waveguides of
type (a) correspond to the SiO2 TiO2 films presently
used by us, while waveguides of type (b) can be realized
with films of higher TiO2 contents or of Si3N4. The
substrates S are glass, silica or oxidized silicon wafers
(Si/SiO2).
Fig. 2 shows the sensitivity constant ON/OdF, for the
adsorption of water on the waveguide surface. For
waveguides of type (a) the thickness range dv ,~ 130200nm yields the highest sensitivities, and luckily
enough the waveguides used for our first experiments
had just such thicknesses. Adsorption of one H 2 0

W. L u k o s z /' Sensors and Actuators B 29 (1995) 37 50

monolayer of thickness (Iv, = 0 . 3 nm on these waveguides induces m a x i m u m effective refractive-index

changes AN ~ (1.2-1.5) x 10 4 for TE0 and TMo
modes. Also shown is the sensitivity constant ?~N/~.dv,
of the difference interferometer, which has its broad
maximum around d v = 2 5 0 n m , where N--NTEo--

0.20

tO
N.
Z
to

/I TM-T~ 0

-0.15

NTMo.

Fig. 3 shows the sensitivity constant ON/Odv, for the

adsorption of protein molecules from an aqueous solution; the sensitivities depend on the refractive index nv,
of the adlayer formed, and a typical value of n v, = 1.45
was assumed in this Figure. However, to avoid any
misunderstanding, we mention that the nv, values of the
protein adlayer can be determined (see Section 6.3).
Again the optimum thickness range for waveguides of
type (a) is roughly dv ~ 100-200 nm if the A N T E o and
ATvM,, effective refractive-index changes are to be measured, and dv ~, 200 250 nm for the difference interferometer.
Fig. 4 shows the sensitivity constants ON/Onc for IO
sensors working as refractometers for samples C having
a refractive index near n c = 1.33, such as aqueous
solutions.
In microporous waveguiding films F the sensor effect
(3) can occur, i.e., an adsorption of analyte molecules inside the pores. This leads to a change
Any = (1 - q) Anp of the film's refractive index, where q
is the packing density of the solid material, (1 - q ) the
relative pore volume and AnN the change in refractive
index inside the pores. During adsorption and capillary

'-'

3.5

"~
tO

41

400

d F (nm)
0.30

U
to
~

T E ~

0 -

to

-0.2.5
0

400

d F (nm)
Fig. 4. C a l c u l a t e d sensitivities ONvEo/~nc, &VMo/~nc for T E o and
T M o modes, a n d sensitivity - ~N/~:n c o f difference interferometer as
a differential reffactometer for a q u e o u s solutions vs. waveguide thickness d v. n c - 1.33; 2 = 633 nm. Top, w a v e g u i d e of type (a); b o t t o m ,
w a v e g u i d e of type (b) as in Fig. 2.

condensation of water in the pores from ambient humid

air, the refractive index np in the pores changes from
np = 1 to ne ~ nliquid; i.e., we have 0 < Anp ~< 0.33 dependent on the pore filling. The effective refractive-index
change is A N = (ON/Onv)Anv. The optical sensitivities
ON/Onv for adsorption from a gaseous medium such as
1.0

TM o - TE o

tO
~.o

-3.5

4OO

400

d F (nm)

d F (nm)
'-~
7

1.0

TE o

to
to

tO

tO
0
~-~

-5

4O0
d F (rim)

Fig. 3. C a l c u l a t e d sensitivities ONTEo/OdF., ONTMo/OdF,for T E o and

T M o modes, a n d sensitivity - OlV/~d F, of difference interferometer for
a d s o r p t i o n of protein molecules from an a q u e o u s solution vs. waveguide thickness d v. A s s u m e d refractive index of protein adlayer
n F, = 1.45; n c = 1.33; 2 = 633 nm. Top, w a v e g u i d e o f type (a); bottom, w a v e g u i d e o f type (b) as in Fig. 2.

400

d F (nm)
Fig. 5. C a l c u l a t e d sensitivities ONTEo/~nF, gNvM,,/~,n F for T E o and
T M o modes, and sensitivity ~N/Onv o f difference interferometer related to a d s o r p t i o n of molecules from a gaseous s a m p l e C inside the
m i c r o p o r o u s w a v e g u i d i n g film F vs. its thickness d F. n c = 1;
2 = 633 nm. Top, w a v e g u i d e of type (a); b o t t o m , w a v e g u i d e of type
(b) as in Fig. 2.

42

W. Lukosz / Sensors and Actuators B 29 (1995) 37 50

air are shown in Fig. 5. For both the TE o and TMo

modes, the sensitivities ON/Onv versus dv monotonically
approach the value one for large waveguide thicknesses.
For the sensor effect (3), the field inside the waveguiding film F is responsible, not the evanescent field as in
sensor effects (1) and (2). Therefore, it is not necessary
to use very thin high-index waveguiding films F in
grating coupler and interferometric IO sensors. But
with the difference interferometer high optical sensitivities ON/#nF are only obtained with thin waveguides. An
additional advantage of very thin waveguides is that the
sensors' response times due to in- and out-diffusion of
the analyte are small.

4. Direct chemical and biochemical sensors

IO chemical and biochemical sensors for the detection and/or quantification of analytes in gaseous or
liquid sample have to be specific or highly selective.
This specificity has to be guaranteed by biochemistry,
while optics has to provide the high sensitivity. The
specificity is provided by a chemically selective or
'chemoresponsive' coating on the waveguide surface.
Such chemoresponsive coatings are also used in other
types of optical (bio)chemical sensors, such as surface
plasmon resonance sensors.
We differentiate between two forms of such
chemoresponsive coatings: (I) extremely thin, preferably
monomolecular coatings and (II) coatings typically
about 100 nm to several micrometres thick.
Coatings of type (I) can be used for affinity sensors,
in particular immunosensors. The waveguide is coated
with a monomolecular layer of receptor molecules that
specifically bind certain ligand molecules. In immunosensors the receptors are antigen (Ag) or antibody
(Ab) molecules, respectively, which bind the corresponding Ab or Ag molecules if these are present in the
sample. Another example of affinity sensors is the binding of (single-stranded) DNA-target molecules as analytes by hybridization to (single-stranded) DNA-probe
molecules (oligomers) immobilized as receptors on the
waveguide surface. The IO sensors respond to the increase in thickness dF or refractive index nv, of the
adlayer F', which consists at first of the receptors alone,
then of the forming Ag Ab complex (see Fig. 6(a)).
The adlayer F' is very thin (about 4 - 1 2 nm) compared
with the penetration depth Azc of the evanescent wave
into the sample C (which is typically Azc ~ 100 nm).
Therefore, the sensitivity constant ON/#dv, discussed
above in Section 3 describes not only direct adsorption
of molecules on the waveguide surface but also this
receptor ligand binding.
For affinity sensors, coatings of type (II) can also be
used. The receptors are then immobilized in a typically
100 nm thick highly porous coating C', such as a hy

,+

+ c z
Ag

__

F
x

(a)

T
(b)

Fig. 6. Waveguides with chemically selective (or chemoresponsive)

coatings as (bio)chemical sensors. S, substrate; F, waveguide; C,
sample containing analyte molecules. (a) Immunosensor with a
monomolecular chemoresponsive coating consisting of immobilized
antigen (Ag) molecules that bind the corresponding antibody (Ab)
molecules. (b) Chemosensors with a typically 0.1 l#m thick
chemoresponsive layer C' whose refractive index no,. is changed by
binding of analyte molecules M.

drogel. The ligands diffuse into the hydrogel C' and bind
to the receptors. Coatings of type (II) can also be used
for chemical sensors, in particular gas sensors. The
chemoresponsive coating C' has to adsorb, chemisorb or
bind selectively analyte molecules M from the sample C.
These processes lead to an increase Anc, of the refractive
index n o of this coating C', which, in turn, leads to an
increase AN = (ON/?mc,) Ano of the effective refractive
index (see Fig. 6(b)). In the special case where the
chemoresponsive coating C' is thicker than the penetration depth Azo of the evanescent field into the coating
C', the sensor actually works as a differential refractometer with a sensitivity constant #N/~n c, which is
given by the 'refractometer sensitivity' ON/Onc discussed
in Section 3 where nc has to be replaced by no,.
The IO direct (bio)chemical sensors essentially measure the change in surface mass density Fv,, i.e., the
mass per unit area (see Ref. [5]); the resolving power
AFmi n is of the order of a few to a fraction of
1 pgmm-2.
Therefore, unspecific adsorption of
molecules from the sample has to be avoided as it can
limit the performance of all sensors. But this is a
problem the IO sensors have in common with all other
types of optical direct (bio)chemical sensors. Another
common problem of integrated optical and other optical
direct sensors is that the analyte contained in the sample
has to be transported by convection and diffusion in the
alloted assay time, e.g., 15 min, to the optical surface.
5. IO sensor configurations (types of IO sensors)

The basic or primary IO sensor effect was explained

in Section 3 in terms of the effective refractive-index
changes AN. These changes AN can be measured by
various optical means. A number of different types of
IO sensors have been developed in which the AN
changes are transformed into readily measurable physical quantities. In the following we concentrate on the
sensor types developed in our group, i.e., the input and
output grating couplers in Section 5.1 and the difference
interferometer in Section 5.2. Other sensor types are
only briefly discussed in Sections 5.3 and 5.4.

43

w. Lukosz / Sensors and Actuators B 29 (1995) 37-50

M1

5.1. Input and output grating couplers

Since the beginning of 'integrated optics', grating

couplers have been used to couple light into and out of
a p l a n a r waveguide. Fig. 7 depicts that in- and outcoupling are related to each other by the reciprocity theorem, which permits reversal of the direction of
propagation of all the light waves. Consequently, inand outcoupling are governed by the same relation
( _+)N =- nair sin ~ / + l;t/A

(5)

where N is the effective refractive index of the guided

mode, n,~r = 1.0003 is the refractive index of air, 2 the
wavelength, A the grating period and l = + 1, _+2 . . . .
the diffraction order. The plus and minus signs on the
left side of Eq. (5) hold for guided modes propagating
in the + x and - x directions, respectively. In the
incoupling situation at is the optimum incoupling angle;
in the outcoupling situation it is the outcoupling angle.
The principle of both input and output grating coupler sensors can be expressed by the difference relation
AN = n.i~ A(sin ~i) ~ r/air COS gl A~I

(6)

which follows from Eq. (5): induced effective refractiveindex changes AN lead to changes Ac~zof the angles ~.
The sample has to cover the waveguide in the grating
area. The quantity AN appearing in Eq. (6) is the
effective refractive-index change induced in the waveguide in the grating area. The effective refractive index N
of the mode outside this grating region is irrelevant;
these parts of the waveguide can but often need
n o t - - b e covered by a protection layer.
We use surface relief gratings with 1/A = 2400 lines
per m m operating in diffraction order l = 1 at visible
wavelengths 2, in particular at ). = 633 or 514 nm. In
the early stages of the work, we used gratings with
1/A = 1200 lines per m m operating in diffraction orders
l = 1 and 2.
In our group, input and output grating coupler
instruments were built. With both instruments, the
angles :~t(t) of the TE0 and TM0 modes in a planar
m o n o m o d e waveguide are measured as functions of
time t, and from these angles the effective refractive

/B

.,ql===

Fig. 7. Principle of input and output grating couplers. F, waveguiding film: S, substrate. The angle ~ is the optimum angle of incidence
in incoupling experiments and is equal to the outcoupling angle if the
same grating is used as an outcoupler. C, sample in cuvene or flow
cell B; PL, protection layer.

/R

I1sM
Fig. 8. Schematic of input grating coupler instrument. F, waveguiding film; S, substrate; R, rotation stage, with vertical axis of rotation.
In an angular scan, the TEo and TMo modes are successivelyexcited
by an s- and a p-polarized laser beam. L, lever arm; MS, micrometer
screw; SM, stepping motor; Cu, cuvene; Laser, He Ne laser
(2- 633 nm); FP, Foster prism; M~ and M2, mirrors (from Ref. [12]).

indices NTEo(t) and

NTMo(t) are

determined from Eq.

(5).
The in/outcoupling angles ~ are defined with respect
to the normal on the waveguide. We determine the
direction of the normal (:~ = 0) experimentally by proceeding as follows: for each type of mode (TEo and
TM0), the modes propagating in the + x and - x
directions are in/outcoupled using the diffraction orders
l = + 1 and - 1 , respectively; the in/outcoupling angles
are ~ and c ~ =
c~, respectively. The normal that
corresponds to the angle e = 0 is the bisector between
the two directions ~ and :~ /- This procedure has to be
performed only once before the start of the sensor
experiment. During the actual sensor experiment (when
the sample is brought in contact with the waveguide) it
suffices to excite only the TE0 and TM0 modes propagating in one, say the + x , direction.
In the first instrument built in our group, the input
grating coupler [11-16], the optimum incoupling angles
~(t) of the TEo and TMo modes are found by measuring the incoupled power during a computer-controlled
electromechanical rotation of the waveguide about a
vertical axis (see Fig. 8). Consequently, the temporal
resolution is rather low at 2.4 s. Data acquisition and
evaluation are performed with a PC. The instrumental
resolution in effective refractive-index changes is
ANmin ~,, 2 10 -6. For further experimental details of
the instrument see Ref. [16]; our instrument is different
from a commercially available (from ASI, Switzerland)
version of the input grating coupler.
The output grating coupler [17 20] was developed as
a fully opto-electronic instrument (see Fig. 9). The
outcoupled TE0 and TM0 modes are focused by a lens
on two separate position-sensitive detectors. The outcoupling angles ~(t) of the TEo and T M o modes are
simultaneously measured with adjustable (if required,
submillisecond) temporal resolution. D a t a acquisition
and evaluation are performed with a PC. The instrumental resolution in effective refractive-index changes is
A N t o i n e 5 x 10-7; further experimental details are
given in Refs. [21, 33].
The resolution ANmm of the grating coupler instrument is related to the interaction length L of the guided
mode in the grating region with the sample. This inter-

44

W. Lukosz

Sensors and Actuators B 29 (1995) 37 50

action length L is determined by the propertms of the

grating, i.e., its physical length L,. in the x-direction, or,
if L~ << L,, by its coupling length Lc, which is inversely
proportional to the modulation of the surface relief
grating. The interaction length L primarily determines
the angular width a N - n,i~d(sin ~) ~ nair cos 0~c~g of
the incoupling efficiency curve versus variable
? 7 - G ~ s i n :~; we have (5]?~2/L. It is important to
note that changes in the in/outcoupling angles can be
measured to a small fraction g of the angular width C%<
Therefore, we express the achievable resolution in effective refractive-index changes as ANmin = g(SN in Ref. [5].
In our experiments, L ~ I mm a n d g ~ 1 0 2 10-3.
If the input coupler grating is illuminated with a
converging beam of light with a sufficiently large spread
c~ of angles of incidence, in the reflected light a dark
line appears under the angle cG i.e., the optimum angle
of incidence. The induced effective refractive-index
changes AN (in the grating region) can be determined
by measuring the change of the angular position of the
dark line. Such a version of the input grating coupler
was set up in another group [22].
The measurement of time-dependent effective refractive-index changes AN(t) can be expected to give some
interesting information in many sensor experiments. In
order to obtain quantitative results (in refractometry and
in real-time monitoring of protein adsorption or of biochemical reactions) we proceed as follows in all measurements with the input and output grating couplers:
(1) Before the actual experiment, at time t ~<0, i.e.,
before the sample gets into contact with the waveguide
surface, we measure the effective refractive-index values
NTEo(0) and NTMo(0); from these two values the thickness dF and refractive index nv of the waveguiding film
F are determined.
inlet
.%

Cu

outlet

~c

M2
WG

~.

BS

n Fq
(a)

,~/4

H.
il

Fig. 10. (a) Schematic of the difference interferometer. WG, waveguide; BS, beam splitter; W~ and W > Wollaston prisms; Dj, photodetectorsj = 1 4; l~, cylindrical lens, l, cylindrical or spherical lens, 27'2,
half-wave plate; ,,/4, quarter-wave plate; ADC, 16-bit analog-to-digital converter; PC, computer. (b) Detailed cross-sectional view of
waveguide WG. F, waveguiding film; S, substrate: PL, protection
layer; C, sample in cuvette B: L, interaction length (from Ref. [23]).

(2) At time t > 0, during the actual experiment, only

the effective refractive-index changes kNveo(t) and
ANTMo(t) have to be measured. Futher data evaluation
starts from the effective refractive-index values
NvEo(t) = NsEo(O) + ANTE.U) and NvMo(t) -- NTMo(0)
+ANsMo(t). By rigorously solving the mode-guiding
equation, the optical parameters of interest are determined, for example, the refractive index nc(t) of the
sample in refractometry, or the thickness dv.(t) and
refractive index nv,(t) of an adsorbed or bound adlayer
F'. This evaluation requires that the individual waveguides are first characterized by the procedure described
in (1) above.

5.2. Difference interJerometer

In our group we developed a third instrument, the
difference or polarimetric interferometer [23,24] (see
Fig. 10). A similar instrument based on the same principle was built later at Hofl'mann-La Roche, Basel (see
Ref. [25]). In a planar waveguide the TEo and TM0
modes are coherently excited by laser light of wavelength 2. Both modes propagate on a common path
down the waveguide. In a waveguide section of length
L, typically L ~ 10-20 mm, both modes interact with
the sample. This interaction causes time-dependent
changes ANTEo(t) and ANrMo(t) of their effective refractive indices. Therefore, a time-dependent phase diffrence
A~)(t ) = AqSTE0(t)-- AOOTMo(t)= 27r(L/,i) A~7(t )

Fig. 9. Schematic of output grating coupler instrument. F, waveguiding film; S, substrate; L, spherical lens of focal length f = 300 mm, l,.,
cylindrical lens; Cu, cuvette; PDS, position-sensitive detector; ADC,
analog-to-digital converter; PC, computer. An effective refractive-index change AN leads to a change of the outcoupling angle Ac~, and,
consequently, to a displacement zXu of the focus (m-line) of the
outcoupled beam on the PSD (from Ref. [21]).

W2

(7)

between the TEo and the TM 0 modes occurs at the end

of the waveguide, where
A_N(t) - ANTEo(t) - ANTMo(t )

(8)

This phase difference A~(t) is the response or output

signal of the difference interferometer; it can be measured with different methods. We measured the phase

W. Lukosz

Sensors and Actuators B 29 (1995) 37-50

difference A(b(t) as a function of time t, using a set-up

comprising a beam splitter, two Wollaston prisms, a
2/2- and a 2/4-plate and four photodetectors as described in more detail in Refs. [23,24]. Four photodetectors ( j = 1-4) measure the intensities
I,.2(t) = I0{1 _+ cos[A~(t) + ~)0]}

(9)

and

I3.4(t) = I;{1 _+ sin[A~(t) + ~0]}

(10)

where Io and Io are proportional to the incoupled

power and ~)0 is a constant phase difference, The phase
difference A~)(t) is determined from the detector outputs I/(t) as follows from the relation
A~)(t) + Aq)0 = arctan{([Ii(t ) + ~(t)][13(t ) - 14(t)])/
([11 (t) -- I2(t)][I3(t) + I4(t)])}

(11)

and from the sign of [Ii(t)-I2(t)] or [I3(t)-I4(t)], a

phase value A~)(t)+ ~0 lying in the interval - z r to +re
is calculated. To this calculated phase, the value 2~rm is
added, where the integer m is unambiguously chosen so
that the time-dependent function A~)((j) is continuous
at successive sampling points tj = j At. Our system permits the choice of any sampling interval At >~ Atmi n =
80 ~ts. In this way, A~(t) is determined, largely independent of laser power fluctuations, of temporal variations
of the incoupling efficiencies and of changes in the
attenuation of the guided modes caused by interaction
with the sample, with an experimental resolution of
A~min/RTr ~ 5 10

4.

Advantageous features of the difference interferometer are:

(i) The 'sensor chip' is just a planar waveguide. Its
fabrication is easy compared to that of IO M a c h Zehnder interferometers discussed in Section 5.3, where
strip waveguides and Y-junctions or directional couplers used as beam splitters and combiners have to be
microstructured.
(ii) Long interaction lengths L from a few millimetres to several centimetres, and consequently a high
resolution, can be realized.
(iii) Because the TEo-and TMo modes are orthogonally polarized their phase difference A~)(t) is easily
measured very accurately.
(iv) The interferometer has a good stability because
the orthogonally polarized beams propagate on a common path from the laser to the phase-measuring set-up.
The difference interferometer can also be called a
common path or 'polarimetric' interferometer since the
basic effect, i.e., the induced phase difference A(b(t), can
be interpreted as a change in polarization of the outcoupled elliptically polarized beam. Its resolution limit,
expressed in effective refractive-index differences A~7, is
A]Vmi n =

g2/L

(12)

45

where
g - A~min/27r

(13)

For an interaction length L = 17mm at the H e - N e

laser wavelength/i = 633 nm, we have ANmin = 2 x 10 -s
i f g = 5 x 10 4.
In our experiments with the difference interferometer, we used planar SiO2 TiO2 waveguiding films first
on glass substrates, then on oxidized silicon wafers
(Si/SiO2) as substrates; the SiO2 buffer layer was about
3.5 lam thick. Compared to glass, the Si/SiO2 substrates
have several advantageous features:
(i) Higher processing temperatures can be used and,
consequently, waveguiding films of different microporosity can be fabricated with the sol gel process.
Waveguides (A) fired at a temperature of about
T ~ 500 C for 1 h were microporous; waveguides (B),
which after this first heat treatment were additionally
fired at T ~ 850 C, are more compact.
(ii) Endfaces of good optical quality (through which
the light is coupled into and out of the waveguide) are
obtained by scribing and cleaving the wafer along a
crystal plane.
(iii) Substrate modes are highly attenuated in the
absorbing silicon. (In endfire coupling, not only the
desired TEo and T M 0 modes but also unwanted 'substrate modes' are normally excited, i.e., light is trapped
in the substrate by total internal reflection and can
propagate down to the photodetectors.)
Since Si/SiO2 substrates are not transparent, such
waveguides cannot be used with grating coupler sensors, where the light is preferably coupled into or out of
the waveguide through the substrate and not through
the sample.
The essential element in the working mechanism of
the difference interferometer is that the TE0 and TM 0
modes interact somewhat differently with the 'measurand', i.e., with the adsorbed or bound adlayer F' in
sensor effect (1), with the change Ant in refractive index
of the liquid sample C in sensor effect (2), and with the
change Any of the refractive index of a microporous
waveguide in sensor effect (3). This leads to the polarization dependence of the sensitivity constants gN/~dv,,
~/V/?~dc, and g/V/~r/v shown in Figs. 2--5.
Figs. 2 and 3 show that both for the adsorption of
water and of proteins on the waveguide surface, the
maximum value of the sensitivity constant (?N/~dv, of
the difference interferometer is reduced by a factor of
0.35-0.4 and of about 0.5, respectively, for waveguides
of types (a) and (b) from the maximum ON/c~dv , values
for the TE o or TM0 modes. The mentioned factors
mean a reduction of the sensitivity of the difference
interferometer compared to that of a M a c h - Z e h n d e r
interferometer. The results shown in Fig. 4 mean that
the difference interferometer, if used as a refractometer,
can attain roughly half the maximum sensitivity of a

46

w. Lukosz / Sensors and Actuators B 29 (1995) .37-5(I

M a c h - Z e h n d e r interferometer. This moderate reduction in the sensitivities is a minor disadvantage of the

difference interferometer; it could, however, easily be
compensated by a corresponding increase of the interaction length L.
The difference interferometer has only a single output signal, the phase A~)(t). It responds both to adsorption of molecules and to refractive-index changes Anc
of a liquid sample, but its response can only be unambiguously interpreted if one of the two sensor effects (1)
or (2) is predominant. One possibility to differentiate
between the two effects is to record the response A~(t)
as a function of time t beginning at the time to when the
sample is brought into contact with the waveguide. The
An c effect can be practically instantaneous, while the
adsorption process, which is either diffusion or reaction
limited, takes some time. However, at high analyte
concentrations it can also be quite fast. Therefore, it is
important that in principle the difference interferometer
can be designed in such a way that it responds to only
one of the two effects, but at the penalty of a reduced
sensitivity.
From Figs. 3 and 4 we conclude that the difference
interferometer measures adsorption only and is insensitive to small cover index changes Anc if the waveguide
has a certain thickness d v = d v, where (SN/
(?nc)]dr-,q. = 0. In this case, the sensitivity 8N/Sdv, for
protein adsorption is smaller by a factor of about 0.6
than its maximum value at the optimum waveguide
thickness dF; it also has a different sign. In
(bio)chemical sensing, the reduced sensitivity may be
acceptable, since Anc changes due to temperature variations and to changes in sample composition could
otherwise lower the detection limit. However, we have
not experimentally verified these theoretical predictions.
On the other hand, the difference interferometer can
be employed as a differential refractometer that is insensitive to a small adsorption of molecules on the
waveguide surface if waveguides of a certain thickness
d r = d r are used, where ( S N / S d v , ) l d . _ d = O . The refractometer sensitivity 8N/Sdc is smaller by a factor of
about 0.5 than its maximum value at a higher waveguide thickness.
Summarizing, we note that insensitivity to one of the
two effects (1) or (2) can be obtained at the penalty of
a reduction in sensitivity for the other effect.
5.3. Two-beam interJerometers

We briefly mention two-beam interferometers used

by other groups. Hartman et al. [26] describe an IO
interferometer, where in a planar waveguide two modes
of the same polarization but of different mode numbers
m ' m (with m, m ' = 0, 1. . . . ), e.g., the TE 0 and TE~
modes, propagate on a common path and interact with
the sample.

M a c h - Z e h n d e r interferometers are well suited as IO

sensors. In one leg of the interferometer, the guided
mode interacts over a length L with the sample, where
typically L = 10-20 ram; the rest of the waveguide is
covered with a protective layer. The interferometer is
normally used in one polarization, i.e., with either TE o
or TM0 modes. The induced effective refractive-index
changes AN produce phase shifts Aq)(t)= 2~(L/
2)AN(t) and these, in turn, give variations of the
intensity l ( t ) = (L~/2){1 + cos[Aq)(t)+ Aq)o]} at the output port. M a c h - Z e h n d e r interferometers can be realized as either hybrid or fully IO devices. In general,
fully IO devices have higher stability; a disadvantage is
the higher fabrication costs of the sensor chips.
In 'hybrid' Mach Zehnder interferometers, beam
splitting and recombination is done in three dimensions
with conventional optical beam splitters. The waveguide is preferably a planar waveguide; endfire coupling
could be used as proposed in Ref. [5]. In a different
version of a hybrid Mach Zehnder, Heideman et al.
[27] used two surface relief gratings on a planar waveguide (separated by a non-corrugated waveguide section of length L) to couple light into and out of an
Si3N4 planar waveguide. In fully IO versions of the
Mach Zehnder interferometer, strip waveguides and
Y-junctions (or 3 dB directional couplers) are used as
beam splitters and combiners. Such interferometers fabricated in glass (by electric-field-assisted silver indiffusion) were used by Fabricius et al. [ 2 8 ] for
refractometry and immunosensor experiments. These
glass waveguides had a gradient-index profile with maximum refractive-index difference n ~ . - n s ~0.1 and a
thickness of about 1 ~tm; consequently, their sensitivity
constants 8N/Sdv, and ~?N/Snc are much lower than for
very thin high-index waveguides.
5.4. Other IO sensors

Bragg reflector gratings, for example, realized as

surface relief gratings of grating period A, can be used
as IO sensors. We demonstrated the basic effect [1,2]
that effective refractive-index changes AN of the median
effective refractive index N in the grating area lead to
corresponding changes A2B of the Bragg wavelength 2~,
where
2NA

l, B

with the diffraction order l = 1, 2 . . . . .

quently
AN
N

--

A2B
- 2B

(14)
and conse-

(15)

The changes in reflectivity R and/or transmission T of

the Bragg reflector are measured at a fixed wavelength
2; alternatively, other readout schemes using wavelength-tunable (diode) lasers are possible.

47

W. Lukosz / Sensors and Actuators B 29 (1995) 37 5(1

In all types of sensors described above, the waveguide was spatially homogeneous, i.e., it had a constant
thickness dv and a constant refractive index nv, and
consequently, a spatially constant effective refractive
index N. We also assumed that the induced effective
refractive-index changes AN are spatially constant in
the grating region of input or output grating couplers
and of Bragg reflectors. In the case of the difference and
M a c h - Z e h n d e r interferometers, AN denotes spatial
mean values averaged over the interaction length L.
Different types of sensors could, in principle, exploit
spatial variations AN(x, y) of the induced effective refractive-index changes in planar waveguides. For example, a linear spatial variation of the effective refractiveindex change in the y-direction
AN(y) = \ ~73'/
leads lo a deflection of a guided mode in a planar
waveguide by the angle fl, where
sin/)' =

(17)

L being the interaction length in the x-direction [29].

With high-index microporous SiO2-TiO2 waveguides,
this effect is easily demonstrated: blowing dry N 2 gas
through a nozzle sideways onto the waveguide surface
in ambient air of normal humidity, the beam is
deflected away by the gas stream into a region where
the waveguide is less 'dry', i.e., has a higher effective
refractive index N(y).
A step towards sensors of higher complexity is the
use of waveguides with spatially varying thickness dr,
and consequently spatially varying effective refractive
index N(x, y). Kunz [30] demonstrated various schemes
of IO sensors using such waveguides.

However, we wish to point out clearly that we have

not developed a quantitative relative-humidity sensor.
We are presently investigating whether the following
approach might be feasible: with a Peltier heater/cooler
in contact with the (Si/SiO2) substrate we cycle the
waveguide's temperature between a lower value
T - ~ A T and a higher value T + A T about the ambient temperature T, and study how the sensor's (actually
a difference interferometer's) response is related to the
relative-humidity value.
We also demonstrated a different version of a humidity sensor with the difference interferometer [24,35].
The waveguide was coated with a porous (evaporated)
SiO 2 adlayer C' about 1 lam thick. The filling of the
micropores and, consequently, the refractive index n c
of the adlayer C', depend on the humidity. The sensor
actually works as a refractometer responding to
changes Ant.. The main advantage of using a porous
adlayer is that it also acts as a filter protecting the
waveguide F itself from dust, thus prolonging its lifetime. We expect that this principle combined with
porous chemoresponsive coatings C' can be successfully
applied to gas sensing.
6.2, Refractometrv

We demonstrated that the output grating coupler

works well as a refractometer even with somewhat
microporous SiO2-TiO 2 waveguides on glass substrates, although more compact waveguides on silica
substrates fired at higher temperatures are preferable.
The instrument was tested with sucrose solutions of
different concentrations c = 0.04-1.0% and with
ethanol methanol mixtures in a continuous flow system. That a residual microporosity of the waveguides is

0.0

6. Results

We discuss results obtained with our input and

output grating coupler instruments and the difference
interferometer rather briefly, but give a complete list of
references.
6.1. Re&tire-humidity sensing

Not only the input but also the output grating

couplers work well as relative-humidity sensors. We
demonstrated this by monitoring the small relative-humidity variations produced by the air-conditioning system in our laboratory simultaneously with both grating
couplers and a conventional hygrometer [18]. Fig. 11
shows how very sensitively the difference interferometer
responds to small relative-humidity changes of ambient
air.

'

'

_~

e~ -0.1
-0.2
~

-0.3
-0.4
-0.5

I ......
I

10

20

' I

30

40

'.v."I'
i

50

60

time (minutes)
Fig. 11. Relative-humidity sensing with a difference interferometer.
The responses of a conventional hygrometer and of the difference
interferometer (phase difference A~(t)) to periodic small relative-humidity changes of ambient air at room temperature are compared.
Sampling time was At = 1 s (from Ref. [23]).

48

W. Lukosz /Sensors and Actuators B 29 (1995) 37-50

1.37
methanol

methanol

U--

1.36"
1.35-

t-

o 1.34-

1.331.32-

ethanol

16o

ethanol

260

360

460

time (see)
Fig. 12. Output grating coupler used as a refractometer for alcohols.
The refractive index nc(t ) of the sample C vs. time t is determined for
repeated exchange of ethanol and methanol (from Ref. [21]).

tolerable is a consequence of the fact that the effective

refractive-index changes ANTEo(t) and ANTM,)(t) of both
the TE0 and the TM 0 modes are measured. From these
two quantities the refractive index nc(t) of the sample is
determined together with the refractive index nv(t) of
the waveguiding film F as a function of time t by
rigorous solution of the waveguiding equation [21].
(The change in the nv(t) values indicates a slow exchange of fluid in the micropores of the waveguide.)
Experimentally a resolution [Anc]min=5 x 10 -5 was
achieved. An example of such a measurement with two
alcohols is shown in Fig. 12.
Also the difference interferometer was successfully
used as a refractometer, e.g., for aqueous sucrose solutions with compact SiO2 TiO2 waveguides of low microporosity on Si/SiO 2 substrates (waveguides of type
(B) fired at T ~ 800 C) [24]. With an experimental
resolution of ANmm ~ 5 x 10 8 and the calculated sensitivity constant of ~N/#nc ~ - 0 . 1 (see Fig. 4), from the
relation ANmin = (ON/~nc)[Anc]mi n, we would expect refractive-index changes of [A//c]mi n ~ 5 10 7 t o be resolvable under ideal circumstances.

6.3. Real-time protein-adsorption studies

We demonstrated that the adsorption of proteins on
the waveguide surface can be monitored in real time.
The adsorbing molecules form an adlayer F'. Working
with the input/output grating coupler instrument, we
are able to determine two optical parameters of the
adlayer F' from the two measured effective refractive-index changes ANTEo(t) and ANTMo(t). Assuming the
adlayer F' to be isotropic, we can determine its thickness dF(t) and its refractive index nF,(t) versus time t,
and, thus, its surface mass density Fv, from the relation
Fv.(t)-- (dn/d[c]) l[nv,(t ) - nc]dv,(t), where dn/d[c]
1.88 10 4 g l 1 is the refractive-index increment of
aqueous protein solutions.

Results on adsorption of human immunoglobulins G

and A (h-IgG and h-IgA, respectively), bovine serum
albumin (BSA), protein A and avidin obtained with the
incoupler were reported in Refs. [11 16].
With the outcoupler instrument, mainly the adsorption of avidin, Neutravidin and streptavidin was studied [21,31]. For example, avidin forms a monolayer F'
on the surface with parameters d~, ~ 4 nm, nv, ~ 1.48
and Fv, ~ 3 ng mm 2.
With both the in/outcoupler simultaneous measurements with the normally used He Ne laser wavelength
2 = 633 nm and the argon-laser wavelength 2 = 514 nm
were made, in order to check the reliability of our
method for determining nF, and d~,. The thickness value
dr, must be independent of 2; the obtained dr. values
agree well for complete monolayers [15,16,31].
With the difference interferometer, also mainly the
adsorption of avidin, Neutravidin and streptavidin was
investigated. From the interferometer's single response,
i.e., from the phase difference A~)(t), only the surface
mass density F v, can be determined; the V~-, value
depends on the assumed value of the refractive index n~,
(see Refs. [34, 35]).
In an improved model the adlayer F' is considered to
be optically anisotropic. The adsorbed protein
molecules are modelled as orientated (in themselves
isotropic) ellipsoids embedded in a homogeneous
isotropic matrix, the aqueous buffer solution. Nonspherical shape and/or orientation are caused by attractive forces extended by the surface. From a
Maxwell Garnett theory, we derived that such an adlayer F' is uniaxially anisotropic with its optical axis
normal to the surface; it is characterized by its thickness
dr, and the ordinary and extraordinary refractive indices nF, o and n~-,,oo [32]. These three optical parameters,
however, cannot be determined from the two measured
quantities ANTE.U) and ANTM,(t) (but 'possibly from
multi-wavelength measurements with monomode
(m = 0) waveguides, or, alternatively, from single-wavelength measurements with thicker, multimode (m = 0
and 1) waveguides).
With the anisotropic model we can explain the following puzzling findings: in the evaluation of the adsorption of a protein monolayer with the isotropic
model, we often obtained thicknesses dv.(t) versus time
t that were unrealistically small (compared to the
molecules' size) at low surface coverages but increased
to plausible values for complete monolayers. The evaluation with the anisotropic model points to an oblate
ellipsoidal shape of the molecules; the layer thickness
dv,(t) is independent of the surface coverage [33].

6.4. Affinity sensing and immunosensing

Perhaps the most interesting result is that certain
biochemical reactions can be monitored in real time,

W. Lukosz ,' Sensors and Actuators B 29 (1995) 3 7 50

i.e., while they happen. They are affinity reactions

between a receptor and a ligand and immunoreactions
between an antigen (Ag) and the corresponding antibody
(Ab), one reaction partner being immobilized on the
waveguide surface, the other one provided in the sample.
With the input grating coupler instrument, we studied
a number of affinity reactions and immunoreactions, for
example, between avidin and biotinylated proteins, such
as biotin L C - B S A or biotin protein A, between h-lgG
and anti-h-IgG and between h-IgG and protein A; the
final results are given in Refs. [13,15,16].
With the output grating coupler and the difference
interferometer we first studied the affinity reaction between avidin (immobilized by adsorption on the waveguide) and biotinylated proteins, such as biotin LC
BSA [21,24,31,34]. Then, in a series of immunosensing
experiments, we made use of the strong adsorption of
avidin on the waveguide surface to immobilize the
biotinylated antibody, a goat anti-rabbit-IgG. Fig. 13
shows the whole experiment as monitored with the
difference interferometer [35]. First, avidin is adsorbed
on the waveguide, the biotinylated goat anti-rabbit-igG
binds to the adsorbed avidin monolayer, and finally the
immunoreaction between the immobilized antibody and
I

-5
Ol
" - -10

~e

10 l~g/ml biotinylated
Goat Anti-Rabbit IgG

10 ixg/ml
Avidin

-20

PBS

PBS

-15

20

40

L
i

60

80

100

120

time (minutes)
0.10

0.05
0.00

500

49

'

450-

4 - 5 gg/ml

4350:

,~~

~ 3o0- /

500ng/ml

250- ~
200<1 150I O0 -

./"

50
0 ~,

15

30

45

60

time (min)

Fig. 14. Immunosensing with the output grating coupler. The

changes in surface mass density At(t) vs. time t are shown as the
antigen (rabbit-IgG) in solution with different concentrations e binds
to the antibody (goat anti-rabbit) immobilized on the waveguide
surface with the same method as in Fig. 13 (from Ref. [33]).
the corresponding antigen, namely, rabbit-IgG takes
place. The same experiment was also studied with the
output grating coupler [33]; but in Fig. 14 we present only the results of the antibody-antigen reaction for different concentrations of the antigen. With
both instruments an antigen concentration
of
c=50ngml
~3x
10 ~0M is clearly detected. With
this experiment and others, we demonstrated that
affinity sensing and immunosensing with subnanomolar
resolution are feasible.
But we have to mention a partly unresolved experimental problem that limits further improvements of the
resolution and accuracy of the IO sensors. The waveguides show a 'drift' effect in aqueous solutions, i.e., a
slow but persistent increase of the measured effective
refractive indices NTE, and NTM,, with time, which is
related to the microporosity of the waveguiding films.
Even the relatively compact, i.e., low porosity, waveguiding SiO:-TiO2 films on silica and Si/SiO2 substrates
show a residual drift. The problem can probably be
resolved by really compact waveguiding films also of
other high-refractive-index materials, or possibly by
suitable organic coatings on a (microporous) waveguide.

t~
Ckl -0.05

7. Conclusions

"~ -0.10
-0.15
-0.20 I
0

20

40

60

80

100

time (minutes)
Fig. 13. R e a l - t i m e m o n i t o r i n g o f affinity r e a c t i o n a n d i m m u n o r e a c t i o n s with the difference i n t e r f e r o m e t e r . T h e p h a s e difference A~)(t)
vs. t i m e t is s h o w n as b i o t i n y l a t e d g o a t a n t i - r a b b i t - l g G as a n t i b o d y is
i m m o b i l i z e d o n the w a v e g u i d e s u r f a c e b y b i n d i n g to p r e v i o u s l y
a d s o r b e d avidin (top), a n d as r a b b i t - l g G as the a n t i g e n b i n d s to the
immobilized antibody (bottom). An antigen concentration of
c=50ngml
1 ~ 3 x 10 m M is clearly d e t e c t e d , while with h _ i g G o f
c o n c e n t r a t i o n c = 1 lag ml ~ n o r e s p o n s e is o b s e r v e d except f o r a
residual drift effect. PBS, p h o s p h a t e buffer s o l u t i o n ( f r o m Ref. [35]).

In this review paper we have described the serendipitous discovery of the IO sensor effect, the general
principle of all IO sensors that respond to induced
changes of the effective refractive index of guided modes,
the results of the theory of sensor sensitivities and the
different types of IO sensors. In particular, the input and
output grating couplers and the difference interferometer
that were built by us were discussed and results obtained
with them. We used the sensors successfully as refractometers. We demonstrated that the adsorption of
proteins on the waveguide surface and, even more
importantly, also biochemical reactions such as affinity

50

W. Lukosz

Sensors and Actuators B 29 (1995) 37 50

reactions (between a receptor and a ligand) and immunoreactions (between antibodies and antigens), where
one reaction partner is immobilized on the waveguide
surface, can be monitored in real time. We also demonstrated the feasibility of direct affinity sensors and
immunosensors with subnanomolar detection limits.
Acknowledgements
The author wishes to thank his co-workers and Ph.D.
studens from whose work the experimental results discussed in Section 6 were taken, namely, Drs Ph.M.
Nellen, D. Clerc and Ch. Stature, who with the technical
support of E. Hausammann and G. Natterer, built the
input and output grating coupler instruments, and the
difference interferometer, respectively.
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