Anal Bioanal Chem (2005) 381: 141–155
DOI 10.1007/s00216-004-2895-4
R EV IE W
Guenter Gauglitz
Direct optical sensors: principles and selected applications
Received: 14 July 2004 / Revised: 11 October 2004 / Accepted: 11 October 2004 / Published online: 11 November 2004
Springer-Verlag 2004
Abstract In the field of bio and chemosensors a large
number of detection principles has been published
within the last decade. These detection principles are
based either on the observation of fluorescence-labelled
systems or on direct optical detection in the heterogeneous phase. Direct optical detection can be measured
by remission (absorption of reflected radiation, opt(r)odes), by measuring micro-refractivity, or measuring
interference. In the last case either Mach–Zehnder
interferometers or measurement of changes in the
physical thickness of the layer (measuring micro-reflectivity) caused, e.g., by swelling effects in polymers (due
to interaction with analytes) or in bioassays (due to
affinity reactions) also play an important role. Here, an
overview of methods of microrefractometric and microreflectometric principles is given and benefits and
drawbacks of the various approaches are demonstrated
using samples from the chemo and biosensor field. The
quality of sensors does not just depend on transduction
principles but on the total sensor system defined by this
transduction, the sensitive layer, data acquisition electronics, and evaluation software. The intention of this
article is, therefore, to demonstrate the essentials of the
interaction of these parts within the system, and the
focus is on optical sensing using planar transducers,
because fibre optical sensors have been reviewed in this
journal only recently. Lack of selectivity of chemosensors can be compensated either by the use of sensor
arrays or by evaluating time-resolved measurements of
analyte/sensitive layer interaction. In both cases
chemometrics enables the quantification of analyte
mixtures. These data-processing methods have also been
successfully applied to antibody/antigen interactions
even using cross-reactive antibodies. Because miniaturiG. Gauglitz
Institute of Physical and Theoretical Chemistry,
University of Tuebingen, Auf der Morgenstelle 8,
72076 Tuebingen, Germany
E-mail: guenter.gauglitz@ipc.uni-tuebingen.de
Tel.: +49-7071-2976927
Fax: +49-7071-295490
sation and parallelisation are essential approaches in
recent years, some aspects and current trends, especially
for bio-applications, will be discussed. Miniaturisation is
especially well covered in the literature.
Keywords Optical Æ Chemosensing Æ Biosensing Æ
Transducer Æ Application
Introduction
Because fibre optical sensors have recently been reviewed in this journal [1], this article covers the principles and applications of planar-type optical sensors, and
covers the essentials of optical sensing in recent research
and development. In parallel to recent key developments
in conventional analytical techniques, some focus of
research has been on biochemical and chemical sensors.
The combination of physical sensors (transducers) with
more or less analyte-selective layers of biochemical or
chemical substrates has introduced selectivity to these
systems. For this reason such arrangements have to be
considered as complete sensor systems containing
transduction principles, the sensitive layer, the signal
processing, and evaluation strategies. Of the huge variety of transduction principles, this paper concentrates on
optical techniques which provide many possibilities of
application of optical principles, either using direct
monitoring of the interaction between an analyte and
this sensitive layer, or including an indicator dye or a socalled labelled system, especially for fluorescence detection.
Because this paper is a review based on a lecture, a
large number of optical principles will be classified, a
survey on sensitive layers that differ in sensitivity,
selectivity, stability, and reversibility will be given, and
the applicability of multivariate data evaluation will be
discussed. Although this classification of the optical
principles attempts to cover most of the optical techniques used and discussion of the sensitive layers intends
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to give a survey of potential materials, the application
must focus on the work of the author’s group, because
otherwise the paper would have exceeded the appropriate length. To cover part of the wide field [2] recent
review articles have been included. However, because of
the numerous publications in this field, this article concentrates on the author’s work.
Optical methods have recently attracted interest
especially for detection of biomolecular interactions and
in highly parallelised systems. When discussing sensor
techniques, it is usually necessary to restrict the scope to
the original definition of a sensor (reversible, allow
continuous monitoring, simple and cheap devices).
However, especially in the modern application of optical
techniques in high-throughput screening and biomolecular interaction studies, the original definition is no
longer valid. Most biochemical interactions are somehow irreversible, and the sensitive layer has to be
regenerated. Nevertheless, the original definition is not
approved any longer by groups doing research work in
this field, but rather implies that modern techniques
based on optical spectroscopy will be applied to a
molecular or biomolecular interaction using detection
principles in combination with the sensitive layer and
data evaluation. A restriction to the original definition
and discussing only ‘‘sensors’’ would limit this article to
conventional applications only and would not enable
introduction of the interesting field of upcoming applications [3, 4].
In accordance with the different components of a
sensor system, in addition to discussion of optical
principles and a survey of sensitive layers, especially
interesting assays in the area of biosensing will be
mentioned; the influence of fluidics will be discussed,
especially when flow-injection analysis is used to support
Fig. 1 Schematic drawing of regular reflection, resulting refraction,
and total internal reflection for angles larger than the critical angle
in the wave guide. The guided wave exhibits an evanescent field
close to the wave guide, which is influenced by absorbing molecules
or changes in the refractive index within the penetration depth of
this evanescent field. Accordingly the coupling of this external field
to the guided field vectors within the wave guide influences the
effective refractive, the transverse electric (TE-) and magnetic
(TM-) modes differently [15]
the sensor system, and finally applications from the wide
fields of chemosensing and biosensing will be listed, and
occasionally discussed in detail. In this context, modern
approaches such as miniaturisation and parallelisation
will be included. Because it has been mentioned that the
sensitive layer must be characterised, some aspects of
spectral ellipsometry and AFM will be added. The
numerous publications in the area of optical sensing
only enable citation of some related publications and
reviews.
Optical principles
The properties of electromagnetic radiation can be
characterised by amplitude, frequency (wavelength),
phase, polarisation state, and time dependence. In
optical spectroscopy absorbance (or transmittance) is
usually monitored, although fluorescence and reflectance
have gained increasing interest. At the beginning of the
development of optical sensors, opt(r)odes were introduced by Lübbers, who used fluorescence measurement
to determine O2, CO2, and pH [5]. Also only simple
colour changes were measured, for example in the
detection of ammonia via pH changes in a urea sensor
[6]. Both approaches used fibres coated at the end with
the polymer in which either a pH-sensitive dye was
embedded or a fluorophore with its fluorescence quenched by, e.g., a gas. By analogy with ion-selective electrodes with doped electrode material this sensitive layer
should bring selectivity and the system was named opt(r)ode. Modern developments of opt(r)odes have been
reviewed elsewhere [1, 7]. The fibre was used just as a
transport system for remote sensing using electromagnetic radiation between the transducer (in this case a
photo diode, the physical sensor) and the sensitive layer
(the polymer film with embedded dyes). This combination of sensitive layer and transduction is called a
chemical sensor. Nowadays, this principle is used in
many applications with typical sensor properties, such as
being reversible and enabling continuous measurement
of gases or liquids [2]. The change in amplitude of the
radiation is monitored in absorbance. Fluorescence
intensity can be measured simply as its dependence on
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Fig. 2 The principle of reflectometric interference spectroscopy
(RIfS) is based on white-light interference according to the given
formula. Changes in the optical thickness of the layer between the
two interfaces causes a change from second reflected beam I2 to I’2
superimposed on I1 and from a destructive interference (at a
specific wavelength: broken line) to a positive constructive
interference. Considering the whole interference pattern a shift is
observed correlating to the amount of change in physical thickness.
IR can be used as a reference. The change in physical thickness is
either caused by swelling of a polymer layer (uptake of analyte) or
by an affinity reaction adding biomolecules to receptor molecules at
the interface
Fig. 3 Arrangements for measurement of RIfS. Combination of
the white-light source with a mini-spectrometer enables the timeresolved recording of interference patterns. An optical switch
makes it possible to monitor up to 32 channels with one
spectrometer in a pseudo-parallel method. It has been demonstrated that even four wavelengths supply enough information [17]
and light emitting diodes could be used
quenching effects or, better, lifetime measurements can
be used [8, 9]. Modern lock-in technologies and phasesensitive measurements to determine the lifetime provide
tools for rapid and inexpensive electronic components
[10]. Therefore fluorescence enables use of simple and
cost-saving devices.
In optodes using absorbance measurements, in principle diffuse reflectance is applied (sometimes called
remission) [5, 11]. Besides the measurement of quenching of fluorescence intensity of a fluorophore by the
analyte or variation of its lifetime, fluorescence anisotropy [12] can be used to determine structural changes or
orientations, a characteristic which is frequently used for
monitoring rotational diffusion processes [13]. Fluorescence correlation spectroscopy gives information on
lateral diffusion and, indirectly, on the increased size of
molecules after interaction even at the single-molecule
level [14].
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The use of regular reflectance introduced a huge
variety of detection principles based on reflectometry
and refractometry. Both use the dependency on the
thickness of the layer and/or the refractive index, which
influences the phase and/or amplitude of the electromagnetic radiation penetrating this layer or being reflected. The use of reflected electromagnetic radiation is
represented schematically in Fig. 1 [15].
Because one part of the radiation is usually reflected
at the interface of a thin layer, whereas the other penetrates the layer and is there reflected at the other interface, these two partial reflected beams can become
superimposed and form an interference pattern, resulting in constructive or destructive interference depending
on the angle of incidence, wavelength, and optical density of the layer, which is given by the product of
refractive index and physical thickness of the layer. The
modulation of this interference pattern, as demonstrated
in Fig. 2, depends on these properties of the layer and
changes sensitively in response to changes in or at this
layer. This simplified version of ellipsometry, called reflectometric interference spectroscopy (RIfS) [16], provides a simple and robust technique in chemosensing
and biosensing as demonstrated later in the applications
section. The principle of the arrangement is given in
Fig. 3.
If polarised light is introduced the information content becomes even larger, because ellipsometry enables
separation of the refractive index and the physical
thickness when using many wavelengths. Ellipsometry
was introduced as far back as in the 1940s [18, 19], and
has regained interest in modern semiconductor and
wafer technology as a simple control technique. On the
other hand, it enables characterisation of sensitive layers
and is used not only to characterise simple polymer
films, but also biopolymers [20, 21]. In refractometry,
minimum changes in the refractive index or transmittance of a medium close to a wave guide influence
radiation guided in the wave guide, because its evanescent field probes this medium resulting in an effective
refractive index. Thus the transverse electrical (TE) and
transverse magnetic (TM) modes of the wave propagating in the wave guide are influenced differently. These
evanescent field techniques open a wide variety of optical detection principles reviewed in [11], such as Mach–
Zehnder [22] interferometer, Young interferometer [23],
grating coupler [24, 25], resonant mirror [26], and surface plasmon resonance devices [27–29]]. Bragg gratings
[30] even enable the set up of arrays or remote sensitive
detection along a fibre.
Because the evanescent field penetrates (with exponential decay) the medium at the interface of the wave
guide by just half the wavelength of the guided radiation, these devices have the advantage of detecting only
effects within this penetration depth (just a few 100 nm)
(Fig. 1).
The various detection principles mentioned for evanescent fields all interrogate the change in effective
refractive index in the wave guide. Interferometer-type
wave guides (e.g. Mach–Zehnder chips) determine the
difference between the phase of two waves travelling in
two arms of the wave guide [22]. The Young interferometer is a similar type of interferometer, the wave guide
arms not reunifying but rather imaging the interference
pattern produced by the two open ends of the wave
guide arms on a CCD [23]. Using both TE and TM
modes enables internal referencing. This has been improved by Lukosz [31] in its mode beat interferometer,
measuring amplitudes and phases of both polarisation
states.
The grating coupler is frequently used to monitor
changes in refractive index [24]. A wave guide layer is
combined with a layer in which a grating is embedded.
The grating constant is influenced by the refractive index
within the adjacent medium. As for interference filters,
this grating condition determines the preferred wavelengths or varies with the angle of incidence. Thus the
guided wave will depend on the gradient in the medium
next to the wave guide. Radiation incident on the grating will be reflected or coupled into the wave guide,
depending on refractive index, wavelength, and angle of
incidence [32]. Either an angle-resolving arrangement or
a CCD camera (avoiding mechanical parts) is used to
monitor the reflected radiation [23]. Bi-diffractive couplers [33] have two gratings with different grating constants superimposed. The outcoupled wave has an angle
different from that of the directly reflected wave. Rather
tricky are gratings embossed in polycarbonate which
take advantage of non-parallel grooves of the grating or
a thickness gradient of the wave guide [34].
Another type of interrogation of the polarisation
status is applied in prism couplers [26]. The radiation
couples out of a prism via the frustrated total internal
reflection of a low refractive index layer (with a thickness
of 1000 nm) into the high-refractive-index wave guide.
Forty-five degree polarisation is chosen, and TM and
TE modes travel in the resonant layer (wave guide:
thickness 100 nm), differently influenced via the evanescent field by changes in the adjacent medium. Thus,
the polarisation state changes in this ‘‘resonant mirror’’
[35].
The best examined evanescent field technique is surface plasmon resonance; the theory and application to
chemo- and biosensing have been reviewed in many
articles [36, 37]. A prism is coated on its base by an
approx. 50-nm metal film. The prism takes care of total
reflectance of incident radiation, which excites this film
to an extent depending on angle of incidence and
wavelength plasmons in resonance at the surface opposite to the wave guide interface adjacent to the medium
of interest. The resonance condition of these plasmons
depends on the refractive index of this medium. Resonance of plasmons reduces the reflected intensity of the
p-polarised light resulting in a ‘‘dip’’ in the reflectance
diagram. Either a prism or a wave guide with a buffer
layer [28] to the metal film is used for achieving total
internal reflectance. This direct optical detection method
has been commercialised the longest [38].
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In recent years, additional reviews have been published on SPR techniques [39–42], the resonant mirror
[43], the grating coupler [44, 45], and Bragg gratings [46].
An interesting approach is a recently published combination of SPR and fluorescence [47–49]
The distance-sensitive effect is also used either in
ATR (attenuated total reflectance) techniques or in
arrangements using labelled molecules (fluorophores)
close to the wave guide (TIRF, see below).
Comparing refractometry and reflectometry, it must
be considered that refractive indices are highly temperature-dependent; therefore evanescent field devices must
either be well referenced in dual-channel instrumentation or thermostatted (down to 0.01 K). Both principles
monitor changes in optical density; in reflectometry,
however, the decrease of refractive index as a result of a
temperature increase is, by chance, nearly compensated
by an increase of the physical thickness of the layer due
to thermal volume expansion. Thus, reflectometry normally does not require thermostatting.
The combination of evanescent field excitation and
labelled systems is used for total internal reflection fluorescence (TIRF) monitoring of the fluorimetric
behaviour of molecules excited by evanescent fields close
to the interface of a wave guide within or at the sensitive
layer [11]. The disadvantage of the necessary labelling of
compounds (costs, expenditure, possibly reduced by
reactivity) is compensated normally by lower limits of
detection when fluorescence is used as detection principle. Accordingly, many modern biosensors use TIRF as
a working principle. Schematic drawings and detailed
explanations of the optical principles, and literature
citations, can be found, e.g., in [11, 49].
Many years ago, Förster introduced the principles of
resonance energy transfer using dipole–dipole interactions of a fluorescent donor dye and an acceptor dye, its
absorption fitting the fluorescence wavelength of the
donor [50]. For very close distances between these two
chromophores (distance d<10 nm, dependence d 6) the
fluorescence of the donor is reduced by this energy
transfer and the fluorescence intensity of the acceptor
increased. Frequently normal quenching effects are
superimposed on this energy transfer. Nowadays, this
principle is used as a detection method in many biomolecular interaction applications requiring two labelled
compounds, with appropriate chromophore properties.
This method turns out to provide a good possibility of
measurement in homogeneous phases, an approach
which is often preferable in bioassays [51]. No washing
steps are necessary. Donor dyes can also be combined
with such quenchers to avoid use of a second labelled
compound [52].
Sensitive layers and assay formats
Selectivity, sensitivity, stability, and reversibility are
requirements for sensor systems which must be provided
in part by the sensitive layers. The user expects a rather
high signal-to-noise ratio, short response times, low
limits of detection, high sensitivity, and—at low
cost—the possibility of using sensors for real samples
also, not just in laboratory applications. As a consequence of ion-selective electrodes, initially semiconductor material was doped and used as a sensing system.
Later, as already mentioned, dyes were embedded in
polymer films. Layers derived from chromatography
(simple polysiloxanes) [53] are used to produce rather
stable layers with high reversibility and very short response times. Even when these polymer layers were
functionalized, however, selectivity remained low and
the limit of detection for gases or liquid stayed in the
ppm range [54].
Because the mesogenic structure of liquid crystals can
vary quite sharply close to phase transition temperatures
[55], some applications of such liquid crystals are also
known, even very simple devices changing colour (e.g.
films for temperature measurement). Microporous
material provides the possibility of introducing selectivity according to the free volume, thus discriminating
molecules by size [56]. Recently, this sieve effect has been
combined with swelling properties of the polymer to
detect gases or liquids, depending on their molecular
dimensions and partition coefficients [57].
Modified biopolymers, on the other hand, have been
used for many years to provide high selectivity and
sensitivity. As reported below, applications have been
based on antigen–antibody interaction, inhibition of
enzymes, DNA/DNA hybridisation, protein–protein
interaction, and many other properties in the field of
membranes and signal cascades. Whereas polymers or
organic-sensitive layers participate in non-specific interaction (lack of selectivity), biomolecules should result in
receptor–ligand specificity. In heterogeneous phases,
however, non-specific effects are typical. For this reason
surface chemistry and modification are extremely necessary requirements when producing an efficient biosensor. The very high binding constant (1015 lmol 1) of
biotin and avidin is frequently used to immobilize biotinylated biochemical molecules on the transducer surface [58]. The disadvantage of such approaches is that
the system is not reversible because of the high binding
constant.
Another approach is the silanisation of glass or
quartz transducers with subsequent covalent binding of
various biopolymers supplying reduced non-specific
binding properties and enabling functionalisation with
ligands or receptors. This silanisation step can be characterised by NMR spectroscopy and ellipsometry [59].
Dextran hydrogels [60] supply a large number of functional sites within the volume [55]. Often, however,
especially when observing protein interactions, nonspecificity is not reduced sufficiently.
Another approach, therefore, is to bind poly(ethylene glycol) [61] of different chain length to the silanised
surface to produce a kind of a polymer brush. Ligands
can be immobilised by means of either amino or carboxy functions. These layers resist non-specific binding
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[62], but have a reduced number of interaction sites,
because they are restricted to the surface and not in the
volume.
Besides these principal approaches, many other ideas
have been realised, e.g. the use of His-tags [63] or the
immobilisation of membrane structures of lipid double
layers [64] to the transducer, to model cell walls. All
these different approaches are intended to reduce nonspecific binding, enable a large number of specific
binding sites, increase the stability of the layer, which is
essential for regeneration strategies, and increase selectivity and sensitivity.
Because, in contrast with simple polymer films, these
sensor types are not reversible, reusability has to be
introduced by use of regeneration strategies. As mentioned in the Introduction, these biosensors cannot be
called ‘‘sensors’’ by definition. However, being reusable,
this regeneration is often regarded as a substitute for the
required reversibility.
Although these biopolymers should increase the stability of biolayers, their stability is not comparable with
that of, e.g., polysiloxane films or microporous systems
[65]. For this reason, for many years supramolecular
structures [66] and biomimetic layers [67] have been a
wide field of research as attempts have been made to
combine the advantages of stability and reversibility
with sensitivity and selectivity. Artificial layers are synthesised with recognition structures comparable with
those of natural biomolecules. Supramolecular structures such as calixarene [68, 69] were tried first to increase selectivity of simple chemosensors. They were
used to separate, e.g., various chlorinated compounds.
Fig. 4 Assays using interaction between biomolecules in homogeneous phase and/or at heterogeneous interfaces. In both assays
thermodynamics (equilibrium constant) and kinetics (association
and dissociation rate constants) determine the interaction. Direct
assays immobilise the receptor at the surface to measure the
analyte, here a binding inhibition assay is demonstrated where
derivatives of the analyte or ligand to be detected are immobilised.
In the preincubation phase receptor and ligand are mixed in the
homogeneous phase and the concentration of non-blocked receptor
molecules is detected via the heterogeneous phase. Large numbers
of interaction sites at the transducer make this process diffusioncontrolled; at low ‘‘loading’’ the kinetics at the heterogeneous
phase can be measured
Another approach was the use of cyclodextrins [70] or
cyclohexapeptide [71] structures. Both could be functionalised to supply either dependence on ionic strength
or even discrimination of enantiomers. Such modified
cyclodextrins or polysiloxanes modified with optically
active amino acids [72], prove the capability of sensors to
measure enantiomers. Similar separation coefficients as
for gas or liquid chromatography have been demonstrated [73].
One of the first approaches used to introduce selectivity by immobilising polynucleotide or peptide sequences to the surface were hybridisation studies.
Meanwhile, PNA (with peptides as a backbone) [74] was
proven to be a better complementary binding system
than DNA because of less repulsion by charges and
better backbone stability; this resulted in stability
against DNases and nucleases. Locked polynucleotides
have recently been demonstrated to be another successful approach [75]. The polynucleotides were compared with PNA and DNA layers [76]. Further interest
has concentrated on lipid membranes as biopolymers,
enabling the construction of a variety of functional
systems (e.g. for transport of proteins [77, 78]).
Learning from nature, another approach is the synthesis of layers of molecular imprinted polymers [79].
The problem of these layers is that stability and selectivity are in reverse proportion to response times. Advances in this field have been reviewed in [80]. Their
potential optical sensing approaches for environmental
and industrial applications have been reviewed in [81].
Because of growing public concern with regard to health
and environmental problems, researchers are using
molecularly imprinted polymers in food and agriculture
[82]. Molecular imprinting technology has recently
emerged to produce biomimetic receptors that challenge
their unnatural counterparts. An overview of this
method and its application can be found in [83]. Despite
all these promising numerous applications and developments, their future perspectives have yet to be validated. Imprinting of the surface instead of the volume is
used in the so-called spreader-bar technique [84], in
which template molecules cause immobilised brushes at
the surface to rearrange around the template to form a
kind of ‘‘well’’ in which only molecules similar to the
Additional review articles
Bragg sensors [113]
Antigen/antibody interaction,
DNA hybridisation [111]
Biomedical [114, 115]
interferometric sensors [117]
Miniaturisation
Cyclodextrins [70]
Cyclohexapeptides [71]
MIPs [79]
Biomolecular interaction (dextran [60],
PEG [61], biotin [58])
Microporous polymers [56]
Hyperbranched polyesters [98]
Microporous polymers [56] liquid crystals
Biopolymer/antigen derivative
Hybridisation
Membrane–peptide interactions [94]
Biomolecular interaction, screening
Polysiloxane films [95]
Biopolymer/inihibitor derivative
Polymer films
Biopolymer/antigen derivative
Screening [107]
Reflectometric interference
spectroscopy [16]
Surface plasmon
resonance [27, 28, 36, 37]
Grating
coupler [24, 25]
Resonant
mirror [26, 35]
Mach–Zehnder
Interferometer [22]
Parallelisation
Parallelisation
Reflectometry
Refractometry
Direct optical detection
Evanescent field
FRET [50, 51]
miniaturisation
Indicator dye
Absorption [11]
Fluorescence [8–10]
TIRF [11, 87, 88]
Labeled systems
Biopolymer/antigen-derivative
in heterogeneous phase
Homogeneous assay,
phase-separation assay [91]
at heterogeneous phase
Polymer films [53],
liquid crystals [55]
Biopolymer/antigen derivative
Biopolymer/antigen derivative
Sensitive layer
Optical principle
Sensor type
Table 1 Survey of optical principles, assay formats, and selected related applications
Using quantum dots [116]
DNA intercalation
Low molecular weight analytes [104, 105]
Fermentation control [106]
Combinatorial synthesis [108],
triazines synthesis [105]
Epitope mapping [109]
Thrombin inhibition [110]
Phosphorylation
Signal cascade
Water analysis [112]
Hydrocarbons [96], aromatic compounds [97]
Volatile organic compounds (VOC),
Alcohols [99], freons [98, 100]
Enantiomers
Amino acids [101]
Enantiomers [102]
Pesticides [103], triazines [104]
Enantiomers [72], Process monitoring
Enantiomers [73], homologous
series of alcohols [92, 93]
Biomolecular interaction DNA [47]
Thrombine inhibitors
Pesticides
Hydrocarbons, aromatic compounds
Environmental analysis: pesticides,
EDCs [87, 89, 90]
Pesticides [88], nuclease assay [91], SNPs
Urea concentration [6]
Application
147
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template will later ‘‘bind’’. All these approaches are
interesting for heterogeneous phase assay.
In chemical sensors simple arrangements are usually
used for gas flow or fluidics. Flow controls are used for
the calibration procedure, for mixing different analyte
concentrations. Simple pumps which enable a change
between solvent and analyte/solvent mixtures at different concentrations are sufficient for the fluidics. In
contrast, biosensor systems use more sophisticated fluidics which enable changes of flow rates, preincubation
times, and different injection cycles between analyte,
analyte derivative, and reagent. These systems must also
provide the possibility of regeneration cycles.
As Fig. 4 shows, various possible assay formats are
known. Preferable is a homogeneous assay which enables direct interaction between the analyte and the reagent. The problem, however, especially in optical
detection is that this interaction must change optical
properties. Colour changes can occasionally be detected,
but with some difficulty. For this reason labelled systems
are used in homogeneous assays. In previous times,
radioactive labelling enabled very low limits of detection, but is nowadays not preferable. Therefore fluorescence labelling is normally used, taking advantage of
either quenching effects or (most often) fluorescence
resonance energy transfer.
For measurements in a heterogeneous phase it would
be preferable to immobilise the reagent in the biopolymer and detect the analyte directly. This is certainly
possible with labelled compounds using quenching effects; for direct optical detection without labelled compounds, however, preferably large analyte molecules
have to be examined. The sensitivity of any kind of
optical detection can be improved by increasing the
analyte mass or volume. Therefore, either a competitive
test scheme (labelled competing with non-labelled analyte) or a so-called binding inhibition test scheme is used.
This represents the following assay type: an analyte
derivative is immobilised in the biopolymer; in a preincubation step the analyte and the reagent are mixed
together; the analyte as a ligand blocks receptor sites,
which can no longer react in this blocked state with the
analyte derivative immobilised to the surface. This approach can be realised with a labelled reagent or direct
optical applications. A large number of interaction sites
in the biopolymer causes transport-limited (diffusionlimited) interaction between non-blocked receptor and
analyte derivative at the biopolymer. The slope of timeresolved measurements of the interaction process becomes linear and can be calibrated to the concentration
of the free receptor in equilibrium in the homogeneous
phase. If one measures in dependence on time during the
preincubation step, even association and dissociation
rate constants in the homogeneous phase can be obtained by simulation and taking account of the superimposition of the various processes [85].
A small number of immobilised analyte derivatives
on the biopolymer enables determination of the kinetics
in the heterogeneous phase by use of association and
dissociation rate constants. This biomolecular interaction analysis (BIA) is well discussed in literature [86].
Equilibrium and rate constants normally differ for
homogeneous and heterogeneous phases, and the
kinetics and thermodynamics are influenced by labelling
by a fluorophore [85]. This must be well considered
when discussing and comparing different biomolecular
interactions. Optical principles and assay types are
summarised in Table 1, with selected typical applications.
Applications
Remission measurements (changes in the absorbance of
reflected light) and fluorescence effects are the most
simple sensing methods. They enable very simple
arrangements which can sometimes cause artefacts.
They do not use the advantages of optics, namely
spectral detection. For this reason, occasionally, e.g. for
measurement of urea, in which the ammonia produced
causes a spectral shift of an indicator dye because of
changes in pH, a spectrum or at least at two wavelengths
have been recorded [6] to overcome artefacts. Intensity
quenching of fluorophores, measurement of anisotropy,
or even fluorescence correlation spectroscopy for singlemolecule detection, result in a wide variety of applications which cannot be reviewed in detail here but which
are the topics of quite a number of review articles [8,
113, 114]. A new approach is the use of quantum dots
[116].
This review concentrates on selected applications by
our group as given in the lecture focussing on some
typical examples. Beginning with chemical sensors and
detection in the gaseous phase, most of the results are
influenced by the humidity of the gaseous phase. This is
also true for polysiloxane as a polymer using the swelling
effect with RIfS. However, modification of these polysiloxane layers can reduce effects of humidity even for
chlorinated hydrocarbons as analytes [97]. As was proven for RIfS, a whole spectrum does not have to be
recorded—even four wavelengths are sufficient for
determination of the swelling effect [17] and perspectives
for highly parallelised detection were opened.
This uptake of analytes from the carrier gas into a
polymer film coated on the transducer or covalently
bound results in swelling of the polymer film which can
be detected by use of interference spectroscopy. Normally, Henry’s law is valid at low concentrations, and
the limit of detection is down to a few ppm. Higher
specific interaction results in Langmuir-type calibration
curves given by saturation effects at increasing concentration. These effects can be classified by separate measurement of refractive index and physical thickness
changes, by use of spectro ellipsometry [118, 119]. These
measurements demonstrate that most effects in nonselective polymer films are caused by changes in physical
thickness which result from swelling, whereas the effect
of changes in the refractive index is negligible. The
149
polymer films used are reviewed in [95]. Various types of
polymer, for example functionalised polysiloxanes [97],
esters, hyperbranched polyesters [98], or dendrimers
have been used for a variety of applications. Mach–
Zehnder chips have also been used to measure volatile
organic compounds (VOC) at low limits of detection
[120–122]. Chemical sensors based on polysiloxane layers have been compared with quartz microbalance,
calorimetric, and capacitance sensors [123]. Applications
of optical detection to chemo gas sensors have been reviewed [54].
The same films can be applied to the detection of
analytes in liquids using RIfS [96]; for this, however, the
polymer must be covalently bound to the transducer via
silanisation and peptide-like binding approaches. By this
means the stability of the films is increased from a few
days up to 3 months, because water can no longer lever
off the polymer film from the transducer [124].
Although the type of binding of the silanes to the
surface has been discussed, NMR studies have also
shown that normally even a tripoid immobilisation is
possible [59]. The properties of the polymers depend on
the cross-linking, therefore not all polymers can be used.
However, so-called microporous systems also have very
good, sensitive layer properties. In this case, microsieve
effects predetermine the feasibility of detection of mol-
Fig. 5 Time-resolved measurements of the interaction between a
mircoporous layer and a homologous series of alcohols (gaseous
methanol, ethanol, propanol, butanol): SPR signal/shift in resonance wavelength) versus alcohol concentration (relative partial
pressure mixed in a gas mixing station) and time. The predicted/
true graph represents the quality of the neural network based
evaluation. Good standard deviation is obtained for the small
alcohols [118]
ecules, which depends on their volume. The uptake of
gases or liquids increases the pore volume, however, thus
also causing swelling effects. However, saturation effects
(Langmuir-type curves) and observed effects on the
changes of refractive index demonstrate the limited
number of interaction sites.
These films are particularly useful in environmental
chemistry and process control; typical applications include monitoring of chlorinated compounds in the waste
water of chemical company production processes,
determination of concentrations of air-conditioner
refrigerants in, e.g., cars, or measurement of other
chlorinated and non-chlorinated hydrocarbons. These
microporous systems even enable separation of two
different refrigerants, R22 and R134a, down to rather
low limits of detection [98] and with a high reproducibility even in mixtures [100, 125].
These microporous systems also enable determination of the concentration of, e.g., homologous alcohols
in mixtures either by using a sensor array or even
applying chemometrics [92, 93]. It turns out that high
selectivity of each element of the sensor arrays for specific compounds in the mixture will not reduce problems
of cross-reactivity, but that this can be achieved by
application of modern tools of chemometrics, for
example neural networks or neural networks in combination with evolutionary strategies. Normally, either the
area or the slope of a signal is measured in equilibrium.
However, the modern tools of direct optical detection
enable the application of time-resolved measurements.
These have the advantage that the signal is measured
during the interaction process at as many time-points as
necessary. An example is given for a mixture methanol,
ethanol, propanol, and butanol. Microsieve effects of
150
microporous systems support the discrimination. An
array of sensors (RIfS) or even a single element (using
surface plasmon resonance) causes a minor percentage
of errors. This has been published for ternary mixtures
[93]. Even a quaternary mixture can be handled, as is
demonstrated for the signals given in Fig. 5 [99]. Even
better results could be achieved by applying chemometrics to the refrigerants, for which the uncertainty is
close to 2% [100].
Functionalisation of polysiloxanes has even enabled
separation of enantiomers. The feasibility was proven in
a comparison of mass-sensitive and reflectometric
methods with Chirasil-Val [72]. Another application is
the use of biomimetic structures based on cyclodextrin
to monitor the anaesthetic sevoflurane. In an alkaline
rebreathing circuit the inhalation anaesthetic degrades
into a least two products; one of these is a chiral halodiether and one of the enantiomers has a different,
narcotic action [126]. By use of a modified Lipodex E
(cyclodextrin derivative) column, the chiral separation
factor obtained at 30C was larger than nine, the same
as in capillary gas chromatography. Comparable values
were obtained in a comparative study of enantioselective
recognition by use of thickness shear mode resonators,
surface acoustic wave sensors, surface plasmon resonance, and reflectometric interference spectroscopy [73],
thus proving that chemosensors using modified cyclodextrins enable enantioselective detection of a halogenated diether.
In this work a far higher separation factor was
achieved than for some molecular imprinted polymers
(MIP) [102]. With MIP, however, rather high separation
factors could also be achieved [127].
In other approaches, surface-bound cyclohexapeptides have been used as elements for molecular recognition of amino acids [101]. The chiral cyclopeptide
libraries can be used as chiral receptors. The cyclohexapeptide was immobilised on the transducer by
means of three lysines; the other three positions were
varied.
Biomimetic structures are also used for label-free,
product-specific monitoring of biotechnological processes used to manufacture the glycopeptide antibiotic
vancomycin. Usually, the pH or oxygen content and the
temperature are controlled during the fermentation
process; only glycose [128] is measured, however,
whereas normally process control is performed by use of
HPLC. A lysine-D-alanine-D-alanine sequence has been
immobilised on the transducer. Use of RIfS enables
detection of vancomycin even during the fermentation
process [106]. This approach led to the thought of
combining a parallel RIfS system with MALDI-TOF
[129], because the measurement of biomolecular interaction by RIfS even includes degradation products
interacting with the peptide sequence immobilised on the
surface.
A typical requirement, especially in biomolecular
interaction, is to measure as small molecules as possible.
As mentioned in the section on optical principles all
label-free detection methods have problems detecting
small ligands at low concentration and with small
binding constants. Therefore SPR technology and RIfS
were tested for such detection. Two results can be referred to for RIfS, first the interaction measurement of
biotin with avidin [104] and the on-line monitoring of
solid-phase peptide synthesis on glass-type surfaces
[105]. Label-free detection is reviewed in [130].
The applications already mentioned all used singlechannel detection. However, considering future detection requirements for combinatorial libraries, parallel
sensing has extreme advantages over other methods
[107]. Also, determination of binding constants together
with association and dissociation rate constants for
antibody–triazine-derivative interactions, with a large
number of calibration steps and replica measurements,
made the need for parallelisation obvious [131]. Combining the idea of using single wavelengths instead of
white light interference, as in the four wavelength set-up
used for cost-effective chemosensing, a parallelised system was developed [132]. Some of the applications of
this will be mentioned here.
Primary screening for lead compounds requires
combinatorial synthesis which should be closely linked
to the drug-discovery process [133]. Until now, the usual
screening procedure uses radioactive or fluorescence-labelled detection techniques directly on the resin beads
after a split-and-combine library synthesis [134]. Because
direct optical detection without labelling is an interesting
alternative, direct label-free RIfS detection on a microtitre plate format using simultaneous imaging by a
CCD camera was applied to the synthesis of a triacine
library [108]. The selectivity of an anti-simazine antibody was evaluated by measuring biomolecular interaction processes with a variety of immobilised triazine
derivatives with different substituents in two positions.
Thus, in a single measurement cycle a library of 36
derivatives was examined. Single-channel RIfS also enabled direct control of the synthesis in one well, proving
the feasibility of this using method to monitor the
interaction of small molecules [105].
Another approach which proves the benefits of
screening is the use of a parallel affinity assay for
thrombin inhibitors in label-free screening HTS detection. By using a binding inhibition assay, the screening
of 384 substances for thrombin activity can be performed within less than 15 min. The optical reproducibility is high enough to support a data quality which
enables parallel quantification of the IC50 values (half of
the receptor sites are blocked) of the library substances
[110]. Screening could also be performed with 5%
DMSO added to the samples; this is relevant in highthroughput screening (HTS) applications in practice in
which pure water cannot be used as a solvent.
Finally epitope mapping of transglutaminase (tTGase) is given as an example for HTS. The enzyme tissue
tTGase has been identified as the major autoantigen in
coeliac disease and an antibody has been developed. To
learn the sequence of amino acids a binding assay with
151
Fig. 6 Epitope mapping to determine the sequence motif of
transglutaminase (tTGase). Twenty-one peptide sequences are
synthesised by combinatorial chemistry and peptide 6 is found to
be a lead structure. When this is immobilised on the transducer,
antibody produces a large increase in layer thickness and buffer
solution results in very low signal. The better the peptide sequence
fits the antibody the smaller the increase in layer thickness and
signal. The measured binding curves are evaluated to give
calibration curves and binding constants (right)
various peptides would be helpful. Therefore by combinatorial chemistry 21 peptides were synthesised as
depicted in Fig 6. A binding inhibition assay is set up. A
lead structure (known from enzyme-linked immunosorbent assay, ELISA) is immobilised in all the wells of a
microtitre plate arrangement. First, buffer is spilled over
the plate and, as can be seen in Fig. 6, no binding signal
is measured using RIfS. If antibody alone is in contact
with the immobilised lead structure a high signal is obtained. Now antibody and the sequences are preincubated and using three replicates these different solutions
are dispensed into the different wells of the microtitre
plate. According to the complementarity of peptide and
antibody sequences the binding pattern in Fig. 6 is
achieved, demonstrating that the lead sequence is not the
best matching sequence. Using the binding curves and
repeating just the interesting measurements in a singlechannel device binding constants can be discriminated
[109]. Grating-coupled surface plasmon resonance
Table 2 Results from measurement of pesticides and endocrine
disruptors by use of TIRF
Analyte
IC50
(lg L 1)
SD (%)a
LOD
(lg L 1)
Atrazine
Atrazine-diset.
Simazine
Isoproturon
Alachlor
2,4D
pcp
Carbofuran
Estradiol
Ethinylestradiol
Estrone
Bisphenol A
0.082
1.337
0.54
1.211
0.53
0.89
22.680
6.409
0.590
1.070
0.145
0.670
2.74
2.74
1.7
1.11
3.98
2.15
1.45
1.28
2.98
3.20
1.18
1.73
0.006
0.039
0.03
0.016
0.07
0.07
1.120
0.024
0.060
0.070
0.006
0.007
a
Standard deviation of blank measurements
equipment is on the way to being commercialised by
HTS Biosystems [135].
Modern spotting technology rather than pipetting
was used for these parallelised measurements [136].
They have advantages over micro arrays and not using
well plates.
Parallelisation is common in fluorescence methods.
Either simple fluorescence techniques with a reader
[137], TIRF [138, 139] or FRET [45] are used. A typical
application is in environmental analysis [89]. Classical
analytical methods to determine pesticides in ground or
waste water need enrichment steps but can discriminate
between different analytes [140]. Therefore ELISA assays have been introduced. These assays do not enable
automated monitoring at low concentrations, however.
TIRF can overcome this problem. At present six spots
on a wave guide are used to set up a binding-inhibition
assay in combination with a flow-injection analysis
system. Analyte derivatives are immobilised, a different
one at each spot. Antibodies are added to the preincubation solution. Missing analyte (the pollution)
causes fluorescence at a specific spot which is read by
means of fibre optics [49]. Some data are given in
Table 2 [87, 141].
Another possibility is the use of FRET in environmental analysis using micro or nanotitre plates [88].
Both methods have been referenced for real water
samples to classical GC–MS results. TIRF and FRET
have been compared [103]. Chemometrics have been
applied to overcome the problem with non-specific
antibodies. For endocrine-disrupting compounds (Table 2) recovery rates could be improved and limits of
detection reduced [90].
To enable the change of analyte derivatives within the
flow-injection system (FIA) without removal of the
transducer, DNA sequences have been immobilised on
the TIRF transducer (auxiliary system). Matching
strands carry the analyte derivative. However, DNA is
not stable enough and reduces the number of interaction
sites by charge repulsion. Therefore instead of DNAstrands the PNA (peptide backbone) was used to improve the system [142].
Miniaturisation is the final topic to be covered [143].
Over the last decade many papers have been published
dealing with miniaturisation, and lab-on-chip techniques
in particular. Micro total analysis systems were devel-
152
Fig. 7 Highly parallelised RIfS set-up for study of interactions
between antibodies and antigens in a binding inhibition assay. A
miniaturised system is used with up to 384 spots on 12·18 mm2. On
the left the real-time binding curves are drawn from the data array;
on the right one of these curves is enlarged
oped many years ago at Ciba Analytical Research. Recently, a new review has been published by one of the
inventors in this field [144]. Miniaturisation, integration,
and systemisation of various types of sensor have been
considered with regard to modern technologies in micro
fabrication; these techniques have found considerable
interest in Japan [145]. The development of miniaturised
immunosensing devices has been reviewed as a small
technology with a large future [146]. Pocket-size analytical equipment based on the lab-on-chip approach has
become available and is intended for use in biomedical
and environmental monitoring [147]. This chip technology is also of interest for any type of DNA chip, and
will enable improved analysis in proteomics; it provides
a type of proteomics-on-a-chip and is a challenge to
couple lab-on-chip with microfluidics and detection
platforms [148]. Although all these approaches face
limits of detection problems, they are the prerequisite of
another very interesting approach in optical sensing
dedicated to enable multi-parameter or multi-analyte
monitoring in parallel. Therefore, miniaturisation usually appears together with parallelisation. Parallelisation
can be increased by reducing spot or well size. As
mentioned above, the use of nanotitre plates takes
advantage of reduced sample volume and the reduced
amount of reagent necessary. An interesting approach is
combination of homogeneous and heterogeneous phase
assays as realised in a so-called phase separation assay.
The wells are coated with gold and the derivative is
immobilised via thiol groups. By this means the
dynamics of the fluorescence signal are increased [91]. In
many applications, however, an FIA system is preferable. Thus, for some time, new approaches for micro
fluidics have been discussed.
So-called hybrid systems supply miniaturised chips
and flow channels and overcome problems with pumps
and valves, which cause some problems in the handling
of biomolecules. The transport equations have to be
solved and diffusion processes simulated to guarantee
perfect mixing [149].
The results of a miniaturised set-up [111] for direct
optical detection with RIfS are shown in Fig. 7. The
time-resolved binding curves of 96 spots for antigen–
antibody interaction are given. Kinetic evaluation is
possible. Currently, however, monitoring of up to 384
binding events is restricted to large molecules.
Conclusions
Optical sensors have proven in the past to be either very
simple and cost-effective devices or enable rather
sophisticated multisensor applications. Because of the
existence of many different optical principles which can
be classified into use of direct optical detection or taking
advantage of labelled compounds, in principle many of
these methods can be applied to a huge number of
applications. It is becoming evident that of the different
sensor principles—electronic, electrochemical, masssensitive, or optical devices—none is generally superior,
but rather the feasibility depends on the application. The
same holds true for the different optical sensor principles. This became obvious when comparing various
refractometric and reflectometric methods in the same
biomolecular interaction study using antibodies and
antigens, and setting up the surface chemistry by the
153
same person. In both cases of two studies the limits of
detection for all the methods examined ranged within
one order of magnitude. Discrimination was achieved
only at the cost of expenditure on apparatus and by
sophistication of the fluidics used [150, 151].
Reliable results can be obtained with either chemosensors or biosensors. The selectivity and limit of
detection are usually better for biosensors. Chemosensors, on the other hand, provide reversibility and greater
stability of the sensitive layer. It turns out that the
quality of the set-up depends not only on the optical
method but also, especially, on this sensitive layer. For
this reason, most of the improvements that can be expected in optical detection methods are in the area of
sensitive layers. This becomes obvious from looking at
developments in biometics or functionalised polymers
[47, 48, 78].
The essential result of these considerations is certainly
that research in sensing requires interdisciplinary
understanding of the detection principles, of the sensitive
layer, of the kinetics and thermodynamics of interaction
processes, and of the fluidics. Thus fundamental research
must be performed to characterise these layers and the
interaction processes to improve the understanding
which is the prerequisite of any optimisation approach.
Whereas laboratory systems often give very good
results and even enable separate determination of concentrations in multianalyte mixtures, the quality of the
overall sensing system normally becomes obvious when
these systems are applied either to real environmental
samples, e.g. waste water, to saline solutions, or, on the
other hand, to blood or sera [152–155]. Sensors turn out
to be a typical modern example of interdisciplinary research, considering multivariate parameter arrays.
Acknowledgements For long years of support of his research the
author has to thank the Deutschen Forschungsgemeinschaft, the
Fond der Chemischen Industrie, the BMBF, the Arbeitsgemeinschaft Industrieller Forschung, the Deutsche Bundesstiftung Umwelt, some European funding and much industrial cooperation.
Details of the funding is acknowledged in the different publications
cited. The author also wants to thank all his coworkers, cited and
not cited, for work achieved, and, especially, Dr Martin Mehlmann
for checking the manuscript.
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