Trends in Analytical Chemistry 156 (2022) 116708

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Trends in Analytical Chemistry

journal homepage: www.elsevier.com/locate/trac

Through the looking-glass - Recent developments in reflectometry

open new possibilities for biosensor applications
Peter Fechner*, Günter Gauglitz, Günther Proll
€t, Auf der Morgenstelle 18, 72076, Tübingen, Germany
Institute of Physical and Theoretical Chemistry, Eberhard Karls Universita

a r t i c l e i n f o a b s t r a c t

Article history: Label-free biosensing has developed from niche application to state of the art biosensing technique
Received 25 January 2022 throughout the past decades. The reason for the raise of acceptance for this technology is the fact, that it
Received in revised form generates valuable kinetic data (on- and off-rates), does not require complicated sample-pre-treatment,
30 May 2022
and can be used in direct test formats. As the acceptance increased, label-free advanced and many
Accepted 3 June 2022
different approaches with the same underlying principles have evolved. This review will take a brief look
Available online 6 June 2022
at the history, and explore the reflectometry family tree, which includes ellipsometry as the main
ancestor, Reflectometric Interference Spectroscopy (RIfS), Biolayer Interferometry (BLI), Total Reflecto-
Keywords:
Biosensor
metric Interference Spectroscopy (TRIS), Spectral Reflectance Imaging Biosensor (SRIB or also IRIS), 1ʎ
Label-free Reflectometry, Arrayed Imaging Reflectometry (AIR), and Oblique-Incidence Reflectivity Difference mi-
Reflectometry croscopy (OI-RD) and highlight extraordinary examples of recent developments and their impact in
Kinetics biomolecular interaction analysis.
Optical transduction © 2022 Elsevier B.V. All rights reserved.
Sensor

1. Introduction particularly high performance (e.g. low detection limits). However,

Understanding the interaction between biomolecules is crucial
for many aspects in modern life sciences, and dysregulation of
these processes often leads to severe diseases. Therefore, biomol-
ecular interaction analysis (BIA) is an important aspect when it
comes to investigate, understand, and modify these processes. BIA
can be carried out in many different ways [1,2]. Label-free ap-
proaches represent an important part in BIA because these tech-
niques require only little sample pre-treatment and the behaviour
of the biomolecular interaction is not altered by labels [3]. Direct
optical detection methods can be divided into refractometric and
reflectometric transduction principles. Even if this boundary seems
to be somewhat blurred in some hybrid systems, in the case of
refractometric dominance of a measurement technique, the
evanescent field is typically influenced by changes in the refractive
index and is thus responsible for the signal generation. Among
others, Surface Plasmon Resonance (SPR) [4e6] or Grating Couplers
[7] are two of the most prominent methods in this group. These
technologies are also commercially very successful and are used in
a large number of applications, not least because of their
* Corresponding author.
E-mail address: peter.fechner@uni-tuebingen.de (P. Fechner).
the measurement of smallest refractive index changes places
highest demands on the thermostatization of the biosensors and
the referencing in order to prevent the negative influence of small
temperature changes during a measurement. This is in contrast to
transduction principles, in which the change in physical layer
thickness also contributes significantly to signal generation. This
review will focus on the latter, namely reflectometric approaches.
Reflectometric approaches can be seen as a special subgroup of
label-free BIA. Although representing a rather small group
compared to the previously described refractometric approaches,
this field has extensively evolved over the last years, which is re-
flected by their commercial success. Furthermore, they will
continue to play a crucial role in the future, not only due to their
commercial success, but also due to some intrinsic advantages
compared to other approaches.
This review will guide you from the theory underlying all
reflectometric approaches to the different realisations that have
evolved over the years and highlight the recent key developments
in each field.
When light interacts with matter, phenomena occur at phase
boundaries that can be described by the complex refractive index.
For example, when a light beam hits a phase boundary, part of the
light is reflected, the rest penetrates into the new medium and that
part of the light which penetrates into the adjacent medium

https://doi.org/10.1016/j.trac.2022.116708
0165-9936/© 2022 Elsevier B.V. All rights reserved.
P. Fechner, G. Gauglitz and G. Proll
changes its direction according to Snellius' law. In the case of non-
absorbing media (e.g. gas or water in the visible light range),
Snellius' equation consists only of real numbers. However, the
polarisation state must be taken into account when calculating the
transmission and reflection coefficients. The reflection and trans-
mission coefficients are described by Fresnel's formulas. Here, the
Fresnel coefficients include the amounts of the respective ampli-
tude ratios of incident and reflected or transmitted light as well as
the phase shifts of the reflected and transmitted partial beams with
respect to the incident light. When comparing the Fresnel co-
efficients for light polarised parallel to the plane of incidence (rp) to
light polarised perpendicular to the plane of incidence (rs), it be-
comes clear that the course of the reflectivities is different. For
example, if we look at the reflectance at an air-glass phase
boundary as a function of the angle of incidence, we notice that rp
passes through a zero point which is why the reflectance at the
corresponding angle is zero for these non-absorbing materials.
From this it can be deduced that in general for non-absorbing
materials the reflected beam is completely s-polarised under
these conditions. This angle is called the Brewster angle. It should
therefore be noted that for reflection and transmission, there is a
phase shift between incident and reflected or transmitted light.
Furthermore, for the transduction principles discussed in this re-
view, it is important to note that when reflecting from a layered
system, the reflections at each individual phase boundary must be
considered. The reflection coefficient of a layered system is there-
fore calculated from the reflection coefficients of the individual
phase boundaries (see Fig. 1). Taking into account all reflections and
transmissions that take place, the result is an infinite number of
partial waves corresponding to an infinite mathematical series.
This multiple reflection of electromagnetic radiation on at least
one thin layer is a typical characteristic in the signal generation of
micro-reflectometric biosensor principles. In general, these optical
transduction principles in biosensing discussed here exploit the
influence on the reflection of electromagnetic radiation at the
interface, including resonance and interference effects. Conse-
quently, these direct optical detection methods all measure the
changes of refractive index n and the physical thickness of an
interaction layer d or respectively changes of the product of both
parameters (n
d) called optical thickness. This distinguishes the
micro-reflectometric methods as a subgroup of direct optical label-
free transduction principles from micro-refractometric methods
(not discussed in this review) which mainly measure changes in the
Fig. 1. Reflection and transmission at a thin-layer system Light coming from the
substrate is reflected (R1) at the interface of substrate/thin layer and transmitted into
the thin layer. At the interface of thin layer/superstrate, light is again partially trans-
mitted (T1) into the superstrate or reflected. This happens multiple times (R2, T2, R3,
T3, …) and as a result, an interference pattern is formed.
2
Trends in Analytical Chemistry 156 (2022) 116708
refractive index which usually use an evanescent field of electro-
magnetic radiation for this purpose. Due to the decrease in the
intensity of the evanescent field with increasing distance from the
surface, the sensitivity of the measurements changes continuously
across the thin film in micro-refractometric methods. In contrast,
the sensitivity of micro-reflectometric methods remains approxi-
mately constant over the entire distance of the sensitive layer
(which is in principle only limited by the coherence length of the
light source).
2. Discussion
The phenomena described above can be utilized in different
ways. We will focus on the possibility to use reflectometry as a tool
in label-free biomolecular interaction analysis. Many of the
following techniques seem related to each other. An example how
closely related some of these approaches in terms of signal gener-
ation are, is shown in Fig. 2.
A basic schematic how each of these approaches can be realised
can be found in the following paragraphs. However, we want to
point out that these illustrations are simplified (e.g. moving parts or
lenses have been removed for clarity). For a more accurate depic-
tion, please refer to the primary literature.
2.1. Ellipsometry
Ellipsometry can been seen as the “most common ancestor” of
the majority of reflectometric methods. Therefore, the theory of the
working principle of an ellipsometric measurement also aids un-
derstanding the measurement principles of the following methods
and the basic working principle is shown in Fig. 3.
Ellipsometry is an optical method for the non-destructive ex-
amination of surfaces and thin organic and inorganic layers and
was originally used primarily in the semiconductor industry. With
this measurement method, the change in the polarisation state of
light is measured when it is reflected from a surface or a layer
system, thus allowing the physical thickness (d) and the refractive
index (n) of a thin layer to be determined independently. For this
purpose, light polarised both parallel and perpendicular to the
plane of incidence (s- and p-polarised partial beams) is irradiated
onto the surface of the thin film where it is reflected after several
reflections within the thin film. The ratio of the resulting ampli-
tudes of the two modes as well as their phase difference result in
two experimental “spectra” depending on the wavelength. The
determined ellipsometric angles J and D (J and D are the relative
amplitude and phase difference for linearly p- and s-polarised light
before and after reflection from the surface of the sample, respec-
tively) are related to the phase shift of the s- and p-polarised partial
beams. In other words, the change in the polarisation state of the
light reflected from the sample can be represented in terms of J
and D which are related to the ratio of the total reflection co-
efficients rp and rs at oblique incidence angles. Considering r as the
reflection ratio and ß as the phase, the fundamental equation of
¼ j rps jeiðbp bs Þ ¼ tanJeiD , where the ellipso-
rp r
ellipsometry is r ¼ rs
metric parameters D and J are related through r. A model is fitted
to this measured data, providing the value for both d and n.
An ellipsometric measurement at one wavelength and angle can
yield a maximum of two unknown quantities. If the number of
unknown parameters is > 2, correspondingly more J and D -values
must be used for the calculation. There are three basic technical
methods to choose from: 1) VAE (Variable Angle Ellipsometry):
Measurements are carried out using a monochromatic light source
at several angles of incidence; 2) SE (Spectroscopic Ellipsometry):
Spectroscopic measurements are carried out at a fixed angle of
P. Fechner, G. Gauglitz and G. Proll
Fig. 2. Signal generation in reflectometry A: If the layer composition of a thin-layer system changes over time, e.g. upon binding of an analyte, the interference spectrum changes.
This change can be either monitored by 1: following the x-direction shift of an extreme point (Biolayer Interferometry (BLI), Reflectometric Interference Spectroscopy (RIfS), and
Total Reflectometric Interference Spectroscopy (TRIS)) or by 2: the intensity change of a single (or small band) wavelength (Spectral Reflectance Imaging Biosensor (SRIB/IRIS) and
1ʎ Reflectometry).
B: Both approaches result in the same information, a time-resolved signal whose intensity changes over time and corresponds to binding events.
Fig. 3. Ellipsometry An example realisation of ellipsometry consists of a light source
(1), and a polarisator (2). The light (dotted line) is then reflected at the substrate (3
dark grey) containing the sensitive layer (3 light grey). Afterwards, it passes an ana-
lysator (4), a monochromator (5) before it is detected by a photomultiplier (6).
incidence; and 3) VASE (Variable Angle Spectroscopic Ellipsom-
etry): Both angle and wavelength are varied. In all cases, data
evaluation is performed by nonlinear regression. At the same time,
the user must always be aware that this evaluation represents a
modelling that can also provide solutions that are not uniquely
determined.
A significant advantage of ellipsometry over several other
reflectometric methods is that polarisation states can be measured,
making this method insensitive to intensity variations of the light
source. Thus, the intensity difference after reflection of s- and p-
polarised light is very small for small film thicknesses or film
thickness changes, but a 10
to 100
larger change in phase shift
D can be determined. However, the model does not yield a defini-
tive physical answer for very thin films, since in the mathematical
solution n and d are strongly correlated. For this reason, and
because it is affected by temperature effects, ellipsometry is pref-
erably used to characterize biosurfaces and is not yet a tool for
normal biosensing.
The merits of classical ellipsometry using silicon wafers as
transducers come to the fore in applications such as those shown by
Ref. [8] in the rapid and sensitive detection of immunoglobulin G
(IgG), where a detection limit of 15 ng/mL could be achieved.
Another affinity biosensor using aptamers as recognition elements
for mercury ions was described by Ref. [9]. However, of particular
interest is the low detection limit for e.g. cardiac troponin I from a
serum of 10 pg/mL that could be achieved by the Rotating Analyser
Ellipsometer (RAE) configuration [10]. In addition to these
3
Trends in Analytical Chemistry 156 (2022) 116708
exemplary classical ellipsometer applications in biosensing, there
has been a clear trend towards multiplexing in recent years. This is
done very successfully by imaging ellipsometry as described for
example in the field of biomarker detection of Carcinoembryonic
Antigen by Ref. [11] or also for further screening of antibodies,
bacterial and viral pathogens [12]. However, the latter publication
also pointed out the trend several years ago that imaging ellips-
ometry can be improved by further combinations of methods. Thus,
there are quite a few application examples in this review that
already include a measurement setup using Total Internal Reflec-
tion (TIRE). For example, in literature there are related articles in
which this variant of ellipsometry is applied to detect surface
markers on exosomes [13] or, in a slightly modified form by
attenuated internal reflection, to detect mycotoxins via aptamer
assays [14], or to quantify Brevetoxin B with a LOD of 0.80 nM using
specific aptamers [15]. Although modifications of ellipsometry with
plasmonic components can no longer be counted as classical
reflectometric methods, it is interesting to see how this plasmonic
ellipsometry can even be used to study brain activity or brain tissue
[16], or, with the aid of plasmonic substrates, to study mesen-
chymal stem cells [17]. A review paper on this topic was published
by Sohrabi [18] in 2021. In addition, various combinations of
ellipsometry with nanomaterials have also been proposed by
Ref. [19], as also shown in a work by Ref. [20] and his group who
were able to study human serum albumin using porous alumina
oxide.
2.2. RIfS
Reflectometric Interference Spectroscopy (RIfS) is also a label-
free optical method, which can be used to measure changes in
the optical thickness (product of n and d) via a change in the
refractive index and physical layer thickness on a sensor surface. In
contrast to ellipsometry, no polarised partial beams are used here,
but a simpler and more robust method originally introduced by
Fabry and Pe rot as a measurement of the reflectance of two
superimposed beams reflected at two interfaces of a thin layer. The
technical realisation is typically based on a multimode fiber optics
(glass or polymer) into which the light of a white light source is
coupled (see Fig. 4). This white light is irradiated without further
spectral selection mainly perpendicularly onto a glass transducer
from the side facing away from the flow cell. The part of the radi-
ation reflected from the transducer or the biolayer is captured by
P. Fechner, G. Gauglitz and G. Proll Trends in Analytical Chemistry 156 (2022) 116708

between drugs and human serum albumin or also to study the

thrombolytic process of drugs [32]. This series of developments
also includes RIfS variants with silicon microwells [33] or even
porous nanostructures [34]. Of particular note, however, is the
work of [35] in which special silicon chips are used that have re-
gions with different silicon dioxide layer thicknesses. This approach
enables multiplexed detection at fixed positions. This technique is
referred to there as White Light Reflectance Spectroscopy (WLRS)
and is used for the detection of mycotoxins. In addition to these
transducer-side improvements, there are also approaches as
Fig. 4. Reflectometric Interference Spectroscopy (RIfS) A traditional realisation of RIfS described in Ref. [36] that attempt to optimize signal generation. In
consists of a white light source (1), which is coupled to an optical fibre (2). The fibre is this example, RIfS is modified towards polarisation RIfS by using p-
index matched to a transparent substrate (3 dark grey) containing the sensitive layer (3
and s-polarised light. This more complex setup is again more
light grey) and then the light (dotted line) guided via the fibre towards the spec-
trometer (4). similar to the principles of ellipsometry but allows to achieve a
detection limit 4e7 times better than with normal RIfS demon-
strated by characterizing molecular adsorption and nanoparticle
the same light guide and directed via a branch (Y-piece) towards adhesion.
the detector, which in this case is typically a diode array spec-
trometer. As a method in biosensing, RIfS is therefore based on the
2.3. BLI
reflection of partial beams of white light at different phase
boundaries of a transducer. A characteristic spectrum is obtained
In contrast to RIfS, Biolayer Interferometry (BLI) does not use
via the interference of the phase-shifted partial beams, which
planar transducers but instead employs so-called fibre tips as
contains information about the optical thickness of the biolayer
consumables on which the interference of partial white light beams
[21]. However, in contrast to ellipsometry, it is not possible to
(caused by reflection at the two phase boundaries of the biolayer)
discriminate between n and d anymore. A detailed discussion of the
ensures signal generation analogous to RIfS (compare Figs. 4 and 5),
theory of Reflectometric Interference Spectroscopy can be found in
therefore the measured signal is the product of n and d, the optical
Ref. [22].
thickness. By combining BLI with the microplate format by the
A major advantage of RIfS is its robustness to temperature var- Bio company (now part of the Sartorius Group), it became
Forte
iations, which is due to the opposing effects of the temperature on n
even possible to determine kinetic and thermodynamic parameters
and d, both of which are largely compensated by the product for-
by moving these fibre tips (so-called orbital flow) within the
mation to the optical thickness. This has been shown, for example,
microplate wells without the use of a flow-injection analysis (FIA)
in the determination of melting curves of double-stranded (hy-
system. This advantage of BLI has contributed to its commercial
bridized) oligonucleotides at the sensor surface [23]. Moreover, the
success as evidenced by the large number of applications. However,
transducers are typically made of glass (or another transparent
one of the remaining limitations, as with RIfS, is the limited mul-
material such as indium tin oxide) and thus allow a variety of
tiplexing capability compared to imaging techniques in combina-
coating methods starting from a silane chemistry with different
tion with microarrays, such as imaging ellipsometry or the 1ʎ
biopolymers/biomolecules. However, the multiplexing capability of
Reflectometry discussed below, for the same reason. Nevertheless,
RIfS is limited by the spectrometer (diode array spectrometer)
BLI has already been successfully used for high-throughput
required for detection. To overcome this limitation, it is possible to
screening due to the automated devices available. For example
measure time resolved the intensities of selected wavelengths
[37], used this technology for kinetic screening of T-cell receptors
serving as bases for the reconstruction of a spectrum. This approach
(TCRs) on peptide major histocompatibility complexes (pMHC). In
can be realised as low-cost sensors [24] or even as an imaging
addition, BLI has also found its way into biotechnological/phar-
device for microplates [25]. However, the time resolution of this
maceutical development, as demonstrated by the work of [38] in
measurement method is limited by the time required for the
which data for antibody self-interaction was obtained in this
sequential measurements of the single wavelengths.
manner to assist lead and candidate selection. There are also
RIfS in its original form was used by Ref. [26] to build a paraoxon
multiple developments that show the use of BLI in diagnostics. For
sensor with the enzyme acetylcholinesterase, achieving a detection
example, in Ref. [39] a good overview is given on biosensor
limit of 9.9,108 M. In the same year, our group published a paper
investigating the adsorption kinetics of proteins on biomaterials
using classical RIfS [27]. An overview of the many applications of
this method can be found in Ref. [3]. As already described for
ellipsometry, the trend for RIfS, in addition to improvements in
transduction, is also to move towards new transducer materials and
amplification methods with the aim of generally improving
analytical performance. This includes, for example, the use of
porous silicon oxide as reusable transducers for the detection of
single nucleotide exchanges in DNA in combination with a Fourier
transform version of RIfS [28] or, in a similar technical realisation,
the quantification of the human growth hormone from blood [29].
Another slight modification of the detection towards a phase-shift
RIfS was proposed by Ref. [30] for the elucidation of bacterial net-
works and their antimicrobial susceptibility. Here, additional silica Fig. 5. Biolayer Interferometry (BLI) BLI is realised by using a white light source (1),
which is coupled to an optical fibre (2). The fibre is connected to a fibre tip (3 dark
microarchitectures are used on the transducer side. Similarly,
grey), which contains the sensitive layer (3 light grey) and is immersed into a
colloidal silica crystal films have also been used for some time [31]. microtitre-plate well. Then the light (dotted line) is guided via the fibre towards the
uses these materials, for example, to monitor the binding affinity spectrometer (4).

4
P. Fechner, G. Gauglitz and G. Proll
methods in the field of neurodegenerative disease diagnostics
including the BLI technology. Even as an on-site method, BLI is used
in the detection of carbamazepine in whole blood [40]. Another
recent application concerns the investigation of the target binding
activity of CRISPR-Cas effector complexes as shown by Ref. [41]. In
addition to these new application developments, BLI is of course
used in classical areas for which the benefits of direct optical bio-
sensing methods have become widely known. As an example, we
refer to small molecule detection [42], which in the case of BLI can
also handle very small molecules (<200Da) [43]. Another general
trend in biosensing is the method combination of interaction
studies and identification via mass spectrometry. Examples of this
application can also be found in literature in combination with BLI
[44]. takes advantage of these two techniques for affinity studies
between macromolecules and for their identification, and the
technique can even be used for complex biological mixtures.
2.4. TRIS
Total Reflectometric Interference Spectroscopy (TRIS) can be
considered as a special variant of RIfS. Just as RIfS, it is based on the
multiple reflections of white light at a transparent thin-layer sys-
tem forming an interference pattern which is monitored over time
using a spectrometer. This interference pattern shift over time is (as
for BLI and RIfS) the change in optical thickness over time. Contrary
to RIfS, it utilizes e as the name implies e total reflection, and
therefore measures the reflection under an angle (see Fig. 6).
Furthermore, this approach uses polarised light to increase the
amplitude of the recorded interference spectrum.
The main advantage of this method is that working under total
reflection condition eliminates the effects of backscattering and
reflection of e.g. a flowcell backside, which would result in un-
wanted noise or intensity offsets. The latter being an issue
considering that working with white light results in quite a large
penetration depth.
Although the method offers some advantages, it is very rarely
applied to biological interaction analysis.
An interesting combination with Axisymmetric Drop Shape
Analysis-Profile (ADSA-P) is presented in Ref. [45], where it is
shown that it can be used to monitor the adsorption of human
serum albumin to a polystyrene coating.
2.5. SRIB/IRIS
The Spectral Reflectance Imaging Biosensor/Interferometric
Reflectance Imaging Sensor (SRIB/IRIS) has undergone a lot of
Fig. 6. Total Reflectometric Interference Spectroscopy (TRIS) TRIS is realised by using a
white light source (1), which is focussed onto a polarizer (2). The beam (dotted line) enters
a prism (3) and is then reflected at a reflective glass substrate (4 dark grey) containing the
sensitive layer (4 light grey). The interference pattern is monitored by a spectrometer (5).
5
Trends in Analytical Chemistry 156 (2022) 116708
changes e not only considering its name e from its first notable
publication in 2008 [46] to today [47]. Accordingly, the realisation
of this method has also been modified throughout these years. In
the beginning, a tuneable laser was used as light source, which has
been replaced with an LED now. The detection is realised by a CCD
camera (see Fig. 7). The main difference of this method to RIfS, is
that in TRIS only a small part of the spectrum is used and therefore
change of reflectivity is measured. The main difference of this
method compared to 1ʎ Reflectometry, is the fact that the illumi-
nation and detection does occur on the frontside of the transducer
(unlike RIfS, TRIS or 1ʎ Reflectometry). This means that a trans-
parent flowcell must be used, and the light beams have to travel
through the sample medium twice, the latter making the method
vulnerable to matrix effects if not properly referenced.
Besides being able to read out protein-microarrays label-free,
the IRIS technology has successfully been used to detect small
molecules [48], DNA/DNA [49] and DNA/protein [50] interactions,
and viruses [51] (the latter, however, not under flow through
conditions).
2.6. 1ʎ Reflectometry
1ʎ Reflectometry, rarely also referenced as “SCORE (Single
Colour Reflectometry)”, is a simplified version of the RIfS. which
uses monochromatic light and therefore also measures reflectivity.
The main difference is that the light source produces (quasi-)
monochromatic light instead of white light. This enables the
method to utilize a simple photodiode instead of an entire spec-
trometer, and consequently parallelization can be achieved by
replacing the fibre optics with an objective and the detector with a
CCD or CMOS camera (see Fig. 8).
However, a drawback of this method is that a successful real-
isation requires a thorough understanding of this method since not
every wavelength chosen is equally suitable for detecting the bio-
molecular interaction of interest. Usually, the layer system of the
transducer can be easily tailored to a commercially available
monochromatic light source to ensure a reliable detection of a
range of biomolecules (e.g. proteins, oligonucleotides, etc.) which
are within the range of molecular weight. For bigger particles (e.g.
viruses, nanoparticles [52], cells, etc.) it might be beneficial to
adjust the system to a different wavelength.
Fig. 7. Interferometric Reflectance Imaging Sensor (IRIS) A monochromatic light
source, e.g. LED (1) is reflected at a beam splitter (2a). The monochromatic light (dotted
line) passes the sensitive layer (3 light grey) before it is reflected at the substrate (3
dark grey) and passes the sensitive layer a second time. After the light passes the first
beam splitter (2a), it is reflected by the second beam splitter or mirror (2b) and
detected by a camera (3).
P. Fechner, G. Gauglitz and G. Proll
Fig. 8. 1ʎ Reflectometry A monochromatic light source, typically LED (1) is used to
illuminate a transparent substrate (4 dark grey) containing a sensitive layer (4 light
grey). Therefore, it is first reflected at a beam splitter (2), then the monochromatic light
(dotted line) passes a ʎ/2 plate (3) before being illuminating the transducer sensitive
layer (4). After the reflected light passes the ʎ/2 plate (3) again, it is guided by beam
splitter (2a) towards its detection by a camera (5).
One of the first realisations was described in Ref. [53] where
biomolecular interaction analysis was performed on transparent
plastics in combination with a simple photodiode read-out.
Single analyte applications have been used to quantify anti-
salmonella antibodies in animal sera [54] and multi-analyte ap-
plications have been achieved by simply moving the detection unit
to a laterally resolved second analyte spot on the same transducer
at the cost of a lower time resolution [55]. Imaging setups using
cameras allow labeldfree HTS screening [56]. While the possible
maximum throughput scales with resolution of the camera it has to
be noted that the practical throughput might be limited by the
availability of high-density arrays [57]. Recent developments
include the usage of polarised light and detection under an angle
for better signal-to-noise ratio [58,59], multiplexing via DMD [60],
and cell monitoring [61].
In summary, 1ʎ Reflectometry has shown to be competitive with
other label-free optical biosensor techniques such as BLI and Sur-
face Plasmon Resonance (SPR), but with better scalability [62]. A
main advantage is the (almost) free scalability of the imaging
approach according to the needs and throughput of an assay.
2.7. AIR
Arrayed Imaging Reflectometry (AIR) is e as the name implies e
Fig. 9. Arrayed Imaging Reflectometry (AIR) The beam (dotted line) of a mono-
chromatic light source, e.g. laser (1) passes a polarizer (2) and then hits a reflective
substrate (3 dark grey) containing a sensitive layer (3 light grey) under a fixed
polarisation and angle. Upon changes in the sensitive layer, the reflected light is
detected by a camera (4).
6
Trends in Analytical Chemistry 156 (2022) 116708
Fig. 10. Oblique-Incidence Reflectivity Difference microscopy (OI-RD) The beam
(dotted line) of a polarized laser (1) passes a photoelastic modulator (2) and a phase
shifter (3) before reflection of a transparent substrate (4 dark grey) containing a
sensitive layer (4 light grey). After passing an analyser (5), the reflected beam is
detected by a photodiode (6).
a reflectometric method based on an elegant realisation of
destructive interference to enhance label-free detection of protein/
protein interactions [63].
The approach is realised by a laser light source which illumi-
nates a Si/SiO2 substrate under a fixed angle and polarisation. This
light is then detected by a camera. But here comes the twist: The
incidence angle, wavelength and layer system of the substrate are
designed in a way which results in near-zero reflectance [64].
Binding of proteins to this substrate then disturbs these zero
reflectance conditions, which increases the amount of reflected
light detected by the camera (see Fig. 9).
This approach has some metrology advantages, such as good
limits of detection [65]. On the other hand, the technique is not as
flexible in its range of analytes, since the transducer substrate
would have to be adjusted to the size of each analyte for optimal
working conditions; in addition, it works under stopped-flow
conditions, therefore rate constants cannot be accessed with this
technique. The obtained affinity constants are comparable to other
label-free techniques [66].
With these boundaries, this technique shines especially in
antibody screening approaches [67,68] and recently in SARS CoV-2
serology [69], the latter showing how this technique allows
discrimination and quantification of antibody levels against
different influenza and corona viruses.
2.8. OI-RD
Oblique-Incidence Reflectivity Difference microscopy (OI-RD)
can be seen as another variant of an ellipsometry approach with a
few modifications to tailor it more to biomolecular interaction
monitoring. In brief, a laser is guided through a photoelastic
modulator and a Pockels cell to illuminate a glass substrate from
the backside. The reflected light passes through an analyser, and its
intensity is recorded by a photodiode with a FT-lock-in (see Fig. 10).
The technique has shown its potential in high throughput pro-
tein micro array screening [70] by detection of 10,000 protein in-
teractions label-free and time-resolved. It has been used for
characterization of oligosaccharide-binding proteins using carbo-
hydrate microarrays [71]. Other applications include the moni-
toring of protein degradation by proteinases [72] or adaption of the
sandwich assay format [73]. Notably, this technique was also used
to monitor binding of antibodies to living cells [74], which shows
the advantage of reflectometric approaches (in contrast to
evanescent field-based techniques) when dealing with large
structures.
Since OI-RD is a scanning approach, this technique naturally
suffers from limited time-resolution when the detection (array)
area increases. Nevertheless, it is still capable to generate data
suitable for the determination of kinetic rate constants [75].
P. Fechner, G. Gauglitz and G. Proll
2.9. Comparison
Table 1
Comparison of the key features of the different approaches.
Ellipsometry RIfS
Spectral þ/ þ þ
Time resolution o þ
Multiplex þ o
Commercially available þ e
Small molecules þ o
Proteins þþ þþ
Cells o þ
3. Conclusion
After having learned about all these types of reflectometric
techniques, approaches, and realisations you might now ask:
Which one is the best? And unsurprisingly and unsatisfactory:
There is no correct answer. A point of reference when deciding in
favour or against a transduction method can be the detection limit.
However, in biological assays this is inevitably linked to the affinity
between receptor and ligand (and many other factors such as flu-
idics, surface loading etc.), so that a comparison will always be
difficult even if a concentration value in mol/L is given. Although a
specification for all methods in terms of the minimum detectable
mass/substance per surface area (e.g. pg/mm2) would provide more
comparability, this value is not always readily available. For this
reason, an attempt has been made in Table 1 to provide an overview
of which areas of application have already been demonstrated with
a method.
One non-negligible trend is surely the progression of imaging
approaches. This development is especially driven by the ongoing
growth of the mobile market and sensor chips for its camera
modules. Therefore, we expect an ongoing improvement of tech-
niques using imaging approaches instead of photodiodes or
spectra. In this context, the intrinsic possibility of referencing to
reference areas on a sensor surface as well as the simple addition of
intra-assay replicates provides added value for users.
However, setting up a successful biosensor device is e apart
from the capabilities of the detection method e largely depending
on other factors such as costs, available surface chemistries and
usability, the latter not including the instrumentation itself, but
also the software and its user-friendliness.
Versatility usually comes at the cost of performance and vice
versa. While highly specialised approaches promise to excel in one
field, e.g. low limits in protein detection with highly optimised layer
substrates using AIR, they lose a lot of performance in other
application fields. The scientist has to decide which approach is
best suited for their field of research.
In general, the micro-reflectometric methods which determine
the optical thickness as the product of n and d as a measurand,
exhibit greater stability to temperature fluctuations. Conversely, a
simpler and more robust thermostatting to a desired temperature
for a biological assay is therefore also possible, since small techni-
cally induced temperature fluctuations (which are insignificant for
reaction performance) do not have a negative effect on the mea-
surement signal. Furthermore, this is a significant advantage of
reflectometry when it comes to miniaturisation, which is manda-
tory for e.g. point-of-care testing (POCT).
BLI, utilizing the well accepted microtiter plate format, being
commercially available and having a well-established software, is
having an intrinsic advantage when it comes to integration in
Trends in Analytical Chemistry 156 (2022) 116708
BLI TRIS IRIS 1ʎ Reflectometry AIR OI-RD
þ e e e e
þ þ þ þþ e þ
þ o þþ þþ þ þþ
þ e þ þ þ e
þ o o þ o o
þþ þþ þþ þþ þþ þþ
o o o þ o þ
existing workflows. However, when it comes to the determination
of kinetic rate constants, which is one of the key advantages of a
label-free technique, flow-through techniques usually generate
more valid constants.
In terms of high throughput, OI-RD and 1ʎ Reflectometry are
very promising approaches while allowing for accurate determi-
nation of kinetic rate constants due to having more sophisticated
fluidics compared to microtiter-plate-based approaches or even
offline incubation. However, it should be noted at this point that the
sole generation of thousands of sensorgrams from a single mea-
surement with such an HTS method also inevitably entails an
evaluation and assessment of this mass of data. It is hoped that new
evaluation programs from bioinformatics and even artificial intel-
ligence methods will be used to develop tools that will help this
biosensor analysis to be used in real life in further complex mea-
surement campaigns.
It will certainly remain exciting to see how this family of micro-
reflectometric biosensor methods will continue to develop. Besides
the already mentioned continuous improvements of available de-
tectors like CMOS cameras, it is the combination of nanomaterials
on the transducer side, partly with new surface chemistries or
immobilization strategies, as described in recent work, which will
enable the processing of more and more complex biological/
biochemical questions. For example, special optical properties of
transducers with particularly favourable surface-to-volume ratios
can be used, which at the same time allow e.g. new and better
stabilisation strategies of complex biomolecules. These trends
again show how interdisciplinary the development of biosensor
methods (not only micro-reflectometric ones) is.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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